ABSTRACT
OBJECTIVE: The first biomarker associated with the rheumatoid arthritis is rheumatoid factor (RF) and since the earliest reports a role has been proposed in the diagnosis and in the prediction of clinical features and outcome. The study of RF isotypes has further attempted to improve diagnostic accuracy and identify specific subgroups of patients. The main objective of this study is to provide an analysis of the literature on the role of RF isotypes in the diagnosis and prognosis of rheumatoid arthritis (RA). METHODS: We performed a systematic literature review and meta-analysis on the role of RF isotypes in RA (only in English, from PubMed, search terms: "rheumatoid factor isotypes", "diagnosis", "prognosis" and "rheumatoid arthritis", last search 31 July 2022, two independent assessment of quality and biases, results included in tables and in the meta-analysis). RESULTS: Thirty-six articles were examined (7517 patients). Testing all RF isotypes with latex test or nephelometry allows for the highest sensitivity (68.6%, 95% CI 66.2% to 71.0%); nonetheless, the determination of IgA isotype provides the highest specificity (91.4%, 95% CI 90.8% to 92.0%) and the highest positive likelihood ratio (7.7, 95% CI 5.7 to 10.4). When testing IgM isotype the highest diagnostic OR (21.7, 95% CI 16.1 to 29.3) is reached. When analysing anti-citrullinated protein antibodies, RF isotype determination increases diagnostic accuracy. On the other hand, these do not provide relevant prognostic information, as results are conflicting. CONCLUSIONS: Testing RF allows the highest sensitivity, while IgA isotype the highest specificity and positive likelihood ratio for RA diagnosis. On the other hand, determination of RF isotypes dose not allow prognostic information, as data are limited and heterogeneous.
Subject(s)
Arthritis, Rheumatoid , Rheumatoid Factor , Humans , Arthritis, Rheumatoid/diagnosis , Immunoglobulin Isotypes/analysis , Anti-Citrullinated Protein Antibodies , Immunoglobulin AABSTRACT
The coronavirus disease (COVID-19) is the global public health challenge currently persisting at a grand scale. A method that meets the rapid quantitative detection of antibodies to assess the body's immune response from natural COVID-19 illness or vaccines' effects is urgently needed. In the present study, an attempt was made to integrate a newly designed spectrometer to the COVID-19 test strip procedure; this augmentation provides the quantitative capacity to a lateral flow immunoassay (LFIA). Optical interpretation of results by quantitative α index, rather than visual qualification, can be done quickly, in 5-10 minutes. The developed product was compared with several other serological IgM/IgG antibody reagents on the market by recruiting 111 participants suspected of having COVID-19 infection from March to May 2020 in a hospital. Taking RT-PCR as the diagnostic gold standard, the quantitative spectral LIFA platform could correctly detect all 12 COVID-19 patients. Concerning RT-PCR negative patients, all three antibody testing methods found positive cases. The optical-based platform exhibited the ability of early detection of immunoglobulins of RT-PCR negative patients. There was an apparent trend that elevation of IgM levels in the acute phase of infection; then IgG levels rose later. It exhibited the risk of a false-negative diagnosis of RT-PCR in COVID-19 testing. The significant detection ability of this new optical-based platform demonstrated clinical potential.
