Understanding the Significance and Implications of Antibody Numbering and Antigen-Binding Surface/Residue Definition.

Monoclonal antibodies are playing an increasing role in both human and animal health. Different strategies of protein and chemical engineering, including humanization techniques of non-human antibodies were applied successfully to optimize clinical performances of antibodies. Despite the emergence of techniques allowing the development of fully human antibodies such as transgenic Xeno-mice, antibody humanization remains a standard procedure for therapeutic antibodies. An important prerequisite for antibody humanization requires standardized numbering methods to define precisely complementary determining regions (CDR), frameworks and residues from the light and heavy chains that affect the binding affinity and/or specificity of the antibody-antigen interaction. The recently generated deep-sequencing data and the increasing number of solved three-dimensional structures of antibodies from human and non-human origins have led to the emergence of numerous databases. However, these different databases use different numbering conventions and CDR definitions. In addition, the large fluctuation of the variable chain lengths, especially in CDR3 of heavy chains (CDRH3), hardly complicates the comparison and analysis of antibody sequences and the identification of the antigen binding residues. This review compares and discusses the different numbering schemes and "CDR" definition that were established up to date. Furthermore, it summarizes concepts and strategies used for numbering residues of antibodies and CDR residues identification. Finally, it discusses the importance of specific sets of residues in the binding affinity and/or specificity of immunoglobulins.

Molecular characterization of a human immunoglobulin G4 antibody specific for the major birch pollen allergen, Bet v 1.

BACKGROUND: Allergen-specific IgG4 antibodies induced by specific immunotherapy are thought to represent a protective immune response. Objective Our aim was the molecular characterization of a human IgG4 antibody (BAB5) specific for the major birch pollen allergen Bet v 1 that was derived from an immunotherapy-treated patient. METHODS: The cDNA coding for BAB5 was obtained by reverse transcriptase-PCR from the BAB5-producing cell line, compared with the germ line sequences and was expressed as a soluble antibody fragment in Escherichia coli. The epitope specificity and cross-reactivity of BAB5 were investigated with recombinant and synthetic Bet v 1 fragments and Bet v 1 homologous allergens from pollen. The ability of BAB5 to block allergic patients IgE was determined by competition experiments and sandwich ELISA. RESULTS: BAB5 is an affinity-matured Bet v 1-specific IgG4 antibody that reacts exclusively with Bet v 1 but not with Bet v 1-related allergens. Unlike an earlier-described monoclonal IgG1-blocking antibody, BAB1, which had been isolated from the same patient, BAB5 did not block allergic patients' IgE reactivity to Bet v 1. CONCLUSION: Our study demonstrates that not all allergen-specific IgG antibodies inhibit IgE recognition of allergens and can contribute to the success of immunotherapy. The epitope specificity and affinity of IgG antibodies but not their isotype are decisive for their protective activity.

[Cloning and expression of ScFv gene against alpha-toxin of Clostridium perfringens type A].

The VH and VL genes from a hybridoma cell line producing mouse McAb against alpha-toxin of Clostridium perfringens type A were amplified by RT-PCR. The VH and VL genes were connected thought a flexible linker (Gly4Ser)3 and the VH-linker-VL (ScFv) gene was cloned into a vector pGEM-T. The ScFv gene consists of 726 bp encoding 242 amino acid residues. Both VH and VL genes were confirmed as functionally rearranged mouse immunoglobulin variable region. According to kabat classed method, the VH and VL gene segments belong to mouse Ig heavy chain subgroup II (B) and kappa light chain subgroup III respectively. The ScFv gene was amplified inserted the expression vector pHOG21 and transformed into E coli XL1-BLUE. The ScFv protein was highly expressed in recombinant strain XL1-BLUE (pHOG-2E3) and the expression level of the ScFv was about 25% of total bacteria protein by SDS-PAGE. The neutralization assay showed that the expressed ScFv protein could neutralize the phospholipase C activities of alpha-toxin.

Molecular characterization of human IgG monoclonal antibodies specific for the major birch pollen allergen Bet v 1. Anti-allergen IgG can enhance the anaphylactic reaction.

We report the molecular characterization of five human monoclonal antibodies, BAB1-5 (BAB1: IgG(1); BAB4: IgG(2); BAB2, 3, 5: IgG(4)), with specificity for the major birch pollen allergen, Bet v 1. BAB1-5 were obtained after immunotherapy and contained a high degree of somatic mutations indicative of an antigen-driven affinity maturation process. While BAB1 inhibited the binding of patients IgE to Bet v 1, BAB2 increased IgE recognition of Bet v 1, and, even as Escherichia coli-expressed Fab, augmented Bet v 1-induced immediate type skin reactions. The demonstration that IgG antibodies can enhance allergen-induced allergic reactions is likely to explain the unpredictability of specific immunotherapy.

