ELISA, also known as enzyme-linked immunosorbent assay, is the basic technique that is carried out to detect and quantify antibodies, proteins, peptides, and hormones in the sample, like blood, serum, plasma, and tissue. Antibodies are the proteins produced in blood in response to the immunogenicity illicit by the specific antigens. It helps in the examination of the presence of antibodies in the body in response to certain infectious diseases. ELISA is a unique analysis in comparison to other antibody-assays as it gives qualitative and quantitative results together with the separation of specific and non-specific interactions taking place via serial binding to solid surfaces, which is typically a polystyrene microtiter plate.

Principle of ELISA Test

The ELISA technique is developed for antigen or antibody detection. Use the appropriate antigen or antibody that is firmly fixed on a solid phase, like the plastic surface of a polyvinyl plate or polystyrene tube. It is also known as "solid-phase immunosorbent assays" (SPIA). When a test sample is put on a microtitre plate, Ag-Ab reactions will occur if Ag or Ab are present in the sample (with immobilised Ab or Ag). The reaction mixture is further supplemented with enzyme-labeled antibody, which will eventually bind with either the test antigen or the Fc region of the test antibody. The enzyme system consists of:

1. An enzyme: horse radish peroxidase, alkaline phosphatase which is labeled or linked, to a specific antibody
2. A specific substrate:

• o-Phenylenediamine dihydrochloride for peroxidase
• P Nitrophenyl Phosphate (PNPP)- for Alkaline Phosphatase

At the end, the substrate is added to the antigen-antibody reaction. The enzyme catalyses the substrate to produce the color end point (a yellow compound in the case of alkaline phosphatase). The amount of antibody or antigen in the test sample is measured using an ELISA reader, and the intensity of the colour is proportionate to that amount.

Characteristics of the ELISA Test

The ELISA test is increasingly used in the detection of antigens or antibodies due to its high sensitivity and simplicity. It uses only microliter volumes of test reagents and is just as sensitive as radioimmunoassay (RIA). The detection of several antibodies and antigens, including hormones, poisons, and viruses, has now been widely used around the globe.

1. The excellent specificity and sensitivity of the ELISA test
2. It is possible to read the results of quantitative ELISA tests visually.
3. A lot of tests can be performed simultaneously. ELISAs are appropriate for use in monitoring and centralised blood transfusion programmes since they are developed specifically for screening large numbers of specimens at once.
4. ELISA reagents are stable and can be distributed in district and rural laboratories, but their usage is restricted to specific optimised conditions as more complex instruments and qualified workers are required to perform the test. The ELISA test has high sensitivity and specificity.

Classification

ELISA tests can be classified into three different categories depending upon the methods used for binding interactions between antibodies and antigens, namely as:

• Indirect ELISA – Antigen is coated to the microtiter well
• Sandwich ELISA – Antibody is coated on the microtiter well
• Competitive ELISA – Microtiter well which is antigen-coated is filled with the antigen-antibody mixture.

Indirect ELISA

Indirect ELISA is a two-step process involving the binding of a primary antibody and a labelled secondary antibody. The first step is the incubation of the primary antibody with the antigen, which is followed by the incubation with the secondary antibody. However, secondary antibodies can induce a cross-reaction, which might result in nonspecific signals. In the indirect ELISA test, the sample antibody is sandwiched between the antigen coated on the plate and an enzyme-labeled, anti-species globulin conjugate. Color is produced by the addition of an enzyme substrate and a chromogenic reagent. The colour production is directly proportional to the amount of bound sample antibody. The colour development in the test wells increases with the amount of antibody in the sample. Indirect ELISA is useful for quantifying total antibody levels in samples.

