Sequence variants influencing the regulation of serum IgG subclass levels.

Immunoglobulin G (IgG) is the main isotype of antibody in human blood. IgG consists of four subclasses (IgG1 to IgG4), encoded by separate constant region genes within the Ig heavy chain locus (IGH). Here, we report a genome-wide association study on blood IgG subclass levels. Across 4334 adults and 4571 individuals under 18 years, we discover ten new and identify four known variants at five loci influencing IgG subclass levels. These variants also affect the risk of asthma, autoimmune diseases, and blood traits. Seven variants map to the IGH locus, three to the Fcγ receptor (FCGR) locus, and two to the human leukocyte antigen (HLA) region, affecting the levels of all IgG subclasses. The most significant associations are observed between the G1m (f), G2m(n) and G3m(b*) allotypes, and IgG1, IgG2 and IgG3, respectively. Additionally, we describe selective associations with IgG4 at 16p11.2 (ITGAX) and 17q21.1 (IKZF3, ZPBP2, GSDMB, ORMDL3). Interestingly, the latter coincides with a highly pleiotropic signal where the allele associated with lower IgG4 levels protects against childhood asthma but predisposes to inflammatory bowel disease. Our results provide insight into the regulation of antibody-mediated immunity that can potentially be useful in the development of antibody based therapeutics.

Immunoparesis defined by heavy/light chain pair suppression in smoldering multiple myeloma shows initial isotype specificity and involves other isotypes in advanced disease.

Smoldering multiple myeloma (SMM) is an asymptomatic and biologically heterogeneous plasma cell disorder, with a highly variable clinical course. Immunoparesis, defined by total immunoglobulin measurements, has been shown to be an independent risk factor for progression to symptomatic disease. The heavy/light chain (HLC) assay allows precise measurement of the polyclonal immunoglobulin of the same isotype, enabling the evaluation of isotype-matched immunoparesis (IMI). In this study, we prospectively characterized immunoparesis, as determined by HLC measurements, in 53 SMM patients. Severe IMI was present in 51% of patients, while severe IP of uninvolved isotypes (HLC IP) was present in 39%. Most of the patients with severe HLC IP presented with severe IMI, but not the other way around. Isotype specificity of immune suppression was suggested by lower relative values of isotype-matched HLC pairs, both for IgG and IgA SMM. Severe IMI was associated with other risk factors for progression while patients with severe IMI and severe HLC IP showed an even higher risk profile. Both severe IMI and severe IgM HLC IP showed a significantly shorter time to progression. Finally, gene expression analysis demonstrated differences in the bone marrow microenvironment between patients with IMI and IMI plus HLC IP, with an increased expression of genes associated with cytolytic cells. In conclusion, our data supports isotype specificity of early immunoglobulin suppression mechanisms. While suppression of both involved and uninvolved isotypes is associated with risk of progression, the later appears to develop with more advanced disease and could be mediated by different mechanisms.

Serum IgM heavy chain sub-isotypes and light chain variants revealed in giant grouper (Epinephelus lanceolatus) via protein A affinity purification, mass spectrometry and genome sequencing.

Two IgM heavy (H) chain sub-isotypes (80 and 40 kDa) and two light (L) chain variants (25 and 30 kDa) were detected in the serum of giant grouper (Epinephelus lanceolatus), purified by ammonium sulphate precipitation followed by protein A affinity chromatography. This method yielded 5.6 mg/mL high purity IgM from grouper serum, with efficiency estimated at 39.5% recovery from crude serum. The H and L chains were identified by SDS-PAGE and mass spectrometry (MS). Nanopore long-read sequencing was used to generate a genomic contig (MW768935), containing Cµ, Cδ loci, VH regions, and a H chain Joining segment. cDNA sequencing of Cµ transcripts (MW768933 and MW768934) were used to polish the genomic contig and determine the exons and introns of the corresponding locus. MS peptide mapping revealed that the 80 kDa H chain consisted of CH1-4 domains while peptides from the 40 kDa H chain only mapped to CH1-2 domains. Our genomic contig showed the Cµ locus has a Cµ1-Cµ2-Cµ3-Cµ4 arrangement on the same strand as the other Ig loci identified in this genomic sequence. Our study corrects the NCBI annotations of the opposing Cµ loci (LOC117268697 and LOC117268550) in chromosome 16 (NC_047006). Further, we identified both κ and λ L chain isotypes in serum IgM. The molecular weight differences observed may result from different combinations of CL and VL genes. Putative IgM sub-isotypes have also been reported in Epinephelus itajara and Epinephelus coioides. The presence of IgM sub-isotypes may be a conserved trait among Epinephelus species.

