Quantitation of activation of the human terminal complement pathway by ELISA

Martin E. Sanders, Mark A. Schmetz, Carl H. Hammer, Michael M. Frank, Keith A. Joiner

Research output: Contribution to journalArticle

14 Scopus citations

Abstract

We have devised an enzyme-linked immunosorbent assay (ELISA) to quantitate fluid phase terminal complement pathway activation. Upon activation to form C5b-9, terminal complement components express neoantigens not present in the unassembled individual components. Expression of one of these neoantigens occurs at the step of C9 activation. C9 neoantigen is present in fluid phase SC5b-9 complexes, membrane-bound MC5b-9 complexes, and in vitro polymerized C9. Under physiologic conditions, the presence of C9 neoantigen indicates that the terminal complement pathway is activated through the terminal component C9. In our assay for C9 neoantigen, we used rabbit antiserum to polymerized C9 rendered specific for C9 neoantigen c determinants by serial absorption with human serum, human C9, and other terminal complement components bound to Sepharose. Using the IgG from this antiserum, we devised a sandwich ELISA to bind SC5b-9 from solution onto polystyrene plates. The ELISA plates were developed with the use of goat antiserum to native C9 epitopes followed by a swine anti-goat IgG-alkaline phosphatase conjugate. Quantitation of SC5b-9 in solution was performed by comparing sample OD to a standard curve generated with human SC5b-9 that was purified from zymosan-activated serum. The assay was sensitive to as little as 100 ng of SC5b-9/ml and should be useful for screening plasma, serum, cerebrospinal fluid, or other biological fluids for the presence of terminal complement pathway activation.

Original languageEnglish (US)
Pages (from-to)245-256
Number of pages12
JournalJournal of Immunological Methods
Volume85
Issue number2
DOIs
StatePublished - Dec 27 1985
Externally publishedYes

Keywords

  • C5b-9 complex
  • ELISA
  • neoantigen
  • terminal complement activation

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

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