HGH ADA (human growth hormone) ELISA

This assay employs the indirect enzyme immunoassay technique. hGH is coated onto a 96 well microplate. Calibrator and test samples are prepared by dilution into assay buffer and are pipetted into the appropriate wells. Antibodies to hGH present in biological matrices are bound to the immobilized hGH. After washing away any unbound substances, antibody-enzyme reagent and a substrate solution is added to the wells for color development. The color development is proportional to the amount of antibodies to hGH present in the calibrator and test samples. The color development is stopped and the intensity of the color is measured.

Coated microtiter plate, 96 wells (12x8 strips) 1 Calibrator diluent 1.8 ml Calibrator (1.15mg/mL) 12 μl 10X wash buffer 50 ml Assay buffer 50 ml 1000X detection reagent 17 μl TMB 12 ml TMB stop solution

Prepare only the appropriate amount of required reagent on the day of use. Store all reagents as per instructions stated on the label. 1. Wash Buffer (1X) Preparation Dilute wash buffer concentrate with deionized water 1/10 before use (for example add 20mL concentrate to 180mL deionized water) . Mix well. 2. Detection Reagent (1X) Preparation: Dilute detection reagent with assay buffer 1/1000 before use (for example add 11μl concentrate to 11ml of assay buffer) . Mix well. The following is an example calibrator curve.

This assay employs the indirect enzyme immunoassay technique. hGH is coated onto a 96 well microplate. Calibrator and test samples are prepared by dilution into assay buffer and are pipetted into the appropriate wells. Antibodies to hGH present in biological matrices are bound to the immobilized hGH. After washing away any unbound substances, antibody-enzyme reagent and a substrate solution is added to the wells for color development. The color development is proportional to the amount of antibodies to hGH present in the calibrator and test samples. The color development is stopped and the intensity of the color is measured

1. Construct a standard curve by plotting the absorbance obtained from each standard against concentration. Use a 4 or 5 parameter curve fit. Alternatively a log-log curve fit may be used. 2. The concentration of the unknowns can be read directly from this standard curve using the absorbance value for each sample. 3. Any sample undiluted or diluted still reading greater than the highest standard should be diluted appropriately with assay buffer and retested. If the samples have been diluted, the concentration determined from the standard curve must be multiplied by the dilution factor.

Dilute calibrators and test samples 1/100 with assay buffer (for example add 5µL of prepared calibrator or sample to 495µL of assay buffer) . Mix well. Do not store diluted samples.