MASS-FIX for the detection of monoclonal proteins and light chain N-glycosylation in routine clinical practice: a cross-sectional study of 6315 patients.

Immunoenrichment-based matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), termed MASS-FIX, offers several advantages over immunofixation for the detection and isotyping of serum monoclonal protein, including superior sensitivity and specificity, the ability to differentiate therapeutic monoclonal antibodies, and the rapid identification of light chain (LC) N-glycosylation. We identified 6315 patients with MASS-FIX performed at our institution since 2018. Of these, 4118 patients (65%) with a wide array of plasma cell disorders (PCD), including rare monoclonal gammopathies of clinical significance, had a positive MASS-FIX. Two-hundred twenty-one (5%) of the MASS-FIX positive patients had evidence of LC N-glycosylation, which was more commonly identified in IgM heavy chain isotype, kappa LC isotype, and in diagnoses of immunoglobulin light chain (AL) amyloidosis and cold agglutinin disease (CAD) compared to other PCD. This cross-sectional study describes the largest cohort of patients to undergo MASS-FIX in routine clinical practice. Our findings demonstrate the widespread utility of this assay, and confirm that LC N-glycosylation should prompt suspicion for AL amyloidosis and CAD in the appropriate clinical context.

Investigation of total immunoglobulin G concentration, heavy chain antibody levels, and neutrophil to lymphocyte ratio in female camels and their newborn calves.

Camels belong to a group of animals, where the structure of placenta does not allow intrauterine transfer of maternal immunoglobulins to the fetus and maternal immunity is exclusively transferred by colostrum to the newborn calf. There are few studies on the passive transfer of maternal immunity in the dromedary camel. This study determined total immunoglobulin G concentration, heavy chain antibody (HCAbs) levels, and neutrophils to lymphocytes ratio (NLR) in female camels and their newborn calves. For this, samples were collected from nine she-camels (blood and colostrum) and their calves (blood). IgG concentration and HCAb level were determined in mother serum and colostrum as well as in calf serum using ELISA. The NLR was calculated after the estimation of relative fractions of neutrophils and lymphocytes in collected blood samples using a blood cell analyzer. Both IgG and HCAbs were higher concentrated in camel colostrum than in mother serum. At parturition and before the first colostrum intake, calf serum did not contain any measurable concentration of IgG and only low levels of HCAbs. After colostrum consumption, a rise in IgG and HCAb levels was observed in calf serum. For total IgG, a minimum was reached on day 30 postnatum. While a significant increase in IgG concentration was seen on day 60 postnatum, no significant rise was measured in HCAbs at that age. Only post-colostrum IgG levels in calf serum correlated positively with IgG levels in mother colostrum. Directly after birth, newborn calves showed significantly higher NLR than their mothers. This indicates a pro-inflammatory nature of the calf immune response. The decrease of the NLR on day 60 postnatum may argue for the maturation of the calf immune response at this age.

Proteomic markers with prognostic impact on outcome of chronic lymphocytic leukemia patients under chemo-immunotherapy: results from the HOVON 109 study.

Despite recent identification of several prognostic markers, there is still a need for new prognostic parameters able to predict clinical outcome in chronic lymphocytic leukemia (CLL) patients. Here, we aimed to validate the prognostic ability of known (proteomic) markers measured pretreatment and to search for new proteomic markers that might be related to treatment response in CLL. To this end, baseline serum samples of 51 CLL patients treated with chemo-immunotherapy were analyzed for 360 proteomic markers, using Olink technology. Median event-free survival (EFS) was 23 months (range: 1.25-60.9). Patients with high levels of sCD23 (>11.27, p = 0.026), sCD27 (>11.03, p = 0.04), SPINT1 (>1.6, p = 0.001), and LY9 (>8.22, p = 0.0003) had a shorter EFS than those with marker levels below the median. The effect of sCD23 on EFS differed between immunoglobulin heavy chain variable gene-mutated and unmutated patients, with the shortest EFS for unmutated CLL patients with sCD23 levels above the median. Taken together, our results validate the prognostic impact of sCD23 and highlight SPINT1 and LY9 as possible promising markers for treatment response in CLL patients.

