IgM Molecules Research Articles

Functional studies with IgM and IgA immunoglobulins: binding to pIgR, FcαμR, FcμR, and CDC activities.

IgMs are the first antibodies produced by the immune system upon encounter of a possible pathogen and are one of five antibody subclasses in humans. For IgG, the most intensively studied antibody class, the N-linked glycosylation site located in the Fc-domain is directly involved in high affinity binding to the respective receptors and initiation ofcorresponding immune response. IgM molecules have five N-glycosylation sites and one N-glycosylation site in the J-chain, which can be incorporated in IgM or IgA molecules. There is only limited knowledge available concerning the function of these N-glycosylations in IgMs. To address this question, we produced IgM molecules lacking a particular N-glycosylation site and tested these variants as well as IgA molecules for binding to the known receptors: the polymeric immunoglobulin receptor (pIgR), the dual receptor for IgA and IgM, FcαμR, and the specific receptor for IgM, FcμR. The single glycosylation sites did not show an impact on expression and multimerization, except for variant N402Q, which could not be expressed. In SPR measurements, no major impact on the binding to the receptors by particular glycosylation sites could be detected. In cellular assays, deglycosylated variants showed some alterations in induction of CDC activity. Most strikingly, we observed also binding of IgA to the FcμR in the same affinity range as IgM, suggesting that this might have a physiological role. To further substantiate the binding of IgA to FcμR we used IgA from different origins and were able to confirm binding of IgA preparations to the FcμR.

Conditional activation of an anti-IgM antibody-drug conjugate for precise B cell lymphoma targeting.

Cancerous B cells are almost indistinguishable from their non-malignant counterparts regarding their surface antigen expression. Accordingly, the challenge to be faced consists in elimination of the malignant B cell population while maintaining a functional adaptive immune system. Here, we present an IgM-specific antibody-drug conjugate masked by fusion of the epitope-bearing IgM constant domain. Antibody masking impaired interaction with soluble pentameric as well as cell surface-expressed IgM molecules rendering the antibody cytotoxically inactive. Binding capacity of the anti-IgM antibody drug conjugate was restored upon conditional protease-mediated demasking which consequently enabled target-dependent antibody internalization and subsequent induction of apoptosis in malignant B cells. This easily adaptable approach potentially provides a novel mechanism of clonal B cell lymphoma eradication to the arsenal available for non-Hodgkin's lymphoma treatment.

Development of a linear ion trap mass spectrometer capable of analyzing megadalton MALDI ions

Detecting megadalton matrix-assisted laser desorption/ionization (MALDI) ions in an ion trap mass spectrometer is a technical challenge. In this study, megadalton protein and polymer ions were successfully measured by MALDI linear ion trap mass spectrometer (LIT-MS) for the first time. The LIT-MS is comprised of a Thermo linear ion trap mass analyzer and a highly sensitive charge-sensing particle detector (CSPD). A newly designed radio frequency (rf) scan mode with dipolar resonance ejection techniques is proposed to extend the mass range of LIT-MS up to one million Thomson (Th). We analyze high mass ions with mass-to charge (m/z) ratios ranging from 100 kTh to 1 MTh, including thyroglobulin, alpha-2-macroglobulin, immunoglobulins (e.g., IgG and IgM), and polymer (∼ 940 kTh) ions. Besides, it is also very challenging for ion trap mass spectrometry to detect megadalton ions at low concentrations. By adopting high affinity carboxylated/oxidized detonation nanodiamonds (oxDNDs) to enrich IgM molecules and form antibody-nanodiamond conjugates, we have successfully reached ∼ 5 nM (5 μg/mL) concentration which is better than that by the other techniques.

