Hi everyone,
I have a doubt regarding standards for ELISA. I don't have purified antigen for marking standards. can I use crude extract in different dilutions as my standards? or if not how to do ELISA without standards. Kindly help me.
It depends what your crude extract is and what you are measuring by ELISA. Let's assume that you are measuring antibody titers to some specific antigen (X) in sera of immunized/infected animals. Let's assume that you have an E. coli strain expressing antigen X that you want to use for your ELISA's. If you do not have a purified antigen X, you can use "crude extracts" of antigen X-expressing E.coli. However, as a background control you will also need to use E.coli that does not express that particular antigen X. In addition, you will need to assay the sera of controls and immunized/infected animals. This is a "crude" method, but if you are just looking for a yes/no antibody response to an antigen X you can do it. It is difficult to answer your question, since you provide very little detail.
If you have everything in the same buffer, and your 'crude extract' is some representation of your 'standard' condition, and you always use the same 'crude extract' you can use serial dilutions of it to compare to the unknown samples. For example if you measure a serum protein and you have 'normal' serum, you can use it instead of the purified protein, however you won't be able to tell exact amounts only % of your 'normal'.
Thank you Vjollca Konjufca and Tunda Hidvegi
i will try it and check. thanks for your valuable suggestion.
Hi, my friends above have given you some good advice, I would only add that you need to make a pool of all your normal that way it tells you some information about the population and how the individuals relate to that particular population. Also use this method for your standards and you should be able to get it published, if you do well enough on the rest of your study.
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