Production of Autoreactive Heavy Chain-Only Antibodies in Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is characterized by the overproduction of high-affinity autoreactive antibodies. Here, we show that more than 65.8% of 222 recombinant antibodies derived from 8 SLE patients can be secreted as heavy chain-only antibodies (HCAbs) when expressed in HEK-293T cells. The secretion of HCAbs follows the conventional endoplasmic reticulum-Golgi apparatus pathway, despite triggering a weaker unfolded protein response (UPR). Many of the purified SLE HCAbs remain autoreactive and have an even higher affinity for dsDNA, Sm, nucleosome, and cardiolipin than HCAbs from healthy individuals. Extended analyses of the CDR3 region and the heavy chain variable (VH) region of HCAb F3 show that the VH region is responsible for IgH secretion, while the CDR3 region determines its reactivity. Such a high frequency of HCAb secretion cannot fully concur with our current understanding of antibody assembly and secretion. The presence of a large proportion of autoreactive HCAbs in SLE reveals a novel mechanism for the generation of autoreactive antibodies in lupus.

Daratumumab plus CyBorD for patients with newly diagnosed AL amyloidosis: safety run-in results of ANDROMEDA.

Although no therapies are approved for light chain (AL) amyloidosis, cyclophosphamide, bortezomib, and dexamethasone (CyBorD) is considered standard of care. Based on outcomes of daratumumab in multiple myeloma (MM), the phase 3 ANDROMEDA study (NCT03201965) is evaluating daratumumab-CyBorD vs CyBorD in newly diagnosed AL amyloidosis. We report results of the 28-patient safety run-in. Patients received subcutaneous daratumumab (DARA SC) weekly in cycles 1 to 2, every 2 weeks in cycles 3 to 6, and every 4 weeks thereafter for up to 2 years. CyBorD was given weekly for 6 cycles. Patients had a median of 2 involved organs (kidney, 68%; cardiac, 61%). Patients received a median of 16 (range, 1-23) treatment cycles. Treatment-emergent adverse events were consistent with DARA SC in MM and CyBorD. Infusion-related reactions occurred in 1 patient (grade 1). No grade 5 treatment-emergent adverse events occurred; 5 patients died, including 3 after transplant. Overall hematologic response rate was 96%, with a complete hematologic response in 15 (54%) patients; at least partial response occurred in 20, 22, and 17 patients at 1, 3, and 6 months, respectively. Renal response occurred in 6 of 16, 7 of 15, and 10 of 15 patients, and cardiac response occurred in 6 of 16, 6 of 13, and 8 of 13 patients at 3, 6, and 12 months, respectively. Hepatic response occurred in 2 of 3 patients at 12 months. Daratumumab-CyBorD was well tolerated, with no new safety concerns versus the intravenous formulation, and demonstrated robust hematologic and organ responses. This trial was registered at www.clinicaltrials.gov as #NCT03201965.

Differential levels of Alpha-1-inhibitor III, Immunoglobulin heavy chain variable region, and Hypertrophied skeletal muscle protein GTF3 in rat mammary tumorigenesis.

Early detection of breast cancer can be best facilitated by the development of precancerous markers. Serum proteins being the sensitive signatures, can be the ideal choice. We previously demonstrated the reduced levels of two serum proteins at a very early stage of tumorigenesis in a breast cancer model, developed in Wistar rats by 7,12-dimethylbenz[a]anthracene (DMBA) administration. Here we report the dysregulation of three more proteins in the serum collected at another early stage (15 weeks) of tumorigenesis in the same model. The proteins were identified (as Alpha-1-inhibitor III (Mug1), Immunoglobulin heavy chain variable region (IGHV), and Hypertrophied skeletal muscle protein GTF3) by MALDI-TOF MS after the screening and fingerprinting of serum samples by one-dimensional (1D) and two-dimensional (2D) electrophoresis respectively. Relative expression analysis of corresponding genes was also carried out, and the results were found as supporting the proteomic findings. In addition, the candidate proteins of the study and their corresponding ribonucleic acids (RNAs) were subjected to homology modelling and docking (using softwares like MODELLER, 3dRNA, Autodock4.0, and GROMACS etc), which revealed the binding sites for carcinogen (DMBA) and its nature of interaction with proteins and RNAs. Moreover, the network analysis by GeneMANIA unraveled the protein/gene functional network in which Mug1, IGHV, and GTF3 are involved. Based on the significant protein and gene expression alterations in early tumorigenesis, these proteins may prove very effective in search for biomarkers for the early detection of mammary cancer. Further, these proteins can also be tried as targets for chemotherapy.

