Explain the role of the secondary antibody and the chromogen

Explain the role of the secondary antibody and the chromogen in an ELISA? Why did you perform 3 tests lot each serum sample? One of the patients had a weak positive test result. Give at least two plausible reasons for a specimen to yield a weak positive result. In your handout or through an online search, read about the pathogen for which your \"patients were being tested. Then, review your patient profiles and results. Identify at least on patient that had results that surprised you. Explain why you found those results surprising.

Solution

Please find the answers below:

Part 1: ELISA is the techniques used for quantitative assessment of analyte present in a sample. Its principle is based upon incubation of sample with primary antibody and then with secondary antibody. The secondary antibody is conjugated with an enzyme, which produces either fluorescnce or generates a color when an appropriate substrate is added to the system. For most of the cases, the detection system is a chromogen which produces a blue-colored product whose absorbance can be read using a spectrophotometer. Thus, the secondary antibody contains the enzyme which can produce a colored product when an appropriate substrate is added. Common examples of enzymes are horse radish peroxidase, alkaline peroxidase etc.

Part 2: While performing ELISA, frequent manual handling of samples takes place. This can increase handling errors and manual problems with the results. In order to avoid such problems, generally triplicates of samples are analysed by ELISA. This also increases replicability of the experiment and adds precision to the tests.

Part 3: The possible reasons why a sample yields low signals are:

Part 4: The analyte to be tested should be properly mentioned by the ELISA kit manufacturer on the hand-out of the kit. The clonicity i.e. monoclonal or polyclonal antibodies used should also be clearly mentioned in the hand out. One of the facts that can yield arbitrary/strange results is presence of a negative signal from an otherwise infected/positive signal. The possible reason for such result can be handling errors, excessively dilute sample, problem with any of the components of the kits etc.