The activation of complement components by aggregates of antibodies and their fragments

The relative consumption of the complement components C1, C2, C3 and C4 from guinea pig serum on addition of rabbit antibody-antigen aggregates, papain digested aggregates, heat aggregated rabbit IgG and Fc has been measured. It has been found that antibody-antigen aggregates are much more efficient with respect to consumption of all four components than heat aggregated IgG which in turn is more efficient than heat aggregated Fc. In the latter two preparations the inefficiency is most marked with C2 and C3 consumption. The results suggest that splitting of C2 may depend on prior association with bound C4b which yields a bound C42 complex to the Fab section of the aggregated antibody.     

Retained antigen-binding activity of Fab alpha fragments of human monoclonal immunoglobulin A1 (IgA1) cleaved by IgA1 protease

Immunoglobulin A1 (IgA1) proteases may be important virulence factors of certain bacteria involved in the pathogenesis of meningitis, gonorrhea, destructive periodontal diseases, and some other infections affecting mucosal membranes. This study evaluated the antigen-binding activity of free Fab alpha fragments released from human myeloma IgA1 by IgA1 protease from Haemophilus influenzae. Six myeloma proteins with antibody activity against streptolysin O, alpha-staphylolysin, or streptococcal hyaluronidase were used. Complete cleavage of the IgA1 myeloma proteins in the hinge region of the heavy chain did not affect their antigen-binding capacity. The titers of neutralizing activity associated with free Fab alpha fragments were not significantly different from those of the intact IgA1 proteins. The retained antigen-binding capacity of cleaved IgA1 is an important factor in the understanding of how IgA1 proteases may interfere with the immune protection of mucosal membranes.     

Activation of complement by human serum IgA, secretory IgA and IgA1 fragments

Activation of the complement (C) system by human IgA was studied. Both subclasses of IgA, IgA1 and IgA2, and secretory IgA were shown to activate C, as determined by deposition of C3 on glutaraldehyde-activated microwells coated with IgA. The activation of the C system occurred in the presence of MgEGTA and not in D-deficient serum. In addition to C3, deposition of properdin (P) but not of C4 was detected. These results indicate that C activation, as determined by measuring deposition of C3 and P, occurred by the alternative pathway (AP). The data further show that the major part of the hinge region, which is deleted in IgA2 as compared with IgA1 and which forms the major structural difference between the two subclasses, is not involved in C activation. Reduction and alkylation destroyed the ability of IgA to activate C, as has also been demonstrated for IgG. In order to define the C activating region of the IgA molecule, several fragments of IgA1 were tested. The four-chain molecules F(ab')2 and F(abc)2 were shown to activate the AP. No activation was observed with the two-chain fragments Fab and Fc. The Fc fragment of IgA also did not activate the CP, as does the Fc fragment of IgG. This indicates that activation of the AP of C by IgA is dependent on the presence of the F(ab')2 fragment. In conclusion: human IgA does activate C by the AP. This activation requires an intact F(ab')2 fragment.     

Complement fixing abilities of IgA myeloma protiens and their fragments: The activation of complement through the classical pathway

Complement fixing abilities of IgA and its fragments chemically aggregated were tested. Both IgA1 and IgA2 myeloma proteins fixed considerable amount of human complement. IgA2 fixed complement more efficiently than IgA1 did. Fc and F(ab′)₂ of IgA1 were more active than undigestived IgA1, and their activities were comparable to that of Fc of IgG, whereas the activity of F(ab′)₂ of IgA2 was weak. Complement component profiles of human serum treated either IgA or its fragments revealed that the classical pathway of the complement system was activated.     

Large scale production and purification of paraquat and desipramine monoclonal antibodies and their Fab fragments

We describe the rapid, large scale purification of Fab fragments from mouse monoclonal antibodies. Antibodies against two clinically important and often fatal toxins, paraquat and desipramine, were isolated from mouse ascites fluid by preparative high performance hydroxylapatite (HPHT) or ion exchange (DEAE) high performance liquid chromatography. A competitive inhibition ELISA was used to determine the cross-reactivity of the antibody with analogs of the antigens. Papain digests of the IgGs were subjected to further HPHT followed by Sephadex G-100 chromatography to yield homogeneous Fab fragment preparations. The high purity of these preparations, demonstrated by SDS polyacrylamide gel electrophoresis, has only been achieved previously by affinity chromatography. Intrinsic association constants for the intact IgG and the Fab fragment--antigen interactions, determined by competitive inhibition ELISA, were similar. This indicates that antigen-binding activity was conserved during the production and purification of the Fab fragments.     

