The use of 4,4'-biphenylbisdiazonium fluoroborate as a coupling agent for passive haemolysis tests

The possibility of use a stable diazonium compound, to replace bisdiazotized benzidin (BDB) for conjugating antigens to red blood cells was investigated. Five different batches of 4,4'-biphenylbisdiazonium fluoroborate (BDF) were prepared and tested in conjugation experiments. The sensitivity to specific lysis of red cells coated with different antigens (EG cells) was assayed by both direct and indirect passive haemolysis tests. Optimal conditions for the preparation of EG cells with different protein antigens were established. With the exception of the concentration of the bifunctional reagent, these conditions were similar to those previously reported for BDB. The antibody content of different anti-protein sera could be determined, with a 10 % error, by using EG cells prepared under optimal conditions in indirect passive haemolysis tests. The amount of antibody detected by this method varied from 7 to 12 ng N Ab/ml, depending upon the nature of the antigen fixed to red cell. The content of BSA in dilute solutions could be estimated by the specific inhibition of the indirect passive haemolysis tests. Testing different samples of BDF maintained at different conditions showed that the compound could be kept at room temperature, during the least four years, without appreciable loss of the conjugating properties, provided it was kept in the dry state and in the dark.