A SENSITIVE ELISA FOR THE CHARACTERIZATION OF 2 FORMS OF CIRCULATING INTERCELLULAR-ADHESION MOLECULE-1 IN HUMAN PLASMA

Nine murine monoclonal antibodies (MAbs) raised against recombinant so luble intercellular adhesion molecule-1 (sICAM-1) were evaluated by me ans of ELISAs, and their neutralizing activity was investigated in two bioassays employing Raji cells activated with phorbol myristate aceta te. Three of the MAbs inhibited autoaggregation of the activated cells and adhesion to sICAM-1 fixed on a plastic plate. Non-neutralizing an tibody SM1-255 is directed to an epitope that was not recognized by th e other eight MAbs. This property enabled us to develop two ELISAs emp loying SM1-255 as a liquid-phase antibody for the quantitation of sICA M-1 circulating (cICAM-1) in human plasma. One assay, employing non-ne utralizing antibody WIS2-11 as a solid-phase, has a sensitivity of two pg/well with coefficients of variation of 3.6-5.8% (within assay) and 5.5-9.5% (between assay). The other assay, employing neutralizing ant ibody WIS5-85 as a solid-phase, has a sensitivity of four pg/well with coefficients of variation of 2.7-7.2% (within assay) and 9.2-11.2% (b etween assay). There was a discrepancy between cICAM-1 levels of human plasma determined by these two assays. All samples showed 2- to 5-tim es higher levels in the assay using WIS2-11 than in that using W1S5-85 . The result from the gel electrophoresis employing Western blotting s uggests that ICAM-1 circulates in at least two molecular forms with mo lecular masses of about 85 and 130 kDa and with different reactivities to W1S2-11 and WIS5-85.