A flow cytometric immunoassay to quantify adsorption of complement activation products (iC3b, C3d, SC5b-9) on artificial surfaces

Abstract

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 flow cytometric analysis of the beads after the bound complement proteins were indirectly fluorescently tagged by use of a monoclonal antibody against a complement protein: C5b-9, iC3b, C3d, C4d, Bb, C3a, and C1q. Calibration 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 micrograms/cm2. Further, the surface bound represented a substantial percentage of the total generated. The iC3b level on polystyrene beads rapidly reached 0.09 micrograms/cm2 and the C3d levels were an order of magnitude less. On agarose beads the iC3b levels continually rose to 0.17 micrograms/cm2 and, as before, the C3d levels were substantially lower. The surface concentration of C4d and Bb on both surfaces were significant but less than 1.0 ng/cm2. There was minimal evidence of C3a and C1q adsorption for any surface. Use of amino-polystyrene beads moderately reduced the level of bound iC3b, C3d, and SC5b-9, whereas carboxylated beads reduced the levels by almost a factor of two. The appreciable amounts of iC3b and SC5b-9 consistently noted on the artificial surfaces tested in this paper suggests that for these two activation products in vitro analysis of material induced complement activation should also include surface analysis.

Deletion ofwboAEnhances Activation of the Lectin Pathway of Complement inBrucella abortusandBrucella melitensis

“…Complement antibodies were added at a concentration of 20 g/ml in PBS-BSA; nonspecific staining and unstained controls received PBS-BSA only. The plate was incubated at 4°C for 30 min (18). After washing and resuspending the samples as before, secondary, FITC-conjugated anti-murine IgG or anti-goat IgG antibody or PBS-BSA (for unstained controls) was added, and the plate was again incubated at 4°C for 30 min.…”

On the binding of complement to solid artificial surfaces in vitro

“…A large portion of the "Binding of complement to solids" - Wetterö et al biomaterial-complement research has utilized conventional radioimmunological or enzyme linked immunosorbent assays (RIA/ELISA) and/or often emphasized fluid phase products like the anaphylatoxins or soluble terminal complexes. However, also the direct surface analysis with regard to surface-bound complement proteins is of utmost importance in biomaterials research [12].…”