Monoclonal antibody, method of production thereof and use thereof

The present invention relates to a monoclonal antibody which specifically recognizes 6,15-diketo-13,14-dihydro-prostaglandin F.sub.1 .alpha. with high sensitivity, a hybridoma producing said monoclonal antibody, a method of establishing said hybridoma and an immunoassay method for 6,15-diketo-13,14-dihydro-prostaglandin F.sub.1 .alpha. using said monoclonal antibody. The monoclonal antibody of the present invention makes it possible to conveniently measure 6,15-diketo-13,14-dihydro-prostaglandin F.sub.1 .alpha., a stable metabolite of prostaglandin I.sub.2 thought of as reflecting the concentration changes therein, with specificity and high sensitivity. It is useful in determining the content of prostaglandin I.sub.2, which exhibits platelet aggregating inhibitory activity, vasodilating activity and other activities, in biological samples.

FIELD OF THE INVENTION 
The present invention relates to a monoclonal antibody against 
6,15-diketo-13,14-dihydro-prostaglandin F.sub.1 .alpha. (also referred to 
as 6,15-DK-13,14-DH-PGF.sub.1 .alpha. for short), a method of production 
thereof and a use thereof. The invention also provides a hybridoma 
producing said monoclonal antibody. 
BACKGROUND OF THE INVENTION 
Prostacyclin (prostaglandin I.sub.2, also referred to as PGI.sub.2 for 
short), a bioactive substance belonging to the arachidonic acid cascade, 
possesses potent platelet aggregation suppressive action, vasodilating 
action and other bioactivities. The bioactivities of PGI.sub.2 are 
antagonistic to thromboxane A.sub.2 (also referred to as TXA.sub.2 for 
short); its measurement in vivo has been much emphasized. However, 
PGI.sub.2 is an unstable substance, whose half-life at 37.degree. C. is 
reportedly about 5 minutes. For this reason, 6-keto-prostaglandin F.sub.1 
.alpha. (also referred to as 6-K-PGF.sub.1 .alpha. for short), which is 
derived from PGI.sub.2 in the absence of enzyme, has been measured in 
place of PGI.sub.2 (ALAN R. BRASH et al., J. Pharmacol. Exp. Ther., 226, 
78 (1983)). 
However, because of their low production rates and short half-lives, the 
concentrations of 6-K-PGF.sub.1 .alpha. in human plasma obtained under 
resting conditions are in the low picogram range (FitzGerald, G. A. et 
al., Circulation, 67, 1174-1177 (1983)). Moreover, sampling-induced 
artifacts and ex vivo synthesis by blood cells make most plasma 
measurements of primary icosanoids totally unreliable (Carlo Patrono et 
al., Advances in Prostaglandin, Thromboxane, and Leukotriene Research, 
Vol. 15, p. 71-73 (1985), edited by O. Hayaishi and S. Yamamoto, Raven 
Press, New York). Therefore, because the values of 6-K-PGF.sub.1 .alpha. 
obtained vary widely among institutions, comparison by absolute values has 
been almost impossible among different institutions [M. Suzuki et al., 
Japanese Journal of Clinical Medicine, Vol. 43, p. 1082 (1985)]. 
One method of circumventing such problems is measurement of metabolites 
that have an extended half-life. Although the 6,15-DK-13,14-DH-PGF.sub.1 
.alpha. metabolite of PGI.sub.2 has a longer half-life than 6-K-PGF.sub.1 
.alpha., it was expected to precisely reflect changes in PGI.sub.2 
concentration [C. Patrono et al., Clinical Research, Vol. 29, p. 276A 
(1981)]. 
The object of the present invention is to provide a means for conveniently 
measuring 6,15-DK-13,14-DH-PGF.sub.1 .alpha., a stable metabolite of 
prostaglandin I.sub.2 thought of as reflecting the concentration changes 
therein, with specificity and high sensitivity. 