Mass Spectrometry-Based Method Targeting Ig Variable Regions for Assessment of Minimal Residual Disease in Multiple Myeloma.

Multiple myeloma is a systemic malignancy of monoclonal plasma cells that accounts for 10% of hematologic cancers. With development of highly effective therapies for multiple myeloma, minimal residual disease (MRD) assessment has emerged as an important end point for management decisions. Currently, serologic assays lack the sensitivity for MRD assessment, and invasive bone marrow sampling with flow cytometry or molecular methods has emerged as the gold standard. We report a sensitive and robust targeted mass spectrometry proteomics method to detect MRD in serum, without the need of invasive, sequential bone marrow aspirates. The method detects Ig-derived clonotypic tryptic peptides predicted by sequencing the clonal plasma cell Ig genes. A heavy isotope-labeled Ig internal standard is added to patient serum at a known concentration, the Ig is enriched in a light chain type specific manner, and proteins are digested and analyzed by targeted mass spectrometry. Peptides from the constant regions of the λ or κ light chains, Ig heavy chains, and clonotypic peptides unique to the patient monoclonal Igs are targeted. This technique is highly sensitive and specific for the patient-specific monoclonal Igs, even in samples negative by multiparametric flow cytometry. Our method can accurately and precisely detect monoclonal protein in serum of patients treated for myeloma and has broad implications for management of hematologic patients.

[Diagnostic performance of κ/λ serum free light chain ratio (Freelite® assay) and IgGκ/IgGλ ratio (Hevylite® assay) as prognostic biomarkers of the evolution of immune thrombocytopenia into a chronic disease in adult patients]. / Performance diagnostique des rapports κ/λ des chaines légères libres sériques (test Freelite®) et IgGκ/IgGλ (test Hevylite®) comme marqueurs pronostiques de chronicisation du purpura thrombopénique immunologique de l'adulte.

INTRODUCTION: Immune thrombocytopenia (ITP) is an acquired hemorrhagic disease due to antiplatelet antibodies, that will become a chronic disease in 70% of adults. Most of chronic ITP patients display clonal restriction of antiplatelet antibodies. To date, there is no biomarker able to predict the evolution of the disease. The objective of the study is to determine whether Hevylite® and/or Freelite® assays are prognostic factors for progression to chronic ITP. METHODS: This is a retrospective, monocentric, prognostic study of a biomarker, performed using frozen samples stored in a serum library. Freelite® and a Hevylite® assays were performed on the samples collected at diagnosis for adult patients with newly diagnosed ITP at the University Hospital of Poitiers between 2014/01/01 and 2017/05/01. To predict the evolution into a chronic disease, a ROC curve analysis was performed on four variables: IgGκ, IgGκ/IgGλ ratio, IgGκ - IgGλ, and κ/λ ratio. RESULTS: Thirty-two patients were included and analyzed. No patient had an abnormal κ/λ ratio. Three patients had an abnormal IgGκ/IgGλ ratio. The following variables IgGκ, IgGκ/IgGλ, IgGκ - IgGλ, and κ/λ ratio were not able to predict progression to chronic ITP in our study. CONCLUSION: This study did not reveal any prognostic value of the Freelite® and Hevylite® tests on the evolution of ITP into a chronic disease.

Cardiac involvement in heavy and light chain amyloidosis: A case report and literature review.

INTRODUCTION: Heavy and light chain amyloidosis is an extremely rare condition. There are few reports referring to the clinical impact of cardiac involvement in heavy and light chain amyloidosis, and the significance of myocardial impairment has not yet been completely explained. PATIENT CONCERNS: A 66-year-old Japanese man was admitted to our hospital presenting with nephrotic syndrome and congestive heart failure. DIAGNOSIS: Kidney and endoscopic gastric mucosal biopsy demonstrated congophilic hyalinization in most of the glomeruli and surrounding vessel walls, which were highly positive for immunoglobulin A and lambda. Finally, the patient was diagnosed as an atypical multiple myeloma with systemic heavy and light chain amyloidosis. INTERVENTIONS: The patient was referred to hematology for further treatment and was moved to another hospital for the administration of chemotherapy using melphalan and dexamethasone. OUTCOMES: The patient was still alive after 15-month follow-up from the initial diagnosis. CONCLUSION: Initial screening and follow-up for cardiac involvement are important for heavy and light chain amyloidosis. Further investigation for the prognosis of heavy and light chain amyloidosis is required to improve the strategies of diagnosis and treatment options for patients with this disease.

