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3-p-Hydroxyphenylpropionic Acid—A Sensitive Fluorogenic Substrate for Automated Fluorometric Enzyme Immunoassays
Abstract
The application of 3-p-hydroxyphenylpropionic acid (HPPA), a fluorogenic substrate of horseradish peroxidase (HRP) to an automated microplate fluorometric enzyme immunoassay is described. Fluorescence intensity of the end product was highly dependent on the pH of the buffer and on the concentrations of the substrate mixture ingredients. The determination of human thyrotropin (TSH) and recombinant hepatitis B surface antigen (rHBsAg) were performed using a fluorometric enzyme immunoassay (FEIA) with HPPA as the substrate, and a colorimetric one with tetramethylbenzidine (TMB) as the chromogenic substrate. The sensitivity of both types of assays proved comparable. The distinct advantage of a fluorometric assay is the possibility to perform a quantitative detection of analyte over a very wide dynamic range. Clinical evaluation of both assays showed good correlation between the FEIA and conventional methods.
... The adaptation to HRP was attempted by Zapata and co-workers using 4-hydroxybenzoic acid (HBA) [5]. The oxidation products of 4-hydroxyphenylpropionic acid (pHPPA) [6], chavicol [7], Amplex red [8] and prochlorperazine [9] did not sensitize their fluorescence measurements in the corresponding experiment setup. The other common drawbacks of aforementioned fluorogenic substrates are their insufficient reaction rates, relatively low stability in air or toward H 2 O 2 in the absence of HRP, tending to cause strong background signals and poor water-solubility [10,11]. ...
A natural product, stilbene glycoside (2,3,5,4’-tetrahydroxydiphenylethylene-2-O-glucoside, TBG), has been evaluated for the first time as a potential substrate for horseradish peroxidase (HRP)-catalyzed fluorogenic reactions. The properties of TBG as a fluorogenic substrate for HRP and its application in a fluorometric enzyme-linked immunosensing system were compared with commercially available substrates such as p-hydroxyphenylpropionic acid (pHPPA), chavicol and Amplex red using Brucella melitensis antibody (BrAb) as a model analyte. The immunosensing body based on HRP-BrAb was constructed by dispersing graphite, BrAg and paraffin wax at room temperature. In a competitive immunoassay procedure, the BrAb competed with HRP-BrAb to react with the immobilized BrAg. In the enzymatic reaction, the binding HRP-BrAb on the sensing body surface can catalyze the polymerization reaction of TBG by H2O2 forming fluorescent dimers and causing an increase in fluorescence intensity. TBG showed comparable ability for HRP detection and its enzyme-linked immunosensing reaction system, in a linear detection ranging of 3.5´10-8~7.6´10-6g/L and with a detection limit of 1.7´10-9 g/L. The immobilized biocomposite surface could be regenerated with excellent reproducibility (RSD=3.8%) by simply polishing with an alumina paper. The proposed immunosensing system has been used to determine the BrAb in rabbit serum samples with satisfactory results.
... The fragments were conjugated with horseradish peroxidase as described previously (3). Substrate reaction with 3-p-(hydroxyphenyl)propionic acid was carried out as described previously (11). Negative controls were prepared with sera from laboratory personnel shown to be negative for toxoplasma-specific IgM by EIA. ...
An easy-to-perform fluorometric enzyme immunocapture assay (FEIA) was developed by Labsystems, Helsinki, Finland, to detect toxoplasma-specific immunoglobulin M (IgM) in dried blood spots. Assay materials were distributed to two sites that have programs in place designed to identify infants born with congenital toxoplasma infection: the Statens Serum Institut, Copenhagen, Denmark, and the New England Regional Newborn Screening Program, Boston, Mass. Each site tested over 700 dried blood samples from healthy newborns to define a cutoff at the 99.5 percentile (5 enzyme immunounits for Copenhagen and 4 enzyme immunounits for Boston). Each site then applied its own cutoff of interpret results for dried blood spots prepared from either adults with serology suggestive of acute infection (Copenhagen) or infants determined to be congenitally infected on the basis of serological criteria (Boston). In Copenhagen, 35 of 38 adult samples were either positive to a small degree or borderline positive for IgA. These samples thus may not represent acute infection. In Boston, of 26 congenitally infected infants, 22 were positive by FEIA. The four infant specimens not positive by FEIA were either negative or borderline positive by the standard Boston assay. These results demonstrate that the IgM FEIA is a potential alternative to other filter paper assay for toxoplasma-specific IgM currently in use for newborns.
