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Binding of a mouse monoclonal IgE (anti‐DNP) antibody to radio‐derivatized polystyrene‐DNP complexes
Author(s) -
Varga Janos M.,
Klein Georg F.,
Fritsch Peter
Publication year - 1990
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.4.9.2347467
Subject(s) - monoclonal antibody , chemistry , polystyrene , antibody , immunoglobulin e , microbiology and biotechnology , immunology , organic chemistry , biology , polymer
Polystyrene (PS) labware products (microtiter plates, test tubes, etc.) show radiation (gamma) dose‐dependent carbodiimide (EDC)‐mediated uptake of carboxyl‐containing molecules such as DNP‐[ 3 H]Gly. The presence of water during irradiation increases, and oxygen decreases the coupling capacity of irradiated PS. The EDC‐mediated attachment of DNP‐Gly to PS is stable because it resists prolonged exposure to acids at low or high salt concentrations; the bond is sensitive to bases and oxidizing agents. Commercially available radiation‐sterilized PS labware products also show elevated EDC‐mediated uptake of DNP‐[ 3 H]Gly. Nonirradiated PS products or non‐PS materials in these studies were inactive. Commercially irradiated PS microtiter plates were coated with DNP‐amino acids in an EDC‐mediated reaction, and the binding of a radiolabeled mouse monoclonal IgE (aDNP) to DNP‐coated plates was studied. 1 ) The minimal ligand (DNP‐Gly) and reagent (EDC) concentrations for plate coating to reach saturating antibody binding were found to be 0.2 mM and 0.2 mg/ml, respectively. High coating densities were suboptimal for antibody binding; 2 ) five to 60 min of coupling times yielded optimal coating densities; 3 ) The binding of antibody to DNP‐Gly‐coated plates reached half‐maximal levels in approximately 40 min; 4 ) Dissociation of antibody from DNP‐Gly and DNP‐Ser‐coated plates was very slow. Intra‐ and interassay coefficients of variation of antibody binding to plates coated with DNP‐Gly under optimal conditions were generally below 3%. We conclude that the PS‐bound DNP produced by this method is recognizable by anti‐DNP antibodies. The optical quality of PS is not affected by radio‐derivatization, and the ligand‐coated plates obtained by these methods are suitable for colorimetric assays. All brands of heavily irradiated PS examined were suitable carriers for EDC‐mediated coupling of DNP‐aminoacids.—V arga , J. M.; K lein , G. F.; F ritsch , P. Binding of a mouse monoclonal IgE (anti DNP) antibody to radio‐derivatized polystyrene‐DNP complexes. FASEB J. 4: 2678‐2683, 1990.