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.
Hi Sandeep
What is your biotinylation protocol? Have you tried to use EZ-Link NSH-PEO Solid Phase Biotinylation Kit of Pierce? I had excellent results with it. No modification to the antibody molecule occurs during this conjugation process.
The physical behavior of the antibody, such as its solubility or aggregation, could be altered by excessive biotinylation of lysine residues. Since biotin is hydrophobic, and biotinylating lysine residues eliminates a surface charge, the protein surface becomes significantly more hydrophobic if multiple biotins are attached. The protocol should be adjusted to reduce the biotinylation stoichiometry.
Hi, Adam and Oscer
Thanks for the reply,
No i have not tried EZ-Link NSH-PEO Solid Phase Biotinylation Kit of Pierce but I tried Sigma kit (NHS Ester Biotin), Hook Biotin PEG2 Amine (G Biosciences), Sulfo Chromo link Water soluble (Solulink), EZ-Link® HPDP-Biotin (Thermo) with different chemistries. But none of above protocols worked. If you think the kit you mentioned will work I will try it. if you have any alternate strategy please suggest.
You haven't really explained the nature of the problem you are having. If the antibody no longer binds to the antigen after biotinylation, it may be because the biotin is being attached to the complementarity determining region. If you are using amine reactive biotinylation reagents, you might try cysteine-directed ones (after reduction of the disulfide bonds) or carbohydrate-directed ones instead. Or, forget about biotinylation and employ protein A or G instead of streptavidin.
Hi Adam,
sorry for the late post I was on leave.
Thanks for the suggestion,
The problem is after biotin conjugation, the antibody can not able to detect the analyte present in the Biorad Immunoassay controls L1, L2 and L3. But the same Mab Biotin is able to detect the standards which is T3 from sigma (used for making the standards) and the T3 present in serum. I tried cysteine-directed ones (after reduction of the disulfide bonds using Ethyl amine ) or carbohydrate-directed ones (G Biosciences kit), I also tried Traut reagent chemistry, TCEP ,SATA , but the problem remained same. I employed Protein A, G and Goat Anti mouse IgG for coating for direct binding of Mab, but it didnt work. I made Protein A biotin conjugate and mixed with native Mab and used in assay but the same result observed. I am really getting nervous. please help.
Make sure that the unbiotinylated antibody works as it did before and that the problem isn't degradation of the antigens or a problem with the detection reagents. It sounds like biotinylation isn't necessarily the problem, since none of the non-biotin approaches you mentioned worked either.
By the way, I've never heard of using ethylamine to reduce disulfides. I don't think that would work.
Have you demonstrated that the antibody is actually biotinylated after each of the modification procedures and measured the stoichiometry?
Do you have a positive control in each experiment?
Dear Adam,
Thanks for the reply.
The antibody works well when coated directly on the ELISA plate. The same antibody didnt worked when used on other format like GAM, Protein A, Protein G and streptavidin coated plates.
Sorry, its a typing mistake i used 2 MEA-HCL for reduction (Make Thermo Scientific) conc used 50mM 2MEA/1ml/5mg Ab
I have demonstrated the antibody by using 1. HABA assay, OD at 280 and 354.
I have tried different ratios (Protein : Biotin )1:10, 1:5, 1:2.5 which gave me 7,3,2 biotin per antibody molecule.
we dont have facility of stoichiometry.
Yes we have control in each experiment the control is the mab coated plate system.
I have attached one excel sheet for your reference.
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