Background-free Detection of Mouse Antibodies on Mouse Tissue by Anti-isotype Secondary Antibodies.

Immunodetection on mouse routinely processed tissue via antibodies raised in mice faces cross-reactivity of the secondary anti-mouse reagents with endogenous immunoglobulins, which permeate the tissue. Various solutions to this problem have been devised and include endogenous Ig block with anti-mouse Fab fragments or directly conjugated primary antibodies. Mouse isotype-specific antibodies, differently from reagents directed against both heavy and light chains, fail to detect endogenous Ig after fixation and embedding, while providing a clean and specific detection system for mouse antibodies on mouse routinely processed tissue.

Functional Homology for Antibody-Dependent Phagocytosis Across Humans and Rhesus Macaques.

Analyses of human clinical HIV-1 vaccine trials and preclinical vaccine studies performed in rhesus macaque (RM) models have identified associations between non-neutralizing Fc Receptor (FcR)-dependent antibody effector functions and reduced risk of infection. Specifically, antibody-dependent phagocytosis (ADP) has emerged as a common correlate of reduced infection risk in multiple RM studies and the human HVTN505 trial. This recurrent finding suggests that antibody responses with the capability to mediate ADP are most likely a desirable component of vaccine responses aimed at protecting against HIV-1 acquisition. As use of RM models is essential for development of the next generation of candidate HIV-1 vaccines, there is a need to determine how effectively ADP activity observed in RMs translates to activity in humans. In this study we compared ADP activity of human and RM monocytes and polymorphonuclear leukocytes (PMN) to bridge this gap in knowledge. We observed considerable variability in the magnitude of monocyte and PMN ADP activity across individual humans and RM that was not dependent on FcR alleles, and only modestly impacted by cell-surface levels of FcRs. Importantly, we found that for both human and RM phagocytes, ADP activity of antibodies targeting the CD4 binding site was greatest when mediated by human IgG3, followed by RM and human IgG1. These results demonstrate that there is functional homology between antibody and FcRs from these two species for ADP. We also used novel RM IgG1 monoclonal antibodies engineered with elongated hinge regions to show that hinge elongation augments RM ADP activity. The RM IgGs with engineered hinge regions can achieve ADP activity comparable to that observed with human IgG3. These novel modified antibodies will have utility in passive immunization studies aimed at defining the role of IgG3 and ADP in protection from virus challenge or control of disease in RM models. Our results contribute to a better translation of human and macaque antibody and FcR biology, and may help to improve testing accuracy and evaluations of future active and passive prevention strategies.

An expanded genetic code facilitates antibody chemical conjugation involving the lambda light chain.

Most of the currently approved therapeutic antibodies are of the immunoglobulin gamma (IgG) κ isotype, leaving a vast opportunity for the use of IgGλ in medical treatments. The incorporation of designer amino acids into antibodies enables efficient and precise manufacturing of antibody chemical conjugates. Useful conjugation sites have been explored in the constant domain of the human κ-light chain (LCκ), which is no more than 38% identical to its LCλ counterpart in amino acid sequence. In the present study, we used an expanded genetic code for site-specifically incorporating Nε-(o-azidobenzyloxycarbonyl)-l-lysine (o-Az-Z-Lys) into the antigen-binding fragment (Fab) of an IgGλ, cixutumumab. Ten sites in the LCλ constant domain were found to support efficient chemical conjugation exploiting the bio-orthogonal azido chemistry. Most of the identified positions are located in regions that differ between the two light chain isotypes, thus being specific to the λ isotype. Finally, o-Az-Z-Lys was incorporated into the Fab fragments of cixutumumab and trastuzumab to chemically combine them; the resulting bispecific Fab-dimers showed a strong antagonistic activity against a cancer cell line. The present results expand the utility of the chemical conjugation method to the whole spectrum of humanized antibodies, including the λ isotype.

Immunoglobulins with Non-Canonical Functions in Inflammatory and Autoimmune Disease States.

Immunoglobulins are known to combine various effector mechanisms of the adaptive and the innate immune system. Classical immunoglobulin functions are associated with antigen recognition and the initiation of innate immune responses. However, in addition to classical functions, antibodies exhibit a variety of non-canonical functions related to the destruction of various pathogens due to catalytic activity and cofactor effects, the action of antibodies as agonists/antagonists of various receptors, the control of bacterial diversity of the intestine, etc. Canonical and non-canonical functions reflect the extreme human antibody repertoire and the variety of antibody types generated in the organism: antigen-specific, natural, polyreactive, broadly neutralizing, homophilic, bispecific and catalytic. The therapeutic effects of intravenous immunoglobulins (IVIg) are associated with both the canonical and non-canonical functions of antibodies. In this review, catalytic antibodies will be considered in more detail, since their formation is associated with inflammatory and autoimmune diseases. We will systematically summarize the diversity of catalytic antibodies in normal and pathological conditions. Translational perspectives of knowledge about natural antibodies for IVIg therapy will be also discussed.

