I coated anti-PSA antibody on PS microspheres (2.5 um) as per the method given here-
https://www.bangslabs.com/sites/default/files/imce/docs/TechNote%20204%20Web.pdf
I used Citrate-phosphate buffer for pH 6.6 (since pI of anti-PSA Ab is b/w 6-6.7), with final pH of buffer=6.4. I used around 60-70 ug of antibody for coating 100 ul of 10% microspheres, which is approx. 50-60% of antibody required for saturation, followed by BSA blocking buffer. When I flowed the microspheres through micro-channels, it bound non-specifically to my control microchannel as well as untreated microchannel surfaces. Could you suggest why this happened, and what can be a possible solution for it?
Consider the zeta potential of the particles. Your channel is charged in the opposite manner to your particles. You need some pre-treatment of the channel to prevent this happening. If you can't change the pH you may need some other stabilizer on the particles. Are you using PBS as buffer?
If you are relying on surface adsorption alone, you are expected to have less than perfect system - pH can change, depending on the buffer system, as you may change the temperature in your downstream procedures.
If you are covalently linking the Ab to beads, rather than relying on just the albumin saturating the remaining sites, throw in some small mono-functional molecules (a huge excess) to block everything. A good example in case of linking through NH2 (a commonly used functional group) is to use excess of ethanolamine.
Best,
Thank you for your answers.
@Alan F Rawle,
I did pre-treatment with PEI solution (followed by 1% glutaraldehyde), but it did not affect the results at all. In another batch I had also used PBS as a buffer in place of citrate-phosphate buffer. What kind of stabilizers can be used here?
@Tausif Alam,
You are suggesting using covalent coupling in place of passive adsorption. What kind of effects are seen when pH changes occur in regard to passive adsorption mechanism.
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