The potentiality of antibody-producing cells. II. Evidence for two antibody molecules of different specificities secreted by micromanipulated bispecific mouse spleen cells.

Bispecific PFC appearing on the 4th day after immunization of mice with sheep erythrocytes conjugated with trinitrophenyl hapten (TNP-SRBC) were identified by their capacity to lyse native SRBC and TNP-conjugated horse erythrocytes (TNP--HoRBC) simultaneously in an open carboxymethylcellulose medium. Individual PFC thus detected, were micromanipulated into two successive media containing the indicators SRBC and TNP--HoRBC. Out of 103 transferred double cells ninety-two (89 per cent) remained double in the second medium and of the ninety-two transferred into the third medium, fifty-four (58 per cent) provoked a double lysis, eighteen (19 per cent) provoked a single lysis and twenty (23 per cent) ceased to lyse. On the other hand, when the second medium contained the indicators and the soluble specific inhibitor (TNP--BSA), out of 156 transferred double PFC, 125 (80 per cent) became single and only nineteen (12 per cent) remained double. Of these cells, 136 were transferred into the third medium (not containing inhibitor) and here again sixty-five (48 per cent) became double, forty-nine (36 per cent) remained single and twenty-two (17 per cent) ceased to provoke any lysis. Other double PFC were micromanipulated from the original revealing medium into two successive media containing only one indicator and the homologous (second medium) or the heterologous third medium) soluble inhibitor (TNP--BSA or soluble SRBC antigen), in order to see whether a soluble inhibitor suppresses only the corresponding specific lysis. Out of 186 double PFC transferred into the media containing the indicator and the corresponding inhibitor 147 (79 per cent) were specifically inhibited, whereas out of 176 double PFC transferred into the third medium (in which about 20 per cent of the cells cease to function) containing one indicator and the unrelated inhibitor, 117 (66-6 per cent) lysed the indicator in spite of the presence of the unrelated inhibitor. Since a specific inhibitor suppresses the lysis of the corresponding indicator, whereas its presence does not interfere with the lysis of the unrelated indicator in the majority of double PFC, it can be concluded that these cells secrete two types of antibody molecules possessing different specificities.