Applications of Protein Microarrays in Biomarker Discovery for Autoimmune Diseases.

Dysregulated autoantibodies and cytokines were deemed to provide important cues for potential illnesses, such as various carcinomas and autoimmune diseases. Increasing biotechnological approaches have been applied to screen and identify the specific alterations of these biomolecules as distinctive biomarkers in diseases, especially autoimmune diseases. As a versatile and robust platform, protein microarray technology allows researchers to easily profile dysregulated autoantibodies and cytokines associated with autoimmune diseases using various biological specimens, mainly serum samples. Here, we summarize the applications of protein microarrays in biomarker discovery for autoimmune diseases. In addition, the key issues in the process of using this approach are presented for improving future studies.

A strategic approach to nonclinical immunogenicity assessment: a recommendation from the European Bioanalysis Forum.

Immunogenicity assays are required to evaluate anti-drug antibody (ADA) responses that can be generated against biotherapeutic modalities. Regulatory guidelines focus on clinical requirements, yet it has become apparent that industry has applied these clinical recommendations for immunogenicity assessment to nonclinical studies in varying degrees. ADAs are an anticipated outcome of dosing a humanized or fully human biotherapeutic into an animal. However, a nonclinical ADA response is rarely predictive of the immunogenic potential in humans. The addendum to ICH S6 recommends that immunogenicity should be explicitly examined where there is: evidence of altered pharmacodynamic activity; unexpected changes in exposure in the absence of a pharmacodynamic marker or evidence of immuno-mediated reactions. The European Bioanalytical Forum has extensively discussed and reached a consensus on a minimal strategic approach of when and what to include for nonclinical immunogenicity assessments. Additionally, this paper recommends a strategy for ADA assay validation and sample analysis for those cases when it is considered necessary to include an immunogenicity assessment in nonclinical toxicology studies.

Pharmacist involvement in clinical assessment and laboratory testing for heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is a rare adverse drug reaction. The anti-PF4 antibody assay (ELISA) is utilized to assist in the clinical evaluation of HIT due to its high negative predictability and wide-spread availability. However, it also associated with false positive results. The 4T score can assist in predicting an individual's risk for HIT and the need for further laboratory testing. This was a single-center prospective observational cohort study. Orders for HIT testing were sent via page to a clinical pharmacist to calculate a 4T score. If low risk, the pharmacist contacted the ordering prescriber to recommend discontinuation of laboratory testing. During the study, a clinical support tool was implemented to assist prescribers with ordering HIT tests. The study was divided into a pharmacist intervention group and a control group. A total of 303 pages were received. One hundred nine were missed due to unavailability of the pharmacist at time of page. A pharmacist reviewed 194 pages and intervened on 132. One hundred seven were scored as low risk, 70 as intermediate risk and 9 as high risk. Pharmacist intervention resulted in discontinuing 64 ELISA and 11 serotonin release assay tests. The clinical support tool resulted in a yearly decrease of HIT testing by 27%. Laboratory cost savings totaled $11,000 but did not include avoidance of laboratory technician or drug cost. Pharmacist involvement in the clinical assessment of HIT and the use of a support tool resulted in the reduction of HIT tests in low risk patients.

Flow-Based Immunosensing Using the Pore Channel of a Porous Membrane As a Reaction Space.

This Letter describes a new rapid and sensitive immunosensing device using the pore space of a porous membrane as the reaction space. A track-etched membrane with uniform cylindrical pores is used as the base substrate of this device. The capture antibodies are covalently and densely immobilized inside the membrane pores by the uniform introduction of poly(acrylic acid) (PAAc) via the plasma graft polymerization technique, followed by the active ester method. This membrane shows excellent antibody retention by covalent binding. The detection test was carried out via a sandwich-type assay, and all reaction steps from the antigen-antibody reaction to the enzyme reaction were conducted by permeating each solution into the pores. The detection test showed a signal comparable to that of the conventional enzyme-linked immunosorbent assay, although the detection time required in the test was shortened to 35 min. The reason for achieving both high sensitivity and short detection time is that the antibody accumulated pore space with high selectivity and promoted contact between the reactants by solution permeation. This report is expected to aid the design of systems for membrane-based devices, which currently have problems associated with sensitivity, rapidity, selectivity, or amount of sample. We further expect that this system could be applied to various diagnostic areas, including point-of-care testing.

