calif and total protein measured at od280 with a kratos spectroflow 757 detector kratos mawah n j a single 125i 1b4 peak composed of coincident od280 and radioactivity tracings characteristically elutes at about 6 minutes 30 seconds following sample injection specific activity of the product is generally about 10 u03bcci u03bcg protein and 97 99 of the counts are precipitable with 10 trichloroacetic acid the binding of this radiolabeled antibody is assessed on human pmns purified on a discontinuous ficoll hypaque gradient english d and anderson b r j immunol methods 5 249 255 1974 and activated with about 100 ng ml phorbol myristate acetate for about 20 minutes at about 37 u00b0 c lo et al j exp med 169 1779 1793 1989 to determine the avidity of antibodies for cd18 molecules on the pmn surface about 1 u00d7 105 activated pmns are incubated in a buffer such as hanks balanced salt solution containing about 20 mm hepes ph 7 2 about 0 14 units aprotinin sigma chemical co and about 2 human serum albumin binding buffer containing about 1 3 ng 125i 1b4 2 8 u00d7 10 11 m in the presence of increasing concentrations of unlabeled 1b4 antibody about 10 7 to 10 15m in about a 300 u03bcl reaction volume for about 1 hour at about 4 u00b0 c with constant.