We are developing the T3 streptavidin based assay. Currently we tried different T3 mab. These mab gives good results when coated directly on ELISA plate. But after biotinylation, mabs behaves differently with Biorad Immunoassay controls. If any one is having experience on this please help. Or suggest some different strategy. We tried biotin conjugation using two different protocols one NHS and other sulfhydryl.
Dear kulkarni
The procedural steps for antibody-fluorophore conjugation and for antibody-biotin conjugation are quite similar. These steps consist of mixing the dye or the biotin-derived reagent at an appropriate molar ratio and pH with the antibody solution and, after incubation, purifying the conjugate from the reaction mixture by size-exclusion chromatography or dialysis. The antibody-dye or antibody-biotin conjugate can then be analyzed to determine the degree of labeling, i.e., the number of moles of dye or biotin per mole of antibody. See Critical Parameters for helpful hints.
Dear Seyyed,
Thanks for the suggestion.
I would like to give some details
1. I tried different conjugation
a. your suggested method
b. using TCEP
c. Using SATA
The molar ration used was 1:10 Ab:Probe
But after conjugation I was not able to get the reactivity with tha analyte, which I was getting only with the Mab.
More specific after biotin conjugation Biorad Immunoassay controls were not picking up.
So please guide me how i can retain the Mab acitvity after conjugation
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