Antibody reagent for detecting dissecting aortic aneurysm and uses thereof

By measuring smooth muscle myosin heavy chain in the blood of a patient using an antibody to the smooth muscle myosin heavy chain, dissecting aortic aneurysm can be diagnosed very easily and rapidly without any special equipment.

BACKGROUND OF THE INVENTION 
1. Field 
The present invention relates to an antibody reagent for detecting 
dissecting aortic aneurysm (recently also called aortic dissection) which 
comprises an antibody to a smooth muscle myosin heavy chain, and use of 
the antibody reagent. 
2. Description of Related Art 
Dissecting aortic aneurysm is a disease with severe chest pains and is 
caused by disruption of the aortic media by blood entering through a 
laceration of the luminal vascular wall. Dissecting aortic aneurysm is 
caused in aorta in most cases but is caused also in branches in some 
cases. As causes for the disease, there are suggested not only the 
degeneration and weakening of the intima (e.g. cystic medionecrosis and 
arterio-sclerosis) but also the extension of aorta, hypertension, etc. 
A typical example of disease with chest pains is acute myocardial 
infarction. Acute myocardial infarction may be diagnosed without much 
difficulty through electrocardiograph change or biochemical blood testing. 
By contrast, in the case of dissecting aortic aneurysm, a specific change 
is hardly observed in an electrocardiograph or blood testing in spite of 
the high lethality of this disease. Therefore, diagnosis of this disease 
requires extreme care. 
As a method for diagnosis of dissecting aortic aneurysm, echo examination, 
CT (X-rays computed tomography), DSA (digital subtraction angiography), 
MRI (magnetic resonance imaging), etc. have been attempted and have 
produced rather good results (Common Disease Series 4: Angina 
Pectoris.multidot.Myocardial Infarction, pp. 310-313, Nankodo K. K.). All 
of these methods, however, require special equipment, and hence are not 
always satisfactory as a method for use in an urgent examination wherein 
the method is required to be always able to be carried out anywhere. 
Accordingly, the present invention is mainly intended to provide a method 
for detecting dissecting aortic aneurysm which is applicable to an urgent 
examination, and a reagent for use in the method. 
SUMMARY OF THE INVENTION 
The present inventors conducted detailed researches with the expectation 
that, in the cases of a disease associated with blood vessel disturbance 
such as dissecting aortic aneurysm, a substance constituting the blood 
vessel is released into blood. Consequently, the present inventors 
concluded that the blood vessel disturbance can be diagnosed by detecting 
smooth muscle myosin heavy chain which is a main protein constituting the 
blood vessel. 
Since smooth muscle myosin heavy chain is biochemically different from 
skeletal muscle myosin heavy chain, cardiac muscle myosin heavy chain and 
non-muscle myosin heavy chain, it is relatively easy to obtain an antibody 
specific for smooth muscle myosin heavy chain. Therefore, an antibody to 
smooth muscle myosin heavy chain was prepared, whereby there has been 
established an assay system which enables detection of smooth muscle 
myosin heavy chain in blood. The smooth muscle myosin heavy chain levels 
in the sera of healthy individuals were measured by this assay system and 
found to be low. On the other hand, as a result of measurement in the sera 
of patients with dissecting aortic aneurysm, there were observed 
significantly higher smooth muscle myosin heavy chain levels which clearly 
reflect the clinical symptoms of the patients. Thus, it was confirmed that 
measuring smooth muscle myosin heavy chain in blood is useful for 
detecting dissecting aortic aneurysm, whereby the present invention has 
been accomplished. 
Accordingly, the present invention relates to an antibody reagent for 
detecting dissecting aortic aneurysm which comprises an antibody to smooth 
muscle myosin heavy chain. 
The present invention also relates to a kit for detecting dissecting aortic 
aneurysm which comprises an antibody reagent to smooth muscle myosin heavy 
chain and a washing solution containing a surfactant. 
The present invention further relates to a method for detecting dissecting 
aortic aneurysm which comprises measuring smooth muscle myosin heavy chain 
in a sample, and detecting dissecting aortic aneurysm on the basis of the 
value obtained. 
The present invention still further relates to use of an antibody to smooth 
muscle myosin heavy chain for detecting dissecting aortic aneurysm.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
(1) Antibody reagent 
The antibody reagent of the present invention is used in a method for 
detecting dissecting aortic aneurysm which comprises measuring smooth 
muscle myosin heavy chain in a test sample according to immunoassay, and 
detecting dissecting aortic aneurysm on the basis of the value obtained. 