Lack of complement activation by human IgA immune complexes.

Complement activation may play an important role in renal injury associated with glomerular deposition of IgA immune complexes. The ability of naturally occurring human IgA immune complexes (IgA-IC) and covalently cross-linked human IgA oligomers (X-IgA) to activate complement were examined in vitro and in vivo. Large-sized IgA-IC were isolated from a patient's serum by affinity purification (Jacalin-Sepharose) and gel chromatography. Stable X-IgA were prepared by chemical cross-linking with a heterobifunctional reagent, N-succinimdyl 3-(2-pyridyl-dithio) propionate (SPDP). Treatment of fresh normal human serum with large amounts of either IgA-IC or X-IgA failed to activate C3. The C3 consumption was measured immunochemically by the decrease of the B antigen on the native C3 and by the generation of iC3b. Addition of these complexes to serum did not result in cleavage of factor B. Administration of human IgA-IC or X-IgA to mice, killed after 6 h, resulted in glomerular deposition of IgA. Despite the presence of intense glomerular IgA deposits no C3 was detected. Collectively, these findings suggest that neither soluble nor renal localized human IgA complexes activate complement.     

Rapid kinetic-based screening of human Fab fragments

Human antibodies able to bind with high affinity and specificity to numerous targets have been successfully identified from Fab phage display libraries. A key step in the library selection screening process is the early characterization of library isolates in order to determine which of these isolates to pursue further. Here we describe a Biacore assay that allows isolated clones expressed as soluble Fab fragments in E. coli to be screened and ranked based on their affinity against the target. The assay takes advantage of our ability to measure Fab concentrations in crude bacterial extracts in Biacore using very high density Protein A chips. The procedure allows up to 100 clones per week to be screened and permits the identification of a small number of high-affinity Fabs from a large batch obtained following library selection or affinity maturation.     

Monoclonal antibodies to distinct epitopes on human IgA and their use to IgA determination

Several monoclonal antibodies (MAbs) against human IgA have been obtained which specifically bind to human myeloma and polyclonal IgA. Three of these MAbs have been purified and studied further. They recognize both IgA subclasses and define three distinct epitopes on the IgA molecule. These MAbs were used to develop a solid-phase radioimmunoassay (RIA) in which one MAb was immobilized and the other two were labeled with 125I. The assay has a sensitivity in the nanogram range. A good correlation was found (r = 0.97, P less than 0.001) when the solid-phase RIA was compared with a commercially available immunodiffusion technique for the determination of IgA levels in serum samples.     

Factors influencing bonding of bromelain agglutinators and their Fab fragments

The complex of bromelain agglutinators and their homologous Fab fragments is dissociated by gel chromatography under certain conditions. When albumin is present as a source of thiol groups, Fab fragments previously treated with N-ethylmaleimide (NEM) will dissociate from the agglutinators (fluid phase). If anti-Rh Fab fragments are bound to Rh-positive erythrocytes, the agglutinates are not dissociated by thiols (cellular phase). Prior to erythrocyte sensitization, the agglutinator site on Fab fragments can be blocked by thiol-disulfide exchange. Once the Fab fragments are coated on erythrocytes, the agglutinator site is more readily available than it was prior to sensitization, as evidenced by inhibition with 0.01 M NEM. The differences between the bonding characteristics of the fluid phase and the cellular phase and the influence of mercaptoalbumin on the agglutinator-Fab complex suggest that the agglutinators are not antibodies.     

