Journal of immunological methods

Publications in this journal

• Article: Optimization of the ligature-induced periodontitis model in mice.

  Toshiharu Abe, George Hajishengallis
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  ABSTRACT: Periodontitis is a prevalent oral inflammatory disease that leads to alveolar bone loss and may exert an adverse impact on systemic health. Experimental animal models are critical tools to investigate mechanisms of periodontal pathogenesis and test new therapeutic approaches. The ligature-induced periodontitis model has been used frequently in relatively large animals, including non-human primates, to assess the host response and its effects on the tooth-supporting tissues (gingiva and bone) under well-controlled conditions. Although mice constitute the most convenient and versatile model for mechanistic immunological research (plethora of genetically engineered strains and immunological reagents), the tiny size of the murine oral cavity has presented technical challenges for ligature placement. In this report, we present a straightforward method for ligating the second maxillary molar tooth, and, moreover, identified the most appropriate sites for evaluating inflammatory bone loss in a valid and reproducible manner. These optimizations are expected to facilitate the use of the mouse ligature-induced periodontitis model and consequently contribute to better understanding of the immunopathological mechanisms of periodontitis.
  Journal of immunological methods 05/2013;
• Article: Optimising the quantification of cytokines present at low concentrations in small human mucosal tissue samples using Luminex assays.

  Emily Staples, Richard James Michael Ingram, John Christopher Atherton, Karen Robinson
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  ABSTRACT: Sensitive measurement of multiple cytokine profiles from small mucosal tissue biopsies, for example human gastric biopsies obtained through an endoscope, is technically challenging. Multiplex methods such as Luminex assays offer an attractive solution but standard protocols are not available for tissue samples. We assessed the utility of three commercial Luminex kits (VersaMAP, Bio-Plex and MILLIPLEX) to measure interleukin-17A (IL-17) and interferon-gamma (IFNγ) concentrations in human gastric biopsies and we optimised preparation of mucosal samples for this application. First, we assessed the technical performance, limits of sensitivity and linear dynamic ranges for each kit. Next we spiked human gastric biopsies with recombinant IL-17 and IFNγ at a range of concentrations (1.5 to 1,000 pg/mL) and assessed kit accuracy for spiked cytokine recovery and intra-assay precision. We also evaluated the impact of different tissue processing methods and extraction buffers on our results. Finally we assessed recovery of endogenous cytokines in unspiked samples. In terms of sensitivity, all of the kits performed well within the manufacturers' recommended standard curve ranges but the MILLIPLEX kit provided most consistent sensitivity for low cytokine concentrations. In the spiking experiments, the MILLIPLEX kit performed most consistently over the widest range of concentrations. For tissue processing, manual disruption provided significantly improved cytokine recovery over automated methods. Our selected kit and optimised protocol were further validated by measurement of relative cytokine levels in inflamed and uninflamed gastric mucosa using Luminex and real-time polymerase chain reaction. In summary, with proper optimisation Luminex kits (and for IL-17 and IFNγ the MILLIPLEX kit in particular) can be used for the sensitive detection of cytokines in mucosal biopsies. Our results should help other researchers seeking to quantify multiple low concentration cytokines in small tissue samples.
  Journal of immunological methods 05/2013;
• Article: ELISA for determination of total coagulation factor XII concentration in human plasma.

  Daniel Elenius Madsen, Johannes Jakobsen Sidelmann, Kathrine Overgaard, Claus Koch, Jørgen Brodersen Gram
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  ABSTRACT: Human blood coagulation factor XII (FXII) is the one chain 80 kDa zymogen form of the active serine protease α-FXIIa, which consists of a heavy and light chain linked by a disulfide bond, the light chain being responsible for the protelytical activity. FXII is the first component of the contact dependent pathway of coagulation, but its physiological role is still subject to debate. In the present study we utilized two monoclonal antibodies against the heavy chain of FXII to establish a sandwich enzyme linked immunosorbent assay (ELISA) for quantification of total FXII concentration in human plasma samples. A unique characteristic of this assay is its equal recognition of FXII and inhibitor bound FXII. This is important, as inhibitor complexes of α-FXIIa are formed in vivo as well as during blood sampling and handling. Validation of the assay demonstrated a high sensitivity, with a limit of detection and quantification of 1.2 ng/mL and 2.6 ng/mL respectively. The coefficients of variation for the repeatability and within-laboratory standard deviations were 2.6% and 5.2% respectively. The reference interval determined from healthy volunteers (n=240) was 10.6 - 43 mg/L.
  Journal of immunological methods 04/2013;
• Article: False-positive immunogenicity responses are caused by CD20(+) B cell membrane fragments in an anti-ofatumumab antibody bridging assay.

