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My colleague, wrote you about problem he is having with an antibody we bought from you, and we got a reply from you (please see below for the original message and your reply). In sum, we bought your anti-beta-1 definsin antibody (AB14421) based on your advertisement recommending this antibody for ELSIA detection of beta-1 defensin in human samples. In our tests using a commercial purified beta-1 defensin in our ELISA assay, we never were able to detect anything with your antibody. In previous email communications, we raised this issue and you provided a complete protocol for the ELISA assay. We followed the protocol and still were not able to detect anything. We followed up by asking for your clarification on the source of the antigen in your assays. In your reply below you now tell us that your ELISA was tested positive on a synthetic peptide generated based on a partial sequence of human beta-1 defensin. Moreover the synthetic peptide contains several amino acid changes from human beta-1 defensin. It is wonderful your antibody can detect this synthetic peptide. However, for you to advertise the antibody for beta-1 defensin ELISA, do you have any data supporting this claim: do you have anything you show me to confirm the antibody can actually detect human beta-1 defensin (native, synthetic or recombinant form of the entire protein)? Obviously, the only reason we (and others) ended up buying this antibody is for use in beta-1 defensin detection, not detection of a synthetic peptide. If you have data that your antibody can detect intact human beta-1 defensin, can you give me detail how this is done (e.g. source of defensin, how much defensin, condition used, and result of dose-response curve). If none of this information is available, please advise what we need to do with the item we bought from you based on what you advertised. Thank you in advance for your prompt attention to this matter.
Asked on Jan 31 2005
Thank you very much for your enquiry. I have contacted the originator of this antibody, and they tested the antibody in ELISA using human beta Defensin 1 (aa1-36) (DHYNCVSSGGQCLYSACPIFTKIQGTCYRGKAKCCK). In this test the antibody had a titer of 1 to 3000. The peptide against which the antibody was made, was a synthetic peptide: YRGKAKC(ACM)G, corresponding to amino acids 28-34 of Human beta 1 Defensin. As your colleague has reported, no signal was obtained using ab14421 in ELISA. We would very much like to investigate this problem, but I do need require some information regarding your protocol. If you could kindly answer the questions below, we will do our best to determine what is going on. Thank you, and I look forward to hearing from you. Also - what was the batch number that you received (it is located on the vial)? 1. Please describe the problem (high background, no signal, non-specific colour development, poor standard curve etc). 2. What type of ELISA are you performing? (Direct ELISA, indirect ELISA, sandwich ELISA etc.) 3. On what material are you testing the antibody in ELISA? •Species? •Cell type? •etc? 3. How did you coat the plates? 4. How did you block the unspecific binding sites? For how long? How did you wash the wells? 5. Primary antibody •Specification (in which species was it raised against)? •At what dilution(s) have you tested this antibody? •Incubation time, wash steps (multiple short washes are more effective than fewer longer wash steps)? 6. Secondary antibody •What secondary antibody are you using? •Specification (in which species was it raised against)? •At what dilution(s) have you tested this antibody? •Incubation, wash steps? •Do you know whether the problems you are experiencing come from the secondary? 7. Which detection system did you use? Substrate? 8. Did you apply positive and negative controls along with the samples? Please specify. What did you use for standards? 9. Optimization attempts •How many times have you tried the ELISA? •Do you obtain the same results every time? •What steps have you altered?
Answered on Feb 03 2005