Binding of human IgA1 and IgA1 fragments to jacalin

Interaction of jacalin, an N-terminal galactose specific lectin, with human IgA1 and IgA1 fragments was investigated. IgA1 and all galactose containing fragments bound to jacalin-Sepharose, including Fab fragments containing only the galNac linked to serine-224 and Fc fragments containing four gal-galNac sequences. These data indicate that both the galNac and gal-galNac sequences can interact with jacalin. Jacalin precipitated IgA1 and the fragments F(abc)2, F(ab')2 and Fc in agar gel and from solutions. It also precipitated Fab' fragments in agar gel. Jacalin did not precipitate Fab fragments significantly. This suggests that, except for the single binding site on the Fab fragments containing the galNac linked to serine-224, jacalin itself also has a limited number of sites to interact with N-terminal galactose residues. ELISA studies revealed that intact IgA1 had a lower jacalin binding capacity than F(abc)2 fragments which lack CH3 domains, than F(ab')2 which lack the CH2 and CH3 domains, and than Fc fragments containing four gal-galNac sequences. This led to the conclusion that part of the galNac or gal-galNac sequences in intact IgA1 molecules are inaccessible to interaction with jacalin. Cleaving the C-terminal domains off may have induced a reorientation of the hinge region structure, including the orientation of the carbohydrate units.     

人源Fab抗体库的构建和抗HBsAg抗体的筛选和鉴定

目的 构建人源Fab抗体文库,筛选抗HBsAg 抗体片段并进行初步鉴定.方法 收集20份临床检验废弃的成人乙肝感染者淋巴细胞,抽提总RNA ,逆转录成cDNA,构建抗乙肝病毒人源免疫型Fab抗体文库.以HBsAg包板进行4轮循环的吸附-洗脱-扩增,挑单克隆用Phage-ELISA、DNA测序筛选阳性克隆,对阳性克隆进行可溶性表达,并用ELISA对其特异性进行鉴定.结果 构建的人源Fab型抗体文库的库容为2.0×108,并具有良好的多样性.经过4轮筛选,成功获得4株能与HBsAg结合的人源抗体克隆,分别命名为hFabHB1、hFabHB2、hFabHB3 和hFabHB4.对其中的hFabHB1进行可溶性表达,ELISA鉴定阳性.结论 成功构建了抗乙肝病毒人源免疫型Fa b抗体文库,从中筛选获得的4株人源Fab抗体片段有望在乙型肝炎的预防和治疗上发挥作用 .     

Aberrant synthesis of antibodies directed at the Fab fragment of IgA in patients with IgA nephropathies

The sera of 37 patients with IgA nephropathy (IgA NP) were assayed for levels of antibodies specific for the Fab fragment of homologous IgA, and the values obtained were compared to antibody levels in a panel of 26 normal volunteers. IgG antibody levels in IgA NP patients were significantly elevated over those of the controls (P less than 0.01); at the same time IgM anti-Fab alpha levels were significantly decreased when compared to the control panel (P less than 0.01). There was no correlation of antibody levels of either isotype with levels of circulating immune complexes; however, IgM antibody levels of IgA NP patients showed a significant negative correlation with severity of renal insufficiency.     

Fab fragments of monoclonal antibodies protect the human acetylcholine receptor against antigenic modulation caused by myasthenic sera

The human cell line TE671 produces large amounts of muscle nicotinic acetylcholine receptor (AChR). TE671 cells were used to determine the specificity of antibodies which can increase the internalization rate of AChR (antigenic modulation) and to test procedures for protecting AChR against this mechanism. The half-life of AChR both in the absence and the presence of anti-AChR antibodies was very similar to that of AChR on human muscle cell cultures. The relative contribution of different anti-AChR antibody fractions to the total antigenic modulation capacity of human myasthenic sera was investigated by competition experiments between Fab fragments of anti-AChR monoclonal antibodies (MoAbs) and intact antibodies (MoAb or myasthenic sera). Fab fragments, which do not induce antigenic modulation, were allowed to shield the corresponding regions of the AChR. Intact antibodies were subsequently added. It was found that protection of the main immunogenic region (MIR), but not of a region on the beta-subunit, essentially blocked the modulatory effect of the intact anti-MIR MoAbs, and approximately 80% of that of myasthenic sera. These data suggest that anti-MIR antibodies are mainly responsible for the loss of human AChR via antigenic modulation. Furthermore the observation that Fab fragments of anti-MIR MoAbs can efficiently protect AChR against antigenic modulation may have therapeutic implications.     

