Differential sensitivity of haemolytic plaque methods at various stages of the immune response

Abstract

A recently described variant of the Ingraham-Bussard carboxymethyl-cellulose (CMC) hemolytic plaque technique has been applied to the study of direct and enhanced plaque formation by CBA mouse spleen cells immunized against sheep red blood cells (SRBC). This is the open CMC technique, in which thin monolayers containing spleen cells, SRBC, CMC, guinea pig complement, tissue culture medium and, where appropriate, specific rabbit anti-mouse globulin sera are held on coverslips under paraffin oil. The method has been compared with the liquid monolayer (Cunningham) plaque technique at various stages of the primary response. It has been found that the open CMC method is up to eight times more sensitive than the liquid monolayer method for direct plaque-forming cells (PFC), and up to 13 times more sensitive for enhanced or IgG-PFC. Moreover, there is a differential sensitivity, as the ratio of open CMC: liquid monolayer plaque counts varies widely with the stage of the immune response. The new method appears especially good for cells found early in a primary response, i.e., at 1.5–2.5 days for the IgM response, and 4–5 days for the IgG response. The special sensitivity appears to apply to all mouse immunoglobulin classes and with both rabbit anti-mouse globulin sera and mouse anti-allotype sera as enhancing agents. It was noted that, with the open CMC method, IgM-PFC levels were already four times background by 24 hr after antigen and reached peak levels of around 400,000 PFC/spleen by 3.75–4 days after antigen. In contrast, IgG-PFC were first detected at 3.5 days after antigen, and their numbers rose very rapidly to approximately 900,000/spleen at day 4 and 2,000,000 PFC/spleen at day 5. It was shown that micromanipulation transfer of either IgG or IgM-PFC to a second monolayer suitable for their revelation resulted in plaques in 96–97% of cases. Direct PFC found in the first 3–4 days of the response were transferred into (a) anti-μ, (b) anti-γ, or (c) anti-κ-containing monolayers. In case (a) most transferred PFC failed completely to make a plaque and the remainder formed plaques at a greatly inhibited rate. In case (b), transfers were successful and plaque formation rate was uninfluenced. In case (c), the majority of cells were inhibited, but a minority (probably cells synthesizing IgM with λ light chains) were uninfluenced. Enhanced PFC were transferred into direct or anti-κ-containing monolayers. In the former case, a ready discrimination into IgG-PFC, unable to cause lysis, and IgM-PFC could be made. In the latter case, most cells re-formed plaques under the enhancing influence of anti-κ. It was noted that most IgM-PFC formed sharp, clear plaques and most IgG-PFC, turbid, fuzzy plaques. Exceptions in both directions, however, occurred. The theoretical implications of the results were discussed.