No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Endogenous interference in imunoassays in clinical chemistry. A rewiev
Abstract
The increasing availability and use of immunoassays in clinical chemistry have revealed a number of endogenous interferences. Solid-phase sandwich immunoassays based on monoclonal antibodies are particularly sensitive to any factor able to bridge immunoglobulins together. Heterophilic immunoglobulin antibodies have been demonstrated in up to 40% of patient samples and to cause spuriously elevated results unless certain precautions are taken. Rheumatoid factors belong to the same category, but their affinity is usually too low to cause significant interference. Immunoscintigraphy generates high-titre anti-immunoglobulin responses causing serious interferences in immunoassays. Recently interfering factors of unknown nature causing nonspecific binding of enzyme-labelled antibodies have been observed. Spuriously decreased values can be caused by complement, which may interfere with antigen-binding to solid phase antibody. The aforementioned and other endogenous interferences in immunoassays are reviewed and methods for their elimination discussed.
... Serum dilution, to some extent can eliminate the complement interference or prozone effect observed in the case of high antibody titers, thus can improve the sensitivity of MIA [21]. On the other hand increased serum dilution can lead to decreased sensitivity [23]. WNV E-MIA was conducted at 1:100 dilution of serum [8,10,11], whereas other MIA employed lower serum dilution (1:20), as it resulted in the low levels of non-specific background with high dynamic range of signal intensities [24,25]. ...
... Another important parameter that can affect the performance of Luminex-based serological assays is the dilution of the serum. Serum dilution, though it contributes to low background signal, can also lead to decrease in sensitivity of the Luminex assay [23] and false-negatives could potentially exist [35]. In the previous studies, WNV E-MIA was conducted at a serum dilution of 1:100 as it provided low background binding and optimal assay results [8,10,11,36]. ...
... This observed change in MFI may be due to the prozone effect or high-dose hook effect as a result of high antibody titers in serum or the complement interference in the NHI serum. Prozone effect and complement interference in the immunoassays can be eliminated by sufficient dilution of the serum [23,37], but this can also lead to decrease in sensitivity of the assay, as observed for IgM antibodies (Figure 2A). Therefore, we determined 1:20 to be the optimal dilution of HI serum for WNV E-MIA that can detect low-titer IgM and IgG antibodies during early and late time-points after infection. ...
Immunopathogenesis studies employing West Nile virus (WNV) mice model are important for the development of antivirals and vaccines against WNV. Since antibodies produced in mice early during WNV infection are essential for clearing virus from the periphery, it is important to detect early and persistent anti-WNV antibodies. ELISA and plaque reduction neutralization tests are traditionally used for detection of anti-WNV antibodies and WNV-neutralizing antibodies, respectively. Although these assays are sensitive and specific, they are expensive and time consuming. Microsphere immunoassays (MIA) are sensitive, specific, allow for high throughput, are cost effective, require less time to perform than other methods, and require low serum volumes. Several assay parameters such as serum heat-inactivation (HI) and dilution can alter WNV MIA sensitivity. We examined the effect of these parameters on WNV E-protein MIA (WNV E-MIA) for the enhanced detection of anti-WNV IgM and IgG antibodies. WNV E-MIA was conducted using serial dilutions of HI and non-HI (NHI) serum collected at various time points from mice inoculated with WNV. HI significantly enhanced detection of IgM and IgG antibodies as compared to NHI serum. WNV IgM and IgG antibodies in HI sera were detected earlier at day 3 and IgM antibodies persisted up to day 24 after infection. HI serum at 1∶20 dilution was found to be optimal for detection of both IgM and IgG antibodies as compared to higher-serum dilutions. Further, addition of exogenous complement to the HI serum decreased the WNV E-MIA sensitivity. These results suggest that serum-HI and optimal dilution enhance WNV E-MIA sensitivity by eliminating the complement interference, thereby detecting low-titer anti-WNV antibodies during early and late phases of infection. This improved MIA can also be readily employed for detection of low-titer antibodies for detection of other infectious agents and host proteins.
... Serum dilution helps eliminate the influence of complement proteins or the prozone effect observed in the case of high antibody titers and thus may improve the sensitivity of MIA [28]. Meanwhile, increased serum dilution can decrease sensitivity [29]. MIA of WNV was performed at a serum dilution of 1:100 [30], whereas other ...
... Serum dilution helps eliminate the influence of complement proteins or the prozone effect observed in the case of high antibody titers and thus may improve the sensitivity of MIA [28]. Meanwhile, increased serum dilution can decrease sensitivity [29]. MIA of WNV was performed at a serum dilution of 1:100 [30], whereas other MIAs used a lower serum dilution (1:20) as this resulted in low levels of nonspecific background signals within a high dynamic range of signal intensities [31]. ...
This work demonstrates the use of a modified mica to concentrate proteins, which is required for proteomic profiling of blood plasma by mass spectrometry (MS). The surface of mica substrates, which are routinely used in atomic force microscopy (AFM), was modified with a photocrosslinker to allow “irreversible” binding of proteins via covalent bond formation. This modified substrate was called the AFM chip. This study aimed to determine the role of the surface and crosslinker in the efficient concentration of various types of proteins in plasma over a wide concentration range. The substrate surface was modified with a 4-benzoylbenzoic acid N-succinimidyl ester (SuccBB) photocrosslinker, activated by UV irradiation. AFM chips were incubated with plasma samples from a healthy volunteer at various dilution ratios (102X, 104X, and 106X). Control experiments were performed without UV irradiation to evaluate the contribution of physical protein adsorption to the concentration efficiency. AFM imaging confirmed the presence of protein layers on the chip surface after incubation with the samples. MS analysis of different samples indicated that the proteomic profile of the AFM-visualized layers contained common and unique proteins. In the working series of experiments, 228 proteins were identified on the chip surface for all samples, and 21 proteins were not identified in the control series. In the control series, a total of 220 proteins were identified on the chip surface, seven of which were not found in the working series. In plasma samples at various dilution ratios, a total of 146 proteins were identified without the concentration step, while 17 proteins were not detected in the series using AFM chips. The introduction of a concentration step using AFM chips allowed us to identify more proteins than in plasma samples without this step. We found that AFM chips with a modified surface facilitate the efficient concentration of proteins owing to the adsorption factor and the formation of covalent bonds between the proteins and the chip surface. The results of our study can be applied in the development of highly sensitive analytical systems for determining the complete composition of the plasma proteome.
... Notably the detectable differences between HI and NHI sera were only significant for several dilutions with high rh-sTREM-1 concentrations in the Radsak ELISA (Fig. 3). Therefore improved detection of sTREM-1 through dilution of serum could be assumed, nevertheless increased serum dilution can also lead to decreased sensitivity 18 . Focusing on the recovery rates and effects of heat-inactivation, we observed analogue results between Radsak and R&D ELISA. ...
... Particularly endogenous factors are difficult to detect and eliminate, since they vary from patient to patient and also from time to time in one patient. Amongst others these endogenous factors include hyperlipidemia and nonesterified fatty acids, crossreacting heterophilic anti-immunoglobulin antibodies and complement 18 . In addition, differences in sample logistics are well-known to have an impact on the outcome of assays, especially for biomarkers that are frequently influenced by complement. ...
Triggering receptor expressed on myeloid cells (TREM)-1 plays an important role in innate immune responses and is upregulated under infectious as well as non-infectious conditions. In addition, a soluble TREM-1 variant (sTREM-1) is detectable in sera or bronchoalveolar-lavage fluids from patients. Currently, various studies are difficult to compare, since the methods of detection by enzyme-linked immunosorbent assays (ELISA) vary among different research groups. In this study, we compared three different s-TREM-1 specific ELISAs and identified individual assay characteristics finding notable differences in sTREM-1 concentrations in part depending on the employed buffers. Investigating potential confounding factors for sTREM-1 detection, serum heat-inactivation (HI) showed improved recovery compared to non-HI (NHI) serum, reproducible by addition of complement and re-heat-inactivation. Hence we identified complement as a heat-sensitive confounder in some sTREM-1 ELISAs. We conclude that it is difficult to directly compare data of several studies, in particular if different ELISAs are engaged. Immunoassays for research use only are in general hampered by lack of standardization. Further standardization is needed until sTREM-1 ELISA is capable for better reproducibility of studies and clinical application.
... Endogenous antibodies produced in this way can bind to different epitopes involving the antigen/analyte, with varying binding affinities. Endogenous antibodies capable of interference in immunoassays are therefore not uncommon and various studies have placed their prevalence at 30-40% in the population [76][77][78]. They probably arise from commonplace activities such as keeping pets, ingesting animal antigens, vaccination, infection, and blood transfusion [34,79]. ...
... However, in some cases, large amounts of low-affinity antibody (which may be in concentrations as high as g/l) or relatively low concentrations of antibody with highbinding affinity can and do overwhelm the analytical system, leading to assay interference and erroneous results; in our laboratory, a figure of 0.5% was found [81] of which 80% were clinically misleading. Furthermore, the prevalence of this type of interference is likely to worsen in the future because of the emergence of immunotherapy [77,[82][83][84][85][86][87] in the treatment of a wide range of conditions and the use of radiolabeled antibodies in diagnosis using immunoscintigraphic procedures [88,89] The most prolific use of monoclonal antibodies is likely to be in the immunotherapy of some viral infections, inflammatory disorders such as rheumatoid arthritis (RA), and in patients with hematological malignancies and solid tumors [82,90] Monoclonal antibodies of murine origin are used for the selective delivery of drugs to cancer cells. Humanization of mouse monoclonal antibodies to give a chimeric molecule reduces but does not eliminate an immune response. ...
The primary role of the clinical laboratory is to report accurate results for diagnosis of disease and management of illnesses. This goal has, to a large extent been achieved for routine biochemical tests, but not for immunoassays which remained susceptible to interference from endogenous immunoglobulin antibodies, causing false, and clinically misleading results. Clinicians regard all abnormal results including false ones as “pathological” necessitating further investigations, or concluding iniquitous diagnosis. Even more seriously, “false-negative” results may wrongly exclude pathology, thus denying patients’ necessary treatment. Analytical error rate in immunoassays is relatively high, ranging from 0.4% to 4.0%. Because analytical interference from endogenous antibodies is confined to individuals’ sera, it can be inconspicuous, pernicious, sporadic, and insidious because it cannot be detected by internal or external quality assessment procedures. An approach based on Bayesian reasoning can enhance the robustness of clinical validation in highlighting potentially erroneous immunoassay results. When this rational clinical/statistical approach is followed by analytical affirmative follow-up tests, it can help identifying inaccurate and clinically misleading immunoassay data even when they appear plausible and “not-unreasonable.”
... Biological samples for these assessments are complex matrices and contain various components that can significantly impact the accuracy of analyte quantification. Interference has been discussed previously in excellent reviews with a focus on the clinical laboratory setting [1][2][3]. Many assays used during drug development are custom developed and less characterized. ...
