Anti-Protein Plaque-Forming Cells Detected with High Efficiency by the Use of Red Cells Coupled to Bovine Serum Globulin through Bis-diazo-benzidine

It has been known for many years that sheep red cells coupled to proteins through diazotized benzidine can be hemolyzed by anti-protein sera (1). However, early attempts to use protein-coupled cells in the hemolytic plaque assay to detect cells producing anti-protein antibodies were unsuccessful (2). The first successful experiments were done with red cells coupled to protein by means of 1-ethyl-3-(3-dimethylamino propyl) carbodiimide HCl (ECDI) (3, 4), but this method has drawbacks in that ECDI is an expensive reagent, and large amounts of protein are required to obtain coupled cells with useful sensitivity. We have now re-investigated the use of benzidine and have found that we can obtain sheep red cells coupled with bovine γ globulin (BGG-SRBC) which are more efficient in detecting anti-BGG plaque-forming cells (PFC) than were any of the preparations we obtained after detailed investigation of the ECDI technique (5). This communication describes the preparation of BGG-SRBC for the PFC assay by the use of bis-diazo-benzidine (BDB) and compares them directly with cells prepared by the use of ECDI with respect to recovery of anti-BGG PFC and the titration of anti-BGG serums by passive hemolysis.