... The screen is also highly selective for one of two possible hydroxylated isomers of 3-PPA. Although an oxygenase can potentially hydroxylate different positions on the aromatic backbone of 3-PPA, only the product hydroxylated at the 4-position, 3-(4-hydroxyphenyl) propionate, generates strong blue fluorescence (emission at 465 nm, 350 nm excitation) when coupled using HRP [23]. HRP does not generate any detectable fluorescence with 3-(2-hydroxyphenyl) propionate as the substrate in an in vitro assay. ...
Oxygenases catalyze the hydroxylation of a wide variety of organic substrates. An ability to alter oxygenase substrate specificities and improve their activities and stabilities using recombinant DNA techniques would expand their use in processes such as chemical synthesis and bioremediation. Discovery and directed evolution of oxygenases require efficient screens that are sensitive to the activities of interest and can be applied to large numbers of crude enzyme samples.
Horseradish peroxidase (HRP) couples the phenolic products of hydroxylation of aromatic substrates to generate colored and/or fluorescent compounds that are easily detected spectroscopically in high-throughput screening. Coexpression of the coupling enzyme with a functional mono- or dioxygenase creates a pathway for the conversion of aromatic substrates into fluorescent compounds in vivo. We used this approach for detecting the products of the toluene-dioxygenase-catalyzed hydroxylation of chlorobenzene and to screen large mutant libraries of Pseudomonas putida cytochrome P450cam by fluorescence digital imaging. Colors generated by the HRP coupling reaction are sensitive to the site of oxygenase-catalyzed hydroxylation, allowing the screen to be used to identify catalysts with new or altered regiospecificities.
The coupled oxygenase-peroxidase reaction system is well suited for screening oxygenase libraries to identify mutants with desired features, including higher activity or stability and altered reaction specificity. This approach should also be useful for screening expressed DNA libraries and combinatorial chemical libraries for hydroxylation catalysts and for optimizing oxygenase reaction conditions.
As early as 1810, it was observed that placing a fresh piece of horseradish root tissue into a solution of guaicum resulted in the development of an intense blue color. It was later determined that this was due to the action of peroxidase enzymes. Peroxidases (E.C. 1.11.1.7) are widely distributed in nature and are found in plants, microorganisms and animals, where they catalyze the oxidation of a variety of substrates utilizing H2O2 as an electron acceptor (Adam et al. 1999). They are typically named after either their sources, e.g., horseradish peroxidase (HRP) and soybean peroxidase (SBP) or their substrates, such as lignin peroxidase (LiP) or cytochrome c peroxidase (CcP).
Ligand-binding assays are ideal for routine bioanalysis, but we reason that the straightforward replacement of the conventional chromogenic horseradish peroxidase substrate, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid, of a routinely used preclinical immunoassay to detect hIgG, with the fluorogenic 3-(4-hydroxyphenyl)propionic acid would broaden the narrow dynamic range. The replacement leads to a sensitivity of 0.47 (minimum required dilution [MRD] 10) and 1.02 (MRD 50) ng/ml, and dynamic ranges of 3.3 (MRD 10) and 3.6 (MRD 50) orders of magnitude, and thereby had improved sensitivity and dynamic range compared with other conventional colorimetric ELISAs, other ligand-binding assay technologies or LC-MS assays. Improvements in sensitivity and dynamic range were achieved for the sera of horse, mice and monkeys without assay optimization.