Research Progress in Molecular Biology of Fish Immunoglobulin M (IgM)

Immunoglobulin (Ig) is a type of globulin produced by B lymphocytes during pathogenic infection of vertebrates. It has immune functions and can realize specific recognition and neutralization of corresponding antigens. As IgM is reported first in fish, IgM is the first antibody produced during immune responses and plays a vital role in systemic and mucosal immune tissues. IgM molecules have two forms: membrane-bound IgM (mIgM) and secreted IgM (sIgM). The latter is produced by plasmacytes and secreted into body fluid, existing as immunological effect molecules. The former embeds into B cytomembrane and exists as an antigen receptor. It binds with assistant molecules to form cell receptor compounds. This study reviews research progress on the structures and production processes of IgM genes in different fish species and the distribution characteristics of IgM on B cells, mediated signal pathways, and functions. It aims to enrich basic theoretical knowledge of fish immunology and provide some scientific references for disease control in fishes.

CD19 Is Internalized Together with IgM in Proportion to B Cell Receptor Stimulation and Is Modulated by Phosphatidylinositol 3-Kinase in Bone Marrow Immature B Cells.

Newly generated immature B cells that bind self-antigen with high avidity arrest in differentiation and undergo central tolerance via receptor editing and clonal deletion. These autoreactive immature B cells also express low surface levels of the coreceptor CD19, a key activator of the PI3K pathway. Signals emanating from both CD19 and PI3K are known to be critical for attenuating receptor editing and selecting immature B cells into the periphery. However, the mechanisms that modulate CD19 expression at this stage of B cell development have not yet been resolved. Using in vivo and in vitro models, we demonstrate that Cd19 de novo gene transcription and translation do not significantly contribute to the differences in CD19 surface expression in mouse autoreactive and nonautoreactive immature B cells. Instead, CD19 downregulation is induced by BCR stimulation in proportion to BCR engagement, and the remaining surface IgM and CD19 molecules promote intracellular PI3K-AKT activity in proportion to their level of expression. The internalized CD19 is degraded with IgM by the lysosome, but inhibiting lysosome-mediated protein degradation only slightly improves surface CD19. In fact, CD19 is restored only upon Ag removal. Our data also reveal that the PI3K-AKT pathway positively modulates CD19 surface expression in immature B cells via a mechanism that is independent of inhibition of FOXO1 and its role on Cd19 gene transcription while is dependent on mTORC1.

Peripheral smear reckons the nature of IgM cold agglutinins: A case‐based study asserting the role of thermal amplitude and clonality

Peripheral smear reckons the nature of IgM cold agglutinins: A case‐based study asserting the role of thermal amplitude and clonality

Immunoglobulin M Monoclonal Gammopathies of Clinical Significance.

Immunoglobulin M monoclonal gammopathy of undetermined significance (MGUS) comprises 15-20% of all cases of MGUS. IgM MGUS is distinct from other forms of MGUS in that the typical primary progression events include Waldenstrom macroglobulinaemia and light chain amyloidosis. Owing to its large pentameric structure, IgM molecules have high intrinsic viscosity and precipitate more readily than other immunoglobulin subtypes. They are also more commonly associated with autoimmune phenomena, resulting in unique clinical manifestations. Organ damage attributable to the paraprotein, not fulfilling criteria for a lymphoid or plasma cell malignancy has recently been termed monoclonal gammopathy of clinical significance (MGCS) and encompasses an important family of disorders for which diagnostic and treatment algorithms are evolving. IgM related MGCS include unique entities such as cold haemagglutinin disease, IgM related neuropathies, renal manifestations and Schnitzler’s syndrome. The diagnostic approach to, and management of these disorders differs significantly from other categories of MGCS. We describe a practical approach to the evaluation of these patients and our approach to their treatment. We will also elaborate on the key unmet needs in IgM MGCS and highlight potential areas for future research.

Analysis of phagocytosis by mIgM+ lymphocytes depending on monoclonal antibodies against IgM of largemouth bass (Micropterus salmoides)

