ON CROSS REACTIONS OF IMMUNE SERA TO AZOPROTEINS

The phenomena observed on absorption of immune sera to azoproteins with heterologous azoantigens lead to the conclusion that in general the sera do not contain a single antibody but antibody fractions somewhat different in their reactivity for heterologous antigens. From the constitution of the azocomponent it follows that these fractions cannot be specific for distinct chemical groups in the molecule. In fact, inhibition experiments showed that in the cases examined the various antibodies are all specifically directed towards the whole molecule of the homologous hapten

ON THE SPECIFICITY OF SEROLOGICAL REACTIONS WITH SIMPLE CHEMICAL COMPOUNDS (INHIBITION REACTIONS)

Experiments are described which confirm the result that the specificity of inhibitory reactions involving substituted aromatic acids is decidedly influenced by the position of the substituent. When antigens with specific groups of very simple constitution are used for the tests, inhibiting effects are obtained also with substances distantly related to those determining the reactivity of the antigens. On the other hand, if antigens are built up from protein and chemical compounds of somewhat higher complexity, the specificity of the inhibition reactions with synthetic crystallized substances is of the same order as that of the usual serum reactions; in other words, it is possible to distinguish such compounds by serological tests as readily as proteins can be differentiated with the aid of precipitating sera

SEROLOGICAL DIFFERENTIATION OF STERIC ISOMERS (ANTIGENS CONTAINING TARTARIC ACIDS) : SECOND PAPER

In continuation of studies on the stereochemical specificity of serum reactions, antigens were examined containing the acyl radicals of the levo-, dextro-, and meso-tartaric acids. It was found that in this case also, immune sera can readily be obtained which differentiate sharply the three antigens identical in every other respect but possessing stereoisomeric groups. Since the tartaric acids by their chemical constitution belong to the same class of substances as sugar acids the results have a bearing upon the question of the specificity of natural antigens containing carbohydrates such as have been described by Avery and Heidelberger

ANAPHYLACTIC SHOCK BY AZODYES. II

From the experiments presented, it follows that the specific precipitation and the production of anaphylactic shock with certain azodyes, as described previously, is due to these substances themselves and is not dependent upon formation of azoproteins by interaction of the dyes with proteins in the test tube or the animal body. Besides these, some other azodyes which in our tests did not give precipitation with corresponding immune sera were also found, in very small quantities, to induce anaphylactic contraction of the uterus of sensitized guinea pigs

ON CROSS REACTIONS OF IMMUNE SERA TO AZOPROTEINS : II. ANTIGENS WITH AZOCOMPONENTS CONTAINING TWO DETERMINANT GROUPS

Azoproteins have been prepared with azocomponents possessing two serologically active groups. On immunization with such antigens immune sera were obtained containing two separate, unrelated antibodies, each specific for one of the two groups and separable by absorption. In other cases one of the two structures was dominant, in that antibodies were formed only towards this and not towards the other grouping. The specificity of the antibodies was in general found to be influenced to some extent by the presence of a second group in the antigen. The relevancy of these observations for antibodies directed against natural antigens has been noted

SEROLOGICAL REACTIONS WITH SIMPLE CHEMICAL COMPOUNDS (PRECIPITIN REACTIONS)

Experiments are described demonstrating the precipitation of azodyes by immune sera prepared by the injection of azoproteins containing the same azo component. These precipitin reactions prove conclusively the view already advanced on the basis of inhibition reactions that antibodies combine specifically with substances of small molecular weight. Although in this respect both phenomena have the same significance, the precipitin reactions with dyes are simpler in that the aid of a protein antigen is eliminated. That specific serological precipitin reactions can take place with substances other than proteins has been amply demonstrated by studies on bacterial antigens (14) (polysaccharides (15)). The present findings show that for the precipitation with immune sera not even a high molecular weight of the reactive substance is required. Factors determining the tendency to separate out from the liquid upon combination with antibody seem to be the colloidal state of the solution and the chemical composition of the substance. With regard to the influence of chemical composition, a striking example is provided by the suberic acid dye which gives particularly strong precipitin reactions, most probably on account of its long aliphatic side chains. The results reported may be of use for studies on the mechanism of serological precipitation