A novel substrate, 4-(p-hydroxystyryl)pyridine (pHSP) was synthesized and firstly used in a horseradish peroxidase (HRP) based fluorometric enzyme-linked immunosensing system. The results of the property investigation of pHSP demonstrate the advantages in stability and reactivity with HRP over conventional substrates such as p-hydroxyphenylacetic acid (p-HPA), Amplex red and chavicol. In a pH 5.8 Britton-Robinson buffer solution, HRP-antibody conjugate (HRP-IgG) could catalyze the oxidation reaction of pHSP by H2O2, and the pHSP was converted to significantly fluorescent dimers. The increase of the fluorescence intensity (excitation: 300 nm, emission: 437 nm) of the HRP enzymatic product is proportional to the concentration of HRP-IgG binding to the Brucella melitensis antigen modified matrix. An enzyme-linked immunosensing method based on this principle was developed. The linear range of determination is 6.3 × 10-11~1 × 10-8 mol·L-1 with the relative standard deviation of 4.1%. The detection limit is 6.3×10-11 mol · L-1. The another obvious advantage of the proposed procedure is the sensitivity deriving from the solution of the insoluble substrate determination.
A natural product, 4-hydroxycinnamic alcohol (HCA) was used as a substrate for HRP in enzyme-linked fluoroimmunoassay. In enzymatic reaction procedure, HRP-Brucella melitensis antibody conjugate (HRP-BrAb) catalyze the polymerization of HCA by H2O2, and the HCA is partly converted to polymers, a fluorescent species. The fluorescence increase of the HRP-enzymatic product at emission of 467 nm (excitation at 315 nm) is proportional to the concentration of HRP-BrAb binding to the Brucella melitensis antigens, which were entrapped in cellulose-paraffin matrix. The linear range of determination for BrAb is 2.7-90 μg/L with the relative standard deviation of 4.6%. The detection limit is 2.7 μg/L. HCA is stable in air and non-toxic to human health. The proposed method can be used for analysis of commercial formulation and plasma sample with satisfactory results.
Enzyme immunoassay (EIA) has been the primary subject of a number of books and reviews, and an important concern of other recent books and reviews. Enzymes are the most versatile and popular class of labeling substances used for immunoassays, and they have an assured future. The invention of immunoenzymatic staining in the mid-1960s is said to have been the beginning of enzyme immunoassay (EIA), and this enabled the preparation of permanent photomicroscopic slides with visualization of specific antigens. In 1971, enzymes were introduced as alternatives to radioisotopes in immunoassays. Enzymes are perhaps the most varied class of labeling substances. About 1990, the most common enzyme label in new immunoassays was horseradish peroxidase, with alkaline phosphatase in second place. The term enzyme-mediated immunoassay encompasses, as well as all assays with enzymes as the labeling substance, assays with labels containing enzyme substrates, enzyme inhibitors, coenzymes, or enzyme cofactors. The operation of such assays depends on the use of one or more enzymes as ancillary reagents. Enzymes are also exploited in many other ways for the benefit of immunoassays. To prepare conjugates with enzyme, the method of conjugation used is particularly mild so as to retain both immuno- and enzymatic activities.
An enzyme-catalyzed reaction system puerarin-horseradish peroxidase (HRP)-hydrogen peroxide, which used puerarin as substrates for HRP, was developed. In enzymatic reaction procedure, HRP catalyze the polymerization of puerarin by H2O2, and the puerarin is partly converted to dimers, a significantly fluorescent species. The increase of the fluorescence of the HRP-enzymatic reaction solution at emission of 478 nm (excitation: 315 nm) is proportional to the concentration of HRP. The enzyme-linked fluoroimmunoassay based on the principle mentioned above and competitive immunoassay model for the determination of Brucella melitensis antibodies (BrAb) was established. The results of the property investigation of puerarin demonstrate the advantages in stability and reactivity with HRP over conventional substrates such as p-hydroxyphenylacetic acid (p-HPA), Amplex red and homovanillic acid. The experimental parameters such as BrAb-HRP, temperature were optimized. The proposed method has been successfully used for analysis of BrAb in rabbit plasma sample. The linear range of determination for BrAb is 1.3~120 ng/mL with the relative standard deviation of 3.8%. The detection limit is 1.3 ng/mL (3σ).
