*4.2.2. Sandwich ELISA*

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

54 Protein-Protein Interaction Assays

results in two forms [12, 29–31]:

*4.2.1. Direct ELISA*

charge exchange.

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

**1.** Qualitative: In quantitative ELISA, just positive or negative results can be mentioned. A cutoff value is adjusted by running known positive and negative samples, and the optical

**2.** Quantitative: Quantitative ELISA is used for the quantification of analyte, and the series of

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

• 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

density of the solution is measured by spectrophotometer.

standards are used and the unknown amount of analyte is measured.

and reagents are provided with each specific kit along with the methodology.

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

Direct ELISA comprised of the following steps:

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


**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.

**9.** The absorbance or fluorescence or electrochemical signal (e.g., current) of samples is measured to determine the presence of antigen and to quantify it (**Figure 2**).

**3.** The plate is washed to remove all the unbound antibodies, and the antigen-antibody (Ag-Ab) complex have a competition with labeled antigen as there are less unbound antibodies and coated antigen needs to attach with antibodies which will be taken from bound Ag-Ab

Protein-Based Detection Methods for Genetically Modified Crops

http://dx.doi.org/10.5772/intechopen.75520

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**5.** A substrate is added, and as a reaction of the enzyme and substrate, color is produced.

**6.** To prevent the eventual saturation of the signal, the stop solution is added to stop the

In some kits, enzyme-linked antigens are used instead of antibodies, and the remaining competition mechanism is same as described earlier; therefore, there will be a competition of

To determine the immune response in the body enzyme-linked immunosorbent assay (ELISA) is being used, which have different methods. These methods are being extensively used to determine analyte in the biological samples of whole blood, serum, urine and other biological fluids. These assays have wide applications in agriculture, industrial, environmental, athletic

**1.** The antigen present in oncology samples. The elevated levels of carcinoembryonic antigen (CEA) and prostate-specific antigen (PSA) can be used for early diagnosis of tumorigenic

**2.** Other disorders and diseases can be diagnosed by immunoassays, for example, antigenic determinants of infectious disease organisms, including fungi, viruses and bacteria and yeast. *Helicobacter pylori*, Mycobacterium tuberculosis, malaria, West Nile Virus, HIV, human papilloma virus (HPV), human chorionic gonadotropin (HCG) for pregnancy, hormones determination, gastrointestinal disorders determination, inherited metabolic diseases determination by enzymes, hexosaminidase as a marker of Tay-Sachs disease and histidase as a marker of histidinemia, tissue damages determination by tissue specific antigen in circulation by determination of creatinine kinase for muscle damage cardiac

**3.** Suitable identification of the foreign protein in genetically modified organisms (GMOs)

**4.** To test the athletes' blood sample for recombinant growth hormone, immunoassays are

widely used in sports anti-doping laboratories (rGH rhGH, GH, hGH).

**4.** Then, the secondary antibody is added which is attached with the enzyme.

complex.

reaction.

antigens instead of antibodies.

and legal/forensic fields [34–38].

processes.

and antibodies.

This assay can be used to determine:

troponine for myocardial infarction.

*4.2.5. Applications of ELISA*

#### *4.2.3. Indirect ELISA*

Indirect ELISA is the same as that of the direct ELISA, only primary antibody has been unlabeled, which is very specific to the antigen and the labeled secondary antibody is added, which is labeled withe enzyme or any other label (**Figure 3**).

#### *4.2.4. Competitive ELISA*

There is a competition of analyte in this ELISA. The procedure is as follows:


**Figure 2.** Sandwich ELISA. This figure shows the antigen is the analyte which is sandwiched between two antibodies, the antibody can be sandwiched between two antigens in the same way.

**Figure 3.** Indirect ELISA. This figure shows the antigen is the analyte to be detected, the primary antibody specific to the antigen is added, secondary antibody is added then, which is labelled with the enzyme and after that chromogenic substrate is added, which reacts with the enzyme and give fluorescence.


In some kits, enzyme-linked antigens are used instead of antibodies, and the remaining competition mechanism is same as described earlier; therefore, there will be a competition of antigens instead of antibodies.
