Comtitive binding immuno-enzymatic assay method - with enzymatic amplification cycle to produce malate

摘要

In a competitive binding assay comprising reaction of substrate (A) and NAD(+) (or deriv.) labelled substrate (A') with a specific binder (esp. antibody) the new feature is that an amplification reaction is carried out using the reactions: A' + G-6-P = A'H + H(+) + 6-phosphogluconate (I) A'H + H(+) + oxaloacetate = A' + malate (II) (G-6-P is glucose-6-phosphate). The first reaction is in presence of G-6-P-dehydrogenase and the second in presence of malate dehydrogenase (MDH), and finally (I) and/or (II) are measured. Measurement of (I) is e.g. by reaction with NADP(+) in presence of (I)-dehydrogenase to give ribulose-5-phosphate (R-5-P) and NADPH and of (II) e.g. by enzymatic reaction with NADP(+) to give pyruvate and NADPH. The reduced coenzyme is measured spectrophotometrically or fluorometrically. The method can be used to assay pharmaceuticals, hormones, proteins, xenobiotic substances, etc. Detection limits are typically 50-100 moles for spectrophotometry or 2.5-10 moles for fluorimetry.