A study of optimal reaction conditions for an assay of the human alternative complement pathway

Keith A Joiner, A. Hawiger, J. A. Gelfand

Research output: Contribution to journalArticle

45 Citations (Scopus)

Abstract

The optimal conditions for performance of a sensitive functional assay for the human alternative complement pathway were studied. The serum dilution causing 50% lysis of rabbit erythrocytes in magnesium EGTA buffer is designated the APH50 titer. Optimal reaction conditions for the assay were pH 7.2, incubation temperature 37° C, incubation time 60 minutes, and magnesium concentration 0.002 M. Lowering the ionic strength of the buffer from 0.150 M to 0.0125 M increased APH50 titers nearly 2.5-fold, but decreased the reproducibility of titers. Significant fluid-phase conversion of C3 at 37°C in low ionic strength buffer was demonstrated by crossed immunoelectrophoresis. Using the optimal reaction conditions, in normal ionic strength buffer the mean APH50 ± SD for 45 normal adults was 25.3 ± 5.7 U/mL. Heparin, an inhibitor of the alternative pathway, decreased APH50 by 50% at a concentration of 100 U heparin/mL serum, and totally abolished alternative pathway hemolytic activity at 1,000 U heparin/mL serum, while lowering CH50 titers to a much lesser degree. When increasing doses of zymosan were used for complement activation in vitro, the per cent APH50 depletion at low doses of zymosan was at least twice the per cent depletion of CH50 or antigenic P, B, and C3. A striking dichotomy between nearly complete APH50 depletion and normal or near normal CH50 and hemolytic C4 levels was documented for a human burn patient and for a baboon infused with a lethal dose of Escherichia coli lipopolysaccharide. Therefore, we documented a substantially greater sensitivity of APH50 than of conventional complement determinations for detecting complement consumption by alternative pathway activators.

Original languageEnglish (US)
Pages (from-to)65-72
Number of pages8
JournalAmerican Journal of Clinical Pathology
Volume79
Issue number1
StatePublished - 1983
Externally publishedYes

Fingerprint

Alternative Complement Pathway
Buffers
Osmolar Concentration
Heparin
Zymosan
Serum
Two-Dimensional Immunoelectrophoresis
Papio
Complement Activation
Egtazic Acid
Magnesium
Lipopolysaccharides
Erythrocytes
Escherichia coli
Rabbits
Temperature

ASJC Scopus subject areas

  • Pathology and Forensic Medicine

Cite this

A study of optimal reaction conditions for an assay of the human alternative complement pathway. / Joiner, Keith A; Hawiger, A.; Gelfand, J. A.

In: American Journal of Clinical Pathology, Vol. 79, No. 1, 1983, p. 65-72.

Research output: Contribution to journalArticle

@article{a745173693854c29850b4ca5bb1d72ee,
title = "A study of optimal reaction conditions for an assay of the human alternative complement pathway",
abstract = "The optimal conditions for performance of a sensitive functional assay for the human alternative complement pathway were studied. The serum dilution causing 50{\%} lysis of rabbit erythrocytes in magnesium EGTA buffer is designated the APH50 titer. Optimal reaction conditions for the assay were pH 7.2, incubation temperature 37° C, incubation time 60 minutes, and magnesium concentration 0.002 M. Lowering the ionic strength of the buffer from 0.150 M to 0.0125 M increased APH50 titers nearly 2.5-fold, but decreased the reproducibility of titers. Significant fluid-phase conversion of C3 at 37°C in low ionic strength buffer was demonstrated by crossed immunoelectrophoresis. Using the optimal reaction conditions, in normal ionic strength buffer the mean APH50 ± SD for 45 normal adults was 25.3 ± 5.7 U/mL. Heparin, an inhibitor of the alternative pathway, decreased APH50 by 50{\%} at a concentration of 100 U heparin/mL serum, and totally abolished alternative pathway hemolytic activity at 1,000 U heparin/mL serum, while lowering CH50 titers to a much lesser degree. When increasing doses of zymosan were used for complement activation in vitro, the per cent APH50 depletion at low doses of zymosan was at least twice the per cent depletion of CH50 or antigenic P, B, and C3. A striking dichotomy between nearly complete APH50 depletion and normal or near normal CH50 and hemolytic C4 levels was documented for a human burn patient and for a baboon infused with a lethal dose of Escherichia coli lipopolysaccharide. Therefore, we documented a substantially greater sensitivity of APH50 than of conventional complement determinations for detecting complement consumption by alternative pathway activators.",
author = "Joiner, {Keith A} and A. Hawiger and Gelfand, {J. A.}",
year = "1983",
language = "English (US)",
volume = "79",
pages = "65--72",
journal = "American Journal of Clinical Pathology",
issn = "0002-9173",
publisher = "American Society of Clinical Pathologists",
number = "1",

