Masayasu Kitagawa1, Yasuo Yagi1, David Pressman1
1Department of Biochemistry Research, Roswell Park Memorial Institute New York State Department of Health From the , 3, Buffalo 3, New York
Tóm tắt
Summary
Anti-Xp antibodies were fractionated by adsorption on immunoadsorbents and elution with haptens.
Different types of immunoadsorbent and various programs of elution with either different concentrations of a particular hapten or different haptens were used. In several cases, the eluted antibody was evaluated further with respect to heterogeneity by re-fractionation.
Some anti-Xp antibodies adsorbed on Xp-RSA-PAS could be eluted by o-substituted benzoate (0.3 M). Others not eluted by the first procedure were eluted by m-substituted benzoate and still others were eluted only by p-substituted benzoate. The observed order of effectiveness of substituents on elution was p-NO2 > p-Cl, m-NO2 > m-Cl > o-NO2 = o-Cl. There were some antibodies that were eluted by m-nitrobenzoate but not by p-chlorobenzoate and other that were eluted by p-chlorobenzoate but not by m-nitrobenzoate.
Very clear evidence was obtained that some of the antibodies could well accommodate an o-substituent on the hapten whereas other antibodies could not.
Each program of elution produced fractions with different properties. This indicated the heterogeneity of the original group of antibodies. However, each one of these fractions, when subjected to refractionation with a second program of elution, reflected the heterogeneity of the original population of antibody molecules going through this same second program.
The elution patterns were also characteristic of the immunoadsorbent. Almost all of the antibodies could be adsorbed on Xp-RSA-PAS, o-ClXp-RSA-PAS and m-NO2Xp-RSA-PAS but were eluted more easily by p-nitrobenzoate from the immunoadsorbents in the order, o-ClXp-, m-NO2Xp and Xp-RSA-PAS. X′-RSA-PAS and Xp-R-PAS failed to adsorb all antibodies and a part of the antibodies which were adsorbed was eluted more easily from these immunoadsorbents than from Xp-RSA-PAS.
The results provide evidence that some antibodies are directed not only to the haptenic group but also to the protein portion surrounding the haptenic group.
The amount of antibody was determined by labeling it with I131 or I125 and measuring the radioactivity. Effort and time could be saved and very close comparisons could be made by using the paired label technique in which one antibody fraction was labeled with I131 and the other labeled with I125 and the two passed simultaneously through the same column.