... A type of interference that deserves special attention are antibodies in the sample [3]. Heterophilic anti bodies have in general broad reactivity and low affinity and are able to crosslink capture and detection antibody leading to falsely high results [4]. ...
During preclinical and clinical studies, immunoassays are used to measure the concentration of the therapeutic antibody, anti-drug antibodies and soluble protein biomarkers. The reliability of these assays is crucial since the results are routinely used for safety assessment and dose selection. Furthermore, soluble protein biomarkers can provide information about target engagement, proof of mechanism, proof of principle and prediction of response. Study samples mostly consist of complex matrices that can exhibit considerable interference, resulting in inaccurate measurements. This perspective discusses the source of interference and strategies to mitigate or eliminate interference in immunoassays used during preclinical and clinical drug development of drugs with a focus on the development of therapeutic antibodies.
... Plapp et al. (18 ) suggested that QC materials be composed of the same matrix as the specimens. With human serum as diluent, problems may arise for the following reasons: (a) the presence of infectious agents that cannot be screened; (b) batch-to-batch variability attributable to endogenous factors (19 ); and (c) a finite shelf life that may fall short of the time spans of clinical studies. To minimize the variability for pharmacokinetic profile studies, a buffer that could be substituted for human serum as an assay diluent was sought. ...
... Therefore, development was focused on the normal concentration of albumin to maintain a total protein content similar to the low end of the HSA reference interval. Weber et al. (19 ) suggested that the addition of bovine serum might eliminate interference of heterophilic antibodies. After comparing the absorbance and slope obtained for bovine serum with those obtained for human serum, we chose 30 mL/L HSA with 100 mL/L FBS as the serum substitute and diluent for AMGN-0007 assays. ...
... Blood collection tubes are not inert containers but have several constituents, including substances in and/or applied to rubber stoppers, tube wall material, surfactants, anticoagulants, separator gels and clot activators that can potentially interfere with immunoassays (Weber, 1990). Many laboratories have converted from glass to plastic collection tubes for convenience. ...
... Albumin may interfere as a result of its high concentration and its ability to bind or release large proportions of ligand. Complement binds to the Fc fragment of immunoglobulins and can block the analyte-specific binding sites of antibodies (Weber et al., 1990). Lysozyme can form a bridge between the solid-phase IgG and the detector antibody (Selby, 1999). ...
... Bilirubin (171 µmol/L) có thể ảnh hưởng tới định lượng acetaminophen và vancomycin. Lipid máu tạo ra độ đục gây chênh lệch kết quả đo phổ và sắc kí, vì vậy cần bị loại bỏ thông qua li tâm siêu tốc; protein niệu có thể ảnh hưởng tới sự gắn của chất phân tích với kháng thể trong quy trình định lượng miễn dịch [16,24,25]. ...
Therapeutic drug monitoring (TDM) and model-informed precision dosing (MIPD) have become effective methods in determining and adjusting dose regimens of various drugs in clinical practice. With the latest development in technology, particularly in computer science, drug individualization has evolved to be more flexible and precise. However, these methods still have certain drawbacks and have not been applied on a grand scale. This review shall present the most basic concept of TDM and MIPD as well as current issues in their application.
... 2,3 According to the current literature, this so-called matrix interference or effect can be caused by different components; blood cells, sample viscosity, or plasma components such as heterophilic antibodies (antibodies produced against poorly defined antigens presenting low affinity and weak binding), 4 human antianimal antibodies (HAAA, high-affinity antibodies generated when the immune system is in contact with animal antibodies), 5 and other plasma proteins such as albumin, lysozyme, fibrinogen, and paraprotein have been reported to cause test interferences. 6 Boscato et al. showed that analyte−antibody binding substances were detected in 40% of studied samples (688 samples), being responsible for 15% interference in assays. 7 Appropriate matrix management is therefore essential to develop reliable bioassays and biosensors; however, this is highly dependent on the molecular analysis platform since the type of reagents (e.g., antibody purity) and the antibody binding conditions (e.g., antibody affinity, diffusion distance, surface interactions) are key contributors to the matrix effect. ...
... The prevalence of hA in serum samples was reported to vary from 3% to 40% [16,22]. However, clinically more important is the prevalence of disturbed PSA levels due to hA in routine PSA assessment. ...
Background
Prostate-specific antigen (PSA) test is of paramount importance as a diagnostic tool for the detection and monitoring of patients with prostate cancer. In the presence of interfering factors such as heterophilic antibodies or anti-PSA antibodies the PSA test can yield significantly falsified results. The prevalence of these factors is unknown.
Methods
We determined the recovery of PSA concentrations diluting patient samples with a standard serum of known PSA concentration. Based on the frequency distribution of recoveries in a pre-study on 268 samples, samples with recoveries <80% or >120% were defined as suspect, re-tested and further characterized to identify the cause of interference.
Results
A total of 1158 consecutive serum samples were analyzed. Four samples (0.3%) showed reproducibly disturbed recoveries of 10%, 68%, 166% and 4441%. In three samples heterophilic antibodies were identified as the probable cause, in the fourth anti-PSA-autoantibodies. The very low recovery caused by the latter interference was confirmed in serum, as well as heparin- and EDTA plasma of blood samples obtained 6 months later. Analysis by eight different immunoassays showed recoveries ranging between <10% and 80%. In a follow-up study of 212 random plasma samples we found seven samples with autoantibodies against PSA which however did not show any disturbed PSA recovery.
Conclusions
About 0.3% of PSA determinations by the electrochemiluminescence assay (ECLIA) of Roche diagnostics are disturbed by heterophilic or anti-PSA autoantibodies. Although they are rare, these interferences can cause relevant misinterpretations of a PSA test result.
... RF has been shown to interfere with free and total thyroid hormone tests [87] as well as TSH [317] and Tg [318]. HAbs have a prevalence of 30-40 percent [319][320][321] and have the potential to interfere with a broad range of methods that use IMA principles [290, 300,306,322]. In recent years assay manufacturers have increased the immunoglobulin blocker reagents added to their tests and this has reduced interference from 2 to 5 percent [290, 297,323]. ...
This chapter reviews how improvements in sensitivity and specificity of thyroid function tests [total and free thyroid hormones, TSH, thyroid autoantibodies (TRAb, TPOAb and TgAb) and thyroglobulin (Tg)] have dramatically improved clinical strategies for detecting and treating thyroid disorders. The review discusses the strengths and limitations of the different methodologies currently used (RIA, IMA and LC-MS/MS) and their propensity for analyte-specific interferences caused by heterogeneity (TSH, TgAb and Tg) or analyte-specific autoantibodies (T4Ab, T3Ab, TSHAb and TgAb). In addition, non-analyte related interferences from heterophile antibodies, including human anti-mouse antibodies (HAMA) and Rheumatoid Factor (RF), and interferences related to the use of Biotin and Streptavidin reagents, are discussed. The review provides an update on collaborations between the International Federation of Clinical Chemistry (IFCC) committee for the standardization of thyroid function tests (C-STFT) and the in-vitro diagnostic (IVD) industry-the goal being to eliminate between-method biases. Although re-standardization of thyroid hormone tests against established reference measurement procedures, and harmonization of TSH tests to the all-method mean has proved effective, recalibration has yet to be implemented by the IVD. Until between-method biases are eliminated, it is not feasible to propose universal reference ranges that would apply across methods. The review contains a comprehensive discussion of the clinical utility of Tg methodology (RIA, IMA or LC-MS/MS), used to monitor patients with differentiated thyroid cancer (DTC). Mechanisms for in-vitro and possible in-vivo TgAb interference with Tg testing are proposed. The methodologic and clinical strengths and weakness of each test are discussed relative to current guidelines.For complete coverage of this and related areas in Endocrinolofy, visit our free web-books, www.endotext.org and www.thyroidmanager.org.
... This increase during storage may be due to a dissociation of the AMH molecule (Rustamov et al., 2012). Another potential cause is interference by complement (Weber, Kapyaho, & Tanner, 1990 In the study by Place and co-workers, using the DSL assay, one spayed bitch was considered having a falsely high AMH concentration, being a statistical outlier (Place et al., 2011). In our laboratory, we have had single case of high concentrations of AMH in neutered males/ spayed female dogs. ...
Contents
During the last decade, analysis of anti‐Müllerian hormone ( AMH ), highly conserved between mammalian species, has contributed to new information in reproductive endocrinology, due to clinically available diagnostic assays. AMH is produced solely in the gonads, in the Sertoli cells of testes and granulosa cells of the ovary, and thus offers possibilities to diagnose physiologic and pathologic conditions involving these organs. This article reviews indications for AMH analysis in cats and dogs, including diagnosing the presence of gonads, and granulosa or Sertoli cell tumours. Diagnostic challenges are addressed. One specific organ, the prostate, is commonly affected by pathologic changes in older dogs. A commercial assay for analysing canine prostatic specific esterase ( CPSE ) enables analysis of CPSE in clinical practice, of potential value in the workup of benign prostatic hyperplasia in male dogs. This is described in this review, as is a new method for analysis of steroids: liquid chromatography‐tandem mass spectrometry LC ‐ MS / MS . Steroids have since long been analysed in studies on reproduction, and LC ‐ MS / MS has the advantage of allowing analysis of panels of multiple steroids from small sample volumes. Altogether, these available methods may give new insights into small animal reproduction and are valuable tools for the practicing veterinarian.
... However, RIA has some major drawbacks including poor inter-batch intermediate precision, and the need to take highly stringent and costly precautions for the use and disposal of radioactive material. In addition, this method based on immunocompetition is highly susceptible to interference and liable to generate cross-reactions with metabolized fragments, circulating receptors and other analogous endogenous proteins 28,29 . False-positive or negative results may therefore occur and the concentrations recorded may not reflect the actual endogenous values due to a lack of specificity and the variability of the origins and purity of the standards used to generate calibration curves 30 . ...
I125 radioimmunoassay (RIA) is currently the standard technique for quantifying cerebrospinal fluid (CSF) orexin-A/hypocretin-1, a biomarker used to diagnose narcolepsy type 1. However, orexin-A RIA is liable to undergo cross-reactions with matrix constituents generating interference, high variability between batches, low precision and accuracy, and requires special radioactivity precautions. Here we developed the first quantitative mass spectrometry assay of orexin-A based on a multiple reaction monitoring (MRM) approach. This method was tested in keeping with the Clinical and Laboratory Standards Institute (CLSI) guidelines and its clinical relevance was confirmed by comparing patients with narcolepsy type 1 versus patients with other neurological conditions. The results obtained using MRM and RIA methods were highly correlated, and Bland–Altman analysis established their interchangeability. However, the MRM values had a wider distribution and were 2.5 time lower than the RIA findings. In conclusion, this method of assay provides a useful alternative to RIA to quantify orexin-A, and may well replace it not only in narcolepsy type 1, but also in the increasing number of pathologies in which the quantification of this analyte is relevant.