Temperature is one factor that influences the performance of immunoassays. Most commonly all incubation steps of the immunoassay are performed at ambient temperature. To systematically study the influence of temperature deviations on assay parameters like signal variation coefficients, limit of detection and measurement range, direct competitive enzyme immunoassays for the determination of two anthropogenic markers, caffeine and carbamazepine, were run at different temperatures above and below room temperature during individual assay steps. The temperature during the competition step had the greatest impact on the assay sensitivity yielding changes in test midpoints by a factor of 10 to 15. For carbamazepine, the test midpoints varied from 36 ng L−1, 108 ng L−1 to 378 ng L−1 employing 4 °C, 21 °C, or 37 °C as incubation temperature, respectively. The edge effect can be minimized resulting in a good plate homogeneity with small variation coefficients, when the assay is performed at ambient temperature.
This report describes a microplate-based enzyme immunoassay with fluorometric detection (FEIA) for the determination of thyroxine (T4) from dried blood spots. The T4 FEIA was developed for newborn screening of congenital hypothyroidism (CH) and for the follow-up of l-thyroxine substitution therapy in CH patients. The detection limit of the proposed method was 8.0 nmol/l blood, with imprecision being from 5.1 to 11.9% (within-run) and from 4,6% to 9.8% (between-run). Highly significant but moderate correlation was observed with the Neonatal T4 RIA of Diagnostic Products (r=0.72, P<0.0005; n=47) and the SpectriaR serum T4 RIA of Orion (r=0.79, P<0.0005; n=89). The overall incubation time of the assay is 3 h. The assay uses the same fluorogenic substrate as the neonatal thyrotropin (TSH) test described earlier [14], thus allowing simultaneous determination of both T4 and TSH.
An immunoaffinity chromatographic (IC) method for the determination of 2,4-dichlorophenoxyacetic acid (2,4-D) and atrazine was developed. The sensitivity was 1 and 0.1 mug l-1 for 2,4-D and atrazine, respectively, the relative standard deviation was in the range 3-8% and the total assay time per sample was 10 min. IC was combined with three detection systems: spectrophotometric, chemiluminescence and fluorescence. The whole procedure can be fully automated and applied to the quasi-continuous control of pesticide contamination and a rapid assay under out-of-laboratory conditions.
Enzyme immunoassays with optical detection are amongst the most widely used bioanalytical tools. We defined seven parameters for the quality assessment of immunoassays that were addressed in a systematic study of direct and indirect immunoassays, using the enzymes horseradish peroxidase (HRP) and alkaline phosphatase (AP), the chromogenic substrates 3,3′,5,5′-tetramethylbenzidine (TMB) and para-nitrophenyl phosphate, and the fluorescent substrates 3-(4-hydroxyphenyl)propionic acid and 4-methylumbelliferyl phosphate. The same monoclonal antibody against caffeine was used throughout the study. The four quality parameters regarding the standard curve were the test midpoint (sensitivity), the measurement range, the relative dynamic range of the signal, and the goodness of fit of the adjusted four-parameter logistic function. All HRP immunoassays showed a higher sensitivity compared to the AP assays. On the basis of all four criteria, it was established that the direct assay format is superior to the indirect format, the immunoassay using HRP TMB fulfilling all requirements best. In a second step, caffeine concentrations in 24 beverage and cosmetics samples were determined and three more quality parameters were assessed with this application. The direct HRP TMB assay showed one of the best intra- and inter-plate precisions and the best accuracy, defined by the correlation of results with those from the chosen reference method liquid chromatography tandem mass spectrometry (LC-MS/MS). Considering all criteria, HRP TMB seems to be the enzyme substrate system of choice preferably used in the direct assay format.