A new risk model to predict time to first treatment in chronic lymphocytic leukemia based on heavy chain immunoparesis and summated free light chain.

BACKGROUND: Chronic lymphocytic leukemia (CLL) is frequently accompanied by immune dysregulation. AIMS: In this multicenter prospective study, we investigated whether heavy + light chains (HLC: IgGκ, IgGλ, IgAκ, IgAκ, IgMκ, IgMλ) and IgG subclasses (IgG1, IgG2, IgG3, and IgG4) could be used as novel prognostic markers of immunoparesis in 105 treatment-naïve patients with CLL. RESULTS: Heavy + light chains immunoparesis of ≥1, ≥2, and ≥3 isotypes was evident in 74 (70%), 58 (55%), and 36 (34%) patients, respectively. Severe HLC immunoparesis was identified in 40 (38%) patients. Of the IgG subclasses, IgG1 and IgG2 were most frequently suppressed, affecting 46 (44%) and 36 (34%) patients, respectively; 63 (60%) patients had low levels of at least one IgG subclass. In multivariate analysis, severe HLC immunoparesis (hazard ratio [HR]: 36.5; P = .010) and ΣFLC ≥ 70 mg/L (HR: 13.2; P = .004) were the only factors independently associated with time to first treatment (TTFT). A risk model including these variables identified patients with 0, 1, and 2 risk factors and significantly different TTFT (P < .001). Patients with two factors represented an ultra-high-risk group with a median TTFT of only 1.3 months. CONCLUSION: The above findings demonstrate the potential for the use of HLC immunoparesis, together with sFLC measurements, as future prognostic biomarkers in CLL.

Conceptual Approaches to Modulating Antibody Effector Functions and Circulation Half-Life.

Antibodies and Fc-fusion antibody-like proteins have become successful biologics developed for cancer treatment, passive immunity against infection, addiction, and autoimmune diseases. In general these biopharmaceuticals can be used for blocking protein:protein interactions, crosslinking host receptors to induce signaling, recruiting effector cells to targets, and fixing complement. With the vast capability of antibodies to affect infectious and genetic diseases much effort has been placed on improving and tailoring antibodies for specific functions. While antibody:antigen engagement is critical for an efficacious antibody biologic, equally as important are the hinge and constant domains of the heavy chain. It is the hinge and constant domains of the antibody that engage host receptors or complement protein to mediate a myriad of effector functions and regulate antibody circulation. Molecular and structural studies have provided insight into how the hinge and constant domains from antibodies across different species, isotypes, subclasses, and alleles are recognized by host cell receptors and complement protein C1q. The molecular details of these interactions have led to manipulation of the sequences and glycosylation of hinge and constant domains to enhance or reduce antibody effector functions and circulating half-life. This review will describe the concepts being applied to optimize the hinge and crystallizable fragment of antibodies, and it will detail how these interactions can be tuned up or down to mediate a biological function that confers a desired disease outcome.

Heavy/light chain assay in the monitoring of multiple myeloma.

Serum protein (SPE) and immunofixation electrophoresis (IFE) have been extensively validated for the routine use of identifying, characterising and quantifying monoclonal proteins. However, accurate quantitation of IgA monoclonal proteins can be difficult when they migrate in to the ß fraction, due to co-migration with transferrin and complement components. The heavy/light chain (HLC) immunoassay is an additional tool for measuring intact immunoglobulin monoclonal proteins. Therefore, we aimed to examine the clinical utility of the HLC assay for the disease monitoring of IgG and IgA multiple myeloma (MM) patients. A total of 177 samples from 30 MM patients (21 IgG and 9 IgA) were analysed retrospectively with median number of six follow up samples per patient (range 3-13). Serum free light chains (sFLC) and HLC were quantified using Freelite and Hevylite immunoassays. Details of M-protein concentration, ß-globulin levels, total immunoglobulin levels and disease treatment response were obtained from the laboratory and patient information system. Passing-Bablok regression analysis was performed to compare (i) M-protein quantification with involved HLC (iHLC) and (ii) total immunoglobulin with summated HLC pairs for each immunoglobulin type (e.g., IgGκ+IgGλ). For 127 IgG MM samples, IgG iHLC levels showed a good correlation with SPE quantification (iHLC y=0.96x+4.9; r=0.917) and summated HLC showed a good correlation with total IgG concentration (summated HLC y=0.94x+5.74; r=0.91). In total, 95/127 (75%) IgG MM follow-up samples had an abnormal HLC ratio and 122/127 (96%) had a positive SPE, probably due to the lower sensitivity of HLC assay in detecting clonality in patients with IgG MM. Consistent with this, one patient assigned a very good partial response by International Myeloma Working Group criteria would be assigned a complete response based on HLC measurements. For 50 IgA MM samples, 42/50 (84%) had an abnormal HLC ratio. Conversely, 50/50 (100%) of M-proteins showed ß fraction migration and were difficult to accurately quantify by SPE. Therefore, M-protein concentration and iHLC did not correlate as well in IgA MM (y=1.9x-8.4; r=0.8) compared to IgG MM. However, there was good correlation between total IgA and summated IgA HLC (IgAκ+IgAλ y=1.35x-0.33; r=0.95). Of the 8/50 (16%) IgA samples with a normal HLC ratio, 6/8 (75%) were consistent with the disease status being in complete remission. Interestingly, in one IgA MM patient, SPE and IFE were negative, but the serum FLC ratio and involved FLC were highly abnormal, consistent with the presence of light chain escape. Our data suggest HLC measurements could add value to the current disease monitoring of MM patients. In IgG MM patients, the M-protein level correlated well with HLC values. The HLC assay complements the serum FLC assay and is especially useful for monitoring of IgA MM patients who display M-proteins migrating in the ß region on SPE.