The phagocytic actives of B cells in fish have been proven in recent years. In this study, five positive hybridomas secreting monoclonal antibodies (MAbs) against largemouth bass IgM were produced. Indirect immunofluorescence assay (IFA) demonstrated that five MAbs could specifically recognize membrane-bound IgM (mIgM) molecule of largemouth bass. Indirect ELISA and Western blotting analysis showed that all the five MAbs had no cross-reactions with the other two teleost IgMs. Flow cytometry analysis (FCM) revealed that the percentages of largemouth bass mIgM+ lymphocytes in head kidney, peripheral blood and spleen were 51.66 ± 0.608%, 16.5 ± 1.235% and 42.92 ± 1.091%, respectively. In addition, the phagocytosis rates of mIgM + lymphocytes ingesting Nocardia seriolae from head kidney, peripheral blood and spleen were calculated to be 5.413 ± 0.274%, 16.6 ± 0.289% and 26.3 ± 0.296%, respectively. The qPCR results of sorted cells indicated that most inflammatory cytokines (IFNγ, IL-1β, IL-2, IL-12β, IL-34, IL-10), chemokine (CXCL12), chemokines receptors (CXCR2, CXCR4) and genes (FcγRⅠa, NCF1, CFL, ARP2/3, CD45, Syk, MARCKS) related to FcγR-mediated phagocytic signaling pathway in phagocytic mIgM+ lymphocytes were up-regulated significantly (P < 0.05). Taken together, the results suggested that the MAb (MM06H) produced in this paper could be used as a tool to study mIgM+ lymphocytes of largemouth bass, and FcγR may participate in the phagocytosis of mIgM+ lymphocytes, which is helpful to further study the role of mIgM+ lymphocytes in innate immunity.

Multimeric antibodies from antigen-specific human IgM+ memory B cells restrict Plasmodium parasites.

Multimeric immunoglobulin-like molecules arose early in vertebrate evolution, yet the unique contributions of multimeric IgM antibodies to infection control are not well understood. This is partially due to the difficulty of distinguishing low-affinity IgM, secreted rapidly by plasmablasts, from high-affinity antibodies derived from later-arising memory cells. We developed a pipeline to express B cell receptors (BCRs) from Plasmodium falciparum-specific IgM+ and IgG+ human memory B cells (MBCs) as both IgM and IgG molecules. BCRs from both subsets were somatically hypermutated and exhibited comparable monomeric affinity. Crystallization of one IgM+ MBC-derived antibody complexed with antigen defined a linear epitope within a conserved Plasmodium protein. In its physiological multimeric state, this antibody displayed exponentially higher antigen binding than a clonally identical IgG monomer, and more effectively inhibited P. falciparum invasion. Forced multimerization of this IgG significantly improved both antigen binding and parasite restriction, underscoring how avidity can alter antibody function. This work demonstrates the potential of high-avidity IgM in both therapeutics and vaccines.

IMMUNE ANTIBODIES IN MOKSHA AND ERZYA ETHNIC GROUPS OF THE REPUBLIC OF MORDOVIA

ABO system's immune antibodies are common, but not as persistent as natural anti-A and anti-B antibodies. They are the result of hyper immunization of an iso- or heteroimmune nature and belong to the IgG class. The study of serum for the presence of immune antibodies of the ABO, Rh system is necessary for the diagnosis of hemolytic disease cases of newborns with ABO, Rh-conflict pregnancy. Clinically significant immune antibodies of erythrocyte systems capable to cause in vivo destruction of red blood cells are of great importance in transfusiology and immunohematology. The most common cause of their occurrence is alloimmunization with red blood cell antigens during childbirth. The aim of this study was to determine the characteristics of the immune anti-erythrocyte antibodies in residents of the Republic of Mordovia belonging to the ethnic groups of Moksha and Erzya. Methods. The cross-sectional study involved 294 people from the Moksha and 387 people and Erzya ethnic groups, randomly selected in 7 districts of the Republic of Mordovia. Detection of IgG anti-A, anti-B ABO systems was performed by inactivation of disulfide bonds in IgM molecules using a 5 % unitiol solution and then by direct agglutination method at indoor temperature with standard red blood cells of group O(I), A(II), and B(III). The study of incomplete anti-erythrocyte antibodies of other systems was performed by indirect Coombs' test using gel technology. Results. The study of immune antibodies of the ABO system revealed from 10.61 to 16.00 % of anti-A antibodies and from 2.27 to 6.38 % of anti-B antibodies in both men and women Moksha and Erzya. We studied immune anti-erythrocyte antibodies: anti-D 0.34 % in Moksha and anti-D 0.26% and anti-Kell 0.26 % antibodies in Erzya. The scale of immunogenicity of red blood cell antigens in the studied groups: A&gt;, B&gt;, D&gt; and K&gt;. The alloimmunization index of transfusion-hazardous antigens was calculated as 0.34 for Moksha and 0.52 for Erzya. Conclusions. Physiologically, the immune system in Moksha and Erzya actively reacts with antibody's production and it is sensitive to immunization by the group of antigens.

