On-chip complement activation adds an extra dimension to antigen microarrays

Antibody profiling on antigen microarrays helps us in understanding the complexity of responses of the adaptive immune system. The technique, however, neglects another, evolutionarily more ancient apparatus, the complement system, which is capable of both recognizing and eliminating antigen and serves to provide innate defense for the organism while cooperating with antibodies on multiple levels. Complement components interact with both foreign substances and self molecules, including antibodies, and initiate a cascade of proteolytic cleavages that lead to the covalent attachment of complement components to molecules in nanometer proximity. By refining the conditions of antibody profiling on antigen arrays we made use of this molecular tagging to identify antigens that activate the complement system. Antigen arrays were incubated with serum under conditions that favor complement activation, and the deposited complement C3 fragments were detected by fluorescently labeled antibodies. We used genetically C3-deficient mice or inhibition of the complement cascade to prove that the technique requires complement activation for the binding of C3 to features of the array. We demonstrate that antigens on the array can initiate complement activation both by antibody-dependent or -independent ways. Using two-color detection, antibody and complement binding to the relevant spots was measured simultaneously. The effect of adjuvants on the quality of the immune response and binding of autoantibodies to DNA with concomitant complement activation in the serum of mice suffering from systemic autoimmune disease was readily measurable by this new method. We propose that measurement of complement deposition on antigen microarrays supplements information from antibody binding measurements and provides an extra, immune function-related fingerprint of the tested serum.     

Immune Complex Signatures of Patients with Active and Inactive SLE Revealed by Multiplex Protein Binding Analysis on Antigen Microarrays

Systemic lupus erythematosus is characterized by dysfunctional clearance of apoptotic debris and the development of pathogenic autoantibodies. While the complement system is also involved in the disease no attempt has been made to generate a comprehensive view of immune complex formation from various autoantigens. We increased the complexity of autoantibody profiles by measuring the binding of two complement proteins, C3 and C4, in addition to two antibody classes, IgG and IgM, to a collection of autoantigens. These complement components covalently bind to those microarray features where antibodies and other serum components induce complement activation. Using this technology, we compared functional serum antibody profiles of control subjects (n = 31) and patients with lupus erythematosus (n = 61) in the active (n = 22) and inactive (n = 39) phase of the disease. Multivariate analysis was applied to identify contributions of binding data on 25 antigens to the discrimination of the study groups. Receiver operating characteristic analysis was used to portray the discriminative property of each measured parameter for each antigen in pairwise group comparisons. Complement C3 and C4 deposition increased on autoantibody targets in spite of the decreased serum complement concentrations, and decreased on other autoantigens, demonstrating the imbalance of complement function in patients with lupus erythematosus. Our observations confirmed previously known markers of disease and showed that C3 and C4 deposition data were at least as powerful as Ig binding data in separating the study groups.     

Tensor‐structured decomposition improves systems serology analysis

Systems serology provides a broad view of humoral immunity by profiling both the antigen‐binding and Fc properties of antibodies. These studies contain structured biophysical profiling across disease‐relevant antigen targets, alongside additional measurements made for single antigens or in an antigen‐generic manner. Identifying patterns in these measurements helps guide vaccine and therapeutic antibody development, improve our understanding of diseases, and discover conserved regulatory mechanisms. Here, we report that coupled matrix–tensor factorization (CMTF) can reduce these data into consistent patterns by recognizing the intrinsic structure of these data. We use measurements from two previous studies of HIV‐ and SARS‐CoV‐2‐infected subjects as examples. CMTF outperforms standard methods like principal components analysis in the extent of data reduction while maintaining equivalent prediction of immune functional responses and disease status. Under CMTF, model interpretation improves through effective data reduction, separation of the Fc and antigen‐binding effects, and recognition of consistent patterns across individual measurements. Data reduction also helps make prediction models more replicable. Therefore, we propose that CMTF is an effective general strategy for data exploration in systems serology.     