In view of the circumstances described above, the present inventors 
conducted investigations with the aim of developing a practical means of 
assaying 6,15-DK-13,14-DH-PGF.sub.1 .alpha., and prepared a monoclonal 
antibody against 6,15-DK-13,14-DH-PGF.sub.1 .alpha. enabling such assay. 
The inventors conducted further investigations based on this achievement 
and developed the present invention. 
SUMMARY OF THE INVENTION 
The present invention relates to 
(1) A monoclonal antibody reacting specifically to 
6,15-diketo-13,14-dihydro-prostaglandin F.sub.1 .alpha., 
(2) A cloned hybridoma which is derived from fusing a spleen cell of a 
mammal immunized with 6,15-diketo-13,14-dihydro-prostaglandin F.sub.1 
.alpha. with a homogenic or heterogenic lymphoid cell, 
(3) A method for producing said cloned hybridoma which comprises fusing the 
homogenic or heterogenic lymphoid cell with the spleen cell from the 
mammal immunized with 6,15-diketo-13,14-dihydro-prostaglandin F.sub.1 
.alpha. and selecting the desired hybridoma, 
(4) A method for producing said monoclonal antibody which comprises 
culturing said cloned hybridoma in either a liquid medium or a mammalian 
abdominal cavity to produce the monoclonal antibody and collecting the 
monoclonal antibody, and 
(5) A method for determining 6,15-diketo-13,14-dihydro-prostaglandin 
F.sub.1 .alpha. which comprises using the monoclonal antibody in the 
above-mentioned item (1).

DETAILED DESCRIPTION OF THE INVENTION 
The antibody against 6,15-DK-13,14-DH-PGF.sub.1 .alpha. of the present 
invention can be prepared by applying a known technique per se. 
Commercially available 6,15-DK-13,14-DH-PGF.sub.1 .alpha. products can be 
used in the present invention (Cayman Company, distributed by Funakoshi 
Pharmaceutical Co., Ltd.). The desired antibody can be obtained by 
inoculating the binding product of 6,15-DK-13,14-DH-PGF.sub.1 .alpha. and 
a high molecular carrier substance, as an immunogen, to a non-human 
warm-blooded animal to form an antibody, and collecting said antibody. 
Binding of 6,15-DK-13,14-DH-PGF.sub.1 .alpha. and the high molecular 
carrier substance for this purpose can be achieved using a known routine 
means [e.g., Hormone and Metabolic Research, Vol. 8, p. 241 (1976)], 
including a method using a dehydrant, such as water-soluble carbodiimide. 
Examples of the high molecular carrier substance include bovine 
thyroglobulin, bovine serum albumin, bovine gamma globulin and hemocyanin. 
The product prepared is dialyzed against water at about 4.degree. C. by a 
routine means, and may be stored after freezing or freeze drying. 
For immunization with the immunogen described above to obtain an antibody, 
laboratory mammalians such as sheep, goats, rabbits, guinea pigs, rats and 
mice are used, and among them rats and mice are preferred for obtaining a 
monoclonal antibody. Concerning the method of immunization, any route is 
acceptable, including subcutaneous, intraperitoneal, intravenous, 
intramuscular and intradermal injection, when mice, for instance, are 
immunized, but it is preferable to inject the immunogen mainly 
subcutaneously, intraperitoneally or intravenously (particularly 
subcutaneously). Also, inoculation intervals, inoculum amounts etc. are 
widely variable and various methods are available, but it is a common 
practice to inoculate the immunogen 2 to 8 times at 2-week intervals and 
use splenocytes 1 to 5 days (preferably 2 to 4 days) after final 
immunization. It is desirable that the inoculum amount be not less than 
0.1 .mu.g (preferably 10 to 30 .mu.g) per mouse as the amount of 
polypeptide for each immunization. 
