Blocking Agent...? - (Jul/06/2010 )
I'm working on sandwich-type ELISA for virus detection (capture antibody captures virus, probe antibody then binds to captured virus, signal molecule then binds to probe antibody, then plate is read) and am having trouble finding the right blocking agent to use.
So far, we've tried combinations of casein, PVP, and gelatin and none seem to give us a signal to noise ratio we can work with. Have any of you used any blocking agents or combinations of blocking agents that I'm missing, beyond simply BSA or something of the like?
I think it may be an issue of insufficient washing, as we only do three wash steps after incubating the virus and two after incubating the probe antibody. However, I'm afraid more washing could disrupt antibody binding to the virus or the walls.
Any suggestions? Thanks in advance!
I'm not sure if it will work for ELISA but I've used cold-fish skin gelatin for IF with excellent results. The stuff is gross but it makes some of my antibodies that have high background with just BSA blocking gorgeous!!
rkay447 on Jul 6 2010, 04:03 PM said:
Do you mean fish skin gelatin gave you excellent results over BSA and porcine gelatin, or just excellent results over BSA? Would there be a big difference between the two types of gelatin?
The gelatin we use is porcine skin gelatin, and comes powdered from our supplier in a plastic jar. I suppose the cold fish gelatin comes liquified, since it probably does not solidify at RT... are there typically antimicrobials in it (like azide)?
there are many many blocking agents. Casein, BSA, gelatin and others. East coast biologics makes 'sea block' and variants. There is also a 'blocking peptide fragment' that is available. Glycine is also used.
I have not heard of PVP for blocking...usually polymers in the assays are used to exclude water and increase ab-ag interaction...background signals also increase. (PEGs and PVPs are known for this)
Extra washes will not 'disrupt' your ab binding. You can add 1-2 more. One variant of washing is to allow the wash solution to sit in the wells for 10-15 seconds before removal.
You can also include a surfactant in your wash if you have not already done so. (ie tween-20 or triton x 100)
you can also block with normal serum (2-5%) from the species from which the secondary antibody is prepared.
I am working on microfluidics ELISA and we use 1% BSA.
Between combinations of casein and PVP with 0-2% gelatin, we found the best results with no gelatin and 0.5% PVP w/v. I have yet to try the cold fish gelatin or Sea Block, but we may be purchasing some soon.
Sea Block sounds like it's fish plasma, through what I could decypher from google-translating the page from Chinese to English. Would this be more effective than simply trying the cold fish gelatin?
I recommend you try a second block between the primary and secondary antibody in the serum of the species that produced the secondary. Most background comes from secondary so this could fix your problem. Try the no gelatin and 0.5% PVP that you've already determined is best, do the primary antibody and then reblock in the sera. Many people use goat anti-rabbit or mouse for secondary so you'll want goat serum. Block in 3-5% serum and then do the secondary. Have you titrated out your primary antibody so that you are using the highest possible dilution? I have an antibody that stains at 1:250 in one hour but with high background but if I stain overnight at 1:750, the background is almost completely gone but I maintain the specific signal. If you haven't done this, you might want to see if a high dilution but with overnight incubation yields better results.
You might also want to look at invitrogen's secondary antibodies. They offer some highly cross-absorbed antibodies that aren't really all that much more expensive and generally give very little background.
it sounds like there is some confusion with non-specific binding and heterophile reaction.
block your plates with your best possible combination.
Coat with primary ab
Block with best agent you have be it fish, bsa, commercial blocking agent etc
Prior to running the test:
Pre-incubate your sample (15 min 37C) with non-specific serum (or non-specific IgGs) of the primary/secondary antibodies. Any heterophile should then be 'blocked'.
Let us know how this works.