Impact of Adopting Routine Luminex-Based Pretransplant Assessment of HLA Antibodies on Clinical Practice and Outcomes in Kidney Transplantation.

BACKGROUND: Accumulating evidence suggests that detection of human leukocyte antigen (HLA) antibodies by solid phase Luminex assays predicts renal allograft outcomes. However, several controversies exist regarding the interpretation, reproducibility, impact and financial feasibility of global utilization of this assay in pretransplant assessment. METHODS: We studied short-term patient-centered outcomes, medical standards of care, and financial plausibility of using Luminex-based screening for HLA antibodies in renal allograft recipients compared to outcomes in nontested patients. RESULTS: We included 1808 patients assessed for transplantation from 2011 to 2018. Luminex-tested patients had lower rates of rejection in the first post-transplant week (OR 0.36, P < .001) and lower odds of antibody-mediated rejection in the first 6 months (OR 0.4, P = .004). Forty-four patients with preformed, donor-specific antibodies were transplanted, and everolimus was introduced into our protocols for low-risk patients based on risk stratification by Luminex results. The number of tests needed to be performed to prevent 1 episode of antibody-mediated rejection in the first 6 months was 28 (P = .004), which was financially plausible. CONCLUSIONS: Routine pre-transplant assessment of HLA antibodies using Luminex assays may allow for better patient-centered, short-term graft outcomes and objective tailoring of immunosuppression at a financially plausible, cost-effective rate.

2013 Banff Criteria for Acute Antibody-Mediated Rejection Are Superior to 2007 Banff Criteria in the Diagnosis and Assessment of Renal Allograft Outcomes.

BACKGROUND: The 2013 Banff meeting updated the requirements for the diagnosis of acute/active antibody-mediated rejection (AAMR) in kidney allografts. There has been speculation that the changes lower the threshold for diagnosing AAMR, and may lead to possible unnecessary and expensive treatment. METHODS: We compared the 2013 Banff classification for AAMR to the previous 2007 Banff to determine if there was an increase in the number of patients receiving a diagnosis of AAMR and if the diagnosis affected allograft survival and post-biopsy 3-month and 6-month creatinine and eGFR values. RESULTS: A total of 212 renal allograft biopsies were compared to both 2007 and 2013 Banff classification requirements for AAMR. Ten patients (11 biopsies) met the 2007 criteria. An additional 15 patients (20 biopsies) met the 2013 criteria. These 2 groups showed no statistically significant demographic differences. By applying the 2013 Banff classification, we observed a 2.5-fold increase in the number of AAMR cases. One-year post-transplant allograft survival was higher in the 2013 group (.85 vs .55) and the 3-month and 6-month post-biopsy creatinine values were significantly lower for the 2013 group (1.6 ± .6 vs 3.3 ± 2.2, P value .01, and 1.7 ± .6 vs 3.4 ± 2.8, P value .03). The 3-month and 6-month eGFR values were higher in the 2013 group, although not statistically significant. CONCLUSIONS: These results suggest that use of Banff 2013 criteria in place of Banff 2007 may result in diagnosing milder and earlier cases of AAMR with the possibility of initiating earlier treatment and improving graft outcomes.

Disposable impedance-based immunosensor array with direct-laser writing platform.