A Novel Platform for the Potentiation of Therapeutic Antibodies Based on Antigen-Dependent Formation of IgG Hexamers at the Cell Surface

IgG antibodies can organize into ordered hexamers on cell surfaces after binding their antigen. These hexamers bind the first component of complement C1 inducing complement-dependent target cell killing. Here, we translated this natural concept into a novel technology platform (HexaBody technology) for therapeutic antibody potentiation. We identified mutations that enhanced hexamer formation and complement activation by IgG1 antibodies against a range of targets on cells from hematological and solid tumor indications. IgG1 backbones with preferred mutations E345K or E430G conveyed a strong ability to induce conditional complement-dependent cytotoxicity (CDC) of cell lines and chronic lymphocytic leukemia (CLL) patient tumor cells, while retaining regular pharmacokinetics and biopharmaceutical developability. Both mutations potently enhanced CDC- and antibody-dependent cellular cytotoxicity (ADCC) of a type II CD20 antibody that was ineffective in complement activation, while retaining its ability to induce apoptosis. The identified IgG1 Fc backbones provide a novel platform for the generation of therapeutics with enhanced effector functions that only become activated upon binding to target cell–expressed antigen.     

Antigen Binding to Secretory Immunoglobulin A Results in Decreased Sensitivity to Intestinal Proteases and Increased Binding to Cellular Fc Receptors

In intestinal secretions, secretory IgA (SIgA) plays an important sentinel and protective role in the recognition and clearance of enteric pathogens. In addition to serving as a first line of defense, SIgA and SIgA·antigen immune complexes are selectively transported across Peyer''s patches to underlying dendritic cells in the mucosa-associated lymphoid tissue, contributing to immune surveillance and immunomodulation. To explain the unexpected transport of immune complexes in face of the large excess of free SIgA in secretions, we postulated that SIgA experiences structural modifications upon antigen binding. To address this issue, we associated specific polymeric IgA and SIgA with antigens of various sizes and complexity (protein toxin, virus, bacterium). Compared with free antibody, we found modified sensitivity of the three antigens assayed after exposure to proteases from intestinal washes. Antigen binding further impacted on the immunoreactivity toward polyclonal antisera specific for the heavy and light chains of the antibody, as a function of the antigen size. These conformational changes promoted binding of the SIgA-based immune complex compared with the free antibody to cellular receptors (FcαRI and polymeric immunoglobulin receptor) expressed on the surface of premyelocytic and epithelial cell lines. These data reveal that antigen recognition by SIgA triggers structural changes that confer to the antibody enhanced receptor binding properties. This identifies immune complexes as particular structural entities integrating the presence of bound antigens and adds to the known function of immune exclusion and mucus anchoring by SIgA.     

Antibody-mediated cell cytotoxicity in a defined system: regulation by antigen, antibody, and complement.

The use of a serum-free environment and target cells carrying defined amounts of radiolabeled antigen allowed a quantitative study of the role of antigen, antibody, and complement on antibody-mediated cell cytotoxicity (AbMC). For lysis to occure, a minimum number of antigen molecules must be present on the target cell. 51Cr release from target cells with lower antigen density requires larger concentration of effector cells and antibodies. Target cell-bound complement, itself unable to mediate cytotoxicity, reduces the number of IgG molecules required for lysis. The antibody and complement, however, have to be bound to the same target cell. Bystander complement-coated erythrocytes, present in the same reaction mixture with IgG-coated targets, are not lysed. Blocking of AbMC is effected only by antigen, either soluble or in immune complexes prepared in antigen excess. Antigen competes at the level of the target cell. Blocking at the level of the effector cell, by use of immune complexes prepared at equivalence or in antibody excess, is difficult to achieve. The large number of cells with Fc receptors contained in mouse spleens may explain this finding. Arming of effector cells by passive binding of immune complexes is poorly effective as a means of obtaining lysis of the target cells. In all situations, the outcome of the reaction is determined by the presence of free antibody-combining sites, alone, or in immune complexes, that are able to combine with the target cell membrane antigen. The requirements for lysis are rather stringent.     