An international multi-center serum protein electrophoresis accuracy and M-protein isotyping study. Part II: limit of detection and follow-up of patients with small M-proteins.

Background Electrophoretic methods to detect, characterize and quantify M-proteins play an important role in the management of patients with monoclonal gammopathies (MGs). Significant uncertainty in the quantification and limit of detection (LOD) is documented when M-proteins are <10 g/L. Using spiked sera, we aimed to assess the variability in intact M-protein quantification and LOD across 16 laboratories. Methods Sera with normal, hypo- or hyper-gammaglobulinemia were spiked with daratumumab or elotuzumab, with concentrations from 0.125 to 10 g/L (n = 62) along with a beta-migrating sample (n = 9). Laboratories blindly analyzed samples according to their serum protein electrophoresis (SPEP)/isotyping standard operating procedures. LOD and intra-laboratory percent coefficient of variation (%CV) were calculated and further specified with regard to the method (gel/capillary electrophoresis [CZE]), gating strategy (perpendicular drop [PD]/tangent skimming [TS]), isotyping (immunofixation/immunosubtraction [ISUB]) and manufacturer (Helena/Sebia). Results All M-proteins ≥1 g/L were detected by SPEP. With isotyping the LOD was moderately more sensitive than with SPEP. The intensity of polyclonal background had the biggest negative impact on LOD. Independent of the method used, the intra-laboratory imprecision of M-protein quantification was small (mean CV = 5.0%). Low M-protein concentration and high polyclonal background had the strongest negative impact on intra-laboratory precision. All laboratories were able to follow trend of M-protein concentrations down to 1 g/L. Conclusions In this study, we describe a large variation in the reported LOD for both SPEP and isotyping; overall LOD is most affected by the polyclonal immunoglobulin background. Satisfactory intra-laboratory precision was demonstrated. This indicates that the quantification of small M-proteins to monitor patients over time is appropriate, when subsequent testing is performed within the same laboratory.

An international multi-center serum protein electrophoresis accuracy and M-protein isotyping study. Part I: factors impacting limit of quantitation of serum protein electrophoresis.

Background Serum protein electrophoresis (SPEP) is used to quantify the serum monoclonal component or M-protein, for diagnosis and monitoring of monoclonal gammopathies. Significant imprecision and inaccuracy pose challenges in reporting small M-proteins. Using therapeutic monoclonal antibody-spiked sera and a pooled beta-migrating M-protein, we aimed to assess SPEP limitations and variability across 16 laboratories in three continents. Methods Sera with normal, hypo- or hypergammaglobulinemia were spiked with daratumumab, Dara (cathodal migrating), or elotuzumab, Elo (central-gamma migrating), with concentrations from 0.125 to 10 g/L (n = 62) along with a beta-migrating sample (n = 9). Provided with total protein (reverse biuret, Siemens), laboratories blindly analyzed samples according to their SPEP and immunofixation (IFE) or immunosubtraction (ISUB) standard operating procedures. Sixteen laboratories reported the perpendicular drop (PD) method of gating the M-protein, while 10 used tangent skimming (TS). A mean percent recovery range of 80%-120% was set as acceptable. The inter-laboratory %CV was calculated. Results Gamma globulin background, migration pattern and concentration all affect the precision and accuracy of quantifying M-proteins by SPEP. As the background increases, imprecision increases and accuracy decreases leading to overestimation of M-protein quantitation especially evident in hypergamma samples, and more prominent with PD. Cathodal migrating M-proteins were associated with less imprecision and higher accuracy compared to central-gamma migrating M-proteins, which is attributed to the increased gamma background contribution in M-proteins migrating in the middle of the gamma fraction. There is greater imprecision and loss of accuracy at lower M-protein concentrations. Conclusions This study suggests that quantifying exceedingly low concentrations of M-proteins, although possible, may not yield adequate accuracy and precision between laboratories.

Defining the right diluent for intravenous infusion of therapeutic antibodies.

