Immunoaffinity purification of 11-dehydro-thromboxane B2 from human urine and plasma for quantitative analysis by radioimmunoassay.
11-Dehydro-thromboxane B2 is now considered to be a reliable parameter of thromboxane A2 formation in vivo. An immunoaffinity purification method was developed for radioimmunoassay of this compound contained in human urine and plasma. Monoclonal anti-11-dehydro-thromboxane B2 antibody was prepared and coupled to BrCN-activated Sepharose 4B. Human urine or plasma was applied to a disposable column of the immobilized antibody. After the column was washed with water, 11-dehydro-thromboxane B2 was eluted with methanol/water (95/5) with a recovery of more than 90%. The purified extract was subjected to a radioimmunoassay utilizing 11-[3H]dehydro-thromboxane B2 methyl ester and the monoclonal anti-11-dehydro-thromboxane B2 antibody. The detection range of the assay was 10-600 fmol (IC50 = 90 fmol). The cross-reactivities of the antibody with thromboxane B2, 2,3-dinor-thromboxane B2, and other arachidonate metabolites were less than 0.05%. These compounds were efficiently separated from 11-dehydro-thromboxane B2 by the immunoaffinity purification. This procedure also allowed the separation of 11-dehydro-thromboxane B2 from unidentified urinary and plasma substances which interfered with the radioimmunoassay. Validity of the results obtained by the radioimmunoassay was confirmed by GC/MS employing selected ion monitoring for quantification.
Hayashi
Y
Y
Department of Biochemistry, Tokushima University School of Medicine, Japan.
Shono
F
F
Yamamoto
S
S
Takasaki
W
W
Nakagawa
A
A
Watanabe
K
K
Yamashita
K
K
Miyazaki
H
H
D000328:Adult; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis; D002846:Chromatography, Affinity / Q000379:methods*; D003429:Cross Reactions; D005260:Female; D006801:Humans; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D011863:Radioimmunoassay; D015203:Reproducibility of Results; D013929:Thromboxane B2 / Q000031:analogs & derivatives* / Q000032:analysis / Q000097:blood / Q000302:isolation & purification / Q000652:urine
D000911:Antibodies, Monoclonal; D013929:Thromboxane B2; C049235:11-dehydro-thromboxane B2