At the moment I'm trying to optimize a competitive Vitamin B12 ELISA. It worked very well for a range between 200 pg-1ng, but unfortunately I need a range of 45pg-1ng. Can you give me any advices to improve the sensitivity and dynamic range of my assay.
I used an assay with 57Co-labeled vitamin B12 mixed with my sample of interest and then adsorption of free unbound vitamin B12 on BSA saturated charcoal to detect vitB12 binding proteins. Quick, easy, sensitive. But I cannot find a commercial source of 57Co-labeled vitB12 anymore.
If anybody has an idea on where to buy labeled vitB12, contact me.
Hello Regis,
We are using HRP-conjugated B12. of any interest for you?
Take care
vincent
Hi Vincent,
If your HRP-B12 is working in competitive ELISA, it means it should be recognized by transcobalamins (Y/N)? If Yes, send me the details of the product.
To your specific problem, I don't know how you are coating your wells/chips but I would go for detergent blocking (i.e. no protein blocking solution).
The molar ratio B12:HRP might be critical. If the current ratio is 1:3 maybe a cross-linking ratio of B12 to HRP of 1:1 is decreasing the competition and improving the sensitivity of your assay. On the other, increasing the molar ratio of B12 relative to HRP might increase the affinity of the conjugate for the coating surface, enable you to lower down HRP concentrations used for competition and increase your sensitivity this way......
As you did your homework and already have a working ELISA, change your ELISA substrate. This is probably the easiest change to try. These are all the general comments that I can do based on the information given in your question.
Good luck.
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