Immunoassay is a powerful technique to identify and quantify biological molecules, which base on the specificity and selectivity of antigen-antibody interaction. Impedance-based immunosensor has recently shown a great potential to provide rapid and label-free detections. However, the conventional impedance-based immunosensors rely on dedicated electrochemical measurement interface which involves expensive fabrication procedures such as gold deposition and photolithography. In this work, we propose an ultra-low-cost and high processing efficiency platform for impedance-based immunosensing. With effortless operations of direct-laser-writing, an impedance-based immunoassay can be fabricated within 5 min in standard laboratories. The as-fabricated devices have shown great stability and a high device-to-device uniformity. In order to further validate impedance sensing system's performance, finite element analysis and impedance equivalent model analysis were performed. The measured data was consistent with the simulation results. With the standard gold electrodes surface bio-functionalization procedures, the disposable immunoassay can detect anti-IgG down to 10 ng/ml.

Label-free real-time detection of biotinylated bovine serum albumin using a low-cost optical cavity-based biosensor.

We have developed a low-cost optical cavity-based biosensor with a differential detection method for point-of-care medical diagnostics. To experimentally demonstrate its label-free real-time biosensing capability, we performed the detection of biotinylated bovine serum albumin (BSA). Streptavidin is introduced into the optical cavity structure and immobilized on 3-aminopropyltriethoxysilane (APTES) coated surface. After rinsing out unbound streptavidin with DI water, biotinylated BSA without any labeling is introduced. A CMOS camera captures the transmitted light of two different wavelengths passing through the optical cavity sensing area in real-time. Then, the differential values are calculated to enhance the responsivity. We successfully demonstrated the label-free real-time detection of biotinylated BSA, and the measurement results matched well with the simulation results. The limit of detection of the optical cavity-based biosensor for the biotinylated BSA detection with the sensing area of 180 µm × 180 µm is estimated to be 2.82 pM, which could be reduced further for a smaller sensing area with the tradeoff of a longer sensing time.

The path to VICTORy - a beginner's guide to success using commercial research antibodies.

Commercial research antibodies are crucial tools in modern cell biology and biochemistry. In the USA some $2 billion a year are spent on them, but many are apparently not fit-for-purpose, and this may contribute to the 'reproducibility crisis' in biological sciences. Inadequate antibody validation and characterization, lack of user awareness, and occasional incompetence amongst suppliers have had immense scientific and personal costs. In this Opinion, I suggest some paths to make the use of these vital tools more successful. I have attempted to summarize and extend expert views from the literature to suggest that sustained routine efforts should made in: (1) the validation of antibodies, (2) their identification, (3) communication and controls, (4) the training of potential users, (5) the transparency of original equipment manufacturer (OEM) marketing agreements, and (5) in a more widespread use of recombinant antibodies (together denoted the 'VICTOR' approach).

Assessment of Humoral, Innate, and T-Cell Immune Responses to Adeno-Associated Virus Vectors.

Adeno-associated virus (AAV)-based gene therapy is being applied to treat a wide array of diseases. Preexisting host immune responses to AAV and immune responses elicited by AAV vector administration remain a problem that needs to be further studied. Here we present a series of protocols to assess immune responses before and after AAV vector administration that are applicable to multiple animal models and phase 1 clinical trials. More specifically, they may be use to evaluate (1) the humoral immune response, through levels of AAV-neutralizing and binding antibodies; (2) the innate immune response, through the acute induction of inflammatory cytokines; and (3) the T-cell immune response, through the activation of transgene- and vector-specific CD8+ and CD4+ T cells.

Quartz crystal microbalance as an assay to detect anti-drug antibodies for the immunogenicity assessment of therapeutic biologics.

