BigPatents India

A process for preparing antibody labelled microspheres using anti CD4 /anti CDS antibody for detecting CD4 and CDS positive cells in the blood which comprises the steps of: Placing 0.5 ml of 2.5% carboxylated microparticles into Eppendorf centrifuge tube (1.5-1.9 ml capacity) and washing twice with sufficient carbonate buffer; resuspending the pellet in phosphate buffer and washing two times in phosphate buffer; the pellet is then resuspended in a 0.625 ml of the 0.02M sodium phosphate buffer, pH 4.5; Adding drop wise 0.625 ml of the 2% carbodiimide solution; Mixing 3-4 hours at room temperature; Centrifuging for 5-6 minutes and removing and discarding supernatant; Resuspending pellet in phosphate buffer; Centrifuging for 5-6 minutes and removing and discarding supernatant; remove unreacted carbodiimide; Resuspending pellet in 1.2ml of borate buffer; Adding 200-400 ug of Anti CD4 / Anti CDS antibody; mixing gently overnight at room temperature on an end-to-end mixer; Adding 50ul 0.25M ethanolamine(2-aminoethanol) and mixing gently for 30 minutes for blocking unreacted sites on the microparticles; Centrifuging for 10 minutes and saving supernatant for protein determination; Resuspending pellet in 1 ml of 10 mg/ml BSA solution in borate buffer; Cap and vortex; Mixing gently for 30 minutes at room temperature for blocking any remaining non-specific protein binding sites; Centrifuging for 5-6 minutes and removing and discarding supernatant; Resuspending pellet in 0.5 ml PBS, pH 7.4 containing 10mg/ml BSA, 5% glycerol, and 0.1 % NaN3 (storage buffer); storing at 4º C.