Therefore, the antibody reagent of the present invention comprises at 
least an antibody specific for smooth muscle myosin heavy chain. 
The antibody used in the antibody reagent of the present invention may be 
either polyclonal or monoclonal so long as it binds specifically to smooth 
muscle myosin heavy chain. The antibody is not particularly limited and 
may be a well-known one. In particular, an antibody having a low 
cross-reactivity with other myosins is preferably used. Specifically, 
smooth muscle myosin heavy chain in a sample can be specifically measured 
by the use of such a monoclonal antibody having less than 3% 
cross-reactivity with other myosins, preferably less than 1%, as is 
described in Examples described hereinafter. 
Such a monoclonal antibody can be easily prepared by properly applying 
conventional methods as described in Examples described hereinafter see, 
for example, Japanese Biochemical Association, "Men-eki Seikagaku Kenkyuho 
(Zoku Seikagaku Jikken Koza 5)", pp. 1-88 (1986); Biochemistry, 27, 
3807-3811 (1988); Eur. J. Biochem., 179, 79-85 (1989); J. Mol. Biol., 198, 
143-157 (1987); J. Biol. Chem., 264, 9734-9737 (1989); J. Biol. Chem., 
264, 18272-18275 (1989); J. Biol. Chem., 266, 3768-3773 (1991); 
Circulation, 88, 1804-1810 (1993)!. 
As the antibody used in the antibody reagent of the present invention, an 
antibody itself may be used. Preferably, an active antibody fragment 
thereof is used, because the fragment can prevent non-specific adsorptions 
thereto. As the active antibody fragment, there may be used any of various 
active antibody fragments retaining characteristics of the antibody, such 
as F(ab')2, Fab' and Fab. These active fragments may be prepared according 
to conventional methods including one comprising limited degradation of a 
purified antibody with a protease such as papain, pepsin and trypsin see, 
for example, Japanese Biochemical Association, "Men-eki Seikagaku Kenkyuho 
(Zoku Seikagaku Jikken Koza 5)", p. 89 (1986)!. 
As the antibody reagent, a dissolved antibody or a freeze-dried antibody 
may be used. If necessary, a modified antibody in a form suitable for a 
measurement system (e.g. an immobilized anitibody or a labeled antibody) 
may be used as the antibody reagent. 
The antibody modification may be carried out according to conventional 
methods. In more detail, as a material for a carrier for preparing the 
immobilized antibody, there may be exemplified synthetic organic 
high-molecular weight compounds such as poly(vinyl chloride)s, 
polystyrenes, styrene-divinylbenzene copolymers, styrene-maleic anhydride 
copolymers, nylons, poly(vinyl alcohol)s, polyacrylamides, 
polyacrylonitriles, polypropylenes, and poly(methylene methacrylate)s 
polysaccarides such as dextran derivatives (e.g. Sephadex), agarose gels 
(e.g. Sepharose and Bio-Gel), and celluloses (e.g. paper disc and filter 
paper); and inorganic high-molecular weight compounds such as glass, 
silica gel, and silicones. These materials may have one or more functional 
groups (e.g. amino group, aminoalkyl group, carboxyl group, acyl group and 
hydroxyl group) introduced thereinto. The material for the carrier 
preferably has a marked protein-binding ability. 
As to the shape of the carrier, there may be exemplified flat plate-like 
carriers (e.g. microtiter plates and discs), granular carriers (e.g. 
beads), tubular carriers (e.g. test tubes), fibrous, filmy or particulate 
carriers (e.g. latex particles), capsular carriers and vesicular carriers. 
A carrier having a shape suitable for a measurement method may be 
selected. Liposomes (single-layer or multilayer lipid films) and the like 
may also be used as the carrier for immobilizing the antibody thereon. 
For binding the antibody to the carrier, there may be employed conventional 
methods such as physical adsorption method, ionic binding method, covalent 
binding method, and entrapping method see, for example, Ichiro Chihata 
"Koteika Koso", Kodansha K. K. (Mar. 20, 1975)!. In particular, the 
physical adsorption method is preferable because of its simplicity. The 
antibody may be bound to the carrier either directly or through another 
substance between them. 
As a labeling agent for preparing the labeled antibody, there may be used, 
for example, radioisotopes (e.g. 32P, 3H, 14C and 125I), enzymes (e.g. 