Figure
Overview on the different enzyme immunoassay formats compared
The properties of resveratrol (3′, 4′, 5-trihydroxystlbene, RST) were for the first time evaluated as a potential substrate
for horseradish peroxidase (HRP)-catalyzed fluorogenic reaction. The properties of RST for use as fluorogenic substrates for
HRP and its application in immunoassays were compared with commercially available substrates such as p-hydroxyphenylpropionic acid (pHPPA), chavicol and Amplex red by a fluoroimmunosensing method in the use of Schistosomia japonicum antibody (SjAb) as a model analyte. The fluoroimmunosensing device was constructed by dispersing Schistosomia japonicum antigen (SjAg), nano-Ag/SiO2 particles and sol-gel at low temperature. In pH 5.8 Britton-Robinson buffer (B-R), HRP-SjAb conjugates can catalyze the polymerization
reaction of RST by H2O2 forming fluorescent dimmers. The increase of the fluorescence intensity of the dimmers product at emission of 462 nm (excitation:
315 nm) is proportional to the concentration of HRP-SjAb binding to the SjAg entrapped in the nano-Ag/SiO2 particles-sol-gel matrix. A competitive binding assay has been used to determine SjAb in rabbit serum with the aid of SjAb
labeled with HRP. Substrate RST showed comparable ability for HRP detection and its enzyme-linked immunosensing reaction system,
in a linear detection ranging of 1.5×10−6–7.3×10−4 g/L and with a detection limit of 1.5×10−6 g/L. The immobilized biocomposites surface could be regenerated by simply polishing with an alumina paper, with an excellent
reproducibility (RSD = 4.7%). The proposed method has been successfully used for analysis of the rabbit serum sample with
satisfactory results.
We describe a novel method for the detection of thyrotropin from dried blood spots using a horseradish peroxidase-labelled sandwich enzyme immunoassay with fluorometric detection. The detection limit of the present assay is 1.25 mIU/l with within-run and between-run imprecision being in the range 5.2 to 11.4%. The results of the assay correlate well with two commercial methods: an enzyme immunoassay (r = 0.93) and a time-resolved fluorescence assay (r = 0.90). The blood spot values also show a good correlation (r = 0.93) with respective values obtained from plasma using a commercial immunoradiometric method.The assay may also be performed colorimetrically with sensitivity similar to the fluorometric assay. However, the latter provides a wider dynamic range with an upper limit of 400 mIU/l while the colorimetric method reaches a plateau at 25 mIU/l. Due to its simplicity and rapid performance (3 h), the fluorometric assay is suitable for the routine screening of congenital hypothyroidism.
Binding of human alpha 2-macroglobulin (alpha 2M) to group G streptococci and to their immunoglobulin G (IgG)-binding proteins (protein G) was investigated. Native alpha 2M bound specifically to strain G-148 with an apparent dissociation constant of (2.2 +/- 1.5) x 10(-9) M. Proteinase-complexed alpha 2M did not compete for the binding sites, and 125I-labelled proteinase-complexed alpha 2M did not bind to the bacteria. Binding of native alpha 2M to the cells was not affected by IgG or protein G consisting of only IgG-binding domains. 125I-labelled recombinant protein G did not bind to native or proteinase-complexed alpha 2M. However, a lysate of G-148 cells inhibited binding of alpha 2M to the bacteria, and immobilized wild-type protein G bound alpha 2M directly from fresh human plasma. In 13 group G streptococcal isolates, IgG-binding proteins were immunologically identified as protein G. In 11 isolates, these molecules reacted also with alpha 2M and human serum albumin (HSA). Western blots (immunoblots) of two wild-type protein G variants revealed identical bands reactive with goat IgG, HSA, and native alpha 2M. Digestion of wild-type protein G with clostripain destroyed in both variants the binding sites for alpha 2M but not for albumin and IgG. N-terminal fragments of protein G (lacking the IgG-binding region) bound both alpha 2M and HSA, whereas a similar HSA-binding peptide lacking the first 80 amino acids did not react with alpha 2M. Our findings are consistent with a specific binding site for native alpha 2M in the N-terminal region of protein G and suggest that binding of alpha 2M via IgG-binding proteins may be a general feature of human group G streptococci.