Therapeutic monoclonal antibodies (mAbs) are commonly administered to patients through intravenous (IV) infusion, which involves diluting the medication into an infusion solution (e.g., saline and 5% dextrose). Using the wrong diluent can cause product aggregation, which may compromise patient safety. We and others have shown that Herceptin® (trastuzumab) and Avastin® (bevacizumab) undergo rapid aggregation upon mixing with dextrose and human plasma in vitro. In this study, we evaluated the compatibility of a panel of 11 therapeutic mAbs with dextrose or saline and human serum. These mAbs were randomly selected for their distinct formulations and IgG isotypes (IgG1, IgG2, IgG4, and Fc-fusion protein). All the mAbs appeared to be compatible with saline and human serum. However, mAbs that were formulated at acidic pH (≤ 6.5) exclusively formed insoluble aggregates upon mixing with dextrose and serum. Such aggregation was not detected for the mAbs that are at neutral pH (7.2-7.5) or in buffers containing sodium chloride. Mass spectrometric analysis revealed that the insoluble aggregates were composed of mAb molecules and several serum proteins (e.g., complement proteins, apolipoprotein, fibronectin) that are characterized by an isoelectric point of pH 5.4-6.7. At proximate pH to the isoelectric point values, those abundant serum proteins appeared to undergo isoelectric precipitation with mAb molecules. Our observations highlight a potential risk of protein aggregation at the blood-IV interface if a diluent is incompatible with a specific mAb formulation. This information has implications in guiding the design of product formulations and the selection of the right diluent for intravenous infusion of therapeutic mAbs.Abbreviations: ADC: antibody-drug conjugate; D5W: 5% dextrose in water; IM: intramuscular; IV: intravenous; LC-MS/MS: liquid chromatography-tandem mass spectrometry; mAb: monoclonal antibody; SC: subcutaneous; pI: isoelectric point.

An Algorithm for Building Multi-State Classifiers Based on Collision-Induced Unfolding Data.

Collision-induced unfolding (CIU) has emerged as a valuable method for distinguishing iso-cross-sectional protein ions through their distinct gas-phase unfolding trajectories. CIU shows promise as a high-throughput, structure-sensitive screening technique with potential applications in drug discovery and biotherapeutic characterization. We recently developed a CIU classification workflow to support screening applications that utilized CIU data acquired from a single protein charge state to distinguish immunoglobulin (IgG) subtypes and membrane protein lipid binding. However, distinguishing highly similar protein structures, such as those associated with biotherapeutics, can be challenging. Here, we present an expansion of this classification method that includes CIU data from multiple charge states, or indeed any perturbation to protein structure that differentially affects CIU, into a combined classifier. Using this improved method, we are able to improve the accuracy of existing, single-state classifiers for IgG subtypes and develop an activation-state-sensitive classifier for selected Src kinase inhibitors when data from a single charge state was insufficient to do so. Finally, we employ the combination of multiple charge states and stress conditions to distinguish a highly similar innovator/biosimilar biotherapeutic pair, demonstrating the potential of CIU as a rapid screening tool for drug discovery and biotherapeutic analysis.

From Rhesus macaque to human: structural evolutionary pathways for immunoglobulin G subclasses.

The Old World monkey, Rhesus macaque (Macaca mulatta, Mm), is frequently used as a primate model organism in the study of human disease and to test new vaccines/antibody treatments despite diverging before chimpanzees and orangutans. Mm and humans share 93% genome identity with substantial differences in the genes of the adaptive immune system that lead to different functional IgG subclass characteristics, Fcγ receptors expressed on innate immune cells, and biological interactions. These differences put limitations on Mm use as a primary animal model in the study of human disease and to test new vaccines/antibody treatments. Here, we comprehensively analyzed molecular properties of the Fc domain of the four IgG subclasses of Rhesus macaque to describe potential mechanisms for their interactions with effector cell Fc receptors. Our studies revealed less diversity in the overall structure among the Mm IgG Fc, with MmIgG1 Fc being the most structurally like human IgG3, although its CH2 loops and N297 glycan mobility are comparable to human IgG1. Furthermore, the Fcs of Mm IgG3 and 4 lack the structural properties typical for their human orthologues that determine IgG3's reduced interaction with the neonatal receptor and IgG4's ability for Fab-arm exchange and its weaker Fcγ receptor interactions. Taken together, our data indicate that MmIgG1-4 are less structurally divergent than the human IgGs, with only MmIgG1 matching the molecular properties of human IgG1 and 3, the most active IgGs in terms of Fcγ receptor binding and Fc-mediated functions. PDB accession numbers for deposited structures are 6D4E, 6D4I, 6D4M, and 6D4N for MmIgG1 Fc, MmIgG2 Fc, MmIgG3 Fc, and MmIgG4 Fc, respectively.