Subject(s)
COVID-19/immunology , Immunoassay/methods , Immunoglobulin Isotypes/analysis , Humans , Pandemics , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Background: Rapidly progressive glomerulonephritis caused by antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is typically characterized as pauci-immune glomerulonephritis. However, immune complex (IC) deposition in the glomerulus has been reported in a growing number of studies. Here, we assess the presence of glomerular immune deposits alongside renal outcome in myeloperoxidase (MPO)-ANCA associated glomerulonephritis (MPO-ANCA GN). Methods: Clinical and histopathologic characteristics of 97 patients with MPO-ANCA GN classified by renal biopsy from January 2008 to December 2019 were extracted retrospectively from electronic medical records. The extent of immune deposits in the kidney (C3, C4, C1q, IgA, IgG, IgM) at diagnosis were analyzed by immunofluorescence (IF). Patients were followed up for a median period of 15 months. The response to treatment and outcomes of renal and histological lesion changes were also assessed. Results: In our study, 41% (40/97) of patients showed positive IF (≥2+) for at least one of the six immunoglobulin or complement components tested. Patients with IC deposits showed higher levels of serum creatinine (p=0.025), lower platelet counts (p=0.009), lower serum complement C3 (sC3) (≤790 ml/L) (p=0.013) and serum IgG (p=0.018) than patients with pauci-immune (PI) deposition at diagnosis. End-stage renal disease was negatively associated with eGFR (HR 0.885, 95% CI 0.837 to 0.935, p<0.0001), platelet count (HR 0.996, 95% CI 0.992 to 1.000, p=0.046) and serum globulin (HR 0.905, 95% CI 0.854 to 0.959, p=0.001). Patients with lower sC3 levels showed a worse renal outcome than the patients with normal sC3 at diagnosis (p=0.003). Analysis of the components of the renal deposits found that patients with IgG deposits exhibited a poorer renal outcome compared to patients that were IgG negative (p=0.028). Moreover, Bowman's capsule rupture occurred less frequently in patients with IgM deposition compared with IgM negative counterparts (p=0.028). Vascular lesions and granuloma-like lesions had been seen more frequently in cases with IgA deposition than those without IgA deposition (p=0.03 and 0.015, respectively). Conclusion: In conclusion, patients with immune complex deposits in the kidney showed less platelet count, lower sC3 and sIgG levels, and higher serum creatinine levels. Patients with low sC3 at initial and with continued low sC3 during the treatment displayed a trend toward poorer kidney survival. Moreover, the IC group showed a worse renal outcome than the PI group, further enforcing the present strategy of introducing complement targeted therapies in AAV.
Subject(s)
Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/immunology , Antibodies, Antineutrophil Cytoplasmic/analysis , Complement System Proteins/analysis , Glomerulonephritis/immunology , Immunoglobulin Isotypes/analysis , Kidney Glomerulus/immunology , Peroxidase/immunology , Adult , Aged , Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/drug therapy , Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/mortality , Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/pathology , Biopsy , Cyclophosphamide/therapeutic use , Disease Progression , Drug Therapy, Combination , Female , Fluorescent Antibody Technique , Glomerulonephritis/drug therapy , Glomerulonephritis/mortality , Glomerulonephritis/pathology , Glucocorticoids/therapeutic use , Humans , Immunosuppressive Agents/therapeutic use , Kidney Failure, Chronic/immunology , Kidney Failure, Chronic/mortality , Kidney Glomerulus/drug effects , Kidney Glomerulus/pathology , Male , Middle Aged , Retrospective Studies , Risk Assessment , Risk Factors , Time Factors , Treatment OutcomeABSTRACT
India is one of the most affected countries by COVID-19 pandemic; but little is understood regarding immune responses to SARS-CoV-2 in this region. Herein we examined SARS-CoV-2 neutralizing antibodies, IgG, IgM, IgA and memory B cells in COVID-19 recovered individual from India. While a vast majority of COVID-19 recovered individuals showed SARS-CoV-2 RBD-specific IgG, IgA and IgM antibodies (38/42, 90.47%; 21/42, 50%; 33/42, 78.57% respectively), only half of them had appreciable neutralizing antibody titers. RBD-specific IgG, but not IgA or IgM titers, correlated with neutralizing antibody titers and RBD-specific memory B cell frequencies. These findings have timely significance for identifying potential donors for plasma therapy using RBD-specific IgG assays as surrogate measurement for neutralizing antibodies in India. Further, this study provides useful information needed for designing large-scale studies towards understanding of inter-individual variation in immune memory to SARS CoV-2 natural infection for future vaccine evaluation and implementation efforts.
