Immunoglobulin Subclasses Research Articles

The importance of IgG glycosylation-What did we learn after analyzing over 100,000 individuals.

All four subclasses of immunoglobulin G (IgG) antibodies have glycan structures attached to the protein part of the IgG molecules. Glycans linked to the Fc portion of IgG are found in all IgG antibodies, while about one-fifth of IgG antibodies in plasma also have glycans attached to the Fab portion of IgG. The IgG3 subclass is characterized by more complex glycosylation compared to other IgG subclasses. In this review, we discuss the significant influence that glycans exert on the structural and functional properties of IgG. We provide a comprehensive overview of how the composition of these glycans can affect IgG's effector functions by modulating its interactions with Fcγ receptors and other molecules such as the C1q component of complement, which in turn influence various immune responses triggered by IgG, including antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). In addition, the importance of glycans for the efficacy of therapeutics like monoclonal antibodies and intravenous immunoglobulin (IVIg) therapy is discussed. Moreover, we offer insights into IgG glycosylation characteristics and roles derived from general population, disease-specific, and interventional studies. These studies indicate that IgG glycans are important biomarkers and functional effectors in health and disease.

Comprehensive evaluation of anti-emicizumab antibodies in acquired hemophilia A: a detailed case study and methodological evaluation

BackgroundAcquired hemophilia A (AHA) is a rare and severe bleeding disorder characterized by autoantibodies inhibiting coagulation factor (F)VIII. Current treatment of AHA involves bypassing agents or FVIII replacement therapy, yet their efficacy is limited in cases of high inhibitor titers. Emicizumab, a humanized bispecific monoclonal antibody, has shown promising hemostatic effectiveness in persons with congenital hemophilia A (HA) and AHA, but a minority of patients developed anti-drug antibodies (ADAs), compromising its efficacy. MethodsWe present a comprehensive characterization of anti-emicizumab antibodies in a patient with AHA experiencing diminished emicizumab efficacy from week 10 of treatment. We developed a method for analysis of anti-emicizumab antibodies, distinguishing immunoglobulin (Ig) subclasses and IgG subtype profiling. ADAs’ neutralizing activity was evaluated using a modified clotting assay. ResultsThe Luminex-based assay demonstrated the presence of anti-emicizumab antibodies, confirmed through competitive analysis. We detected anti-emicizumab IgG antibodies in the patient’s plasma between week 16 and 29. IgG1 and IgG2 subclasses were identified. Longitudinal analysis showed IgG isotype antibodies against both emicizumab and FVIII. Anti-emicizumab antibodies exhibited noninhibitory activity, confirmed by a modified Bethesda assay. Moreover, no accelerated clearance of emicizumab was observed in plasma samples from the patient. ConclusionThis study presents the first documented case of anti-emicizumab antibodies in AHA. Our study provides insights into ADA development against emicizumab, emphasizing the need for monitoring tools. The developed method enables comprehensive evaluation of ADAs, aiding in personalized treatment strategies. These findings contribute to understanding emicizumab's immunogenicity profile in AHA, facilitating its optimized clinical use.

MOG CNS Autoimmunity and MOGAD.

At one time considered a possible form of neuromyelitis optica (NMO) spectrum disorder (NMOSD), it is now accepted that myelin oligodendrocyte glycoprotein (MOG) antibody (Ab)-associated disorder (MOGAD) is a distinct entity from either NMO or multiple sclerosis (MS) and represents a broad spectrum of clinical phenotypes. Whereas Abs targeting aquaporin-4 (AQP4) in NMO are pathogenic, the extent that anti-MOG Abs contribute to CNS damage in MOGAD is unclear. Both AQP4-specific Abs in NMO and MOG-specific Abs in MOGAD are predominantly IgG1, a T cell-dependent immunoglobulin (Ig) subclass. Key insights in neuroimmunology and MOGAD pathogenesis have been learned from MOG experimental autoimmune encephalomyelitis (EAE), described 2 decades before the term MOGAD was introduced. MOG-specific T cells are required in MOG EAE, and while anti-MOG Abs can exacerbate EAE and CNS demyelination, those Abs are neither necessary nor sufficient to cause EAE. Knowledge regarding the spectrum of MOGAD clinical and radiologic presentations is advancing rapidly, yet our grasp of MOGAD pathogenesis is incomplete. Understanding both the humoral and cellular immunology of MOGAD has implications for diagnosis, treatment, and prognosis.