An Inflammatory Story: Antibodies in Tuberculosis Comorbidities.

Mycobacterium tuberculosis (Mtb) resides in a quarter of the world's population and is the causative agent for tuberculosis (TB), the most common infectious reason of death in humans today. Although cellular immunity has been firmly established in the control of Mtb, there is growing evidence that antibodies may also modulate the infection. More specifically, certain antibody features are associated with inflammation and are divergent in different states of human infection and disease. Importantly, TB impacts not just the healthy but also those with chronic conditions. While HIV represents the quintessential comorbid condition for TB, recent epidemiological evidence shows that additional chronic conditions such as diabetes and kidney disease are rising. In fact, the prevalence of diabetes as a comorbid TB condition is now higher than that of HIV. These chronic diseases are themselves independently associated with pro-inflammatory immune states that encompass antibody profiles. This review discusses isotypes, subclasses, post-translational modifications and Fc-mediated functions of antibodies in TB infection and in the comorbid chronic conditions of HIV, diabetes, and kidney diseases. We propose that inflammatory antibody profiles, which are a marker of active TB, may be an important biomarker for detection of TB disease progression within comorbid individuals. We highlight the need for future studies to determine which inflammatory antibody profiles are the consequences of comorbidities and which may potentially contribute to TB reactivation.

Isotype dependent on-column non-reversible aggregation of monoclonal antibodies.

Monoclonal antibodies of the IgG2 and IgG4 isotype were found to exhibit an increased propensity for displaying two-peak elution profiles during cation exchange chromatography. In some cases, this two-peak elution profile also resulted in the formation of non-reversible mAb aggregates. Comparison of IgG1, IgG2, and IgG4 molecules with the same variable region reveals that the two-peak behaviour is predominantly mediated by the constant region and most likely the lower CH1, hinge and upper CH2 regions of the mAb. Furthermore, comparison of the behaviour of two different IgG4 molecules, reveals that the degree of non-reversible aggregate formation, whilst facilitated by the isotype format, is mediated primarily by the variable region of the molecule. As well as the properties of the mAb molecule itself, the chemistry and structure of the cation exchange resin was also found to have an effect, with the two-peak elution profile being more pronounced with polymer-grafted resins such as Capto S Impact and Eshmuno CPX. These results combined support the theory that binding of IgG2 and IgG4 mAbs to cation exchange resins usually occurs through at least two mechanisms mediated by the structural features of the constant region of IgG2s and IgG4s. One of these mechanisms is not only stronger than the other, but also can lead to a conformational change in the molecule. This conformational change can occur in both variable and constant domains of the antibody. This transitory unfolded state can in turn lead to non-reversible aggregation of some mAb molecules.

Generation of assay positive controls for detection of isotype anti-drug antibodies for immunogenicity monitoring.

AIM: Development of drug-related surrogate positive controls for isotype anti-drug antibodies assay remains challenging. Efforts on antibody engineering or chemical crosslinking have been made. However, multiple epitope recognition, purity and stability are often of concern. To tackle these challenges, we used LC-SPDP/SMPB crosslinking to conjugate polyclonal anti-drug IgG and human immunoglobulin isotype (hIgE, hIgA, hIgM or hIgG4) with optimized conditions. RESULTS: The final product was a hybrid of anti-drug IgG cross-linked to human isotype immunoglobulin through stable thioether bond. The characteristics of the hybrids and their performance as assay positive controls were further evaluated. CONCLUSION: The results demonstrate that LC-SPDP/SMPB chemical crosslinking method is suitable for generating stable hybrids to be used as assay positive controls in isotype anti-drug antibodies assay.

Molecular cloning, characterization and expression of immunoglobulin D on pathogen challenge and pathogen associated molecular patterns stimulation in freshwater carp, Catla catla.