When splenocytes are used as lymphocyte source, provided that the spleen is 
excised, it is desirable to previously conduct partial blood sampling to 
confirm increased blood antibody titer, and then make a fusion experiment. 
In cell fusion of the above-mentioned lymphocytes and a lymphoblast line, 
immunized mouse lymphocytes (particularly those derived from splenocytes), 
for instance, are fused with an appropriate lymphoblast line such as a 
myeloma cell of the same or different animal species (preferably the same 
species) having a marker for hypoxanthine-guanine-phosphoribosyl 
transferase deficiency (HGPRT.sup.--) or thymidine kinase deficiency 
(TK.sup.--). A fusogen, such as Sendai virus or polyethylene glycol (PEG), 
is used for this fusion. It is of course possible to add dimethyl 
sulfoxide (DMSO) or another fusion promoter. PEG with an average molecular 
weight of about 1000 to 6000 is used at a concentration of about 10 to 80% 
and at a treating time of 0.5 to 30 minutes. As a preferable set of 
conditions, efficient fusion can be achieved by treatment with 35 to 55% 
PEG6000 for 4 to 10 minutes. The fused cell (hybridoma) can be selectively 
proliferated using hypoxanthine-aminopterin-thymidine medium [HAT medium; 
Nature, Vol. 256, p. 495 (1975)] or another medium. 
Mouse sera and the culture supernatant of proliferated cells can be 
screened for production of the desired antibody; antibody titer screening 
can be carried out as follows. Specifically, antibody titer can be 
determined by radioimmunoassay (RIA method), enzyme immunoassay (EIA 
method) and other methods, which are widely modifiable. As a preferred 
mode of assay, the method based on EIA is described below. To anti-mouse 
immunoglobulin, previously immobilized to a solid phase by a conventional 
method (the use of a 96-well microtiter plate as a solid phase is 
advantageous because it allows rapid assay using a plate reader etc.), the 
subject culture supernatant or mouse serum is added, followed by reaction 
at constant temperature ("constant temperature," as used hereinafter, 
means 4.degree. to 40.degree. C.) for a given period. The reaction product 
is then thoroughly washed, after which enzyme-labeled 
6,15-DK-13,14-DH-PGF.sub.1 .alpha. is added, followed by reaction at 
constant temperature for a given period. After the reaction product is 
thoroughly washed, an enzyme substrate is added, followed by reaction at 
constant temperature for a given period, after which the coloring product 
can be measured by absorbance or fluorescence intensity. 
With respect to cells in the wells in which proliferation occurred on the 
selection medium, antibody activity against 6,15-DK-13,14-DH-PGF.sub.1 
.alpha. was present and specific activity was noted, it is desirable to 
conduct cloning by limiting dilution analysis and other means. The 
supernatant of the cloned cells is screened in the same manner as above to 
increase the number of cells with high antibody titer, whereby hybridoma 
clones producing a monoclonal antibody are obtained. 
The hybridoma thus cloned is proliferated in liquid medium or a mammalian 
abdominal cavity. For example, said monoclonal antibody can be obtained 
from culture broth after cultivation for 2 to 10 days, preferably 3 to 5 
days, in a liquid medium, such as that prepared by adding 0.1 to 40% 
bovine serum to RPMI-1640 medium. An antibody with a titer much higher 
than that obtained with cell culture supernatant can be obtained in large 
amounts with high efficiency by inoculating the cloned hybridoma to the 
abdominal cavity of an appropriate mammal, such as a mouse, to allow cell 
proliferation, and then collecting ascitic fluid. For this purpose, in the 
case of a mouse, for instance, 1.times.10.sup.4 to 1.times.10.sup.7, 
preferably 5.times.10.sup.5 to 2.times.10.sup.6 hybrid cells are 
intraperitoneally or otherwise inoculated to BALB/c or another line of 
mice previously inoculated with mineral oil etc., ascitic fluid being 
collected 7 to 20 days (preferably 10 to 14 days) later. From the ascitic 
fluid, in which the antibody was produced and accumulated, the desired 
monoclonal antibody can easily be isolated as pure immunoglobulin by 
ammonium sulfate fractionation, DEAE-cellulose column chromatography or 
another method. 