Inhibition of immune precipitation by complement

Normal human complement serum (NHS) inhibited precipitin reactions between tetanus toxoid and human or rabbit anti-tetanus toxoid IgG antibody, between bovine serum albumin (BSA) and rabbit anti-BSA IgG antibody, and between hen egg albumin and rabbit anti-egg albumin IgG antibody. Ethylene-diaminetetraacetic acid (EDTA) prevented this inhibition. Mg-ethyleneglycol-bis(aminoethyl)-tetra-acetic acid-(EGTA) also prevented the inhibition except with lower concentrations of antibody and antigen. Therefore, the inhibition of immune precipitation seemed to occur mainly through the classical pathway of complement activation. The alternative pathway was usually dispensable, but it augmented the inhibition. Guinea pig complement serum (NGS) was less effective than NHS in inhibiting immune precipitation. Guinea pig serum deficient in C4 (C4DGS) did not inhibit the immune precipitation. Mouse complement serum was effective for inhibiting precipitation, and C5-deficient serum was as effective as normal serum. Therefore, the inhibition of immune precipitation is considered to occur by activation of complement up to the step of C3. The size of the soluble immune complexes formed in the presence of NHS varied depending on the concentrations of antibody and antigen, even when the ratio of antigen to antibody was constant. On incubation at 37 degrees C immune precipitation was inhibited by 1/2 dilution of NHS for 2 to 3 hr and then gradually increased to the level in the absence of complement. When the immune complexes were formed in the presence of serum containing complement, fragments of C4 and C3 were incorporated into the soluble immune complexes. The C3 fragments incorporated into the soluble complexes were C3b, iC3b, C3c, and C3d, some of which were bound covalently with heavy chains of IgG antibody molecules. Some of the covalent linkages between C3 fragments and IgG seemed to be destroyed by alkali treatment, but not by hydroxylamine treatment. The formation of covalent bonds between IgG and C3 and probably C4 was essential for inhibition of immune precipitation, because inhibitors of their formation, such as putrescine, cadaverine, and salicylhydroxamic acid, effectively prevented the inhibition of precipitation. When antigen and antibody reacted in the presence of mixtures of various combinations of isolated complement components, C1, C4, C2, and C3 showed maximal inhibition of immune precipitation, whereas factors I and H had little effect.     

Immune deposits formed in situ by a monoclonal antibody recognizing a new intrinsic rat mesangial matrix antigen

We describe a unique mesangial matrix component of the rat glomerulus identified by a murine monoclonal antibody. The antigen is present exclusively in the glomerular mesangium and cannot be detected in other rat tissues by indirect immunofluorescence techniques or following pretreatment of tissue sections with acid urea or other nonionic detergents. Specific immunoprecipitation of the solubilized antigen yields a single peptide with an apparent m.w. of 81,000 when analyzed by discontinuous SDS-PAGE. This mesangial matrix component is collagenase resistant and trypsin sensitive. Perfusion of an isolated kidney preparation with this antibody results in direct binding of the mouse immunoglobulin to its mesangial antigen. Passive administration of the monoclonal antibody to Lewis rats results in characteristic electron dense deposits within the mesangial matrix that can be visualized ultrastructurally as early as 3 days. The immune deposits form without the activation of rat complement and persist for longer periods than those that develop after the planting of aggregated proteins or preformed immune complexes. Experimental animals that received either a monoclonal antibody specific for laminin or a non-kidney binding preparation did not develop such immune deposits at any time during the course of the autologous phase of the immune process. The results obtained in this study indicate that electron dense immune deposits can develop in the mesangium with the participation of a unique intrinsic matrix component and specific circulating monoclonal antibodies by an in situ mechanism of immune complex formation.     

High-throughput phosphotyrosine profiling using SH2 domains

Protein tyrosine phosphorylation controls many aspects of signaling in multicellular organisms. One of the major consequences of tyrosine phosphorylation is the creation of binding sites for proteins containing Src homology 2 (SH2) domains. To profile the global tyrosine phosphorylation state of the cell, we have developed proteomic binding assays encompassing nearly the full complement of human SH2 domains. Here we provide a global view of SH2 domain binding to cellular proteins based on large-scale far-western analyses. We also use reverse-phase protein arrays to generate comprehensive, quantitative SH2 binding profiles for phosphopeptides, recombinant proteins, and entire proteomes. As an example, we profiled the adhesion-dependent SH2 binding interactions in fibroblasts and identified specific focal adhesion complex proteins whose tyrosine phosphorylation and binding to SH2 domains are modulated by adhesion. These results demonstrate that high-throughput comprehensive SH2 profiling provides valuable mechanistic insights into tyrosine kinase signaling pathways.     

