[ANALYSIS OF EFFECTIVENESS OF USINGJIgY FROM CHICKEN IN SANDWICH METHOD OF HBsAg TESTING].

AIM: Study antigen-binding ability of polyclonal antibodies (PCA) of chicken compared with monoclonal -antibodies (MCA) of mice in the model of interaction with HBsAg. MATERIALS AND METHODS: Mice MCA 18C8 and MKA F3/F4 (IgG) were used, effective in enzyme immunoassay sandwich method of HBsAg determination (with a minimal detection dose of 0.017 ng/ml), and affinity purified anti-HBsAg PCA of chicken (IgY), obtained from 2 immunized birds (PCA No. 1 and PCA No. 2). The ability of antibodies to bind HBsAg was evaluated by analytical sensitivity (slope of binding curve) of solid-phase enzyme immunoassay system using mice MCA and chicken PCA. RESULTS: PCA No. 2 has provided a statistically significant 40% increase of analytical sensitivity, compared with <

[Effect of pH of Adsorption Buffers on the Number and Antigen-Binding Activity of Monoclonal Antibodies Immobilized on the Surface of Polystyrene Microplates].

The change in the concentration and antigen-binding activity of 28 monoclonal antibodies was studied after their adsorption on the surface of polystyrene microplates in buffers with different pH values (1.0, 2.8, 7.5, 9.6, and 11.9). We used 16 clones to the HIV p24 protein and 12 clones to the surface antigen of Hepatitis B Virus. The binding efficiency of adsorbed antibodies to the labeled antigen was evaluated by the slope of the linear region of the binding curve to the concentration axis. It was shown that the antigen-binding activity of six antibodies (21.5%) statistically significantly increased after adsorption at pH 2.8 and 11.9 as compared to pH 7.5 and 9.5. The maximum amount of antibodies was found to be adsorbed on the solid surface at pH 7.5. The analysis of the binding of 125I-HBs-antigen to adsorbed antibodies made it possible to evaluate the concentration of active antibodies on the polystyrene surface. It was shown that the increase in the antigen-binding activity was due to an increase in the proportion of antibodies with retained activity after adsorption at pH 2.8 and 11.9. Under these conditions, about 20% of the antibodies retained their antigen-binding activity, and 6% did so after immobilization at pH 7.5.

Analysis of serotonin transporter in human platelets by immunoblotting using site-specific antibodies.

We have produced a panel of site-specific antibodies recognizing different regions of the human serotonin transporter (SERT). This panel included: 1) monoclonal antibodies 23C5 (mAbs 23C5) to the C-terminal region (amino acid residues 597-630); 2) polyclonal antibodies (pAbs) to the N-terminal region (amino acid residues 69-83); 3) pAbs to the region (amino acid residues 86-100) in the beginning of the first transmembrane domain (TMD). The antibodies were produced using recombinant proteins and synthetic peptides (containing certain sequences of SERT) as antigens. These antibodies were purified by affinity chromatography, conjugated to horseradish peroxidase (HRP), and used for immunoblotting analysis of SERT in extracts of human platelets. Sodium dodecyl sulfate extracts were prepared under conditions preventing non-specific proteolytic degradation of the proteins. In platelet extracts, all antibodies were able to detect the 67 kD protein, apparently corresponding to full-length SERT molecule (its theoretical mass is about 70 kD). These antibodies also detected several polypeptides of smaller size (56, 37, 35, 32, 22, and 14 kD), apparently corresponding to N-terminal, C-terminal, and non-terminal SERT fragments. Specificity of immunostaining was confirmed by preincubation of HRP-labeled anti-SERT antibodies with excess of corresponding antigen, which resulted in disappearance of protein band staining. It is suggested that SERT undergoes a programmed proteolytic cleavage (processing) resulting in formation of several SERT-derived polypeptides of smaller size. It is possible that one of the cleaved SERT species is required for serotonin transport activity. Possible sites for specific proteolysis may be located in the region near TMD1 and in the intracellular loop between TMD4 and TMD5.