Molecular modeling and preclinical evaluation of the humanized NR-LU-13 antibody.

A mouse-human chimeric monoclonal antibody (chNR-LU-13), specific for the EGP40 pancarcinoma antigen, was humanized through three-dimensional molecular modeling. Humanization of the chNR-LU-13 antibody is expected to enhance its use for patients undergoing immunotherapy. On the basis of the observed amino acid sequence identity, chNR-LU-13 complementary determining regions (CDRs) of the V(L) and V(H) regions were grafted onto the human anti-DNA-associated idiotype immunoglobulin clone, R3.5H5G'CL. Ten amino acids residues within the humanized framework were back-mutated to their corresponding chNR-LU-13 sequence, because they were predicted to disrupt the canonical classification of the CDRs or were within 5 A of a CDR. Synthesis of the V(L) and V(H) regions was accomplished by recursive PCR, and the dual-chain expression vector p451.C4 was positioned under control of the CMV(P+E). We observed by competitive ELISA that the recombinant humanized NR-LU-13 (huNR-LU-13) IgG1 antibody exhibited an indistinguishable immunoreactivity profile when compared with the murine monoclonal antibody (muNR-LU-10). The huNR-LU-13 antibody was effective in mediating both antibody-dependent cellular cytotoxicity and complement-mediated cytotoxicity when assayed against either the breast carcinoma cell line, MCF-7, or the colon adenocarcinoma cell line, SW1222. Biodistribution studies using i.v. coinjected 131I-muNR-LU-10 and 125I-huNR-LU-13 confirmed that the huNR-LU-13 specifically targets to the tumor in athymic BALB/c mice bearing the SW1222 human tumor xenograft. Humanization of the chNR-LU-13 antibody is expected to eliminate an undesired human antimouse antibody response, allowing for repeated i.v. administration into humans.

Purification and characterization of IgG1 and IgG2 from buffalo (Bubalus bubalis) serum and colostrum.

Buffalo IgG1 and IgG2 were purified from serum and colostrum using salt precipitation, dialysis, gel filtration and ion-exchange chromatography. Their purity was monitored by immunodiffusion and immunoelectrophoresis using anti-heavy chain specific sera and SDS-PAGE. Selective binding of IgG2 to protein-A was used to remove IgG2 from IgG1 preparations. The IgG1 and IgG2 had a molecular mass (Mr) of 162.0 and 161.5 kD, respectively and were found to consist of heavy (H) and light (L) chains. The H and L chains had Mr of 58 and 24 kD, respectively. Reduction-alkylation followed by gel filtration was used for the isolation of H and L chains. While intact H chains were obtained, the L chains appeared to be cleaved into 14 kD molecules and smaller fragments. The mean hexoses content of the serum IgG1 and IgG2 was 1.81 +/- 0.02% and 0.70 +/- 0.02%, respectively. The corresponding values for colostral IgG1 and IgG2 were 1.76 +/- 0.01% and 0.78 +/- 0.08%. Both the IgG subclasses activated homologous complement. These results suggest that buffalo and cattle IgG subclasses have many common characteristics and minor differences.

Quality control criteria for quantitative enzyme-linked immunosorbent assay of porcine immunoglobulins A and M.

Using radioimmunoassay methods, quality control criteria were applied to monoclonal antibodies produced to measure porcine immunoglobulins by quantitative ELISA. Porcine IgM and IgA were purified to homogeneity and were used to produce murine hybridomas that secreted antibodies against IgM, IgA, and immunoglobulin light chains. A competitive ELISA was developed to measure IgM, and a sandwich ELISA was used to quantify IgA in serum and colostrum. Both ELISA were tested for specificity, accuracy, sensitivity, and precision. Monoclonal antibodies were specific for porcine IgM or IgA in serum and colostrum, and competitive and sandwich ELISA fulfilled all validation criteria.

Biosynthetic incorporation of [75Se]selenomethionine: a new method for labelling lymphocyte membrane antigens.

A novel approach for radiolabelling lymphocyte membrane antigens is described. This technique is based on the use of the gamma-emitting amino acid analogue [75Se]selenomethionine. Human HLA-A, B, C and DR heavy and light chains and mouse Ia antigens were efficiently labelled by this technique and were precipitated with monoclonal antibodies. Approximately the same radioactivity was incorporated into the HLA-A, B, C chains whether [75Se]selenomethionine, [35S]methionine or [3H]leucine were used as precursors. Easily detectable as a gamma-emitter, [75Se]selenomethionine thus constitutes a useful biosynthetic label of lymphocyte surface antigens. The same method was used to label immunoglobulins produced by hybridomas and to determine the nature of the secreted light chains.