Revue Fran?aise de Transfusion et Immuno-hématologie最新文献

In this trial, we have tested four monoclonal antibodies related to the Lutheran System. These antibodies were studied by serological methods with a panel of red cells of Lutheran common phenotypes and weak variants.

Glycophorins of human erythrocytes have been extensively studied and the structure of three of them is fully (glycophorins A and C) or almost fully (glycophorin B) known [1, 2]. Glycophorins span the erythrocyte membrane and their NH₂-terminal domains exposed at the cell surface are heavily glycosylated. Glycophorin A occurs in two genetically determined forms carrying at NH₂-terminal end blood group M and N antigenic determinants. Glycophorin B (blood group Ss glycoprotein) has the structure of NH₂-terminal region (a.a. residues 1–26) identical to glycophorin A of blood type N, and also shows a high degree of homology with glycophorin A in the internal portion of the molecule, whereas glycophorin C has a different amino acid sequence. The knowledge of structure and orientation in the membrane and genetic differentiation of glycophorins facilitate elucidation of the fine specificity of anti-glycophorin antibodies.

The 30 anti-glycophorin-antibodies obtained were tested by agglutination of untreated and modified erythrocytes, immunoblotting, and binding to glycophorin A in microtiter plate ELISA. Moreover, inhibition of antibodies by untreated and modified glycophorin A preparations was studied. The methods used were described in detail in our recent publications [3, 5].

The antibodies could be divided into groups (Table I), depending on specificity. The 19 antibodies recognized epitopes located at the NH₂-terminal end of glycophorin A that could be easily shown by specific or distinctly preferable reactivity with blood group M (8 MoAbs) or N (11 MoAbs) antigen. The antibodies with anti-N specificity also reacted with glycophorin B. Among the remaining blood group MN-unrelated antibodies, 4 were specific for glycophorin A, 3 recognized epitopes common for glycophorins A and B, 2 reacted to glycophorin C, and the specificity of 2 antibodies could not be clearly established. The antibodies in each group differed in sub-specificity and antigen-binding properties.

This group of mouse monoclonal reagents comprised 31 different antibodies, 19 in the form of culture supernatants and 12 as ascitic fluids. The isotypes of antibodies in supernatant form were determined using a sandwich ELISA immunodot assay, before performing initial serological and physicochemical characterisation by microplate haemagglutination. Culture supernatants were titred without prior dilution, whereas ascitic fluids and purified supernatants were initially diluted 1 : 50 and 1 : 10 respectively in isotonic saline containing 1 % (w/v) BSA before titration. In order to determine their optimal reaction parameters, each antibody was titred at 22° C against selected M + N −, M + N + and M − N + cells at 3 different pH values − 5.5, 7.0 and 8.5. Those giving negative reactions were subjected to an indirect antiglobulin test and were then retested at 37° C and 4° C using optimal pH. Some antibodies were later found to require pretreatment with neuraminidase to induce haemagglutination.

Each antibody was subsequently tested, at optimal pH and temperature, with the same M + N −, M + N + and M − N + cells, pretreated with the proteolytic enzymes ficin, trypsin and chymotrypsin plus the exoglycosidase, neuraminidase from Vibrio cholerae. Where necessary, more extensive pH studies or sequential enzyme pretreatment of cells were performed before the antibodies were divided into 3 groups - those with anti - M - like specificity, anti - N - like specificity or those showing no specificity for M - or N - antigens. Each group was then tested with selected variant cells and those with apparent anti-carbohydrate specificities were subjected to absorption with synthetic oligosaccharides linked to inorganic carriers (SYNSORBS).

A method developed in this paper allows to measure the amount of pepsin in the intraveinous immunoglobulin preparation.