Dear, I'm developing an in-house indirect ELISA to detect the present of antibodies in the serum against target protein of the pathogen. The setting of my ELISA is that I have the target protein coating overnight. Then, the serial dilutions of the serum from each donor are added, following by detection abs and substrate. The problem is that the low serum dilution (high concentration of serum) of some donors give lower signal than the high serum dilution (low concentration of serum). I try to discuss this as it could be from the matrix effect but I'm not sure if this could be the factor since I always blame myself for any nonsense errorness :'). Thank you in advance for the comment
You may be observing the Hook Effect, which occurs with certain immunoassay protocols when the substance to be detected is present at a very high concentration.
https://en.wikipedia.org/wiki/Hook_effect
Mostly Poor ELISA Standard Curve with low TMB O.D comes from degradation of coating in ELISA plate. But Conjugate degradation, washing and TMB sensitivity may lead to low signal ELISA. you can find more here
https://diagnopal.ca/elisa-standard-curve-problems-and-elisa-curve-troubleshooting/
Sudeep Kumar Maurya I'm sorry for not giving the clear information. Firstly, I have the antigen coated on the well. The following day I block the well with blocking solution before adding the serum from each human donor. Next I use the detection ab, which is the goat anti-human IgG that conjugate to the HRP. Finally I add the substrate, which is the ABTS to it. The antigen is supposed to specifically bind to serum from human mainly as it is the antigen from human pathogen. Thank you for the explanation of yours, I also think in that way too.
Adam B Shapiro Thank you so much, I guess I ever learnt this in the class but I forgot that it can be this phenomenon. Thank you so much again ^^
Akbar Khadem-Sadegh Thank you for the reply :), I suspect the reason to be from that too as it could be from errorness during process. I'll try to optimize it
It is good Idea, I think for ElISA optimization doing Checker- Board will save your time and give you better idea about your reaction component. Remember to use analyte concentratio in highest biological level. See the link
https://diagnopal.ca/elisa-set-up-with-checker-board-optimization/
Moangsom, I have had this problem before. It is not due to Hook effect because you doing Indirect ELISA. It probably is because of some High affinity and low concentration of IgA. Also, look for ELISA interference and false negative results.
you can find here more:
https://diagnopal.ca/what-is-interference-and-cross-reaction-in-elisa/
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