Autoantibodies to ACE2 and immune molecules are associated with COVID-19 disease severity

BackgroundIncreased inflammation caused by SARS-CoV-2 infection can lead to severe coronavirus disease 2019 (COVID-19) and long-term disease manifestations. The mechanisms of this variable long-term immune activation are poorly defined. One feature of this increased inflammation is elevated levels of proinflammatory cytokines and chemokines. Autoantibodies targeting immune factors such as cytokines, as well as the viral host cell receptor, angiotensin-converting enzyme 2 (ACE2), have been observed after SARS-CoV-2 infection. Autoantibodies to immune factors and ACE2 could interfere with normal immune regulation and lead to increased inflammation, severe COVID-19, and long-term complications.MethodsHere, we deeply profiled the features of ACE2, cytokine, and chemokine autoantibodies in samples from patients recovering from severe COVID-19. We measured the levels of immunoglobulin subclasses (IgG, IgA, IgM) in the peripheral blood against ACE2 and 23 cytokines and other immune molecules. We then utilized an ACE2 peptide microarray to map the linear epitopes targeted by ACE2 autoantibodies.ResultsWe demonstrate that ACE2 autoantibody levels are increased in individuals with severe COVID-19 compared with those with mild infection or no prior infection. We identify epitopes near the catalytic domain of ACE2 targeted by these antibodies. Levels of autoantibodies targeting ACE2 and other immune factors could serve as determinants of COVID-19 disease severity, and represent a natural immunoregulatory mechanism in response to viral infection.ConclusionsThese results demonstrate that SARS-CoV-2 infection can increase autoantibody levels to ACE2 and other immune factors. The levels of these autoantibodies are associated with COVID-19 disease severity.

Distinctive antibody responses to Mycobacterium tuberculosis in pulmonary and brain infection.

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), remains a global health burden. While M. tuberculosis is primarily a respiratory pathogen, it can spread to other organs, including the brain and meninges, causing TB meningitis (TBM). However, little is known about the immunological mechanisms that lead to differential disease across organs. Attention has focused on differences in T cell responses in the control of M. tuberculosis in the lungs, but emerging data point to a role for antibodies, as both biomarkers of disease control and as antimicrobial molecules. Given an increasing appreciation for compartmentalized antibody responses across the blood-brain barrier, here we characterized the antibody profiles across the blood and brain compartments in TBM and determined whether M. tuberculosis-specific humoral immune responses differed between M. tuberculosis infection of the lung (pulmonary TB) and TBM. Using a high throughput systems serology approach, we deeply profiled the antibody responses against 10 different M. tuberculosis antigens, including lipoarabinomannan (LAM) and purified protein derivative (PPD), in HIV-negative adults with pulmonary TB (n = 10) versus TBM (n = 60). Antibody studies included analysis of immunoglobulin isotypes (IgG, IgM, IgA) and subclass levels (IgG1-4) and the capacity of M. tuberculosis-specific antibodies to bind to Fc receptors or C1q and to activate innate immune effector functions (complement and natural killer cell activation; monocyte or neutrophil phagocytosis). Machine learning methods were applied to characterize serum and CSF responses in TBM, identify prognostic factors associated with disease severity, and define the key antibody features that distinguish TBM from pulmonary TB. In individuals with TBM, we identified CSF-specific antibody profiles that marked a unique and compartmentalized humoral response against M. tuberculosis, characterized by an enrichment of M. tuberculosis-specific antibodies able to robustly activate complement and drive phagocytosis by monocytes and neutrophils, all of which were associated with milder TBM severity at presentation. Moreover, individuals with TBM exhibited M. tuberculosis-specific antibodies in the serum with an increased capacity to activate phagocytosis by monocytes, compared with individuals with pulmonary TB, despite having lower IgG titres and Fcγ receptor-binding capacity. Collectively, these data point to functionally divergent humoral responses depending on the site of infection (i.e. lungs versus brain) and demonstrate a highly compartmentalized M. tuberculosis-specific antibody response within the CSF in TBM. Moreover, our results suggest that phagocytosis- and complement-mediating antibodies may promote attenuated neuropathology and milder TBM disease.