The primordial immunoglobulin class, IgD, was the first non-IgM isotype discovered in teleosts. The crucial roles of IgM and IgZ in imparting systemic and mucosal immunity, respectively, in various fish species have been widely established. However, the putative function of a unique IgD isotype during pathogenic invasions has not been well explored. The present study reports the existence of an IgD ortholog in freshwater carp, Catla catla, and further evaluates its differential expression profile in response to bacterial, parasitic and viral antigenic exposure and pathogen associated molecular patterns (PAMPs) stimulation. The IgD of C. catla (CcIgD) cDNA sequence was found to encode 226 amino acids and confirmed homology with heavy chain delta region of Cyprinidae family members. Phylogenetic analysis of CcIgD exhibited greatest similarity with Ctenopharyngodon idella. qRT-PCR analysis revealed significant upregulation (P < 0.001) of IgD gene expression in kidney with respect to other tissues at 24 hr post-Aeromonas hydrophila challenge. CcIgD gene expression in skin was enhanced following Streptococcus uberis infection and in blood following Argulus infection and inactivated rhabdoviral antigen stimulation. Further, the treatment of bacterial and viral products (PAMPs) also triggered significant (P < 0.05) increases in CcIgD mRNA expression in kidney. These findings indicate the functional importance of teleost IgD in orchestrating tissue specific neutralization of antigens on stimulation with different pathogens and PAMPs.

Exploring Site-Specific N-Glycosylation of HEK293 and Plant-Produced Human IgA Isotypes.

The full potential of recombinant Immunoglobulin A as therapeutic antibody is not fully explored, owing to the fact that structure-function relationships of these extensively glycosylated proteins are not well understood. Here monomeric IgA1, IgA2m(1), and IgA2m(2) variants of the anti-HER2 antibody (IgG1) trastuzumab were expressed in glyco-engineered Nicotiana benthamiana plants and in human HEK293-6E cells. All three IgA isotypes were purified and subjected to biophysical and biochemical characterization. While no differences in assembly, antigen binding, and glycosylation occupancy were observed, both systems vary tremendously in terms of glycan structures and heterogeneity of glycosylation. Mass-spectrometric analysis of site-specific glycosylation revealed that plant-produced IgAs carry mainly complex-type biantennary N-glycans. HEK293-6E-produced IgAs, on the contrary, showed very heterogeneous N-glycans with high levels of sialylation, core-fucose, and the presence of branched structures. The site-specific analysis revealed major differences between the individual N-glycosylation sites of each IgA subtype. Moreover, the proline-rich hinge region from HEK293-6E cell-derived IgA1 was occupied with mucin-type O-glycans, whereas IgA1 from N. benthamiana displayed numerous plant-specific modifications. Interestingly, a shift in unfolding of the CH2 domain of plant-produced IgA toward lower temperatures can be observed with differential scanning calorimetry, suggesting that distinct glycoforms affect the thermal stability of IgAs.

The Use of Multiple Reaction Monitoring on QQQ-MS for the Analysis of Protein- and Site-Specific Glycosylation Patterns in Serum.

In recent years, high-throughput glycomics approaches have been developed and applied to either complete biofluids, cell lysates or tissues, or proteins isolated thereof. However, during such analyses the N-glycan are released from the protein backbone and therefore site- and protein-specific information is lost. There exists a need for high-throughput methods that allow quantification of site- and protein-specific glycosylation patterns from complex biological mixtures. We here describe the use of a multiple reaction monitoring mass spectrometry based method for the generation of glycopeptide profiles of the nine high abundance glycoproteins IgG, IgA, IgM, haptoglobin, alpha-1-antitrypsin, alpha-2-macroglobulin, alpha-1-acid glycoprotein, transferrin, and complement C3. We show that the sample preparation can be performed at the 96-well level, and using a 17-min gradient on a RP-UPLC-QQQ instrument, 96 samples can be analyzed within 3 days.

Revisiting the structural basis and energetic landscape of susceptibility difference between HLA isotypes to allergic rhinitis.

The human leukocyte antigen class II (HLA II) molecules are implicated in the immunopathogenesis of allergic rhinitis (AR). The HLA II contains three allelic isotypes HLA-DR, -DQ, and -QP that exhibit considerably different susceptibility to AR. Here, we investigated the structural basis and energetic landscape of the susceptibility difference between the three HLA II isotypes to AR by combining computational analysis and experimental assay. Multiple sequence alignment revealed a low conservation among the three subtypes with sequence identity of ∼10% between them, suggesting that the peptide repertoires presented by HLA-DR, -DP and -DQ are not overlapped to each other, and they may be involved in different immune functions and dysfunctions. Structural analysis imparted that the antigenic peptides are rooted on the peptide-binding groove of HLA molecules and hold in a PPII-like helical conformation. Subsequently, the interaction behavior of 17 AR allergen-derived peptides with HLA-DR, -DP and -DQ was investigated using a statistics-based quantitative structure-activity relationship (QSAR) predictor. It was found a significant difference between the binding capabilities of these antigenic peptides to HLA-DR and to HLA-DP/-DQ; the former showed a generally higher affinity than the latter with p-value of 0.02 obtained from 2-tailed Student's t-test. The computational findings were then confirmed by HLA II-peptide stability assay, which demonstrated that the AR allergen-derived peptides have a high in vitro selectivity for HLA-DR over HLA-DP/-DQ. Thus, the HLA-DR isotype, rather than HLA-DP and -DQ, is expected to associate with the pathological process of AR.

