GCSF ADA (granulocyte colony stimulating factor) ELISA

This assay employs the indirect enzyme immunoassay technique. GCSF 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 GCSF present in biological matrices are bound to the immobilized GCSF. 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 GCSF 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 ultra-pure water 1/20 before use (for example, add 50mL wash buffer concentrate to 450mL ultra-pure 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. 3. Calibrator Preparation: Dilute the calibrator from 1.15mg/ml down to 11.5µg/ml by pipetting 5µl of calibrator stock into 495µl assay buffer. Label "Cal. Int." Mix well. Prepare calibrators with concentrations ranging from 1440ng/ml to 45ng/ml. The following is an example calibrator curve

This assay employs the indirect enzyme immunoassay technique. GCSF 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 GCSF present in biological matrices are bound to the immobilized GCSF. 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 GCSF 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. 2. The concentration of the unknowns can be back calculated directly from this standard curve using the absorbance value for each sample.

Dilute calibrators and test samples 1/50 with assay buffer (for example add 5µl of prepared calibrator or sample to 245µl of assay buffer) . Mix well. Do not store diluted samples.