Binding of a mouse monoclonal IgE (anti-DNP) antibody to radio-derivatized polystyrene-DNP complexes.
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-[3H]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-[3H]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.
Varga
J M
JM
Department of Dermatology, University of Innsbruck, Austria.
Klein
G F
GF
Fritsch
P
P
D019297:2,4-Dinitrophenol; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D004140:Dinitrophenols / Q000276:immunology*; D005720:Gamma Rays; D005998:Glycine; D006863:Hydrogen-Ion Concentration; D007073:Immunoglobulin E / Q000276:immunology*; D007700:Kinetics; D011137:Polystyrenes; D014475:Uncoupling Agents
D000911:Antibodies, Monoclonal; D004140:Dinitrophenols; D011137:Polystyrenes; D014475:Uncoupling Agents; D007073:Immunoglobulin E; D019297:2,4-Dinitrophenol; D005998:Glycine