.beta.-galactosidase, peroxidase, alkaline phosphatase, 
glucose-6-phosphate dehydrogenase, catalase, glucose oxidase, lactate 
oxidase, alcohol oxidase and monoamine oxidase), 
coenzymes.multidot.prosthetic groups (e.g. FAD, FMN, ATP, biotin and 
heme), fluorescein derivatives (e.g. fluorescein isothiocyanate and 
fluorescein thiofulvamyl), Rhodamine derivatives (e.g. tetramethyl 
Rhodamine B isothiocyanate), fluorescent dyes such as umbelliferone and 
1-anilino-8-naphthalenesulfonic acid, luminol derivatives (e.g. luminol, 
isoluminol and N-(6-aminohexyl)-N-ethylisoluminol), and colloidal metal 
particles of gold, silver, platinum, a compound or any of these metals, or 
the like. 
A method for binding the labeling agent to the antibody may be properly 
selected from conventional methods as described in text books e.g. "Zoku 
Seikagaku Jikken Koza 5, Men-eki Seikagaku Kenkyuho", Tokyo Kagaku Dojin 
K. K., pp. 102-112 (1986)!. 
(2) Detection kit 
The detection kit of the present invention is used in a method for 
detecting dissecting aortic aneurysm which comprises measuring smooth 
muscle myosin heavy chain in a test sample according to immunoassay, and 
detecting dissecting aortic aneurysm on the basis of the value obtained. 
Therefore, the kit is characterized by comprising at least the 
above-mentioned antibody reagent of the present invention as a constituent 
reagent and a surfactant-containing washing solution added thereto as 
another constituent reagent. 
As the antibody reagent in the detection kit of the present invention, an 
antibody reagent having a shape suitable for an immunoassay method adopted 
in the kit (e.g. an immobilized antibody or a labeled antibody) may be 
properly selected from the above-exemplified antibody reagents and 
incorporated into the kit. 
The surfactant contained in the washing solution is not particularly 
limited so long as it is water-soluble. In particular, an amphoteric 
surfactant or a nonionic surfactant is preferable. A specific example of 
the amphoteric surfactant is egg yolk lysolecithin. Specific examples of 
nonionic surfactant are Tween series surfactants (e.g. Tween 20, Tween 40, 
Tween 60, Tween 80 and Tween 85), Span series surfactants (e.g. Span 20, 
Span 80, Span 85 and Span 80), Brij series surfactants (e.g. Brij 35 and 
Brij 58), and (n) p-t-octyl phenyl ether series surfactants (e.g. Triton 
CF-10, Triton N-101, Triton X-100, Triton X-114, Triton X-305, Triton 
X-405 and Nonidet P-40). The amount of the surfactant added is suitably 
0.003% (w/v) or more. 
The washing solution containing such a surfactant is incorporated into the 
detection kit of the present invention and used in the assay, whereby 
smooth muscle myosin heavy chain in a sample can be measured with higher 
sensitivity. 
As reagents other than those described above, reagents suitable for a 
measurement system may be properly selected from those usually used in the 
assay method (e.g. standard antigen solutions, an enzyme solution, 
substrate solution, reaction-stopping solution, and diluent for sample) 
and may be incorporated into the detection kit. 
The assay method adopted in the detection kit of the present invention is 
not particularly limited so long as it is a conventional method adopted in 
immunoassay. There may be adopted any assay method such as competitive 
method, sandwich method, agglutination method, blot overlay method, 
immunochromatograph method and the like. 
The details of the assay method adopted is referred to, for example, the 
following articles: 
(a) Hiroshi Irie "Radioimmunoassay, second series", Kodansha K. K. (May 1, 
1979); 
(b) Eiji Ishikawa, et al. "Koso Men-eki Sokuteiho" 2nd. ed., IGAKU SHOIN 
Ltd. (Dec. 15, 1982); 
(c) "Immunoassay for Clinical Examinations--Techniques and Application", 
Rinsho Byori, extra issue, special edition No. 53, Rinsho Byori Kankokai 
(1983); 
(d) "Cyclopedia of Biotechnology", CMC K. K. (Oct. 9, 1986); 
(e) JP-B 6-43998, JP-A 55-15100, JP-B 7-60159 and JP-A 63-25553; 
(f) "Methods in ENZYMOLOGY Vol. 70" (Immunochemical techniques (Part A)); 
(g) "Methods in ENZYMOLOGY Vol. 73" (Immunochemical techniques (Part B)); 
(h) "Methods in ENZYMOLOGY Vol. 74" (Immunochemical techniques (Part C)); 
(i) "Methods in ENZYMOLOGY Vol. 84" (Immunochemical techniques (Part D: 
Selected Immunoassay)); and 
(j) "Methods in ENZYMOLOGY Vol. 92" (Immunochemical techniques (Part E: 
Monoclonal Antibodies and General Immunoassay Methods)). 
