Antigen-independent binding of rat immunoglobulins in a radioimmunoassay. Solutions to an unusual background problem

Research output: Contribution to journalArticle

Abstract

A high non-specific binding of immunoglobulins to plastic surfaces was noted with a number of rat sera, when tested in an indirect 125I-labeled protein-A assay for detection of cell-surface-bound rat immunoglobulins of various classes and IgG subclasses. This type of non-specific binding was found with all types of Ig. The degree of binding varied with the type of test plate used and fluctuated with time among sera drawn sequentially from the same donors. Coating test wells with fetal calf serum supplemented with BSA, gelatin or fibrinogen did not eliminate the reactions. The immunoglobulins bind directly to the polystyrene, and not to antigens present in fetal calf serum or autoantigens in rat serum. Two different approaches were used to eliminate the nonspecific reaction. When living cells were used as target antigens, exclusively cell-bound radioactivity was eluted with the non-ionic detergent Nonidet P40, which solubilizes the cell membrane without breaking the protein-A/rabbit IgG, rabbit IgG/rat Ig, or rat Ig/plastic interactions. When rat serum antibodies are tested on target antigens adsorbed on non-tissue culture grade plates, non-specific binding may be avoided by including 0.05% Tween 20 in the incubation mixture.

Details

Authors
Organisations
Research areas and keywords

Subject classification (UKÄ) – MANDATORY

  • Immunology in the medical area

Keywords

  • plastic binding of Ig, non-specific Ig binding in RIA, cell surface antigens, binding test, rat Ig (sub)classes
Original languageEnglish
Pages (from-to)17-25
JournalJournal of Immunological Methods
Volume66
Issue number1
Publication statusPublished - 1984
Publication categoryResearch
Peer-reviewedYes