Development of a Monoclonal Antibody Specific to the IgM Heavy Chain of Bighead Catfish (Clarias macrocephalus): A Biomolecular Tool for the Detection and Quantification of IgM Molecules and IgM+ Cells in Clarias Catfish.

Catfish is a commonly-cultivated freshwater fish in Thailand and many Southeast Asian countries. The molecular data obtained for the IgM heavy chain (IgMH) of catfish have been useful for distinguishing monoclonal antibodies (mAbs). A mAb specific to Cμ1 of the IgMH of catfish (IgMHCμ1 mAb) was developed in a rabbit model using sequence information from bighead catfish (Clarias macrocephalus). The IgMHCμ1 mAb strongly recognized the IgM heavy chain of the tested catfish, namely, bighead catfish, African catfish (Clarias gariepinus) and their hybrid (C. macrocephalus × C. gariepinus), in immunological Western blot analysis and competitive ELISAs. Additionally, the IgMHCμ1 mAb successfully recognized IgM+ cells by detecting IgM molecules in both secreted and membrane-bound forms in peripheral blood leukocytes (PBLs). The IgMHCμ1 mAb was further used to quantify the percentage of IgM+ cells among PBLs through flow cytophotometry. The IgM+ cell percentages of healthy bighead catfish, African catfish and their hybrid were 38.0–39.9%, 45.6–53.2%, and 58.7–60.0%, respectively. Furthermore, the IgMHCμ1 mAb showed no cross-reactivity with the IgM of zebrafish. These findings suggest that this mAb can be used as an immunological tool for monitoring the health, immune status, and immune development of cultivated Clarias catfish.

Characterization of B1-cells during experimental leukomogenesis.

Bovine leukemia causes a significant polyclonal expansion of CD5+, IgM+ B lymphocytes, known as persistent lymphocytosis (PL), in approximately 30% of infected cattle. However, it is not yet clear what happens to this subpopulation of B cells in the early period of infection of animals. Quantitative characterization of IgM+ and CD5+ B cells during the immune response, which can provide important information on the mechanisms of lymphocyte priming in BLV infection. The experiment used BLV-negative calves of black-motley breed at the age of 8 months (n = 11). Animals (n = 8) were intravenously injected with blood of a BLV-positive cow. Control calves (n = 3) were injected with saline. Studies were performed before and after infection on days 5, 7, 14, 21, 28 and 65 of the immune response. The determination of the number of B-lymphocytes in the blood was carried out by the method of immunoperoxidase staining based on monoclonal antibodies to IgM, CD5. As a result of the studies, it was found that the level of CD5+ B cells increases on the 14th day of the primary immune response, characterized by polyclonal proliferation of CD5+ B cells, which are the primary target for BLV. Our research data confirm that in the lymphocytes of experimentally infected cattle, surface aggregation of IgM and CD5 molecules on B-lymphocytes is absent. It is known that the wave-like nature of IgM synthesis, which was shown in previous studies, depends on a subpopulation of B1 cells. After 7 days of the immune response, IgM+ and CD5+ cells do not correlate, which shows their functional difference. The increase in CD5+ cells is probably not associated with B cells, but with T cells differentiating under the influence of the virus. A subset of B1 cells is the primary target of cattle leukemia virus. The 65th day of the immune response is characterized by the expansion of IgM+ B cells, a decrease in the number of CD5+ cells and a uniform distribution of receptors around the perimeter of the cells.