ON THE SEROLOGICAL SPECIFICITY OF PEPTIDES

With the idea that studies on the serological properties of peptides may ultimately aid in the understanding of the precipitin reactions of proteins, antigens have been prepared containing aminobenzoylated dipeptides, namely glycyl-glycine, glycyl-d, l-leucine, d, l-leucyl-glycine and d, l-leucyl-d, l-leucine. These four antigens were found to be different serologically, their specificity depending on the structure of the terminal amino acid carrying the free carboxyl group, and to a less degree also on the second amino acid. The results were obtained by means of precipitin and inhibition tests. Analogies to observations on enzyme specificity are discussed

ON THE SEROLOGICAL SPECIFICITY OF PEPTIDES. III

Experiments are described dealing with immune sera to pentapeptides and peptide amides. Absorption and inhibition tests gave no indication of the presence in the immune sera of special antibodies for portions of a peptide molecule but the antibodies appeared to be specific for an entire pentapeptide even though the sera contained qualitatively different fractions. Marked disparity was found between the reactions of peptides and corresponding amides indicating differences between acid and other polar groups in their influence on serological specificity

SEROLOGICAL DIFFERENTIATION OF STERIC ISOMERS

The tests presented in Tables I, II, and III show that only in the higher concentration cross reactions do take place, and that there is definite specificity of the two sorts of immune sera for the homologous antigens. Thus it is easy to differentiate the l- and d-antigens in dilutions 1:100 and upwards. The occurrence of cross reactions can readily be ascribed to the fact that the l- and d-acids present in the two antigens are identical in every respect but the position of the groups connected with the asymetric carbon atom. The i-antigen reacts with both sorts of immune sera as could be expected since it must consist of a mixture of equal parts of l- and d-antigen. The reactions of the i-antigen appear to be only slightly weaker than those of the homologous ones owing to the fact that the intensity of the reactions diminishes but slowly with increasing dilution of the antigens. It is also to be considered that small differences cannot be judged very accurately. Tests with two l- and four d-immune sera not recorded in the tables confirmed the results already discussed. Considering that ferments are known to be adapted ordinarily to one type of steric isomers it may be worth noting that antibodies were formed by the same species of animals for optical antipodes. From the results summarized in Tables IV,a and IV,b one sees that the l- and d-immune sera also differentiate clearly between the l- and d-acids when they are not diazotized and not combined with protein. The l-acid inhibits much more the precipitation of the l-antigen by the homologous immune serum than the d-acid and the converse effect occurs if the inhibiting action is tested on the precipitation of d-antigen by d-immune serum. The inactive phenyl (para-aminobenzoylamino) acetic acid behaved in such tests as a mixture of l- and d-acids, i.e., it acted markedly in both cases, more than the heterologous and less than the homologous acid. The experiments reported bring a definite proof for the view that the steric configuration of antigenic groups is one of the factors determining serological specificity. In the particular case under consideration the mere difference in the position of H and COOH as indicated in the following formulas sufficed to alter the reactivity. See PDF for Equation. The fact that steric isomers are acted upon selectively by immune sera may be supposed to play a significant part in the serological specificity of carbohydrates such as those discovered in bacterial antigens

SEROLOGICAL STUDIES ON AZOPROTEINS : ANTIGENS CONTAINING AZOCOMPONENTS WITH ALIPHATIC SIDE CHAINS

A study was made of the specificity of artificial compound-antigens containing aliphatic chains. Striking specificity was exhibited in the reactions of compounds with short chains containing a carboxyl group; for instance succinic acid could be differentiated from malonic or glutaric acid which contain one fewer and one more carbon atom respectively. With the substances containing longer chains, reactions were observed which, although specific to a certain extent, appear to depend mainly on the general physicochemical properties of long aliphatic chains. With a limited number of substances the influence of substituents —halogen, OH, NH2—on the serological specificity was investigated. By means of inhibition reactions it was possible to demonstrate the serological specificity of cis-trans isomers such as maleic and fumaric acid