Indirect ELISA. To detect IgM antibodies against a specific pathogen, we use coated plates from a commercial kit (validated in case of IgG detection) and we replace the conjugate with an anti-goat IgM antibody. Despite changing of antibody concentration, absorption of a specific site with BSA-based or casein-based buffers, and increased wash steps, field -control samples (derived from negative exploitation) give high background absorbance (0.9 in some cases).
A detail: the pathogen coated in those plates is not pure but derived from cultures in embryonic eggs. Can anyone tell me why? Does anyone have suggestions on how to decrease background?
add 1 or 2% on normal serum from the specie of the anti-goat IgM antibody
have you tried adding blocking Abs to the sample buffer, what about immunoglobulin inihibiting reagent?
What is your blank? I assume buffer?
Go on to work with casein-based buffer, but your conjugate must be anti Ig M(heavy chains). Not all IgM mol. or Ig with light chans. Good luck.
Use filtered (3%) casein and block or apply onto wells 3x for 10-15 minutes each.
Hi. It may not relevant, but I have had the experience with goat IgG that I get a high background if I use bovine proteins in my blocking solution. I am not sure how similar the bovine and the goat antibodies are, but I then use a fraction V purified BSA and the background is almost gone. I have not used other alternatives for blocking solutions, but I have once read that gelatine could be used as blocking agent. I hope this can help you. Good luck.
You can try to add detergent (for example Tween 20 at final concentration 0.05%) in the solution of anti-goat IgM Ab.
If the conjugate you are using is anti-goat IgM antibody, I presume your primary antibody was raised in a goat. Goat antibodies are a bit "sticky" and that is why you have high background absorbance or noise. Perhaps you can try adding detergent to your washing buffer, for example Tween 20 at a concentration of 0.05%. During washing you may have to soak the wells at a time which you can optiomize. See how it goes.
You can try skimmed milk powder instead of BSA as a blocking agent. Besides, reducing background noise, it is cheaper also.
Dear Marcella,
In the case of IgM, you have to choose carefully the secondary antibody. Those that recognize whole IgM (which is a pentamer) generate high background because of the interference with the light chains of other antibodies (are shared by all immunoglobulin types). This can be reduced by using secondary antibodies that react either with one IgM µ (mu) heavy chain or with the IgM pentameric Fc5µ fragment.
I hope this may help you.
Kind regards,
Carmen
Dear all, lots of suggestions. Thanks. Will have meeting today and will try new strategies. Bovine-derived compounds are certainly a problem. we ran out an essay by comparing negative other -species sera and bovines gave high absorbance. Keep you updated.
Have never tried that but I am thinking about protein-A-HPR. It seems the binding between protein-A and goat IgM is weak. But I may be wrong.
I think the problem might be your samples - I also encountered high backgrounds with antisera when they were not prepared appropriate and contain "sticky stuff".Try to dilute the samples further (at least 1:200), this worked fine for me. I usually also increase the antigen concetration to keep the signal the same, but thats not possible with the commercial ELISA I guess - good look !
IgM is not more complicated than any other antibody class. In this antibody class you will find olny much more low-affinity antibodies which have often a broad specificity. To exclude thoses from binding to your antigen, just use for the sample dilution a buffer with increased salt concentration: e.i. PBS, 0.01% Tween 20, 0.3 mol/L NaCl.
The increase salt concentration will lead to a mild conformation change in IgM and so you will detect antibodies with higher affinities only.
The method with increased salt conentration (if you are using some more steps up to 1.5 mol/L) was in the 80th a method for a rough estimation of the affinity.
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