I would like to know how to identify ELISA data are positive or negative
Hello Aly, Colour development and intensity, depending on nature of substrate (TMB: 3,3’,5,5’-tetramethylbenzidine or pNPP: p-nitrophenyl-phosphate) are gold standards for detection of positive results which normally should develop after 15 min. ELISA data is typically graphed with optical density vs log concentration to produce a sigmoidal curve. Known concentrations of antigen are used to produce a standard curve and the data is used to measure the concentration of unknown samples by comparison to the linear portion of the standard curve. In fact, it is the relatively long linear region of the curve that makes the ELISA results accurate and reproducible. The unknown concentration can be determined directly on the graph or with curve fitting software which is typically found on ELISA plate readers.
First select which substrate you are using to develop colour. For test (depending upon direct , indirect, sandwich etc. ELISA) you have to plot standard graph. Decide the minimum concentration giving the results with different concentrations and also see for its linearity with positive sample. Run the same with negative samples and decide the best concentration of bio molecule to be considered or can differentiate from negative results as positive. At each time run both positive and negative serum samples and run each sample in triplicate to ensure the results.
Hello Aly,
Basically, ELISA is an antigen-antibody reaction, in order to plot the stand graph we prefer specific antibody against the antigen. But yes, there are so many different types of ELISA could be performed based upon the question you are targeting (for example, in my case I have coated the plate with peptide, and I was expecting that to bind with one of the protein of my interest). There are so many binding assay are also possible to validate you result, such as Co-Immunoprecipitation (Co-IP). But since ELISA is very sensitive you must troubleshoot your result (as per the suggestion given above) if you are not getting the result as expected.
Hope I could address it properly.
Good luck
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