... Analytical interference in immunoassays should be considered whenever unexpected results are obtained (Davies, 2013;Hoofnagle and Wener, 2009;Selby, 1999;Tate and Ward, 2004;Weber et al., 1990). Despite being a well-recognized issue in mainly solid phase immunoassays, relatively few scientific publications have described or suggested complement interference as a cause of inaccurate (mainly falsely low) results in clinical laboratory assays (Börmer, 1989;Borque Fig. 2. Bland-Altman (relative difference) plot showing the relative CMV IgG difference (%) between the EDTA treated and untreated sera on VIDAS. ...
In this study we showed that complement factors are responsible for assay interference in the VIDAS CMV IgG assay. Three different serum treatments were applied to show the cause of interference: heat treatment (56°C), adding cobra venom factor and adding EDTA. Elimination of complement interference by EDTA treatment of serum was prospectively evaluated on 215 CMV IgM positive samples and a sensitivity increase of the VIDAS CMV IgG assay was noticed. On average the CMV IgG level increased 100% after EDTA treatment of the serum. In paired serum samples from 38 patients we could show that serum treatment with EDTA can make the CMV IgG level changes more obvious in recent CMV infections. Since the CMV IgG avidity II assay on VIDAS depends on the determination of CMV IgG, the CMV IgG avidity was also evaluated in this study but only a limited effect of the complement interference was observed.
... Interf erence in modern immunoassays caused by heterophilic antibodies (HA) is well documented [1]. According to various published reports, in the general population these antibodies can be found in up to 40% of clinical samples [2]. In many cases, these antibodies have nonspecific affinities and do not interfere with different immunoassays. ...
Heterophilic antibodies are human immunoglobulins directed against various animal antigens. They can produce false-positive results in the analysis of different tumor markers, including prostate-specific antigen. This interference can lead to misdiagnosis, unnecessary tests, and overtreatment in some cases. We present herein the case of a 52-year-old man with repeated spurious elevation of prostate-specific antigen, reaching levels of 108.7 ng/mL, that were suspected to be caused by heterophilic antibodies. The interference was solved by changing the analysis technique. Real values of prostate-specific antigen were less than 1 ng/mL.
... The most frequently described interfering mechanisms are those caused by heterophilic antibodies, human anti-mouse antibodies, rheumatoid factors, and complement [reviewed in Refs. (19,20 )]. Introduction of blocking agents (e.g., mouse serum or IgG) is often useful in eliminating this type of interference (21,22 ). ...
Background: Cardiac troponin I (cTnI) is a sensitive marker of cardiac injury, but cTnI assays, like other immunoassays, are susceptible to interferences. We evaluated the presence of interfering substances by measuring the recovery of cTnI added to samples from volunteers and from patients with acute coronary syndromes (ACS).
Methods: We added a ternary complex of human cardiac troponin (30–500 μg/L) or cTnI from serum to samples from healthy volunteers and ACS patients. We measured cTnI with a two-site sandwich time-resolved immunofluorometric assay using two antibodies against epitopes in the central stable part of cTnI. We also analyzed 108 heparin-plasma samples from 16 ACS patients with this assay, with an assay based on four antibodies, and with two commercial cTnI assays, AxSYM and ACS:180.
Results: In samples from both healthy persons and ACS patients, recoveries for our assay were 1–167% (range). Recoveries were increased by addition of an antibody with an epitope in the N-terminal region of cTnI to the solid phase and an antibody with an epitope in the C-terminal region as a second detection antibody. In 2 of 16 patients with ACS, normal cTnI concentrations found when measured with the original assay demonstrated clinically abnormal (up to 10-fold higher) results with the additional N- and C-terminal antibodies in the early phase of infarction. Both commercial cTnI assays also demonstrated clinically misleading, falsely low cTnI concentrations.
Conclusions: Some yet unidentified, variable component, present in the blood from healthy volunteers and ACS patients, interferes with the binding of antibodies against epitopes in the central part of cTnI used in two commercial assays. Our approach to supplement the mid-fragment cTnI antibodies with antibodies in the N- and C-terminal parts of the molecule in an experimental assay represents a step in resolving this interferent.
... Since antibodies cross the placenta, these HAbs have the potential to interfere with the diagnostic accuracy of neonatal screening tests (596). Although the prevalence of interfering antibodies can be as high as 30-50 percent, manufacturers typically develop their assays to include reagents that block or neutralize most interferences (459,(597)(598)(599). The prevalence of such interference is difficult to estimate employing present methodologies but is currently estimated to range between 0.03 and 3.0 percent (457,598). ...
... Also, as Ca 2+ ions are needed to activate the complement the use of EDTA plasma prevents the interference caused by the complement. (Kapyaho et al., 1989;Weber et al., 1990). ...
... This method not only measures fPSA and PSA in complex with a 1 -ACT on an equimolar basis, but also overcomes some problems inherent in assays based on intact monoclonal antibodies [6]. The use of an equimolar measurement was reported to reduce false positive and false negative results by eliminating the effects of heterophilic antibodies, and complement and rheumatoid factors [7,18]. In our study, with non-equimolar tPSA measurement performed by monoclonal antibodies, approximately one third of patients with BPH had to undergo a biopsy of the prostate. ...
... This method not only measures fPSA and PSA in complex with a 1 -ACT on an equimolar basis, but also overcomes some problems inherent in assays based on intact monoclonal antibodies [6]. The use of an equimolar measurement was reported to reduce false positive and false negative results by eliminating the effects of heterophilic antibodies, and complement and rheumatoid factors [7,18]. In our study, with non-equimolar tPSA measurement performed by monoclonal antibodies, approximately one third of patients with BPH had to undergo a biopsy of the prostate. ...
... This method not only measures fPSA and PSA in complex with a 1 -ACT on an equimolar basis, but also overcomes some problems inherent in assays based on intact monoclonal antibodies [6]. The use of an equimolar measurement was reported to reduce false positive and false negative results by eliminating the effects of heterophilic antibodies, and complement and rheumatoid factors [7,18]. In our study, with non-equimolar tPSA measurement performed by monoclonal antibodies, approximately one third of patients with BPH had to undergo a biopsy of the prostate. ...
... This method not only measures fPSA and PSA in complex with a 1 -ACT on an equimolar basis, but also overcomes some problems inherent in assays based on intact monoclonal antibodies [6]. The use of an equimolar measurement was reported to reduce false positive and false negative results by eliminating the effects of heterophilic antibodies, and complement and rheumatoid factors [7,18]. In our study, with non-equimolar tPSA measurement performed by monoclonal antibodies, approximately one third of patients with BPH had to undergo a biopsy of the prostate. ...
... However, more sensitive assays (e.g., immunoradiometric assay (IRMA) ( Zucchelli et al., 2007) may need to be validated for reliable measurement of plasma ANP concentrations in healthy horses and horses with mild elevation of ANP concentrations. The three assays were all designed to be used with an extraction step to eliminate interferences due to non-specific protein binding, binding to platelets, haemolysis or lipaemia, or naturally occurring AB directed against the detecting AB or the analyte ( Richards et al., 1987;Weber et al., 1990;Ezan and Grassi, 2000). However, in one study on human plasma samples, conventional extraction methods lead to an extraction recovery of only 52%, whereas immunoextraction methods increased the extraction recovery of 92% ( Clerico et al., 1990). ...
Measurement of atrial/A-type natriuretic peptide (ANP) concentrations may be of use for assessment of cardiac disease, and reliable data on the analytic performance of available assays are needed. To assess the suitability for clinical use of commercially available ANP assays, intra-assay and inter-assay coefficient of variation and dilution parallelism were calculated for three immunoassays (RIAPen, RIAPhoen, and an ELISAPen) using blood samples from healthy and diseased horses to cover a wide range of ANP concentrations. Further, agreement between assays was assessed using linear regression and Bland-Altman analyses. For all assays, precision was moderate but acceptable and dilution parallelism was good. All assays showed analytic performance similar to other immunoassays used in veterinary medicine. However, the results from the three assays were poorly comparable. Our study highlights the need for an optimised species-specific assay for equine samples.
... In addition to accuracy problems, endogenous interference presents an immunochemistry-related peculiarity (for review, see [6,7]). The presence of interfering heterophilic antibodies especially is considered to be very common. ...
Although identical monoclonal antibodies from the same source are used, the analytical performance of commercial assays for the measurement of the tumour markers CA 15-3 and CA 19-9 may be quite different. In the present study immunoluminometric (ILMA, LIA-mat, Byk-Sangtec) and immunomagnetic (MSA, Technicon Immune 1 ® , Bayer Diagnostics) assays for CA 15-3 and CA 19-9 are compared. Comparisons with the microparticle enzyme immunoassay (MEIA, Abbott) were also performed. MSA exhibited an excellent interassay-precision for the two analytes with coefficients of variation (CVs) between 2 and 4%, while the CVs with the ILMA ranged between 5 and 8%. In the CA 19-9 determination the regression analysis yielded MSA = 0.927 * ILMA 2.131 kU/l; r = 0.975; S(y.x) = 21.3 kU/l; n = 72). Comparison studies with the MEIA showed regression factors for MSA and ILMA in the order of 1.13 and 1.27, respectively. With the ILMA, samples exhibiting CA 19-9 concentrations above the dilution limit yielded results that deviated up to 2.6-fold from the comparison methods. A falsely low result was observed with the ILMA in a liver-transplant patient. The investigation of the CA 15-3 methods using sera from patients with mammary carcinoma relapse (n = 156) yielded different regression lines between MSA and ILMA results depending on the concentration range. CA 15-3 values obtained with the MSA agreed well with the MEIA results. With the ILMA, dilution linearity was not found to be satisfying within and above the measuring range.
... Tako donji detekcioni limit (LLD) predstavlja najni`u PSA vrednost koja se razlikuje od nule sa 95% pouzdano{}u pod idelanim anal-iti~kim uslovima. Ra~una se kao srednja vrednost plus dve standardne devijacije iz 20 ili vi{e ponovljenih analiziranja puferskog rastvora ili uzorka seruma koji ima koncentraciju PSA »nula« (35,36). ...