Because of their biological origins, therapeutic biologics can trigger an unwanted deleterious immune response with some patients. The immunogenicity of therapeutic biologics can affect drug efficacy and patient safety by the production of circulating anti-drug antibodies (ADA). In this study, quartz crystal microbalance (QCM) was developed as an assay to detect ADA. Etanercept (Enbrel®) was covalently grafted to dextran-modified QCM surfaces. Rabbits were immunized with etanercept to generate ADA. Results showed the QCM assay could detect purified ADA from rabbits at concentrations as low as 50 ng/mL, within the sensitivity range of ELISA. The QCM assay could also assess the ADA isotype. It was shown that the ADA were composed of the IgG isotype, but not IgM, as expected. Furthermore, it was shown that QCM surfaces that had been used to detect ADA could be regenerated in glycine-HCl solution and reused. The QCM assay was also demonstrated to detect ADA in crude serum samples. Serum was collected from the rabbits and analyzed before and after etanercept immunization. ADA were clearly detected in serum from rabbits after immunization, but not in serum before immunization. Serum from patients administered with etanercept for rheumatoid arthritis (RA) treatment was also analyzed and compared to serum from healthy donors. Sera from 10 RA patients were analyzed. Results showed one of the RA patient serum samples may have ADA present. In conclusion, QCM appears to be a viable assay to detect ADA for the immunogenicity assessment of therapeutic biologics.

FIGHTING FOR THE FUTURE OF ANTIBODIES.

Nathan Blow looks at how efforts to create guidelines and scoring systems could change the way you buy antibodies.

Inhibition of interleukin-5 induced false positive anti-drug antibody responses against mepolizumab through the use of a competitive blocking antibody.

Mepolizumab, a humanized IgG1 monoclonal antibody that blocks native homodimeric interleukin-5 (IL-5) from binding to the IL-5 receptor, has recently been approved for treatment of severe eosinophilic asthma. Our initial immunogenicity assay method for phase I and II studies utilized a bridging electrochemiluminescence format with biotin and ruthenium-labelled mepolizumab linked by anti-drug antibodies (ADA). We discovered that IL-5 significantly increased in dosed subjects from a phase II study and that the increased IL-5 was in the form of a drug-bound complex. We demonstrated that the elevated drug-bound IL-5 produced false-positive response in the in vitro ADA assay, in which drug-bound IL-5 dissociated and then bridged mepolizumab conjugates to yield positive signal. To eliminate the IL-5 interference, we compared two strategies: a solid-phase immunodepletion of IL-5 and an in-solution IL-5 immunocompetition. We identified the best competitive antibody for each purpose. We found both methods demonstrated similar effectiveness in reducing the false positive signal in IL-5 spiked samples; however, the in-solution immunocompetition for IL-5 had fewer false positives in study samples. Additionally, the in-solution immunocompetition method was experimentally simpler to execute. We modified the ADA assay by adding a pre-treatment step with a mepolizumab competitive anti- IL-5 antibody. Using this new method, we retested clinical samples from two phase II studies (MEA112997 and MEA114092). The confirmed ADA positive incidence was reduced from 29% and 61% to 1% and 8% with the modified in-solution immune inhibition method. Target interference is a fairly common problem facing immunogenicity testing, and target-induced false positive cannot be distinguished from true ADA response by the commonly used drug competitive confirmation assay. The approach and method used here for resolving target interference in ADA detection will be useful for differentiating between a true ADA response and target induced false positive as well as similar challenges in other programs.

A high-value cost conscious approach to minimize heparin induced thrombocytopenia antibody (HITAb) testing using the 4T score.