(the articles (f) to (j) are published by Academic Press). 
The detection kit of the present invention is further explained below by 
taking the case of sandwich method which is a suitable method among assay 
methods. As the kit, the following kit may be exemplified. 
Kit A: 
1 immobilized first antibody; 
2 second antibody; 
3 labeled anti-immunoglobulin antibody; 
4 antigen of a known concentration; and 
5 washing solution (containing a surfactant). 
Labeled second antibody may be used in place of both the second antibody 
and the labeled anti-immunoglobulin antibody of kit A. As such a kit, the 
following kit B is exemplified. 
Kit B: 
1 immobilized first antibody; 
2 labeled second antibody; 
3 antigen of a known concentration; and 
4 washing solution (containing a surfactant). 
A kit comprising such constituent reagents is suitable for, for example, 
rapid assay for obtaining measurement results within one hour. 
In addition, when biotin-avidin method is employed, the following kit C is 
exemplified. 
Kit C: 
1 immobilized first antibody; 
2 biotinylated second antibody; 
3 labeled avidin; 
4 antigen of a known concentration; and 
5 washing solution (containing a surfactant). 
In the above-mentioned kits, needless to say, the term "antibody" means an 
antibody to smooth muscle myosin heavy chain, and the term "antigen" means 
smooth muscle myosin heavy chain. The first antibody and the second 
antibody may be capable of recognizing either the same antigenic 
determinant or different antigenic determinant on smooth muscle myosin 
heavy chain. 
A method for measuring smooth muscle myosin heavy chain in a test sample 
using any of the above-mentioned detection kits is not different at all 
from those adopted in the case of using other conventional detection kits 
in which sandwich method is employed. In detail, an immobilized antibody 
reagent is reacted with the test sample, and B/F separation is carried out 
if necessary, after which a labeled antibody reagent is reacted with the 
reaction product (two-step method). Alternatively, an immobilized antibody 
reagent, the test sample and a labeled antibody reagent are reacted at the 
same time (one-step method). In either case, after the reaction(s), smooth 
muscle myosin heavy chain in the sample may be detected or quantitated by 
a conventional method suitable for a labeling agent used for preparing the 
labeled antibody. 
(3) Detection method 
The detection method of the present invention comprises measuring smooth 
muscle myosin heavy chain in a test sample, and detecting dissecting 
aortic aneurysm on the basis of the value obtained. 
The test sample used for the measurement is blood collected (with the lapse 
of time) from a patient suspected of suffering from dissecting aortic 
aneurysm, namely, a patient complaining of chest pain, or a fraction 
obtained by fractionating the blood (e.g. serum). If necessary, the test 
sample may be used in the measurement after having been diluted with a 
suitable buffer solution such as PBS. 
It is sufficient that smooth muscle myosin heavy chain in a blood sample is 
detected or quantitated using the above-mentioned antibody reagent or 
detection kit of the present invention. 
When it is revealed as a result of the measurement that the smooth muscle 
myosin heavy chain level in the blood sample is significantly higher than 
the smooth muscle myosin heavy chain average level in blood from normal 
individuals, it is diagnosed that the patient from whom the blood sample 
has been collected may be suffering from dissecting aortic aneurysm with a 
very high probability, and the patient should be subjected to more 
detailed examinations. When the smooth muscle myosin heavy chain level in 
the blood sample is not statistically different from that of blood from 
normal individuals, it is diagnosed that the patient from whom the blood 
sample has been collected is highly likely to be free from dissecting 
aortic aneurysm, and the patient should be subjected to reexaminations 
from a different viewpoint. 
EXAMPLES 
The present invention is specifically illustrated with the following 
examples, which should not be construed as limiting the scope of the 
invention. 
Example 1 
Preparation of Various Kinds of Myosins 
Human uterus smooth muscle myosin, human aorta smooth muscle myosin, human 
platelet myosin and human skeletal muscle myosin were provided by Dr. 