In modern laboratory medicine there is a growing tendency for finding out the most specific and most sensitive biomarkers as indicators of a disease and diagnostic means for the follow-up treatment and therapeutical results. Detection of sensitive biomarkers was speeded up by world companies as manufacturers of diagnostic reagents. Today, the determination of some biochemical parameters is performed by immunodetermination which uses monoclonal antibodies and different markers, and thus achieving the desired high specificity of determination. The selections of biomarkers which are the most important for the clinical practice and which will give the best diagnostic information, requires large researches including analytical, clinical and economic properties. Thanks to statistical methods, especially to ROC analysis, the so-called cutoff-values are determined. On the basis of these values differentiation between sick people and healthy individuals is possible. In other words, the evaluation of clinical specificity requires also inclusion of patients whose clinical picture reflects the disease for which a marker is tested. The development and choice of biomarkers is also helped by IFCC with suggestion of the best procedures and preparation of reference materials. The use of selected biomarkers must be profitable in respect of the price and diagnostic information. In the paper the principles of selection, use, diagnostic and prognostic significance of biomarkers is discussed. The following biomarkers served as examples: cardiovascular (troponin T and I, myoglobin, CK-MB mass), bone (deoxypyridinolin, osteocalcin, C-terminal propeptide collagen, type I, osteo-alkaline-phosphase), malignant (PSA) and for pancreas diseases (lipase, PAM).
Thyroglobulin (Tg) is the large (660 kDa) glycoprotein that is synthesized exclusively by thyroid follicular cells and serves as the scaffold for thyroid hormonogenesis. Since thyroid tissue is the only source of circulating Tg, serum Tg measurement is the cornerstone of post-operative biochemical monitoring for recurrence in patients with follicular-derived (differentiated) thyroid cancer (DTC). However, technical limitations negatively impact the clinical utility and interpretation of Tg measurements, irrespective of whether the Radioimmunoassay, (RIA), Immunometric assay, (IMA) or Liquid Chromatography Tandem Mass Spectrometry, (LC-MS/MS) class of method is used. There are intrinsic sensitivity differences between the different methodologies as well as between assays from the same methodologic class. Methods also differ in specificity for detecting the heterogeneous Tg isoforms in serum, resulting in wide between-method discordances that preclude changing methods while monitoring DTC patients. The Tg autoantibodies (TgAb) that are present in approximately 25% of DTC patients remain a persistent problem, causing interference with primarily the IMA class of methods, but also potentially influencing the clinical utility of RIA and LC-MS/MS measurements by in-vitro and/or in-vivo mechanisms. This chapter will discuss the biological factors and technical limitations that impact the clinical utility of serial Tg monitoring of DTC patients with and without TgAb present.
Background
Interfering antibodies in human serum and plasma are known to react with mammalian antibodies in immunoassays and cause false‐positive test results. Although this phenomenon was recently shown in companion animals, knowledge regarding immunoassay interference in veterinary medicine is very limited.
Objectives
The aims of this study were to set up a species‐independent immunoassay procedure to detect interference in serum samples, to screen for interference in a cross‐section of canine and feline patient samples from an animal hospital, and to determine if the detected interference could be neutralized using an immunoassay based on nonmammalian reagents.
Methods
A 2‐site sandwich‐type interference assay was set up using commercially available mouse reagents. A total of 369 serum samples from 320 dogs and 263 samples from 218 cats were analyzed using the interference assay. Multiple samples were submitted from 36 dogs and 39 cats. Nineteen samples identified as interference‐positive were analyzed in an assay using chicken antibodies.
Results
Interference was detected in samples from 28 dogs (9%) and 10 cats (5%) screened with the interference assay. Except for 1 cat, consistent results were obtained for all 75 dogs and cats that submitted more than 1 sample. The interference was eliminated when analyzed in the chicken‐based assay (P < .001).
Conclusions
Substances with reactivity toward mouse IgG can be detected in serum samples from dog and cat patients using a 2‐site interference assay. The detected substances are most likely interfering antibodies, possibly originating from immunization with other mammalian species.
Objective
Macroprolactinemia detection is important to avoid unneccessary tests and overtreatment. High prolactin levels require routine screening and clinicians must be aware of macroprolactinemia frequency encountered with the method in use. In this study we aimed to determine the macroprolactinemia rate in our laboratory.
Methods
Prolactin results of different patients analysed on two different immunoassay systems within two consecutive years were evaluated. Analyses were performed on Beckman Coulter UniCel
Results
For the samples analysed on DxI800 (n=14,958) hyperprolactinemia frequency was 8.1% (n=1208). One of 138 samples submitted for macroprolactin analysis was positive, while three of them were in the gray-zone. For the samples analysed on Cobas
Conclusion
A difference was found between two immunoassay systems used in our laboratory in terms of macroprolactinemia rate. However, inability of simultaneous analyses on both systems, lack of evaluation with gel filtration chromatography, and heterophile antibody blocking tube were the limitations.
Thyroglobulin, the major protein product of thyroid follicular cells, is not produced by other cells in the body. Two principal immunoassay formats exist to measure thyroglobulin and are affected in different ways by the presence of thyroglobulin antibodies and other interfering substances. Most laboratories currently use double antibody (sandwich) methods, which can reliably detect lower concentrations of thyroglobulin, can measure higher values without dilution, and have lower likelihood of interference from cross-reactive substances. These assays will give falsely low results in the presence of thyroglobulin antibodies, but can produce falsely increased results in the presence of heterophile antibodies or rheumatoid factor. Older competitive immunoassays have a limited ability to detect low concentrations of thyroglobulin, must be diluted at high concentrations, and are potentially subject to interference from similar molecules in the circulation. In the presence of circulating anti-thyroglobulin, results are more commonly falsely increased than decreased; however, interference from heterophile antibodies and rheumatoid factor is usually minimal or absent. Recently developed mass spectrometry-based assays have the potential to reduce or eliminate these interferences, but assays currently available are not as sensitive as the best immunoassays and they have not been validated in clinical studies. While preoperative thyroglobulin has limited clinical utility, thyroglobulin measurements are commonly used to detect residual thyroid tissue or tumor in the follow-up of patients with differentiated thyroid cancer who lack thyroglobulin antibodies. Increased thyroglobulin while on thyroid hormone suppression indicates a high likelihood of residual or metastatic thyroid cancer. In addition, undetectable thyroglobulin while on thyroid hormone suppression after remnant ablation or a negative whole-body iodine scan or TSH-stimulated thyroglobulin is relatively sensitive as a sole test. TSH-stimulated thyroglobulin is a sensitive marker of residual thyroid tissue and may be positive in persons with no evident residual radioiodine uptake; in some of these patients, subsequent stimulated thyroglobulin measurements become undetectable on repeat testing.
Background:
Analysis of serum beta-hCG aids diagnosis and treatment of intrauterine pregnancies, ectopic gestations, and gestational trophoblastic neoplasia. beta-hCG concentrations are specific for trophoblastic tissue, thus are rarely questioned.
Cases:
An 18-year-old nullipara had bleeding and a positive beta-hCG. Ultrasound identified no pregnancy. She passed tissue and stopped bleeding. Serum beta-hCG remained elevated despite uterine curettage and three courses of methotrexate. Results of urine beta-hCG were negative, as was reference laboratory serum assay. A 31-year-old nullipara had a spontaneous abortion, but serum beta-hCG remained elevated. Uterine curettage found secretory endometrium, yet elevated serum beta-hCG persisted. Urine beta-hCG was negative, as was reference laboratory serum assay.
Conclusion:
Patients with histories incongruent with serum beta-hCG findings should have urine beta-hCG analysis.
Thyroglobulin, an approximately 670-kDa glycopeptide, is the major protein product of thyroid follicular cells; its rate of synthesis is increased by thyrotropin (TSH). After synthesis, it is modified by the attachment of iodine to selected tyrosine residues, which undergo rearrangement to form iodothyronines, particularly thyroxine (T4) and, to a lesser extent, thysonine (T3). Other modifications of thyroglobulin also occur, including glycation and sulfation (1). The degree of TSH stimulation affects the extent of branching of carbohydrate side chains (2). There is variable processing of thyroglobulin, creating a family of proteins with different molecular structures around a common core peptide backbone. Interestingly, there is less variability in thyroglobulin structure in thyroid cancer than in other thyroid diseases (3). Reduced iodine content in thyroglobulin exists in patients with thyroid malignancy (4), which can lead to different recognition by monoclonal antibodies (5). This structural heterogeneity creates a challenge for thyroglobulin immunoassays, and results often differ significantly when using different thyroglobulin methods (6).
IntroductionSerum Versus Plasma and Glass Versus Plastic TubeSuitability of Serum Separator Gel Tubes for Blood Collection for Therapeutic Drug MonitoringImpact of Documenting Dosing Time and Other Factors that Affect Results of Therapeutic Drug MonitoringOther Preanalytical Factors that may Affect Test ResultsEffect of High Bilirubin, Hemolysis, and High Lipid on Therapeutic Drug MonitoringConclusions
References
This overview of lateral-flow immunochromatographic assays covers the development of the first of such tests for pregnancy, their general mode of operation and the role of, and interplay between, their different components. Emerging materials and issues related to the wider range of biofluids these devices are now designed to work with are also discussed. This assay platform has existed for almost 20 years and despite efforts to improve upon it, its ability to produce an accurate and rapid result in the hands of both professional and naïve user 8 will ensure that its spectrum of applicability widens.Keywords:immunoassay;latex;antibody;nitrocellulose;gold sol;lateral flow;adsorption;protein;urine;heterophile;blood;urine
Hyperlipidemia is an important public health problem with increased incidence and prevalence worldwide. Current clinical biomarkers, triglyceride, total cholesterol, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol lack the necessary specificity and sensitivity and only increase significantly after serious dyslipidemia. Therefore, sensitive biomarkers are needed for hyperlipidemia. Hyperlipidemia-specific biomarkers would improve clinical diagnosis and therapeutic treatment at early disease stages. The aim of metabolomics is to identify untargeted and global small-molecule metabolite profiles from cells, biofluids, and tissues. This method offers the potential for a holistic approach to improve disease diagnoses and our understanding of underlying pathologic mechanisms. This review summarizes analytical techniques, data collection and analysis for metabolomics, and metabolomics in hyperlipidemia animal models and clinical studies. Mechanisms of hypolipemia and antilipemic drug therapy are also discussed. Metabolomics provides a new opportunity to gain insight into metabolic profiling and pathophysiologic mechanisms of hyperlipidemia.
Abstract Although prostate specific antigen (PSA) is widely used in the discrimination of benign prostatic
hyperplasia (BPH) and prostate cancer, its diagnostic value is controversial due to an appreciable false positive
rate. In the present study, we compared a recently introduced assay method, equimolar PSA measurement,
to non-equimolar PSA measurement and also determined the diagnostic value of percent free PSA with
changing total PSA (tPSA) measurements. Between April 1999 and December 2001, the sera of 61 patients
with BPH and 41 with prostate cancer were examined. Total PSA and free PSA was determined using the Immulite 2000 assay system, whereas equimolar tPSA measurement was performed using Bayer PSA Q for the Chiron ACS 180 system. Comparative analysis of the two different assays revealed better diagnostic sensitivity and specificity values for equimolar tPSA measurement, which in turn would have led to 10% of the patients avoiding an unnecessary biopsy. Additionally, percent free PSA with the changing denominator of tPSA assays showed that the free PSA/equimolar tPSA ratio was the best tumor marker among the studied forms of PSA. It was concluded that equimolar tPSA measurement using recombinant Fab fragments is superior to the classical measurements with monoclonal antibodies, and that the use of percent free PSA with the equimolarly measured tPSA has better sensitivity and specificity in the discrimination of benign and malignant diseases of the prostate.