Protocol:

1. Coat the microtiter plate with antigen: Pour 50 µl of antigen solution (a coating agent) into each well.
2. Seal the coated plates with plastic wrap and incubate them in an incubator for two hours at 37 °C.
3. Uncover the microtiter plate after incubation, then pour the solution into a container.
4. Washing Step: After removing the coating solution, wash the plate twice by adding 200 µl of PBS to each well. By pipetting, the solutions or washes are removed. Remove the last few drops by patting the dish with a piece of paper towel.
5. Blocking step: Add 200 µl of blocking buffer to the coated wells and incubate for 30 min at room temperature to block the last of the protein-binding sites.
6. Dispose of the solution and carry out the washing procedure. Flip the microplate gently onto the paper towel. Add 50 μl of antibody solution using micropipette from the vial to the wells.
7. Cover the plate with plastic wrap and incubate at room temperature for two hours. Discard the liquid, then place dry absorbent paper on the plate's bottom.
8. Repeat the washing step.
9. Repeat the blocking step.
10. Repeat the washing step.
11. Add 50 µl of the secondary antibody reagent to each well.
12. Cover the micro-titer plate with plastic wrap and let it dry at room temperature for 2 hours.
13. Repeat the washing process.
14. Add 75 µl of the substrate solution to each well of the microtiter plate.
15. Cover the microtiter plates with plastic wrap and incubate at room temperature for one hour. Add 25 µl stop solution (0.5 M NaOH) from the vial to the wells on the microtiter plates.
16. Using a microtiter plate reader to measure NPP hydrolysis, use a 405-nm filter.

Sandwich ELISA

A sandwich ELISA quantifies the presence of antigen between the two layers of antibodies, i.e., the capture and detection antibodies. At least two antigenic sites must be present on the target antigen to optimise its binding to antibodies. In sandwich ELISA, either monoclonal or polyclonal antibodies can be used as the capture and detection antibodies. Monoclonal antibodies can quantify even a minute difference in antigen by recognising a single epitope. A polyclonal antibody is frequently used as the capture antibody to pull down as much antigen as possible. Sandwich ELISAs remove the sample purification step before analysis and enhance sensitivity (2–5 times more sensitive than direct or indirect). Sandwich ELISAs increase sensitivity by skipping the sample purification step before analysis (2–5 times more sensitive than direct or indirect). (Figure 2)

Coating with Capture Antibody

1. Coat the capture antibody to the wells of a PVC microtiter plate at a concentration of 1–10 μg /mL in carbonate/bicarbonate buffer (pH 9.6).
2. Seal the plate with adhesive plastic and incubate it at 4°C overnight.
3. Remove the coating solution, then wash the plate twice with 200 µl of PBS placed in each well of the microtiter plate. Discard the solution by flicking the plate over a sink. Remove the remaining drops by patting the dish with a piece of paper towel.

Incubation with Detection and Secondary Antibody

1. Add 100 µl of diluted detection antibody to each well of the microtiter plate.
2. Wrap the plate with adhesive plastic and let it dry at room temperature for two hours.
3. Use a PBS buffer to clean the plate four times.
4. Add 100 µl of conjugated secondary antibody to the blocking buffer before use.
5. Cover the microtitre plate with adhesive plastic and incubate at room temperature for a couple of hours.
6. Wash the plate with PBS buffer four times

Detection

The two most used enzymes for ELISA assays are alkaline phosphatase (ALP) and horse radish peroxidase (HRP).

ALP Substrate

For the majority of applications, P-nitrophenyl-phosphate (pNPP) is the most often employed substrate. After 15 to 30 minutes of incubation at room temperature, check the nitrophenol's 405 nm yellow colour, and halt the reaction by adding an equal volume of 0.75 M NaOH.

HRP Chromogens

The substrate for HRP is hydrogen peroxide. Hydrogen peroxide cleavage is coupled to oxidation of a hydrogen donor which later changes color during reaction.

TMB (3,3',5,5'-tetramethylbenzidine)

Add the TMB solution to each well, allow it to stand for 15 to 30 minutes, then add an equal volume of the stopping solution (2 M H2SO4). The optical density should me measured at 450 nm.

Calculations

Always run ELISA tests on duplicate or triple samples. This will help in providing adequate data for the results' statistical validation. There are now many computer programmes that help in ELISA result processing. For each set of identical standards and samples, calculate the average absorbance values. Duplicates should lie within the 20 % of mean.

