Coupling to Microparticles

Besides Proteins, DNA, lectins and many other species have been coupled to microparticles. Coupling to microparticles may be achieved by passive adsorption or covalent attachment. Most techniques using passive adsorption technology report four to six months of bead stability. This technology is the basis for most commercialized latex assays. The passively-bound proteins may eventually are lost from the surface of the particle. Some applications demand covalent techniques. Two major areas include materials with low affinity for polystyrene and cases where a component of the assay will displace passively adsorbed material. Many attempts to couple DNA have been successful only with covalent coupling or with a covalently coupled linking group. Surfactant is a notorious example of a material that can displace proteins from the bead’s surface. If surfactant is required as an additive in the assay, covalent coupling procedures are recommended.

Adsorbing Protein on Beads

Plain Beads

1. Initial Buffer: 0.1M Borate Buffer, pH 8.5
2. Suspend in buffer, spin down and resuspend 2 or 3 times
3. Suspend in borate buffer
4. Add protein and mix end-to-end overnight
5. Spin and save supernatant for protein determination
6. Resuspend in BSA in appropriate buffer and spin down twice
7. Resuspend in PBS, pH 7.4, containing BSA and glycerol (storage buffer)
8. Protein bound directly on surface

Coupling by Glutaraldehyde

Amino Functional Beads

1. Suspend in 0.02M PBS, pH 7.4 buffer, spin down and resuspend 2 or 3 times.
2. Suspend in PBS
3. Suspend in 8% glutaraldehyde in PBS, pH 7.4, and mix 4-6 hrs. end to end
4. Wash to remove excess glutaraldehyde and resuspend in PBS buffer
5. Add protein and mix end-to-end overnight
6. Spin and save supernatant for protein determination
7. Resuspend in 0.2M Ethanolamine in PBS, mix for 30 minutes
8. Spin and discard supernatant
9. Resuspend in BSA in appropriate buffer and spin down twice
10. Resuspend in PBS, pH 7.4, containing BSA and glycerol (storage buffer)
11. Protein bound 5 carbon atoms from surface of blue dyed beads and 11-12 carbon atoms from surface of amino beads

For 0.5 ml of an aqueous 2.5% microparticle suspension, 200-400 micrograms protein and 1ml 0.2M Ethanolamine in PBS are used.

Amino functional beads couple proteins with glutaraldehyde: Glutaraldehyde is more stable than the carbodiimide reagents used with carboxylate beads. Coupling with glutaraldehyde results in the proteins being bound 11-12 carbon atoms away from the surface of the bead versus 2-3 carbon atoms as in the case of carboxylate beads coupled using carbodiimide.

Protein Coupling Troubleshooting:

Problem	Solution
Clumping prior to use	Sonication
Clumping after procedure Carbodiimide addition causes clumping Glutaraldehyde addition causes clumping Protein addition causes clumping Washing causes clumping	Isolate which step causes clumping Add slowly, agitate beads, increase bead concentration Add slowly, agitate beads, increase bead concentration Increase protein concentration Add surfactant or reduce washing steps
Low binding	Move pH of binding closer to protein isoelectric point
Variable coating	Use pure water-no contaminants
Coating, but no reaction	Optimize pH away from isoelectric point
Centrifuge not practical	Use membrane filtration for small particles
Nonspecific adsorption	Use alternative for BSA (glycine, casein)
Small proteins bound but not reactive	Use a crosslinking agent to extend coupling away from the surface of the bead
Long-term storage leaches protein	Try covalent attachment or lyophilize final product