Keywords Prostate specific antigen, Equimolar measurement, Benign prostatic hyperplasia, Prostate cancer
Background:
Although substantial advances have been made in ligand-binding assays (LBA) for biotherapeutics in the past decade, there are still gaps that need to be addressed, especially in the context of understanding matrix effect and its root causes. Critical and in-depth characterization of matrix effect can provide valuable knowledge of the LBA limitations for proper results interpretation.
Results:
This article illustrates several strategic approaches with regard to identifying the root cause of matrix effect and practical solutions, including recognizing the confounding factors associated with matrix effect, selection of proper reagents to avoid matrix effect, and a systematic approach in dealing with matrix effect in method development and validation.
Conclusion:
These strategic approaches have enhanced the management of matrix effect in LBA.
Immunochemical methods, methods that employ antibodies as analytical reagents, have revolutionized laboratory medicine. Although the noncovalent bound between analyte in biological specimen and complementary antibody in reagent is specific, positive or negative interferences are possible. Interfering substances can be present in both specimen and reagents, respectively. Most interfering substances are inherent in patients’ blood under physiological and pathophysiological conditions, especially under diagnostic and therapeutic procedures. Some interferences are similar to those in chemical analyses and some are typical only for immunoassays (hook effect ; cross-reactivity with structurally similar or identical epitopes ; heterophile antibodies ; anti-animal antibodies ; autoantibodies ; the matrix effect). The main characteristic of all immunoassays is that the reagent which discovers or quantifies the target analyte contains the specific antibody. Results higher than real values are recorded due to a low antibody specificity. Results can also be influenced by pre-analytical factors or assay formulations. One should suspect interferences a) after obtaining an unacceptable result ; b) if there is non-linearity during dilution ; c) if there is no agreement with other test results or clinical data ; d) if different immunoassays in determination of the same analyte provide significantly different results. It is necessary to think about present predictable and always possible unpredictable and unrecognizable interferences. Unawareness and non-recognition of interferences could lead to diagnostic errors, unnecessary laboratory tests, inadequate and unnecessary treatment and therapy. There is no simple and practical way to identify interferences in specimens before analysis. Whenever interference is suspected, there are a few possibilities to solve them, e.g. serial dilution, pre-treatment with blocking reagent, use of more specific methods. At the same time a proper information upon pre-analytical diagnostic and therapeutic procedures is needed for accurate interpretation of the results. In this process, consistent communication and compliance between laboratory professionals and clinicians are required.
Failure to match assay matrices with samples in immunoassays can result in incorrect sample values being reported. For multiplex assays this presents particular problems, due to the need to find a matrix suitable for all the analytes. Here, we describe strategies adopted to overcome matrix problems identified in establishing a cytokine multiplex assay in human plasma. Standard analytes were diluted in plasma samples to identify representative plasma for assay development. Horse sera were screened to evaluate potential interference before using to adjust a matrix to match plasma samples. Suitability of the matrix match was confirmed by evaluating recovery of known concentrations of analytes from plasma. Individual plasmas modified the assay signal for some analytes to a variable extent, particularly for IL-1α and IL-1β. Addition of horse serum to assay buffer improved matching to plasma samples, although endogenous MCP-1 activity was apparent in one sample. Matching of plasma and assay matrices allowed recoveries within 10% to 20% of the expected values, unless the samples contained atypical interfering activity. Attention to choice of samples and diluent used for assay development is particularly important for measurement of sample analytes in cytokine multiplex assays.
The existence of insulin-like growth factors (IGFs) in blood was fi rst recognized
by Wiliam D. Salmon Jr. and Wiliam H. Daughaday in 1956. Examining
the role of pituitary-regulated growth-stimulating substances, these
authors demonstrated that a growth hormone (GH) dependent factor in serum
could stimulate labeled sulfate (35SO4) incorporation into chondroitin sulfate
(cartilage) in vitro. In line with their fi ndings, Salmon and Daughaday initially
designated this factor as ‘sulfation factor’. Comparative studies were also
performed in vivo. Hypophysectomy of rats markedly reduced labeled sulfate
incorporation into chondroitin sulfate of epiphyseal cartilage, whereas injections
of pituitary extracts and purifi ed bovine GH effectively restored 35SO4
incorporation. The observations that the direct effect of bovine GH on
costal cartilage was minimal, whereas serum from hypophysectomized rats
treated with bovine GH stimulated 3H-thymidine incorporation into cartilage
led to the postulation that GH utilized the intermediary substance sulfation
factor.
The success of analytical methods in clinical chemistry has led to a sense of security in the value of laboratory results. This is largely justified, as evidenced by the quality of laboratory performance assessed by external assurance schemes. Nevertheless, it is not widely recognised among clinicians that some biochemical tests are more prone to interference from unusual serum constituents than others—and that quality assurance schemes can do little about this.
An important example of this is tests carried out by immunoassays based on the recognition of molecules by antibodies. The antibodies are largely derived from animal sources and are typically used for measuring hormones, tumour markers, cardiac troponins, and therapeutic drugs and for viral serology.
The design of assays has evolved enormously since the discovery of immunoassay by Berson et al in 1956,1 and it is now a major analytical tool in clinical laboratories worldwide, allowing relatively minute (picomole (10−12)) amounts of analytes to be measured with unrivalled precision.
Interference …
Immunoassays are used daily by clinical endocrinologists to refute or confirm a diagnosis, or to follow up the course of a treatment. Immunoassays have become increasingly sensitive, specific and reproducible, so that clinicians have great confidence in their results. However, they do not always yield correct results because interferences still occur. In the first part of this review, immunoassay interferences are described and their mechanisms explained: matrix effects, specificity defaults, interferences from antibodies and binding proteins, or the hook effect. The hormones most frequently concerned by these pitfalls are reviewed, even as strategies used to prevent and minimize interferences. In the second part, the consequences of failure to recognize an interference are reviewed: unjustified clinical decisions (wrong diagnosis, treatment, surgery) and, more exceptionally, publication in the scientific literature of erroneous results. Clinicians may sometimes inform laboratories of an increased risk of interference. In general, clinicians are in the best position to fully validate test results; because only they know all the medical data about their patients. If there is any suspicion of discrepancy between clinical and laboratory data, they must alert the laboratory. This is the only way to better detect and, if possible, eliminate interferences and their regrettable outcomes.
Among the endogenous interferents affecting assay results, the most common are bilirubin, hemoglobin, lipids, and paraproteins.
These interferents may affect therapeutic drug monitoring (TDM), drugs of abuse (DAU) testing, and toxicology assays of any
format where the sample is used directly for analysis without any pretreatment of specimen. Immunoassays are commonly used
in clinical laboratories where analyte-specific antibody or binding agents are used to estimate the analyte concentration
in the specimen. Some enzyme and chemistry assays are also utilized in TDM and DAU analysis. Such assays use various types
of signals, the most common being colorimetry, fluorimetry, and chemiluminescence. Assays may be prone to interference depending
on the format or label used. Commercial assay kits report the result of such interference in the kit inserts (up to levels
of >20 mg/dL bilirubin, >500 mg/dL hemoglobin, and >1000 mg/dL lipids). The interference is caused by the optical, fluorescent,
or chemiluminescent properties of these interferents. Thus, bilirubin interferes by its absorption and fluorescence properties,
hemoglobin by its absorption, fluorescence and chemiluminescence properties, and lipids interfere mostly from their light-scattering
(turbidity) properties. Bilirubin and hemoglobin may also interfere because of side reactions in the assay. Modern auto-analyzers
can detect all three interferents and flag the results. Flagged results should be carefully reviewed for the accuracy. Both
hypo- and hyper-proteinemia can affect assay results. Paraproteins interfere in many assays by precipitating out during the
sample blanking step thus producing false results. Another source of interference may be from probe (sample or reagents) or
reaction cuvettes carryover contamination in random-access auto-analyzers. If the validity of test results is questioned,
the assay should be repeated either by removing the interferent from the sample or by using different method which is known
to suffer less from that type of interference.
The use of sandwich immunoprocedures based on monoclonal antibodies has improved the quaIity of immunological determinations of human chorionic gonadotropin (hCG), but results from external quality assessment schemes (EQAS) show, that there is a need for further improvement. Although the present hCG standard certified by WHO (Third International Standard) is generally used by kit manufacturers, results for different procedures may give highly variable results. This is probably due to differences in antibody specificity and to nonspecific interference. During the last decade, immunoprocedures specifically measuring low concentrations of free a (hCGa) and D (hCGD) subunits have become available, and these are becoming clinically important [l, 2, 3, 4, 51.
Cross-reactivity in immunoanalysis. The lack of specificity is a major limitation in immunoanalysis. Cross-reactivity dependson the type of antibodies used (monoclonal, polyclonal), assay configuration, and relative concentrations of cross-reactive compounds. Cross-reactivity may be evaluated by different methods, which are presented and discussed.
BACKGROUND
Prostate-specific antigen (PSA) is the most important tumor marker in prostate cancer diagnosis and follow-up. Its catabolism by the liver has not influenced its use as a prostate marker until the recent report of a significant increase in a man and a woman with acute hepatitis. In addition, PSA was detected in liver tumor extracts, which warranted its evaluation in liver cytolysis and hepatocellular carcinoma. In this study, PSA was evaluated in a cohort of both sexes presenting either acute hepatitis or hepatocellular carcinoima.METHODS
Forty-two patients with acute hepatitis (21 male patients, 21 female patients) and 54 patients with hepatocellular carcinoma (31 male patients, 23 female patients) were tested for PSA by equimolar immunoassay (Abbott AxSYM Total PSA, Abbott Diagnostics, Rungis, France) and for relevant liver biological parameters (alpha-fetoprotein, alanine aminotransferase, aspartate aminotransferase, total bilirubin, and prothrombin rate).RESULTSPSA was undetectable in all the female patients and was consistent with age in the males (PSA median and range in acute hepatitis, 0.36 μg/l (range, 0.05–1.3); in hepatocellular carcinoma, 0.36 μg/l (range, 0.02–3.9)). It did not correlate with alpha-fetoprotein and aminotransferases.CONCLUSIONS
Our results confirm the well-established reliability of PSA, and show that PSA remains a valid prostate marker in patients with acute hepatitis and hepatocellular carcinoma. Prostate 41:258–262, 1999. © 1999 Wiley-Liss, Inc.