Specific T-cell subsets have a role in anti-viral immunity and pathogenesis but not viral dynamics or onwards vector transmission of an important livestock arbovirus.

Bluetongue virus (BTV) is an arthropod-borne Orbivirus that is almost solely transmitted by Culicoides biting midges and causes a globally important haemorrhagic disease, bluetongue (BT), in susceptible ruminants. Infection with BTV is characterised by immunosuppression and substantial lymphopenia at peak viraemia in the host. In this study, the role of cell-mediated immunity and specific T-cell subsets in BTV pathogenesis, clinical outcome, viral dynamics, immune protection, and onwards transmission to a susceptible Culicoides vector is defined in unprecedented detail for the first time, using an in vivo arboviral infection model system that closely mirrors natural infection and transmission of BTV. Individual circulating CD4+, CD8+, or WC1+ γδ T-cell subsets in sheep were depleted through the administration of specific monoclonal antibodies. The absence of cytotoxic CD8+ T cells was consistently associated with less severe clinical signs of BT, whilst the absence of CD4+ and WC1+ γδ T cells both resulted in an increased clinical severity. The absence of CD4+ T cells also impaired both a timely protective neutralising antibody response and the production of IgG antibodies targeting BTV non-structural protein, NS2, highlighting that the CD4+ T-cell subset is important for a timely protective immune response. T cells did not influence viral replication characteristics, including onset/dynamics of viraemia, shedding, or onwards transmission of BTV to Culicoides. We also highlight differences in T-cell dependency for the generation of immunoglobulin subclasses targeting BTV NS2 and the structural protein, VP7. This study identifies a diverse repertoire of T-cell functions during BTV infection in sheep, particularly in inducing specific anti-viral immune responses and disease manifestation, and will support more effective vaccination strategies.

NanoBERT: a deep learning model for gene agnostic navigation of the nanobody mutational space.

Nanobodies are a subclass of immunoglobulins, whose binding site consists of only one peptide chain, bestowing favorable biophysical properties. Recently, the first nanobody therapy was approved, paving the way for further clinical applications of this antibody format. Further development of nanobody-based therapeutics could be streamlined by computational methods. One of such methods is infilling-positional prediction of biologically feasible mutations in nanobodies. Being able to identify possible positional substitutions based on sequence context, facilitates functional design of such molecules. Here we present nanoBERT, a nanobody-specific transformer to predict amino acids in a given position in a query sequence. We demonstrate the need to develop such machine-learning based protocol as opposed to gene-specific positional statistics since appropriate genetic reference is not available. We benchmark nanoBERT with respect to human-based language models and ESM-2, demonstrating the benefit for domain-specific language models. We also demonstrate the benefit of employing nanobody-specific predictions for fine-tuning on experimentally measured thermostability dataset. We hope that nanoBERT will help engineers in a range of predictive tasks for designing therapeutic nanobodies. https://huggingface.co/NaturalAntibody/.

Селективный дефицит иммуноглобулина А в сочетании с селективным дефицитом иммуноглобулина G4 у взрослого пациента

Aim. To demonstrate the possibility of developing primary immunodeficiency with impaired antibody synthesis in an adult patient and discuss the algorithm for laboratory diagnostics and treatment tacticsto demonstrate the possibility of developing primar hypoimmunoglobulinemia after a course of imunosupressive therapy. Key points. Primary immunodeficiencies are a group of diseases associated with monogenic mutations. They do not have a typical clinical picture. A clinical observation is presented, when a selective deficiency of immunoglobulin A in combination with a selective deficiency of the subclass of immunoglobulin G was detected in a patient with chronic bronchopulmonary pathology. Clinical and laboratory criteria for diagnosis and treatment tactics are discussed. Сonclusion. Primary immunodeficiencies are often hidden by infectious masks. It is necessary to study serum immunoglobulins, with their normal levels or selective deficiency of immunoglobulin A, to additionally investigate the content of subclasses of immunoglobulin G. Keywords: primary immunodeficiency, selective immunoglobulin A deficiency, immunoglobulin G subclass deficiency.

