U.S. patent application number 12/349928 was filed with the patent office on 2011-10-27 for methods for detection or measurement of viruses.
Invention is credited to Katsumi Aoyagi, Kumiko Iida, Tatsuji Kimura, Chiharu Ohue, Shintaro Yagi.
Application Number | 20110262892 12/349928 |
Document ID | / |
Family ID | 27329006 |
Filed Date | 2011-10-27 |
United States Patent
Application |
20110262892 |
Kind Code |
A1 |
Aoyagi; Katsumi ; et
al. |
October 27, 2011 |
METHODS FOR DETECTION OR MEASUREMENT OF VIRUSES
Abstract
A method for treating a virus-containing sample, characterized
by treatment of a virus-containing sample with a treatment solution
containing (1) an anionic surfactant and (2) an amphoteric
surfactant, nonionic surfactant or protein denaturant; a virus
assay method using said treating method; a method for treating a
virus-containing sample, characterized by treatment of a
virus-containing sample with a treatment solution containing (1) a
chaotropic ion and (2) an acidifying agent; a virus assay method
using said treating method; a virus assay method, characterized in
that a virus antigen and a virus antibody are measured based on
their binding to their probe in the presence of a surfactant with
an alkyl group of 10 or more carbon atoms and a secondary, tertiary
or quaternary amine, or a nonionic surfactant, or of both of them;
and a monoclonal antibody and a hybridoma producing the same for
carrying out said method.
Inventors: |
Aoyagi; Katsumi; (Iruma-gun,
JP) ; Ohue; Chiharu; (Iruma-gun, JP) ; Iida;
Kumiko; (Iruma-gun, JP) ; Kimura; Tatsuji;
(Iruma-gun, JP) ; Yagi; Shintaro; (Iruma-gun,
JP) |
Family ID: |
27329006 |
Appl. No.: |
12/349928 |
Filed: |
January 7, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09269897 |
Apr 2, 1999 |
7776542 |
|
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PCT/JP98/03476 |
Aug 4, 1998 |
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12349928 |
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Current U.S.
Class: |
435/5 ;
530/387.9 |
Current CPC
Class: |
G01N 33/5767 20130101;
G01N 2333/02 20130101; G01N 33/56983 20130101; G01N 2333/18
20130101 |
Class at
Publication: |
435/5 ;
530/387.9 |
International
Class: |
C12Q 1/70 20060101
C12Q001/70; C07K 16/08 20060101 C07K016/08 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 4, 1997 |
JP |
9-209515 |
Aug 4, 1997 |
JP |
9-209522 |
Jul 31, 1998 |
JP |
10-218136 |
Claims
1. A method for a treating virus-containing sample, characterized
by treatment of a virus-containing sample with a treatment solution
containing (1) an anionic surfactant and (2) an amphoteric
surfactant, nonionic surfactant or protein denaturant.
2. A method for treating a virus-containing sample, characterized
by treatment of a virus-containing sample with a treatment solution
containing (1) an anionic surfactant, (2) an amphoteric surfactant
and (3) a nonionic surfactant or protein denaturant.
3. A method for treating a virus-containing sample, characterized
by treatment of a virus-containing sample with a treatment solution
containing (1) an anionic surfactant, (2) an amphoteric surfactant,
(3) nonionic surfactant and (4) a protein denaturant.
4. A method according to any one of claims 1 to 4, wherein said
treatment solution further contains urea, an imidazole
ring-containing compound or an indole ring-containing compound.
5. A method according to claim 4, wherein said imidazole
ring-containing compound is imidazole, histidine, imidazoleacrylic
acid, imidazolecarboxyaldehyde, imidazolecarboxamide,
imidazoledione, imidazoledithiocarboxylic acid,
imidazoledicarboxylic acid, imidazolemethanol, imidazolidinethione,
imidazolidone, histamine or imidazopyridine.
6. A method according to claim 4, wherein said indole
ring-containing compound is tryptophan, indoleacrylic acid, indole,
indoleacetic acid, indoleacetic hydrazide, methyl indoleacetate,
indolebutyric acid, indoleacetonitrile, indolecarbinol,
indolecarboxyaldehyde, indolecarboxylic acid, indoleethanol,
indolelactic acid, indolemethanol, indolepropionic acid,
indolepyruvic acid, indolyl methyl ketone, indomycin,
indoleacetone, indomethacin, indoprofen or indolamine.
7. A method for treating a virus-containing sample, characterized
by treatment of a virus-containing sample with a treatment solution
containing (1) a chaotropic ion and (2) an acidifying agent.
8. A method for treating a virus-containing sample, characterized
by treatment of a virus-containing sample with a treatment solution
containing (1) a chaotropic ion, (2) an acidifying agent and (3) a
nonionic surfactant.
9. A method according to any one of claims 1 to 8, wherein said
virus is a virus which forms virus particles having a structure
comprising a structural protein encapsulating genomic RNA or DNA
and a membrane protein or lipid membrane surrounding it.
10. A method according to claim 9, wherein said virus is hepatitis
C virus (HCV), hepatitis D virus, hepatitis E virus, hepatitis G
virus, hand-foot-and-mouth disease virus, a flavivirus (yellow
fever virus, West Nile virus, Japanese encephalitis virus, dengue
virus), a togavirus (alpha-virus, rubivirus, arterivirus, rubella
virus), a pestivirus (hog cholera virus, bovine diarrhea virus), a
paramyxovirus (parainfluenza virus 1, 2, 3, 4, canine distemper
virus, Newcastle disease virus, RS virus, rinderpest virus, simian
parainfluenza virus, measles virus, mumps virus), an orthomyxovirus
(human influenza virus, avian influenza virus, equine influenza
virus, swine influenza virus), a rhabdovirus (rabies virus,
vesicular stomatitis virus), a picornavirus (poliovirus, Coxsackie
virus, echovirus, bovine enterovirus, porcine enterovirus, simian
enterovirus, mouse encephalitis virus, human rhinovirus, bovine
rhinovirus, equine rhinovirus, foot and mouth disease virus,
hepatitis A virus), a coronavirus (human coronavirus, avian
infectious bronchitis virus, mouse hepatitis virus, porcine
transmissible gastroenteritis virus), an arenavirus (lymphocytic
choriomeningitis virus, lassa virus, Korean hemorrhagic fever
virus), a retrovirus (HTLV: human adult leukemia virus, HIV: AIDS
virus, feline leukemia sarcoma virus, bovine leukemia virus, Rous
sarcoma virus), a reovirus (rotavirus), a calcivirus (Norwalk
virus), a bunyavirus (renal syndrome hemorrhagic fever virus), a
phyllovirus (Ebola virus, Marburg virus), hepatitis B virus (HBV),
a pox virus (vaccinia virus, alastrim virus, cowpox virus, smallpox
virus), a parvovirus (human parvovirus, porcine parvovirus, bovine
parvovirus, canine parvovirus, feline leucopenia virus, Aleutian
mink disease virus), a papovavirus (papilloma virus, polyoma
virus), adenovirus, a herpes virus (herpes simplex virus,
cytomegalovirus, chickenpox herpes zoster virus, EB virus, equine
herpes virus, feline herpes virus, Marek's disease virus) or
African swine cholera virus.
11. A method according to any one of claims 1 to 10, wherein said
virus is hepatitis C virus (HCV) or hepatitis B virus (HBV).
12. A virus assay method, characterized by using a sample treating
method according to any one of claims 1 to 10 and reacting it with
a probe which specifically recognizes a virus antigen, for
detection or quantitation of the presence of the virus antigen.
13. A hybridoma cell line selected from the group consisting of
HC11-11 (FERM BP-6005), HC11-14 (FERM BP-6006), HC11-10 (FERM
BP-6004), HC11-3 (FERM BP-6002) and HC11-7 (FERM BP-6003).
14. A monoclonal antibody produced by a hybridoma selected from the
group consisting of HC11-11 (FERM BP-6005), HC11-14 (FERM BP-6006),
HC11-10 (FERM BP-6004), HC11-3 (FERM BP-6002) and HC11-7 (FERM
BP-6003).