Heparin Induced Thrombocytopenia (HIT) is a serious complication from administration of heparin products. The 4T score is a validated pre-test probability tool to screen for HIT in hospitalized patients. As the negative predictive value (NPV) is very high further testing for HIT in patients with a low score can be avoided. Our objective was to determine trends at our hospital with respect to utilization of HIT antibody (HITAb) testing and evaluate economic burden from unnecessary HIT testing. A retrospective cohort review was performed on patients age 18 and above admitted to a tertiary care center from February 2013 to December 2014 who underwent HITAb testing. Surgical ICU patients were excluded. Patients were stratified into low, intermediate, and high risk for HIT based on the 4T model. Statistical analysis was performed using Chi square and regression models. Of 150 patients that underwent HITAb testing, 134 met inclusion criteria. 73 were male (54.47 %) and mean age was 55.50 ± 17.27 years. 81 patients had a low 4T score 0-3. Analysis of testing trends showed 60.44 % of patients were tested for HITAb despite being low risk using the 4T model. Only three patients with low 4T score were positive on confirmatory SRA testing (NPV 96.29 % CI 95 = 89.56-99.23 %). Expenditure due to inappropriate testing and treatment was estimated at $103,348.13. The majority of HITAb testing was found unnecessary based on the investigator calculated 4T score. We propose implementation of an electronic medical record (EMR) based calculator in order to reduce unneeded tests and reduce use of costlier alternative anticoagulants.

General Assessment of Humoral Activity in Healthy Humans.

The humoral immune system is network of biological molecules designed to maintain a healthy homeostatic equilibrium. Because antibodies are an abundant and highly specific effector of immunological action, they are also an important reservoir of previous host exposures. Antibodies may play a major role in early detection of host challenge. Unfortunately, few practical methods exist for interpreting the information stored in antibody variable regions. Immunosignatures use a microarray of thousands of random sequence peptides to interrogate antibodies in a broad and unbiased fashion. The pattern of binding between antibody and peptide is reproducible. Once the system has been trained on a disease cohort, blinded samples can be reliably predicted. Although immunosignatures of both chronic and infectious disease have been extensively tested, less has been done to demonstrate how healthy immunosignatures change over time or between individuals. Here, we report the results of a study of immunosignatures of healthy persons over brief (12 h sampled once per hour), intermediate (32 days sampled once per day), and long (5 years sampled once every year) time spans. Using this information, we were also able to detect intentional and unintentional immunological perturbations in the form of a vaccine and an infection, respectively. Our findings suggest that, even with the variability inherent in healthy immunosignatures, a single person's immunosignature will remain constant over time. Over this healthy signature, vaccines and infections create subsignatures that are common across multiple people, even subsuming healthy fluctuations. These findings have implications for disease monitoring and early diagnosis.

Immunogenicity assessment of biotherapeutic products: An overview of assays and their utility.

Biotherapeutic products (BTPs) are the fastest growing medicines in the pharmaceutical market. Despite their clinical success, the immunogenicity of BTPs continues to be a major concern. Assessment of immunogenicity as well as appropriate interpretation of immunogenicity data is therefore, of critical importance for defining safety profile of these products for the purpose of their licensure and use. In the past decade, much progress has been made towards how immunogenicity should be studied. This article reflects the content of the brief presentation on principles of methods used for immunogenicity assessment and their merits and limitations given at the first World Health Organization (WHO) implementation workshop on rDNA derived biotherapeutic products held in the Republic of Korea in May 2014 to support the case studies on immunogenicity presented and discussed during the workshop. The purpose of this article is to provide an overview of the methods used for assessing immunogenicity of biotherapeutic products (BTPs) and the most important considerations in interpreting results in the context of regulatory overview of these products.

Detección de anticuerpos circulantes en suero para el diagnóstico de dermatosis ampollar IgA lineal / Detecção de anticuerpos circulantes em suero para diagnóstico de dermatose ampollar IgA linear

Objetivo: realizar una revisión, apreciación crítica y síntesis de la evidencia disponible sobre la validez diagnóstica de la detección de anticuerpos circulantes para el diagnóstico de dermatosis ampollar IgA lineal. Metodología: se realizó una búsqueda de evidencia en las bases de datos: MEDLINE, EMBASE, la Librería Cochrane y LILACS. Adicionalmente, se hizo se indagó por estudios locales a través del motor de búsqueda Google. Dos evaluadores de manera independiente, tamizaron las referencias obtenidas, resolviendo las discrepancias por consenso. Resultados: se identificaron 20 publicaciones. Con los resultados obtenidos, no fue posible identificar revisiones sistemáticas de la literatura ni estudios de validez diagnóstica de la IFD. Se hizo una preselección de 2 estudios observacionales descriptivos que no cumplieron los cirterios de inclusión. Conclusiones: con los resultados de las búsquedas de evidencia realizadas no es posible evaluar la utilidad de a detección de anticuerpos circulantes para el diagnóstico de la dermatosis ampollar IgA lineal.(AU)