  Keguan Chen, Jerry G Page, Ann M Schwartz, Thomas N Lee, Stephen L Dewall, Daniel J Sikkema, Catherine Wang
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  ABSTRACT: An electrochemilumescent (ECL) bridging assay to detect anti-ofatumumab antibodies (ADA) in human serum samples was developed and validated. Using this assay format, clinical samples were first screened to identify potential ADA positive samples, which were then further tested by adding excess drug, confirming the positive signals as drug specific. However, when the method was implemented into clinical studies for ADA testing, a high positive rate was observed in the pre-dose samples collected from patients with chronic lymphocytic leukemia (CLL). Since the positive signals were not associated with ofatumumab (Ofa) treatment, and diminished after treatment, it was suspected that matrix interference might be responsible, resulting in false-positive responses. We performed a series of experimental investigations to identify, characterize, minimize or eliminate the possible false-positive responses. One possible source was identified to be CD20(+) (the target of Ofa) present on cell membrane fragments (CMFs). The false-positive responses caused by CD20(+) CMFs could be reduced by solid-phase immunodepletion, ultracentrifugation, or inhibited by adding another anti-CD20 antibody (rituximab). As a consequence, the ADA method was modified to minimize the matrix interference caused by CD20(+) CMFs and, then, validated for sample testing.
  Journal of immunological methods 04/2013;
• Article: Enhancement of Antibody Fragment Secretion into the Escherichia coli Periplasm by Co-expression with the Peptidyl Prolyl Isomerase, FkpA, in the Cytoplasm.

  Raphael Levy, Kiran Ahluwalia, David J Bohmann, Hoa M Giang, Lauren J Schwimmer, Hassan Issafras, Nithin B Reddy, Chung Chan, Arnold H Horwitz, Toshihiko Takeuchi
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  ABSTRACT: Improper protein folding or aggregation can frequently be responsible for low expression and poor functional activity of antibody fragments secreted into the E. coli periplasm. Expression issues also can affect selection of antibody candidates from phage libraries, since antibody fragments displayed on phage also are secreted into the E. coli periplasm. To improve secretion of properly folded antibody fragments into the periplasm, we have developed a novel approach that involves co-expressing the antibody fragments with the peptidyl prolyl cis-trans isomerase, FkpA, lacking its signal sequence (cytFkpA) which consequently is expressed in the E. coli cytosol. Cytoplasmic expression of cytFkpA improved secretion of functional Fab fragments into the periplasm, exceeding even the benefits from co-expressing Fab fragments with native, FkpA localized in the periplasm. In addition, panning and subsequent screening of large Fab and scFv naïve phage libraries in the presence of cytFkpA significantly increased the number of unique clones selected, as well as their functional expression levels and diversity.
  Journal of immunological methods 04/2013;
• Article: Coupled Affinity and Sizing Chromatography: A novel in-process analytical tool to measure titer and trend Fc-protein aggregation.

  Martin Lemmerer, Anne Serdakowski London, Alexandra Panicucci, Cristina Gutierrez-Vargas, Michael Lihon, Philippe Dreier
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  ABSTRACT: The expanding use of monoclonal antibodies in the biopharmaceuticals industry has brought the need for new analytical tools. We have developed a coupled affinity and gel-filtration high-performance liquid chromatography method to simultaneously analyze titer and quality of monoclonal antibodies. Before this assay, available analytical methods for protein aggregation required highly purified proteins. This assay can qualitatively describe a protein from a clarified cell culture solution by trending protein aggregation over time while measuring protein titer. It can be used to assess proteins in both early and late stage culture due to its dynamic range and sensitivity. This assay is a sensitive technique that overcomes the time limitations of previous approaches. It provides an essential tool to accomplish process optimization.
  Journal of immunological methods 04/2013;
• Article: Adapting Dried Blood Spot Sampling for an Anti-Therapeutic Antibody Immunogenicity Assay.