Detection of a natural antibody targeting the shed ectodomain of BP180 in mice

BackgroundPemphigoid diseases are characterized by subepidermal blister formation accompanied by autoantibodies targeting skin component molecules, such as BP180. It is suggested that an epitope–phenotype correlation exists among autoantibodies recognizing BP180. However, it is unclear which regions of BP180 are likely targets for autoantibodies. ObjectiveTo elucidate the portions of BP180 where antibodies tend to react under the breakdown of immune tolerance. MethodsWe immunized mice with full-length mouse BP180 (mBP180) to produce anti-mBP180 antibodies. Using the immunized mice, hybridoma cells were established to produce anti-mBP180 antibodies. We analyzed the characteristics of the anti-mBP180 antibodies that were produced in terms of epitopes, immunoglobulin subclasses, and somatic hypermutations. ResultsHybridoma cells derived from immunized mice with full-length mBP180 produced antibodies targeting the intracellular domain (IC) and the shed ectodomain (EC) of mBP180. Using the domain-deleted mBP180 recombinant protein, we revealed that monoclonal anti-mBP180 EC antibodies react to neoepitopes on the 13th collagenous region of cleaved mBP180, which corresponds to the epitopes of linear IgA bullous dermatosis antibodies in human BP180. Furthermore, the subclasses of these antibodies could be distinguished by epitope: The subclass of the anti-mBP180 IC monoclonal antibodies was IgG, whereas that of the anti-mBP180 EC antibodies was IgM. Of note, a clone of these IgM mBP180 EC antibodies was a germline antibody without somatic hypermutation, which is also known as a natural antibody. ConclusionThese data suggest that mice potentially have natural antibodies targeting the neoepitopes of cleaved mBP180 EC.

Immunotherapies in MuSK-positive Myasthenia Gravis; an IgG4 antibody-mediated disease.

Muscle-specific kinase (MuSK) Myasthenia Gravis (MG) represents a prototypical antibody-mediated disease characterized by predominantly focal muscle weakness (neck, facial, and bulbar muscles) and fatigability. The pathogenic antibodies mostly belong to the immunoglobulin subclass (Ig)G4, a feature which attributes them their specific properties and pathogenic profile. On the other hand, acetylcholine receptor (AChR) MG, the most prevalent form of MG, is characterized by immunoglobulin (Ig)G1 and IgG3 antibodies to the AChR. IgG4 class autoantibodies are impotent to fix complement and only weakly bind Fc-receptors expressed on immune cells and exert their pathogenicity via interfering with the interaction between their targets and binding partners (e.g. between MuSK and LRP4). Cardinal differences between AChR and MuSK-MG are the thymus involvement (not prominent in MuSK-MG), the distinct HLA alleles, and core immunopathological patterns of pathology in neuromuscular junction, structure, and function. In MuSK-MG, classical treatment options are usually less effective (e.g. IVIG) with the need for prolonged and high doses of steroids difficult to be tapered to control symptoms. Exceptional clinical response to plasmapheresis and rituximab has been particularly observed in these patients. Reduction of antibody titers follows the clinical efficacy of anti-CD20 therapies, a feature implying the role of short-lived plasma cells (SLPB) in autoantibody production. Novel therapeutic monoclonal against B cells at different stages of their maturation (like plasmablasts), or against molecules involved in B cell activation, represent promising therapeutic targets. A revolution in autoantibody-mediated diseases is pharmacological interference with the neonatal Fc receptor, leading to a rapid reduction of circulating IgGs (including autoantibodies), an approach already suitable for AChR-MG and promising for MuSK-MG. New precision medicine approaches involve Chimeric autoantibody receptor T (CAAR-T) cells that are engineered to target antigen-specific B cells in MuSK-MG and represent a milestone in the development of targeted immunotherapies. This review aims to provide a detailed update on the pathomechanisms involved in MuSK-MG (cellular and humoral aberrations), fostering the understanding of the latest indications regarding the efficacy of different treatment strategies.

Anti-Islet Autoantibodies in Type 1 Diabetes.