Subject(s)
Antibodies, Neutralizing/analysis , Antibodies, Viral/analysis , B-Lymphocytes , COVID-19/immunology , Spike Glycoprotein, Coronavirus/immunology , Adolescent , Adult , Aged , B-Lymphocytes/cytology , B-Lymphocytes/immunology , COVID-19/epidemiology , Humans , Immunity, Humoral , Immunoglobulin Isotypes/analysis , India/epidemiology , Male , Middle Aged , Pandemics , Young AdultABSTRACT
Background: There is limited information on the functional neutralizing capabilities of breastmilk SARS-CoV-2-specific antibodies and the potential adulteration of breastmilk with vaccine mRNA after SARS-CoV-2 mRNA vaccination. Methods: We conducted a prospective cohort study of lactating healthcare workers who received the BNT162b2 vaccine and their infants. The presence of SARS-CoV-2 neutralizing antibodies, antibody isotypes (IgG, IgA, IgM) and intact mRNA in serum and breastmilk was evaluated at multiple time points using a surrogate neutralizing assay, ELISA, and PCR, over a 6 week period of the two-dose vaccination given 21 days apart. Results: Thirty-five lactating mothers, median age 34 years (IQR 32-36), were included. All had detectable neutralizing antibodies in the serum immediately before dose 2, with significant increase in neutralizing antibody levels 7 days after this dose [median 168.4 IU/ml (IQR 100.7-288.5) compared to 2753.0 IU/ml (IQR 1627.0-4712.0), p <0.001]. Through the two vaccine doses, all mothers had detectable IgG1, IgA and IgM isotypes in their serum, with a notable increase in all three antibody isotypes after dose 2, especially IgG1 levels. Neutralizing antibodies were detected in majority of breastmilk samples a week after dose 2 [median 13.4 IU/ml (IQR 7.0-28.7)], with persistence of these antibodies up to 3 weeks after. Post the second vaccine dose, all (35/35, 100%) mothers had detectable breastmilk SARS-CoV-2 spike RBD-specific IgG1 and IgA antibody and 32/35 (88.6%) mothers with IgM. Transient, low intact vaccine mRNA levels was detected in 20/74 (27%) serum samples from 21 mothers, and 5/309 (2%) breastmilk samples from 4 mothers within 1 weeks of vaccine dose. Five infants, median age 8 months (IQR 7-16), were also recruited - none had detectable neutralizing antibodies or vaccine mRNA in their serum. Conclusion: Majority of lactating mothers had detectable SARS-CoV-2 antibody isotypes and neutralizing antibodies in serum and breastmilk, especially after dose 2 of BNT162b2 vaccination. Transient, low levels of vaccine mRNA were detected in the serum of vaccinated mothers with occasional transfer to their breastmilk, but we did not detect evidence of infant sensitization. Importantly, the presence of breastmilk neutralising antibodies likely provides a foundation for passive immunisation of the breastmilk-fed infant.
Subject(s)
Antibodies, Neutralizing/analysis , Antibodies, Viral/analysis , BNT162 Vaccine/administration & dosage , Milk, Human/immunology , Adult , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , BNT162 Vaccine/analysis , BNT162 Vaccine/blood , Cohort Studies , Female , Health Personnel , Humans , Immunoglobulin Isotypes/analysis , Immunoglobulin Isotypes/blood , Infant , Lactation , Milk, Human/chemistry , Prospective StudiesABSTRACT
INTRODUCTION: Antibody mediated rejection is the leading cause of kidney transplant failure. Not all antibodies are harmful and some may be protective. Immunoglulin Gs, of which there are four subtypes, are detected by single antigen bead testing. The aims of this study were to characterise the IgG subclass profiles for class I HLA-specific antibodies in an uncensored post-transplant population and to determine the underlying relationship between reactivity patterns and MFI cut-offs with the pan-IgG assay. METHODS: Patients were recruited to the study who were transplanted in our centre between 2009 and 2014. Prospectively stored post-transplant serum initially underwent a Labscreen Mixed assay and those positive for class I HLA-specific antibody underwent standard SAB testing, EDTA, 1 in 10 dilution and IgG subclass modifications using the Luminex platform. A total of 4947 bead reactions from 51 patients were analysed. RESULTS: A 1 in 10 dilution was used as a comparator pan-IgG assay for summed subclass and individual subclass linear regression analyses. Using a dilution to standard assay ratio we characterised all reactions for prozone potential i.e. how likely there is to be inhibition related to complement complex formation. We stratified samples into degrees of association and were able to determine suggested MFI thresholds of Log 5.35 for the dilution assay and Log 5.05 for the summed subclass assay when considering a Log MFI of 6.9 (1000) in the standard assay. Using individual subclass dominant reactions (>70%) we were able to determine linear relationships between the 1 in 10 dilution pan-IgG assay and the individual subclass assays (excluding prozone potential reactions for IgG1/3) enabling us to suggest Log MFI thresholds of 5.03, 3.58, 4.3 and 4.05 respectively for IgG1-4. DISCUSSION: We recommend a 1 in 10 dilution as the optimum pan-IgG comparator assay for a subclass analysis. We advocate the utilisation of the summed subclass assay to determine overall relationships and potential subclass failures. Following others, we recommend serum pre-treatment of the subclass assays to mitigate prozone. We suggest cut-offs for each IgG subclass which should be used with caution given the many inhibitory influences which may include competitive inhibition for bead binding, IgM and IgA interference and under-representation of specific subclasses on the bead panel.