  Yuhong Xiang, Mackenzie Welch, Lakshmi Amaravadi, Christopher Stebbins
  Journal of immunological methods 04/2013;
• Article: Relationship between macrophage differentiation and the chemotactic activity toward damaged myoblast cells.

  Masataka Uchida, Eri Oyanagi, Motohiko Miyachi, Akira Yamauchi, Hiromi Yano
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  ABSTRACT: We investigated the effect of macrophage differentiation on the chemotactic activity to invade local damaged myoblasts using in vitro models of muscle injury. We found that: 1) the chemotactic activity of macrophages toward areas of damaged myoblasts might be induced more by live myoblasts than dead them, 2) the chemotactic activity of macrophages is not due to velocity, but depends on the directionality toward damaged myoblasts cells, and 3) macrophage differentiation strongly influence their chemotactic activity toward damaged myoblasts cells through the expression of CCR2 and/or F-actin.
  Journal of immunological methods 04/2013;
• Article: In vivo depletion of leukocytes and platelets following injection of T cell-specific antibodies into mice.

  Lionel Loubaki, Tony Tremblay, Renée Bazin
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  ABSTRACT: In vivo T cell depletion experiments are widely used to establish the role of these cells in a variety of immunological processes. Different clones of monoclonal antibody targeting the CD3 molecular complex (mainly 145-2C11 and 17A2) have been successfully used for T cell depletion. In the present work, we assessed the specificity of monoclonal antibody-mediated CD3 T cell depletion in mouse peripheral blood. We showed that treatment of BALB/C mice with monoclonal antibodies (clones 145-2C11 and 17A2) efficiently deplete T cells in vivo, but also lead to a substantial reduction in B cell, granulocyte and platelet counts. In contrast, T cell depletion using a combination of anti-CD4 and anti-CD8 antibodies was efficient and produced less deleterious effects on other blood cell populations. Therefore, the results obtained from T cell depletion experiments using anti-CD3 antibodies must be interpreted with caution prior to draw definitive conclusions on the role of T cells in a given immunological process.
  Journal of immunological methods 04/2013;
• Article: Determination of B cell epitopes and Evaluation of Antigen Capture ELISA for the earlier diagnosis of CHIK Virus using anti-rCHIK E1 rabbit Antibodies.

  Krishna Kammara Yathi, Salini Bhasker, Mohankumar Chinnamma
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  ABSTRACT: Chikungunya fever caused by an alpha virus has been generally considered as self limiting and non fatal. Recent reports on Chikungunya infection indicate high mortality rates due to the severity of the viral infection. For the early diagnosis of CHIK virus, the incubation period required for the development of antibodies in the serum of patients was a constraint for antigen based ELISA. The results of the present study demonstrates the development and evaluation of the antigen capture ELISA using recombinant anti-CHIK rabbit antibodies and anti-CHIK human antibody for more specific and rapid detection of CHIK viral antigen. A comprehensive bioinformatics analysis of the amino acid sequence of CHIK E1 protein was done for determining the antigenic residues, predominant B cell epitopes and their properties. Rabbit antibodies against recombinant CHIK E1 antigen was developed and purified. Antigen capture ELISA was done in 104 CHIK patient serum samples using anti-rCHIK E1 rabbit antibodies and anti-CHIK human antibodies. The highest rate of sensitivity (96 %) and specificity (100 %) was observed in the assay data and it highlights the accuracy of the test as a clinical diagnostic tool. No cross reactivity was observed with samples of Dengue patients. Apart from the development and evaluation of the ELISA test, the dominant epitopes identified in the recombinant CHIK E1 protein sequence can be exploited for the development of a subunit Chikungunya vaccine.
  Journal of immunological methods 04/2013;
• Article: Approaches for the simultaneous detection of thiamphenicol, florfenicol and florfenicol amine using immunochemical techniques.

