**4.5. Cloned enzyme donor immunoassay**

In this type of Immunoassay, in which two types of enzymes are being used which can be active only when they combine together [45]. The one enzyme is conjugated with the same type of specific analyte to be determined and this enzyme complex is called as analyteenzyme-fragment conjugate. The other enzyme attaches to the specific antibody. The analyteenzyme-fragment conjugate is unable to assemble with the other enzyme, if it is attached to the antibody. For this purpose, the antibody should be displaced from the enzyme.

Therefore, when the analyte to be determined present in the serum is mixed with the analyteenzyme-fragment conjugate and antibody-enzyme. There is a competition between the analyte in the serum and the analyte-enzyme-fragment conjugate. If the concentration of analyte is high in the serum, then, it will attach with the antibody-enzyme, and the enzyme will be free to attach with the analyte-enzyme-fragment conjugate to give enzyme activity with the substrate. It means the higher the concentration of analyte in the serum, the higher will be enzyme activity and vice versa.

#### **4.6. Lateral flow immunochromatographic assays**

Simple devices (Strips) are being used to detect the analyte of interest in the sample without the need of any equipment. A widely used such tests are home pregnancy test, HCV, HBV diagnostic test, and so on. Immunoassays have the ability to be broadly executed on a business scale for the recognition of proteins in food [10, 46–49]. The test is used to detect Bt-GM crops for the expression of insecticidal crystal protein (ICP) of *Bacillus thuringiensis*. One-step lateral flow tests, which are also called immunochromatographic strips (ICS) or dipstick tests, have been a popular platform for qualitative rapid visual tests, which use colloidal gold conjugate to generate signals.

In previous methodologies, QuickStix lateral flow test devices employ the same immunoassay principles as the plate format, but coat the antibodies and other reagents on a nitrocellulose membrane rather than on the inside of test wells or tubes. Nitrocellulose (NC) membranes have been the first choice of device manufacturers for over 20 years. A test strip assay device, in which a mobile conjugate labeled with colloidal labels such as gold, can be deposited on a chromatographic medium, and after reaction with an analyte, thus transported with the solvent to a test zone. The labeled mobilizable detection reagent reacts with an analyte, and the resulting product migrates with the liquid sample as the sample progresses to the test zone. During manufacturing, after the unlabeled binding agent is added to and immobilized in the test zone, the remainder of the test strip material is treated with blocking agents, in order to block any remaining binding sites. The zone where the mobilizable labeled reagent is located is often referred to as the "labeling zone," but can be referred to as the "reversible immobilization zone" or "mobilization zone" while the analyte is reacting with the mobilized labeled reagent, the liquid sample and mobilized labeled reagent migrates further within the porous carrier to the detection zone, where reagent that binds the same analyte is fixed or immobilized, usually in the form of a line. The important aspects of antibody pairs include steric separation of epitopes, an adequate titer of stocks, high affinity, high specificity, high avidity and purity.

The benefits of immunochromatographic tests include user-friendly format, very short time to get a test result, long-term stability over a wide range of climates and relatively inexpensive to make. These features make strip tests ideal for applications such as home testing, rapid point of care testing and testing in the field for various environmental and agricultural analytes. It is limited to diagnostic screening applications only. Furthermore, the achievable sensitivity is a factor of about 10–100 poorer than an instrumented laboratory immunoassay, restricting the technology's utility to relatively high abundance analytes only. Some of the more common lateral flow tests currently on the market are tested for pregnancy, strep throat [50], Chlamydia and human brucellosis [51]. Lateral flow assays have been used extensively as diagnostic tools for monitoring of toxins (**Figure 4**).

**Figure 4.** Lateral flow method.

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

**4.4. Immunoscreening**

58 Protein-Protein Interaction Assays

with radioactive compounds or enzymes [44].

**4.5. Cloned enzyme donor immunoassay**

enzyme activity and vice versa.

jugate to generate signals.

**4.6. Lateral flow immunochromatographic assays**

Type-IV hypersensitivity to chemicals, metals and environmental toxins such as molds can be determined by an immunoassay called as a memory lymphocyte immunostimulation assay (MELISA). The test determines the harmful substance in the blood, which is causing allergic reactions, but it will not measure the amounts of toxic substances. Two research articles showed MELISA had many false positive results, while one subsequent study showed that it is very reliable, specific and sensitive method to detect the metals in metal allergic patients [39–43].

It is a method to determine the proteins produced by genes inserted into expression vectors. For this, antiserum should be available and the secondary antibody should also be labeled

In this type of Immunoassay, in which two types of enzymes are being used which can be active only when they combine together [45]. The one enzyme is conjugated with the same type of specific analyte to be determined and this enzyme complex is called as analyteenzyme-fragment conjugate. The other enzyme attaches to the specific antibody. The analyteenzyme-fragment conjugate is unable to assemble with the other enzyme, if it is attached to

Therefore, when the analyte to be determined present in the serum is mixed with the analyteenzyme-fragment conjugate and antibody-enzyme. There is a competition between the analyte in the serum and the analyte-enzyme-fragment conjugate. If the concentration of analyte is high in the serum, then, it will attach with the antibody-enzyme, and the enzyme will be free to attach with the analyte-enzyme-fragment conjugate to give enzyme activity with the substrate. It means the higher the concentration of analyte in the serum, the higher will be

Simple devices (Strips) are being used to detect the analyte of interest in the sample without the need of any equipment. A widely used such tests are home pregnancy test, HCV, HBV diagnostic test, and so on. Immunoassays have the ability to be broadly executed on a business scale for the recognition of proteins in food [10, 46–49]. The test is used to detect Bt-GM crops for the expression of insecticidal crystal protein (ICP) of *Bacillus thuringiensis*. One-step lateral flow tests, which are also called immunochromatographic strips (ICS) or dipstick tests, have been a popular platform for qualitative rapid visual tests, which use colloidal gold con-

In previous methodologies, QuickStix lateral flow test devices employ the same immunoassay principles as the plate format, but coat the antibodies and other reagents on a nitrocellulose membrane rather than on the inside of test wells or tubes. Nitrocellulose (NC) membranes have been the first choice of device manufacturers for over 20 years. A test strip assay device,

the antibody. For this purpose, the antibody should be displaced from the enzyme.