Subject(s)
HLA Antigens/immunology , Histocompatibility Antigens Class I/immunology , Immune Sera/chemistry , Immunoglobulin G/analysis , Immunoglobulin Isotypes/analysis , Immunosorbent Techniques , Kidney Transplantation , Complement System Proteins/metabolism , Cross Reactions , Humans , Immune Sera/metabolism , Isoantigens/immunology , Microspheres , Transplant RecipientsABSTRACT
BACKGROUND: The study investigated the concentration of IgA, IgM and IgG in colostrum, transitional and mature milk and the effect of parity, age, BMI and family income on secreted immunoglobulin contents of human milk. METHODS: Sequential samples of colostrum, transitional and mature milk were collected from 38 women. Enzymelinked immunosorbent assay was used to analyse the immunoglobulin concentrations. RESULTS: The study revealed that IgA was the dominant immunoglobulin and mean concentration in colostrum, transitional and mature milk was 5.92 ± 1.50 g/L, 3.85 ± 0.64 g/L and 3.72 ± 0.68 g/L, respectively. Both IgA and IgM levels of colostrum decreased significantly in both transitional (P = 0.000) and mature milk (P = 0.000), while the concentration of IgG rises significantly in them (colostrum vs. transitional milk, P = 0.000; and colostrum vs. mature milk P = 0.011). While maternal age, BMI and family income had no significant influence on the immunoglobulin levels at different stages of lactation, parity showed significant influence on IgG (P = 0.03) and IgM (P = 0.05) levels of transitional milk and IgA level of colostrum (P = 0.05). CONCLUSION: The findings suggest that immunoglobulin composition in breast milk is strongly associated with stage of lactation and is likely to be more susceptible to parity than BMI and socioeconomic characteristics.
Subject(s)
Colostrum/chemistry , Immunoglobulin Isotypes/analysis , Maternal Age , Milk, Human/chemistry , Mothers/statistics & numerical data , Parity , Socioeconomic Factors , Adolescent , Adult , Bangladesh , Ethnicity/statistics & numerical data , Female , Humans , Pregnancy , Young AdultABSTRACT
B cells are important in the pathogenesis of many, and perhaps all, immune-mediated diseases. Each B cell expresses a single B cell receptor (BCR)1, and the diverse range of BCRs expressed by the total B cell population of an individual is termed the 'BCR repertoire'. Our understanding of the BCR repertoire in the context of immune-mediated diseases is incomplete, and defining this could provide new insights into pathogenesis and therapy. Here, we compared the BCR repertoire in systemic lupus erythematosus, anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, Crohn's disease, Behçet's disease, eosinophilic granulomatosis with polyangiitis, and immunoglobulin A (IgA) vasculitis by analysing BCR clonality, use of immunoglobulin heavy-chain variable region (IGHV) genes and-in particular-isotype use. An increase in clonality in systemic lupus erythematosus and Crohn's disease that was dominated by the IgA isotype, together with skewed use of the IGHV genes in these and other diseases, suggested a microbial contribution to pathogenesis. Different immunosuppressive treatments had specific and distinct effects on the repertoire; B cells that persisted after treatment with rituximab were predominately isotype-switched and clonally expanded, whereas the inverse was true for B cells that persisted after treatment with mycophenolate mofetil. Our comparative analysis of the BCR repertoire in immune-mediated disease reveals a complex B cell architecture, providing a platform for understanding pathological mechanisms and designing treatment strategies.
