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I´ll answer the questions below. But I´m afraid we misunderstood each other. I ordered recombinant adiponectin (ab13882-100 lot 71252)and used it as standard in our ELISA. The lyophilized adiponectin was reconstituted as described in the product sheet: 0.1 M Acetat buffer pH 4 and after reconstitution we immediately diluted the adiponectin to a concentration of 10µg/ml in PBS to neutral pH. Than we used the diluted adiponectin as standard material in our ELISA, which functions very well with recombinant adiponectin of other providers. So there is no problem with the ELISA itself, but with your adiponectin. What do you think about reconstitution procedure - it seems to me rather rough, is adiponectin not solvable in neutral PBS? I would like to try this way, if you can provide another aliquot of adiponectin. 1. Please describe the problem (high background, no signal, non-specific colour development, poor standard curve etc). your recombinant adiponectin gives no signal - adiponectin of other sources does! 2. What type of ELISA are you performing? (Direct ELISA, indirect ELISA, sandwich ELISA etc.) sandwich elisa 7. Which detection system did you use? Substrate? Streptavidin-POD and TMB 8. Did you apply positive and negative controls along with the samples? Please specify. What did you use for standards? yes and our positive human serum controls also work very well!
Asked on Nov 17 2004
Thank you for your reply. First of all, please ignore the invoice until we sort this problem out. I have looked through the details you have forwarded to us and unfortunately from point 3. to 6. on the ELISA Questionnaire I can't find the answers. Although I understand that your ELISA assay work nicely with your positive control, the extra information regarding samples, blocking, dilution of the primary antibody etc would have been very important for us to be able to identify the source of the problem. You have carried out a Sandwich type of ELISA. Have you actually coated the wells with the primary antibody first or with the samples and/or the recombinant protein? How much recombinant protein have you used? In what concentration range have you tested the protein? I would also like to know the primary antibody you used. Is it monoclonal or polyclonal antibody? We look forward to hearing from you soon.
Answered on Nov 23 2004