We compared a nephelometric method and a radial immunodiffusion (RID) assay for the measurement of retinol-binding protein in samples of serum from children with malignancies. The mean (+/- standard deviation) retinol-binding protein concentration as measured by the Behring Nephelometer was 31.0 +/- 15.6 mg/l; the mean by RID was 31.2 +/- 15.7 mg/l. This difference was not statistically significant by Student's t test (P = 0.6), and the correlation coefficient (r) was 0.87. Thus, the Behring Nephelometer method measures retinol-binding protein rapidly and as accurately as radial immunodiffusion.
We designed and synthesized 2-[6-(4′-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF) and 2- [6-(4′-amino)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (APF) as novel fluorescence probes to detect selectively highly reactive oxygen species (hROS)
such as hydroxyl radical (⋅OH) and reactive intermediates of peroxidase. Although HPF and APF themselves scarcely fluoresced, APF selectively and dose-dependently
afforded a strongly fluorescent compound, fluorescein, upon reaction with hROS and hypochlorite (−OCl), but not other reactive oxygen species (ROS). HPF similarly afforded fluorescein upon reaction with hROS only. Therefore,
not only can hROS be differentiated from hydrogen peroxide (H2O2), nitric oxide (NO), and superoxide (O
) by using HPF or APF alone, but −OCl can also be specifically detected by using HPF and APF together. Furthermore, we applied HPF and APF to living cells and
found that HPF and APF were resistant to light-induced autoxidation, unlike 2′,7′-dichlorodihydrofluorescein, and for the
first time we could visualize −OCl generated in stimulated neutrophils. HPF and APF should be useful as tools to study the roles of hROS and−OCl in many biological and chemical applications.
A highly sensitive sandwich enzyme immunoassay (EIA) for human growth hormone (hGH) was developed. hGH to be assayed was incubated with an anti-hGH IgG-coated polystyrene ball, and then the polystyrene ball after washing was incubated with anti-hGH Fab -peroxidase conjugate. Peroxidase activity bound to the polystyrene ball was correlated to the amount of hGH to be assayed. Polystyrene balls were coated by physical adsorption with rabbit anti-hGH IgG which was not affinity-purified. Rabbit anti-hGH Fab which had been affinity-purified was coajugated with horseradish peroxidase using N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate.The sensitivity was 0.06 pg of hGH per tube or 6 pg/ml of serum when 0.01 ml of serum samples was used. No significant interference was observed in the presence of 8 ng/tube of human prolactin, 8 μU/tube of human thyroid-stimulating hormone, 50 mU/tube of human luteinizing hormone, 10 mU/tube of human follicle-stimulating hormone, or 5 U/tube of human chorionic gonadotropin. The recoveries of hGH added to human sera were 96.2-121 %. The coefficients of within-assay and between-assay variations were 3.6-10.0 % and 5.6-9.8 % respectively. The regression equation and coefficient for correlation to radio-immunoassay (RIA) were Y(EIA)=1.04X(RIA)-0.60 and 0.98 (n=137), respectively.