Serum Exosomes from Newborn Piglets Restrict Porcine Epidemic Diarrhea Virus Infection.

Exosomes are vehicles in the body fluid that participate in many biological processes, especially immune responses. In this study, we employed comparative proteome analysis to investigate the roles of serum exosomes during viral infection in neonates using porcine epidemic diarrhea virus (PEDV), a devastating enteric virus in newborn piglets, as a model virus. Serum exosomes were first isolated from newborn piglets infected with PEDV or mock-infected newborn piglets, followed by label-free LC-MS/MS-based comparative quantitative proteomic analysis. Among the 441 detected proteins, 10 complement proteins were found in the serum exosomes, and significantly decreased expression levels of the C3, C6, and CFB complements were measured in PEDV-infected serum exosomes compared to those in mock-infected serum exosomes. After confirmation by Western blot, we then investigated the function of these exosomes in PEDV infection and discovered that exosomes from mock-infected newborn piglets restricted PEDV infection. However, this inhibition disappeared after the exosomes were heat-inactivated, suggesting that complements are key antiviral molecules. Our findings improve the understanding of antiviral responses mediated by exosomes in neonatal piglets and facilitate the discovery of novel antiviral drugs.

Multi-isotype Glycoproteomic Characterization of Serum Antibody Heavy Chains Reveals Isotype- and Subclass-Specific N-Glycosylation Profiles.

Antibodies are critical glycoproteins that bridge the innate and adaptive immune systems to provide protection against infection. The isotype/subclass of the antibody, the co-translational N-glycosylation on the CH2 domain, and the remodeling of the N-linked glycans during passage through the ER and Golgi are the known variables within the Fc domain that program antibody effector function. Through investigations of monoclonal therapeutics, it has been observed that addition or removal of specific monosaccharide residues from antibody N-glycans can influence the potency of antibodies, highlighting the importance of thoroughly characterizing antibody N-glycosylation. Although IgGs usually have a single N-glycosylation site and are well studied, other antibody isotypes, e.g. IgA and IgM, that are the first responders in certain diseases, have two to five sites/monomer of antibody, and little is known about their N-glycosylation. Here we employ a nLC-MS/MS method using stepped-energy higher energy collisional dissociation to characterize the N-glycan repertoire and site occupancy of circulating serum antibodies. We simultaneously determined the site-specific N-linked glycan repertoire for IgG1, IgG4, IgA1, IgA2, and IgM in individual healthy donors. Compared with IgG1, IgG4 displayed a higher relative abundance of G1S1F and a lower relative abundance of G1FB. IgA1 and IgA2 displayed mostly biantennary N-glycans. IgA2 variants with the either serine (S93) or proline (P93) were detected. In digests of the sera from a subset of donors, we detected an unmodified peptide containing a proline residue at position 93; this substitution would strongly disfavor N-glycosylation at N92. IgM sites N46, N209, and N272 displayed mostly complex glycans, whereas sites N279 and N439 displayed higher relative abundances of high-mannose glycoforms. This multi-isotype approach is a crucial step toward developing a platform to define disease-specific N-glycan signatures for different isotypes to help tune antibodies to induce protection. Data are available via ProteomeXchange with identifier PXD010911.