Ultrasensitive enzymatic radioimmunoassay using a fusion protein of protein a and neomycin phosphotransferase II in two-chamber-well microtiter plates
✍ Scribed by H.-D. Hunger; Chr. Flachmeier; G. Schmidt; G. Behrendt; Ch. Coutelle
- Book ID
- 102985114
- Publisher
- Elsevier Science
- Year
- 1990
- Tongue
- English
- Weight
- 955 KB
- Volume
- 187
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
A new sensitive method for antigen detection employing a phosphorylation reaction is described using human serum albumin as a model. The antigen is initially bound to the surface of polystyrene microtiter plates and reacted with an antibody (rabbit). A microbiologically produced bifunctional fusion protein of protein A and neomycin phosphotransferase II (NPT II) serves as a second immunological reagent by virtue of its protein A component. The detection is based on the phosphorylation of an aminoglycoside antibiotic by the NPT II moiety of the fusion protein using [gamma-32P]ATP as a cosubstrate. This reaction is performed in solution and the evaluation is accomplished by dotting aliquots of the reaction mixture onto phosphocellulose paper, washing with water, and autoradiography. Microtiter plates with a specially designed 10 microliter-volume reaction chamber are particularly advantageous for this procedure. The sensitivity of detection is currently 10 fg (1 pg/ml) of antigen.