A FLOW CYTOMETRIC IMMUNOASSAY TO QUANTIFY ADSORPTION OF COMPLEMENT ACTIVATION PRODUCTS (IC3B, C3D, SC5B-9) ON ARTIFICIAL SURFACES

Crosslinked agarose microspheres and various polystyrene microspheres were analyzed for complement components after incubation with serum at 37 degrees C for times up to 2 h. Quantification involved direct now cytometric analysis of the heads after the bound complement proteins w ere indirectly fluorescently tagged by use of a monoclonal antibody ag ainst a complement protein: C5b-9, iC3b, C3d, C4d, Bb, C3a, and C1q. C alibration with fluorescein microbead standards demonstrated that the membrane attack complex (SC5b-9) was surface bound on all surfaces and that the surface concentration gradually increased to levels as high as 0.5 mu g/cm(2). Further, the surface bound represented a substantia l percentage of the total generated. The iC3b level on polystyrene bea ds rapidly reached 0.09 mu g/cm(2) and the C3d levels were an order of magnitude less. On agarose beads the iC3b levels continually rose to 0.17 mu g/cm(2) and, as before, the C3d levels were substantially lowe r. The surface concentration of C4d and Bb on both surfaces were signi ficant but less than 1.0 ng/cm(2). There was minimal evidence of C3a a nd C1q adsorption for any surface. Use of amino-polystyrene beads mode rately reduced the level of bound iC3b, C3d, and SC5b-9, whereas carbo xylated beads reduced the levels by almost a factor of two. The apprec iable amounts of iC3b and SC5b-9 consistently noted on the artificial surfaces tested in this paper suggests that for these two activation p roducts in vitro analysis of material induced complement activation sh ould also include surface analysis. (C) 1997 John Wiley & Sons, Inc.