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glutinin (HA) is now being widely employed in assay of rubella antibody by hemagglutination-inhibition test in place of the time-requiring procedures for infectivity neutralization (Parkman et al, 1964). Trials to demonstrate HA in rubella infected primary green monkey kidney cells, Vero cells (an established cell line from green monkey kidney, Yasumura and Kawakita, 1963, Liebhaber, Riordan and Horstmann, 1967) and an rabbit cornea cell line SIRC cells (Leerhð“y, 1966) revealed that only Vero cells produce HA of a low level when the infected cultures were carefully maintained for more than 20 days. In experiments concerning HA production by rubella virus, we used maintenance medium without supplement of calf serum, because it usually contains inhibitory sub stance (s) to rubella HA (Stewart et al, 1967). The removal of calf serum from the culture medium immediately after infection, resulted in a spontaneous degradation of cells within a week, and consequently in a low yield of HA. In order to improve the viability of BHK-21 cells in the absence of calf serum and to obtain better yields of HA : clonal selections of the cells were performed after the method described by Puck, Marcus and Cieciura (1956). The results are presented in this communication. M33 strain of rubella virus had been passed 32 times through green monkey kid ney cells, twice through cynomolgus monkey kidney cells and 16 generations through BHK-21/WI-2 cells (Vaheri et al, 1965), before supplied from Dr. Ohtawara of this laboratory. The virus was passed four times or more in BHK-21/WI-2 cells before use in this experiment. The virus seed was a maintenance media of BHK-21/WI-2 cells pooled on the 5-6 day of infection with rubella virus. It was divided into small vials after a clarification by low speed centrifugation, and stored at 80 C. BHK-21/WI-2 cells, passage history of which was unknown, had been maintained in this laboratory for more than one year by Dr. Ohtawara. For growth and maintenance of BHK-21 cells, Earle's balanced salt solution con taining 0.5 % lactalbumin hydrolyzate, 0.1 % yeast extract, 100 ƒÊg/ml of streptomycin, 100 units/ml of penicillin and 5 % calf serum was used. The cells were grown in a medium containing calf serum until a confluent monolayer was formed. Then, they were inoculated with virus at the highest possible multiplicity of infection (moi), placed for 2 hr at room temperature, washed well with phosphate buffered saline (PBS) and fed with culture medium without calf serum. HA titration was performed in test tubes. Serial twofold dilutions of HA were made in 0.2 ml of PBS of pH 7.4 containing 0.1 % bovine serum albumin (Armour's fraction V). Two-tenths ml of goose red blood cells (RBC) suspension in PBS (OD at 490 mƒÊ , 1 cm light path =0.45) was added to each tube and incubated at 7 C for 1 hr. HA units were determined as a reciprocal of the highest dilution that gave complete aggluti-

ESTABLISHMENT OF A CLONE OF BHK-21 CELLS FAVORABLE TO RUBELLA HEMAGGLUTININ PRODUCTION