15. A kit, assay kit or diagnostic reagent for determining the
presence or absence of a virus in a sample, which is for use in an
immunoassay method according to claim 12 and comprises an anionic
surfactant.
16. A kit, assay kit or diagnostic reagent for determining the
presence or absence of a virus in a sample, which is for use in an
immunoassay method according to claim 12 and comprises a monoclonal
antibody according to claim 14.
17. A kit, assay kit or diagnostic reagent for determining the
presence or absence of a virus in a sample, which is for use in an
immunoassay method according to claim 12 and comprises a chaotropic
agent.
18. A kit, assay kit or diagnostic reagent for determining the
presence or absence of HCV in a sample, which is for use in an
immunoassay method according to claim 12 and comprises a monoclonal
antibody produced by hybridoma HC11-14 (FERM BP-6006), HC11-10
(FERM BP-6004) or HC11-11 (FERM BP-6005).
19. A diagnostic kit according to any one of claims 15 to 17 which
further includes urea, an imidazole ring-containing compound or an
indole ring-containing compound.
20. A diagnostic kit according to claim 19, wherein said imidazole
ring-containing compound is imidazole, histidine, imidazoleacrylic
acid, imidazolecarboxyaldehyde, imidazolecarboxamide,
imidazoledione, imidazoledithiocarboxylic acid,
imidazoledicarboxylic acid, imidazolemethanol, imidazolidinethione,
imidazolidone, histamine or imidazopyridine.
21. A diagnostic kit according to claim 19, wherein said indole
ring-containing compound is tryptophan, indoleacrylic acid, indole,
indoleacetic acid, indoleacetic hydrazide, methyl indoleacetate,
indolebutyric acid, indoleacetonitrile, indolecarbinol,
indolecarboxyaldehyde, indolecarboxylic acid, indoleethanol,
indolelactic acid, indolemethanol, indolepropionic acid,
indolepyruvic acid, indolyl methyl ketone, indomycin,
indoleacetone, indomethacin, indoprofen or indolamine.
22. A virus assay method characterized by measurement of a virus
antigen based on its binding with a probe in the presence of a
surfactant with an alkyl group of 10 or more carbon atoms and a
secondary, tertiary or quaternary amine, or a nonionic surfactant
with a hydrophilic/lipophilic balance (HLB) of 12-14.
23. A method according to claim 22, wherein said surfactant having
an alkyl group and a secondary, tertiary or quaternary amine is a
surfactant with an alkyl group of 10-20 carbon atoms and a tertiary
or quaternary amine.
24. A method according to claim 22 or 23, wherein said tertiary or
quaternary amine surfactant is dodecyl-N-sarcosinic acid, a cetyl
or dodecyltrimethylammonium salt,
3-(dodecyldimethylammonio)-1-propanesulfonic acid, a
dodecylpyrimidium salt or decanoyl-N-methylglucamide (MEGA-10).
25. A method according to either of claim 23 or 24, wherein said
nonionic surfactant is polyoxyethylene isooctyl phenyl ether or
polyoxyethylene nonyl phenyl ether.
26. A method according to any one of claims 22 to 25, wherein said
virus antigen probe is an antibody for the virus antigen.
27. A method according to any one of claims 22 to 26, wherein said
virus is a virus which forms virus particles having a structure
comprising a structural protein encapsulating genomic RNA or DNA
and a membrane protein or lipid membrane surrounding it.
28. A method according to claim 27, wherein said virus is hepatitis
C virus (HCV), hepatitis D virus, hepatitis E virus, hepatitis G
virus, hand-foot-and-mouth disease virus, a flavivirus (yellow
fever virus, West Nile virus, Japanese encephalitis virus, dengue
virus), a togavirus (alpha-virus, rubivirus, arterivirus, rubella
virus), a pestivirus (hog cholera virus, bovine diarrhea virus), a
paramyxovirus (parainfluenza virus 1, 2, 3, 4, canine distemper
virus, Newcastle disease virus, RS virus, rinderpest virus, simian
parainfluenza virus, measles virus, mumps virus), an orthomyxovirus
(human influenza virus, avian influenza virus, equine influenza
virus, swine influenza virus), a rhabdovirus (rabies virus,
vesicular stomatitis virus), a picornavirus (poliovirus, Coxsackie
virus, echovirus, bovine enterovirus, porcine enterovirus, simian
enterovirus, mouse encephalitis virus, human rhinovirus, bovine
rhinovirus, equine rhinovirus, foot and mouth disease virus,
hepatitis A virus), a coronavirus (human coronavirus, avian
infectious bronchitis virus, mouse hepatitis virus, porcine
transmissible gastroenteritis virus), an arenavirus (lymphocytic
choriomeningitis virus, lassa virus, Korean hemorrhagic fever
virus), a retrovirus (HTLV: human adult leukemia virus, HIV: AIDS
virus, feline leukemia sarcoma virus, bovine leukemia virus, Rous
sarcoma virus), a reovirus (rotavirus), a calcivirus (Norwalk
virus), a bunyavirus (renal syndrome hemorrhagic fever virus), a
phyllovirus (Ebola virus, Marburg virus), hepatitis B virus (HBV),
a pox virus (vaccinia virus, alastrim virus, cowpox virus, smallpox
virus), a parvovirus (human parvovirus, porcine parvovirus, bovine
parvovirus, canine parvovirus, feline leucopenia virus, Aleutian
mink disease virus), a papovavirus (papilloma virus, polyoma
virus), adenovirus, a herpes virus (herpes simplex virus,
cytomegalovirus, chickenpox herpes zoster virus, EB virus, equine
herpes virus, feline herpes virus, Marek's disease virus) or
African swine cholera virus.
29. A method according to any one of claims 22 to 28, wherein said
virus is hepatitis C virus (HCV) or hepatitis B virus (HBV).
Description
TECHNICAL FIELD
[0001] The present invention relates to methods of detecting or
measuring viruses and reagents therefor.
BACKGROUND ART
[0002] Currently, various methods of detecting viruses have been
used to detect the presence of infectious viruses in blood or blood
products, and to identify the presence of viruses in patients with
diseases. However, these methods are not always highly sensitive or
specific though the sensitivity and the specificity may vary with
the type of virus to be detected. Even when they are sensitive and
specific enough, they are often expensive and require lengthy
procedures as in the culture and isolation of a virus. As a
background to the present invention, type C hepatitis (hepatitis C)
will be mentioned in detail below.
[0003] The causative agent of hepatitis C had long been unknown,
but when the gene of the virus was cloned (Science 244: 359-362,
1989) and a diagnostic method by antibody measurement using a
recombinant antigen generated based on said gene was developed
(Science 244: 362-364, 1989; Japanese Patent Publication (Kohyo) 2
(1990)-500880), it was found that hepatitis C is an infectious
disease whose causative agent is hepatitis C virus (HCV) that is
transmitted through the blood and blood products as its main route
of infection. With the development of the so-called second
generation antibody testing method in which a recombinant core
antigen and a recombinant NS3 antigen have been added, it is now
possible to identify virtually all HCV patients by testing their
serum. This has made it possible to eradicate almost all HCV
infections transmitted though blood donations in Japan.
[0004] However, as for other common viral infections such as by the
human immunodeficiency virus (HIV), there is a period of time until
the appearance of antibodies after infection, or the so-called
window period in which a virus is unidentifiable by existing
testing methods. This means that the risk of secondary infection is
still present, due to blood-borne components that cannot be
identified by antibody testing methods, in areas where
blood-selling is legal or in some regions of Japan. The antibody
testing method also has a drawback in that it cannot distinguish a
person who has recuperated from an infection and a person who is in
the active stage of infection because of its principle of
testing.
[0005] Interferon (IFN) is currently used for the treatment of
hepatitis C. Some researchers insist, however, that the efficacy of
the therapy can be evaluated by only measuring the antibody titer
of HCV because the titer declines 6 months after elimination of HCV
by IFN. However, since the antibody titer starts to decline only
after the reduction of antigen stimulation or several months after
the elimination of antigen, it is impossible to determine whether
IFN administration resulted in the elimination of HCV, at a desired
timing and accuracy, by the antibody testing alone. Hence, in order
to monitor the therapy, it is necessary to detect HCV per se in
addition to the HCV antibody.
