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A procedure for the routine and simultaneous laboratory detection of IgG antibodies produced in humans in the course of various infectious diseases is described. The procedure, based on dot-enzyme-linked immunosorbent assay (ELISA), used single nitrocellulose strips onto which several antigens were dotted in close proximity. Optimal conditions were specified that allowed the unequivocal and simultaneous detection of IgG antibodies specifically directed against Toxoplasma gondii, rubella virus, cytomegalovirus, and herpes simplex virus type 1 and type 2 antigens. This technique has proved to be simultaneously specific, sensitive, and reliable, and it has been applied to prenatal screening of sera from pregnant women. It is suggested that this technique should also be used for the screening of large numbers of sera under field trial conditions.

The tyrosyl residues of tetanus toxin can be specifically nitrated by tetranitromethane. The introduction of NO2 groups into the toxin molecule is followed by a rapid decrease in toxicity. Although tryptophyl residues are differently involved and some aggregation occurs, these two phenomena do not take any significant part in the toxicity loss. The immunological reactivity in vitro is preserved. However, it is modified when a certain amount of NO2 groups have been introduced. The thus-formed derivative no longer gives a reaction of identity with the native toxin. Furthermore, when a sufficiently large number of NO2 groups have been introduced, two lines of precipitation are seen. Antisera raised in rabbits with a few nitro-derivatives of the toxin exhibit a low avidity. Precipitating power with pure toxin of these antisera is lower than with a normal antitetanus serum. The capability of tetanus toxin to give rise to neutralizing antibodies is not impaired until 33 NO2 groups have been introduced, then it gradually decreases until it finally disappears. Modification of the toxin by nitration allowed us to discriminate three groups of determinants. In particular, the results presented are further substantiated by those given by 3-aminotyrosyl derivatives of tetanus toxin.

The amino acid analysis of tetanus toxin as purified from bacterial extracts or from culture filtrates of Plectridium tetani, was carried out. The results that have been obtained support the assumption of the identity of both types of toxin. Tetanus toxoid amino acid composition was comparatively determined. Significant decreases in the amounts of some amino acids were found. These deficits are accounted for within the reactions with formaldehyde which lead to the formation of Mannich-like derivatives. The presence of 6 free SH-groups on the molecule of toxin could be substantiated. Two of these groups are difficult to reveal. The existence of two disulfide bridges was established. These results are discussed, in particular, with regard to the expression of the toxicity. The presence of only one N-terminal amino acid residue could be revealed.