Production and characterization of recombinant human anti-HBs Fab antibodies

Recombinant human Fab antibodies were generated with different reactivities against the hepatitis B virus surface (HBs) antigen. To isolate the antibodies, a method was used that combined transformation of human B cells by Epstein-Barr virus (EBV) infection with a primer-vector system developed for isolating DNA fragments of human Ig Fab portions. With this method, monoclonal and oligoclonal cell lines producing anti-HBs antibodies were established and three anti-HBs Fab antibodies were isolated from two of these cell lines. From analysis of affinity characteristics, immunohistochemical activity, and cytolysis activity, these three Fab antibodies were classified into three different groups. The first group had high affinity for HBs, the second had the ability to kill HBV-infected cells, and the third was applicable to immunohistochemical staining with HBV-infected cells. The combined effect of these antibodies was also investigated by complement-dependent cytotoxicity assay.     

Evidence of IgG-mediated enhancement of the antibody response in vivo without complement activation via the classical pathway

The complement (C) dependency of IgG-mediated enhancement of the antibody response was investigated by immunizing mice with trinitrophenyl-coupled keyhole limpet hemocyanin (TNP-KLH) and either a C-activating TNP-specific monoclonal IgG2a antibody (Hy-1.2) or a mutant, non-C-activating variant of Hy-1.2 (M12). Hy-1.2 as well as M12 efficiently enhanced the anti-KLH response, although Hy-1.2 was more active. In addition, also a naturally non-C-activating TNP-specific IgG1 antibody enhanced the response to TNP-coupled bovine serum albumin. Moreover, C-activating IgG could enhance the antibody response in mice depleted of C3 by treatment with cobra venom factor. These findings suggest that the classical pathway of C activation is not required for IgG-mediated enhancement.     

Characterization of human monoclonal autoantibody Fab fragments against oxidized LDL

Oxidized low-density lipoprotein (oxLDL) is a key autoantigen in atherosclerosis. The genetic structures and pathogenic roles of autoantibodies against this protein remain to be established. In this study, we cloned several monoclonal IgG autoantibody Fab fragments specific for oxLDL from peripheral blood lymphocytes of atherosclerosis patients, using phage display technology. The sequences of their variable regions were determined at the cDNA level. The closest germline counterparts for the heavy chains belonged to the V(H)3 or V(H)1 family. The sequences and lengths of complementarity-determining regions (CDR)3-V(H) were diverse, and frequent mutations of positively charged amino acids (particularly arginine) over entire V(H) and V(L) sequences were observed. It is proposed that anti-oxLDL autoantibody formation is driven by antigens. Among the Fabs, P2-8 and P3-175 bound to both MDA-LDL and Cu-oxLDL, and inhibited the uptake of oxLDL by macrophages, suggesting the epitope(s) recognized by the Fabs is a part of ligands on oxLDL that is involved in uptake by macrophage scavenger receptor. These human autoantibody Fabs require detailed investigation to ascertain their potential as agents for clinical applications.     

Allergenic components of the mRNA-1273 vaccine for COVID-19: Possible involvement of polyethylene glycol and IgG-mediated complement activation

Following the emergency use authorization of the mRNA-1273 vaccine on the 18^th of December 2020, two mRNA vaccines are in current use for the prevention of coronavirus disease 2019 (COVID-19). For both mRNA vaccines, the phase III pivotal trials excluded individuals with a history of allergy to vaccine components. Immediately after the initiation of vaccination in the United Kingdom, Canada, and the United States, anaphylactic reactions were reported. While the culprit trigger requires investigation, initial reports suggested the excipient polyethylene glycol 2000 (PEG-2000)—contained in both vaccines as the PEG-micellar carrier system—as the potential culprit. Surface PEG chains form a hydrate shell to increase stability and prevent opsonization. Allergic reactions to such PEGylated lipids can be IgE-mediated, but may also result from complement activation-related pseudoallergy (CARPA) that has been described in similar liposomes. In addition, mRNA-1273 also contains tromethamine (trometamol), which has been reported to cause anaphylaxis to substances such as gadolinium-based contrast media. Skin prick, intradermal and epicutaneous tests, in vitro sIgE assessment, evaluation of sIgG/IgM, and basophil activation tests are being used to demonstrate allergic reactions to various components of the vaccines.