How many times have you observed differences in reactivity of your coated reagents when assessing lot-to-lot reproducibility of the raw microspheres ? Same conditions of coating, same antibody at the same concentration, same volume of particles, same...

And what about the surface?

One critical concern is to consider the number of molecules immobilised per unit of surface, rather than per number or per volume of particles. Diameter and percentage of solid (this information is indicated precisely on the Certificate of Analysis) vary from one particle to the other, providing differences in the surface available for coupling.

You only need to know the surface area of a spherical particles : S = pD²

and the number of particles per ml :

E is the percentage of solid
d the density of the particle (1.059 g/cm³ for polystyrene)
D the diameter in µm

100 µl of a 10% solid suspension of K080 particles (diameter 0.800 µm) yield a total surface of :

S x N = 708.2 cm²

Adding 0.16 mg of a monoclonal antibody (IgG, M.W. = 150 000 g/mol) to this suspension gives a theoritical surface coverage of : 1.5 pmol/cm²

As a comparison, adding the same amount of antibody to the same volume of particles (100 µl) at 10% solid and of diameter 0.75 µm gives, in the same experimental conditions, a surface coverage of : 1.4 pmol/cm²
The variation (6.6%) between the two reagents is likely to generate unvaluable effects.

Which coverage?

For Latex Agglutination Tests involving an antibody as a coated protein, most optimisations lead to an antibody coverage within the range [0.5 - 5 pmol/cm²].