  Terence L Fodey, Suja E George, Imelda M Traynor, Philippe Delahaut, D Glenn Kennedy, Christopher T Elliott, Steven R H Crooks
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  ABSTRACT: Thiamphenicol and florfenicol are antibacterial agents permitted for use as veterinary drugs in animals used for food production. However, as the EU has established maximum residue limits for both and the metabolite florfenicol amine, there is a requirement to monitor animal food products for their residues. In this study antisera were generated which can simultaneously detect thiamphenicol, florfenicol and florfenicol amine in an immunoassay. Details of the various coupling techniques employed to prepare immunogens and enzyme labels are provided and the antibodies produced have been assessed, in homologous and heterologous ELISA formats, with respect to sensitivity and specificity. It was found that while the antisera raised to thiamphenicol and florfenicol generally performed better in a heterologous set up, those raised to florfenicol amine were not only less affected by the assay format but also produced the most sensitive antibodies to all three target analytes. Antisera matched previous sensitivity (IC50<1ngmL(-1)) but had improved cross-reactivity (>100%) to thiamphenicol and florfenicol.
  Journal of immunological methods 04/2013;
• Article: Multiplexed immunoassay to assess Shigella-specific antibody responses.

  Robert W Kaminski, Kristen Clarkson, Alexis A Kordis, Edwin V Oaks
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  ABSTRACT: Infection with Shigella spp. results in bacillary dysentery and a systemic and mucosal antibody response. The immune response is directed at multiple antigens, including LPS and the invasin plasmid antigen (Ipa) proteins, and is capable of conferring short-term, serotype-specific protection. Both live-attenuated and several subunit vaccine approaches have focused on inducing a pronounced mucosal immune response directed to the same antigens recognized after natural infection. Traditionally, Shigella-specific antibody responses are measured using ELISA. Although ELISAs are robust immunological assays, limited sample volume and assay costs often precludes its use for immunological evaluation against multiple antigens. To overcome these shortcomings, a novel assay has been developed to simultaneously measure specific antibody levels to six Shigella antigens, including three serotype-specific LPS preparations and three conserved protein antigens in a Luminex™-based system. Coupling methods were optimized to covalently link recombinant Ipa proteins (IpaB, IpaC, and IpaD) and purified LPS from S. sonnei, S. flexneri 2a, and S. dysenteriae 1 to unique bead sets. The antigen-coupled beads maintained stable reactivity with monoclonal antibodies (mAbs) for 6weeks (protein) to 3months (LPS). The specificity of the Luminex assay was similar to an ELISA, with the multiplexed assay providing a larger dynamic range. Comparable levels of antigen-specific reactivity were attained in monoplex or multiplex formats indicating limited interference. The correlations (R(2)) between the ELISA and the multiplexed assay along with the repeatability and reproducibility of the assay were very high. Minor changes in species-specific conjugated secondary antibodies allowed the optimized multiplexed assays to be used to assess antigen-specific antibodies in serum or blood eluted from filter paper isolated from mice and guinea pigs highlighting applicability of the assay for seroepidemiology and pre-clinical/clinical vaccine studies.
  Journal of immunological methods 04/2013;
• Article: Isolation and analysis of single cells from the mouse heart.

  Alexander R Pinto, Anjana Chandran, Nadia A Rosenthal, James W Godwin
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  ABSTRACT: The adult mouse heart is comprised of a highly heterogeneous cell population. Isolation and effective cellular and molecular analysis of various cell types is critical for understanding cardiac development, homeostasis and disease. Moreover, strategies to isolate and analyze the complex inflammatory and tissue remodeling cell types that follow cardiac injury is particularly important for development of strategies to improve cardiac repair. Here we describe in detail how non-cardiomyocytes can be successfully isolated from the mouse heart. In addition, we describe how these isolation methods can be effectively coupled with flow cytometry, fluorescence activated cell sorting and/or magnetic-labeling to analyze and enrich cells for subsequent cellular or molecular analyses.
  Journal of immunological methods 04/2013;
• Article: Single Domain antibody-Alkaline Phosphatase Fusion Proteins for Antigen Detection- Analysis of Affinity and Thermal Stability of Single Domain Antibody.

