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
Preparation of Conjugate
A conjugate is the covalent complex of IgG and an enzyme. The coupling of HRP is described below:
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.
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