[0006] It was difficult to establish a method of directly detecting
the virus particle (virus antigen) of HCV because blood levels of
the virus are very low as compared to other viruses such as
hepatitis B virus (HBV) and because the virus cannot be propagated
in vitro or using an animal etc. as a host. Therefore, instead of
detecting the virus antigen, methods of detecting the genomic RNA
of the virus were developed such as the polymerase chain reaction
(PCR) method (Science 230: 1350-1354, 1985) and the branched-chain
DNA probe method. But, the method of detecting viral genomes have
several problems when compared to the method of detecting virus
antigens.
[0007] First, it has been pointed out that since the substance to
be detected is RNA that is not very stable during storage, the
procedure of freezing and thawing of serum may cause a reduction in
the measured value. Thus, the serum samples to be tested must be
stored more carefully than when they are used in other assay
methods. Utmost care must also be taken in transportation of the
samples.
[0008] Although the testing methods that involve the use of a PCR
method are the most sensitive for detecting gene fragments, they
have problems in that : reverse transcription from a genomic RNA to
a template DNA is often accompanied by losses, which therefor
requires great skills to obtain an accurate quantitative value,
and: since amplification is an important principle in the methods,
a high incidence of false-positives may occur in case of
contamination, and thus the processing of a large volume of samples
at one time is impossible. Furthermore, even those methods which
are postulated to be a simple procedure take 2 hours or more for
pretreatment of samples and are complicated since repeated
procedures of centrifugation and the like are required. In
addition, such complicated procedures lead to increased chances of
contamination and thereby increased chances of obtaining
false-positive results. On the other hand, the branched-DNA probe
method is low in detection sensitivity and besides takes about 20
hours before obtaining test results (Igaku to Yakugaku [Medicine
and Pharmacology] 31: 961-970, 1994), and hence the method leaves
much to be desired in terms of sensitivity and processing time.
[0009] In order to solve the above-mentioned problems associated
with the methods of detecting viral genomes, methods were developed
that involve the direct detection of a virus antigen. As shown in
Japanese Unexamined Patent Publication (Kokai) No. 8 (1996)-29427,
a method was developed that detects the core antigen of HCV in the
serum using monoclonal antibody specific for the core antigen. As
has been reported in Tanaka et al., Journal of Hepatology 23:
742-745, 1995, and Fujino et al., Igaku to Yakugaku [Medicine and
Pharmacology] 36: 1065-1070, 1996, methods of detecting the core
antigen in the serum have been shown to have a clinical usefulness
as do the above-mentioned methods of detecting the viral genome.
However, there are still several major problems that need be solved
as in the methods of detecting the viral genome.
[0010] One such problem is that the sensitivity, compared to the
PCR method, is so low that it cannot be used as a final test method
of serum screening. Tanaka et al., Journal of Hepatology 23:
742-745, 1995, indicated that the detection limit is
10.sup.4-10.sup.5 copies/ml of HCV RNA. Fujino et al., Igaku to
Yakugaku [Medicine and Pharmacology] 36: 1065-1070, 1996, reported
that the method has shown a positive rate of 67% on 102 sera of the
patients before treatment with chronic hepatitis C who were found
to be RNA positive by the most sensitive detection method of CRT
(competitive reverse transcription)-PCR method. That is, in terms
of sensitivity, the method lags far behind the most sensitive
CRT-PCR method.
[0011] Furthermore, the complicated procedure of treating samples
for measurement, and the long time it takes, pose problems when it
is used in screening. Thus, the method requires a multi-step
procedure for sample (serum) treatment comprising: a polyethylene
glycol treatment (4.degree. C., 1 hr) for the concentration of
virus particles and the removal of serum components; centrifugation
(15 min); the removal of supernatants; urea treatment; the alkali
treatment (37.degree. C., 30 min); the addition of the neutralizing
agent and the like. In addition, the process of dispersing, with
urea, the precipitate having an increased viscosity due to the PEG
treatment requires great skill. In order to obtain a reproducible
result, therefore, great skill is required and, besides, a minimum
of 2 hours of treatment is necessary. Furthermore, such processes
as centrifugation, supernatant removal, etc. are not amenable to
automation and render the simultaneous treatment of a large number
of samples very difficult. Thus, from a viewpoint of ease of
handling as well, the method is not suited for applications that
require the treatment of a large volume of samples as in screening
tests.
[0012] On the other hand, the virus antigen detection system is
superior to the highly sensitive PCR method in the following
points. Thus, it is very tolerant to contamination because it
involves no procedure of excessive amplification in the detection
step. Furthermore, since it is intended to detect antigen protein
that is relatively stable instead of poorly stable RNA, it requires
no excessive care in the storage of samples, it does not require
special equipment such as the deep freezer that is needed for
samples to be detected by PCR, and the transportation of the
samples is also easier.
[0013] These features are suitable for applications in which a
large number of samples is measured as in the blood industry or
health checkup testing. However, because the disclosed method of
detecting the core antigen, as indicated above, is not amenable to
automation and is low in sensitivity so that it cannot be a gold
standard in applications that require high sensitivity such as in
the blood industry, it cannot be applied to tests that handle a
large number of samples such as screening, and cannot make the best
use of its advantageous features over the PCR method. Furthermore,
clinically useful assay methods must always face the challenges of
sensitivity, specificity, reproducibility, ease of handling, and
low cost, and sustained efforts are needed to satisfy these
challenges as much as possible. With regard to detection of virus
antigens other than HCV, especially for use in screening handling a
large number of samples, there are many methods that are not put
into practical use because they are low in sensitivity, as compared
to the PCR method, or the desired antigen could not be fully
exposed.
DISCLOSURE OF THE INVENTION
[0014] It is an object of the present invention to provide a method
for detecting various virus antigens, including a method for
detecting HCV antigen, that is suitable for treating a large number
of samples as in screening in the blood industry and health
checkups. In other words, the object of the present invention is to
provide the detection system for various virus antigens including a
method of detecting HCV antigen that has a sensitivity and
specificity equivalent to those of the PCR method, that permit
simple pretreatment, or that can be easily automated without
pretreatment. Preferred embodiments of the present invention will
now be explained hereinbelow with a main reference to HCV.
[0015] According to the first embodiment (1) of the present
invention, there is provided a means to detect or determine HCV by
disrupting the virus particle, fully exposing the virus antigen,
disrupting antibodies, if present, against the virus antigen, and
detecting or determining the virus antigen.
[0016] Thus, the present invention provides (I) a method for
treating a virus-containing sample, characterized by treatment of a
virus-containing sample with a treatment solution containing (1) an
anionic surfactant and (2) an amphoteric surfactant, nonionic
surfactant, or protein denaturant.
[0017] The present invention also provides (II) a method for
treating a virus-containing sample, characterized by treatment of a
virus-containing sample with a treatment solution containing (1) an
anionic surfactant, (2) an amphoteric surfactant and (3) a nonionic
surfactant or protein denaturant.
[0018] The present invention also provides (III) a method for
treating a virus-containing sample, characterized by treatment of a
virus-containing sample with a treatment solution containing (1) an
anionic surfactant, (2) an amphoteric surfactant, (3) a nonionic
surfactant and (4) a protein denaturant.
[0019] The present invention also provides (IV) a virus assay
method characterized by using a sample treating method according to
any one of (I) to (III) and reacting a sample with a probe which
specifically recognizes a virus antigen, for detection or
quantitation of the presence of the virus antigen.
[0020] The present invention also provides a kit, assay kit or
diagnostic reagent for determining the presence or absence of a
virus in a sample, which is for use in the above (IV) immunoassay
method and comprises an anionic surfactant.
[0021] The present invention also provides a kit, assay kit or
diagnostic reagent for determining the presence or absence of a
virus in a sample, which is for use in the above (IV) immunoassay
method and comprises a monoclonal antibody described
hereinbelow.