Subject(s)
Immune System Diseases/immunology , Immunoglobulin Isotypes/analysis , Immunoglobulin Isotypes/immunology , Receptors, Antigen, B-Cell/analysis , Receptors, Antigen, B-Cell/immunology , Adult , Aged , Clone Cells/cytology , Clone Cells/immunology , Humans , Immunoglobulin A/analysis , Immunoglobulin A/immunology , Immunoglobulin Class Switching/immunology , Immunoglobulin G/analysis , Immunoglobulin G/immunology , Middle Aged , Young AdultABSTRACT
The aim of the present study was to compare concentrations of IgG, IgA, IgM and IgD in both serum and saliva samples from smoking and non-smoking individuals using a protein microarray assay. The findings were also compared to previous studies. Serum and saliva were collected from 48 smoking male individuals and 48 age-matched never-smoker male individuals. The protein microarray assays for detection of human IgG, IgM, IgA and IgD were established and optimized using Ig class-specific affinity-purified goat anti-human Ig-Fc capture antibodies and horseradish peroxidase (HRP)-conjugated goat anti-human Ig-Fc detection antibodies. The Ig class specificity of the microarray assays was verified, and the optimal dilutions of serum and saliva samples were determined for quantification of Ig levels against standard curves. We found that smoking is associated with reduced IgG concentrations and enhanced IgA concentrations in both serum and saliva. By contrast, smoking differentially affected IgM concentrations-causing increased concentrations in serum, but decreased concentrations in saliva. Smoking was associated with decreased IgD concentrations in serum and did not have a significant effect on the very low IgD concentrations in saliva. Thus, cigarette smoking differentially affects the levels of Ig classes systemically and in the oral mucosa. Although there is variation between the results of different published studies, there is a consensus that smokers have significantly reduced levels of IgG in both serum and saliva. A functional antibody deficiency associated with smoking may compromise the body's response to infection and result in a predisposition to the development of autoimmunity.
Subject(s)
Autoimmunity , Cigarette Smoking/immunology , Immunoglobulin Isotypes/analysis , Saliva/chemistry , Adult , Cigarette Smoking/blood , Humans , Immunoglobulin Isotypes/immunology , Male , Middle Aged , Mouth Mucosa/chemistry , Mouth Mucosa/immunology , Non-Smokers , Protein Array Analysis , Saliva/immunology , Smokers , Young AdultABSTRACT
Background Free light chains (FLC) have been proposed as diagnostic biomarkers in the cerebrospinal fluid (CSF) of patients with inflammatory central nervous system (CNS) diseases. However, which method to use for determining an intrathecal FLC synthesis has not yet been clarified. The objective of this study was to compare the diagnostic performance of CSF FLC concentration, FLC quotient (QFLC), FLC index and FLC intrathecal fraction (FLCIF). Methods κ- and λ-FLC were measured by nephelometry under blinded conditions in CSF and serum sample pairs of patients with clinically isolated syndrome (CIS; n = 60), multiple sclerosis (MS; n = 60) and other neurological diseases (n = 60) from four different MS centers. QFLC was calculated as the ratio of CSF/serum FLC concentration, the FLC index as QFLC/albumin quotient and the percentage FLCIF by comparing QFLC to a previously empirically determined, albumin quotient-dependent reference limit. Results CSF FLC concentration, QFLC, FLC index and FLCIF of both the κ- and λ-isotype were significantly higher in patients with CIS and MS than in the control group, as well as in oligoclonal bands (OCB) positive than in OCB negative patients. Each parameter was able to identify MS/CIS patients and OCB positivity, however, diagnostic performance determined by receiver operating characteristic (ROC) analyses differed and revealed superiority of FLC index and FLCIF. Conclusions These findings support the diagnostic value of FLC measures that correct for serum FLC levels and albumin quotient, i.e. blood-CSF barrier function.
Subject(s)
Immunoglobulin Light Chains/analysis , Immunoglobulin Light Chains/cerebrospinal fluid , Immunoglobulins/analysis , Adult , Austria , Cross-Sectional Studies , Demyelinating Diseases/immunology , Diagnostic Tests, Routine/methods , Female , Germany , Humans , Immunoglobulin Isotypes/analysis , Immunoglobulin Isotypes/blood , Immunoglobulin Isotypes/cerebrospinal fluid , Immunoglobulin Light Chains/blood , Immunoglobulin kappa-Chains/blood , Immunoglobulin lambda-Chains/blood , Immunoglobulins/cerebrospinal fluid , Male , Middle Aged , Multiple Sclerosis/immunology , Nervous System Diseases/immunology , ROC CurveABSTRACT
BACKGROUND: The quantitative suspension array technology (qSAT) is a useful platform for malaria immune marker discovery. However, a major challenge for large sero-epidemiological and malaria vaccine studies is the comparability across laboratories, which requires the access to standardized control reagents for assay optimization, to monitor performance and improve reproducibility. Here, the Plasmodium falciparum antibody reactivities of the newly available WHO reference reagent for anti-malaria human plasma (10/198) and of additional customized positive controls were examined with seven in-house qSAT multiplex assays measuring IgG, IgG1-4 subclasses, IgM and IgE against a panel of 40 antigens. The different positive controls were tested at different incubation times and temperatures (4 °C overnight, 37 °C 2 h, room temperature 1 h) to select the optimal conditions. RESULTS: Overall, the WHO reference reagent had low IgG2, IgG4, IgM and IgE, and also low anti-CSP antibody levels, thus this reagent was enriched with plasmas from RTS,S-vaccinated volunteers to be used as standard for CSP-based vaccine studies. For the IgM assay, another customized plasma pool prepared with samples from malaria primo-infected adults with adequate IgM levels proved to be more adequate as a positive control. The range and magnitude of IgG and IgG1-4 responses were highest when the WHO reference reagent was incubated with antigen-coupled beads at 4 °C overnight. IgG levels measured in the negative control did not vary between incubations at 37 °C 2 h and 4 °C overnight, indicating no difference in unspecific binding. CONCLUSIONS: With this study, the immunogenicity profile of the WHO reference reagent, including seven immunoglobulin isotypes and subclasses, and more P. falciparum antigens, also those included in the leading RTS,S malaria vaccine, was better characterized. Overall, incubation of samples at 4 °C overnight rendered the best performance for antibody measurements against the antigens tested. Although the WHO reference reagent performed well to measure IgG to the majority of the common P. falciparum blood stage antigens tested, customized pools may need to be used as positive controls depending on the antigens (e.g. pre-erythrocytic proteins of low natural immunogenicity) and isotypes/subclasses (e.g. IgM) under study.
