Tetanus antitoxin titration by haemagglutination

Abstract

The behaviour of sheep cells sensitized with tetanus toxins and toxoids in the presence of antitoxin has been investigated.Sensitized sheep cells were agglutinated in the presence of tetanus antitoxin. The agglutinin titres of such direct agglutinin systems were roughly proportional to the antitoxin content of the sera with some marked exceptions. The discrepancies were associated with the presence of non-specific agglutinins (antibodies other than specific antitoxin) and with the immunization history of the horses providing the sera.When sensitized cells were used as indicators of the presence of free antitoxin in toxin or toxoid-antitoxin mixtures, the correlation with in vivo tests was quite good at the L+ level of test, but not so reliable at the 0·01 L+ level of test.Sensitized cells preserved with formalin were found to be more reliable and the results with such materials were more reproducible than those obtained with freshly prepared sensitized cells.Cells sensitized with different concentrations of antigen gave different titres by direct agglutination with simple dilution of antitoxin; this difference was very much less in haemagglutination inhibition tests.The LA dose (the volume of toxin or toxoid which when mixed with one unit of antitoxin gave a + + ± end-point) varied according to the sensitivity of the suspension used and the purity of the components of the reacting mixtures.Prozones (zones of agglutination inhibition in the region of antibody excess) were found to be associated with the immunization history of the horses from which the sera were obtained. In general the more thorough the immunization the longer the prozones found. The length of prozones was proportionately increased with decrease in the concentration of sensitizing antigen. Possible reasons for the behaviour of certain antitoxins in these tests have been discussed.I am indebted to Prof. M. Raynaud of the Pasteur Institute, Garches, for the supply of tetanus toxoids of high purity.I am very grateful to Mr Charles Newman for considerable technical assistance.