Abstract
The aim of this research was to study the application and effectiveness of Enzyme Linked Immuno Receptor Assay (ELIRA) method for understanding the bioactivity of human Growth Hormone (hGH) in micro-titer plates. For this purpose, rabbit hepatocyte microsomes which contained hGH receptors were used for coating of ELISA micro-titer plates. Then hGH was interacted with coated receptors. Fractions of bounded complexes were identified by antibodies in an Enzyme-based substrate detection system. Different assay conditions such as: buffers, blocking agents, temperatures and times of incubation were analyzed. Our result indicated that, the carbonate coating buffer was not effective in receptor coating in ELIRA. Overnight incubation of hGH and hGH receptors in HEPES assay buffer and BSA blocking resulted in the lower linearity and correlations (R2 = 0.46 to 0.85). However, 3 h incubation in Tris-HCl assay buffer at 30°C resulted in higher linearity and correlations (R2 = 0.95 to 0.97). Finally, the coating of microwells by 250 μg/ml of microsome membranes in Tris buffer at 30°C for 3 hr and blocking by skim milk resulted to the best linearity and higher correlation, (R2 = 0.985) and lower detection limit about 2 ng/ml of bioactive hGH.
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