Methodology for Amino Acids and Proteins

Enzyme Linked Immunosorbant Assay (ELISA)

The ELISA technique is used for a semiquantitiative determination of the concentration of certain antigens/antibodies. It is already used in medicine to detect the antigen or antibodies in serum samples. At present, it has it has application in immunodiagnosis of several infectious diseases.

In developed countries, ELISA kit has become a market commodity for early diagnosis of plant diseases. It is also being standardized to measure the plant growth regulators at the level of parts per billion. The general technique is described here and for each specks purpose it has to be standardized.

The ELISA technique was first introduced in early 1970s by Engvall and Perlmann. The principle underlying the double antibody sandwich technique of ELISA is described below.

The antibodies against the antigen to be measured are adsorbed to a solid support, in most cases a polystyrene microtiter plate. The support after coating with antibody is washed. The antigen is now added and binds to the adsorbed antibodies. Then, an enzyme-linked antibody molecule called the conjugate is added which also bind to the antigen. A chromogenic substrate for the enzyme is added and the colored product generated is measured. The intensity of the color is proportional to the bound enzyme and thus to the amount of the bound antigen. Hence, the intense of the color produced by a series of standard antigens allows the calculation of the amount of antigen in an unknown sample.

» Flat-bottomed Polystyrene Microtitere Plates with 96 Wells
» Micropipettes (Gilson/Finnpipettes) 0-250mL
» Multi-channel Pipette 0-250mL for pipetting of all reagents (if available)
» ELISA Reader (Multiscanphotometer), if available
» 0.1M Carbonate Buffer (pH9.6): Prepare 0.1M Na2CO3 solution and adjust to pH 9.6 with NaOH. The chemicals should be of the highest quality and water double distilled.
» Wash Solution: Mix 90mL Tween 80 with 910mL water.
» BST: 0.2% (w/v) Bovine serum albumin, 0.01% Tween 80 and 0.9% (w/v) sodium chloride in distilled water.
» Substrate Solutions: It depends upon the enzyme that is coupled to the conjugate. Two widely used enzymes in ELISA technique are horseradish peroxidase (HRP) and alkaline phosphatase.   For HRP, there are two substrate solutions and are prepared as below:
» Solution 1 Dissolve 80mg 5 amino-salicylic acid (purple-red brown color) in 100mL 0.05M potassium phosphate buffer (pH 6.0) containing 0.001M EDTA. Add 20mL H2O2 (30%) and mix.
» Solution 2   Mix 24.3mL 0.1M citric acid
25.7mL 0.2M Na2HPO4
40mg ortho-phenylenediamine (yellow color)
40mL H2O2 (30%)
» Stop Solution
» 0.3M NaOH (in the case of solution 1) or 1M H2SO4 (in the case of solu­tion 2) is used.
(When alkaline phosphatase is the enzyme coupled, the substrate is then p-nitrophenyl phosphate and is released as yellow colored p-nitrophenol).
» A diluted solution of IgG against the antigen to be measured. The dilution is usually 1500-2500 folds depending on the titre of IgG. Dilutions are made in 0.1M carbonate buffer.
» Antigen solutions to be tested and standard antigen solutions.
» Enzyme (HRP) labeled diluted IgG solution. As a rule the conjugate solution has to be diluted 500-2000 times in BST.

The Double Antibody Sandwich Technique
Pipette 150mL of the diluted IgG solution to each of the wells of a microtitre plate manually or using multichannel pipette. Cover the plate and incubate overnight at room temperature.
Wash the plates with wash solution. The wells can be emptied by tapping the plate over a sink and then beating the plate upside down against a filter paper. The plates can be stored several months, covered
and cooled. An appropriate volume of wash solution is pipetted into the wells and left for a couple of minutes. The wells are then emptied as described above. Washing is repeated a few times to ensure good results.
After washing add 100mL BST to each well.
Add 100mL of an antigen solution to be tested to the first well of each row.
Mix carefully and thoroughly.  Avoid air bubbles or splashing of small drops.
Take 100mL from the first wells and transfer them to the second wells in each row. Repeat the mixing procedure (step 5). Take 100mL from the second wells and add to the third wells and so on. By this way a two-fold dilution series from wells 1-12 is created. Finally, remove the 100mL excess from the last wells.
Incubate the plate for 2h at 37°C, to allow the antigen bind to the coated antiserum, and then wash thoroughly.
Add 100mL of the diluted conjugate solution to each well and incubate for 2h at 37°C, then wash the plate thoroughly.
Add 100mL of substrate solution to each well and incubate for l-2h at 37°C in the dark.      :
Stop the reaction by adding 100mL of stop solution.
Read the titre of the antigen solutions. This can be done by either using an ELISA Reader or visually by observing the last well that still gives some color with the naked eyes.

Preparation of Conjugate
A conjugate is the covalent complex of IgG and an enzyme. The coupling of HRP is described below:
Dissolve 5mg of HRP in 1mL 0.3M Na2CO3 (pH 8.1). This solution should be prepared fresh.
Add 0.1mL of 1% fluorodinitrobenzene in pure ethanol. If the HRP used is not pure, a precipitate may be formed that must be removed by centrifugation (10min, 18,000rpm).
Mix thoroughly and incubate for 1h at room temperature.
Add 1mL of 0.16M ethylene glycol, mix and incubate for another hour at room temperature. The total volume is now 2.1mL.
Dialyze the mixture against 0.01M sodium carbonate buffer (pH 9.5) for 25h. The buffer should be changed at least three times.
Add IgG dissolved in 0.01M sodium carbonate buffer (pH 9.5) to the peroxidase aldehyde solution in the following ratio: one volume of IgG solution to one volume activated peroxidasealdehyde or 5mg purified IgG protein to 3mL peroxidase solution.
Mix well and incubate 2-3h but not longer at room temperature. If any precipitate is formed, clarify by centrifugation (10min, 10,000rpm).
Dialyze extensively against 0.01M phosphate buffer (pH 7.2) containing 0.9% NaCl at 4°C. Store the conjugate in a refrigerator or freezer in small aliquots and use once only.

Purification of IgG fraction from whole serum:
» Mix 100mL serum with 200mL of 0.06M sodium acetate (pH 4.6). The final pH of the mixture should be 4.8.
» Add 8.2mL (for rabbit serum) of caprylic acid dropwise at room temperature. The volume of caprylic acid (6.8-8.2mL) needed to precipitate IgG varies from sera to sera depending upon the source.
» Stir for 30 min and remove the precipitate (10,000rpm 10 min).
» Dialyze the IgG fraction against 0.9% NaCl solution and store after lyophilization.
The procedure described above can be changed in many ways. The antigen can be coated directly to the wells instead of antibody and proceeded as described.
The experiment is wrong when many or all wells develop the same amount of color. In such cases, the problem can be overcome by using freshly prepared solutions.
ELISA is of late called Enzyme Immuno Assay (EIA).

1. Wim Gaastra (1984) In: Methods in Molecular Biology Vol 1 Proteins (Ed J M Walker) Humana Press New Jersey p 349.
2. Manual on 'Techniques in Molecular Biology' (Proteins) (1986) Workshop held at the Department of Biochemistry, Tamil Nadu Agric Univ Coimbatore p 72.