The monoclonal antibody in the present invention may be immunoglobulin or a 
fraction thereof [e.g., F(ab').sub.2, Fab' or Fab]. 
In the immunochemical assay for 6,15-DK-13,14-DH-PGF.sub.1 .alpha. in the 
present invention, the competitive method is usually used. The competitive 
method is defined as a method in which a given amount of a subject 
solution containing an unknown amount of 6,15-DK-13,14-DH-PGF.sub.1 
.alpha. and a given amount of marker-labeled 6,15-DK-13,14-DH-PGF.sub.1 
.alpha. are subjected to competitive reaction with a given amount of the 
corresponding antibody, and the activity of the antibody-bound marker or 
unbound marker is determined, to determine the amount of 
6,15-DK-13,14-DH-PGF.sub.1 .alpha. in the subject solution. 
As an example application of the enzyme immunoassay method according to the 
present invention, the use of horseradish peroxidase (hereinafter also 
referred to as HRP for short) as a marker is specifically described below, 
which is not to be construed as limitative. 
1: To the subject solution, add a given amount of HRP-labeled 
6,15-DK-13,14-DH-PGF.sub.1 .alpha. and mix. 
2: Add an antibody in an amount corresponding to the given amount of the 
labeled 6,15-DK-13,14-DH-PGF.sub.1 .alpha., mix and carry out a 
competitive reaction. 
3: To the reaction product obtained in 2, add a solid phase on which is 
fixed a foreign antibody against the immunoglobulin of the animal used to 
prepare the antibody of 2 (hereinafter referred to as anti-seed antibody 
for short), and carry out reaction at constant temperature for a given 
period. 
4: Wash the solid phase thoroughly and determine the HRP activity. 
5: Perform procedures 1 through 4 above on a given amount of standard 
solution and draw a standard curve. 
6: Apply the HRP activity obtained with the unknown amount of assay subject 
(subject solution) to the standard curve to determine the amount of 
6,15-DK-13,14-DH-PGF.sub.1 .alpha. in the assay subject. 
Examples of subject solutions include biological samples such as those of 
blood plasma, serum, urine, spinal fluid and tissue extracts. 
Examples of markers include enzymes, radioisotopes, fluorescent substances 
and luminescent substances. 
Examples of preferable radioisotopes include .sup.125 I, .sup.131 I, .sup.3 
H and .sup.14 C. The enzyme is preferably stable and of high specific 
activity; examples of such enzymes include 1) carbohydrases [e.g., 
glycosidases (e.g., .beta.-galactosidase, .beta.-glycosidase, 
.beta.-glucurosidase, .beta.-fructosidase, .alpha.-galactosidase, 
.alpha.-glucosidase, .alpha.-mannosidase), amylases (e.g., 
.alpha.-amylase, .beta.-amylase, isoamylase, glucoamylase, Taka amylase A) 
cellulase, lysozyme], 2) amylases (e.g., urease, asparaginase), 3) 
esterases [e.g., choline esterases (e.g., acetylcholine esterase), 
phosphatases (e.g., alkaline phosphatase), sulfatase, lipase], 4) 
nucleases (e.g., deoxyribonuclease, ribonuclease), 5) iron-porphyrin 
enzymes (e.g., catalase, peroxidase, cytochrome oxidase), 6) copper 
enzymes (e.g., tyrosinase, ascorbic acid oxidase), and 7) dehydrogenases 
(e.g., alcohol dehydrogenase, malic acid dehydrogenase, lactic acid 
dehydrogenase, isocitric acid dehydrogenase). Examples of fluorescent 
substances include fluorescamine and fluorescence isothiocyanate. Examples 
of luminescent substances include luminol, luminol derivatives, luciferin 
and lucigenin. Of these markers, enxymes, particularly peroxidase can be 
used especially advantageously. 
