The Soluble Protective Antigen and the Histamine-Sensitizing Factor of Bordetella Pertussis

Abstract

A simple method is presented for the isolation of the protective and histamine-sensitizing factor (HSF) activities of Bordetella pertussis. HSF was used as a presumptive “tracer” for protective activity in developing the method, based on the close association of these activities. Ten to 30 g of acetone-dried cells were repeatedly blended with glass microbeads and renewed diluent through 5 to 26 cycles. The pooled lysates, clarified at 37,000 × G for 3 hr, were treated with 35% SAS and the precipitate dialyzed. This fraction was high in HSF activity. Since HSF precipitated at low salt, and was excluded from Sephadex G200, it is probably a large molecule. Loss of activity on treatment with trypsin, as well as behavior with ammonium sulfate, indicated it is probably proteinaceous. The method gave an 8- to 17-fold increase in the specific HSF and protective activities on a nitrogen basis. These activities correlated in the main fractions, thus confirming the work of others indicating that they are either due to the same substance, or are inseparable by a variety of methods. The probability that the activity yield would depend on the efficiency with which cell stromas could be made to undergo particle size reduction in relation to the threshold of centrifugal selection was discussed. Also discussed was the relative significance for side reactions in human immunization of HSF on the one hand, and the nonspecific macromolecular substances eliminated by fractionation on the other.