anti-avidin antibodies in normal sera

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Re: anti-avidin antibodies in normal sera

Postby komayyamok » Aug 08 2018 11:41 am

BioMiha wrote: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, PhenQ 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

How to make these things happens ?
komayyamok
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