Paroxysmal nocturnal hemoglobinuria

Erythrocyte acetylcholinesterase deficit analyzed by immunoassay and fluorescence-activated sorting

William Stephen Brimijoin, P. I. Hammond, R. M. Petitt

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

An immunodisplacement assay based on a specific, solid-phase monoclonal antibody was designed to measure acetylcholinesterase in tissue extracts. Sample enzyme content was determined from the competitive reduction of binding of a purified acetylcholinesterase standard, with a detection limit of 5 ng or less. Washed erythrocyte membranes from six normal subjects averaged 1.8 units of acetylcholinesterase activity and 0.45 μg of acetylcholinesterase content per milligram of total protein. Enzyme activity and content in samples from three patients with paroxysmal nocturnal hemoglobinuria (PNH) were reduced approximately in parallel, by as much as 70%. The residual cholinesterase had almost the same homospecific activity as the normal enzyme and was bound with equivalent affinity by six different antibodies. Therefore, the cholinesterase defect was dominated by enzyme loss rather than by structural abnormalities affecting enzyme function. Fluorescence-activated sorting of antibody-labeled erythrocytes revealed a bimodal population distribution. Up to 66% of the PNH cells lacked cholinesterase, and the rest had a near-normal enzyme content. Inasmuch as enzyme-deficient cells represent the complement-sensitive population, cell sorting may help in assessing clinical status and, perhaps, in developing new therapeutic modalities for PNH.

Original languageEnglish (US)
Pages (from-to)522-529
Number of pages8
JournalMayo Clinic Proceedings
Volume61
Issue number7
StatePublished - 1986
Externally publishedYes

Fingerprint

Paroxysmal Hemoglobinuria
Acetylcholinesterase
Immunoassay
Erythrocytes
Fluorescence
Enzymes
Cholinesterases
Competitive Binding
Tissue Extracts
Antibodies
Erythrocyte Membrane
Vulnerable Populations
Limit of Detection
Monoclonal Antibodies
Demography

ASJC Scopus subject areas

  • Medicine(all)

Cite this

Paroxysmal nocturnal hemoglobinuria : Erythrocyte acetylcholinesterase deficit analyzed by immunoassay and fluorescence-activated sorting. / Brimijoin, William Stephen; Hammond, P. I.; Petitt, R. M.

In: Mayo Clinic Proceedings, Vol. 61, No. 7, 1986, p. 522-529.

Research output: Contribution to journalArticle

@article{2b6104561aa548818dfcab53bb4a11dc,
title = "Paroxysmal nocturnal hemoglobinuria: Erythrocyte acetylcholinesterase deficit analyzed by immunoassay and fluorescence-activated sorting",
abstract = "An immunodisplacement assay based on a specific, solid-phase monoclonal antibody was designed to measure acetylcholinesterase in tissue extracts. Sample enzyme content was determined from the competitive reduction of binding of a purified acetylcholinesterase standard, with a detection limit of 5 ng or less. Washed erythrocyte membranes from six normal subjects averaged 1.8 units of acetylcholinesterase activity and 0.45 μg of acetylcholinesterase content per milligram of total protein. Enzyme activity and content in samples from three patients with paroxysmal nocturnal hemoglobinuria (PNH) were reduced approximately in parallel, by as much as 70{\%}. The residual cholinesterase had almost the same homospecific activity as the normal enzyme and was bound with equivalent affinity by six different antibodies. Therefore, the cholinesterase defect was dominated by enzyme loss rather than by structural abnormalities affecting enzyme function. Fluorescence-activated sorting of antibody-labeled erythrocytes revealed a bimodal population distribution. Up to 66{\%} of the PNH cells lacked cholinesterase, and the rest had a near-normal enzyme content. Inasmuch as enzyme-deficient cells represent the complement-sensitive population, cell sorting may help in assessing clinical status and, perhaps, in developing new therapeutic modalities for PNH.",
author = "Brimijoin, {William Stephen} and Hammond, {P. I.} and Petitt, {R. M.}",
year = "1986",
language = "English (US)",
volume = "61",
pages = "522--529",
journal = "Mayo Clinic Proceedings",
issn = "0025-6196",
publisher = "Elsevier Science",
number = "7",

}

TY - JOUR

T1 - Paroxysmal nocturnal hemoglobinuria

T2 - Erythrocyte acetylcholinesterase deficit analyzed by immunoassay and fluorescence-activated sorting

AU - Brimijoin, William Stephen

AU - Hammond, P. I.

AU - Petitt, R. M.

PY - 1986

Y1 - 1986

N2 - An immunodisplacement assay based on a specific, solid-phase monoclonal antibody was designed to measure acetylcholinesterase in tissue extracts. Sample enzyme content was determined from the competitive reduction of binding of a purified acetylcholinesterase standard, with a detection limit of 5 ng or less. Washed erythrocyte membranes from six normal subjects averaged 1.8 units of acetylcholinesterase activity and 0.45 μg of acetylcholinesterase content per milligram of total protein. Enzyme activity and content in samples from three patients with paroxysmal nocturnal hemoglobinuria (PNH) were reduced approximately in parallel, by as much as 70%. The residual cholinesterase had almost the same homospecific activity as the normal enzyme and was bound with equivalent affinity by six different antibodies. Therefore, the cholinesterase defect was dominated by enzyme loss rather than by structural abnormalities affecting enzyme function. Fluorescence-activated sorting of antibody-labeled erythrocytes revealed a bimodal population distribution. Up to 66% of the PNH cells lacked cholinesterase, and the rest had a near-normal enzyme content. Inasmuch as enzyme-deficient cells represent the complement-sensitive population, cell sorting may help in assessing clinical status and, perhaps, in developing new therapeutic modalities for PNH.

AB - An immunodisplacement assay based on a specific, solid-phase monoclonal antibody was designed to measure acetylcholinesterase in tissue extracts. Sample enzyme content was determined from the competitive reduction of binding of a purified acetylcholinesterase standard, with a detection limit of 5 ng or less. Washed erythrocyte membranes from six normal subjects averaged 1.8 units of acetylcholinesterase activity and 0.45 μg of acetylcholinesterase content per milligram of total protein. Enzyme activity and content in samples from three patients with paroxysmal nocturnal hemoglobinuria (PNH) were reduced approximately in parallel, by as much as 70%. The residual cholinesterase had almost the same homospecific activity as the normal enzyme and was bound with equivalent affinity by six different antibodies. Therefore, the cholinesterase defect was dominated by enzyme loss rather than by structural abnormalities affecting enzyme function. Fluorescence-activated sorting of antibody-labeled erythrocytes revealed a bimodal population distribution. Up to 66% of the PNH cells lacked cholinesterase, and the rest had a near-normal enzyme content. Inasmuch as enzyme-deficient cells represent the complement-sensitive population, cell sorting may help in assessing clinical status and, perhaps, in developing new therapeutic modalities for PNH.

UR - http://www.scopus.com/inward/record.url?scp=0022483046&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0022483046&partnerID=8YFLogxK

M3 - Article

VL - 61

SP - 522

EP - 529

JO - Mayo Clinic Proceedings

JF - Mayo Clinic Proceedings

SN - 0025-6196

IS - 7

ER -