Subject(s)
Antibodies, Protozoan/analysis , Immunoglobulin Isotypes/analysis , Malaria, Falciparum/epidemiology , Malaria, Falciparum/prevention & control , Plasmodium falciparum/immunology , Serologic Tests/methods , Immunoglobulin E/analysis , Immunoglobulin G/analysis , Immunoglobulin M/analysis , Malaria Vaccines/immunology , Seroepidemiologic StudiesABSTRACT
BACKGROUND: Quantitative suspension arrays are powerful immunoassays to measure antibodies against multiple antigens in large numbers of samples in a short time and using few microliters. To identify antigen targets of immunity for vaccine development against complex microbes like Plasmodium falciparum, such technology allows the characterization of the magnitude and antigenic specificity of Ig isotypes and subclasses that are important for functional responses. However, standardized assays are not widely available. METHODS: We developed six quantitative suspension array assays to measure IgG1, IgG2, IgG3, IgG4, IgM and IgE specific to multiple P. falciparum antigens. Secondary and tertiary antibodies, as well as human purified antibodies for standard curves, were tested among several commercially available sources. Positive and negative controls included plasmas from malaria hyper-immune African adults and from malaria-naïve European adults, respectively. Reagents were selected and optimal antibody and test sample dilutions established according to sensitivity, specificity and performance of the standard curves. The variability between replicates and plates was assessed with 30 test samples and controls. RESULTS: Assays were able to detect P. falciparum antigen-specific antibodies for all isotypes and subclasses in samples from malaria-exposed individuals, with low background signal in blank wells. Levels detected in malaria-naïve individuals were overall low except for IgM. For the IgG2 and IgE assays, a triple sandwich was required for sensitivity. Standard curves with 5-parameter logistic fit were successfully obtained in all assays. The coefficients of variation for measurements performed in different days were all <30%, and <5% when comparing duplicates from the same plate. CONCLUSION: The isotype/subclass assays developed here were sensitive, specific, reproducible and of adequate quantification dynamic range. They allow performing detailed immuno-profiling to large panels of P. falciparum antigens to address naturally- and vaccine-induced Ig responses and elucidate correlates of malaria protection, and could also be applied to other antigenic panels.
Subject(s)
Antibodies, Protozoan/analysis , Antigens, Protozoan/immunology , Immunoglobulin Isotypes/analysis , Malaria Vaccines/immunology , Malaria, Falciparum/diagnosis , Plasmodium falciparum/immunology , Serologic Tests/methods , Adult , Black People , Humans , Observer Variation , Reference Standards , Reproducibility of Results , Sensitivity and Specificity , White PeopleABSTRACT
For research groups needing to isotype a significant number of antibodies on a fairly regular basis, the protocol outlined here provides a cost-effective option. In this simple sandwich ELISA, the monoclonal antibody is first captured with antibodies that discriminate between heavy-chain isotypes and then is detected with antibodies specific for each light chain. This combination ensures that free light chain secreted by hybridomas does not yield a false-positive result, because in a true positive both the capture and detecting antibodies bind the monoclonal antibody.