  Jinny L Liu, Daniel Zabetakis, Audrey Brozozog Lee, Ellen R Goldman, George P Anderson
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  ABSTRACT: Single domain antibody (sdAb)-alkaline phosphatase (AP) fusion proteins have been demonstrated to be useful immunodiagnostic reagents for bio-threat agent detection. The bivalent nature of sdAb-AP fusion proteins significantly increases effective affinity and thus the sensitivity of detection, but the thermal stability of the fusion protein had not been explored. This property is critical for the development of immunoassays for use in austere environments. In this study four sdAb with specificity for MS2 phage coat protein (CP) were expressed as fusions with AP in order to evaluate the thermal stability and affinity of the resulting constructs. The melting temperature (Tm) of the sdAb and sdAb-AP fusion proteins was measured by a combination of circular dichroism (CD), differential scanning calorimetry (DSC) and Fluorescence-based Thermal Shift assay. Binding kinetics were assessed using surface plasmon resonance (SPR). Our results indicated that the AP fusion protein did not increase the Tm or enhance thermal stability of the sdAb, but did provide the expected increase in binding affinity as compared to the original sdAb.
  Journal of immunological methods 04/2013;
• Article: An Extended Range Generic Immunoassay for Total Human Therapeutic Antibodies in Preclinical Pharmacokinetic Studies.

  Colin M Hall, Josh T Pearson, Vimal Patel, Larry C Wienkers, Robert J Greene
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  ABSTRACT: Bioanalytical support of discovery programs for human monoclonal antibody therapies involves quantitation by immunoassay. Historically, preclinical samples have been analyzed by the traditional Enzyme-Linked Immuno-Sorbent Assay (ELISA). We investigated transferring our generic ELISA for quantitating human IgG constructs in preclinical serum samples to an automated microfluidics immunoassay platform based on nanoscale streptavidin bead columns. Transfer of our immunoassay to the automated platform resulted in the anticipated reduction in analysts' time required for manual manipulation (ELISA) but also a substantial increase in the dynamic range of the immunoassay. The generic nature and wide dynamic range of this automated microcolumn immunoassay permits bioanalytical support of novel therapeutic candidates without the need to develop new, specific assay reagents and minimizes the chances that sample reassays will be required due to out of range concentration results. Improved process efficiencies and enhanced workflow during the analysis of preclinical PK samples enables high throughput assessment of human monoclonal antibody leads in early discovery programs.
  Journal of immunological methods 04/2013;
• Article: A simple skin blister technique for the study of in vivo transmigration of human leukocytes.

  Lisa Davidsson, Lena Björkman, Karin Christenson, Mikael Alsterholm, Charlotta Movitz, Fredrik B Thorén, Anna Karlsson, Amanda Welin, Johan Bylund
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  ABSTRACT: The study of human leukocytes is almost exclusively conducted using cells isolated from peripheral blood. This is especially true for neutrophils, despite the fact that these cells are of main (pathological) importance in extravascular tissues upon e.g., infection and/or tissue damage. The journey from circulation to tissue is typically associated with a number of cellular changes, making the cells primed, or hyper-responsive, and in many aspects distinct from the cells present in circulation. Models to obtain in vivo transmigrated leukocytes from human tissue are available, but not widely used. We here describe an easy-to-use model for the study of local inflammation, stemming from limited tissue damage, which can be used to isolate viable and functional leukocytes. The model is based on the generation of aseptic skin blisters, formed by the application of negative pressure, and allows for investigations of the cellular infiltrate as well as of soluble mediators present in the exudate. We believe that this method, combined with modern analysis equipment suitable for small volumes and cell numbers, could be of great use for increasing our understanding of the nature and function of leukocytes that have left circulation and transmigrated to inflamed tissues.
  Journal of immunological methods 04/2013;
• Article: Peripheral blood natural killer (NK) cell function in healthy adults assessed using the target-induced NK loss (TINKL) assay.

  Hilary S Warren, Fan Wu, Peggy L Horn, David B Pyne, Nicholas P West, Allan W Cripps
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  ABSTRACT: In this technical note we provide data useful for the clinical application of the target-induced Natural Killer (NK) loss (TINKL) assay. The TINKL assay is a sensitive flow cytometry-based assay for measuring NK cell function. Loss of NK cells from the lymphocyte gate occurs following culture with K562 (the prototypic target cell for natural killing) and antibody-coated target cells (for antibody-dependent killing). By analysis of multiple samples of PBMC from single donors we document the intra- and inter-experimental variability of the assay. The intra-experimental coefficient of variation (CV) was on average 11% for natural killing and 3% for antibody-dependent killing, compared to 14% and 9% respectively for the inter-experimental variation. Analysis of a 123 normal healthy adults shows large variability in the functional capacity of NK cells in the population both for natural killing (CV 33%) and antibody-dependent killing (CV 27%).
  Journal of immunological methods 04/2013;
• Article: Preventing adsorption of immunoglobulin G to solid surface using poloxamer 407 eliminates artefactual stimulation of neutrophils.