Introduction Selection of Assay Reagents Assay Optimization, Development, and Validation Clinical Application Summary Acknowledgments References
The colorimetric and chemiluminescent determination of alkaline phosphatase activity in a direct enzyme immunoassay of the competitive type for determination of estradiol-17β (E2) in human plasma or serum were compared. In this assay biotin-labeled E2 and E2 from standard or patient samples compete for free antibody-binding sites. Antibody-bound E2-biotin is then reacted with streptavidin-alkaline phosphatase. Enzyme activity in the bound fraction was detected either with the chromogenic substrate p-nitrophenyl phosphate (pNPP) or with the chemiluminescent substrate disodium 3-(4-methoxyspiro l,2-dioxetane-3,2′-(5′-chloro)-tricyclo[3.3.1.13,7]decan-4-yl)phenyl phosphate (CSPD). Contrary to expectations, the use of CSPD for chemiluminescent endpoint determination of alkaline phosphatase did not improve assay performance. Both the limit of detection of analyte E2 (160 vs. 73 pmol ml−1) and overall precision (C.V., %) were less favourable with CSPD than with pNPP. These results and the high cost of CSPD make this chemiluminescent substrate less suited for endpoint determination in competitive EIA for haptens.
A preparative extraction step using disposable C18 low-pressure chromatography columns greatly improved the specificity of a commercially prepared digoxin radioimmunoassay (RIA). Elution solvents were isopropanol/water (15/85 by vol), which extracted most immunoreactive digitalis-like factors and metabolites, and methanol, which extracted digoxin for RIA. Many different digoxin RIA kits could be used. The coefficients of variation for replicates and duplicates were 4.6% and 5.2%. Analytical recoveries of digoxin standards in serum of 1.0, 0.5, and 0.1 microgram/L were 96%, 95%, and 88%, respectively. Serum digoxin was assayed by this method in 200 patients, 47 of whom were studied by HPLC-RIA. Values correlated better with "true digoxin" by HPLC-RIA (r = 0.93) than did values found by direct assay (r = 0.63). The mean for the isopropanol fraction as a percentage of the mean direct RIA value was higher for the 21 dialysis-dependent patients than was that found for the 179 nondialysis patients (P less than 0.004). The method is suggested as being most useful when metabolites or digitalis-like factors are known to be often high and values for digoxin are disproportionate to the dose.
Xenogeneic antibodies can survive food processing procedures with their biological activity intact and even enhanced. These antibodies can be absorbed from the human gut, and will function both as antigens and antibodies in the human immune system. Antibodies to bovine gamma globulins (BGG) have been detected in human sera and the family of anti-BGG antibodies must include anti-idiotypic antibodies, very low doses of which can influence the immune response. The hypothesis is that the human immune system may be primed by low-level exposure to xenogeneic antibodies specific for those human allergens which are ubiquitous in the farm environment, such as pollens, mites, and moulds, the result being a deleterious and inappropriate response on subsequent exposure to these allergens. Dairy products are the most important source of xenogeneic antibodies in the western diet, and the hypothesis may partly explain the association between cow's milk and allergies to substances other than milk proteins.
We verified that antibody-binding substances in serum that interfere in two-site immunoassays involving murine antibodies are heterophilic antibodies. Incubation of serum containing heterophilic antibodies and a murine monoclonal antibody to human choriogonadotropin (hCG) leads to formation of a series of soluble immune complexes. We investigated the recognition of hCG by reagent antibody in the presence of heterophilic antibodies and found this recognition to be diminished. Consequently, about 30% of serum samples containing heterophilic antibodies falsely appear to contain increased concentrations of hCG. The effect on analyte recognition probably results from steric inhibition of hCG binding to complexed antibody. Heterophilic antibodies detected with a murine antibody also bound immunoglobulin from several other species but did not bind all of those tested.
The aim of the study was to develop an enzyme linked immunosorbent assay (ELISA) for measuring thyroglobulin (Tg) in human serum and to evaluate the influence of serum thyroglobulin auto-antibodies (TgAb) on the ELISA. The sensitivity of the ELISA was 2.1 micrograms/l. Serum Tg levels in healthy controls were from less than 2.1 to 55.5 micrograms/l (n = 46) (95% reference range). With serum Tg concentrations between 19.6 to 90 micrograms/l the within-assay coefficient of variation (CV) was from 4.5 to 6.6% (n = 12) and the between-assay CV from 8.5 to 10.5% (n = 6). The recovery from 20 to 89 micrograms Tg/l serum was from 93 to 101%. There was significant correlation between serum Tg concentrations measured by the ELISA and a RIA method in healthy controls (r = 0.85, n = 46, p less than 0.001) and in patients with differentiated thyroid carcinoma (r = 0.97, n = 28, p less than 0.001). The TgAb interfered with the serum Tg determination both in the ELISA and in the RIA method. The assay is simple and easy to perform, and the equipment is inexpensive and useful for large-scale serum Tg measurements as an alternative to RIA.
To study lipid interference in steroid radioimmunoassays in which dextran-coated charcoal is used as the separating agent, we tested triolein and phosphatidylcholine as model hydrophobic and amphipathic lipids, respectively. Addition of either caused distortion of the standard curve to an extent that was inversely related to the polarity of the steroid molecule. Both lipids form a dispersion that entraps steroid molecules. When we increased the charcoal concentration, the effect of phosphatidylcholine addition was eliminated for assays of both polar and nonpolar steroids. In contrast, the effect from triacylglycerol was not corrected, particularly in assays of nonpolar steroids. We also studied mixtures of lipids mimicking the mixture of lipids extracted from plasma of normolipemic and hyperlipemic indviduals. The degree of lipemia that can be tolerated differs from assay to assay, and primarily varies directly with the polarity of the steroid being assayed.
A young woman with amenorrhea had a consistently positive pregnancy test result (serum radioimmunoassay measurement of β-human chorionic gonadotropin hormone). No fetal or placental tissue was found after uterine curettage and exploratory laparotomy. The false-positive pregnancy test result was due to heterophilic antibovine and antigoat antibodies in the patient's serum. These antibodies interfered with radioimmunoassays using goat antibodies. This case shows that serum heterophilic antibodies can interfere with immunoassays and result in unnecessary diagnostic procedures and/or unnecessary treatment.
(JAMA 1982;248:2489-2490)
• Retrospective analysis of 108 patients who received indium 111–labeled murine monoclonal antibodies for imaging of cancer was performed. Most patients had operative procedures for colorectal carcinoma following completion of scintiscanning. Eleven patients had markedly elevated carcinoembryonic antigen (CEA) levels postoperatively without evidence of residual or recurrent disease. The laboratory method of measuring CEA levels was a commercially available double mouse monoclonal antibody enzyme immunoassay. It was postulated that the unexplained elevation of CEA was a reflection of the presence of human anti—mouse antibody (HAMA) induced by the administration of radiolabeled mouse antibody. A competitive assay for HAMA was undertaken by incubation of these patients' sera with a high dose of nonspecific mouse immunoglobulin prior to CEA determinations, and subsequent CEA levels were normal. The presence of HAMA was confirmed by a noncompetitive solid-phase enzyme immunoassay in 73% of tested patients who received murine monoclonal antibodies for imaging. Identification of artifactual CEA elevations is important in the treatment of cancer patients.
(Arch Surg 1988;123:1242-1246)
Serum antibodies to cow milk proteins and ovalbumin were measured quantitatively. Food hypersensitivity of the immediate type was determined to be present or absent by skin tests and double-blind food challenges. Elevated levels of antibodies to milk proteins in sera characteristic of infants fed cow milk were found to decline with age, so that sera from children who were 6 to 15 years of age (inclusive), not hypersensitive to food, had significantly lower levels than the infants. In contrast, sera from age-matched children, who were shown to have hypersensitivity to some food, were found to have levels of antibodies to milk proteins as elevated as in infancy. Hypersensitivity was not necessarily to milk but often to some other food. This persistence of greater antibody production to milk throughout childhood in those hypersensitive to some food indicates a fundamental difference from those without hypersensitivity to food, either in permeability, in immunological reactivity of the gut or in development of immunological unresponsiveness. Implications for pathogenesis of clinical disorders are discussed.
Serum levels of free and total insulin as well as total C-peptide immunoreactivity (C-peptide and proinsulin) and C-peptide were measured in insulin-treated diabetics with circulating insulin antibodies by the addition of polyethylene glycol (PEG) before and after acidification. PEG resulted in complete precipitation of insulin antibodies from serum and made it possible to measure free insulin in the supernatant. Incubation of serum at 37δ C. for two hours before addition of PEG resulted in values for free insulin that probably resembled the in-vivo levels most closely. The same method could also be used to remove proinsulin bound to circulating insulin antibodies and permitted the measurement of C-peptide in the supernatant.
Clinical studies using this approach indicate that combined measurements of serum free and total insulin and C-peptide provide information that is helpful in understanding the contribution of endogenous and exogenous insulin to the course and metabolic control of insulin-requiring diabetic patients.
We examined the effect of endogenous free fatty acids (FFA) on the measurement of free thyroxin (FT4) by five different methodologies represented in 16 different assays in a large number of patients with nonthyroidal illness (NTI). Some, but not all, one-step (analog) FT4 RIAs negatively correlated with FFA concentration. All two-step FT4 RIAs, equilibrium dialysis FT4, and the dialyzable (free) fraction of T4 positively correlated. In contrast, a binding-rate-based FT4 RIA, FT4 indices based on T3 macroaggregated albumin uptake, and T4/TBG ratios did not correlate. We also analyzed the FT4-FFA relationship with a second, more sensitive approach by correlating test results with FFA/albumin molar ratio as an estimate of the "excess" (nonalbumin bound) FFA. We found that all FT4 RIAs, equilibrium dialysis FT4, FT4 indices based on T3 uptake, the dialyzable fraction of labeled T4 in equilibrium dialysis, the fraction of labeled T4 bound to solid phase antibody in the binding-rate-based RIA, and T3 uptake correlated with the FFA/albumin molar ratio. This FFA dependency was comparable among all the various techniques and was relatively small. Thus, increases or decreases in FT4 results due to varying FFA (and albumin) concentrations are highly likely with most currently available methods (only the T4/TBG ratio did not reveal FFA-dependency), but the magnitude of changes varies with the "excess" FFA.
The binding of serum thyrotropin (TSH) to plastic beads coated with a monoclonal antibody to human TSH was inhibited unless EDTA was present during the incubation. The inhibitory factor in serum was heat labile, and its effect could be abrogated by the addition of human albumin-anti-albumin immune complexes. Subsequently it was shown that the antibody-coated beads were able to bind the first component of complement, C1q, and that this binding was inhibited by addition of albumin-anti-albumin complexes. The results show that a surface coated with a monoclonal murine antibody is able to bind complement, and that binding of complement may interfere in solid-phase immunometric assays.
