anti-avidin antibodies in normal sera

Go to the profile of biomiha
Feb 14, 2011
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Hi all,

I am curious if anyone has similar experience. I am observing a lot of anti-avidin (or more specificaly NeutrAvidin from Pierce) antibodies in normal human sera. I am trying to devise an assay that uses a biotinylated antigen but I can't seem to loose the background signal. I don't want to dilute my target sera too much so as to be able to observe low affinity specific Abs as well.
Thanks for the feedback.
Miha

14 Comments

Go to the profile of mt scientist
mt scientist 7 months ago

Could you pre-treat your samples with bead-bound avidin to clear the pre-existing reactive antibodies?

Go to the profile of mdfenko
mdfenko 7 months ago

what are you seeing that indicates anti-avidin?

are you sure it is not biotin in the serum?

Go to the profile of biomiha
biomiha 7 months ago

I titered normal human sera to determine the cutoff for the assay (with and without the biotinylated antigen). From some sera I get a bizzarely high signal after I develop with anti-human-IgG-HRP.

Go to the profile of mdfenko
mdfenko 7 months ago

anti-human igg should recognize all of the igg present in human serum. the iggs account for a large fraction of the proteins present in serum.

also, if the person(s) from whom the serum is collected takes a regular vitamin supplement then they most likely will have elevated, yet variable, free biotin levels in their serum. you can check the serum for soluble vitamin levels.

Go to the profile of biomiha
biomiha 7 months ago

Thanks for your input mdfenko. However, I am not entirely sure why free biotin levels would influence my problem of anti-avidin antibodies. I have read in a paper that in older people circulating IgGs are biotinylated to a certain extent which could mean that I am actually detecting b.IgGs binding to avidin. I am not entirely sure what is the extent of this and how to check.

Go to the profile of relaxin
relaxin 7 months ago

Could you give us more details on what you are doing? Why do you need normal serum in the assay? It seems you only need streptavidin-conjugated HRP for detection. There is a protein in serum (beta-microseminoprotein) that is known to bind IgG.

Go to the profile of biomiha
biomiha 7 months ago

Ok, to clarify. I am trying to devise an assay to measure the total IgG and different IgG subtypes of antibodies to the Fab fragment of a therapeutic antibody. I want to incubate the biotinylated Fab fragment with the sera I am testing and bind the complexes to plate-immobilized NeutrAvidin. To validate a bioassay it is customary to test a number of normal human sera (negative control) to see if there is any non-specific binding, to determine what dilution of the test sera is appropriate and to determine the cutoff value for positivity. In a different type of experiment, for example, where I immobilized a recombinant protein, I could dilute the sera 1/20 and saw no more than an OD(450 nm) of 0.1 for the negative sera. Therefore, I diluted the test sera 1/20 and determined the cutoff for positivity as the OD(450 nm) that exceeds the mean +3xSD of the negative sera. I want to do the same here, but I observe ODs well above 0.5 for some 1/100 diluted negative sera, which was not expected. I read in one paper that anti-avidin antibodies are present in normal human sera with a log-normal distribution, however I was not expecting such a high response. Perhaps it could be something used to prepare the NeutrAvidin or something else, I have no idea. I am going to compare the results with streptavidin coated plates, although I fear it,ll be even worse. All feedback is much appreciated.
Miha

Go to the profile of Roberto Rosati
Roberto Rosati 7 months ago

Would you like to check if your detection problem is similar to the one described in >this paper<?

Go to the profile of biomiha
biomiha 7 months ago

Thank you r.rosati! This seems to be a common phenomenon. Unlike the authors of the paper I cannot express my Fab fragment with a His tag.

Go to the profile of Roberto Rosati
Roberto Rosati 7 months ago

What about labeling the FAB fragment with another haptene, like digoxigenin, and use an anti-dig antibody? (I'm saying dig because in FISH, I use both dig and bio-labelled probes).

Go to the profile of biomiha
biomiha 7 months ago

Thanks for the suggestion. But, sadly, the HAMA (human anti-mouse antibody) response in normal sera is even worse. I had thought about substituting avidin with a biotin binding mAb, but as I said it's even worse. Has anyone ever attempted conjugating a His-tag peptide to a protein (not expressing the His tag)?

Go to the profile of Roberto Rosati
Roberto Rosati 7 months ago

You could pre-treat with a biotin solution... At saturation concentrations, all avidin fixed to the plate will be bound to biotin, and if you wash away the biotin and add the serum, if the problem was from endogenous b-Ab, it should give no signal.

Go to the profile of tje
tje 7 months ago

Hi, I was wondering whether you ever resolved or improved on this problem? I am now using a similar set-up and also see surprisingly high IgG responses from both healthy and patient sera to my neutravidin control alone (no captured antigen). I am using fairly standard stringent ELISA buffers, blocking and washing procedures - so I feel this may be genuine anti-streptavidin antibodies in sera - which may only be removed by a cross-adsorbtion step (say with NA-beads).
Thanks in advance TJ

Go to the profile of savimakwo
savimakwo 7 months ago

How you make these sera which can normally antibodies