A hybrid cell line secreting monoclonal antibodies to human alpha- fetoprotein (AFP) was produced by fusion of a mouse myeloma cell line with spleen cells from a BALB/c mouse immunized with human AFP. The affinity constant of the antibody was about 1.7 X 10(9) l/mol. When clones were grown in vitro, the highest concentration of specific antibody in the culture medium was 25 microgram/ml. A clone was transplanted intraperitoneally into pristine-primed BALB/c recipients. Ascites developed within 3-4 weeks of transplantation, and the maximal antibody concentration in the ascitic fluid was 2.5 mg/ml. The immunoglobulin fraction of ascitic fluid was coupled to cyanogen bromide-activated Sepharose and used for affinity chromatography of AFP. AFP containing less than 1% contaminating proteins was obtained by passing amniotic fluid through the column and eluting the adsorbed AFP with 4 mol/l urea. The monoclonal antibody was used for radioimmunoassay (RIA). The sensitivity obtained was 50 microgram/l, which is adequate for certain clinical applications.
beta-D-galactosidase from Escherichia coli and horseradish peroxidase were evaluated as labels of Fab' in dose-response curves for human alpha-fetoprotein and human chorionic gonadotropin by sandwich enzyme immunoassay technique using fluorogenic substrates for enzyme assay. The non-specific binding of Fab'-peroxidase conjugates to IgG-coated polystyrene balls was less than that of Fab'-beta-D-galactosidase conjugates, and the affinity-purified Fab'-peroxidase conjugates gave more sensitive dose-response curves for these antigens than the corresponding beta-D-galactosidase conjugates. However, a large quantity of Fab'-peroxidase conjugates was required and a longer incubation was necessary for the peroxidase assay, since the peroxidase assay was much less sensitive than the beta-D-galactosidase assay. Other advantages and disadvantages of the two enzymes are discussed.
The use of an enzyme as a label has a number of advantages over the use of other labels in both immunohistochemistry and immunoassay. Immunofluorescence techniques are not suitable for ultrastructural research on cells, and ferritin-labeled antibodies allow only electronmicroscopic studies. By contrast, enzyme-labeled antibodies permit localization of cellular antigens in relation to tissue structures under light microscope and also demonstration of cellular antigens at an ultrastructural level by electronmicroscopy. Antibody or antibody fragments labeled with enzymes of small molecular weight can more readily permeate cells of tissue sections than ferritin-labeled antibodies. The color of tissue sections prepared by immunoenzymatic techniques is stable for years, while immunofluorescence of tissue sections decreases rapidly when exposed to light. Radioisotope-labeled reagents decay with time; there are health hazards due to radio isotopes; and disposal of radioactive waste is becoming increasingly difficult. By contrast, enzyme-labeled antigens and antibodies are stable for months or even years and there are no problems either of health hazards or of waste disposal with appropriate choice of enzymes and substrates. Under favourable conditions, enzyme immunoassays may be even more sensitive than radioimmunoassays (1). Enzyme-labeled antigens and antibodies have found increasing use during the past decade, reinforced by improvements in enzyme-labeling methods.
beta-D-Galactosidase from Escherichia coli and horseradish peroxidase were compared as labels of anti-human ferritin Fab' by sandwich enzyme immunoassay technique using fluorogenic substrates for enzyme assay. The anti-human ferritin Fab'-peroxidase conjugates gave lower nonspecific bindings and higher specific bindings than the corresponding Fab'-beta-D-galactosidase conjugates. As a result, the former provided more sensitive dose response curves for human ferritin than the latter. However, the peroxidase conjugates were required in a larger quantity, since peroxidase assay was much less sensitive than beta-D-galactosidase assay.
p-Hydroxyphenyl compounds [3-(p-hydroxyphenyl)propionic acid, p-hydroxyphenethyl alcohol, hordenine, p-ethylphenol, 3-(p-hydroxyphenyl)-1-propanol, p-n-propylphenol, and p-hydroxyphenyllactic acid] were recently found to be excellent fluorogenic substrates for the horseradish peroxidase-mediated reaction with hydrogen peroxide. A very rapid and sensitive method for the fluorometric assays of hydrogen peroxide and the peroxidase was established by using 3-(p-hydroxyphenyl)propionic acid as the best of these substrates; hydrogen peroxide can be assayed precisely in amounts as small as 0.1 nmol, with peroxidase activity as low as 7.8 μU.