Anti-islet autoantibodies serve as key markers in immune-mediated type 1 diabetes (T1D) and slowly progressive T1D (SPIDDM), also known as latent autoimmune diabetes in adults (LADA). Autoantibodies to insulin (IAA), glutamic acid decarboxylase (GADA), tyrosine phosphatase-like protein IA-2 (IA-2A), and zinc transporter 8 (ZnT8A) are currently employed in the diagnosis, pathological analysis, and prediction of T1D. GADA can also be detected in non-diabetic patients with autoimmune diseases other than T1D and may not necessarily reflect insulitis. Conversely, IA-2A and ZnT8A serve as surrogate markers of pancreatic β-cell destruction. A combinatorial analysis of these four anti-islet autoantibodies demonstrated that 93-96% of acute-onset T1D and SPIDDM cases were diagnosed as immune-mediated T1D, while the majority of fulminant T1D cases were autoantibody-negative. Evaluating the epitopes and immunoglobulin subclasses of anti-islet autoantibodies help distinguish between diabetes-associated and non-diabetes-associated autoantibodies and is valuable for predicting future insulin deficiency in SPIDDM (LADA) patients. Additionally, GADA in T1D patients with autoimmune thyroid disease reveals the polyclonal expansion of autoantibody epitopes and immunoglobulin subclasses. Recent advancements in anti-islet autoantibody assays include nonradioactive fluid-phase assays and the simultaneous determination of multiple biochemically defined autoantibodies. Developing a high-throughput assay for detecting epitope-specific or immunoglobulin isotype-specific autoantibodies will facilitate a more accurate diagnosis and prediction of autoimmune disorders. The aim of this review is to summarize what is known about the clinical significance of anti-islet autoantibodies in the pathogenesis and diagnosis of T1D.

The Very Low IgE Producer: Allergology, Genetics, Immunodeficiencies, and Oncology.

Opposite to other immunoglobulin (Ig) classes and subclasses, there is no consensus on the definition of normal levels of serum total IgE. However, longitudinal studies on birth cohorts produced growth charts of total IgE levels in helminth-free and never atopic children and defining the normal ranges of total serum IgE concentration at the individual, rather than population, level. Accordingly, very 'low IgE producers' (i.e., children whose tIgE level belong to the lowest percentiles) became atopic while keeping their total IgE levels in a range considered 'normal' if compared to the general age-matched population but 'abnormally high' if projected on the tIgE growth chart against the trajectory of that child's own percentile levels. In 'low IgE producers', the IgE-specific activity, i.e., the ratio between allergen-specific and total IgE, is more important than the absolute specific IgE levels to confirm causality between allergen exposure and allergic symptoms. Patients with allergic rhinitis or peanut anaphylaxis but low or undetectable allergen-specific IgE levels must therefore be reconsidered considering their total IgE levels. Low IgE producers have been also associated with common variable immunodeficiency, lung diseases, and malignancies. A few epidemiological studies have shown a higher risk of malignancies in very low IgE producers, leading to a debated hypothesis proposing a novel, evolutionistic-relevant function for IgE antibodies for antitumor immune surveillance.

IgG1-Dominant Antibody Response Induced by Recombinant Trimeric SARS-CoV-2 Spike Protein with PIKA Adjuvant.

Recombinant trimeric SARS-CoV-2 Spike protein with PIKA (polyI:C) adjuvant induces potent and durable neutralizing antibodies that protect against multiple SARS-CoV-2 variants. The immunoglobulin subclasses of viral-specific antibodies remain unknown, as do their glycosylation status on Fc regions. In this study, we analyzed immunoglobulins adsorbed by plate-bound recombinant trimeric SARS-CoV-2 Spike protein from serum of Cynomolgus monkey immunized by recombinant trimeric SARS-CoV-2 Spike protein with PIKA (polyI:C) adjuvant. The results showed that IgG1 was the dominant IgG subclass as revealed by ion mobility mass spectrometry. The average percentage of Spike protein-specific IgG1 increased to 88.3% as compared to pre-immunization. Core fucosylation for Fc glycopeptide of Spike protein-specific IgG1 was found to be higher than 98%. These results indicate that a unique Th1-biased, IgG1-dominant antibody response was responsible for the effectiveness of PIKA (polyI:C) adjuvant. Vaccine-induced core-fucosylation of IgG1 Fc region may reduce incidence of severe COVID-19 disease associated with overstimulation of FCGR3A by afucosylated IgG1.

