The quantitation of coupled bead antibody by enzyme-linked immunosorbent assay

Quantitation of antibody coupled to a derivatized polystyrene bead through a bifunctional cross linker can be accomplished by a competitive enzyme-linked immunosorbent assay (ELISA) method. This sensitive method is less subject to interference than other protein assay methods such as bicinchoninic acid (BCA) or Lowry. The competitive ELISA method consists of incubating the coupled bead with a (20/80) weight ratio of goat anti mouse kappa alkaline phosphatase/goat anti mouse kappa (GAMKAP/GAMK) for 1.5 hours at 37 degrees C, washing, adding p-nitrophenyl phosphate (PNPP) substrate, and reading the absorbance at 405/450 nm. A standard curve is established with radiolabeled antibody beads for microgram quantitation.