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. 1983 Mar;14(3):191–195. doi: 10.1007/BF00205359

The use of cellular immunoadsorbents to prepare polyclonal antibodies that distinguish between antigens derived from human melanoma cells and autologous lymphocytes

Peter J Kelleher 1, Herbert L Mathews 1, George E Moore 1,3, Percy Minden 1,✉
PMCID: PMC11039103  PMID: 6601511

Abstract

Cellular immunoadsorbents were employed to isolate xenogeneic antibodies that reacted with a restricted group of antigens on human melanoma cells. Melanoma cells and autologous lymphoid cells were grown in tissue culture. Cellular immunoadsorbents were prepared by coupling formalin-treated melanoma and lymphoid cells to diethylaminoethyl cellulose. Rabbits were immunized with melanoma cells and antisera were passed sequentially through immunoadsorbents made of fetal bovine serum, and lymphoid cells. Unbound effluents were then passed through an immunoadsorbent prepared with melanoma cells. Antibodies binding to melanoma cells were eluted and their reactivity to melanoma-derived antigens was tested using a solid-phase radioimmunoassay. Antigens for this assay were NP-40 lysates of melanoma and lymphoid cells and fetal bovine serum. Radioactive Staphylococcal protein A was used to detect binding by the antibodies to the test antigens.

The effects of formalin-fixation and storage of melanoma and lymphoid cells were studied. Storage of fixed melanoma cells for periods up to 4 months did not affect their capacity to bind antibodies. A single exposure of formalin-fixed cells to a low-pH elution buffer which was followed by neutralization did not affect binding by these cells. Antibodies isolated in this manner were of the IgG class and reacted with antigens derived from melanoma cells but not from autologous lymphocytes or fetal bovine serum. This study demonstrated the feasibility of using cellular immunoadsorbents to prepare xenogeneic polyclonal antibodies with a high degree of reactivity to antigens derived from human melanoma cells.

Keywords: Cellulose, Melanoma, Fetal Bovine Serum, Tissue Culture, Polyclonal Antibody

Footnotes

Abbreviations used in this paper: TAA, tumor-associated antigens; 125I-SPA, radiolabeled Staphylococcal protein A; FBS, fetal bovine serum; PBS, phosphate-buffered saline; BB, borate buffer; SPA, solid-phase assay; anti-mel, antibodies to melanoma cells; anti-mel(FBS), anti-mel absorbed with FBS; PAM, ‘purified’ anti-mel(FBS); IgG, immunoglobulin G; PVC, polyvinyl chloride.

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