Top Ways Of Improving The Accuracy Of ELISA Tests

ELISA or enzyme-linked immunosorbent assay is a popular test for detecting and measuring the amount of antibodies present in the blood. It is generally administered to establish the presence of certain antibodies that are indicative of infections present in the body. Among the diseases that ELISA tests are used to diagnose are HIV, Lyme disease, Rocky Mountain spotted fever, pernicious anemia, rotavirus, syphilis, squamous cell carcinoma, varicella-zoster virus, and toxoplasmosis. Usually, a medical practitioner will suggest the ELISA test as a screening tool if the patient has symptoms that are associated with the above-mentioned diseases or even to rule out these conditions.

blood-test

The easy availability of ELISA test kits commercially has made it very easy to administer the test; however, there are still quite a few common mistakes that prevent you from getting accurate results. Some simple tips on how to prevent errors from ruining the test:

Customize the Test Protocol

Carefully peruse all the detailed instructions provided in the manual of the ELISA test kit, and then compose a brief personalized protocol specifying the various steps. By doing this you will be able to avoid searching the manual for important information while you are conducting the test. Preparing the note is also an excellent method of running through the test protocol so that no surprises are encountered during the actual experiment.

Arrange Samples In the Order of Usage

It is a good test procedure to make a template showing where all the samples are on a standard 96-well plate as this saves you the trouble of labeling the plate itself. You can take things a step further by arranging the required samples in the order marked on the template before actually adding them to the plate. The process makes it so much easier for you to keep track of what has been added so the chances of committing mistakes are automatically reduced.

Add a Background with High Contrast

The problem of adding samples or reagents that lack color to the wells is that it may be difficult to establish whether it has been added or not when the plate is on the bench. By using a black sheet of paper or similar you can easily create a dark background below the plate making it simpler to see if a reagent has already been added to a particular well in the plate.

Achieve Pipetting Consistency

You could end up with large data variations due to even very small pipetting differences and this could make the results more undependable. For example, it can be quite difficult to keep track that the exact volume of the reagent has been added to each of the wells as this involves making sure that even the last drop in the tip of the pipette has been dispensed. The reading of the test can also be thrown off due to the bubble accumulation that occurs when the same pipette tip is used. Even tiny differences in the delivery of the solution volumes that can occur due to variance in the wetting level in the pipette tips that occurs with successive use can reduce the accuracy of the test results. To ensure consistency, it is recommended that the tips be changed.

Deploy the Spectrophotometer in Case of ELISA Reader Failure

In a worst case scenario, it may happen that you discover the ELISA reader is not functioning after you’ve set up the assay. The data can still be rescued by using a spectrophotometer. The minimum cuvettes are to be used to read the standards series and the standard curve will have to be manually plotted since the instrument will not perform an automatic analysis. The next step comprises reading the samples well-wise with cuvettes that are on a standard specification, and then the readings need to be plotted against a standard curve. In the case of not having the minimum cuvettes, regular volume cuvettes can be used after diluting the samples and the standards equally.

While many of these suggestions may seem common sense, they may be of great help to beginners who generally tend to end up wasting a lot of time and effort due to these mistakes.

Author bio: James Leland is the chief pathologist of a reputed multi-specialty clinic that carries out a large number of ELISA tests. James has a number of published articles in medical and pathology journals, including one on the detection and measurement of ERG antibody that is recommended reading for pathology students.

Leave a Reply