Of 1585 consecutive serum samples referred for thyroid function testing, 14 gave erroneously high values from a two-site immunoenzymometric assay for thyrotrophin. The addition of mouse or newborn calf serum to the assay failed to correct the interference. In serum samples from 11 patients who were available for follow up a higher concentration of mouse, but not of horse, sheep or rabbit serum reduced the interference. The interference was associated only with assay systems which employed horseradish peroxidase but not 125I as a label. Addition of mouse serum and anti-IgM to the assay reagents successfully removed the interference.
We have previously reported that human anti-mouse IgG antibody (HAMA) can cause false-positive and false-negative results in "sandwich"-type monoclonal antibody (MAb) assays. To eliminate HAMA interference in "sandwich"-type MAb assays, we investigated the use of MAb on solid-phase, vinylidene fluoride floccules, which we have previously used as a solid-phase second antibody for RIA. The simple procedure effectively removes greater than 95% of HAMA from the most positive serum we have obtained from patients hyperimmunized to murine MAb, and it allows for accurate quantification of carcinoembryonic antigen. The solid-phase complex, added to blood, effectively removes HAMA and (or) "HAMA-type" heterophilic antibody from the sera or plasma.
This study sought to determine whether patients with rheumatoid arthritis (RA) were immunologically sensitised to dietary protein (DP). Using an enzyme linked immunosorbent assay (ELISA), antibodies to milk and wheat proteins were measured in 93 unselected out-patients with classical or definite RA. Of these 93, 53 had raised levels of IgG antibodies to one or both dietary proteins (DP). In the DP antibody positive group, 48 patients (90%) also had raised levels of IgA rheumatoid factor (measured by ELISA) while only 7 (17%) of the 40 DP antibody negative patients had detectable IgA RF; P less than 0.02. There was no association between IgM rheumatoid factor and dietary protein antibodies. These results demonstrate that in RA, raised levels of IgA RF are associated with an increased IgG response to antigens which enter the body through the gastrointestinal tract. A breakdown in gastrointestinal tolerance to dietary antigens may play a role in the immunopathogenesis of RA in these patients who might therefore benefit from dietary manipulation.
Recent research has demonstrated the presence of endogenous compounds in blood and urine that crossreact with antibodies raised against digoxin. Given the widespread therapeutic use of digoxin and its being monitored clinically by immunoassay, such digoxin-like immunoreactive compounds pose significant diagnostic and interpretive problems. Serum levels of this factor(s) approaching therapeutic digoxin levels have been found in digoxin-free patients in renal failure, pregnant women, and newborns. The compound is incompletely characterized; however, existing data suggest that it is a small, neutral, nonpeptidic compound. In serum it is highly protein bound, and alterations in this binding appear to give rise to the false-positive assay results. The urinary form is probably conjugated. Digoxin-like immunoreactive substances may play a role in volume homeostasis and appear associated with essential and pregnancy-induced hypertension. If such roles are primary, measurement of digoxin-like immunoreactive substances may prove to be of value in and of itself.
The mean concentration of free thyroxin (FT4) in serum, as determined by direct equilibrium dialysis, was decreased in patients with chronic renal failure (CRF) and increased in patients with various other nonthyroidal illnesses (NTI). The mean concentration of dialyzable free triiodothyronine (FT3) in serum was equally low in both groups of patients. Patients with CRF of various etiology but a similar degree of renal failure as estimated from serum creatinine assay had very similar concentrations of FT4 and FT3 in their serum. Mean thyroxin (T4) and triiodothyronine (T3) concentrations in serum were decreased in CRF and NTI, whereas the mean reverse-T3 concentration in serum was normal in CRF and increased in NTI. T4-binding globulin and albumin were markedly decreased in CRF and NTI; T4-binding prealbumin was increased in CRF and decreased in NTI. The mean concentration of nonesterified free fatty acids (FFA) in serum was increased in NTI but not in CRF. The weak, but significant, positive correlation observed between FT4 and FFA in serum (r = 0.34, P less than 0.01) in NTI indicates that the increase in serum FT4 in this group of patients could be an effect, at least in part, of FFA competing with T4 for binding sites on serum proteins. The stronger correlation detected between the serum FT4 concentration and the FFA/albumin molar ratio in serum (r = 0.60, P less than 0.001) demonstrates the importance of a low albumin concentration for expression of the effect of FFA on FT4 in severe systemic illnesses.
Three patients who had falsely elevated serum TSH concentrations (initial values, 30.5, 74, and greater than 50 mU/L) in a mouse monoclonal immunoradiometric assay are reported. Two patients were treated for hypothyroidism inappropriately, and one underwent unnecessary diagnostic testing. Immunoaffinity chromatography of serum from one patient indicated that the serum TSH level was truly low. Addition of mouse serum or immunoglobulin G (IgG) or absorption of patient serum with solid phase-bound mouse IgG-1 reduced the TSH content in the serum of the three patients to undetectable levels. Blocking studies revealed that all patients had antibodies directed at mouse IgG-1, the subclass of mouse antibody present in the assay kit. The serum of one patient who had autoimmune disease with elevated serum Igs had much broader species cross-reactivity than that of another patient who had known exposure to rats and mice. We hypothesize that such antimouse antibodies can arise either from endogenous autoimmunity or exogenous animal exposure. Serum TSH elevations also were found when the serum samples were tested in other mouse monoclonal immunoassays, underscoring the fact that antibody interference can potentially affect many assays used in endocrinology and other areas of medicine to make major diagnostic and therapeutic decisions. Clinicians must be aware of such interactions; relatively simple laboratory maneuvers can differentiate true from false results in assays of this type.
Retrospective analysis of 108 patients who received indium 111-labeled murine monoclonal antibodies for imaging of cancer was performed. Most patients had operative procedures for colorectal carcinoma following completion of scintiscanning. Eleven patients had markedly elevated carcinoembryonic antigen (CEA) levels postoperatively without evidence of residual or recurrent disease. The laboratory method of measuring CEA levels was a commercially available double mouse monoclonal antibody enzyme immunoassay. It was postulated that the unexplained elevation of CEA was a reflection of the presence of human anti-mouse antibody (HAMA) induced by the administration of radiolabeled mouse antibody. A competitive assay for HAMA was undertaken by incubation of these patients' sera with a high dose of nonspecific mouse immunoglobulin prior to CEA determinations, and subsequent CEA levels were normal. The presence of HAMA was confirmed by a noncompetitive solid-phase enzyme immunoassay in 73% of tested patients who received murine monoclonal antibodies for imaging. Identification of artifactual CEA elevations is important in the treatment of cancer patients.
Sandwich-type immunoassays in which mouse monoclonal antibodies are used are subject to positive interference by heterophile antibodies present in human serum. Manufacturers now customarily add nonspecific mouse immunoglobulins to absorb the heterophile antibodies and eliminate such interference. We describe the case of a patient who had spurious increases in thyrotropin concentration in serum despite use of the mouse immunoglobulins included in the assay kit. This resulted in a puzzling clinical picture, a workup, and treatment. We demonstrate that the observed increases in thyrotropin were markedly reduced by including additional amounts of mouse immunoglobulins of the appropriate class, subclass, and fragment type in the assay mixture.
The interaction between immune aggregates and complement (C) was investigated. Solid-phase immune aggregates were prepared by coating microwells with heat-aggregated bovine serum albumin (BSA) followed by rabbit anti-BSA antibody. The immune aggregates were reacted with human serum or citrated plasma at 37 degrees C. The binding of C3 components was investigated with biotinylated F(ab')2 antibodies to C3c and C3d and avidin-coupled alkaline phosphatase. The form of the incorporated C3, whether C3b-iC3b or C3dg, can be deduced from the response with these two antibodies. The maximal binding of C3b-iC3b to the immune aggregates was observed within 5 min of incubation with serum or citrated plasma. The conversion to C3dg was evident by a decrease in bound anti-C3c concomitant with increasing anti-C3d reactivity within about 10 min of incubation. When the classical C pathway activation was inhibited, the binding of C3b-iC3b was delayed by 20-30 min, whereas stopping of the alternative pathway did not influence the initial kinetics of the reaction. The addition of human red blood cells had no measurable influence on the degradation of bound C3b-iC3b. 125I-labelled anti-BSA antibody bound to the solid-phase BSA was not released during the C3 incorporation. The incorporation of C3b into the immune aggregates was mediated equally well by serum and by citrated plasma. The incorporation of C3b-iC3b into immune complexes (IC) is thought to be responsible for the C-mediated solubilization (CMS) of IC. Citrated plasma, however, exerted no CMS capacity when measured by a radiometric assay. The CMS capacity of serum was inhibited by citrate, but could then be restored by adding Ca2+ and Mg2+, whereas no CMS could be demonstrated with citrated plasma to which divalent metal ions were added.
Using a modified "two-site" immunoradiometric assay, termed an "interference assay," we have demonstrated the occurrence of non-analyte antibody-binding substances in approximately 40% of serum samples. These substances multivalently bind antibodies from any of several species at a site other than the antigen-binding site. Using a two-site immunoradiometric assay for human choriogonadotropin, we have investigated their effect on analyte quantification. In this system these antibody-binding substances mimic the presence of analyte; when analyte is actually present, they can also cause over- or underestimates. Addition of excess nonspecific immunoglobulin from an appropriate species eliminates this interference. However, the amount of nonspecific immunoglobulin added to an assay system may not always be enough to block interference in all samples.
The concentration of FFA in normal human plasma in vivo generally ranges between 0.2 and 0.7 meq/liter; slightly higher concentrations have occasionally been reported in patients who are seriously ill. To determine whether such FFA concentrations may increase the concentration of free T4 in serum, we added increasing amounts of oleic acid to pooled normal human serum (with known FFA content) and measured free T4 by equilibrium dialysis. Total FFA up to 3 meq/liter in normal serum, representing an FFA to albumin molar ratio of about 5:1, had little or no effect on the free T4 concentration, while higher FFA concentrations progressively increased free T4. This same molar ratio of FFA to albumin had to be exceeded to cause a significant increase in the free T4 concentration in diluted serum and in serum from patients with nonthyroid illness. Serum from which more than 95% of the albumin had been removed by chromatography with Affi-Gel blue was much more sensitive to the effects of FFA on free T4. This enhanced sensitivity was reversed by readdition of albumin to the serum, and the addition of albumin to normal serum resulted in diminished effects of FFA on free T4. These results indicate the following: physiological concentrations of FFA do not significantly increase the free T4 concentration in normal human serum; when FFA reach supraphysiological concentrations in serum (in vitro) and the higher affinity FFA-binding sites on albumin become saturated (apparently at an FFA to albumin molar ratio of approximately 5:1), the excess FFA interact with other serum proteins, including thyroid hormone-binding globulin, and thereby increase the free T4 concentration; the concentration of albumin (or other FFA binders) must be considered when evaluating the observed effects of FFA. To explore the relevance of these findings to the hypothesis that FFA may inhibit the binding of T4 to plasma proteins in patients with nonthyroid illness, we measured plasma FFA concentrations in 11 severely ill patients hospitalized in the intensive care unit. We found a mean plasma FFA concentration of 0.45 +/- 0.11 (+/- SEM) mEq/liter and a mean serum albumin concentration of 2.39 +/- 0.29 g/dl in these patients. Their mean plasma FFA to albumin molar ratio was 1.53 +/- 0.41. Since the FFA to albumin molar ratio must exceed about approximately 5:1 before a significant increase in the serum free T4 concentration occurs, these results suggest that FFA do not commonly influence the circulating free T4 concentration in vivo, even in severely ill patients.
