*4.1.1. Method*

The following steps are required to perform radioimmunoassay:


\*Radioimmunoassay can be performed as same as the sandwich ELISA method (see sandwich ELISA), the difference is that in ELISA, enzyme is linked with secondary antibody, while in this sandwich radioimmunoassay, radioactive compound is used.

## **4.2. Enzyme-linked immunosorbent assay (ELISA)**

ELISA is a type of immunoassay, and the principle behind its working is the same as that of immunoassay, just it is a wet-lab based assay that uses solid phase enzyme and that is why it is also called as enzyme immunoassay (EIA). ELISA is considered as a quality control test in industries and diagnostic tests in hospitals. ELISA falls under the category of ligand binding assays as it involves the binging of antibody and antigen. When labeled antigens or antibodies get attached to substrates, they make a reaction which causes a change in color, and this color is used as a signal. This substrate to enzyme linkage was developed by Stratis Avrameas and G.B. Pierce. As it is very necessary to wash away the unnecessary or unbound chemicals after each reaction, so that is why the bound antigen-antibody complex should be fixed to the surface of the container with the help of immunosorbent, and this method was developed by Jerker Porath and Wide in 1966. In 1971, a group of different scientists Bauke van Weemen and Anton Schuurs, in the Netherlands, and Eva Engvall and Peter Perlmann, in Sweden, independently published papers describing the methods of ELISA/EIA. Usually, chromogenic reporters and substrates are used, which give observable change in color according to the amount of antigen-antibody complex. In ELISA, a solid phase which is physically immobilized is used to absorb certain components of the liquid phase which has the analyte to be detected. Different reagents and solutions are added, incubated and washed off, and in the end, some optical changes take place which are measured by spectrophotometer at specific wavelength. If the antigen is present in the liquid to be diagnosed/detected, then the labeled antibody is added and vice versa, and then, the substrate is added which reacts with the enzyme of labeled antigen or antibody and then stop solution is added to stop the reaction, and the color change is measured at specific wavelengths by ELISA reader. ELISA can give results in two forms [12, 29–31]:

• Bovine serum proteins or casein is added to the wells, which are nonreactive in order to

Protein-Based Detection Methods for Genetically Modified Crops

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• Primary antibody having attached enzyme is added, and it binds with the antigen.

change will be there and higher will be the analyte in the liquid to be tested.

• A substrate is added, which changes the color of the solution by reacting with enzymes.

• The higher the concentration of primary antibody in the solution, the higher the color

• The major disadvantage of the direct ELISA is that when antigen is to be measured from serum, antigen mobilization become difficult due to many other proteins present in the

Sandwich ELISA is a type of immunosorbent assay in which one antigen is sandwiched between the two antibodies or one antibody is sandwiched between two antigens for more

**2.** Nonspecific sites on solid surface are blocked by bovine serum albumin, casein or any

**3.** The sample containing antigen is applied to the plate and which is captured by antibody.

**8.** A substrate is added, and the enzyme reacts with the substrate and gives a color which is

**Figure 1.** Direct ELISA. This figure shows the direct ELISA in which the analyte (antigen) to be determined is attached with the labelled antibody and then chromogenic substrate is added which reacts with the enzyme and gives fluorescence.

serum. Sandwich or indirect ELISA becomes more suitable in that case (**Figure 1**).

cover that portion of plastic which is not covered by antigen.

specific reactions. The procedure of the ELISA is given below [32, 33]:

**1.** A known amount of antibody is bound to a fixed surface.

**4.** The unbound antigens are washed away by washing solution

**5.** The secondary antibody is added which is also labeled with enzymes.

**7.** The sandwich is formed having two antibodies and one antigen inside.

*4.2.2. Sandwich ELISA*

other such neutral solution.

**6.** The unbound antibodies are washed away.

proportional to the amount of antigen.


Different kits are also available in the market for each type of ELISA according to the application and requirement. Mostly, the basic principle and methodology are the same. Procedures and reagents are provided with each specific kit along with the methodology.

Following are the four different types of ELISA and their methodologies:

#### *4.2.1. Direct ELISA*

Direct ELISA comprised of the following steps:

• A liquid solution having analyte to be detected is added to the microtiter plate, one sample per well of the plate. The plate has the solid/plastic phase, which absorbs the analyte by charge exchange.

