An efficient enzyme immunoassay for glutamate using glutaraldehyde coupling of the hapten to microtiter plates.
In order to coat microtiter plates for enzyme immunoassays (EIAs), amino acids and other haptens are usually coupled to larger protein molecules. The formation of such conjugates is not always reproducible. This may lead to inconsistent hapten-protein stoichiometries, unfavorable orientation of the hapten on the protein and/or well-to-well variation in the concentration of the available hapten. In the assay described here the excitatory amino acid (EAA) Glu is coupled directly to polystyrene microtiter wells with GA. Each step of the assay was tested for maximum efficiency. The resulting EIA with Glu as a competitor gave excellent reproducibility (coefficient of variation = 5.87%), an EC50 of 2.02 X 10(-5) M and a detection limit of 1.26 X 10(-6) M. This EIA method is generally useful for a variety of antisera to amino acids and small peptides and a wide range of competing substances. It can be used to characterize the conformational requirements for antigen binding, to assay for glutamate or to identify compounds with glutamate-like structure in unknown solutions.
Department of Cell Biology and Anatomy, University of North Carolina, Chapel Hill 27599-7090.
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Ordronneau
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Abdullah
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Petrusz
D000409:Alanine / Q000032:analysis; D001224:Aspartic Acid / Q000032:analysis; D001667:Binding, Competitive; D003429:Cross Reactions; D005971:Glutamates / Q000032:analysis*; D018698:Glutamic Acid; D005976:Glutaral; D006863:Hydrogen-Ion Concentration; D007124:Immunoenzyme Techniques; D015203:Reproducibility of Results; D013997:Time Factors
D005971:Glutamates; D001224:Aspartic Acid; D018698:Glutamic Acid; D000409:Alanine; D005976:Glutaral