Application of an alpha-sidechain length-specific monoclonal antibody to immunoaffinity purification and enzyme immunoassay of 2,3-dinor-6-keto-prostaglandin F1 alpha from human urine.
2,3-Dinor-6-keto-prostaglandin F1 alpha (2,3-dinor-6-keto-PGF1 alpha) is a major urinary metabolite of PGI2 (prostacyclin) and one of the most reliable parameters of PGI2 production in vivo. A mouse was immunized with 2,3-dinor-6-keto-20-carboxy-PGF1 alpha conjugated to bovine serum albumin for preparation of a monoclonal antibody which recognized the difference in the alpha-sidechain length of 6-keto-PGF1 alpha and its 2,3-dinor-metabolite. A sensitive and specific enzyme immunoassay was developed by the solid-phase competition method with 2,3-dinor-6-keto-20-carboxy-PGF1 alpha labeled with peroxidase protein. The detection range of the assay was 14-1200 fmol (IC50 = 120 fmol). The cross-reactivities of the antibody with 6-keto-PGF1 alpha, 6,15-diketo-13,14-dihydro-PGF1 alpha, and other arachidonate metabolites were less than 0.01%. An immunoaffinity column was prepared by coupling the anti-2,3-dinor-6-keto-PGF1 alpha antibody to BrCN-activated Sepharose 4B. Human urine was applied to an octadecylsilyl silica cartridge, and the extract was applied to the immunoaffinity column. This procedure allowed an efficient separation of 2,3-dinor-6-keto-PGF1 alpha from unidentified urinary substances which interfered with immunoassay. Validity of the results obtained by the enzyme immunoassay was confirmed by GC/MS employing selected ion monitoring for quantification.
Hiramatsu
M
M
Diagnostic Research Laboratory, Ono Pharmaceutical Co. Ltd, Osaka, Japan.
Hayashi
Y
Y
Yamamoto
S
S
Hayashi
A
A
Sawada
M
M
Hamanaka
N
N
Yamashita
K
K
D015121:6-Ketoprostaglandin F1 alpha / Q000031:analogs & derivatives* / Q000276:immunology / Q000652:urine; D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D002417:Cattle; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D007124:Immunoenzyme Techniques; D051379:Mice; D011863:Radioimmunoassay; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; D015121:6-Ketoprostaglandin F1 alpha; C028290:2,3-dinor-6-ketoprostaglandin F1alpha