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EIA technique is generally used in medicine to detect the antigen or antibodies in serum samples and also in immunodiagnosis of several infectious diseases. It is used for semiquantitative determination of the concentration of certain antigens/antibodies. The ELISA technique was first introduced in early 1970s by Engvall and Perlmann.
Principle:
The antibodies against the antigen to be measured are absorbed to a solid support, in most cases a polystyrene microtiter plate. After coating the support with antibody it is washed. The antigen is now added and this now binds to the absorbed antibodies. An enzyme linked antibody molecule called conjugate, when added also binds to the antigen.
When a chromogenic substrate for the enzyme is added, the coloured product becomes suitable enough to be measured. The intensity of the colour is proportional to the bound enzyme and thus to the amount of bound antigen. Hence from the intensity of colour produced by a series of standard antigens, the amount of antigen in an unknown sample can be calculated.
Requirements:
1. Micropipettes: 0-25 µl.
2. Multichannel pipettes: 0-250 µl.
3. Flat bottomed polystyrene microtitre plates with 96 wells.
4. ELISA Reader (Multiscan photometer).
5. 0.1M C03 buffer (pH 9.6):
Prepare 0.1M Na2C03 solution and adjust pH 9.6 with NaOH.
6. Wash solution:
90 ml Tween 80+910 ml distilled water.
7. BST:
0.2% (W/V) Bovine serum albumin, 0.01% Tween 80 and 0.9% (W/ V) NaCl in distilled water.
8. Substrate solutions depend on the enzyme that is coupled to the conjugate. Two commonly used enzymes in ELISA are horse radish peroxidase (HRP) and alkaline phosphatase.
9. For HRP two substrate solutions are required.
They are prepared as follows:
Solution 1:
80 mg of 5 amino salycilic acid (purple-red brown colour) in 100 ml 0.05 M potassium phosphate buffer (pH 6.0) containing 0.001 (M) EDTA. Add 20 ml H2O2 (30%) and mix.
Solution 2:
Mix 24.3 ml 0.1 (M) citric acid 25.7 ml 0.2 (M) Na2HPO4 and 50.0 ml water plus 40.0 mg ortho-phenylene diamene (yellow colour) and 40 µl H2O2 (30%).
10. Stop solution:
0.3 (M) NaOH in case of solution 1 and 1.0 (M) H2SO4 in case of solution 2. (When alkaline phosphatase is the enzyme coupled, the substrate is p- nitrophenyl phosphate which is released as yellow coloured p-nirtophenol).
11. Diluted solution of IgG against the antigen to be measured. Depending on the titre of IgG, dilution is made. Usually 1500-2500 fold 0.1 (M) carbonate buffer is used for dilution.
12. Antigen solutions- both standard and the ones to be tested.
13. Enzyme (HRP) labelled diluted IgG solution. Conjugate solutions should be diluted to 500-2000 times in BST.
Procedure:
Double Antibody Sandwich Technique:
1. Pipette out 150 µl of diluted IgG solution to each of the wells of the microtitre plate using multichannel pipette. Cover the plate and incubate at room temperature overnight.
2. Wash the plates with wash solution, a few times and add 100 µl BST to each well.
3. To the first well of each row add 100 µl of an antigen solution to be tested and mix carefully and thoroughly avoiding air bubbles and splashing of droplets.
4. Transfer 100 µl from this first well and transfer to the second wells in each row.
5. Repeat mixing as in first well and transfer 100 µl from 2nd to 3rd, 3rd to 4th etc. Thus a two-fold dilution series from wells 1 to 12 is prepared. Finally the excess 100 µl should be removed from the last wells.
6. Incubate the plate at 37°C for 2 hours to allow the antigen to bind to the coated antiserum and then wash thoroughly.
7. Add 100 µl of diluted conjugate solution to each well and incubate at 37°C for two hours and then wash the plate thoroughly.
8. Add 100 µl of substrate solution to each well and incubate at 37°C in dark for one to two hours.
9. Stop the reaction by adding 100 µl of stop solution.
10. Read the titre of antigen solutions on an ELISA reader or visually observe the last well that still gives some colour.
Preparation of Conjugate:
A conjugate is the covalent complex of IgG and an enzyme.
The coupling of HRP is as follows:
1. Prepare fresh solution of 5 mg of HRP in one ml of 0.3 (M) Na2CO3 (pH 8.1).
2. Add 0.1 ml of 1% fluorodinitrobenzene in pure ethanol (a precipitate may be formed if the HRP used is not pure and this must be removed by centrifuging at 18,000 rpm for 10 minutes). Mix thoroughly and incubate at room temperature for one hour.
3. Add one ml of 0.16 (M) ethylene glycol, mix and incubate at room temperature for another hour (Total volume now is 2.1 ml).
4. Dialise this mixture against 0.01 (M) Na2C03 buffer (pH 9.5) for 25 hours. Change the buffer at least three times.
5. Add IgG dissolved in 0.01 (M) Na2C03 buffer (pH 9.5) to the peroxidase aldehyde solution as follows:
Add one volume of IgG solution to one volume of activated peroxidase aldehyde or 5 mg of purified IgG protein to 3 ml peroxidase solution.
6. Mix well and incubate for 2-3 hours (not longer) at room temperature. In case if precipitate is formed, centrifuge at 10,000 rpm for 10 minutes and clarify.
7. Dialise extensively against 0.01 (M) PO4 buffer (pH 7.2) containing 0.9% NaCl at 4°C. Store the conjugate in a refrigerator or freezer in small aliquots and use only once.
Purification of IgG fraction from the whole serum:
1. Mix 100 ml serum + 200 ml 0.06 (M) sodium acetate (pH 4.6). The final pH of mixture should be 4.8.
2. Add 8.2 ml (for rabbit serum) of caprylic acid drop by drop at room temperature. The volume of caprylic acid (6.8-8.2 ml) needed to precipitate IgG, varies from sera to sera depending upon the source.
3. Stir for 30 minutes and centrifuge at 10,000 rpm for ten minutes to remove the precipitate.
4. Dialise the IgG fraction against 0.9% NaCl solution.
This procedure can be changed in many ways. The antigen can be coated directly to the wells, instead of antibody and proceed as above.
If all wells develop the same amount of colour, the experiment is wrong. Then the problem can be overcome by using freshly prepared solutions.