Matsumura (Saga Medical School). Human cardiac muscle myosin was purified 
according to the method of Yazaki (Circ. Res., 36:208, 1975). The purity 
of these myosins was assessed by SDS-PAGE, after which the protein were 
quantatively determined according to the method of Lowry (J. Biol. Chem., 
193:265-275, 1951) with bovine serum albumin as a standard. All the 
myosins were composed of heavy chain and light chain. 
Example 2 
Preparation of Monoclonal Antibodies 
1) Preparation of monoclonal antibody-producing hybridomas 
BALB/c mice aged 6 to 8 weeks were immunized intraperitoneally with 25 to 
50 .mu.g of human uterus smooth muscle myosin emulsified with complete 
Freund's adjuvant, 4 to 7 times at 2- to 4-week interval. After the final 
immunization, the mouse with highest titer was given intravenously 10 
.mu.g of human uterus smooth muscle myosin in saline. 
Three days after the final immunization, the spleen was removed from this 
mouse, and splenocytes in the spleen were mixed with mouse myeloma cells 
P3-X63-Ag8-U1 (P3U1) (ATCC CRL-1597) in the ratio of 10:1. The resulting 
mixture was centrifuged to obtain pellets, to which 1 ml of a RPMI1640 
solution containing 50% polyethylene glycol was slowly added to carry out 
cell fusion. Thereto was further added RPMI1640 medium to make a total 
volume of 10 ml, followed by centrifugation. The pellets thus obtained 
were resuspended in RPMI1640 medium containing 10% fetal calf serum (FCS) 
so that the cell density of P3U1 might be 3.times.10.sup.4 cells/0.1 ml, 
and the suspension was dispensed in 0.1 ml alquots into a 96-well 
microtiter plate. 
After 24 hours, 0.1 ml of HAT medium was added to each well, after which 
one-half of the medium in each well was replaced by fresh HAT medium every 
3 to 4 days. 
On the 7 to 10th day after the cell fusion, the culture supernatant was 
sampled and dispensed in 50 .mu.l aliquots into a 96-well polyvinyl 
chloride (PVC) plate previously coated with human uterus smooth muscle 
myosin and blocked with 3% gelatin, and was incubated at room temperature 
for 1 hour. After washing the plate three times with PBS, a solution 
prepared by diluting biotinylated horse anti-mouse IgG (Vector 
Laboratories) in 500-fold with PBS containing 1% bovine serum albumin 
(BSA) was dispensed in 50 .mu.l aliquots into each well in the plate and 
allowed to stand at room temperature for 1 hour. After washing three times 
with PBS, a solution prepared by diluting peroxidase-avidin D (Vector 
Laboratories) in 2000-fold with PBS containing 1% BSA was dispensed in 50 
.mu.l aliquots into each well in the plate and allowed to stand at room 
temperature for 15 minutes. After washing three times with PBS, 200 .mu.l 
of a substrate solution (containing 0.25 mg/ml of 4-aminoantipyrine, 0.25 
mg/ml of phenol and 0.4 M of hydrogen peroxide) was added, and the 
reaction solution was subjected to coloration at room temperature. 
Absorbance at 550 nm was measured by means of a microplate photometer, and 
on the basis of the value obtained, hybridomas producing a monoclonal 
antibody which is reactive specifically with human uterus smooth muscle 
myosin were selected. 
The hybridoma cells thus selected were cloned by limiting dilution to 
establish 5 clones of hybridoma (1H6, 4E12, 9A12, 9D7 and 10G2) capable of 
producing an antibody to human uterus smooth muscle myosin. Of these, 
hybridoma 1H6 and hybridoma 4E12 were deposited as SMHMW1H6 and SMHMW4E12 
under the Budapest Treaty, respectively, in Bioengineering Research 
Institute, Agency of Industrial Science and Technology, Ministry of 
International Trade and Industry, and given Accession Numbers FERM BP-4829 
and FERM BP-4830, respectively, under the date of Oct. 13, 1994. 
In the above experiment, the proportion of the desired monoclonal 
antibody-producing hybridomas obtained relative to the number of wells is 
as shown in Table 1. 
TABLE 1 
______________________________________ 
Proportion of monoclonal antibody-producing hybridomas 
Number of wells 
Number of wells 
positive to containing Total number 
specific antibody 
proliferated cells 
of wells 
______________________________________ 
11 653 940 
______________________________________ 
2) Preparation and purification of monoclonal antibodies 
Next, cells of each clone established were cultured and then 
intraperitoneally administered to mice previously given pristane, in a 
number of 3.times.10.sup.6 per mouse. After about 2 weeks, 5 ml of ascitic 
fluid was collected from each mouse. 