}

TY - JOUR

T1 - A study of optimal reaction conditions for an assay of the human alternative complement pathway

AU - Joiner, Keith A

AU - Hawiger, A.

AU - Gelfand, J. A.

PY - 1983

Y1 - 1983

N2 - The optimal conditions for performance of a sensitive functional assay for the human alternative complement pathway were studied. The serum dilution causing 50% lysis of rabbit erythrocytes in magnesium EGTA buffer is designated the APH50 titer. Optimal reaction conditions for the assay were pH 7.2, incubation temperature 37° C, incubation time 60 minutes, and magnesium concentration 0.002 M. Lowering the ionic strength of the buffer from 0.150 M to 0.0125 M increased APH50 titers nearly 2.5-fold, but decreased the reproducibility of titers. Significant fluid-phase conversion of C3 at 37°C in low ionic strength buffer was demonstrated by crossed immunoelectrophoresis. Using the optimal reaction conditions, in normal ionic strength buffer the mean APH50 ± SD for 45 normal adults was 25.3 ± 5.7 U/mL. Heparin, an inhibitor of the alternative pathway, decreased APH50 by 50% at a concentration of 100 U heparin/mL serum, and totally abolished alternative pathway hemolytic activity at 1,000 U heparin/mL serum, while lowering CH50 titers to a much lesser degree. When increasing doses of zymosan were used for complement activation in vitro, the per cent APH50 depletion at low doses of zymosan was at least twice the per cent depletion of CH50 or antigenic P, B, and C3. A striking dichotomy between nearly complete APH50 depletion and normal or near normal CH50 and hemolytic C4 levels was documented for a human burn patient and for a baboon infused with a lethal dose of Escherichia coli lipopolysaccharide. Therefore, we documented a substantially greater sensitivity of APH50 than of conventional complement determinations for detecting complement consumption by alternative pathway activators.

AB - The optimal conditions for performance of a sensitive functional assay for the human alternative complement pathway were studied. The serum dilution causing 50% lysis of rabbit erythrocytes in magnesium EGTA buffer is designated the APH50 titer. Optimal reaction conditions for the assay were pH 7.2, incubation temperature 37° C, incubation time 60 minutes, and magnesium concentration 0.002 M. Lowering the ionic strength of the buffer from 0.150 M to 0.0125 M increased APH50 titers nearly 2.5-fold, but decreased the reproducibility of titers. Significant fluid-phase conversion of C3 at 37°C in low ionic strength buffer was demonstrated by crossed immunoelectrophoresis. Using the optimal reaction conditions, in normal ionic strength buffer the mean APH50 ± SD for 45 normal adults was 25.3 ± 5.7 U/mL. Heparin, an inhibitor of the alternative pathway, decreased APH50 by 50% at a concentration of 100 U heparin/mL serum, and totally abolished alternative pathway hemolytic activity at 1,000 U heparin/mL serum, while lowering CH50 titers to a much lesser degree. When increasing doses of zymosan were used for complement activation in vitro, the per cent APH50 depletion at low doses of zymosan was at least twice the per cent depletion of CH50 or antigenic P, B, and C3. A striking dichotomy between nearly complete APH50 depletion and normal or near normal CH50 and hemolytic C4 levels was documented for a human burn patient and for a baboon infused with a lethal dose of Escherichia coli lipopolysaccharide. Therefore, we documented a substantially greater sensitivity of APH50 than of conventional complement determinations for detecting complement consumption by alternative pathway activators.

UR - http://www.scopus.com/inward/record.url?scp=0020682488&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0020682488&partnerID=8YFLogxK

M3 - Article

VL - 79

SP - 65

EP - 72

JO - American Journal of Clinical Pathology

JF - American Journal of Clinical Pathology

SN - 0002-9173

IS - 1

ER -