Binding of 6,15-DK-13,14-DH-PGF.sub.1 .alpha. and marker can be achieved by 
a conventional method, such as the use of a condensing agent, e.g., 
water-soluble carbodiimide. 
The monoclonal antibody of the present invention makes it possible to 
conveniently measure 6,15-diketo-13,14-dihydro-prostaglandin F.sub.1 
.alpha., a stable metabolite of prostaglandin I.sub.2 thought of as 
reflecting the concentration changes therein, with specificity and high 
sensitivity. It is useful in determining the content of prostaglandin 
I.sub.2, which exhibits platelet aggregating inhibitory activity, 
vasodilating activity and other activities, in biological samples. 
The monoclonal antibody of the present invention hardly reacts with other 
arachidonic acid metabolite, for example, 6-keto-PGF.sub.1 .alpha., 
2,3-dinor-6-keto-PGF.sub.1 .alpha., 6-keto-PGE.sub.1, PGB.sub.2, 
PGD.sub.2, PGE.sub.1, PGE.sub.2, PGF.sub.1 .alpha., PGF.sub.2 .alpha., 
TXB.sub.2, 11-dehydro-TXB.sub.2, 15-keto-PGF.sub.2 .alpha., 
13,14-dihydro-15-keto-PGE.sub.1 or 13,14-dihydro-15-keto-PGF.sub.2 
.alpha.. 
To carry out a specific immunochemical assay based on the competitive 
method, a sample containing an unknown amount of 
6,15-DK-13,14-DH-PGF.sub.1 .alpha., for instance, is reacted with a given 
amount of labeled 6,15-DK-13,14-DH-PGF.sub.1 .alpha. and a given amount of 
monoclonal antibody, after which a solid phase, previously bound with 
anti-seed antibody physically or chemically, is added to react. Then, 
usually, after washing thoroughly the solid the marker activity bound to 
the solid phase is measured. When the marker is a radioisotope, its 
activity is measured using a well counter or a liquid scintillation 
counter. When the marker is an enzyme, a substrate is added, and after a 
while enzyme activity is determined by a colorimetric or fluorometric 
method. Whether the marker is a fluorescent or luminescent substance, each 
is measured by a known method. 
When a biological sample, particularly a serum or plasma sample, is assayed 
for a trace amount of 6,15-DK-14,14-DH-PGF.sub.1 .alpha. with high 
precision, sample pretreatment is often necessary. Examples of sample 
concentrating procedures include the extraction concentration method using 
an organic solvent, the method using a cartridge type column of 
octadecylsilane (ODS) and the method using an antibody-bound affinity 
column. Of these methods, that using a packed cartridge with ODS or 
another organic silicon immobilized thereon is commonly widely used. 
Specifically, when the sample is a body fluid such as plasma, serum or 
urine, it is applied to the cartridge after being adjusted to acidic pH. 
In the case of an organ or tissue section, it is extracted with an 
appropriate organic solvent, such as alcohol or chloroform, after which it 
is adjusted to acidic pH and then applied to the cartridge. After such a 
sample solution is passed through the cartridge for adsorption, it is 
washed and then eluted with an appropriate eluent, such as alcohol, ethyl 
acetate, acetonitrile or a hydrate thereof, to elute the adsorbed target 
substance. The eluate thus obtained is then evaporated to dryness either 
in N.sub.2 gas or under reduced pressure. The residue is then dissolved in 
a buffer; the resulting solution, as a subject solution, is usually 
subjected to RIA, EIA or another immunochemical assay. 
Hybridoma Dk 501, obtained in Example 1 below, has been deposited under 
accession number FERM BP-3464 at the Fermentation Research Institute of 
the Agency of Industrial Technology, Ministry of International Trade and 
Industry, since Jun. 25, 1991, under the Budapest treaty and has also been 
deposited under accession number IFO 50342 at the Institute for 
Fermentation, Osaka (IFO), since Jun. 28, 1991. 
