Mouse Monoclonal Antibodies with Anti-A, Anti-B and Anti-A,B Specificities; Some Superior to Human Polyclonal ABO Reagents (1)
Using BALB/c mice, a finaI high-dose booster schedule was usually followed when soluble blood group substances were used as antigens. With red cells, a shorter schedule with two consecutive intraperitoneal injections without adjuvants was mostly adopted. No.2 protocol can be used in future studies because of its efficacy.
Manual tests were performed using 0.1 ml undiluted culture medium or ascitic fluid diluted in 0.9% NaCl and 0.05 ml 5% red cell suspensions which were incubated at 22C, either on tiles for 20 min or in tubes for 1 h. Reactions were read visually against a well-lit background, and positive reactions were graded from (+) to 4 +. A total of seven hybridomas were selected for further study: three that produced anti-A, two anti-B and two anti-A, B. Six hybridomas produced IgM antibodies and one was an IgG3 antibody.
The addition of salts to the monoclonal antibodies increases the rate of the agglutination
reaction, which remains constant over a relatively wide range, but causes dramatically reduced avidity outside certain limits. Isotonic saline (0.15M) is generally used for blood grouping purposes, but it is well known that the rate of the reaction between polyclonal IgG antibodies and antigen is increased by a reduction of ionic strength from 0.15 to 0.03 M.