A SENSITIVE IMMUNOASSAY FOR HUMAN GROWTH HORMONE (hGH) IN URINE USING ALKALINE PHOSPHATASE LABELED ANTIBODY AND CHEMILUMINSCENT SUBSTRATE

Document Type : Original Article

Authors

1 Department of Biochemistry, faculty of medicine, Alfatah University

2 department of Clinical Biochemistry, Manchester, Royal Infirmary

Abstract

The assay of human Growth hormone (hGH) in urine offer potential advantages in serum. The measurement of hGH in urine remains an analytical challenge as concentrations are low and many samples assay at the limit of detection of the assay used. From the analysis of the assay kinetic parameters of a two- site immunoradiometric assay for hGH, we predicted that residual signal was available at analyte concentration below those detectable with I125. We therefore sought to increase the sensitivity of the assay system by an alternative label. The assay was modified to make use of alkaline phosphatase as label instead of I125; we chose to detect the alkaline phosphatase label using as substrate the chemiluminescent substrate adamantly,4-methoxy -4- (3-phosphatephenyl) - Spiro (1,2-dioxetane-3,2`- adamantine) designates as PPD (Diagnostic Products). The assay was shown to be linear from 0.1 to 100 pg growth hormone per tube. This is equivalent to an hGH concentration of 0.05 to 50 pg / ml in a 2 ml of urine sample. Each standard was assayed 10 times to generate an Ekins plot of hGH concentration against standard deviation (Figure 3). Extrapolation to zero concentration gave a detection limit of 0.04 pg/ ml of growth hormone. Different urine samples (n= 17) gave recoveries of  93.9%± 4% at a growth hormone level of 12.5 pg / ml and 93%± 6%, (n= 10) at the level of 2.5 pg / ml. A total of 23 samples were analyzed by Netria hGH (IRMA) on dialysed urine and by the assay described. There was good agreement (r= 0.980) between both assays.

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