Altered B cell immunoglobulin signature exhibits potential diagnostic values in human colorectal cancer

Antibody-secreting B cells have long been considered the central element of gut homeostasis; however, tumor-associated B cells in human colorectal cancer (CRC) have not been well characterized. Here, we show that the clonotype, phenotype, and immunoglobulin subclasses of tumor-infiltrating B cells have changed compared to adjacent normal tissue B cells. Remarkably, the tumor-associated B cell immunoglobulin signature alteration can also be detected in the plasma of patients with CRC, suggesting that a distinct B cell response was also evoked in CRC. We compared the altered plasma immunoglobulin signature with the existing method of CRC diagnosis. Our diagnostic model exhibits improved sensitivity compared to the traditional biomarkers, CEA and CA19-9. These findings disclose the altered B cell immunoglobulin signature in human CRC and highlight the potential of using the plasma immunoglobulin signature as a non-invasive method for the assessment of CRC.

КЛИНИКО-ФАРМАКОЛОГИЧЕСКИЕ ОСОБЕННОСТИ ПРИМЕНЕНИЯ ПРЕПАРАТОВ НОРМАЛЬНОГО ИММУНОГЛОБУЛИНА ЧЕЛОВЕКА В ПРАКТИКЕ ВРАЧА

Immunostimulating (immunomodulatory) drugs belong to the group of immunotropic drugs, the therapeutic effect of which is associated with a predominant effect on the human immune system. Medicines based on human normal immunoglobulin are immunostimulatory drugs of natural origin. In the practice of a doctor, immunoglobulins are made from human plasma and are more often used in the form for intravenous and subcutaneous administration. Immunoglobulins differ in the ratio of various subclasses of immunoglobulins G (IgG), as well as immunoglobulin class A (IgA), individual immunoglobulins are enriched with antibodies of the IgM classes, and specific immunoglobulins contain a significantly larger amount of IgG class antibodies against certain pathogens than standard immunoglobulins. Approaches to the classification of immunoglobulins, the mechanism of action, indications, contraindications, as well as clinical and pharmacological features of the use of human normal immunoglobulin, drugs available on the market in the Republic of Belarus are described, recommendations are given for their use in clinical practice. The possibilities of using immunoglobulins as a replacement (for immunodeficiency states, agammaglobulinemia) and immunomodulatory (for autoimmune diseases) therapy are presented. The possibility of using intravenous immunoglobulin for the treatment of Kawasaki syndrome, idiopathic thrombocytopenic purpura, Guillain-Barré syndrome, autoimmune polyneuropathy, diseases arising in oncological, surgical and obstetric practice, as well as for complex therapy of sepsis, including in newborns, is considered. Timely appointment of immunoglobulins in optimal doses will reduce the duration of therapy in patients with immunosuppression of various origins, reduce bleeding in immune thrombocytopenia.

Assessment of donor specific IgG Anti-HLA subclasses before and after kidney transplantation in Upper Egypt: A Single Center Study

