Preparation and Properties of Anti-carbohydrate Antibodies

The specificity of the antibodies was also verified by hapten-inhibition tests, the results of which are shown in Fig. 8. In these tests decreasing amounts of antigen were used with the pure antibodies and with antibodies that had been treated with potential inhibitors for 2 h. Agar diffusion was used to observe the rate of formation of precipitin bands with the various amounts of antigen. A comparison of the amount of antigen required to 

Electrophoresis and electrofocusing, coupled with agar diffusion, were devised to further characterize the antibodies. The results are presented in Fig. 9. Whereas the antibodies yield single bands on electrophoresis, multiple bands were obtained on electrofocusing. The latter results show that the preparations contain multimolecular proteins. The proteins form 

To determine the nature of monosaccharide residues in an anticarbohydrate antibody, a sample of 2 mg of anti-Gal antibodies is hydrolyzed in 0.2 mL of 0.2 M HC1 by heating in a boiling water-bath for 2 h. 

Diffusion in agar performed by the conventional method shows a strong reaction between the antibody and Man-BSA, but not with BSA (Fig. 12, plates A). However, both compounds give precipitin complex with anti-BSA serum. Oxidation of the antigens with periodate no longer gives a precipitin reaction with the anti-mannose antibodies, but has no effect on the anti-BSA antibodies. Hapten-inhibition results are also shown in Fig. 12, plates C and D. These tests are conducted with the purified antibody 

The inhibition of precipitin formation by 1-thio-D-mannose and derivatives is shown in the results recorded in Plates C, D, E, and F of Fig. 13. The sulfur derivatives, p-nitrophenyl 1-thio-D-mannopyranoside (well Ii) and ethyl 1-thio-D-mannopyranoside (well I3), caused a marked decrease in the amount of precipitin formation, in comparison to that obtained with the native antibodies (Well A,). However, mannose did not decrease the amount of precipitin formation (well I2). This compound did not bind to the combining site of the antibody. Apparently, the thio group at position 1 of the mannose is required for the binding to occur. 

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