[0022] According to the first embodiment (2) of the present
invention, there is provided a means to detect or determine a virus
by disrupting the virus particle, fully exposing the virus antigen,
disrupting antibodies, if present, against the virus antigen and
detecting or determining the virus antigen.
[0023] Thus, the present invention provides (V) a method for
treating a virus-containing sample, characterized by treatment of a
virus-containing sample with a treatment solution containing (1) a
chaotropic ion and (2) an acidifying agent.
[0024] The present invention further provides (VI) a method for
treating a virus-containing sample, characterized by treatment of a
virus-containing sample with a treatment solution containing (1) an
chaotropic ion, (2) an acidifying agent and (3) a nonionic
surfactant.
[0025] The present invention further provides (VII) a virus assay
method, characterized by using a sample treating method according
to the above (V) and (VI) and reacting a sample with a probe which
specifically recognizes a virus antigen, for detection or
quantitation of the presence of the virus antigen.
[0026] The present invention further provides a kit, assay kit or
diagnostic reagent for determining the presence or absence of a
virus in a sample, which is for use in the above (VII) method and
comprises a chaotropic agent.
[0027] The present invention further provides a kit, assay kit or
diagnostic reagent for determining the presence or absence of HCV
in a sample, which is for use in the above (VII) method and
comprises a monoclonal antibody produced by a hybridoma HC11-14
(FERM BP-6006), HC11-10 (FERM BP-6004) or HC11-11
(FERM-BP-6005).
[0028] According to the second embodiment of the present invention,
there is provided a method to detect or determine a virus antigen
during the window period in which antibodies against said virus
have not yet been generated. In this method, the disruption of the
virus particle to expose the virus antigen is sufficient and there
is no need to disrupt antibodies against the virus antigen in the
blood.
[0029] Thus, the present invention provides a virus assay method
characterized by measurement of a virus antigen based on its
binding with a probe in the presence of a surfactant with an alkyl
group of 10 or more carbon atoms and a secondary, tertiary or
quaternary amine, or a nonionic surfactant with a
hydrophilic/lipophilic balance (HLB) of 12-14, or of both of
them.
[0030] The present invention further provides a hybridoma cell line
selected from the group consisting of HC11-11 (FERM-BP-6005),
HC11-14 (FERM BP-6006), HC11-10 (FERM BP-6004), HC11-3
(FERM-BP-6002), and HC11-7 (FERM-BP-6003).
[0031] The present invention also provides a monoclonal antibody
produced by a hybridoma cell line selected from the group
consisting of HC11-11 (FERM BP-6005), HC11-14 (FERM BP-6006),
HC11-10 (FERM BP-6004), HC11-3 (FERM BP-6002), and HC11-7 (FERM
BP-6003).
[0032] Furthermore, HCV which is an RNA virus, and HBV which is a
DNA virus, are viruses which form virus particles having a
structure comprising a structural protein encapsulating genomic RNA
or DNA and a membrane protein or lipid membrane surrounding it. In
either embodiment, by using a treating method of the present
invention, there is provided detection or determination of a virus
characterized by disrupting a virus particle of not only HCV or HBV
but also a virus having similar a structure thereto, by fully
exposing the virus antigen, and by detecting or determining said
antigen.
BRIEF DESCRIPTION OF DRAWINGS
[0033] FIG. 1 is a graph showing the effect of concentration of
added SDS on sample treatment. Sera from normal healthy human
subjects (normal) and HCV-RNA-positive panel sera 13 and 50 were
used.
[0034] FIG. 2 is a graph showing the effect of concentration of
added CHAMPS on sample treatment. Sera from normal healthy human
subjects (normal) and HCV-RNA-positive panel sera 13 and 50 were
used.
[0035] FIG. 3 is a graph showing the effect of concentration of
added urea on sample treatment. Sera from normal healthy human
subjects (normal) and HCV-RNA-positive panel sera 13, 44, and 50
were used.
[0036] FIG. 4 is a graph showing the effect of temperature of added
Triton X100 on sample treatment. Sera from normal healthy human
subjects (normal) and HCV-RNA-positive panel sera 13, 44, and 50
were used.
[0037] FIG. 5 is a graph showing the effect of temperature during
sample treatment. Sera from normal healthy human subjects (normal)
and HCV-RNA-positive panel sera 13, 44, and 50 were used.
[0038] FIG. 6 is a graph showing the dilution standard curve and
the detection limit of a sandwich assay system in which a standard
panel serum 50, defined as 1 U/ml, was serially diluted and
subjected to a sample treating method, and then was measured using
a monoclonal antibody of the present invention.
[0039] FIG. 7 is a graph showing the dilution standard curve and
the detection limit of a sandwich immunoassay system in which a
standard panel serum 50, defined as 1 U/ml, was serially diluted
and subjected to a sample treating method, and then was
measured.
[0040] FIG. 8 shows an immunological activity of core antigen in
fractions obtained by fractionation with a gel filtration column of
the panel serum 13 that was subjected to sample treating method.
The molecular weight is about 150 kD and about 68 kD for IgG and
albumin, respectively.
[0041] FIG. 9 is a graph showing a correlation between the activity
of core antigen released and the amount of HCV-RNA determined using
Amplicore HCV Monitor (PCR method) of a PCR-positive sample which
was subjected to a sample treating method of the present
invention.
[0042] FIG. 10 is a graph showing the effect of concentration of
added guanidine chloride on sample treatment. Sera from normal
healthy human subjects (normal) and HCV-RNA-positive panel sera 13
and 50 were used.
[0043] FIG. 11 is a graph showing the effect of concentration of
added Triton X100 on sample treatment. Sera from normal healthy
human subjects (normal) and HCV-RNA-positive panel sera 13 and 50
were used.
[0044] FIG. 12 is a graph showing the effect of concentration of
added Tween 20 on sample treatment. Sera from normal healthy human
subjects (normal) and HCV-RNA-positive panel sera 13 and 50 were
used.
[0045] FIG. 13 is a graph showing the effect of temperature during
sample treatment. Sera from normal healthy human subjects (normal)
and HCV-RNA-positive panel sera 13 and 50 were used.
[0046] FIG. 14 is a graph showing the dilution standard curve and
the detection limit of a sandwich immunoassay system in which a
standard panel serum 50, defined as 1 U/ml, was serially diluted
and subjected to a sample treating method, and then was
measured.
[0047] FIG. 15 shows an immunological activity of core antigen in
fractions obtained by fractionation with a gel filtration column of
the panel serum 13 that was subjected to sample treating method.
The molecular weight is about 150 kD and about 68 kD for IgG and
albumin, respectively.
[0048] FIG. 16 is a graph showing a correlation between the
activity of core antigen released and the amount of HCV-RNA
determined using Amplicore HCV Monitor (PCR method) of a sample
which was subjected to a sample treating method of the present
invention and which tested positive by Amplicore HCV Monitor (PCR
method).
[0049] FIG. 17 shows a standard curve obtained by determination of
recombinant hepatitis B virus (HBV) core antigen according to the
present invention.
BEST MODE FOR CARRYING OUT THE INVENTION
[0050] The subject viruses of the present invention are viruses
which form virus particles having a structure comprising a
structural protein encapsulating genomic RNA or DNA and a membrane
protein or lipid membrane surrounding it.
[0051] Representative examples of the above viruses having RNA as a
genome include hepatitis C virus (HCV) and HCV-related viruses.