A mixture of the ascitic fluid and an equal volume of 1.5 M glycine-HCl 
buffer (pH 8.9) containing 3 M sodium chloride was passed through a 
Protein A Sepharose CL-4B column (Pharmacia Fine Chemicals) which had been 
equilibrated with the same glycine-HCl buffer as above. After washing the 
column with a sufficient volume of the same glycine-HCl buffer as above, 
antibody was eluted with 0.1 M citrate buffer (pH 6.0). The eluate was 
dialyzed against PBS and the purity of the antibody was confirmed 
according to SDS-polyacrylamide gel electrophoresis (SDS-PAGE), whereby a 
purified monoclonal antibody was obtained. 
Example 3 
Properties of Monoclonal Antibodies 
1) Isotype 
A culture supernatant of each hybridoma was added to a 96-well PVC plate 
previously coated with human uterus smooth muscle myosin and blocked with 
3% gelatin, and the antibody was screened for isotype using a MonoAb-ID 
EIA kit (Zymed Laboratories). 
The results are as shown in Table 2. 
TABLE 2 
______________________________________ 
Isotypes of monoclonal antibodies 
Hybridoma 
1H6 4E12 9A12 9D7 10G2 
______________________________________ 
Isotype IgGl/.kappa. 
IgGl/.kappa. 
IgGl/.kappa. 
IgGl/.kappa. 
IgGl/.kappa. 
______________________________________ 
2) Analysis for specificity by Western blotting 
Each monoclonal antibody was analyzed for specificity according to Western 
blotting. 
Human uterus smooth muscle myosin (1 mg/ml) was heat-treated at 100.degree. 
C. for 5 minutes together with an equal amount of a reducing solution. The 
thus treated protein was subjected to SDS-PAGE by means of a mini-gel 
electrophoresis apparatus (Marysol K. K.) at 10 mV for about 3 hours using 
a 10% separating gel and a 5% stacking gel. Blotting was carried out as 
follows. The protein was transferred to a nitrocellulose membrane by means 
of a blotting apparatus for mini-gel (Marysol K. K.) by electrical current 
supply at 37 V for about 18 hours. The nitrocellulose membrane was cut 
into strips along the migration lines and some of them were subjected to 
protein staining with Amid black. The other strips were blocked with 3% 
gelatin and subjected to reaction with a culture supernatant of each 
hybridoma at room temperature for 1 hour. 
These strips were washed twice with 20 mM Tris-500 mM NaCl buffer (pH 7.5) 
containing 0.05% Tween 20 (T-TBS) for 10 minutes, and then subjected to 
reaction with a 1/500 dilution of biotinylated horse anti-mouse IgG 
(Vector Laboratories) at room temperature for 1 hour. Thereafter, the 
strips were washed twice with T-TBS for 10 minutes and then subjected to 
reaction with a 1/2000 dilution of peroxidase-avidin D (Vector 
Laboratories) at room temperature for 15 minutes. Subsequently, the strips 
were washed twice with T-TBS for 10 minutes, subjected to coloration with 
a color development solution containing 30 mg of HRP color development 
reagent (Bio-Rad Laboratories), 10 ml of methanol, 50 ml of TBS and 30 
.mu.l of a 30% aqueous hydrogen peroxide solution, and then washed with 
distilled water. 
As a result of the protein staining with Amid black, there were observed 
five bands of 200 K (uterus smooth muscle myosin heavy chain), 140 K (a 
fragment of uterus smooth muscle myosin heavy chain), 70 K (a fragment of 
uterus smooth muscle myosin heavy chain), 20 K (uterus smooth muscle 
myosin light chain) and 17 K (uterus smooth muscle myosin light chain). 
According to Western blotting, it was confirmed that all the monoclonal 
antibodies react with human uterus smooth muscle myosin heavy chain, but 
that none of them react with human uterus smooth muscle myosin light 
chains. 
Example 4 
Detection Kit in Sandwich Method 
1) Preparation of biotinylated antibodies 
Each of the above-mentioned monoclonal antibodies was dialyzed against a 
0.1 M sodium hydrogencarbonate solution, and the dialyzed solution was 
concentrated to 2 mg/ml with Centriflow (Amicon). Biotin (long arm) NHS 
reagent (Vector Laboratories) was dissolved in dimethylformamide to a 
concentration of 10 mg/ml, after which 20 .mu.l of the dilution was mixed 
with 1 ml of the above-mentioned antibody solution, and the reaction was 
carried out at room temperature for 2 hours. The reaction was terminated 
with 5 .mu.l of ethanolamine, and the reaction solution was dialyzed twice 
against PBS to obtain a biotinylated antibody. The biotinylated antibody 
was diluted to 1 .mu.g/ml with PBS containing 1% BSA, to obtain a 
biotinylated antibody solution. 