Donor specific antibodies (DSAs) are known as the leading cause of antibody mediated rejection (AMR), graft loss in kidney transplant (KT) recipients. DSAs characteristics, as immunoglobulin (Ig) classes, subclasses, and strength, are important to assess the immunological risk, early prediction of AMR, and therefor proper management. This longitudinal, case control study included 32 KT recipients at Assiut University Urology Hospital and 10 age and sex matched normal subjects as the control group. Total IgG, its subclasses and anti-human leukocyte antigen (anti-HLA) panel reactive antibody (PRA) were detected pre-transplantation (pre-TX), at 6-12- and 24-36-months post-TX. Rejection occurred in 4 recipients, 3 of them had high total IgG, IgG1 and/or IgG3. IgG2 and IgG4 were normal in all recipients. There were preformed anti-DSAs antibodies in 3/32 recipients (9.4%). Of these, two recipients became negative with no rejection occurred. The third recipient had high post-TX mean fluorescence intensity (MFI) and AMR occurred. The pre-TX PRA was negative in 29/32 recipients (90.6%). The PRA was negative in 8/29 recipients (27.6%) and the remaining 21/29 recipients (72.4%) developed de novo DSAs post-TX (MFI < 3000->10000). Rejection occurred with both low and high MFI. In 11 recipients, anti-HLA class I and II were not different between pre-TX, 3-6- and 24-36 months post-TX with no rejection occurred. The frequency and median levels of total IgG, IgG1 and IgG3 were increased in all recipients 24-36 months post-TX when compared with their levels pre-TX and 6-12 months post-TX in the 11 recipients and with the control group. The graft survival time significantly decreased in recipients with positive post-TX class I PRA. In conclusion, preformed DSAs may persist post-TX or turn negative. De novo DSAs developed post-TX even in non-sensitized recipients. Serum total IgG, IgG1 and IgG3 frequency increase 2-3 years post-TX.

Antiphospholipid Antibody Testing for Anti-cardiolipin and Anti-β2 Glycoprotein I Antibodies Using Chemiluminescence-Based Panels.

Antiphospholipid (antibody) syndrome (APS) is a prothrombotic condition with increased risk for thrombosis and pregnancy-related morbidity. In addition to clinical criteria related to these risks, APS is characterized by the persistent presence of antiphospholipid antibodies (aPL), as detected in the laboratory using a potentially wide variety of assays. The three APS criteria-related assays are lupus anticoagulant (LA), as detected using clot-based assays, and the solid-phase assays of anti-cardiolipin antibodies (aCL) and anti-β2 glycoprotein I antibodies (aβ2GPI), with immunoglobulin subclasses of IgG and/or IgM. These tests may also be used for the diagnosis of systemic lupus erythematosus (SLE). In particular, APS diagnosis/exclusion remains challenging for clinicians and laboratories because of the heterogeneity of clinical presentations in those being evaluated and the technical application and variety of the associated tests used in laboratories. Although LA testing is affected by a wide variety of anticoagulants, which are often given to APS patients to prevent any associated clinical morbidity, detection of solid-phase aPL is not influenced by these anticoagulants, and this thus represents a potential advantage to their application. On the other hand, various technical issues challenge accurate laboratory detection or exclusion of aPL. This report describes protocols for the assessment of solid-phase aPL, specifically aCL and aβ2GPI of IgG and IgM class by means of a chemiluminescence-based assay panel. These protocols reflect tests able to be performed on the AcuStar instrument (Werfen/Instrumentation Laboratory). Certain regional approvals may also allow this testing to be performed on a BIO-FLASH instrument (Werfen/Instrumentation Laboratory).

IgE and IgG4 Epitope Mapping of Food Allergens with a Peptide Microarray Immunoassay.

Peptide microarrays are a powerful tool to identify linear epitopes of food allergens in a high-throughput manner. The main advantages of the microarray-based immunoassay are as follows: the possibility to assay thousands of targets simultaneously, the requirement of a low volume of serum, the more robust statistical analysis, and the possibility to test simultaneously several immunoglobulin subclasses. Among them, the last one has a special interest in the field of food allergy, because the development of tolerance to food allergens has been associated with a decrease in IgE and an increase in IgG4 levels against linear epitopes. However, the main limitation to the clinical use of microarray is the automated analysis of the data. Recent studies mapping the linear epitopes of food allergens with peptide microarray immunoassays have identified peptide biomarkers that can be used for early diagnosis of food allergies and to predict their severity or the self-development of tolerance. Using this approach, we have worked on epitope mapping of the two most important food allergens in the Spanish population, cow's milk, and chicken eggs. The final aim of these studies is to define subsets of peptides that could be used as biomarkers to improve the diagnosis and prognosis of food allergies. This chapter describes the protocol to produce microarrays using a library of overlapping peptides corresponding to the primary sequences of food allergens and data acquisition and analysis of IgE and IgG4 binding epitopes.

Rational Design and Development of SARS-CoV-2 Serological Diagnostics by Immunoprecipitation-Targeted Proteomics.

