Highly sensitive time-resolved fluoroimmunoassay of human immunoglobulin E by using a new europium fluorescent chelate as a label.
A new europium fluorescent chelate, 4,4'-bis(1",1",1",2",2",3",3"-heptafluoro-4",6"-hexanedione-6"-yl) - chlorosulfo-o-terphenyl (BHHCT)-Eu3+, was used as a label for highly sensitive time-resolved fluoroimmunoassay of human IgE. Two assay formats were employed in the analysis. In the first format, an immunoconjugate of rabbit anti-human IgE antibody-human IgE-biotinylated goat anti-human IgE antibody-BHHCT-Eu(3+)-labeled SA (or BHHCT-Eu(3+)-labeled BSA-SA; BSA, bovine serum albumin; SA, streptavidin) was used for measurement. The method gives the detection limits of 3.6 x 10(-2) IU/ml (labeled SA) and 1.1 x 10(-2) IU/ml (labeled SA-BSA). In the second format, an immunoconjugate of goat anti-human IgE antibody-human IgE-rabbit anti-human IgE antibody-biotinylated goat anti-rabbit IgG antibody-BHHCT-Eu(3+)-labeled SA (or BHHCT-Eu(3+)-labeled BSA-SA) was used for measurement. The detection limits of these methods are 3.0 x 10(-3) IU/ml (labeled SA) and 1.5 x 10(-3) IU/ml (labeled BSA-SA). The above detection limits are one to two orders of magnitude lower than those of the conventional radioimmunoassay and enzyme immunoassay. The CV of the present method is less than 7%, and the recovery is in the range of 85-105% for serum samples.
Yuan
J
J
Department of Chemistry, Waseda University, Tokyo, Japan.
Wang
G
G
Kimura
H
H
Matsumoto
K
K
D000818:Animals; D002614:Chelating Agents / Q000378:metabolism; D005063:Europium / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D015200:Fluoroimmunoassay / Q000379:methods*; D006588:Hexanones; D006801:Humans; D007073:Immunoglobulin E / Q000032:analysis*; D007202:Indicators and Reagents; D011817:Rabbits; D013730:Terphenyl Compounds; D005466:Fluorocarbons
D002614:Chelating Agents; D005063:Europium; D005456:Fluorescent Dyes; D006588:Hexanones; D007073:Immunoglobulin E; D007202:Indicators and Reagents; D013730:Terphenyl Compounds; C110071:4,4'-bis(1'',1'',1'',2'',2'',3'',3''-heptafluoro-4'',6''-hexanedione-6''-yl)chlorosulfo-o-terphenyl; D005466:Fluorocarbons