Principle of different Serological Techniques Direct ELISA (antigen screening) The method was invented concurrently in 1971 by Engvall and Perlmann and Van Weemen and Schuurs, and it paved the way for additional ELISA kinds. The direct ELISA approach is ideal for measuring the quantity of antigens with a high molecular weight. The antibody or antigen is directly coated on the plate's surface. The measurement is possible thanks to an enzyme-tagged antibody or antigen. After incubation, the unbound antigens or antibodies are removed from the media by washing. The suitable substrate is then added to the medium in order to generate a signal via colouring. The quantity of antigen or antibody is determined by measuring the signal. (Aydin, 2015) Indirect ELISA Lindstrom and Wager, who were inspired by the direct ELISA approach, invented the technology in 1978. This approach was used to measure swine IgG, according to the researchers. The indirect technique gets its name from the fact that it is another antibody in the medium that identifies and separates the antigen to be tested, rather than the primary antibody. The diseased serum is added to the antigen-coated wells and the plates are incubated in this way. Antibodies produced against antigens in the sick serum plaque form an antigen–antibody complex during this incubation. A secondary antibody that detects the antibody in the serum and is tagged with the enzyme is added to make the antigen– antibody complex visible. After that, the enzyme's substrate is introduced to the medium to generate colour, and the concentration is calculated. This approach of detecting antigens is more widely employed in endocrinology. (Aydin, 2015) Sandwich ELISA The Sandwich Elisa Principle is based on the detection of antigens using sandwich ELISA. Antibody is coated on the microtiter well in this approach. The antigen-antibody complex is formed when a sample containing antigen is put to the well and allowed to react with the antibody linked to the well. Following the washing of the well, a second enzyme-linked antibody specific for a different antigen epitope is introduced and allowed to react with the bound antigen. Preparing a surface on which a known quantity of antibody is attached is part of the Sandwich Elisa Protocol. Incubate the plate at 37 degrees for the antigencontaining sample. Wash the plate to eliminate any unbound antigen. Rapid Diagnostic Techniques Rapid diagnostic tests (RDTs) are a sort of point-of-care diagnostic, which means they're designed to give patients diagnostic findings quickly and easily while they're still at the hospital, screening site, or other health care provider. Receiving a diagnosis at the point of care reduces the need for multiple visits to receive diagnostic results, improving diagnostic specificity and increasing the likelihood that the patient will receive treatment, reducing reliance on presumptive treatment, and lowering the risk that the patient will become sicker before a correct diagnosis is made. Rapid tests are utilized in a number of settings, including residences, primary care clinics, and emergency rooms, and many don't require laboratory equipment or medical knowledge. There are a variety of platforms that are often utilized to create fast diagnostic tests. The following table summarizes the relative utility of common RDT platforms. Because all of the reactants and detectors are contained in the test strip, lateral flow tests are the simplest sort of RDT. They need very little experience with the test and no equipment to execute. The sample is put in a sample well and migrates through the zone where the antigen or antibody is immobilized in a lateral flow test. After a specified length of time has passed, the findings are read. A flow-through test, which yields findings even faster than lateral flow tests, requires an additional wash and buffer phase, limiting its mobility and stability. The binding of carrier particles and target analytes into visible clumps, as viewed using a microscope or with the naked eye, is the basis of an agglutination test. However, if the particles' affinity is poor, the test findings may be inconclusive. RDTs in the dipstick format (with multiple antigen binding sites) function by dipping the dipstick into a sample. To avoid non-specific analyte binding, the dipstick is rinsed and incubated. In low-resource point-of-care settings, these extra processes may restrict its utility. Microfluidics, sometimes known as "laboratories on a chip," is a rapidly developing field of fast diagnostics. These tests would use electrochemical sensors and incorporate all detectors and reactants in a single portable cassette. Western Blotting Western blotting separates proteins by size and uses an antibody to label the protein of interest. Western blotting (also known as Protein Immunoblotting) is a commonly used analytical technique for detecting particular proteins in a sample. It uses an antibody to precisely recognize its antigen. It separates distinct proteins in a given sample using SDSpolyacrylamide gel electrophoresis (e.g. to separate native proteins by 3-D structure or denatured proteins by the length of the polypeptide). The separated proteins are then blotted or deposited onto a matrix (usually nitrocellulose or PVDF membrane) and stained with antibodies (probes) specific to the target protein. The expression details of the target proteins in the supplied cells or tissue homogenate might be acquired by assessing the location and intensity of the specified response. Due to the high resolution of the gel electrophoresis and the immunoassay's exceptional specificity and sensitivity, Western blotting analysis may identify target proteins as little as 1ng. In the domains of molecular biology, biochemistry, immunogenetics, and other molecular biology disciplines, this approach is applied.