Detección de anticuerpos circulantes en suero para el diagnóstico de epidermólisis ampollar adquirida / Detection of circulating antibodies in serum for the diagnosis of acquired blister epidermolysis

Objetivo: realizar una revisión, apreciación crítica y síntesis de la evidencia disponible sobre la validez diagnóstica de la detección de anticuerpos circulantes para el diagnóstico de epidermolisis ampollar adquirida. Metodología: se realizó una búsqueda de evidencia en las bases de datos: MEDLINE, EMBASE, la Librería Cochrane y LILACS. Adicionalmente, se hizo se indagó por estudios locales a través del motor de búsqueda Google. Dos evaluadores de manera independiente, tamizaron las referencias obtenidas, resolviendo las discrepancias por consenso. Resultados: se identificaron 49 publicaciones. Con los resultados obtenidos, no fue posible identificar revisiones sistemáticas de la literatura ni estudios de validez diagnóstica de la IFD. Se hizo una preselección de 7 estudios observacionales. Fue incluido 1. Se presentan los datos descriptivos sobre la positividad de las pruebas usadas para detectar anticuerpos circulantes en personas con sospecha clínica de epidermólisis ampollar adquirida. Conclusiones: con los hallazgos presentados en este informe, no es posible conocer el desempeño de las pruebas para detectar anticuerpos circulantes en suero de pacientes con sospecha clínica de epidermolisis ampollar adquirida.(AU)

Detección de anticuerpos circulantes en suero para el diagnóstico de pénfigo / Detection of circulating antibodies in serum for the diagnosis of pemphigus

Objetivo: realizar una revisión, apreciación crítica y síntesis de la evidencia disponible sobre la validez diagnóstica de la detección de anticuerpos circulantes para el diagnóstico de pénfigo. Metodología: se realizó una búsqueda de evidencia en las bases de datos: MEDLINE, EMBASE, la Librería Cochrane y LILACS. Adicionalmente, se hizo se indagó por estudios locales a través del motor de búsqueda Google. Dos evaluadores de manera independiente, tamizaron las referencias obtenidas, resolviendo las discrepancias por consenso. Resultados: se identificaron 105 publicaciones. Con los resultados obtenidos, no fue posible identificar revisiones sistemáticas de la literatura ni estudios de validez diagnóstica. Se hizo una preselección de 4 estudios observacionales descriptivos que fueron excluidos. Conclusiones: con los hallazgos de las búsquedas de evidencia, no es posible evaluar la utilidad de las pruebas usadas en de la detección de anticuerpos circulantes en suero para el diagnóstico de pénfigo.(AU)

Assays and strategies for immunogenicity assessment of biological agents.

Some patients with chronic inflammatory diseases either do not respond to or lose their initial responsiveness to Tumor Necrosis Factor (TNF) inhibitor therapy. In these patients, the clinical response after switching to another anti-TNF drug suggests that lack of response is not related to the therapeutic target itself but immunogenicity. All biologics are potentially immunogenic and can induce the development of antidrug antibodies (ADAs). ADA formation is associated with lower serum drug levels, infusion reactions, and loss of response. Analytical methods for ADA detection include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), surface plasmon resonance, and electrochemiluminescence. Currently, RIA and ELISA are the preferred methods due to a combination of reproducibility, sensitivity, and cost but have some limitations. There is no single available assay that has all pros and no cons, and therefore the use of more methods for the assessment of samples is a high priority.