Alain E. Bussard

A convenient enzyme-linked immunosorbent assay for testing whether monoclonal antibodies recognize the same antigenic site. Application to hybridomas specific for the β2-subunit of Escherichia coli tryptophan synthase

Seven hybridoma clones, producing antibodies directed against the beta 2-subunit of Escherichia coli tryptophan synthase, have been obtained from mouse cells. To test whether the corresponding monoclonal antibodies recognize different epitopes on beta 2, an ELISA double antibody binding system has been developed and is reported here. The antigen is first coated onto a microtitration plate. Two monoclonal antibodies are then added either separately or simultaneously, and the amount of bound antibody is quantitatively measured by use of immunoglobulin (rabbit anti-mouse IgG) linked to beta-galactosidase. Additivity of the bound enzymatic activity is observed when the monoclonal antibodies bind to distinct epitopes. Using this test, it is shown that, of the 7 anti-beta 2 monoclonal antibodies obtained, 5 recognize the same antigenic site and the 2 others recognize a second site.

Precommitment of normal mouse peritoneal cells by erythrocyte antigens in relation to auto-antibody production.

WE have reported that peritoneal cells (PCs) from unstimulated mice can, after 2 h of incubation in the appropriate medium, start to secrete anti-sheep erythrocyte (SRBC) antibodies1,2. We extended this observation by showing that PCs cultured in standard conditions for 4–6 d in the absence of SRBC, can form numerous haemolytic plaques, demonstrable by the ordinary techniques of local haemolysis (in Agarose, in liquid layer or in cellulose gum3). The immunological nature of this plaque-forming activity was established by the following criteria: complement dependency, inhibition by anti-mouse IgM serum and immunological specificity. Thus, we concluded3,4 that PCs of normal adult mice had been stimulated previously by an unknown immunogen sharing common or cross-reacting determinants with SRBC, and that tissue culture conditions would derepress the built-in capacity of these cells to produce antibodies. Treatment of autologous erythrocytes by the proteolytic enzyme bromelain revealed that normal mouse spleen cells regularly produce antibodies against their own erythrocytes5,6. This led us to investigate the behaviour of normal PCs towards SRBC or mouse red blood cells (MRBCs) treated by bromelain (BrMRBCs). We report here that mouse PCs in culture develop a plaque-forming activity against isologous BrMRBC as well as against SRBC, and we show that these two types of erythrocytes share some common antigen.

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 scientific revolution

Science, Thomas Kuhn argued in The Structure of Scientific Revolutions (1962), proceeds at two different paces. One is what he called “normal science”, which professionals, the general public, the press and politicians generally understand as “research firmly based upon one or more past achievements that some particular scientific community acknowledges for a time as supplying the foundation for its further practice.” This stepwise progression towards a better understanding of Nature, by building on established knowledge, has been described in a myriad of textbooks, dictionaries and scientific papers. However, Kuhn distinguishes this form of knowledge creation from so‐called “puzzle‐solving science”. The latter results from anomalies—experimental observations or other evidence—which do not fit into the widely accepted theoretical framework of how Nature functions. Puzzle‐solving science, according to Kuhn, can therefore trigger a scientific revolution as scientists struggle to explain these anomalies and develop a novel basic theory to incorporate them into the existing body of knowledge. After an extended period of upheaval, in which followers of the new theory storm the bastions of accepted dogma, the old paradigm is gradually replaced. Perhaps the best example of such a paradigm shift in science is the Copernican revolution in cosmology: the move from a geocentric to the heliocentric view of our solar system. Curiously, although Aristarches had already laid the seeds of heliocentrism in the third century BC, it took another 18 centuries before Nicolaus Copernicus proposed that the Earth moves around the sun and not vice versa. Many anomalies, such as the orbit of Mars, were already known at that time, but the power of the Aristotelian dogmas, including the geocentric view of the universe, was too strong to be overcome easily. Truly speaking, however, the notion of a paradigm, as defined by Kuhn, does not have exactly the same meaning in …

Establishment and characterization of a permanent murine hybridoma secreting monoclonal autoantibodies.

Hybridization between plasmocytoma cells from Balb/C mice (X63-Ag8) and peritoneal cells from NZB mice, which secrete, in large amounts autoanibodies directed against bromelin-treated mouse red blood cells, have been achieved. Clones of hybridoma secreting permanently autoantibodies were isolated. The biochemistry and the immunochemistry of these monoclonal antibodies have been studied.

