Separation of the Phase I Agglutinogen of H. Pertussis from Toxic Components
Authors: Flosdorf Earl W. , Kimball Anne C. 
Published: January 2023

Summary
A method of separation of the agglutinogen from phase I, H. pertussis is described. About ten per cent of all the original agglutinogen present in the organisms may be concentrated into a fraction which represents but one per cent of the total solid content of the original sonic extract of the organisms. In the purified form the agglutinin-absorbing fraction elicits agglutinins in rabbits; upon intracutaneous testing it yields an immune response in rabbits which have been immunized with whole phase I organisms or the purified fraction. The agglutinogen has been freed of toxins; in normal animals marked primary toxicity is absent as determined by survival of animals after intravenous injection and by intracutaneous testing. This is in contrast with the primary toxicity found present when whole organisms or less pure fractions are used. The purification also results in a fraction showing specificity of intradermal response in rabbits immunized with phase I as contrasted with phases III and IV, H. pertussis and with parapertussis and unrelated organisms. Chemical studies indicate that the agglutinogen is probably protein in nature but one showing unusual characteristics, e.g., a high degree of thermostability over a wide range of pH. The extremely thermolabile toxin of Evans and Maitland has been detected. This thermolabile toxin is precipitated at pH = 4.5 without detectable loss of activity and may thereby be separated from the agglutinogen except that it adsorbs some agglutinogen. The agglutinogen is very readily adsorbed on all precipitates produced by lowering the pH, as well as on bacterial filters.
Note: Since going to press a very interesting article has appeared by Dr. Mary Lee Wood, “A Filterable Toxic Substance in Broth Cultures of B. Pertussis,” Jour. Immunol., 1940, 39, 25. From the properties described it would appear that it is non-antigenic TLT which is the toxic component of the filtrate. As suggested by Dr. Wood, this component would appear to exist on the surface of the organism from which it passes into the filtrate. From the quantitative relations of the work of Evans and Maitland and from the fact that we have separated TLT equally well from extracts of organisms that were either unwashed or well washed before disintegration, this toxin must also exist throughout the organism as well as at the surface.
More recently, D. G. Evans has reported (Jour. Path. Bact., 1940, 51, 49) this endotoxin to be antigenic when extracted and formolized, but not while in the intact organisms as injected into experimental animals or as in the parasite causing disease in man (no antitoxin found in convalescent serum). He has obtained neutralization of the intracutaneous reaction in rabbits previously injected with the extract. We, like Evans, have not observed neutralization in rabbits immunized with live whole organisms, even following immunization sufficiently intense to yield sera of agglutinative titer of 1:100,000. Our SX-I-immunized rabbits, accordingly, were not so tested. For an answer to this seemingly anomalous situation, where a part would appear to be greater than the whole, further investigation is indicated. If TLT is antigenic, it may be that it is but very slightly so, and that the subcutaneous route of immunization, in this case, is favorable so that antitoxin is produced in amounts which are detectable. Whatever the explanation, however, immunization of children against a toxin would not appear to be the method of choice when agglutinative immunization is available against a non-flagellated organism, occurring as a single type, to halt invasion initially. Toxic immunization would guard merely against one of the actions of the invaders should invasion occur.