Complement-Activating IgM Enhances the Humoral but Not the T Cell Immune Response in Mice

IgM antibodies specific for a certain antigen can enhance antibody responses when administered together with this antigen, a process believed to require complement activation by IgM. However, recent data show that a knock-in mouse strain, Cμ13, which only produces IgM unable to activate complement, has normal antibody responses. Moreover, the recently discovered murine IgM Fc receptor (FcµR or TOSO/FAIM3) was shown to affect antibody responses. This prompted the re-investigation of whether complement activation by specific IgM is indeed required for enhancement of antibody responses and whether the mutation in Cµ13 IgM also caused impaired binding to FcµR. The results show that IgM from Cµ13 and wildtype mice bound equally well to the murine FcµR. In spite of this, specific Cμ13 IgM administered together with sheep red blood cells or keyhole limpet hemocyanine was a very poor enhancer of the antibody and germinal center responses as compared with wildtype IgM. Within seconds after immunization, wildtype IgM induced deposition of C3 on sheep red blood cells in the blood. IgM which efficiently enhanced the T-dependent humoral immune response had no effect on activation of specific CD4⁺ T cells as measured by cell numbers, cell division, blast transformation, or expression of the activation markers LFA-1 and CD44 in vivo. These observations confirm the importance of complement for the ability of specific IgM to enhance antibody responses and suggest that there is a divergence between the regulation of T- and B-cell responses by IgM.     

Quantitative immunocytochemical characterization of mononuclear phagocytes. I. Monoblasts, promonocytes, monocytes, and peritoneal and alveolar macrophages

The purpose of the present study was to compare the phenotype of tissue macrophages with that of their precursors in the bone marrow and blood. The phenotype was determined on the basis of the quantitative binding of monoclonal antibodies to cell-surface antigens (antigen F4/80, complement receptor III, Fc receptor II, Ia antigen, common leukocyte antigen, and Mac-2 and Mac-3 antigens) on individual mononuclear phagocytes. Monoclonal antibody binding to cells, detected by the biotin-avidin immunoperoxidase procedure, was quantitated by cytophotometric determination of the amount of enzyme reaction product on cells. The results of this quantitation are expressed as the median of the specific absorbance per unit of cell-surface area (0.25 micron2) and per cell. Shortly after collection of the mononuclear phagocytes, binding of all monoclonal antibodies except those directed against the common leukocyte and Mac-2 antigens to peritoneal macrophages was enhanced compared with binding to blood monocytes; for alveolar macrophages we found reduced binding of monoclonal antibodies F4/80 and M1/70 (complement receptor III) and enhanced binding of monoclonal antibodies with specificity for the common leukocyte antigen and Mac-2 and Mac-3 antigens. The results obtained with cultured mononuclear phagocytes show that during the development from monoblast to tissue macrophages, monoclonal antibody binding to the various types of mononuclear phagocyte, expressed per unit of cell-surface area, was not significantly altered except that of M3/38 (Mac-2 antigen) to peritoneal macrophages and that of F4/80 and M1/70 (complement receptor III) to alveolar macrophages. Expressed on a per cell basis, the results show an increase in the binding of all monoclonal antibodies except those directed against the Fc receptor II and Mac-3 antigen during the development from promonocytes to peritoneal macrophages; binding of most monoclonal antibodies to alveolar macrophages was considerably lower than that to blood monocytes. It is concluded that the expression of the various cell-surface antigens alters during mononuclear phagocyte differentiation. The expression changed also during culture, although distinct patterns of alteration could not be distinguished.     

Bead Arrays for Antibody and Complement Profiling Reveal Joint Contribution of Antibody Isotypes to C3 Deposition

The development of antigen arrays has provided researchers with great tools to identify reactivities against self or foreign antigens from body fluids. Yet, these approaches mostly do not address antibody isotypes and their effector functions even though these are key points for a more detailed understanding of disease processes. Here, we present a bead array-based assay for a multiplexed determination of antigen-specific antibody levels in parallel with their properties for complement activation. We measured the deposition of C3 fragments from serum samples to reflect the degree of complement activation via all three complement activation pathways. We utilized the assay on a bead array containing native and citrullinated peptide antigens to investigate the levels of IgG, IgM and IgA autoantibodies along with their complement activating properties in serum samples of 41 rheumatoid arthritis patients and 40 controls. Our analysis revealed significantly higher IgG reactivity against the citrullinated fibrinogen β and filaggrin peptides as well as an IgA reactivity that was exclusive for citrullinated fibrinogen β peptide and C3 deposition in rheumatoid arthritis patients. In addition, we characterized the humoral immune response against the viral EBNA-1 antigen to demonstrate the applicability of this assay beyond autoimmune conditions. We observed that particular buffer compositions were demanded for separate measurement of antibody reactivity and complement activation, as detection of antigen-antibody complexes appeared to be masked due to C3 deposition. We also found that rheumatoid factors of IgM isotype altered C3 deposition and introduced false-positive reactivities against EBNA-1 antigen. In conclusion, the presented bead-based assay setup can be utilized to profile antibody reactivities and immune-complex induced complement activation in a high-throughput manner and could facilitate the understanding and diagnosis of several diseases where complement activation plays role in the pathomechanism.     

