b 1 b 2 etc are prepared with each containing unlabelled human mb ck with known amounts such that there would be a 100 60 40 and 20 inhibition of binding respectively used to obtain the control standard inhibition curve 3 five 5 vials marked c 1 c 2 etc are prepared with each containing the same known amount of human bb antiserum 4 six 6 vials marked d 1 d 2 etc are prepared with each containing the same known amount of ria buffer 400 u03bcl containing 1 6 m tris 20 mm mercaptoethanol or other suitable reducing agent 5 pg of rabbit gamma globin and 0 05 of bovine serum albumin at a ph of 7 4 1 vials c 1 c 2 c 3 c 4 are mixed with b 1 b 2 b 3 b 4 vials and d 1 d 2 d 3 and d 4 vials respectively and incubated for 30 minutes with gentle shaking at 4 u00b0 c and referred to as ab 1 ab 2 etc 2 following the above incubation steps vials a 1 a 2 a 3 a 4 are added respectively to the appropriate ab vials 1 2 3 4 and incubated for six 6 hours at 4 u00b0 c with gentle shaking 3 contemporaneously with steps 1 and 2 the c.