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 Citations (Scopus)

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

Fingerprint

Immune Sera
Complement Activation
Enzyme-Linked Immunosorbent Assay
Goats
Serum
Complement Membrane Attack Complex
Zymosan
Polystyrenes
Sepharose
Alkaline Phosphatase
Cerebrospinal Fluid
Epitopes
Swine
Immunoglobulin G
Rabbits
Membranes
anti-IgG
In Vitro Techniques

Keywords

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

ASJC Scopus subject areas

  • Biotechnology
  • Immunology

Cite this

Quantitation of activation of the human terminal complement pathway by ELISA. / Sanders, Martin E.; Schmetz, Mark A.; Hammer, Carl H.; Frank, Michael M.; Joiner, Keith A.

In: Journal of Immunological Methods, Vol. 85, No. 2, 27.12.1985, p. 245-256.

Research output: Contribution to journalArticle

Sanders, Martin E. ; Schmetz, Mark A. ; Hammer, Carl H. ; Frank, Michael M. ; Joiner, Keith A. / Quantitation of activation of the human terminal complement pathway by ELISA. In: Journal of Immunological Methods. 1985 ; Vol. 85, No. 2. pp. 245-256.
@article{ed133185eb0247968fcff412bb17c3aa,
title = "Quantitation of activation of the human terminal complement pathway by ELISA",
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.",
keywords = "C5b-9 complex, ELISA, neoantigen, terminal complement activation",
author = "Sanders, {Martin E.} and Schmetz, {Mark A.} and Hammer, {Carl H.} and Frank, {Michael M.} and Joiner, {Keith A}",
year = "1985",
month = "12",
day = "27",
doi = "10.1016/0022-1759(85)90135-8",
language = "English (US)",
volume = "85",
pages = "245--256",
journal = "Journal of Immunological Methods",
issn = "0022-1759",
publisher = "Elsevier",
number = "2",

}

TY - JOUR

T1 - Quantitation of activation of the human terminal complement pathway by ELISA

AU - Sanders, Martin E.

AU - Schmetz, Mark A.

AU - Hammer, Carl H.

AU - Frank, Michael M.

AU - Joiner, Keith A

PY - 1985/12/27

Y1 - 1985/12/27

N2 - 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.

AB - 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.

KW - C5b-9 complex

KW - ELISA

KW - neoantigen

KW - terminal complement activation

UR - http://www.scopus.com/inward/record.url?scp=0022347969&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0022347969&partnerID=8YFLogxK

U2 - 10.1016/0022-1759(85)90135-8

DO - 10.1016/0022-1759(85)90135-8

M3 - Article

C2 - 2416846

AN - SCOPUS:0022347969

VL - 85

SP - 245

EP - 256

JO - Journal of Immunological Methods

JF - Journal of Immunological Methods

SN - 0022-1759

IS - 2

ER -