[0052] HCV-related viruses include hepatitis D virus, hepatitis E
virus, hepatitis G virus, hand-foot-and-mouth disease virus, a
flavivirus (yellow fever virus, West Nile virus, Japanese
encephalitis virus, dengue virus), a togavirus (alpha-virus,
rubivirus, arterivirus, rubella virus), a pestivirus (hog cholera
virus, bovine diarrhea virus), a paramyxovirus (parainfluenza virus
1, 2, 3, 4, canine distemper virus, Newcastle disease virus, RS
virus, rinderpest virus, simian parainfluenza virus, measles virus,
mumps virus), an orthomyxovirus (human influenza virus, avian
influenza virus, equine influenza virus, swine influenza virus), a
rhabdovirus (rabies virus, vesicular stomatitis virus), a
picornavirus (poliovirus, Coxsackie virus, echovirus, bovine
enterovirus, porcine enterovirus, simian enterovirus, mouse
encephalitis virus, human rhinovirus, bovine rhinovirus, equine
rhinovirus, foot and mouth disease virus, hepatitis A virus), a
coronavirus (human coronavirus, avian infectious bronchitis virus,
mouse hepatitis virus, porcine transmissible gastroenteritis
virus), an arenavirus (lymphocytic choriomeningitis virus, lassa
virus, Korean hemorrhagic fever virus), a retrovirus (HTLV: human
adult leukemia virus, HIV: AIDS virus, feline leukemia sarcoma
virus, bovine leukemia virus, Rous sarcoma virus), a reovirus
(rotavirus), a calcivirus (Norwalk virus), a bunyavirus (renal
syndrome hemorrhagic fever virus), a phyllovirus (Ebola virus,
Marburg virus), and the like.
[0053] Representative examples of the above viruses having DNA as a
genome include hepatitis B virus HBV) and HBV-related viruses.
HBV-related viruses include a pox virus (vaccinia virus, alastrium
virus, cowpox virus, smallpox virus), a parvovirus (human
parvovirus, porcine parvovirus, bovine parvovirus, canine
parvovirus, feline leucopenia virus, Aleutian mink disease virus),
a papovavirus (papilloma virus, polyoma virus), adenovirus, a
herpes virus (herpes simplex virus, cytomegalovirus, chickenpox
herpes zoster virus, EB virus, equine herpes virus, feline herpes
virus, Marek's disease virus), African swine cholera virus, and the
like.
[0054] In addition to the above, there are many pathogenic viruses
known and there are many unidentified viruses present. It is clear
that if such viruses have a structure described above comprising a
structural protein encapsulating genomic RNA or DNA and a membrane
protein or lipid membrane surrounding it, they can be released in a
form suitable for immunoassay using the sample treating method of
the present invention.
[0055] Embodiments for carrying out the present invention will now
be explained below referring to HCV. Since blood levels of HCV are
10.sup.2 copies/ml to 10.sup.6 copies/ml which are lower than those
of HBV (10.sup.9 copies/ml), a very high sensitivity is required
for an assay to detect the virus antigen.
[0056] Generally, in a detection method represented by an
immunological method that uses antibody as a probe, possible
methods to enhance detection sensitivity include I) an increase in
the number of the antigen molecules to be detected, II) an increase
in the number of molecules of the probe, for example antibody, that
binds to the antigen, III) a reduction in nonspecific reactions
that define detection sensitivity caused by the binding of the
probe, for example antibody, with a substance other than the
antigen, and IV) an increase in the detection limit of a label for
use in the detection, and an appropriate combination of these
methods would enable an increase in sensitivity.
[0057] As a method to increase the number of antigen molecules,
I-1) an increase in the amount of sample is most easily conceived.
But, since the maximum amount to be added in a commonly used
reaction system (for example, a 96-well immunoplate) cannot exceed
about 300 .mu.l I-1), a concentration method to increase the number
of molecules to be added to the reaction system has been used.
[0058] In order to increase the number of probes, for example
antibody molecules, that bind to the antigen, the most readily
conceived means includes II-1) an increase in the number of
epitopes to be recognized using multiple probes, for example
antibodies, and II-2) an increase in the number of antibodies bound
per unit time by increasing the affinity (affinity and avidity) of
the probe, for example antibody, with the antigen. Incidentally,
possible methods to enhance the affinity of, for example, antibody
include a method of changing the composition of the buffer in the
reaction system, a method of altering the probe, and a method of
combining these. II-3) Methods are also conceived in which many
antigens are captured by binding a large number of antibodies to
the carrier having a wide surface area such as beads, magnetic
particles, etc. to expand the reaction area with a limited amount
of antigen.
[0059] In the case of infectious diseases, human antibodies having
a high affinity of binding to antigen are expected to be present in
the sample. Accordingly, it is expected that the epitopes of these
antibodies overlap with those of the probes, for example
antibodies, to be used in the detection, resulting in a competitive
reaction that causes a reduction in the number of antibodies to be
used for detection. It is, therefore, anticipated that a reduction
in these interfering antibodies in the sample leads to an increase
in the number of antibody molecules for use in detection that bind
to antigen (II-3).
[0060] It is indeed difficult to generalize the methods of reducing
nonspecific reactions, but strategies are conceived that reduce
nonspecific reactions III-1) to reduce nonspecific reactions by
increasing the affinity (affinity and avidity) of the probe, for
example antibody, with the antigen by changing the composition of
the buffer solution, III-2) to remove the causative agent of the
nonspecific reactions, and the like.
[0061] Possible methods to enhance detection sensitivity include:
IV-l) to employ a label (a radioisotope, etc.) having a high
detection sensitivity; IV-2) to amplify signals by employing an
enzyme or a catalyst as a label; IV-3) to change an enzyme
substrate into one having a higher sensitivity; IV-4) to amplify
signals from an enzymatic reaction or a chemical reaction by an
electrical or a mechanical means; IV-5) to increase the number of
labels per antibody; IV-6) to enhance the sensitivity of the
instrument used for signal detection, and the like.
[0062] Analysis of the steps of pretreatment in the disclosed
method for detecting the HCV core antigen revealed that the method
comprises the step of concentrating the antigen by adding
polyethylene glycol to the sample which is then centrifuged to
recover HCV as a precipitate (I-2) and simultaneously removing part
of the serum components (II-2), followed by the step of
resuspending the precipitate in a solution containing urea and the
alkali agent to inactivate human antibody present therein thereby
releasing core antigen from HCV (II-3), and the step of adding a
solution containing a nonionic surfactant (Triton X100) and a
neutralizing agent to prepare a solution which is to be reacted
with the monoclonal antibody.
[0063] As described above, centrifugation and resuspension of the
precipitate are procedurally complicated steps and require great
skill. Thus, a goal of the present invention is a core antigen
detection system that resolves the above problems concerning
procedures.
[0064] The identity of HCV per se has not been elucidated yet. But,
based on the genomic structure, the structures of related virus
particles, and general information on viruses, it is estimated that
an HCV particle has a genomic RNA that is packed within the core
antigen, which in turn is encapsulated by a coat protein comprising
E1 and E2/NS1 antigens that are anchored to a lipid membrane
surrounding the above packing.
[0065] It is therefore necessary to remove the coating to thereby
permit the binding of a probe, for example an antibody, to be used
for detection of said core antigen in order to detect core antigen.
Furthermore, it has been reported that the virus particle in the
blood takes a complex structure in which the particle is surrounded
by LDL (low density lipoprotein) etc., and since antibodies against
the coat protein are also present, it is estimated that the virus
particle may be present as an immune complex with an anti-coat
protein antibody. Thus, in order to increase the number of antigen
molecules to be detected, it is important to efficiently remove
from the virus particle the coating and contaminants surrounding
the virus particle, and to efficiently release the core antigen
molecules.
[0066] The same holds true for viruses other than HCV and the
structural proteins of viruses must be efficiently released.
[0067] Thus, the present invention relates to a treating method
that brings a virus antigen in a sample (serum) to a state suitable
for detection using a probe, without concentrating the antigen by a
complicated procedure such as centrifugation.
[0068] Furthermore, since a human antibody may be present, as
described above, at a high titer that competes with a probe, for
example antibody, for binding, a procedure to remove said antibody
is important to enhance sensitivity.
[0069] Thus, one embodiment of the present invention relates to a
treating method that easily releases virus antigens in a sample,
concurrently inactivating human antibody that may be present in the
sample.
[0070] By using the treating method of the present invention, virus
antigens in a sample is released from a virus particle or an immune
complex in a form suitable for forming an immune complex with a
probe such as antibody, and by simultaneously inactivating human
antibody present in the sample that interferes with the detection
reaction, a highly sensitive detection can be readily attained by
an immunoassay using a probe such as antibody.
