Coupling of the primary amino groups of protein molecules to carboxyl groups on a latex particle using a water soluble carbodiimide (WSC) is a common procedure. The reaction proceeds rapidly through an active ester intermediate.

The following procedure outlines both a simple one step method and a two step method that we have used with excellent results for coupling avidin, streptavidin, BSA, other proteins, and antibodies to CML latex. The protocol is for a 1µm CML latex at 1% solids. This reaction can easily be scaled up or down to fit individual needs. For example if a different particle size is used then the amounts of WSC and antibody (Ab) can be calculated from :-

For example we suggest for a 1µm particle you use 5mg of protein for 100mg of particles.

For 100mg of 0.3µm CML particles we would need :-

And for 100mg of 4.3µm CML particles we would need :-

Protocol

1. Pipette 2.5 ml (40 mg/ml) CML latex microspheres and dilute with 10 mL MES buffer.
2. Centrifuge the mixture to sediment the particles; ~3,000G for ~20 min.
3. Remove supernatant and re-disperse the pellet in 10 ml MES buffer.
4. Centrifuge again and remove the supernatant from the particles.
5. Re-suspend pellet in 5 ml MES buffer, being sure to completely suspend microsphere particles.

Coupling; One Step Procedure

1. To the latex suspension, add the following:
   1. 2ml of EDAC in MES (i.e.100 mg of EDAC). Note: this solution should be freshly prepared.
   2. 3 ml (i.e.5 mg of Ab) Ab solution (this is ~200% of that required for a monolayer)
2. Incubate latex/protein mixture with gentle mixing at room temperature for 3 to 4 hours.
3. Centrifuge to separate the protein-labeled latex particles from unbound protein.
4. Remove and retain supernatant for protein determination*.
5. Resuspend the pellet in 10 ml PBS.
6. Centrifuge again to sediment the particles.
7. Repeat steps 7-8 twice more for a total of 3 washes.
8. Re-suspend the final latex in 10 ml Storage Buffer giving a final concentration of 1% solids. Store at 4°C until used. Do not freeze.

* At this step, the supernatant can be reserved and a protein determination made. Any residual protein in the supernatant can be subtracted from the original amount added. The remainder is coated on the particles. The only method for protein determination compatible with latex microspheres is the Micro BCA Protein Determination Kit from Pierce.

Glycine is used in the storage buffer to fill any reactive sites on the microsphere surface which have not been covered by the protein. This is to reduce non-specific binding. Bovine Serum Albumin (BSA) may be used for the same purpose if desired. The NaN3 is present as a biocide. If the latex is kept sterile, NaN3, which is not compatible with cell or tissue culture, can be omitted.

Coupling; Two Step Procedure

Antibody Solution Protein of your choice at 1mg/mL in PBS

1. To the latex suspension, add 2ml of 50mg/ml EDAC in MES (freshly prepared)
2. Incubate latex/EDAC mixture with gentle mixing at room temperature for15 min.
3. Centrifuge to sediment the latex particles and remove the supernatant.
4. Re-suspend the pellet in 10 ml PBS buffer.
5. Repeat steps 3-4 twice more for a total of 3 washes.
6. After the third wash, re-suspend the activated particles in 5ml of PBS.
7. Take 5ml of the protein in PBS solution and add the 5ml of particle dispersion.
8. Incubate latex/protein mixture with gentle mixing at room temperature for 3 to 4 hours.
9. Remove and retain supernatant for protein determination*.
10. Re-suspend the pellet in 10 ml PBS.
11. Centrifuge again to sediment the particles.
12. Repeat steps 7-8 twice more for a total of 3 washes.
13. Re-suspend the final latex in 10 ml Storage Buffer giving a final concentration of 1% solids. Store at 4ºC until used. Do not freeze.