A hemolytic assay for the estimation of functional mannose-binding lectin levels in human serum.
A simple assay was developed to estimate functional mannose-binding lectin (MBL) levels in serum based on the principle of yeast-induced bystander lysis of chicken erythrocytes (ChE). The assay is sensitive to inhibition by ethylene glycol bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) (which allows alternative pathway activation), ethylene diamine tetraacetic acid (EDTA), mannose, N-acetylglucosamine and C1 esterase inhibitor (C1-INH), whereas it was not inhibited by galactose. A high-titer human anti-mannan antibody-containing serum with 0.06 microg MBL/ml gave a functional signal corresponding to 0.12 microg equivalents MBL/ml, indicating that anti-mannan antibodies are poorly hemolytic in the assay. The assay is well suited for the large-scale testing of patient samples for a functional MBL pathway of complement activation.
Kuipers
Saskia
S
Eijkman-Winkler Center for Microbiology, Infectious Diseases, and Inflammation, University Medical Center Utrecht G04.614, Heidelberglaan 100, The Netherlands. skuipers@labmicta.nl
Aerts
Piet C
PC
Sjöholm
Anders G
AG
Harmsen
Theo
T
van Dijk
Hans
H
D003174:Complement C1 Inactivator Proteins / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D006461:Hemolysis; D006801:Humans; D037601:Mannose-Binding Lectin / Q000037:antagonists & inhibitors / Q000097:blood*; D050606:Mannose-Binding Protein-Associated Serine Proteases; D012697:Serine Endopeptidases / Q000378:metabolism
D003174:Complement C1 Inactivator Proteins; D037601:Mannose-Binding Lectin; D050606:Mannose-Binding Protein-Associated Serine Proteases; D012697:Serine Endopeptidases