A flow cytometric immunoassay to quantify adsorption of complement activation products (iC3b, C3d, SC5b-9) on artificial surfaces
✍ Scribed by Gemmell, Cynthia H.
- Publisher
- John Wiley and Sons
- Year
- 1997
- Tongue
- English
- Weight
- 129 KB
- Volume
- 37
- Category
- Article
- ISSN
- 0021-9304
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✦ Synopsis
Crosslinked agarose microspheres and various polystyrene microspheres were analyzed for complement components after incubation with serum at 37°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 g/cm 2 . Further, the surface bound represented a substantial percentage of the total generated. The iC3b level on polystyrene beads rapidly reached 0.09 g/cm 2 and the C3d levels were an order of magnitude less. On agarose beads the iC3b levels continually rose to 0.17 g/cm 2 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/cm 2 . There was minimal evidence of C3a and C1q adsorption for any surface. Use of aminopolystyrene 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.