Subject(s)
Antibodies, Monoclonal/analysis , Antibodies, Monoclonal/classification , Enzyme-Linked Immunosorbent Assay/methods , Immunoglobulin Isotypes/analysis , Animals , RodentiaABSTRACT
AIM: Development of drug-related surrogate positive controls for isotype anti-drug antibodies assay remains challenging. Efforts on antibody engineering or chemical crosslinking have been made. However, multiple epitope recognition, purity and stability are often of concern. To tackle these challenges, we used LC-SPDP/SMPB crosslinking to conjugate polyclonal anti-drug IgG and human immunoglobulin isotype (hIgE, hIgA, hIgM or hIgG4) with optimized conditions. RESULTS: The final product was a hybrid of anti-drug IgG cross-linked to human isotype immunoglobulin through stable thioether bond. The characteristics of the hybrids and their performance as assay positive controls were further evaluated. CONCLUSION: The results demonstrate that LC-SPDP/SMPB chemical crosslinking method is suitable for generating stable hybrids to be used as assay positive controls in isotype anti-drug antibodies assay.
Subject(s)
Antibodies/analysis , Chromatography, Gel , Immunoglobulin Isotypes/analysis , Antibodies/chemistry , Antibodies/metabolism , Antigen-Antibody Complex/analysis , Enzyme-Linked Immunosorbent Assay , Humans , Immunoglobulin Isotypes/chemistry , Maleimides/chemistry , Pharmaceutical Preparations/chemistry , Pharmaceutical Preparations/metabolism , Sulfides/chemistryABSTRACT
BACKGROUND: Knowledge of human IgG subclass antibody responses to various allergens has been hampered by a lack of reliable standardized assays. The aim here was to develop quantitative immunoassays for human IgG1, IgG2, and IgG3 antibodies using ImmunoCAP® technology and to evaluate their application. METHODS: Enzyme conjugates with isotype-specific monoclonal antibodies and calibrators composed of purified myeloma paraproteins were developed for each assay and used together with other standardized assay reagents for the Phadia® 100 instrument. The calibrators were adjusted to the international reference preparation IRP 67/86. The assays were characterized and used together with other standard ImmunoCAP assays to measure antibodies to various allergens in preliminary studies. RESULTS: The new assays had limits of quantitation of 1.0 (IgG1), 4.6 (IgG2), and 0.04 mgA/L (IgG3), and coefficients of variation of <20%. Only some minor cross-reactivity with IgG2 was observed for the specific IgG1 assay. The specific IgG2 assay showed a bias for the allotype G2m(23) and compensation factors were used to adjust the measured concentrations accordingly. Preliminary studies indicated a strong and stable IgG4 antibody response to ß-lactoglobulin in healthy individuals, a high IgG1 and even higher IgG2 antibody response to house dust mite in sensitized and nonsensitized subjects, and a mixed IgG subclass response to venom allergens in allergic patients with increasing IgG4 antibody levels during venom immunotherapy. CONCLUSIONS: The new research assays are valuable tools for immunological studies, enabling the characterization of antibody profiles using a standardized approach, and facilitating data interpretation and the comparison of results across studies.
Subject(s)
Allergens/immunology , Antibody Specificity/immunology , Immunoassay/methods , Immunoglobulin G/analysis , Immunoglobulin Isotypes/analysis , Adult , Animals , Antibodies, Monoclonal/immunology , Bee Venoms/immunology , Humans , Immunoglobulin G/classification , Immunoglobulin G/immunology , Immunoglobulin Gm Allotypes/immunology , Immunoglobulin Isotypes/immunology , Lactoglobulins/immunology , Pyroglyphidae/immunology , Wasp Venoms/immunologySubject(s)
Electrophoresis, Capillary , Myeloma Proteins/analysis , Transferrin/analogs & derivatives , Aged , Case-Control Studies , Humans , Immunoglobulin A/analysis , Immunoglobulin Isotypes/analysis , Immunoglobulin M/analysis , Incidental Findings , Monoclonal Gammopathy of Undetermined Significance/metabolism , Monoclonal Gammopathy of Undetermined Significance/pathology , Retrospective Studies , Transferrin/analysisABSTRACT
AIM: Biologics development often requires multiple immunoassays to evaluate both assay reagents and potential drug candidates resulting in extensive analytical development. METHODOLOGY: We developed a semi-universal, 5-layer platform assay on Gyrolab using secondary antispecies or anti-isotype-specific capture and detection antibodies. We applied the assay to several multivalent vaccines. RESULTS: Method performance exhibited a median accuracy of 110%, reproducibility of 9% CV and intermediate precision of 11% CV. System suitability criteria were met for 92.5% of the samples and only one out of 31 replicate samples exhibited a %CV greater than 20%. CONCLUSION: The semi-universal Gyrolab assay allowed assay development without reagent labeling. The format could also be translated into a plate-based assay.
