**3. Classification of immunoassays**

of proper strategies for detection. These strategies are for the most part in view of the study

For approval of a scientific strategy, the testing objective must be characterized and execution qualities must be illustrated. Execution qualities incorporate exactness, extraction proficiency, accuracy, reproducibility, affectability, specificity and strength. The utilization of approved strategies is essential to guarantee acknowledgment of results delivered by diagnostic research facilities [9]. The greater part of protein detection techniques depends on immunoassays. Protein detection techniques can possibly recognize the nearness of a particular GM quality and to give the total measurement of the level of transgene expression. Protein identification strategies are exceedingly reasonable for checking particular GM attributes amid treatment of

Here are the details of immunoassays being used for the detection of genetically modified

An immunoassay is a biological test that identify and quantify the micro- or macromolecules with the help of antigen or antibody, and the molecule to be detected is called as an analyte. Specific antigens can be stimulated by specific immune responses and as a result of an immune response in the body, antibodies are produced, which are proteins, and they have a sense to find the presence of any foreign antigen in the body. Immunoassays vary in formats. Multiple steps are involved in these assays where reagents are being added and then extra reagents are washed away. Multistep assays are often called heterogeneous immunoassays or separation immunoassays [10]. A few immunoassays can be performed by mixing the samples and reagents and are nonseparation immunoassays or homogeneous immunoassays. The vital component of an immunoassay is an antibody which has a high specificity for the target molecule (antigen), and the area on antigen where antibody attaches is called as an epitope. Standards or calibrators of known concentration are being used to quantify the unknown concentration of analyte. These detections of antigen or antibody take place with the help of labels attached to the antigen or antibody. Many labels are detectable as either they produce a color change in a solution, emit radiations or can be induced to emit light or fluorescence

crude items, gave the protein is communicated in the piece of the plant being tried.

under UV light. The most common used labels for immunoassays are the enzymes.

of pathogens, proteins and other proteins in blood samples [14].

In the 1950s, the first immunoassay was developed by the Solomon Berson Rosalyn Sussman Yalow. In 1977, Yalow received the Nobel Prize for her work and came in the list of second American women who won this award [11, 12]. In the 1960s [13], the immunoassay became more simple with the discovery of chemically linked enzymes to the antibodies, and later in 1983 [14], Professor Anthony Campbell from Cardiff University introduced acridinium ester in immunoassay that used its own light. This immunoassay helped to quantify a wide range

of the novel proteins or DNA.

50 Protein-Protein Interaction Assays

proteins.

**2.1. History**

**2. Immunoassays**


#### **3.1. Competitive, homogeneous immunoassays**

In competitive homogenous immunoassay, there is a competition between labeled and bound analyte (bound to the antibody) with unlabeled and unbound analyte in the sample. As a competition, the unlabeled and unbound analyte displace the labeled and bound analyte and get them attached in place, while the detached labeled analyte then give fluorescence, and this fluorescence is measured, which is proportional to the amount of unlabeled and initial unbound analyte in the sample.

## **3.2. Competitive heterogeneous immunoassay**

In heterogeneous assay, there is a competition between bound and labeled analyte (bound to the antibody) with unbound and unlabeled analyte, the difference from competitive homogenous assay is that the labeled unbound/displaced analyte is separated by washing, and the remaining labeled and bound analyte is measured.

#### **3.3. One-site noncompetitive immunoassays**

In this immunoassay, the amount of unknown analyte in the sample is measured by adding the labeled antibodies. The labeled antibodies get attached with the analyte in the sample, and the extra labeled unbound antibodies are washed away, so, only labeled and bound antibodies are present in the sample, the intensity of fluorescence of these antibodies is measured, which is proportional to the 3.4. amount of unknown analyte in the sample.

#### **3.4. Two-site noncompetitive immunoassays**

In this immunoassay, there is an antibody present on a site, and the analyte in a sample is added to the antibody get attached, and then second antibody is added which is attached with the label. If the specific analyte is not present in the solution, the second antibody will not attach. Then, the fluorescence of the labeled antibody is measured, which is directly proportional to the amount of analyte in the sample. It is very important to consider that there are washing steps after every reaction, so extra materials are always washed away. The other thing very important is that what type of labels are attached and how the fluorescence/signal is measured. The details of the labels are given as follows:

#### *3.4.1. Radioactive isotopes*

To produce a radioimmunoassay (RIA), radioactive isotopes can be added into the immunoassay reagents and the radiations emitted by bound antigen–antibody complex can be determined by the conventional methods. RIA is considered as the earliest developed immunoassay, and they are not used frequently nowadays because of the hazards of radioactivity [16, 17].

**3.** Memory lymphocyte immunostimulation assay (MELISA)

**5.** Cloned enzyme donor immunoassay (CEDIA)

**8.** Surround, optical fiber immunoassay (SOFIA)

radioactive compounds are being used [25–28].

to label the known amount of antigen

antigen from the antibody binding sites

with antibody binding sites.

The following steps are required to perform radioimmunoassay:

this sandwich radioimmunoassay, radioactive compound is used.

**2.** A known amount of antibody is mixed with these radiolabelled antigens.

**3.** Labeled antigen and unlabeled antibody get attached by their binding sites.

**9.** Ultra sensitive antibody detection by agglutination-PCR

RIA is an extensive method in which radioactive labels are used in a stepwise manner and as it is very specific and sensitive method which require a special equipment. Another such method is called immunoradiometric assay (IRMA) in which radiolabels are used in an immediate manner rather in steps. Radioallergosorbent test (RAST) is used to determine the allergen in case of allergy. It is the cheapest method to perform immunoassay. Although it is the cheapest method to perform immunoassay, it needs special licensing and precautions as

Protein-Based Detection Methods for Genetically Modified Crops

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

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**1.** Gamma-radioactive isotopes of iodine, for example, 125-I, attached to the tyrosine are used

**4.** A sample of serum having the same antigen of unknown amount is added to the mixture.

**5.** A competition between labeled ("hot") and unlabeled ("cold") antigen is built to attach

**6.** When the concentration of unknown antigen is increased, it starts to displace the labeled

**7.** The displaced labeled antigens and bound antigen–antibody complexes got separated, and the radioactivity of displaced radiolabelled antigens is measured by Gamma Counter.

\*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

**4.** Immunoscreening

**6.** Lateral flow test

**7.** Magnetic immunoassay (MIA)

**10.** CD/DVD-based immunoassay.

**4.1. Radioimmunoassay**

*4.1.1. Method*