Standard Curve Plot

Plot the mean absorbance (y axis) versus the protein concentration to create a standard curve for the target protein (x axis). Obtain a best-fit curve between the graph's points (there are many suitable computer software programmes that help in graph plotting). Each sample's target protein concentration can be calculated using the standard curve. This is normally done via curve-plotting software. This will further provide you with an equation to determine the concentration (x) from an absorbance (y) in the standard curve's range. The samples should be diluted or concentrated prior to the ELSA test in order to get accurate results. When assessing the results for these samples, the concentration determined from the standard curve must be multiplied by the dilution factor.

Coefficient of Variation

The coefficient variation (CV) is the ratio of the standard deviation σ to the mean µ:

Cv= σ/µ

This is expressed as a percentage of deviation from the mean and measures errors in the results. Larger errors are indicated by a larger variance. Using the ELISA data, several computer programmes can determine the CV values.

High CV May Result From:

Pipette tips should be sealed to the pipette before use to prevent inaccurate pipetting and guarantee that the proper volume of liquid is drawn up.

Reagent splashing between wells

contamination of reagents or screen samples by bacteria or fungi

Reagent contamination

Variations in temperature across the plate; make sure the plates are incubated in a temperature-controlled space away from draughts.

Make sure the plates are always covered during the incubation steps if some of the wells are drying out.

Spike Recovery

The impact of sample components on antibody detection of the antigen is assessed by spike recovery. For instance, many proteins included in tissue culture supernatant may prevent antibody binding and raise the signal-to-noise ratio, which will cause the target concentration to be underestimated. Protein spikes at predetermined concentrations are added to both the sample matrix and a reference diluent. Results from the sample matrix and the standard diluent are contrasted, and the assay is used to quantify the spiked protein. The sample matrix is regarded as valid for the assay process if the findings are identical. If the recovery is different, something in the sample matrix is preventing the analyte from being detected.

Direct Competitive ELISA Assay

In a competitive enzyme-linked immunosorbent assay, the purified antigen competes with the antigen present in the test sample for binding to an antibody that has been immobilised in microtiter plate wells. It is also known as "inhibition ELISA." The same concept works with the antibodies present in the test sample and the purified antibodies for the immobilised antigen. A direct ELISA assay uses labelled antigen or antibody, while indirect assay configurations use reporter-labeled secondary antibodies. The concentration of small-molecule antigens in a complex sample can be determined by competitive ELISA. In the direct ELISA, antigen-specific capture antibodies are adsorbed onto the microtiter plate prior to incubating with either known standards or unidentified test samples. Labeled antigen can only attach to the capture antibody when neither the antigen standard nor the antigen in the test samples have occupied the antibody's binding site. Unbound labeled or unlabeled antigens are removed and substrate is added. The amount of reporter-labeled antigen bound to antibody depends on the concentration of antigen in the standard or test sample, producing a signal that is inversely proportional to the antigen concentration in the sample. Therefore, less tagged antigen is bound to the capture antibody, and, as a result, the resultant signal is weaker with higher antigen concentrations in the test sample. (Figure 3)

Advantages of the ELISA Assay:

1. ELISA results provide an accurate diagnosis of a specific disease as they involve the use of two antibodies.
2. This is applicable for complex samples as the antigen does not need to be purified in order to be detected.
3. Results are highly responsive, as both direct and indirect methods are available.
4. Provide quick and rapid results.
5. The possible detection range of ELISA includes quantitative, semi-quantitative, standard curve, qualitative, calibration curve, etc.
6. It is easy to perform and does not require the presence of radioactive material.

Applications of ELISA

1. Detection of antigens and antibodies in the sample
2. Identification of any food allergens present in the food sample.
3. To calculate the serum antibody concentration for a viral test.
4. During a COVID-19 outbreak, rapid kits are used to identify the presence of antibodies in a blood sample so as to assess the spread of the disease.
5. ELISA kits are widely used in the diagnosis of Ebola, pernicious anaemia, AIDS, rotavirus, Lyme disease, syphilis, toxoplasmosis, the Zika virus, carcinoma of the epithelial cells, and many other pathologies.

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