  Iwan Kustiawan, Ninotska I L Derksen, Theo Rispens
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  ABSTRACT: To study the effect of polyclonal intravenous immunoglobulin G (IVIG) on neutrophils in vitro, adsorption of immunoglobulin G (IgG) to solid surfaces has to be prevented, because IgG bound to the solid surface can activate neutrophils through activating FcγRs. In this study we demonstrate that poloxamer 407, a non ionic surfactant, at low concentration (0.05%) prevented the adsorption of high concentrations of IgG (5mg/ml) better than other blocking agents without interfering with the interaction of IgG with the neutrophils. Poloxamer 407 is therefore a suitable blocking agent to prevent the interaction of immunoglobulin to the solid surface in cell-based in vitro experiments.
  Journal of immunological methods 03/2013;
• Article: Biomarkers for Non-Human Primate Type-I Hypersensitivity: Antigen-Specific Immunoglobulin E Assays.

  Darcey Clark, Faith Shiota, Carla Forte, Padma Narayanan, Daniel T Mytych, M Benjamin Hock
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  ABSTRACT: Immunoglobulin E (IgE) is the least abundant immunoglobulin in serum. However, development of an IgE immune response can induce IgE receptor-expressing cells to carry out potent effector functions. A reliable antigen-specific IgE biomarker method for use in non-human primate studies would facilitate (i) confirmation of type-I hypersensitivity reactions during safety toxicology testing, and (ii) a better understanding of non-human primate models of allergic disease. We cloned and expressed a recombinant cynomolgus monkey IgE molecule in order to screen a panel of commercially available detection reagents raised against human IgE for cross-reactivity. The reagent most reactive to cynomolgus IgE was confirmed to be specific for IgE and did not bind recombinant cynomolgus monkey IgG1-4. A drug-specific IgE assay was developed on the MSD electrochemiluminescent (ECL) platform. The assay is capable of detecting 10 ng/mL drug-specific IgE. Importantly, the assay is able to detect IgE in the presence of excess IgG, the scenario likely to be present in a safety toxicology study. Using our ECL assay, we were able to confirm that serum from cynomolgus monkeys that had experienced clinical symptoms consistent with hypersensitivity responses contained IgE specific for a candidate therapeutic antibody. In addition, a bioassay for mast cell activation was developed using CD34(+)-derived cynomolgus monkey mast cells. This assay confirmed that plasma from animals identified as positive in the drug-specific IgE immunoassay contained biologically active IgE (i.e. could sensitize cultured mast cells), resulting in histamine release after exposure to the therapeutic antibody. These sensitive assays for type-I hypersensitivity in the NHP can confirm that secondary events are downstream of immunogenicity.
  Journal of immunological methods 03/2013;
• Article: Novel Data Analysis Methods to Overcome Cut Point Challenges and Enable Comprehensive Assessment of Antidrug Binding Activity in Confirmatory Assays.

  Alvydas Mikulskis, Dave Yeung, Weiping Chen, Devangi Mehta, Lakshmi Amaravadi
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  ABSTRACT: Immunogenicity assessments in response to drug treatment are commonly performed using a tiered approach strategy. All samples are initially tested in a screening assay followed by the evaluation of the screening positive samples in a confirmatory assay. Percent inhibition of signal intensity by the competing unlabeled drug in a confirmatory assay is typically used to measure the specificity of antidrug binding activity in samples, and has been successfully applied to most immunogenicity assays. However, the percent inhibition approach may not be suitable in cases where broadly distributed and high percent inhibition values are observed in drug-naïve subjects or when persistent operator-dependent differences in assay performance are encountered. Herein, we present the case studies faced with such challenges and provide appropriate solutions by introducing two novel data analysis methods: (1) Reference Delta, and (2) Reference Percent Inhibition, - in which relative-to-baseline signal inhibition is calculated for each sample. These novel methods significantly improve the confirmatory assay's ability to detect the samples positive for antidrug antibodies (ADA), especially when challenges are encountered using the traditional percent inhibition approach. Furthermore, both methods can be implemented in parallel with the percent inhibition method, enabling not only confirmation of ADA specificity, but also providing additional insights about the relevance of this antidrug binding activity to drug treatment.
  Journal of immunological methods 03/2013;