A biotin-avidin sandwich ELISA for quantification of intact complement component C9. The sera from hereditary C9 deficient individuals completely lack C9.

A two-site sandwich ELISA method was developed for quantitating intact C9 protein using MoAb P40 (anti-C9b antibody). This antibody reacted with monomeric C9 but not with polymerized C9. MoAb P40 was used as a capture antibody and MoAb X195 (anti-C9a antibody) as a detection antibody. This method is highly sensitive and can detect approximately 0.5 ng/ml of native C9. No cross-reactivities of either C6, C7, or C8 were observed even at concentrations of 10 micrograms/ml per component. In addition, this method allows for measurement of only intact C9 molecules, eliminating the interference of polymerized C9 or inactivated C9. Using this assay, no C9 at all was detected in sera from inherited C9 deficient individuals, including both healthy blood donors and patients with meningococcal meningitis; although by hemolytic assay, C9 levels were reported to be less than 0.2% those of NHS. Therefore, this two-site sandwich ELISA method can replace the hemolytic assay, and is especially useful for measuring small amounts of C9 in serum.