EXAMPLES 
The present invention is hereinafter described in more detail by means of 
the following examples, but the invention is not limited to them. 
EXAMPLE 1 (Establishment of Hybridoma) 
A solution of 500 .mu.g of 6,15-DK-13,14-DH-PGF.sub.1 .alpha. in 80% 
dioxane (300 .mu.l), 750 .mu.g of 
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 500 .mu.g 
of N-hydroxysuccinimide were reacted at room temperature for 3 hours, 
after which 1 ml of purified water was added, followed by three cycles of 
extraction with 2 ml of ethyl acetate. The extract was then evaporated to 
dryness in nitrogen gas. To the dry product, 3 mg of bovine serum albumin 
(BSA) in solution in 1 ml of physiological saline was added, followed by 
20 hours of reaction at 4.degree. C.; the reaction product was then 
dialyzed against physiological saline. This reaction product was used as 
an immunogen. In each of five BALB/c mice, a solution of 100 .mu.g of a 
conjugate of 6,15-DK-13,14-DH-PGF.sub.1 .alpha. and BSA in 0.5 ml of 
physiological saline, containing 0.5 ml of Freund's complete adjuvant 
suspended therein, was intraperitoneally injected. Two to 12 weeks later, 
50 .mu.g of a conjugate of 6,15-DK-13,14-DH-PGF.sub.1 .alpha. and BSA in 
solution in 0.1 ml of physiological saline was injected for booster 
immunization in each mouse via the tail vein. At 3 days following final 
immunization, mouse splenocytes were taken out for cell fusion. 
The collected splenocytes (1.0.times.10.sup.8) and myeloma cells FO 
(1.0.times.10.sup.7) were mixed in RPMI-1640 medium and centrifuged at 
1,000 rpm for 5 minutes. To the resulting sediment, 1 ml of 50% 
polyethylene glycol 1500 was gradually added at 37.degree. C. over a 
1-minute period, and the cells were allowed to fuse. After 7 ml of 
RPMI-1640 medium at 37.degree. C. was added over a 5-minute period, the 
mixture was centrifuged. The resulting fused cells were diluted with HAT 
medium and dispensed to 96-well microplates at 0.1 ml per well, after 
which they were cultivated with half the HAT medium replaced with fresh 
HAT medium at intervals of 2 to 3 days. After 7 to 14 days, hybridoma 
proliferation occurred in all 288 wells, 25% of which (72/288) proved to 
contain anti-6,15-DK-13,14-DH-PGF.sub.1 .alpha. antibody produced therein. 
The cells from the wells with the highest antibody titer were cloned by 
limiting dilution analysis. Specifically, 10.sup.5 /well BALB/c mouse 
thymocytes, as feeder cells, and 1/well hybridoma were added to the wells 
and cultivated in HT medium. This procedure was repeated in two cycles. 
The clone which produced the desired antibody most stably and most richly 
was selected via cloning and named Dk 501 (IFO 50342, FERM BP-3464). 
The antibody titer of the clone was determined as follows. To a microplate 
with an anti-mouse immunoglobulinG (IgG) antibody immobilized thereon, 100 
.mu.l of the hybridoma culture supernatant was added, followed by reaction 
at room temperature for 1 hour. After plate washing, peroxidase-labeled 
6,15-DK-13,14-DH-PGF.sub.1 .alpha. was added, followed by reaction at room 
temperature for 1 hour. After plate washing again, a solution of the 
enzyme substrate o-phenylenediamine was added, and its absorbance 
determined, to obtain the antibody titer of the hybridoma culture 
supernatant. 