Development of monoclonal antibody against IgM of large yellow croaker (Larimichthys crocea) and characterization of IgM+ B cells

In the present study, a monoclonal antibody (mAb) against large yellow croaker IgM was produced by immunizing mice with purified large yellow croaker serum IgM. Western blotting showed that this mAb could specifically react with the heavy chain of large yellow croaker serum IgM. Indirect immunofluorescence assay (IFA) analysis suggested that the resulting mouse anti-IgM mAb could recognize membrane-bound IgM (mIgM) molecules of large yellow croaker. This mouse anti-IgM mAb also can be used for sorting of large yellow croaker IgM+ B cells through the magnetic-activated cell sorting (MACS) method, which was further confirmed by RT-PCR analysis of specific marker genes for B cells. Flow cytometry analysis showed that the percentages of IgM+ B cells in head kidney, spleen and peripheral blood lymphocytes were 29.00 ± 1.58%, 33.00 ± 1.64%, and 16.50 ± 2.39%, respectively. Additionally, the phagocytosis rates of IgM+ B cells for 0.5 μm beads in head kidney, spleen and peripheral blood were calculated to be 7.56 ± 0.58%, 4.053 ± 0.62% and 23.17 ± 2.26%, respectively, while only 2.36 ± 0.23%, 1.16 ± 0.44% and 6.41 ± 0.45 of IgM+ B cells in these three tissues ingested 1 μm beads. Taken together, our data demonstrated that the mouse anti-IgM mAb produced in this study could be used as a tool to characterize IgM+ B cells and to study functions of IgM in large yellow croaker.

Evaluation of interferon β-1b therapy effects on the level and abnormal nature of immunoglobulin G in patients with multiple sclerosis

Aim : to study and evaluate the interferon β-1b (INF) therapy efficiency on the level and abnormal nature of immunoglobulin G in patients with multiple sclerosis (MS). Materials and methods . This scientific work was performed at the Neurology Department and the Department of Histology, Cytology and Embryology of theLvivNationalMedicalUniversity named after Danylo Halytsky and in theLvivRegionalResearchCenter for the Study of Multiple Sclerosis and Other Demyelinating Diseases on the basis of theLvivRegionalClinicalHospital. The inclusion criteria for patients were age between 18 and 65 years as well as definite diagnosis of MS according to McDonald Diagnostic Criteria for MS (2010). 32 patients with MS and 23 practically healthy persons were enrolled in the study. After a written informed consent obtaining from the patient there was a peripheral venous blood sampling using a blood-collection tube system Vacuette. Serum samples were prepared for antibodies to myelin basic protein (MBP) determination and identification of immunoglobulin glycosylation by means of lectin-immunoenzymatic assay. Results. Elevated levels of anti-MBP IgG and IgM in the treatment-naive MS patients were determined in comparison with the healthy persons. At the same time, higher exposure of anti-inflammatory sialic acid residues on IgG molecules and associated native immune complexes were observed in the group of patients who received INF therapy as well as higher exposure of fucosylated tri-mannose residues on measles IgG molecules and associated native immune complexes in comparison with the untreated patients. Conclusions: The blood serum of MS patients who received INF therapy were characterized by lower levels of IgG and IgM antibodies to MBP and higher exposure of sialic acid terminal residues on IgG and IgM molecules in comparison with the untreated patients. It is reasonable to assume that INF therapy could lead to anti-inflammatory native IgG complexes formation and / or decrease in IgG and IgM autoantibodies against the nervous system components, as it has been shown for autoantibodies to MBP.

Exploring the Unique Selectivity of Hydrophobic Cation Exchanger Nuvia cPrime for the Removal of a Major Process Impurity: A Case Study with IgM.

Mixed-mode chromatography is becoming an important tool for downstream process purification, as it provides the selectivity and robustness unmatched by conventional singlemode chromatographic methods. The joint action of multiple functionalities present on the ligands of mixed-mode chromatography matrices effectively enhances the separation of target molecules from impurities. Using Nuvia cPrime as an example, we elucidate the separation principles of hydrophobic cation exchange mixed-mode chromatography and its difference from traditional strong cation exchangers. We have developed a Nuvia cPrime based polish purification step specifically for the removal of a major process contaminant, which has an isoelectric point similar to that of the target monoclonal IgM molecule. Additional purification was accomplished using a second mixed-mode chromatography column packed with Ceramic Hydroxyapatite. The monoclonal IgM prepared with this new process fully retained its biological activity and was free of high molecular weight aggregates, a product quality that was not achievable in previous attempts using traditional ion exchange or hydrophobic interaction chromatography.

