Hi, I am biotinylating my monoclonal antibodies(mAbs) (both IgGs & IgMs) with biotin(EZ-Link® Sulfo-NHS-LC-Biotin) from Pierce Biotechnology, Inc. However, the manual I attached below contains information only for IgGs. According to the manual, I used a molar ratio between biotin to mAb of 20:1. My protein conc. was 1 mg/ml prior to biotinylation. For my calculations, I used the MW of IgG = 150,000 (mentioned in the manual) and MW of IgM = 900,000 (from http://www.piercenet.com/method/antibody-structure-classes).
I managed to successfully biotinylate all of my mAbs (IgGs & IgMs), but, a big problem is the high background of the mAbs after biotinylation (OD~0.6). I have tried redialysis in PBS for about 3 more times to get rid of the excess biotins but only slightly reduced the background. (All of my mAbs are in PBS prior to biotinylation).
My system is an indirect ELISA (Coated antigen, biotinylated mAb, then streptavidin-HRP) whereas my control (which I used to check for the background) is (Coated BSA, biotinylated mAb, then streptavidin-HRP).
So, does anyone have any ideas on how to decrease the background? Please let me know if you need more information and I really appreciate all the answers :)
Thank you very much.
Agree with points made by Mehdi. Also, did you optimize monoclonal concentration? If too high you get a significant background. Optimize using doubling dilutions of you biotinylated antibody on positive and negative (BSA) coated wells.
Dear Pongpawan, Your protocol for biotinylation is acceptable. of course I think MW of IgG is around 150 kd and for IgM is around 750 kd.
Anyway, your background has two main origin. First nonspecific binding, which you can reduce it with optimizing incubation time, wash buffer concentration, pH and detergent content. But second origin related to hapten Ag. you mentioned to coated Ag but not clear it is small hapten or large immunogen. if it is hapten, you have to know about converting hapten to immunogen. maybe hapten-BSA used for immunogen making, and you block the wells after Ag coating, with BSA. So it is obvious for high background. in this situation you must change control well and blocking solution.
Finally if you give some more detail It may be help you more.
best
mehdi
Agree with points made by Mehdi. Also, did you optimize monoclonal concentration? If too high you get a significant background. Optimize using doubling dilutions of you biotinylated antibody on positive and negative (BSA) coated wells.
I agree with all the above comments. I would like to add here that in addition to titrating the biotinylated antibody, please also do a titration of streptavidin-HRP conjugate. Because of very high affinity of biotin and streptavidin binding, one get away with a very low concentration of streptavidin conjugate compared to secondary antibody-enzyme conjugates. Also try using "Low Cross Buffer" from Candor for diluting the biotinylated antibody. I have seen reduced backgrounds using this buffer especially while doing serology assays.
I agree with Neeraja about trying some different buffer in which diluting your primary and secondary antibody. I am not familiar with the "Low Cross Buffer", but usually a solution of PBS Tween-20 0.05-0.1% also can work.
Respect to IgM Mab is very very dificult to make a indirect ELISA that working well. The IgM are very big, it washed very bad and always remained small amount into the well (that after are amplified by streptavidin-HRPO). If you get that your ELISA with IgM Mab working well, please, tell me.
Respect to the IgG Mab-biot is more strange. Probably something wrong during biotinylation. I´ll would make the process again. Other possibility is precipitated the Mab-bioit with 50% SO4 (NH4)2 saturated to eliminate the free biotin
I agree with all the previous comment. I would just add a further tip: increase the number of washing after the incubation with the biotinylated MAb and after the streptavidin HRP conjugate (make 7 - 8 washings after each step). It usually reduces significantly the background in our ELISA assays.
I also agree with previous comments but also have noticed that prior to dialysis, it is very useful to block the excess of free biotin by incubating with 2% Tris/Glycine during 30 minutes with gentle agitation at room temperature. Thsi greatly diminishes the background.
Neutravidin-HRPO conjugate may give lower background than streptavidin-HRPO conjugate.
Have you tried to dilute your biotinylated abs in Biotin-free BSA buffer? This should minimize your Background binding. Try 1% Biotin-free BSA in a TRIS buffer.
Vielen dank! I'll give it a try.
Luisa,
won't the glycine react with the tagged biotin that may result in biotin inactivation?
I also strongly suspect Biotin in the BSA preparation being the culprit for the background. Go for certified biotin free BSA or use another protein like OVA for background determination. Of course it might be useful to know if a carrier protein has been used and which.
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