What is the Elisa Test

What is the Elisa Test?

ELISA is an acronym for enzyme-linked immunoassay. It is a laboratory test that is frequently used to identify antibodies in the bloodstream. An antibody is a protein generated by the body’s immune system when it recognizes dangerous components, known as antigens, that it considers harmful to the body. Antibodies and antigens are combined to create a measurable outcome in the detection of these products.

The same method is also known by other names, such as enzyme immunoassay (EIA). An ELISA test involves immobilizing the antigen (target macromolecule) on a solid surface (microplate) and then combining it with an antibody linked to a reporting enzyme. The reporter enzyme activity is measured by incubating it with the proper substrate to create a quantifiable result, which is then measured. A highly specific antibody-antigen interaction is the most important aspect of an ELISA test.

What Does the ELISA Test Measure?

In a nutshell, the ELISA test measures antibodies, antigens, proteins, allergens, and glycoproteins in a blood sample and is used in the diagnosis of different health conditions. 

Diagnosis of HIV infection, pregnancy testing, syphilis, Lyme disease, rotavirus, and Rocky Mountain spotted fever are only a few examples. ELISA assays are typically performed in 96-well plates, allowing for the measurement of numerous samples in a single experiment.

To ensure the antibody or antigen clings to the surface, the plates used must be specific absorbent plates such as NUNC Immuno Plates. Each ELISA test detects a different antigen, and kits for a wide range of antigens are readily available.

The fundamental benefit of ELISA is its great sensitivity and specificity, which allows it to detect target molecules at pictogram levels. Because of the simple and less demanding experimental procedures, it is commonly employed for high-throughput screening.

Target molecules can be quantified by ELISA in a range of samples, including serum, plasma, urine, saliva, cell, or tissue extracts, and so on.

Getting the Sample for Analysis

The first step in the ELISA test is the acquisition of a blood sample. First, an antiseptic will be applied to your arm by a physician or laboratory technician. Then a tourniquet, or band, will be wrapped around your arm to put pressure on your veins and force them to bulge with blood. A needle will then be inserted into one of your veins to take a little blood sample. The needle will be removed once enough blood has been collected, and a little bandage will be placed to stop any bleeding.

To limit blood flow, you’ll be advised to apply pressure to the place where the needle was entered for a few minutes. Although this process should be painless, your arm may throb a bit afterward. 

 The blood sample will be taken and submitted to a facility for analysis. The technician will place the sample on a petri dish containing the exact antigen for the ailment for which you are being tested at the lab. If the sample has antibodies to the antigen in your blood, the two will bind together. The technician will check this by putting an enzyme in a petri dish and watching how your blood reacts with the antigen.

If the color of the contents of the dish changes, you may have the condition. The presence and amount of antibodies can be determined by the amount of change caused by the enzyme.

How Does the Elisa Test Work?

Direct ELISA in medical labs is commonly performed in a 96-well microtiter plate by a chemical process resulting in the covalent attachment of antigen, which is usually a protein, through its free amino groups. 

The test then entails probing each coated well with an antibody specific for the target antigen, probing that bound antibody with a second antibody specific for the first antibody’s stable region, and detecting the results using a variety of methods.

ELISA enzymes include horseradish peroxidase (HRP) and alkaline phosphatase, with substrates including tetramethylbenzidine (TMB) and 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS). To establish a biologically acceptable range of detection, double or triplicate sampling is often suggested, with varying concentrations of the sample utilized.

You can use a goat-anti-rabbit IgG antibody also to investigate the specificity of an antibody-antigen response and compare the binding of this antibody serum to IgGs in chicken, cow, horse, and rabbit serum in this experiment. To help with this, anti-rabbit IgG has been covalently bonded to peroxidase, which catalyzes a color-producing reaction, allowing you to examine the data without the use of specialized equipment.

The reagents and testing equipment for the experiment will be included in the ELISA Immunoassay kit, which can be used twice by eight groups or once by 16 groups of individuals. Before beginning the lab, make sure that each group has read the instructions that came with their supplies and has all of the materials listed in the experiment guide.

Microtitration plates are only utilized in high-throughput screening and must be handled automatically. For optimal results, some enzyme substrates, such as those that produce fluorescent or chemiluminescent signals, may require opaque plates.

The included microtitration plate should be numbered. These plates are typically designed to hold 96 wells across their surface, but they will be given a 1/4 piece of a conventional plate for the sake of this experiment, resulting in a plate with 24 wells.

For easy tracking of the experiments, these plates should be labeled with the numbers one through six across the top of the grid and the letters A to D down the left-hand side.

Next will be the antigen absorption, which will involve successive, ten-fold dilutions of the sera, followed by the plate being left undisturbed for twenty minutes to allow enough time for the proteins to be adsorbed to the well surfaces.  After that, TBS-gelatin will be used to block non-bound serum protein sites on the plastic.

Now, use pipettes to properly discard the liquids in each well when this process is completed, preparing them for the antibody response. From here, add goat anti-rabbit IgG peroxidase to each well and spin the plate to ensure that the antibody solution reaches all surfaces at the bottom of each well.

For the antibody to adequately attach to the immobilized IgG, the plate must be left alone for another 20 minutes. After that, TBS-gelatin will be used again, and the surplus solution will be removed as before.

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