Sandwich ELISA

Aim: To determine concentration of antigen by Sandwich ELISA method.
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.

In this method, two antibodies that can bind to two different epitopes on the same antigen are required. One of the antibodies s immobilized on a microtitre well and is referred to as capture antibody and the other antibody is labeled with a suitable enzyme [eg. (HRP), alkaline phosphatase (ALP) etc.] and is referred to as labeled antibody. Sample (standard and test) containing the antigen is allowed to react with the immobilized antibody. After the well is washed, labeled antibody is added and allowed to react with the bound antigen. Unreacted labeled antibody is washed out and the enzyme bound to solid support is estimated by adding a chromogenic substrate. The colour developed is measured spectrophotometrically, which is directly proportional to the antigen concentration.
Materials Required:
Glassware: Measuring cylinder, Test tubes.
Reagent: Distilled water.
Other Requirements: Blotting paper, Micropipette, Tips.

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 30ml with 1X PBST. Use this to dilute standard antigen, test sample and HRP labeled antibody.
Preparation of dilutions of standard antigen: Concentration of reconstituted standard antigen is 0.4 mg/ml, dilute this to get a range of concentrations using sample diluent, as follows:
Dilutions Conc. Of Std. Antigen
10 µl of 0.4 mg/ml (stock) 800 ng/ml (a)
+ 5000 µl of sample diluent
500 µl of (a) + 500 µl diluent 400 ng/ml (b)
500 µl of (b) + 500 µl diluent 200 ng /ml (c)
500 µl of (c) + 500 µl diluent 100 ng/ml (d)
500 µl of (d) + 500 µl diluent 50 ng/ml (e)
500 µl of (e) + 500 µl diluent 25 ng/ml (f)
500 µl of (f) + 500 µl diluent 12.5 ng/ml (g)
500 µl of (g) + 500 µl diluent 6.25 ng/ml (h)

Preparation of working concentration of test samples: After reconstitution, dilute each test sample individually by mixing 2ml of the sample with 2 ml of sample diluent.
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

Note:
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.

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 1µg/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.
8. Add 200µl of standard antigen, diluted test samples and PBST to the coated wells.
9. Incubate at room temperature for 30 minutes.
10. Discard the well contents, fill the wells with IX PBST,allow it to stand for 3 minutes, discard the contents.Repeat this step two more times.

Addition of HRP labeled antibody
11. Dilute 5 μl of 1000X Antibody-HRP conjugate with 5 ml of sample diluent to get 1X concentration.
12. Add 200 μl of 1X HRP labeled antibody to all the wells.
13. Incubate at room temperature for 30 minutes.
14. Discard the well contents and rinse the wells 3 times with 1X PBST. (As in step 10).

Addition of substrate and measurement of absorbance
15. Dilute required amount of 10X TMB/H2O2 (substrate) solution to 1X using distilled water.
16. Add 200 μl of 1X substrate to each well.
17. Incubate at room temperature for 10 minutes
18. Add 100 μl of 1X stop solution to each well.
19. Transfer the contents of each well to individual tubes containing 2 ml of 1X stop solution.,
20. Prepare substrate blank by adding 200μl or 1X substrate solution to 2.1 ml of 1X stop solution.
21. Read the absorbance at 450 nm after blanking the spectrophotometer with substrate blank, record your readings in observation table:

Calculation of antigen concentration in test sample:
22. Calculate the average A450 for each of the samples (standard and test)
23. Plot A450 of standards on Y axis (linear scale) versus the concentration of antigen in ng/ml on X axis (log scale) on a semi-log graph sheet. (Refer Fig 2).
24. From the standard curve, determine the concentration of antigen in the test samples.

Calculation:
Calculate the concentration of antigen in mg/ml, in each of the test samples as follows:
Concentration of antigen i the sample
= Concentration in ng/ml (from the graphic X Dilution factor
106
T1 = 2 X 10-3 mg/ml
Result:From the standard curve, report the concentration of antigen In each of the test samples as follows: