A sensitive ELISA for the characterization of two forms of circulating intercellular adhesion molecule-1 in human...

Nine murine monoclonal antibodies (MAbs) raised against recombinant soluble intercellular adhesion molecule-1 (sICAM-1) were evaluated by means of ELISAs, and their neutralizing activity was investigated in two bioassays employing Raji cells activated with phorbol myristate acetate. Three of the MAbs inhibited autoaggregation of the activated cells and adhesion to sICAM-1 fixed on a plastic plate. Non-neutralizing antibody SM1-255 is directed to an epitope that was not recognized by the other eight MAbs. This property enabled us to develop two ELISAs employing SM1-255 as a liquid-phase antibody for the quantitation of sICAM-1 circulating (cICAM-1) in human plasma. One assay, employing non-neutralizing 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 antibody 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% (between assay). There was a discrepancy between cICAM-1 levels of human plasma determined by these two assays. All samples showed 2- to 5-times higher levels in the assay using WIS2-11 than in that using WIS5-85. The result from the gel electrophoresis employing Western blotting suggests that ICAM-1 circulates in at least two molecular forms with molecular masses of about 85 and 130 kDa and with different reactivities to WIS2-11 and WIS5-85.