Hans P. Kocher

Monoclonal autoantibodies against mouse red blood cells: a family of structurally restricted molecules.

Cultured mouse peritoneal cells from unstimulated mice developed plaque-forming activity against isologous bromelain-treated erythrocytes. Several IgM monoclonal autoantibodies obtained by fusion of peritoneal cells from NZB or CBA origin with BALB/c myeloma cells were purified by affinity chromatography on trimethyl ammonium (TMA) column on the basis of their cross-reactivity with TMA, phosphorylcholine (PC) or choline haptens. Binding affinity for PC hapten was of the order of 10(3) M-1. Idiotypic studies with a polyclonal rabbit anti-idiotypic reagent revealed strong cross-reactions with all hybridoma autoantibodies thus far tested. In addition, the rabbit anti-idiotypic serum detected idiotypes or cross-reactive idiotypes in the sera of NZB and CBA as well as BALB/c mice. N-terminal amino acid sequence analyses of three hybridoma autoantibodies from NZB mice and one from CBA mice were carried out. The sequences of the first 32 residues of the four heavy chains showed that three were identical while one had one amino acid interchange; they belong to the VHIII-subgroup. The light chains were identical in the first 35 residues with the exception of a substitution at position 3 in two light chains and are members of the VK-9-subgroup. These results entirely support the idiotypic data. These monoclonal autoantibodies from NZB and CBA mice although isolated and eluted from PC-related haptens do not have any apparent structural nor idiotypic relationship to PC-specific antibodies. Idiotypic and V-region N-terminal sequence data suggest that these autoantibodies constitute a highly restricted family of molecules likely to be encoded by unique germ-line genes which may be expressed as such or as somatic variants in different mouse strains.

A Cocktail of Three Monoclonal Antibodies Significantly Increases the Sensitivity of an Enzyme Immunoassay for Human Granulocyte-Macrophage Colony-Stimulating Factor

A sensitive and specific two-site ELISA was developed for human granulocyte-macrophage colony-stimulating factor (huGM-CSF) based on monoclonal antibodies (mAbs) which have been selected for high affinities and different epitope specificities. Using a cocktail of three mAbs, both for coating and, in their labeled form, for detection, a major increase in sensitivity was achieved (20-fold) compared to a two-site assay employing two different mAbs (one for coating and one for detection). The assay is as sensitive as the most sensitive biological assays. Recombinant mammalian cell expressed and natural huGM-CSF can be reliably detected down to 100 pg/ml (7 pmol/l). In contrast to conventional bioassays, the ELISA is highly specific for huGM-CSF and does not detect other human lymphokines. Results from quantification of recombinant and natural huGM-CSF in ELISA and bioassay correlate.

The immune response of BALB/c mice to phosphorylcholine is restricted to a limited number of VH- and VL-isotypes

Abstract To study the diversity of the anti-phosphorylcholine (PC)§ repertoire in BALB/c mice, hybridomas were produced by cell fusion of immune spleen cells of normal or T 15 idiotypically suppressed mice with a myeloma cell line. Idiotypic suppression was achieved by injection of anti-PCBMP T 15 idiotypic serum into newborn mice. Thirteen homogeneous PC-specific hybridoma-derived antibodies of classes IgM and IgG were isolated and analysed by partial N-terminal amino acid sequence determination of the variable (V) regions of their heavy (H) and light (L) chains. Eleven hybridoma antibodies displayed V H -regions of isotype V H−4 like T 15 and all other PCBMPs, whereas two belonged to a new V H isotype, designated V H−12 , not previously encountered in any PCBMP or mouse V H -region known so far. The sequences of the variable regions of the L-chains expressed the V K−8 -, V K−22 - and V K−24 -isotypes described in PCBMPs M 603, M 167 and T 15 respectively. One hybridoma antibody expressed a L-chain of the V K−15 -isotype, which probably originated from the immunoglobulin of the parental myeloma cell line used for cell fusion. The data suggest that a limited number of V H - and V L -isotypes are being used to generate the PC-specific repertoire in BALB/c mice and that the majority of the hybridoma antibodies contain V H −V L association pairs known from PCBMPs.

Anti-cyclosporine monoclonal antibodies and their anti-idiotopic counterpart: Structure and biological activity

In order to study the structural and functional mimicry of an antigen by anti-idiotypic antibodies, we generated anti-idiotopic monoclonal antibodies (anti-Id mAbs) against a mAb (R45-45-11) with specificity for the immunosuppressive cyclic undecapeptide cyclosporine (Cs; Sandimmune). Three out of five anti-Id mAbs inhibited the binding of Cs to the anti-Cs mAb R45-45-11. All anti-Id mAbs cross-reacted only with one (anti-Cs mAb V45-271-10) out of 19 anti-Cs mAbs. The anti-Cs mAb V45-271-10 recognizes an epitope on the Cs molecule which is very similar to that recognized by R45-45-11. R45-45-11 and V45-271-10 differ only by one amino acid in the variable region. The anti-Id mAbs which recognize combining site-associated idiotopes (Ids) reverse the blocking effect of the anti-Cs mAb R45-45-11 on Cs immunosuppression in vitro. The sequences of the variable regions of heavy and light chain of one anti-Id mAb were determined. X-ray analysis of the corresponding Fab fragment, either alone or complexed with the Fab fragment of the Id, is currently in progress.

Comparison of Secreted and Membrane-Bound Human Immunoglobulins M and D

Abstract Structural data obtained with human membrane-bound (um) and secreted (us) immunoglobulin M heavy chains suggest that 1) um has an apparent molecular size slightly larger than that of us, 2) um only could be efficiently labeled with a hydrophobic reagent, and 3) amino acids released at the C-termini of the chains are different. The data confirm and extend recent observations in human and murine systems by showing that human um chain contains a hydrophobic segment, consistent with its anchorage into the lipid bilayer of the plasma membrane and a C-terminal amino acid sequence different from that of us chain. Preliminary data on the polypeptide structure of membrane-bound and intracellular Fc fragments of human IgD suggest that, in contrast to the murine δ chains, human δ chain is made up of three constant domains as in human IgD myeloma proteins.

Biosynthesis and structure of membrane and secretory immunoglobulins

SummaryAlmost all of the bodys extracellular immunoglobulin (Ig) is derived from Ig-secreting plasma cells of lymphoid tissues. The secreted material is a heterogeneous mixture of different classes and specificities. Lymphoid tissues also contain a large number of essentially non-secretory cells — B lymphocytes — which bear Ig firmly associated with their plasma membranes. Ig molecules thus exist in two functionally different forms, as membrane-bound antigen receptors on the surface of B lymphocytes on the one hand, and as humoral secreted Ig antibodies on the other. On B cells, membrane-bound heavy chains have an apparent mol. wt. slightly larger than that of secreted heavy chains from plasma cells. Membrane-bound but not secreted heavy chains bind detergents, thus suggesting the presence of a hydrophobic region in membrane-bound heavy chains, which is absent in secreted heavy chains. Most investigations have dealt with immunoglobulin M. The two types of IgM heavy chains differ at their carboxy termini. Recent investigations at the nucleic acid level demonstrate that membrane-associated µ chains contain a 41-residue hydrophobic tail adjacent to the last constant domain, whereas secretory µ chains contain a 20-residue hydrophilic tail. At the present time, evidence is accumulating that all membrane-bound Ig heavy chain classes may contain similar hydrophobic structures necessary for anchorage of the molecules into the lipid bilayer.