Subject(s)
Antibodies/analysis , Biological Therapy/methods , Enzyme-Linked Immunosorbent Assay , Vaccines/immunology , Animals , Fluorescent Dyes/chemistry , Immunoglobulin G/analysis , Immunoglobulin Isotypes/analysis , Mice , Reproducibility of Results , Vaccines/analysisABSTRACT
BACKGROUND: Antibodies anticardiolipin (aCL) and anti-ß2-glycoprotein I (aß2GPI) are two of three laboratory criteria of antiphospholipid syndrome (APS). All of assays of antiphospholipid antibodies (aPL), coagulation assays as well as ELISAs, show methodological shortcomings, that affect their sensitivity and specificity. Therefore, we decided to validate these parameters for a new chemiluminescent examination (CLIA). METHODS: aCL and aß2GPI antibodies were measured by ELISAs (AIDA, Bad Kreuznach, Germany) and aß2GPI with CLIA kits (Werfen, Barcelona, Spain). RESULTS: When we evaluated both assays, the coefficient of variation for CLIA was slightly lower (9.04 - 12.74%) than for ELISA (11.05 - 15.3%) and the LOD was 0.2 U/L. The dilution series showed significant linearity for all CLIA methods, aCL IgG, aCL IgM, aß2GPI IgG, and aß2GPI IgM (0 - 3000 U/L), and method comparison studies revealed good agreement with the currently used ELISA (Kappa values ranging 0.534 - 0.936) without determination of aß2GPI IgG. The determination aß2GPI IgG by CLIA method shows higher positivity in 31 samples. These new aCL IgG, aCL IgM, aß2GPI IgG, and aß2GPI IgM tests have excellent analytical characteristics and allow fully automated and simultaneous analysis on an analyzer. CONCLUSIONS: Chemiluminescent determination of an automated analyzer can improve the fundamental parameters of tests such as reproducibility between laboratories.
Subject(s)
Antibodies, Anticardiolipin/analysis , Antiphospholipid Syndrome/diagnosis , Luminescence , beta 2-Glycoprotein I/immunology , Antiphospholipid Syndrome/immunology , Enzyme-Linked Immunosorbent Assay , Humans , Immunoglobulin Isotypes/analysis , Reproducibility of Results , Sensitivity and Specificity , beta 2-Glycoprotein I/antagonists & inhibitorsABSTRACT
BACKGROUND: Dengue laboratory diagnosis is essentially based on detection of the virus, its components or antibodies directed against the virus in blood samples. Blood, however, may be difficult to draw in some patients, especially in children, and sampling during outbreak investigations or epidemiological studies may face logistical challenges or limited compliance to invasive procedures from subjects. The aim of this study was to assess the possibility of using saliva and urine samples instead of blood for dengue diagnosis. METHODOLOGY/PRINCIPAL FINDINGS: Serial plasma, urine and saliva samples were collected at several time-points between the day of admission to hospital until three months after the onset of fever in children with confirmed dengue disease. Quantitative RT-PCR, NS1 antigen capture and ELISA serology for anti-DENV antibody (IgG, IgM and IgA) detection were performed in parallel on the three body fluids. RT-PCR and NS1 tests demonstrated an overall sensitivity of 85.4%/63.4%, 41.6%/14.5% and 39%/28.3%, in plasma, urine and saliva specimens, respectively. When urine and saliva samples were collected at the same time-points and tested concurrently, the diagnostic sensitivity of RNA and NS1 detection assays was 69.1% and 34.4%, respectively. IgG/IgA detection assays had an overall sensitivity of 54.4%/37.4%, 38.5%/26.8% and 52.9%/28.6% in plasma, urine and saliva specimens, respectively. IgM were detected in 38.1% and 36% of the plasma and saliva samples but never in urine. CONCLUSIONS: Although the performances of the different diagnostic methods were not as good in saliva and urine as in plasma specimens, the results obtained by qRT-PCR and by anti-DENV antibody ELISA could well justify the use of these two body fluids to detect dengue infection in situations when the collection of blood specimens is not possible.