2) Preparation of immobilized antibody 
The anti-(smooth muscle myosin heavy chain) monoclonal antibody (4E12) was 
diluted to 10 .mu.g/ml with PBS, and the dilution was dispensed in 50 
.mu.l aliquots into a 96-well plate (H type, Sumitomo Bakelite Co., Ltd.) 
and allowed to stand overnight at 4.degree. C. The plate was washed three 
times with PBS containing 0.05% Tween 20, after which 0.5% skim milk was 
dispensed thereinto in 300 .mu.l aliquots, and the plate was allowed to 
stand at room temperature for 1 hour. The skim milk solution was removed 
to obtain an immobilized antibody reagent. 
3) Preparation of other reagents and a kit 
Smooth muscle myosin heavy chain standard solutions 
The standard solutions were prepared by diluting human aorta smooth muscle 
myosin to a concentration of 25, 12.5, 6.25, 3.125, 1.563, 0.781 or 0.391 
ng/ml in terms of the heavy chain with PBS containing 1% BSA. 
Washing solution 
The solution was prepared by dissolving Tween 20 in PBS to a concentration 
of 0.05% (w/v). 
Enzyme-labeled avidin solution 
The avidin solution was prepared by diluting peroxidase-labeled avidin D 
(A-2004, Vector Laboratories) in 5,000-fold with PBS containing 1% BSA. 
Substrate solution 
The solution was prepared by dissolving 3,3',5,5'-tetramethylbenzidine 
dihydrochloride (TMBZ) and hydrogen peroxide in 0.2 M citrate buffer (pH 
3.8) to concentrations of 0.3 mM and 0.005%, respectively. 
Enzyme reaction stopping solution 
1N sulfuric acid was used. 
The above-mentioned reagents were combined into a kit, whereby the 
detection kit of the present invention was prepared. 
Example 5 
1) Calibration curve 
To each well containing the immobilized antibody reagent (4E12) was fed 100 
.mu.l of PBS containing 1% BSA and then 50 .mu.l of each smooth muscle 
myosin heavy chain standard solution, and stirred, followed by standing at 
room temperature for 4 hours. Each well was washed three times with the 
washing solution, fed with 50 .mu.l of the biotinylated antibody (1H6) 
solution, and then allowed to stand at room temperature for 30 minutes. 
Thereafter, each well was washed three times with the washing solution, 
fed with 50 .mu.l of the enzyme-labeled avidin solution, and then allowed 
to stand at room temperature for 15 minutes. Subsequently, each well was 
washed three times with the washing solution, fed with 100 .mu.l of the 
substrate solution, and then allowed to stand at room temperature for 10 
minutes, whereby a color was developed. The reaction was stopped by adding 
100 .mu.l of the enzyme reaction stopping solution to each well, and 
absorbance at 450 nm was measured with a microplate photometer. The 
calibration curve thus obtained is shown in FIG. 1. 
2) Effect of surfactants 
As a result of investigating the concentration of Tween 20 in the washing 
solution in the procedure described in 1) above, it was proved that the 
measuring sensitivity can be markedly enhanced at a concentration of 
0.003% (w/v) or more. 
Effect of various surfactants other than Tween 20 was compared with that of 
Tween 20. In detail, washing solutions were prepared by dissolving each of 
the various surfactants in PBS to a concentration of 0.05% (w/v), and by 
the use of each washing solution, calibration curves were obtained in the 
same manner as above. As a result, it was found that as shown in FIGS. 3 
to 7, the presence of any of amphoteric surfactants and nonionic 
surfactants in the washing solutions brings about a marked effect as in 
the case of Tween 20. 
3) Reproducibility 
Using three samples (A, B and C), within-run reproducibility and 
between-day reproducibility were investigated according to the procedure 
as described in 1) above. As a result, it was found that as shown in FIGS. 
3 and 4, CV values less than 10% are obtained in the case of both 
reproducibilities, namely, it was proved that the assay shows good 
reproducibility. 