Local hemolysis plaque assay using a new method of coupling antigens on sheep erythrocytes by glutaraldehyde

Abstract Horseradish peroxidase, beef pancreatic ribonuclease, bovine serum albumin and human gamma globulins were fixed to the surface of sheep red blood cells (SRBC) by glutaraldehyde, permitting specific immune lysis of the coated erythrocytes by anti-protein antibodies. Sensitivity to lysis was evaluate by microtitration test. Antigen-coated SRBC were then used in the local hemolysis assay carboxyl-methyl-cellulose gum or in liquid medium, with lymphoid cells from popliteal lymph nodes of rabbits immunized 7–13 days before. Numerous direct plaques was obtained with SRBC modified by each of the two methods described. Monkey anti-rabbit IgG antibodies enhanced plaque number in some experiments, but it was found that rabbit 7S antiboodies also caused direct hemolysis in this system. The coating procedure required small amounts of antigen and coupling reagent. Reproducible results were obtained when preparations of the same age were used. Coated serythrocytes stored in phosphate buffered saline were stable for at least 8 days, but sensitivity to specific lysis oincreased with storage. With the two coating procedures described, the method could be adapted to many diffrerent antigens.

Immunochemical characterization of the auto antibodies produced by mouse peritoneal cells in culture.

Abstract The formation of anti-sheep red blood cells (SRBC) and anti-bromelinized mouse red blood cells (BrMRBC) plaques of hemolysis (PFC) by mouse peritoneal cells (PC) in culture has been studied. A very high number of PFC (up to 10%) could be detected after 4–5 days of culture. In most instances the same cells produced anti-SRBC and anti-BrMRBC antibodies, indicating a high degree of cross reactivity between antigenic determinants of the two types of erythrocytes. Autoradio-immunoelectrophoresis radioactively ( 14 C leucine) tagged tissue culture fluid or cell extracts showed the PC in culture actively synthesized IgG and IgM. A certain amount of these Ig was characterized as anti-SRBC and anti-BrMRBC antibodies. It is suggested that the spontaneous formation of anti-SRBC plaques by PC from non-immunized mice is due to autoimmunization by antigen(s) present on the mouse own erythrocytes.

The participation of trimethylammonium in the mouse erythrocyte epitope recognized by monoclonal autoantibodies

Monoclonal IgM anti-erythrocyte autoantibody produced by a NZB-derived hybridoma has been found to react with trimethylammonium-containing compounds. Such compounds are able to prevent the lysis of bromelain-treated mouse erythrocytes (BrMRBC) by those autoantibodies. Using a column of insolubilized betaine hydrazine (BH) the monoclonal anti-erythrocyte antibody has been specifically retained. Elution of this antibody was accomplished by 0.15 M choline (Ch).

Specificity of antierythrocyte autoantibodies secreted by a NZB-derived hybridoma and NZB peritoneal cells

Abstract The specificity of monoclonal IgM antierythrocyte autoantibody produced by a NZB-derived hybridoma and the specificity of autoantibodies produced by uninduced NZB peritoneal cells in culture were determined. Supernatant fluids from cultures of hybridoma and peritoneal cells reacted in direct hemagglutination assays with bromelin-treated mouse erythrocytes, and, to a lesser extent, with sheep red blood cells; no agglutination was observed with intact mouse red blood cells or human O + erythrocytes. These results suggest the presence of previously characterized anti-HB, but not anti-X or cold reactive autoantibodies, with a cross-reaction between antigenic constituents on sheep and bromelin-treated mouse erythrocytes. Specificity was affirmed by neutralization of agglutination or of direct hemolysis of bromelin-treated mouse erythrocytes with partially purified SEA-HB, the soluble plasma analog of the erythrocyte-bound HB autoantigen. Plaque formation in direct plaque-forming cell assays by both hybridoma and peritoneal cells was specifically inhibited by SEA-HB. These results demonstrate that NZB-derived hybridoma as well as NZB peritoneal cells secrete anti-HB autoantibody, an autoantibody that spontaneously appears in the serum of NZB as well as other strains of mice.

Relationship between choline derivatives and mouse erythrocyte membrane antigens revealed by mouse monoclonal antibodies. I. Anticholine activity of anti-mouse erythrocyte monoclonal antibodies.

Different clones of mouse hybridomas, derived from the fusion of unstimulated mouse peritoneal cells with mouse myeloma cells, producing IgM monoclonal antibodies directed against the membrane of bromelain-treated mouse erythrocytes (MRBC(Br)) have been previously established. We have recently shown that one of these hybridomas produce, in ascites, antibodies cross-reacting with phosphorylcholine derivatives (trimethylammonium (TMA) derivatives). In this work the cross-reactivity for TMA derivatives of the monoclonal antibodies produced by 4 anti-MRBC(Br) hybridomas have been studied at the cell level (plaque-forming cells). Phosphorylcholine, choline bromide and p-aminophenyl-trimethylammonium were found to be potent specific inhibitors of plaque formation (anti MRBC(Br)). The hemolytic activities of ascites and tissue culture supernatants were studied and their inhibition by TMA derivatives was determined. Immunoglobulins from ascites purified on TMA immunoadsorbent column were analyzed by two-dimensional gel electrophoresis, their spectrotype was compared to the spectrotype of immunoglobulins from tissue culture supernatants from the same hybridoma radioactively tagged by internal incorporation of [14C]leucine. It could be shown without ambiguity that the PTMA column retained an IgM with the same characteristics as the IgM secreted in vitro.