Competitive ELISA.

Aim: To determine concentration of antigen by antigen capture ELISA or competitive ELISA.
Principle: ELISA or enzyme linked immunosorbent assay is a sensitive laboratory method used to detect the presence of antigens (Ag) or antibodies (Ab) of interest in a wide variety of biological samples. These assays require an immunosorbent i.e., antigen or antibody immobilized on solid surface such as wells of microtitre plates or membranes.

Antigen capture ELISA method is the most useful immunosorbent assay for detecting antigen, since it is 2-5 fold more sensitive than those assays in which antigen is directly bound onto the solid phase. In this assay, constant and limiting amount of antibody is immobilized onto a solid support. A fixed amount of labeled antigen [i.e., antigen coupled with enzyme like Horse radish peroxidase (HRP), alkaline phosphatase (ALP) etc.] is added and allowed to compete with unlabeled antigen (standard or test sample) for the immobilized antibody. The amount of labeled antigen bound is then estimated by a suitable assay for the label. The amount of labeled antigen that binds is inversely proportional to the amount of unlabeled antigen in the reaction mixture. Thus, the estimate of label in the well decreases with increase in the antigen concentration in the standard or test sample.

Materials Required:
Glassware: Measuring cylinder, Test tubes.
Reagent: Distilled water.
Other Requirements: Blotting paper, Micropipette, Tips.

Note:
Read (he entire procedure before starting the experiment.
Bring all the buffers to room temperature before starting the assay.
Dilute only required amount of buffers to 1X with distilled water, before use.
Use 24 wells per experiment.
Reconstitute samples of antibody, standard antigen and test samples with distilled water; volume as mentioned on their respective labels. Store at 4°C and use within 3 months.
Blocking buffer: BSA in PBST.
Coating buffer: Carbonate bicarbonate buffer.
P6ST; Phosphate buffered saline - Tween.
Stop solution: Sulphuric acid.

Prepare the reagents as indicated below before starting each experiment:
Preparation of sample diluent: Take 1 ml of blocking buffer and make up the volume to 30'ml with 1X PBST. Use this to dilute standard antigen and HRP labeled antigen.
Preparation of dilutions of standard antigen: Concentration of reconstituted standard antigen is 1 mg/ml, dilute this to get a range of concentrations using sample diluent, as follows:
Dilutions Conc. Of Std. Antigen
20 µl of 1 mg/ml (stock) 40 µg/ml (a)
+ 480 µl of sample diluent
200 µl of (a) + 800 µl diluent 8µg/ml (b)
500 µl of (b) + 500 µl diluent 4 µg /ml (c)
500 µl of (c) + 500 µl diluent 2 µg/ml (d)
500 µl of (d) + 500 µl diluent 1 µg/ml (e)
500 µl of (e) + 500 µl diluent 0.5 µg/ml (f)
500 µl of (f) + 500 µl diluent 0.25 µg/ml (g)
500 µl of (g) + 500 µl diluent 0.125 µg/ml(h)

Preparation of working concentration of test samples: After reconstitution, dilute each test sample individually by mixing 10ml of the sample with 2 ml of sample diluent (Dilution is 200 times).
Preparation of Reagents:

Reagents Vol. to be Vol. of distilled
Taken water to be added
1oxtmb/h2o2 0.6ml 5.4ml
10X PBST 10ml 90ml
5X Stop solution 12 ml 48 ml

Procedure:
Day 1: Coating of wells with antibody
1. Concentration of reconstituted antibody is 0.1 mg/ml, dilute with coating buffer, i.e., mix 50 µl of stock with 4.95 ml of coating buffer, to get working concentration of 1 µg/ml.
2. Pipette 200 µI of diluted (1X) antibody into each microtitre well (24 wells). Tap or shake the wells to ensure that the antibody solution is evenly distributed over the bottom of each well.
3. Incubate the microtitre wells overnight at 4°C.
Day 2: Blocking the residual binding sites on the wells
4. Discard the well contents. Rinse the wells with distilled water three times,draining out the water after each rinse.
5. Fill each well with 200 µl of blocking buffer and incubate at room temperature for 1 Hour.
6. Rinse the wells three times (as in step 4) with distilled water. Drain out the water completely by tapping the wells on a blotting paper.

Addition of antigen to the wells
7. Prepare standard antigen dilutions as indicated page above.
8. Add 100µl of standard antigen, diluted test samples and PBST to the coated wells as indicated in fig 1 (in duplicates).
Addition of HRP labeled antigen
9. Prepare 1X HRP labeled antigen using sample diluent, i.e., mix 3 mI of the stock (1000X) with 3 ml of sample diluent.
10. Add 100 v\ of 1X HRP labeled antigen to all the wells.
11. Incubate at room temperature for 30 minutes.
12. Discard the well contents; fill the wells with 1X PBST, allow it to stand for 3 minutes,
discard the contents. Repeat this step two more times.
Addition of substrate and measurement of absorbance
13. Dilute required amount of 10X TMB/H2O2 (substrate) solution to 1X using distilled Water.
14. Add 200 µI of 1X substrate to each well.
15. Incubate at room temperature for 10 minutes.
16. Add 100 ml of 1X stop solution to each well.
17. Transfer the contents of each well to individual tubes containing 2 ml of 1X stop solution.
18. Prepare substrate blank by adding 200//t of 1X substrate solution to 2.1 ml of 1X stop solution.Read the absorbance at 450 nm after blanking the spectrophotometer with substrate blank and record the readings as follows:

Calculation of antigen concentration
1. Calculate the average Ag for each of the samples (standard and test)
2. Plot A450 values of standards (b to h) on Y axis (linear scale) versus the concentration of antigen in µg/ml on X axis (log scale) on a semi-log graph sheet.
3. From the standard curve, determine the concentration of antigen in each of the test samples.
4. Calculate the concentration of antigen in, mg/ml, in each of the test samples as follows:
Concentration of antigen in the sample
= Concentration in µg/ml (from the graph) X Dilution factor
103
T1= 1.8 mg/ml


Result:From the standard curve, report the concentration of antigen in each of the test samples as follows: