Determination of Substance Concentration by Competitive ELISA (zt)

In this protocol, the concentration of a substance of interest in a sample is determined by Enzyme-Linked Immunosorbant Assay (ELISA). Antigen and the substance will compete for specific antibodies. Concentration can be determined by comparison of the blocking effect on plates of antigen using an unknown substance with mixtures containing serial dilutions of known amounts of substance and standard concentration of antibodies. It may be helpful to examine the standard protocol for ELISA before performing this method.

Procedure:
1. Coat Nunc Immuno-Module Plates with Antigen Stock Solution (50 μl/well) for at least 24 hr at room temperature, over the weekend at 4°C, or overnight at 37°C.

2. Remove the antigen and wash the wells three times with PBS.

3. Block the wells with 1% BSA/PBS (100 μl/well) for at least 1 hr at 37°C.

4. Remove the 1% BSA/PBS by flicking the plate.

5. Wash the wells three times with PBS/Tween

6. Set up a competition assay between the antibody and the competing substance:

Prepare 1:2 serial dilutions of the competitive substance covering the range of 480 ng to 0.4 ng/ml. Add 50 μl of each dilution to 25 μl of 3-fold strength titer of antibody (i.e., a solution 3-fold the titer giving 50% inhibition on direct ELISA, see piundefined461" >Protocol ID#461), in sealed tubes.

7. Incubate overnight at room temperature.

8. Set up a competition between the sample containing an unknown concentration of competitive substance and the antibody as above.

9. Wash the wells of the plate four times with 200 μl/well of PBS/Tween.

10. Add 50 μl/well of antiserum (diluted appropriately with PBS) and incubate for 2 hr.

11. Wash the wells of the plate four times with 200 μl/well of PBS/Tween.

12. Add 50 μl/well of the Peroxidase-Conjugated Secondary Antibody (diluted 1:1000 with 1% BSA/PBS) and incubate for 2 hr.

13. Wash the wells of the plate four times with 200 μl PBS/Tween.

14. Add 50 μl of Peroxidase Substrate and incubate for 1 to 5 min in the dark at room temperature. Check the reaction for a color change. The wells should turn blue.

15. Read the absorbance at 450 nm in a plate-reading spectrophotometer.

16. Plot the standards against the absorbance and determine the unknown concentrations by linear regression.

Solutions:
Peroxidase Substrate 1:100 dilution of TMB Stock in Citrate/Acetate Buffer
TMB Stock 10 mg/ml 3,3',5,5'-Tetramethylbenzidine TMB in DMSO
Citrate/Acetate Buffer Citrate/Acetate Buffer, pH 6.0 can be stored frozen at -20°C.
Titrate 0.1 M Sodium Acetate with 0.1 M Citric Acid to a final pH of 6.0.
PBS pH 7.2
2.7 mM KCl
4.3 mM Sodium Phosphate Dibasic (Na2HPO4)
1.8 mM Potassium Phosphate Monobasic (KH2PO4)
137 mM NaCl