P131 Using DTT as a tool to remove background in HLA antibody identification

Aim The goal of this study is to investigate the effectiveness of DTT as a tool to remove excessive IgM, in Single Antigen Bead assays, which can lead to high negative control values. Methods In the present study, nine sera (class I) and seven sera (class II), with NC MFIs ⩾1000 MFI, were tested using One Lambda SAB Assay, neat vs. DTT, Adsorb-out, Adsorb-out x 2 and DTT & Adsorb-out. Sera were tested using the manufacturer’s protocol. Sera were DTT treated by combining 5 μL 50 mM DTT to 195 μL sera. This mixture incubated at 37 °C for 15 min. All sera were pretreated with EDTA. For this study, an elevated NC is defined as an MFI ⩾1000 (two sera demonstrated borderline positivity (953 & 816)). Results With class I, all but one DTT treated serum, demonstrated a decrease in NC MFI. The outlier serum NC MFI demonstrated a decrease but was slightly above acceptable level (1790 MFI → 1083 MFI). The sera were also treated with Adsorb-out, with all resulting NC MFIs >1000. Additionally, eight NC MFIs either did not decrease or increased following Adsorb-out. These sera were treated either with Adsorb-out x2 or DTT & Adsorb-out. Four sera continued to have NC MFIs >1000. Regarding class II DTT treated sera, all but two showed a NC MFI decrease. Of those two, both demonstrated a decrease vs. neat but not 1000 and three showed an increase. The sera were treated with Adsorb-out x 2 or DTT & Adsorb-out. Of these sera, two achieved acceptable levels. Conclusions DTT is a commonly used reducing reagent which is efficient at cleaving exposed disulfide bonds, thus eliminating IgM-based interference. This treatment, in regards to SAB testing, is performed to remove interfering IgM molecules. DTT is also able to obviate C1 interference but does so poorly. EDTA prevents complement interference far more efficient through Ca2+ chelation. Based on our results, we will further investigate the use of DTT as an effective treatment on sera with a high NC MFI which are not amendable to a single treatment of Adsorb-out.

Mouse Antibody of IgM Class is Prone to Non-Enzymatic Cleavage between CH1 and CH2 Domains

IgM is a multivalent antibody which evolved as a first line defense of adaptive immunity. It consists of heavy and light chains assembled into a complex oligomer. In mouse serum there are two forms of IgM, a full-length and a truncated one. The latter contains μ’ chain, which lacks a variable region. Although μ’ chain was discovered many years ago, its origin has not yet been elucidated. Our results indicate that μ’ chain is generated from a full-length heavy chain by non-enzymatic cleavage of the protein backbone. The cleavage occurred specifically after Asn209 and is prevented by mutating this residue into any other amino acid. The process requires the presence of other proteins, preferentially with an acidic isoelectric point, and is facilitated by neutral or alkaline pH. This unique characteristic of the investigated phenomenon distinguishes it from other, already described, Asn-dependent protein reactions. A single IgM molecule is able to bind up to 12 epitopes via its antigen binding fragments (Fabs). The cleavage at Asn209 generates truncated IgM molecules and free Fabs, resulting in a reduced IgM valence and probably affecting IgM functionality in vivo.

Heterogeneity of Memory Marginal Zone B Cells.

