The use of passive haemolysis in the quantitative estimation of anti-protein antibodies

Abstract

The conditions for the establishment of a method for the quantitative assay of antiprotein antibodies by the use of passive haemolysis were studied by using an anti-BSA—BSA system as a model.

When haemolytic assaying of antibody was carried out under standard conditions with a fixed concentration of optimally sensitized red cells, in the presence of excess complement (10 C′H₅₀) reproducible titrations could be performed to detect 0.12 μg. N Ab with an error of ±10 per cent.

Factors affecting the specific lysis of optimally sensitized cells were studied and some parameters fixed. The amounts of coated erythrocytes and complement and the length of time of reaction were seen to influence the final degree of lysis given by a fixed amount of antibody.

The conditions of optimal sensitization of erythrocytes with protein antigen by the use of bis-diazotized benzidine (BDB) are indicated. It was found that, when conjugation was carried out at 0° with proper amounts of a standardized cell suspension, BDB and antigen, the resulting coated cells were highly sensitive to immune haemolysis and highly resistant to `spontaneous' lysis.