Current design of serological tests utilizes conservative immunoassay approaches and is focused on fast and convenient assay development, throughput, straightforward measurements, and affordability. Limitations of common serological assays include semiquantitative measurements, cross-reactivity, lack of reference standards, and no differentiation between human immunoglobulin subclasses. In this study, we suggested that a combination of immunoaffinity enrichments with targeted proteomics would enable rational design and development of serological assays of infectious diseases, such as COVID-19. Immunoprecipitation-targeted proteomic assays allowed for sensitive and specific measurements of NCAP_SARS2 protein with a limit of detection of 313 pg/mL in serum and enabled differential quantification of anti-SARS-CoV-2 antibody isotypes (IgG, IgA, IgM, IgD, and IgE) and individual subclasses (IgG1-4 and IgA1-2) in plasma and saliva. Simultaneous evaluation of the numerous antigen–antibody subclass combinations revealed a receptor-binding domain (RBD)-IgG1 as a combination with the highest diagnostic performance. Further validation revealed that anti-RBD IgG1, IgG3, IgM, and IgA1 levels were significantly elevated in convalescent plasma, while IgG2, IgG4, and IgA2 were not informative. Anti-RBD IgG1 levels in convalescent (2138 ng/mL) vs negative (95 ng/mL) plasma revealed 385 ng/mL as a cutoff to detect COVID-19 convalescent plasma. Immunoprecipitation-targeted proteomic assays will facilitate improvement and standardization of the existing serological tests, enable rational design of novel tests, and offer tools for the comprehensive investigation of immunoglobulin subclass cooperation in immune response.

Comparative Detection of Immunoglobulin Isotypes and Subclasses against Toxoplasma gondii Soluble Antigen in Serum and Colostrum Samples from Puerperal Women.

Background: Toxoplasma gondii is an obligate intracellular parasite that can infect several species, including humans, and can cause severe damage to the fetus when the infection occurs during pregnancy. The environment and/or food contamination are critical to spreading the infection. Human milk is rich in nutrients and bioactive elements that provide growth and development of the immune system of the newborn. All isotypes of immunoglobulins are present in human colostrum and they are produced from systemic or local sources. Breastfeeding protects the infant against various pathogens, but there is no conclusive study to detect IgG subclasses in colostrum against T. gondii. Therefore, the aim of this study was to detect and evaluate the presence of antibody isotypes against T. gondii in paired samples of serum and colostrum. Methods: The study included 283 puerperal patients. ELISA (Enzyme-Linked Immunosorbent Assay) for detection of anti-T. gondii-specific IgM, IgA, and IgG isotypes and IgG1, IgG3, and IgG4 subclasses were conducted on paired samples of serum and colostrum. Results: It was found that 45.9%, 6.0%, and 2.1% of serum samples and 45.2%, 7.1%, and 2.1% of colostrum samples were positive for IgG, IgM, and IgA, respectively. Specific IgG1, IgG3, and IgG4 were positive, respectively, in 98.5%, 54.6%, and 44.6% of serum samples, in contrast with 56.9%, 78.5%, and 34.6% of colostrum samples. Thus, the predominant reactivity of IgG subclasses against T. gondii was IgG1 in serum and IgG3 in colostrum. The higher percentage of positive samples and higher levels of anti-T. gondii IgG3 antibodies were observed in colostrum, when compared to serum samples, suggesting a local production of this subclass. IgG3 and IgG1 subclasses presented different percentages of positivity in serum and colostrum. Only the IgG1 subclass showed a significant correlation between the levels of anti-T. gondii in serum and colostrum, suggesting that IgG1 in breast milk comes from a systemic source. IgG4 showed a similar percentage of positivity in both sample types, but no significant correlation was observed between their levels. Conclusion: Colostrum presents representative levels of IgM, IgA, IgG1, IgG3, and IgG4 antibodies specific to T. gondii. The detection of these antibodies presents the potential for diagnostic application of colostrum samples to better identify the diagnostic status of T. gondii infection, especially during the acute phase. In addition, breastfeeding can also be a possible source of protective antibodies for the newborn against toxoplasmosis, an anthropozoonosis maintained by environmental infection, which interferes in the public health of many countries.