Has everyone had trouble with ELISA which is a low absorbance value including standard and positive control? I've already tried to prepare a new reagent, check the expiration date, etc. but the result still doesn't work. What should I suppose to do? or any suggestion about this?
Hello Suphasuta K.
Besides the steps that you have taken, you need to check for the following.
1. Have you given too many washes than what has been recommended?
2. Have you used the washer system, if yes, then is the system free of microbial contaminants?
3. Have you kept your plate for a short incubation time than what has been recommended?
4. Were the reagents and plate kept at room temperature at least 2-3 hours before starting the assay?
5. Just confirm that you have read the plate at the right wavelength.
6. Is your plate reader functioning well?
7. Has the detection reagent been too dilute?
8. Have you coated insufficient amount of antigen, or have you used less concentrated antibody?
9. You may check the history of the kit usage namely, how many times the kit has been removed from the refrigerator, or has the kit been exposed to temperatures at which it was not supposed to be.
10. Was the plate stored in sealed bag with a desiccant to maintain stability?
Hope these above questions may help you solve your problem!
Best.
This site has a plan for possible low OD.
https://www.sinobiological.com/category/elisa-troubleshooting-low-od-readings
If you did all, try to read in another equipment, maybe you need to change the lamp or there is an internal problem.
Hello Suphasuta K.,
I hope you might have carefully followed protocol and conducted the ELISA as per the instructions provided in the company manual of the kit. I guess that the poor color development in the ELISA plate could have resulted the low absorbance value including standard and positive control. If it is so, instead of searching the underline causes simply replace the kit in order to save your time as well as precious experimental samples.
Best
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