Dirofilaria immitis: diethylcarbamazine-induced anaphylactoid reactions in infected dogs

The immunopathogenesis of the anaphylactoid Mazzotti reactions has been studied by comparing physiologic and immunologic aspects of diethylcarbamazine-induced shock in Dirofilaria immitis infected dogs with antigen induced anaphylaxis in infected and uninfected controls. Filarial antigen, specific host IgG antibody, and C1 and C3 complement levels were quantitatively measured over time in relation to the levels of histamine and prostaglandin D2 in the blood and changes in mean blood pressure. D. immitis antigen injected into uninfected dogs having no detectable IgG antibody to D. immitis or Toxocara canis produced a rapid drop in blood pressure that paralleled a drop in C1 and C3 levels and an increase in prostaglandin D2. Antigen injected into infected dogs with IgG antibody produced a similar drop in blood pressure and complement and increase in prostaglandin D2 which differed from the uninfected group only in the slower clearance of antigen from the blood. Diethylcarbamazine alone produced no measurable changes in blood pressure or complement in uninfected hosts. Diethylcarbamazine, however, administered into skin test positive infected dogs, produced a temporally slower but quantitatively similar loss in blood pressure, drop in complement, and increase in prostaglandin D2 and histamine to that induced by antigen injection. Complement activation and immune complex formation are initiated by antigen release, and subsequent vasoactive mediator release leads to shock with prostaglandin D2 being quantitatively higher in blood than is histamine.     

Profiles of human serum antibody responses elicited by three leading HIV vaccines focusing on the induction of Env-specific antibodies

In the current report, we compared the specificities of antibody responses in sera from volunteers enrolled in three US NIH-supported HIV vaccine trials using different immunization regimens. HIV-1 Env-specific binding antibody, neutralizing antibody, antibody-dependent cell-mediated cytotoxicity (ADCC), and profiles of antibody specificity were analyzed for human immune sera collected from vaccinees enrolled in the NIH HIV Vaccine Trial Network (HVTN) Study #041 (recombinant protein alone), HVTN Study #203 (poxviral vector prime-protein boost), and the DP6-001 study (DNA prime-protein boost). Vaccinees from HVTN Study #041 had the highest neutralizing antibody activities against the sensitive virus along with the highest binding antibody responses, particularly those directed toward the V3 loop. DP6-001 sera showed a higher frequency of positive neutralizing antibody activities against more resistant viral isolate with a significantly higher CD4 binding site (CD4bs) antibody response compared to both HVTN studies #041 and #203. No differences were found in CD4-induced (CD4i) antibody responses, ADCC activity, or complement activation by Env-specific antibody among these sera. Given recent renewed interest in realizing the importance of antibody responses for next generation HIV vaccine development, different antibody profiles shown in the current report, based on the analysis of a wide range of antibody parameters, provide critical biomarker information for the selection of HIV vaccines for more advanced human studies and, in particular, those that can elicit antibodies targeting conformational-sensitive and functionally conserved epitopes.     

Fluorescent antibody study of diseased, end-stage human kidneys.

Forty cases of diseased kidneys at end-stage were studied by fluorescent antibody technique in search for viral etiology of glomerulonephritis and other renal diseases. Among these 40 cases, 12 (30%) were ascribed to immune complex disease because of detection of immunoglobulins and complement in glomeruli of the same kidney specimen. In 8 cases (20%) only complement was detected in glomeruli. In the remaining 50% neither complement nor immunoglobulin deposit was found in glomeruli. The etiologies of the latter cases remain unknown. Of 12 cases of kidney disease of immune complex origin, hepatitis virus type B surface antigen was detected in 2 cases. In these 2 cases the magnitude of immune complex deposits with complement was greater than that of other cases. Other than hepatitis B virus antigen, no other viruses including Coxsackieviruses, ECHO viruses, and HSV-1 could be detected by indirect fluorescent antibody techniques. The proportion of complement deposit to the deposition of complement with immune complex in the diseased kidneys at end-stage was calculated and statistically analyzed.     