The marginal zone (MZ) is largely composed of a unique subpopulation of B cells, the so-called MZ-B cells. At a molecular level, memory B cells are characterized by the presence of somatically mutated IGV genes. The earliest studies in the rat have documented the presence of hapten-specific MZ-B cells after immunization in the MZ. This work later received experimental support demonstrating that the IGHV-Cµ transcripts expressed by phenotypically defined splenic MZ-B cells (defined as CD90negIgMhighIgDlow B cells) can carry somatic hypermutation. However, only a minor fraction (< 10%-20%) of these MZ-B cells is mutated and is considered to represent memory B cells. Memory B cells can either be class-switched (IgG, IgA, IgE), or non-class-switched (IgM) B cells. B cells in the MZ are a heterogeneous population of cells and both naïve MZ-B cells; class switched and unswitched memory MZ-B cells are present at this unique site in the spleen. Naïve MZ-B cells carry unmutated Ig genes, produce low-affinity IgM molecules and constitute a first line of defense against invading pathogens. Memory MZ-B cells express high-affinity Ig molecules, directed to (microbial) antigens that have been encountered. In this review, we report on the memory compartment of splenic MZ-B cells in the rat to provide insights into the origin and function of these memory MZ-B cells.

P275 Using positive control bead MFI in HLA antibody identification as a quality measure

Aim This study is focused to determine the practicality of using the internal positive control bead as a qualitative measure in One Lambda’s Single Antigen Bead assay. Methods In the present study, forty samples (n = 40) were tested using One Lambda (Canoga Park, CA) Single Antigen Bead (SAB) Assay for HLA Class I and fifty-one samples (n = 51) were tested for HLA Class II antibody specificities using manufacturer’s suggested protocol. To treat serum with DTT, 5μL of 50 mM DTT was added to 195 μL of patient serum and incubated at 37 °C for 15 min. Results Forty samples were assayed to determine the identification of anti-HLA class I antibodies and 52 samples were tested to determine the identification of anti-HLA class II antibodies. For this study, 1000 mean fluorescence intensity (MFI) was considered positive. Data listed as in Table. Of the + to = beads, half were borderline, demonstrating low reactivity in an untreated sample but being 200–300 MFI from the positive threshold following treatment. A similar proportion followed the opposite path. Of the = to + group, half demonstrated borderline reactivity, just under the positive threshold. Following DTT treatment, the average MFI rose by only a few hundred (200–300) MFI. Conclusions Dithiothreitol treatment is a commonly used reducing reagent used to denature disulphide bonds in IgM molecules to remove background or prevent steric hindrance. This treatment, in regards to SAB testing, is performed to increase the positive control MFI. DTT is efficient at cleaving exposed disulfide bonds, thus eliminating IgM-based interference. DTT is also able to obviate C1 interference but does so poorly. EDTA prevents complement interference far more efficiently through Ca2+ chelation. Based on our results, we question the utility of using DTT to increase the positive control bead MFI. Due to lot-to-lot variability in the density of the IgG molecule on the surface of the positive control bead, One Lambda’s published positive control bead cutoff is sufficient to determine if the assay is valid. Download : Download high-res image (47KB) Download : Download full-size image

Selective IgM Deficiency: Clinical and Laboratory Features of 17 Patients and a Review of the Literature

Primary selective IgM deficiency (sIgMD) is a primary immunodeficiency with unclear pathogenesis and a low number of published cases. We reviewed clinical and laboratory manifestations of 17 sIgMD patients. Serum IgM, IgG, and its subclasses, IgA, IgE, antibodies against tetanus toxoid, pneumococcal polysaccharides and Haemophilus influenzae type b, isohemagglutinins, and T and B lymphocyte subsets, expressions of IgM on B cells and B lymphocyte production of IgM were compared with previously reported case reports and a small series of patients, which included 81 subjects in total. We found that some patients in our cohort (OC) and published cases (PC) had increased IgE levels (OC 7/15; PC 21/37), decreased IgG4 levels (OC 5/14), very low titers of isohemagglutinins (OC 8/8; PC 18/21), increased transitional B cell counts (OC 8/9), decreased marginal zone B cell counts (OC 8/9), and increased 21low B cell counts (OC 7/9). Compared with the PC (20/20), only two of five OC patients showed very low or undetectable production of IgM after stimulation. A majority of the patients had normal antibody production to protein and polysaccharide antigens, basic lymphocyte subset counts, and expression of surface IgM molecules on B cells. Low IgM levels are associated with various immunopathological disorders; however, pathogenic mechanisms leading to decreased IgM serum level in selective IgM deficiency remain unclear. Moreover, it is difficult to elucidate how strong these associations are and if these immunopathological conditions are primary or secondary.