[0071] According to the first embodiment (1) of the present
invention, a probe such as antibody for use in detection may be any
probe, as long as it binds to the virus antigen in a specific
manner, it has a certain high affinity, and it does not induce
nonspecific reactions when added to the reaction system. For
example, in the detection of a HCV core antigen, as described in
Example 4, one of the probes used in the primary reaction
preferably contains a probe that can recognize and bind to the
C-terminal of the HCV core antigen. The C-terminal of the core
antigen as used herein means a sequence from 81 to 160 of the
sequence shown in SEQ ID NO: 2 or a part thereof. It can also
contain a probe for the N-terminal of the HCV core antigen. The
N-terminal of the core antigen as used herein means a sequence from
10 to 70 of the sequence shown in SEQ ID NO: 2 or a part
thereof.
[0072] According to the second embodiment (2) of the present
invention, a probe such as antibody for use in the detection may be
any probe, as long as it binds to the virus antigen in a specific
manner, it has a certain high affinity, and it does not induce
nonspecific reactions when added to the reaction system. For
example, in the detection of the HCV core antigen, one of the
probes used in the primary reaction preferably contains a probe
that can recognize and bind to the N-terminal of the HCV core
antigen. The N-terminal of the core antigen as used herein means a
sequence from 10 to 70 of the sequence shown in SEQ ID NO: 2 or a
part thereof. It can also contain a probe for the C-terminal of the
HCV core antigen. The C-terminal of the core antigen as used herein
means a sequence from 81 to 160 of the sequence shown in SEQ ID NO:
2 or a part thereof.
[0073] In any of the above embodiments, any molecule that has a
high specificity and affinity for the core antigen can be used as a
probe, including: a monoclonal antibody obtained by immunizing an
experimental animal such as a mouse, a rabbit, a chicken, a goat,
sheep, a bovine, etc., a monoclonal antibody produced by a
hybridoma obtained by the fusion of a myeloma cell with a spleen
cell that was isolated from an immunized individual, a monoclonal
antibody produced by a spleen cell or leukocyte in the blood
immortalized by the EB virus, and an antibody produced by a human
or a chimpanzee infected with HCV; a recombinant antibody produced
by a cell transformed with a recombinant antibody gene generated by
combining a gene fragment of a variable region obtained from the
cDNA or chromosomal DNA of immunoglobulin of a mouse, a human,
etc., a gene fragment of the variable region constructed by
combining a part of the cDNA or chromosomal DNA of immunoglobulin
with an artificially constructed sequence, a gene fragment of the
variable region constructed using an artificial gene sequence, or a
gene fragment of the variable region constructed by a gene
recombinant technology using the above as building blocks, with a
gene fragment of the immunoglobulin constant region; a phage
antibody generated by the fusion of a gene fragment described above
of the variable region with a structural protein of, for example a
bacteriophage, a recombinant antibody produced by a cell
transformed with a recombinant antibody gene generated by combining
a gene fragment described above of the variable region with part of
another suitable gene fragment, for example myc gene, a probe
produced by artificially introducing a variable region into the
trypsin gene, a probe obtained by artificially altering a molecule
that specifically binds to the protein such as receptor, a probe
constructed by the combinatorial chemistry technology, and the
like.
[0074] The present invention further provides the step of treating
a sample with a treatment solution capable of releasing a virus
antigen from a virus particle or an immune complex and of
simultaneously inactivating even a human antibody present in the
sample that interferes with the detection reaction in order to
generate a state suitable for forming an immune complex of the
above virus antigen and a probe thereof such as an antibody from a
sample containing the virus antigen, and an assay method and an
assay kit for detection and quantitation of the released core
antigen by an immunoassay using a probe such as antibody.
The Sample Treatment Solution and the Sample Treating Method
Provided by the Present Invention
[0075] Samples as used herein include biological fluids such as
whole blood, plasma, serum, urine, saliva, cerebrospinal fluid,
liver tissue and the like.
[0076] According to the present invention, the most important
requirement is a method of treating a virus antigen such as the
core antigen in a sample so as to generate a state suitable for a
binding reaction with the probe such as monoclonal antibody without
the complicated processing of a sample. Thus, in order to increase
the number of antigen molecules, it is important to efficiently
release the virus antigen such as the core antigen contained in a
virus particle.
[0077] As has already been known for sodium dodecyl sulfate (SDS)
polyacrylamide gel electrophoresis (SDS-PAGE), most proteins are
denatured by heat treatment in the presence of SDS and thereby
molecules other than the covalently-bound ones are converted into
monomers. Thus, the addition of a treatment agent comprising an
anionic surfactant such as SDS causes disruption of viruses as well
as the denaturing of antibodies against the virus antigen such as
the core antigen in the sample, enabling the release of the virus
antigen such as the core antigen in the sample. This was also
confirmed for the HCV core antigen as shown in Example 7, that is,
when the core antigen in a HCV-infected sample treated with a
treatment agent containing SDS was subjected to a molecular weight
analysis using gel filtration, it was detected at a position that
is theoretically predicted to be the position of the monomer.
[0078] As reported by Kashiwakuma et al., J. Immunological Methods
190: 79-89, 1996, when the core antigen isolated by SDS-PAGE from a
sample comprising an extract of a cell expressing recombinant HCV
is detected using a Western blot analysis, the immunological
activity is detected at a position believed to be that of the
monomer. It is readily understood by a person skilled in the art
that the addition of a denaturant comprising SDS to a sample causes
efficient release of antigens and an increase in the number of
antigen molecules.
[0079] As is well known, however, anionic surfactants such as SDS
have a very strong protein-denaturing effect so that when added to
a reaction of immune complex formation with the antibody they also
denature the antibody and thereby disrupt the function resulting in
the reduction in sensitivity. It is also known that the structure
of epitopes are destroyed by the treatment with an anionic
surfactant causing a weakened bonding with the antibody and a
reduced sensitivity. In order to remove the factors responsible for
the reduction in sensitivity, the denaturing effect following SDS
treatment need to be weakened by some means or other.
[0080] It is known that surfactants comprising anionic surfactants
may be removed by such means as dialysis, ultra-filtration, gel
filtration, electrophoresis, ion exchange, precipitation, membrane
transfer, etc. The fact that, as described above, antigens can be
detected by a Western blot method or gel filtration method
indicates that an antigen-antibody reaction may be effected using a
certain procedure following the SDS treatment. However, these
methods require both time and complex procedures, which is not
suitable for the purpose of the present invention.
[0081] By diluting with an excess amount of the reaction solution,
it is indeed possible to reduce the denaturing effect to a
negligible level that does not affect the reaction, but the method
cannot be applied to the methods such as an immunoassay that
involves the use of microtiter wells in which the amount of samples
to be added is limited. In this regard, it is evident that these
methods are not suitable for the purpose of the present
invention.
[0082] Thus, the inventors of the present inventors have
investigated, in the first embodiment of the present invention,
whether the addition of a treatment agent comprising an anionic
surfactant and some additive could reduce the denaturing effect by
the anionic surfactant to a level in which the probe such as
antibody is not affected, and, at the same time, enhance the
releasing effect of the core antigen by the anionic surfactant.
[0083] The inventors of the present invention have found that the
addition of a treatment agent containing a surfactant other than an
anionic surfactant such as SDS can weaken the denaturing effect of
SDS on the immobilized antibody and, as a result, can enhance
sensitivity as compared to the addition of a treatment agent
containing SDS alone. The inventors have also found that when the
agents that weaken the hydrogen ion bonding such as a surfactant
other than SDS and urea are added to the treatment agent containing
an anionic surfactant such as SDS, similar effects were observed,
and that the release of the core antigens from the virus particles
and the inactivation of the anti-core antigen antibody in the
sample were enhanced with a result that the release of the core
antigens was further enhanced. The inventors have also found that
the detection of the core antigen with a higher sensitivity was
attained by a heat treatment after the addition of a treatment
agent containing SDS and other surfactants, and have completed the
present invention.