A serological phenomenon causing aberrant results with monoclonal immunoenzymetric assays (IEMA's) is reported. Two different commercial IEMA kits detected low levels of choriogonadotropin (hCG) in the serum of a non-pregnant woman. These assays detected between 32 and 55 IU/l of serum hCG over a 3-wk period; however, an RIA for beta-subunit and two monoclonal immunoradiometric assays (IRMA's) detected no hCG (less than 5 IU/l). An IEMA measurement of creatine kinase MB isozyme was also elevated. Antisera to either human immunoglobulin or specifically to human IgM, added to the serum prior to assay, substantially decreased these IEMA reactions. Addition of either mouse serum or purified mouse IgG totally abolished them. It is concluded that these spurious reactions were most likely caused by an IgM antibody which binds to native and enzyme-labelled mouse IgG, but not to iodinated IgG.
Different nephelometric assay systems for quantitation of C-reactive protein (CRP) were compared with radial immunodiffusion (RID) and tested for their susceptibility to interference by serum IgM rheumatoid factor (RF). In 3 nephelometric assays, RF was found to elevate CRP values. Sera with high RF content from patients with rheumatoid arthritis gave significantly higher CRP values by nephelometric assay than by RID; the addition of purified RF to RF-negative sera increased CRP values markedly; and removal of RF from sera corrected falsely elevated CRP values. This interference by RF is explained by the action of human RF as a (secondary) antibody reacting with complexed mammalian IgG anti-human CRP in the assay. In this way the nephelometric signal is enhanced to give falsely elevated CRP values. In contrast, the gel diffusion RID method does not suffer from this non-specific interference.
A method is described for measuring insulin antibodies in insulin treated subjects. The antibodies are characterized by maximum binding capacity and affinity constant. Endogenous and therapeutic insulin interferes in insulin antibody assay, and it is removed by dextran charcoal treatment at pH 3.5. Endogenous total and free insulin may be measured by a simple extension of the method.
A method for the determination of free, active insulin in the sera of insulin-treated diabetics is described. This involved radioimmunoassay after extraction of free insulin with polyethylene glycol. Recovery tests with cold insulin showed 73 per cent recovery of free insulin and no recovery of bound insulin.
The fasting free insulin levels were slightly lower in the patients than in normal persons; exceptions were some patients with complications expected to cause insulin resistance at the peripheral tissue level. Free insulin levels did not correlate with total insulin levels, antibody titers, insulin requirements or conditions of insulin treatment.
A very slight increase of insulin was observed after glucose loading in insulin-treated patients, but a marked increase of the free insulin level followed by an exaggerated increase in the total insulin level was observed in a patient with the insulin autoimmune syndrome.
The diurnal changes of the free insulin suggested the dynamic states of this fraction and its usefulness for determining control of diabetes with insulin.
With the proliferation of immunoassays used in the clinical laboratory, we have come to realize that there are many factors which can interfere with these assays. Some of these, like exogenous radioactivity, will only interfere with one type of immunoassay--the radioimmunoassay. Other interferences will affect all types of immunoassays, although perhaps to differing degrees. This article will survey some of the more common types of interfering substances which may affect immunoassays, including tube types, serum factors, and the interferences which are specifically due to the type of label used in the assay.
The presence of a T4-binding autoantibody resulted in a falsely high value for free T4 and a falsely low one for total T4 in a patient who was clinically euthyroid. Values for serum T3 and thyrotropin were normal. Such results should alert the analyst to the possibility that the patient's serum contains autoantibodies.
Measurement of serum C-reactive protein (CRP) concentration is useful in monitoring the progress of chronic inflammatory diseases. Rheumatoid factor, by its interaction with the Fc portion of IgG, has the potential to interfere with solid-phase immunoassays for CRP and other serum proteins. To determine the effect of RF on a solid-phase enzyme immunoassay for CRP we compared assays employing whole antisera or F(ab')2 fragments. In 92 sera with RF latex titres ranging from 1/80 to 1/81,920, no correlation was found between RF concentrations and CRP measurements. CRP concentrations measured by use of whole antisera (mean +/- standard error of the mean, 59.7 +/- 5.7 mg/l, n = 92) were lower than those measured with F(ab')2 fragments (62.5 +/- 5 mg/l), indicating that exaggeration of CRP measurements did not occur in RF containing sera under the conditions of the assay. Our results show that in the CRP-ELISA, interference from RF was precluded by the high serum dilutions employed. At lower serum dilutions RF binding was detected. Consequently, in solid-phase enzyme immunoassays at lower serum dilutions the presence of RF may lead to false positive results and exaggerated measurements.
A solid-phase immunosorbent radioassay for the detection of circulating antibodies to protein hormones is described. The assay is based on the binding of the homologous 125I-labelled antigen to the antibodies which are then bound to anti-IgG antibodies covalently coupled to Sepharose. It can easily be applied as a complement to any radioimmunoassay for the detection of circulating antibodies to the ligand measured. The assay system avoids falsely elevated values due to interference of high serum concentrations of the antigen. The assay was applied to measure antibodies to FSH, LH, TSH, GH, prolactin, insulin and thyroglobulin (Tg). Among patients with chronic thyroiditis Tg antibodies were found in 100% of the sera. In diffuse toxic goitre 73% of the patients had detectable Tg antibodies. Insulin antibodies were present in 82% of the sera from patients with insulin treated diabetes. No antibodies were found against the other protein hormones tested.
Nine sera with falsely elevated results in a TSH radioimmunoassay were studied. Sera dilution and gel filtration showed that the immunoreactive TSH was different from the standard and labeled TSH. It coeluted with the immunoglobulin fraction of the serum. Rabbit serum decreased or emphasized the artifact according to the dose. Falsely elevated results appeared only in radioimmunoassays using a double antibody method for the separation of free and bound labeled hormone. FSH, LH, and beta HCG radioimmunoassays using the double antibody method underwent the same disturbance as the TSH assay in these patients. Our results suggest that the artifact may be due to the presence in certain human sera of anti-rabbit IgG antibodies, interfering in the radioimmunoassay by inhibition of the binding of first and second antibodies.
A case of false hyperthyrotropinemia was investigated. The serum of the patient contained heterophilic antibodies against rabbit immunoglobulins secondary to immunization with rabbit serum. Some vaccines against viral or bacterial diseases contain animal serum capable of inducing heterophilic antibodies in man. This technical problem can be prevented by the routine addition of control rabbit serum or immunoglobulins to the TSH RIA.
A young man with amenorrhea had a consistently positive pregnancy test result (serum radioimmunoassay measurement of beta-human chorionic gonadotropin hormone). No fetal or placental tissue was found after uterine curettage and exploratory laparotomy. The false-positive pregnancy test result was due to heterophilic antibovine and antigoat antibodies in the patient's serum. These antibodies interfered with radioimmunoassays using goat antibodies. This case shows that serum heterophilic antibodies can interfere with immunoassays and result in unnecessary diagnostic procedures and/or unnecessary treatment.
We previously reported spuriously high values for thyrotropin (TSH), presumably owing to an antibody in human serum that reacts with both reagent rabbit antibodies in an immunoradiometric assay (IRMA). We used this IRMA to measure TSH. Five of 20 sera from laboratory animal handlers showed spuriously high values. When we added 2 mL of nonimmune rabbit serum per liter to the labeled IRMA rabbit antibody reagent and reassayed the five affected specimens, the results were within the reference interval. Smaller additions partly corrected the TSH values, but nonimmune sera of eight other species had no effect. Substitution of goat solid-phase antibody decreased, but did not eliminate, the increases in TSH in three of the five affected sera. Chromatographic properties, results of rheumatoid factor testing, and measurement of human anti-rabbit immunoglobulin suggest that the interference is ascribable to an antibody of the IgG class that reacts with rabbit antibody. Evidently, antibody interference with IRMA procedures may be common in certain populations. It can be avoided by including nonimmune serum corresponding to the species used to produce reagent antibody.
The determination of prostatic acid phosphatase and beta choriogonadotropin with RIA methods were subjected to interference by lipid in hyperlipidemia.
This chapter discusses the various aspects of particle counting immunoassay (PACIA). The PACIA is based on the principle that the number of free particles decreases during the agglutination reaction. It is found that, by counting the unagglutinated particles, with a blood cell counter, it is possible to evaluate the extent of the reaction in a much more sensitive and precise way, than by the naked eye. The PACIA is automated on the basis of the continuous-flow system. The rather strong agitation required to accelerate the agglutination process can be obtained in a mixing coil submitted to continuous vibration. The intra- and interassay precisions were determined, using four or five samples, with concentrations, covering most of the range of the calibration curve. The Ab content of a rabbit antimyelin basic protein (MBP) antiserum used as a calibrator for both PACIA and a solid phase radioimmunoassay was estimated, by the Farr precipitation technique, using 125I-labeled MBP. The sensitivity of PACIA to the small changes in the levels of immune complexes is described, by the results of a study performed on 486 samples of human cord blood, using only rheumatoid factor (RF) inhibition.
To study lipid interference in steroid radioimmunoassays in which dextran-coated charcoal is used as the separating agent, we tested triolein and phosphatidylcholine as model hydrophobic and amphipathic lipids, respectively. Addition of either caused distortion of the standard curve to an extent that was inversely related to the polarity of the steroid molecule. Both lipids form a dispersion that entraps steroid molecules. When we increased the charcoal concentration, the effect of phosphatidylcholine addition was eliminated for assays of both polar and nonpolar steroids. In contrast, the effect from triacylglycerol was not corrected, particularly in assays of nonpolar steroids. We also studied mixtures of lipids mimicking the mixture of lipids extracted from plasma of normolipemic and hyperlipemic indviduals. The degree of lipemia that can be tolerated differs from assay to assay, and primarily varies directly with the polarity of the steroid being assayed.
Biochemical characterization of digoxin-like immunoreactive substances
- S Sadrzadeh
- Mh
- F F Vmcenzi
- A Dasgupta
- S Amhad
- M Kenny
- Kuzuya H.
- Käpyaho K.