A new liquid-phase enzyme immunoassay for rabbit IgM antibodies and its application for comparing the specificity of IgM and IgG antibodies contained in the same antiserum.

A new liquid-phase enzyme immunoassay (EIA) has been developed to compare the specificities of IgM and IgG antibodies when they are both present in the same serum sample and directed towards a simple hapten. The hapten viomycin (VM) was used as the model antigen and antibodies to VM were raised in rabbits. In the immunoassay VM, labelled with the enzyme galactosidase as marker (VM-GAL), was mixed with rabbit anti-VM serum. First IgM anti-VM antibodies bound to VM-GAL were precipitated with a guinea pig anti-rabbit IgM serum and galactosidase activity was measured in the precipitate. Then IgG anti-VM antibodies bound to VM-GAL were precipitated from the supernatant with a goat anti-rabbit IgG serum and enzyme activity was measured in this precipitate. The guinea pig anti-rabbit IgM and goat anti-rabbit IgG were specific for IgM and IgG respectively and did not appear to cross-react. Nine analogues of VM were used as inhibitors in this immunoassay to compare the specificities of IgM and IgG antibodies for determinants on VM. The results suggest that recognition of the fine structure of VM by IgM is less strict than recognition by IgG.

Chromatographic analysis of antituberculosis drugs in biological samples.

Numerous chromatographic and non-chromatographic methods of analysis for anti-tuberculosis drugs and metabolites in biological tissues have been discussed in this review. Depending upon the analytical methodology selected, limits of detection range from microgram to picogram levels. A number of examples have been given of the correlation between different types of assay procedures. The metabolism and pharmacokinetics have been described along with some of the commonly associated problems of sample collection and storage.

Enzyme immunoassay of viomycin. New cross-linking reagent for enzyme labelling and a preparation method for antiserum to viomycin.

A new cross-linking reagent of the hetero-bisfunctional type, a N-(maleimidobenzoyloxy)-succinimide (MBS) was prepared and used for enzyme labelling of viomycin under mild aqueous conditions by a two-step process. In the first step a maleimide residue was selectively introduced onto the N1-amino group of viomycin with a limited amount of MBS. The second step consisted of thioether formation between the maleimide residue and free thiol groups of beta-D-galactosidase. An antiserum to viomycin was raised in rabbit by immunization with a viomycin-BSA conjugate. The conjugate was prepared by protecting N6-amino group of viomycin with an acetyl group and succinylating the N1-amino group, activating the carboxyl group by a mixed anhydride method and coupling it with the amino groups of bovine serum albumin (BSA). The specificity of the antiserum was proved by an enzyme immunoassay based on the competition between viomycin and its enzyme conjugate toward diluted solutions of the antiserum. By use of the viomycin-enzyme conjugate and the antiserum to viomycin, enzyme immunoassay of viomycin was successfully performed by the competitive binding procedure with the double-antibody method, and 0.1 to 4 ng of the antibiotic could be detected.