[0084] The anionic surfactants other than SDS that can be used for
the treatment of samples include sodium cetyl sulfate or other
alkyl sulfate esters, alkyl sulfonates such as sodium dodecyl
sulfonate, alkyl allyl sulfonates, and the like. The surfactants
other than the anionic surfactants that can be added include
amphoteric surfactants, for example CHAPS
(3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), CHAPSO
(3-[(cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate),
dodecyl-N-betaine, 3-(dodecyldimethylammonio)-1-propanesulfonate;
nonionic surfactants, for example polyoxyethylene isooctylphenyl
ethers such as Triton X100, polyoxyethylene nonylphenyl ethers such
as NP 40, polyoxyethylene sorbitol esters such as Tween 80,
polyoxyethylene dodecyl ethers such as Brij 58, and octyl
glucoside, with an amphoteric surfactant such as CHAPS and an
nonionic surfactant such as Triton X100 being preferred. It is also
advantageous to add an agent (protein denaturant) that disrupts
higher structures of proteins such as urea, thiourea, and the
like.
[0085] Concentrations preferably used in the treatment are: 0.5% or
greater for SDS; 0.1% or greater for CHAPS; 1M or greater for urea;
0.1% or greater and 0.75% or smaller for Triton X100.
[0086] The temperature used for the treatment of samples may be any
temperature commonly used in the laboratory, i.e. between 4.degree.
C. and 100.degree. C., but when a nonionic surfactant is added care
should be taken as to its cloud point. Preferably a temperature of
37.degree. C. or greater is employed and the treatment at a
temperature of 50-60.degree. C. that is commonly used for the
inactivation of the serum is more effective.
Removal of Interference by Hemoglobin
[0087] When serum etc. is used as a sample for measurement, red
blood cells contained in said sample undergo hemolysis during the
above pretreatment and hemoglobin is released, and the denatured
hemoglobin may interfere with measurement by binding to the virus
antigen such as the HCV core. Thus, in the first embodiment of the
present invention, it is preferred to remove the interference with
measurement by capturing the heme in the hemoglobin. As an additive
for this purpose, we have found that the addition of at least one
of urea and a compound containing an imidazole ring is
preferred.
[0088] As the imidazole ring-containing compounds, there may be
mentioned imidazole, histidine, imidazoleacrylic acid,
imidazolecarboxyaldehyde, imidazolecarboxamide, imidazoledione,
imidazoledithiocarboxylic acid, imidazoledicarboxylic acid,
imidazolemethanol, imidazolidinethione, imidazolidone, histamine,
imidazopyridine, and the like.
[0089] As the indole ring-containing compounds, there may be
mentioned tryptophan, indoleacrylic acid, indole, indoleacetic
acid, indoleacetic hydrazide, indoleacetic methyl ester,
indolebutyric acid, indoleacetonitrile, indolecarbinol,
indolecarboxaldehyde, indolecarboxylic acid, indoleethanol,
indolelactic acid, indolemethanol, indolepropionic acid,
indolepyruvic acid, indolyl methyl ketone, indolmycin,
indoleacetone, indomethacin, indoprofen, indoramine, and the
like.
[0090] The amount added is preferably 0.5M to 5M for urea, 5 mM to
50 mM for indoleacrylic acid, and 0.05M to 0.5M for the other
additives.
[0091] On the other hand, membrane proteins such as the HCV coat
protein do not dissolve spontaneously unless they are treated to
that end. In order to dissolve a protein having a hydrophobic
portion in water, the method of converting a hydrophobic portion
into a hydrophilic portion with a surfactant is well known. It is
known, however, that certain salts such as guanidine chloride have
a property of making refractory proteins water-soluble. Ions
produced from salts (chaotropic agents) having such a property are
called chaotropic ions, and as the anionic ions, guanidine ions,
thiocyanate ions, iodine ions, periodate ions, perchlorate ions,
and the like are known. Salts that generate these ions have been
used for solubilization of refractory proteins. It was estimated
that chaotropic ions have a function of efficiently releasing the
antigens from the virus particles.
[0092] When a chaotropic ion is added, however, the secondary
structure of proteins is disrupted causing the destruction of the
epitope structure. Thus, when a probe such as antibody is added for
the reaction of immune complex formation in the presence of a
chaotropic ion as it is, binding with the antibody is weakened and
the sensitivity decreases, which are thought to pose a serious
problem.
[0093] On the other hand, the denaturing effect of chaotropic ions
is mostly reversible, so that by weakening ionic strength by
dialysis or dilution the denatured structure temporarily returns to
the original structure. This poses another problem associated with
the use of a treatment agent such as a chaotropic ion. That is,
according to the desired treating method of the present invention,
not only the virus particles present in the sample are efficiently
released, but the high-affinity antibody that binds to the antigen
present in the sample must be inactivated at the same time. Thus,
solubilization with a chaotropic ion does not provide an adequate
inactivation of the high-affinity antibody present in the sample,
and, it is believed, the antibody adversely affects
sensitivity.
[0094] Thus, the treating methods that employ chaotropic ions have
two conflicting problems: in the condition in which a chaotropic
ion can destroy a structure, the antigen-antibody reactions are
inhibited, and on the other hand the effect of a chaotropic ion
alone is not sufficient to inactivate antibodies that interfere
with reactions in the sample, and in the condition in which the
antigen-antibody reactions are not inhibited, contaminating
antibodies can inhibit the reactions.
[0095] In order to solve these conflicting problems it is necessary
to find a condition in which the epitopes of the antigen are
destroyed reversibly and the functions of the contaminating
antibodies in the sample are destroyed irreversibly.
[0096] As to the conditions in which antibody is inactivated, an
alkali treatment, an acid treatment and the like are known. The
acid treatment of serum can cause false-positive results since the
treatment irreversibly denatures some of serum proteins resulting
in the formation of precipitates that in most cases hinder
pipetting after the treatment of samples, and precipitates that
engulfed the denatured proteins are adsorbed to the solid phase at
the time of measurement and thereby may be detected as a density.
In addition, another problem arises because when the antigen of
interest is nonspecifically engulfed in the precipitate, the amount
of antigen that reacts with the probe decreases resulting in a
decrease in sensitivity.
[0097] The inventors of the present invention have found that the
acid treatment combined with the guanidine treatment can resolve
the problems associated with the acid treatment such as precipitate
formation and the conflicting problems associated with the
guanidine treatment, and thereby have completed the present
invention. We have also found that it is further preferred to add a
surfactant to the treatment agent comprising a chaotropic ion such
as guanidine and an acidifying agent. As the acidifying agent,
hydrochloric acid, sulfuric acid, acetic acid, trifluoroacetic
acid, trichloroacetic acid, and the like are preferred.
[0098] As the surfactant, an amphoteric surfactant such as CHAPS
(3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), CHAPSO
(3-[cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate),
dodecyl-N-betaine, 3-(dodecyldimethylammonio)-1-propanesulfonate,
or the like, and a nonionic surfactant such as a polyoxyethylene
isooctylphenyl ether, for example Triton X100; a polyoxyethylene
nonylphenyl ether, for example NP 40; a polyoxyethylene sorbitol
ester, for example Tween 20; a polyoxyethylene dodecyl ether, for
example Brij 58; octyl glucoside, or the like is preferred.
Furthermore, an agent such as urea that partially destroys a higher
structure of proteins by weakening hydrogen ion bonding may be
added therein.
[0099] Especially, it is more preferred to use guanidine
hydrochloride at 2 M or greater, Triton X100 at 2% or greater, and
Tween 20 at 0.02% or greater at a temperature of 4.degree. C. to
45.degree. C.
[0100] In any of the embodiments, it is evident that a virus
antigen can be released in the form of a probe, i.e. a state
suitable for the so-called immunoassay that uses antibody as a
probe, from the sample containing virus particles having a
structure similar to that of HCV or HBV by using the treating
method of the present invention. Viruses having a structure similar
to that of HCV or HBV as used herein are viruses that form virus
particles having a structure composed of proteins in which the
genomic RNA or DNA has been packed and the membrane protein or the
lipid membrane surrounding it. The viruses include, for example,
flaviviruses that are related to HCV, retroviruses such as human
immunodeficiency virus (HIV), and the like. Furthermore, those
having DNA as the genome like HBV are also included when they have
a similar structure.