Peroxidase-labeled 6,15-DK-13,14-DH-PGF.sub.1 .alpha. was prepared as 
follows. A solution of 200 .mu.g of 6,15-DK-13,14-DH-PGF.sub.1 .alpha. in 
80% dioxane (300 .mu.l), 300 .mu.g of 
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 200 .mu.g 
of N-hydroxysuccinimide were reacted at room temperature for 3 hours, 
after which 1 ml of purified water was added, followed by three cycles of 
extraction with 2 ml of ethyl acetate. The extract was then evaporated to 
dryness in nitrogen gas. To the dry product, 1 mg of peroxidase in 
solution in 0.2 ml of physiological saline was added, followed by 20 hours 
of reaction at 4.degree. C.; the reaction product was purified by gel 
filtration with Superose 12. 
EXAMPLE 2 (Production of Monoclonal Antibody) 
To BALB/c mice, previously treated by intraperitoneal administration of 0.5 
ml of pristane, 5.times.10.sup.6 Dk 501 hybrid cells in suspension in 0.5 
ml of RPMI-1640 were intraperitoneally injected. 10 to 14 days later, the 
retained ascitic fluid was collected and purified using Protein-A 
Cellulofine, to yield the monoclonal antibody Dk 501. The yield of the 
monoclonal antibody Dk 501 was 15 mg per ml of ascitic fluid. 
EXAMPLE 3 (Specificity of Monoclonal Antibody) 
The isotype of the monoclonal antibody (Dk 501) established in Example 1 
was determined to be IgG.sub.1 by the Ouchterlony method. Its binding 
specificity was determined by examining its cross reactivity against other 
arachidonic acid metabolites by enzyme immunoassay. The results are shown 
in Table 1. This monoclonal antibody hardly reacted with other arachidonic 
acid metabolites. This monoclonal antibody is therefore identified as a 
monoclonal antibody which specifically reacts to 
6,15-DK-13,14-DH-PGF.sub.1 .alpha.. 
TABLE 1 
______________________________________ 
Arachidonic acid metabolite 
Cross reactivity (%) 
______________________________________ 
6,15-diketo-13,14-dihydro-PGF.sub.1 .alpha. 
100 
6-keto-PGF.sub.1 .alpha. 
0.28 
2,3-dinor-6-keto-PGF.sub.1 .alpha. 
0.03 
6-keto-PGE.sub.1 0.03 
PGB.sub.2 0.02 
PGD.sub.2 0.001 
PGE.sub.1 0.009 
PGE.sub.2 0.004 
PGF.sub.1 .alpha. 0.009 
PGF.sub.2 .alpha. 0.002 
TXB.sub.2 0.001 
11-dehydro-TXB.sub.2 
0.002 
15-keto-PGF.sub.2 .alpha. 
0.63 
13,14-dihydro-15-keto-PGE.sub.1 
0.37 
13,14-dihydro-15-keto-PGF.sub.2 .alpha. 
0.30 
______________________________________ 
The enzyme immunoassay described above was conducted as follows. 100 .mu.l 
of either a standard 6,15-DK-13,14-DH-PGF.sub.1 .alpha. solution or a 
standard solution of another arachidonic acid metabolite, 100 .mu.l of a 
solution of peroxidase-labeled 6,15-DK-13,14-DH-PGF.sub.1 .alpha. and 100 
.mu.l of a solution of the mouse monoclonal 
anti-6,15-diketo-13,14-dihydro-prostaglandin F.sub.1 .alpha. antibody (Dk 
501) obtained in Examples 1 and 2 were mixed, and a solid phase bound with 
anti-mouse IgG antibody was added, followed by reaction at 4.degree. C. 
for 20 hours. After plate washing, 500 .mu.l of a 0.1M citrate buffer 
containing 0.02% hydrogen peroxide and 0.26% o-phenylenediamine was added, 
followed by reaction at room temperature for 1 hour. After 1 ml of 1N 
sulfuric acid was added to terminate the reaction, absorbance at 492 nm 
was determined. 
