ELISA or Enzyme-Linked Immuno Solvent Assay is a quantitative and qualitative detection technique that relies on antigen-antibody reactions. ELISA can detect antibodies or antigens, which can be viral, bacterial, hormones, or proteins. An antigen is immobilized on a plate in an ELISA assay and then associated with a detection-friendly molecule, such as an enzyme or fluorophore. Multi-well plates with 96 or 384 wells usually serve as the ideal platform for ELISA assays because of their sturdy surface.

There are four primary types of ELISA that include Direct ELISA, Indirect ELISA, Sandwich ELISA, and Competitive ELISA.

Direct ELISA:

In Direct ELISA, the target antigen immobilized on the multi-well plate's surface is detected by an antigen-specific antibody directly coupled to detecting molecules.

Indirect ELISA:

Indirect ELISA uses a two-step detection process to attach a primary antibody specific to the antigen to the target. A secondary antibody labeled with tags is then added to detect the primary antibody, which is produced against the species of the primary antibody host.

Sandwich ELISA:

It is the most commonly used ELISA format that requires two specific antibodies for an antigen. One antibody is coated on the multi-well plate's surface, which helps immobilize the antigen. The second antibody, due to its conjugation, assists in the antigen's detection.

Competitive ELISA:

Competitive ELISA, like the previous formats, can be changed to meet the competitive format. Competitive ELISA assays measure the antigen concentration by detecting signal interference.

ELISA Conduction Evaluation and Sources of Error

To establish the total human IgG concentration in plasma samples, the Sandwich ELISA procedure was used. In Sandwich ELISA, two separate antibodies for the same target antigen are used to detect the proteins. The plasma samples were exposed to a capture antibody that was immobilized in wells and then blocked with a buffer.

If the plasma sample contains the target antigen, it will bind to the capture antibody in the well, which is then rinsed to remove any unbound antigens. The next step involves adding an enzyme-linked antibody (detection antibody) to the well, which binds to the antigen. After several rinses are performed to remove any unbound detection antibodies, the substrate (chromogen) is added to the well. The colour development confirms the existence of the antigen, and a sulfuric acid solution is added to prevent any further reaction.

Several washing steps are necessary to carry out the procedure efficiently, and it is necessary to make sure the washing buffer is removed, and the plate is blotted with tissue paper to remove any leftover residue after the washing. It is also essential to use micropipettes appropriately and consistently because any inconsistency will alter the results. When conducting the procedure, it is important to ensure that the capture antibody is not scratched while pipetting.

The following report entails the analysis and discussion of results that were generated during a workshop. The experiment produced data which was used to calculate the coefficient of variation (CV) after running samples in triplicate. The aim for the CV was to be less than 20%, however, Sample 4 and 5 exceeded the desired value, indicating inconsistencies and inaccuracies in the results. Potential reasons for this deviation included incorrect pipetting, reagent spillage, and cross-contamination.

To estimate the concentration of the IgG, a standard curve equation was used in the form of y = 0.0092x + 0.2035. The equation shows the correlation between absorbance and concentration respectively. In order to calculate the concentration of the sample, we used the equation in the form of x = (y - 0.2035)/0.0092. Samples 1, 2, 3, 4, and 5 were tested and their respective concentrations were calculated. Sample 2, 4, and some of the calculations showed a negative value, which indicated an out of range result. To adjust for this, dilution or concentration was necessary to bring the sample reading within the scope of the range.

References were used to provide supplementary information, including Sandwich Elisa methodology, principles of ELISA, and a Sandwich Elisa protocol.

Ultimately, the workshop results showed that while some of the results were accurate, some were not, which indicates that close attention must be paid to factors that influence sample quality throughout the experimental process.