I am developing an immunoassay and am at a proof of concept phase. Since we are regularly experimenting with different ideas to find the best solution, using antibodies is being expensive. We would like to use a cheaper alternative that can give a good approximation to the actual Ab-Ag system.
I heard that avidin-biotin model can be used for this purpose. I would like to know if anyone has used this model previously, and what sort of streptavidin and biotin conjugate should be used for this?
Yes, the best boning model may be the interaction of desthiobiotin to streptavidin with Ka about 6 log lower than biotin, or about log Ka of 9, comparable to many antibodies. Desthiobiotin or even activated derivatives like NHS esters can be made by published procedures and are also commercially available for conjugation to proteins.
Hi Rohan
Herman is correct. Streptavidin has a very high affinity for bioin - higher than many Abs for their Ag, so you need to keep that in mind.
Regards, Rosemary
Desthiobiotin is made by removal of sulfur with Raney nickel and may contain residual biotin that may interfere in immunoassays. Check with vendors for purity. Or better yet remove even traces by selective binding to a small amount of immobilized SAv in a column or by batch process, then lyophilizing the unabsorbed pure desthiobiotin.
Thank you Herman. Would you suggest if the desthiobiotin be used as capture (& primary) Ab or used as an antigen/analyte?
I suggest that biotin - streptavidin should not be employed to mimic an actual antigen-antibody system. The affinity of the latter is much lower and the variation between various systems is endless. You should be able getting not too expensive ready couples and try more than one, e.g. immunoglobulin (Ig) - anti-Ig; BSA - anti-BSA; OVa - anti-OVA; DNP anti-DNP and such.
- Badges
- Science method
- Similar topics
- Medicine
- Immunology
- Immunology Techniques
- Immunoassay
- ELISA