Purification of antibodies to membrane antigens of hamster cells transformed by sv40 and binding of the radioiodinated, purified antibodies to intact cells

Eli Kedar*, Aaron Aaronov, Nathan Goldblum, Dov Sulitzeanu

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

7 Scopus citations

Abstract

Antisera to membrane antigens of SV40‐transformed hamster kidney cells were prepared by injecting a membrane fraction of HK7 cells into rabbits. Purified antibodies were prepared by adsorption of the IgG fraction of the antisera onto, and by elation from, living or glutaraldehyde (GA) fixed HK7 cells. Purification from GA‐treated cells yielded highly satisfactory purified antibody preparations, the reactivity of which could be demonstrated in fluorescent antibody tests. These preparations were radioiodinated with 125I to a specific activity of the order of 10μc/μg and their activity was assayed by ability to bind to living HK7 cells. The labelled antibodies maintained their binding ability practically unchanged for at least 6 months. They could be further purified by an additional adsorption‐elution cycle, using living cells. The doubly purified, radioiodinated antibodies, had a high affinity for HK7 cell membranes: 43% of the radioactivity became bound when 0.3 ng eluate was reacted with 1 × 106 cells, as compared to 8% bound to normal kidney cells. The capacity of the purified, labelled antibodies to bind to intact cells was tested under a variety of conditions. Percent binding increased steeply with cell number and was inversely related to the reaction volume. Binding was faster at 37° C than at 4° C. Non‐specific binding of radioactivity was prevented by carrying out the reaction in the presence of protein. Cells from young cultures bound the labelled antibodies more efficiently than cells from old cultures. The purified, radiolabelled antibodies to the neoplastic cell membrane cross‐reacted to a significant degree with normal hamster liver cells. Antibodies to tumor specific antigens could not be detected by the binding tests, but the presence of such antibodies was suggested by the results of autoradiographic experiments. When treated with labelled antibodies and subjected to autoradiography, only the viral transformed hamster or mouse cells, but not the non‐transformed cells, became labelled. The GA‐treated cells were a highly efficient immunoadsorbent for the purification of antibodies against cell membrane antigens. They maintained their integrity for prolonged periods, were resistant to the reagents used in the elution procedure and could be used repeatedly, up to three times.

Original languageEnglish
Pages (from-to)536-547
Number of pages12
JournalInternational Journal of Cancer
Volume9
Issue number3
DOIs
StatePublished - 15 May 1972

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