The following summarises the findings elicited from the investigation, the significance we attach to the results and the conclusions we have drawn:

A careful study has been made of the reputed Wassermann reaction exhibited by theserum of certain normal animals with a view to throwing light on the nature of the reacting substance or principle in syphilitic serum.

The heated (55° C.) serum of various normal adult animals may fix complement along with the antigen used in the Wassermann test, e.g. rabbit, ox, sheep, horse, mouse, cat, dog, macacus, pig.

The complement-fixation reaction may be associated with the flocculation effect similar to that of syphilitic serum.

These reactions apparently represent a natural or normal property of the serum and do not appear to depend on any pathological condition.

Certain species, including man, are characterised by negative reactions, e.g. white rat, guinea-pig, frog.

The serum of the white rat in the unheated state, however, yields a definite but weakly positive flocculation reaction which is annulled on heating the serum to 55° C., showing that this species is not devoid of the particular principle. In some species there is great uniformity in the occurrence of both reactions (e.g. rabbit, ox, sheep, horse). Such uniformity has led us to regard the reaction as a natural one. In other species there is less regularity and both reactions in certain individuals may be quite negative. In certain animals there may be dissociation of the two reactions: the Wassermann reaction may be negative while the flocculation effect is positive (e.g. pig, pigeon, fowl, cat): the reverse may occur, the Wassermann reaction being positive, the flocculation test negative (e.g. dog, mouse, rabbit).

Certain positively reacting species are characterised by uniformly weak reactions, e.g. mouse.

The Wassermann reaction in normal animals is always limited in degree, contrasting in this respect with the very marked effects obtained with the serum from cases of active syphilis.

In species in which adult animals exhibit positive reactions, very young animals, e.g. up to 3–8 weeks of age, react negatively. The reacting power (observed in rabbits) is thereafter progressive in development and is parallel with the development of a natural antibody (anti-sheep haemolysin). This parallelism supports the view that these reactions are due to antibody-like principles in the serum.

No constant difference in thermostability of the reacting substances can be established between normal animal serum and the serum in syphilis. The degree of thermostability seems to vary slightly among individuals. The flocculating property is more stable than the complement-fixing principle both in animals and in syphilis.

These reactions in normal animals seem to be homologous with the corresponding reactions in syphilis. Our observations suggest that the syphilis serum reactions are due to antibody-like substances homologous with antibodies natural to certain species and widely distributed among animals.

The Wassermann and flocculation reactions in individual rabbits tested at intervals are relatively constant.

Marked augmentation of the Wassermann reaction has been produced experimentally in rabbits: (a) by immunisation with heterophile antigen, as originally shown by Taniguchi; (b) by experimental infection with B. tuberculosis. The effects of other non-specific agents have also been studied but marked alterations have not been observed.

Repeated subcutaneous injection of alcohol in rabbits abolishes the Wassermann property. The injection of certain other chemical substances also produces a weakening influence. Parallel effects are observed on the antisheep haemolysin.

An analytical study has been made of the Wassermann and flocculating substances in the serum of normal animals and human syphilitic serum. For this purpose carbon-dioxide fractioning of the serum has been used. The results have been recorded and differences have been elicited in the behaviour of the serum fractions between normal animals and human syphilitics. These differences probably depend on the total content of the reacting substances in the serum and their distribution in the serum fractions.

The carbonic-acid-insoluble fraction of normal human serum and the serum of negatively reacting animals even after heating at 55° C. may yield a weak complement-fixation effect with the Wassermann antigen. This property is “masked” in whole serum. It is lost on heating above 60° C. and is more marked in the unheated fraction.

The deviation effect produced by the unheated fraction is contributed to by an independent complement-deviation reaction with the alcohol present in the antigen suspension but is still manifest with alcohol-free antigen preparations, showing it is partly a true reaction with the lipoids proper. This effect of the carbonic-acid-insoluble fraction has not been paralleled in the flocculation test unless in certain exceptional instances.

These observations seem to indicate that the principle in serum responsible for the Wassermann reaction is present normally in minimal amount and in a masked state even in those animals which are negative reactors when tested in the usual way. It might be supposed, therefore, that the diagnostic reaction is due to the non-specific augmentation of this natural antibody-like substance.

The influence of various non-specific agents and different infective conditions on the content of the reacting substances in the serum of normal animals requires further investigation.

The complement-deviation reaction of fresh serum with diluted alcohol (substituted for antigen) has been studied in animals and man. The fact has been elicited that the heated carbonic-acid-insoluble fraction of the serum of animals (positive in the usual Wassermann test) and of syphilitic sera yields, along with diluted alcohol, a definite though weak complement-deviation reaction. This may also occur with heated whole serum. This effect is absent from heated normal human serum and the heated serum of “Wassermannnegative” animals. This reaction shows some analogy with the Wassermann reaction and the alcohol of the Wassermann antigen suspensions seems to be a contributory factor to the total complement-fixation in the Wassermann test. If heated serum is used, no fallacy is introduced in the diagnostic test even when the concentration of the alcohol is 1: 12.

The dissociation of the Wassermann and flocculating properties in syphilitic sera is evidenced as a result of carbon-dioxide fractioning—the soluble fraction being more active in the flocculation test, the insoluble moietybeing more active in the Wassermann test, though in some cases the dissociation is less obvious and may be quite absent.

It is suggested from the observations recorded that the Wassermann syphilis reaction represents an increase of a “lipoidophile” antibody naturally present in the serum in minimal amount and in a masked state, which in syphilis is non-specifically augmented in an analogous manner to the augmentation of the natural anti-sheep haemolysin by heterologous stimuli.