Precipitation of Pertussis Immunogen with Benzathene and Proflavine

Abstract

Two years ago I described a method for obtaining soluble preparations of pertussis mouse protective antigen (immunogen) by lysing the bacterial cells with sodium deoxycholate¹. I now wish to report the partial purification of these extracts, using the organic bases benzathene and proflavine as precipitating agents. In a typical experiment with the former, deoxycholate lysate of pertussis derived from a cell concentration of 320 opacity units (OU) per ml. was treated dropwise at pH. 7.4–7.5 at room temperature with a 14 per cent (w/v) solution of benzathene (dibenzylethylene diamine acetate). The mixture was shaken continuously and its optical density monitored until maximum density, indicative of maximum precipitation, was reached, after which 10 per cent excess of benzathene was added. There was an inverse relationship between the concentration of bacterial lysate and the amount of benzathene needed for maximum precipitation. Thus 1.0 ml. of 40 OU/ml. of lysate required 3.25 mg benzathene, 1.0 ml. of 160 OU/ml. needed only 1.5 mg, 1.0 ml. of 320 OU/ml. needed 0.7 mg, and 1.0 ml. of 800 OU/ml. required 0.37 mg. After 2 h the precipitate was centrifuged off and resuspended in tris–chloride buffer. Table 1 shows the mouse protective activity^2–4 of the benzathene precipitate and of the supernatant from the benzathene precipitation compared with a reference vaccine. It is clear that all detectable immunogen was in the insoluble fraction, which was found also to contain virtually all the histamine-sensitizing factor (HSF) and the pyrogen. Gravimetric estimations showed that the benzathene–pertussis precipitate represented 7 per cent of the dry weight of the original live cells.