Acid-induced aggregation propensity of nivolumab is dependent on the Fc.

Nivolumab, an anti-programmed death (PD)1 IgG4 antibody, has shown notable success as a cancer treatment. Here, we report that nivolumab was susceptible to aggregation during manufacturing, particularly in routine purification steps. Our experimental results showed that exposure to low pH caused aggregation of nivolumab, and the Fc was primarily responsible for an acid-induced unfolding phenomenon. To compare the intrinsic propensity of acid-induced aggregation for other IgGs subclasses, tocilizumab (IgG1), panitumumab (IgG2) and atezolizumab (aglyco-IgG1) were also investigated. The accurate pH threshold of acid-induced aggregation for individual IgG Fc subclasses was identified and ranked as: IgG1 < aglyco-IgG1 < IgG2 < IgG4. This result was cross-validated by thermostability and conformation analysis. We also assessed the effect of several protein stabilizers on nivolumab, and found mannitol ameliorated the acid-induced aggregation of the molecule. Our results provide valuable insight into downstream manufacturing process development, especially for immune checkpoint modulating molecules with a human IgG4 backbone.

Molecular cloning of IgZ heavy chain isotype in Catla catla and comparative expression profile of IgZ and IgM following pathogenic infection.

Immunoglobulins serve as a crucial arm of the adaptive immune system against detrimental pathogenic threats in teleosts. However, whether the novel Ig isotype IgZ is present in the Indian major carp, Catla catla, has not yet been elucidated. The present study reports the presence of IgZ ortholog in C. catla (CcIgZ) and further demonstrates its comparative tissue specific expression with IgM (CcIgM) in response to bacterial and parasitic stimulation. The putative 139 amino acid sequence of IgZ heavy chain cDNA of C. catla showed homology with IgZ constant domains of other teleosts. Phylogenetic analysis of the predicted IgZ transcript sequence clustered with previously identified IgZ heavy chain sequences of Cyprinidae family members. The inductive expression profiles of IgZ and IgM genes were evaluated in immunologically relevant tissues at 24, 48 and 72 hr post infection with Aeromonas hydrophila, Streptococcus uberis and Argulus sp. Both CcIgZ and CcIgM were expressed most strongly in the kidneys of healthy fish. Basal expression of CcIgM transcript was higher than that of CcIgZ in all the examined tissues. Stimulation with bacteria triggered significant increase of IgZ in the intestine (P < 0.001) and spleen (P < 0.01), whereas IgM was relatively up-regulated in blood (P < 0.001) after stimulation with each of the three pathogens assessed. The study is the first to report identification of IgZ in C. catla. Further, it provides insights into the differential expression profiles of IgZ and IgM isotypes against various pathogenic infection in C. catla, which may facilitate better prophylaxis again such infections.

Isotype-specific glycosylation analysis of mouse IgG by LC-MS.

With mice being the top model organism in immunology and with Fc glycosylation being increasingly recognized as important modulator of antibody function, the time has come to take a look at the glycosylation of mouse IgG isotypes. Tryptic glycopeptides of mouse IgG1, IgG2, and IgG3 differ in mass and so these three isoforms can be easily discriminated by MS. Commercial IgG contained a rare IgG1 variant but no IgG3, which, however, was found in sera of C57BL/6 and BALB/c mice. These strains deviated with regard to IgG2a and IgG2b alleles. The Ig2a B allele was not observed in any of the four samples investigated. All a/c isotypes contain the same glycopeptide sequence, which deviates from that of IgG2b by containing Leu instead of Ile. The Leu/Ile glycopeptide variants were separated by RP chromatography and the order of elution was determined. The major glycoforms on all isotypes were fucosylated with no and one galactose (GnGnF and GnAF) followed by fully galactosylated AAF and smaller amounts of mono- and disialylated N-glycans. In the commercial serum pool, the relative ratios of glycans differed between isotypes. Sialic acid exclusively occurred as N-glycolylneuraminic acid. Fucosylation was essentially complete. No bisected and no α1,3-galactosylated glycans were found.