* A buffer should be chosen which has a pH value close to the isoelectric point of the protein. This will maximise the protein density on the particle surface. Best results are obtained in terms of reactivity towards antigen if the pH is a little way away from the isoelectric point as this will avoid too compacted a conformation of the antibody.

The following procedure outlines both a simple one step method for the attachment by physical adsorption of avidin, streptavidin, BSA, other proteins, and antibodies to carboxyl/sulfate latex. The protocol is for a 1µm carboxyl/sulfate 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 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 carboxyl/sulfate particles we would need :-

And for 100mg of 4.3µm carboxyl/sulfate particles we would need :-

Protocol

1. Pipette 2.5 ml (40 mg/ml) 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.

Protein coating of these hydrophobic carboxyl microspheres can be successfully achieved by either passive adsorption or covalent coupling using the active ester intermediate with a water soluble carbodiimide.

Coupling by Passive Adsorption

1. To 5ml of the antibody solution in MES buffer (i.e.5 mg of Ab) add 5ml of the latex (this is ~200% of that required for a monolayer). This order of addition will ensure the best coating of the particles with the least possibility of aggregation.
2. Incubate latex/protein mixture with gentle mixing at room temperature overnight.
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; One Step Procedure

WSC EDAC (1 Ethyl 3-(3-Dimethyl Amino Propyl) Carbodiimide HCl)

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.