Inferring Epitopes of a Polymorphic Antigen Amidst Broadly Cross-Reactive Antibodies Using Protein Microarrays: A Study of OspC Proteins of Borrelia burgdorferi

Epitope mapping studies aim to identify the binding sites of antibody-antigen interactions to enhance the development of vaccines, diagnostics and immunotherapeutic compounds. However, mapping is a laborious process employing time- and resource-consuming ‘wet bench’ techniques or epitope prediction software that are still in their infancy. For polymorphic antigens, another challenge is characterizing cross-reactivity between epitopes, teasing out distinctions between broadly cross-reactive responses, limited cross-reactions among variants and the truly type-specific responses. A refined understanding of cross-reactive antibody binding could guide the selection of the most informative subsets of variants for diagnostics and multivalent subunit vaccines. We explored the antibody binding reactivity of sera from human patients and Peromyscus leucopus rodents infected with Borrelia burgdorferi to the polymorphic outer surface protein C (OspC), an attractive candidate antigen for vaccine and improved diagnostics for Lyme disease. We constructed a protein microarray displaying 23 natural variants of OspC and quantified the degree of cross-reactive antibody binding between all pairs of variants, using Pearson correlation calculated on the reactivity values using three independent transforms of the raw data: (1) logarithmic, (2) rank, and (3) binary indicators. We observed that the global amino acid sequence identity between OspC pairs was a poor predictor of cross-reactive antibody binding. Then we asked if specific regions of the protein would better explain the observed cross-reactive binding and performed in silico screening of the linear sequence and 3-dimensional structure of OspC. This analysis pointed to residues 179 through 188 the fifth C-terminal helix of the structure as a major determinant of type-specific cross-reactive antibody binding. We developed bioinformatics methods to systematically analyze the relationship between local sequence/structure variation and cross-reactive antibody binding patterns among variants of a polymorphic antigen, and this method can be applied to other polymorphic antigens for which immune response data is available for multiple variants.     

Removal of glomerular deposits induced by either preformed immune complexes or by a chronic immune complex model in NZB/W mice

The solubilization and removal of defined glomerular immune complex deposits by excess antigen was examined in NZB/W female mice. Glomerular deposits were induced by administering preformed immune complexes to young (2 to 4 mo) mice before they naturally acquired deposits from endogenous disease and to old (7 mo) mice with deposits from naturally acquired disease. The administration of excess antigen specifically removed deposits of preformed immune complexes in both groups. This was associated with a reduction in circulating large latticed complexes containing more than two antigen and two antibody molecules (greater than Ag2Ab2). Established deposits in old mice therefore did not interfere with removal of newly induced deposits of preformed immune complexes. Glomerular deposits were also induced in young mice by a chronic human serum albumin (HSA) immune complex model. The antigen in immune deposits induced by 2 wk of chronic antigen administration was solubilized and was removed within 48 hr of administering excess antigen. Circulating antibodies to the antigen were also reduced by excess antigen. Glomerular deposits of mouse immunoglobulin and complement were not significantly reduced by excess antigen but remained more intense than in mice of comparable age given preformed complexes. Thus deposits of other antigen antibody systems and possibly endogenous disease were induced by the chronic HSA immune complex model in NZB/W mice. However, defined antigen deposits within deposits containing multiple antigen antibody systems can clearly be removed by administering excess antigen.     

A Rapid Method to Characterize Mouse IgG Antibodies and Isolate Native Antigen Binding IgG B Cell Hybridomas

B cell hybridomas are an important source of monoclonal antibodies. In this paper, we developed a high-throughput method to characterize mouse IgG antibodies using surface plasmon resonance technology. This assay rapidly determines their sub-isotypes, whether they bind native antigen and their approximate affinities for the antigen using only 50 μl of hybridoma cell culture supernatant. Moreover, we found that mouse hybridomas secreting IgG antibodies also have membrane form IgG expression without Igα. Based on this surface IgG, we used flow cytometry to isolate rare γ2a isotype switched variants from a γ2b antibody secreting hybridoma cell line. Also, we used fluorescent antigen to single cell sort antigen binding hybridoma cells from bulk mixture of fused hybridoma cells instead of the traditional multi-microwell plate screening and limiting dilution sub-cloning thus saving time and labor. The IgG monoclonal antibodies specific for the native antigen identified with these methods are suitable for in vivo therapeutic uses, but also for sandwich ELISA assays, histology, flow cytometry, immune precipitation and x-ray crystallography.