TABLE 3 
______________________________________ 
Within-run reproducibility 
Measured 
value Mean CV 
ng/ml ng/ml SD % 
______________________________________ 
A 1 1.4 1.3 0.1 6.5 
2 1.3 
3 1.2 
4 1.3 
5 1.2 
B 1 4.9 5.2 0.2 4.6 
2 5.0 
3 5.1 
4 5.5 
5 5.3 
C 1 16.0 15.9 0.2 1.1 
2 15.8 
3 16.2 
4 15.8 
5 15.8 
______________________________________ 
TABLE 4 
______________________________________ 
Between-day Reproducibility 
Measured 
value Mean CV 
ng/ml ng/ml SD % 
______________________________________ 
A 1 1.1 1.1 0.1 4.9 
2 1.1 
3 1.2 
4 1.2 
5 1.1 
B 1 5.0 5.2 0.2 4.6 
2 5.1 
3 5.6 
4 5.3 
5 5.0 
C 1 14.0 15.8 1.1 6.9 
2 15.9 
3 16.8 
4 15.5 
5 16.6 
______________________________________ 
4) Dilution linearity 
According to the procedure as described in 1) above, the sera of three 
patients (M11, M25 and M32) were diluted with the standard solution (0 
ng/ml), and the dilution linearity was investigated to reveal that as 
shown in FIG. 8, lines obtained in all the cases are linear those starting 
from the zero point. Thus, it was made clear that good dilution linearity 
can be attained. 
5) Addition and recovery 
According to the procedure as described in 1) above, the standard solutions 
were added to the sera of patients (samples M14 and M24), and the recovery 
of smooth muscle myosin heavy chain was investigated to reveal that as 
shown in Table 5, smooth muscle myosin heavy chain can be recovered in an 
amount which is substantially the same as the adding amount. 
TABLE 5 
______________________________________ 
Addition-and-recovery test 
Added Observed Recovery 
Recovery 
Sample (ng/ml) (ng/ml) (ng/ml) 
(%) 
______________________________________ 
M14 0.0 2.4 
1.3 3.5 1.1 89.8 
5.0 7.2 4.8 95.7 
20.0 18.7 16.3 81.5 
M24 0.0 1.8 
1.3 3.0 1.2 96.3 
5.0 7.0 5.3 105.3 
20.0 18.6 16.9 84.3 
______________________________________ 
6) Cross-reactivity 
Cross-reactivity with various myosins was investigated according to the 
procedure as described in 1) above. As a result, it was confirmed that as 
shown in FIG. 9, the monoclonal antibodies react with uterus smooth muscle 
myosin similarly with aorta smooth muscle myosin but hardly react with any 
of skeletal muscle myosin, cardiac muscle myosin and platelet myosin 
(non-muscle myosin). 
7) Measurement in sera of healthy individuals 
The smooth muscle myosin heavy chain levels in the sera of 75 normal 
individuals were measured according to the procedure as described in 1) 
above to reveal that the mean thereof was 0.9 ng/ml and the standard 
deviation 0.9 ng/ml. 
8) Measurement in sera of patients 
According to the procedure as described in 1) above, there was measured the 
smooth muscle myosin heavy chain level in each of serum samples collected 
from two patients with dissecting aortic aneurysm with the lapse of time 
from the onset of the disease. As a result, marked elevation of the smooth 
muscle myosin heavy chain level was confirmed at the onset of the disease 
as shown in FIGS. 10 and 11, indicating that the measurement of smooth 
muscle myosin heavy chain is useful for detection or diagnosis of 
dissecting aortic aneurysm. In this case, confirmative diagnosis of 
dissecting aortic aneurysm was conducted by operation. 
INDUSTRIAL AVAILABILITY 
The present inventors found for the first time that dissecting aortic 
aneurysm can be detected by measuring smooth muscle myosin heavy chain in 
a test sample. Moreover, since immunoassay is employed as a detection 
method in the present invention, the method of the present invention does 
not require any special equipment and is applicable to urgent examination 
for dissecting aortic aneurysm, unlike conventional methods. 
In addition, the antibody reagent and detection kit of the present 
invention are absolutely necessary for practicing the detection method of 
the present invention. Furthermore, when there are used at least one 
monoclonal antibody and a washing solution containing a specific 
surfactant, smooth muscle myosin heavy chain in a test sample can be 
detected with higher sensitivity, so that the detection method of the 
present invention is applicable to not only the urgent examination but 
also progress observation during and after treatment.