Hi Swati,

I would say that your go-to method would be to bind the biotinylated IgG in an ELISA plate and then detect it with streptavidin*HRP.

COATING

The coating protocol changes slightly if you are analysing (i) a monoclonal antibody (mAb) of which you have a purified antigen or (ii) a polyclonal preparation or a mAb for which you don't have the purified antigen.

(i) If you have the purified antigen recognised by your mAb of interest

, then I would recommend to coat an ELISA plate overnight with your antigen, about 100 ng/well in a volume of 50 µl per well. The buffer in which you need to resuspend the antigen varies from protein to protein, so I would recommend to check the literature. However, generally PBS or bicarbonate buffer work just fine.

(ii) If you are testing a polyclonal preparation or don't have the purified antigen then you might go ahead and coat the plate with an anti-IgG. Be sure to choose a species-specific preparation, that is if you are testing human IgG for biotinylation, use an anti-human IgG to coat the plate. Probably you can coat with less than 100 ng/well if you don't have enough anti-human IgG and you know you are coating with high-affinity antibody. However, 100ng/well will do the job just fine so don't worry diluting more than that if you don't want to overcomplicate the protocol. PBS should be fine.

In either scenario, incubate the plate overnight at 4˚C.

BLOCKING

The day after, discard the contents of the well and wash the plate with a spray water bottle a couple of times. Then, add PBS-BSA 1% and incubate for 1 hour at 37 ˚C.

BIOTINYLATED ANTIBODY

Discard contents. Add serial dilutions of the biotinylated IgG to each well of the plate. In the past, I always started with 20µg/ml of biotinylated antibody in the first well (Assuming no loss of protein during the biotinylation process) and proceeded with serial dilutions 1:5 down a plate column, for a total of eight points. Make sure to include a negative control of unbiotinylated antibody in a side column.

Incubate 1 hour at 37˚C.

WASH

After incubation, discard contents of the wells and wash five times with PBS-Tween20 0.05%. Make sure that after the final wash no wash buffer is left into the plate. You can do that by pounding the plate on a few sheets of absorbent paper.

INCUBATE ST*HRP

Add HRP-Streptavidin to each well. I used to work with the product N100 by Thermo Fisher but I am sure you can find good quality products from other providers too. Dilute ST*HRP as indicated by the company, dispense 50µl/well and incubate for 1h at 37˚C.

WASH

As before.

DEVELOP PLATE

After washing, add 50µl/well of TMB substrate. There are plenty in the market, but I generally worked with Sureblue TMB substrate by KPL. Incubate for 10 minutes at 37˚C.

BLOCK PLATE

WITHOUT discarding the contents of previous step, add 50µl 1M sulfuric acid to the well. The blue wells will turn yellow. Read results at 450 nm.

Hope this helps.

Cheers,

Giacomo

THANK YOU Giacomo Gorini !

If the biotinylation is not the major aim of your project, I would not test the biotinylation by ELISA, since it is not quantitative anyway. If you want to verify your conjugation level, MALDI-TOF-MS is the most suitable method. The mass shift will give you the number of attached biotins. In most cases, it is sufficient to use your antibody in your anticipated assay. If it works, your biotinylation obviously was OK. BTW: Biotinylations are successful in most cases, if you used a standard protocol, e.g. from the manufacturer.