The detection limit of this enzyme immunoassay using the monoclonal 
antibody was 0.5 pg/tube (1.3 fmol), indicating very high sensitivity 
(FIG. 1). In FIG. 1, Bo in B/Bo is the enzyme activity bound to the solid 
phase in the absence of 6,15-DK-13,14-DH-PGF.sub.1 .alpha.; B is the 
enzyme activity bound to the solid phase in the presence of 
6,15-DK-13,14-DH-PGF.sub.1 .alpha.. 
EXAMPLE 4 (Assay of Blood Plasma for 
6,15-diketo-13,14-dihydro-PGF.sub.1.alpha.) 
(1) Preparation of Immobilized Antibody Column 
After inclusion in a dialytic membrane, 50 mg of 
anti-6,15-diketo-13,14-dihydro-PGF.sub.1.alpha. monoclonal antibody (Dk 
501) was dialyzed against 0.1M carbonate buffer (pH 8.5) at 4.degree. C. 
for 24 hours. The antibody was immobilized by a known method as follows. 5 
g of Sepharose 4B, previously activated with cyanogen bromide, was washed 
with 1000 ml of 1 mM hydrochloric acid and then with 100 ml of 0.1M 
carbonate buffer (pH 8.5). To this washed cyanogen-bromide-activated 
Sepharose 4B, the dialyzed monoclonal antibody was added, followed by 
stirring at room temperature for 3 hours. After blocking the unreacted 
active groups using 0.1M Tris buffer (pH 8.5), the mixture was washed with 
purified water and methanol. The resulting monoclonal-antibody-bound 
Sepharose 4B was diluted with Sepharose 4B to an antibody content of 300 
.mu.g per ml of gel. The diluted antibody-bound Sepharose 4B, in an amount 
of 0.7 ml as of gel volume, was packed in a polypropylene column (Sepacol 
Mini PP, produced by Seikagaku Kogyo). The column was washed with 0.1M 
phosphate-buffered saline (pH 7.5) and then stored at 4.degree. C. 
(2) Plasma Sample Pre-Treatment Using the Immobilized Antibody Column 
After washing the immobilized antibody column with 5 ml of a 0.1M 
phosphate-buffered saline (pH 7.5) containing 0.05% Triton X-100, a plasma 
sample, whether or not supplemented with various concentrations of a 
standard preparation of 6,15-diketo-13,14-dihydro-PGF.sub.1.alpha., was 
applied to the column. After the column was washed with 5 ml of a 0.1M 
phosphate-buffered saline (pH 7.5) containing 0.05% Triton X-100, 15 ml of 
purified water and 35 ml of a mixture of methanol and purified water 
(3:7), elution was conducted with 5 ml of 95% methanol. The resulting 
eluate was evaporated to dryness under nitrogen gas, after which it was 
dissolved in 0.5 ml of 0.1M phosphate-buffered saline to yield an enzyme 
immunoassay sample. 
FIG. 2 shows the validity of the present assay method based on plasma 
sample pre-treatment using the immobilized antibody column. As seen in 
FIG. 2, the experimentally found value increased 1, 2, 3 and 4 fold as the 
amount of plasma sample applied to the column was increased 1, 2, 3 and 4 
fold, indicating a good linearity in simple regression analysis (FIG. 
2(A)). A good linearity was also obtained in the experiment with plasma 
samples supplemented with the standard preparation, a recovery rate of 
over 90% on average obtained (FIG. 2(B)). 
The following references, which are refered to for their disclosures at 
various points in this application, are incorporated herein by reference. 
1. ALAN R. BRASH et al., The Journal of Pharmacology and Experimental 
Therapeutics, Vol. 226, p.78 (1983) 
2. FitzGeld, G. A. et al., Circulation, 67, 1174-1177 (1983) 
3. Carlo Patrono et al., Advances in Prostaglandin, Thromboxane, and 
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