Exposure of Virus Antigen
[0101] According to the second embodiment of the present invention
which relates to a method of detecting the virus antigen in a
sample collected during the window period, antibody to the virus
antigen has not been formed yet and so the disruption of the virus
particle to expose the virus antigen is sufficient and there is no
need to destroy antibodies present in the sample. Thus,
pretreatment of samples described above is not necessary and the
presence of a virus particle-disrupting agent to expose the virus
particle is sufficient. Especially, the virus particle-disrupting
agent is essential for the virus antigens present in the virus
particle.
[0102] It is believed that virus particles in general have a
structure in which a nucleic acid as the genome and a core antigen
form a complex forming a particle and said particle is coated by a
coat comprising a lipid membrane and an envelope protein. It is
also believed that in the blood they are present in the form of a
complex with a low density lipoprotein, an antibody to the virus,
and the like. Thus, a probe cannot recognize or bind to the virus
antigens, specifically the antigens in the virus particle, with the
particles as they are present in the blood. In order to detect the
virus antigens, therefore, they must be treated by, for example,
removing these structures surrounding the virus particle so that
the virus particle can be recognized by a probe.
[0103] Thus, the present invention also provides a reaction
condition under which the virus antigen in the virus particle
contained in the sample is exposed so as to be recognized by the
probe for recognizing the virus particle, a method of the reaction
comprising the system of performing the reaction, and a reagent
containing the system of performing the reaction.
[0104] A reaction system suitable for antigen detection in the
system provided by the present invention comprises a condition
which is mild enough to retain the function of the antibody against
the epitopes of the virus antigen but which can fully expose the
area recognized by the antibody, a virus antigen-recognizing probe,
from the virus particle which is a complicated structure present in
the sample.
[0105] For HCV, it has already been demonstrated that the core
antigen can be detected by treating the virus particles isolated by
ultra-centrifugation (Takahashi et al., 1992, J. Gen. Virol, 73:
667-672) and HCV particles precipitated by aggregation with
polyethylene glycol using a nonionic surfactant such as Tween 80 or
Triton X100 (Kashiwakuma et al., 1996, J. Immunological Methods,
190: 79-89). In the former, however, the detection sensitivity is
not high enough and there remains a question as to whether the
antigen has fully been exposed. In the latter also, the antibody
has been inactivated by the addition of another treatment agent,
and there is no mention of the effect of the surfactant per se.
[0106] According to the present invention, the conditions were
first investigated centering on the surfactant. Accordingly, it was
found that by using a composition based on the surfactant, an
efficient detection of the antigen in the virus particle was
attained, without employing any procedure of pretreatment such as
centrifugation or heating, by only diluting the sample in the
reaction solution.
[0107] It is necessary to effectively extract the virus antigens
from the virus particles, and to suppress interactions with a
variety of substances in the serum, thereby to provide a condition
under which the probe can efficiently react with the antigen. As an
effective surfactant used in this case, there may be mentioned a
surfactant having both an alkyl radical of 10 or more carbons and a
secondary, tertiary, or a quaternary amine in one molecule, or a
nonionic surfactant.
[0108] In the above surfactant having an alkyl radical and a
secondary, tertiary, or a quaternary amine, the alkyl group is
preferably a straight-chain alkyl group and the number of carbon
atoms therein is preferably 10 or greater, and more preferably 10
to 20. As the amine, a tertiary or quaternary amine (ammonium) is
preferred. The specific surfactants include dodecyl-N-sarcosinic
acid, dodecyl trimethyl ammonium, cetyltrimethyl ammonium,
3-(dodecyldimethylammonio)-1-propane sulfonate,
3-(tetradecyldimethylammonio)-1-propane sulfonate, dodecyl
pyridinium, cetyl pyridinium, decanoyl-N-methyl glucamide
(MEGA-10), dodecyl-N-betaine, and the like. Dodecyl-N-sarcosinic
acid and dodecyl trimethyl ammonium are preferred.
[0109] As the nonionic surfactant mentioned above, those having a
hydrophilic-lipophilic balance of 12 to 14 are preferred, and
polyoxyethylene isooctylphenylethers such as Triton X100 and Triton
X114, or polyoxyethylene nonylphenylethers such as Nonidet P40,
Triton N101, and Nikkol NP are preferred.
[0110] According to the present invention, the above two types of
surfactants may be used alone, but combined use of them is more
preferable and a synergistic effect can be obtained by the combined
use.
[0111] Additional components that change the aqueous environment
such as urea may be added.
Monoclonal Antibody as a Probe in the Present Invention
[0112] The gene fragment of the structural protein of HCV as used
herein means a gene fragment containing the core region of the
structural protein of HCV and a DNA fragment having at least a base
sequence encoding a polypeptide containing an amino acid sequence
from 1 to 160 from the N-terminal of HCV. Specifically, it is a
gene fragment comprising a base sequence encoding the amino acid
sequence of SEQ ID NO: 2.
[0113] The polypeptide having the activity of HCV antigen as used
herein means a fusion polypeptide or a polypeptide that
immunologically reacts with the anti-HCV antibody, and can be used
as an antigen for constructing a hybridoma and a monoclonal
antibody obtained therefrom of the present invention. Specifically,
it is a polypeptide having the activity of the HCV antigen
comprising the amino acid sequence of SEQ ID NO: 1 or a polypeptide
having the activity of the HCV antigen comprising a portion of the
amino acid sequence of SEQ ID NO: 1, or a polypeptide having an
additional amino acid sequence attached to the N-terminal or
C-terminal thereof.
[0114] The monoclonal antibody of the present invention against the
above fusion polypeptide and the polypeptide having amino acid
sequences as shown in SEQ ID NO: 3-6 can be readily constructed by
a person skilled in the art. The production of monoclonal antibody
by a hybridoma is well known. For example, BALB/c mice may be
periodically immunized intraperitoneally or subcutaneously with a
fusion polypeptide or polypeptide (hereinafter referred to as the
present antigen) mentioned above as a single antigen or as an
antigen combined with BSA, KLH, or the like, singly or in a mixture
with an adjuvant such as Freund's complete adjuvant. When antibody
titer in the serum has increased, the present antigen is
administered to the tail vein as a booster. After the spleen has
been aseptically isolated, it is fused with a suitable myeloma cell
line to obtain a hybridoma. This method can be carried out
according to the method of Kohler and Milstein (Nature 256:
495-497, 1975).
[0115] The hybridoma cell line obtained by the above method may be
cultured in a suitable culture liquid, and the hybridoma cell lines
producing the antibodies that exhibit specific reactions to the
present antigen are selected and cloned. For the cloning of the
antibody-producing hybridomas, there may be employed the soft-agar
method (Eur. J. Immunol. 6: 511-5198, 1976) in addition to the
limit dilution method. The monoclonal antibodies thus produced are
purified by such methods as column chromatography using protein
A.
[0116] In addition to the above monoclonal antibodies, molecules
used as a probe may be generated. For example, recombinant antibody
has been described in detail in a review by Hoogenboon (Trends in
Biotechnology, 15: 62-70, 1997).
Detection System using a Probe
[0117] The monoclonal antibodies produced according to the present
invention are used as test reagents for the detection and
quatitation of HCV structural proteins in an enzyme-linked
immunosorbent assay, an enzymeimmunoassay, an enzyme immunodot
assay, a radioimmunoassay, an aggregation-based assay, or another
well known immunoassay. When labeled antibodies are used for the
detection, fluorescent compounds, chemiluminescent compounds,
enzymes, chromogenic substances, and the like may be used as the
labeled compounds.
[0118] For example, when a sandwich reaction system-based method is
used to detect the virus antigen in a sample (serum), the
diagnostic kit to be used comprises one or more monoclonal
antibodies coated onto the solid support (for example, an inner
wall of a microtiter well), one or more monoclonal antibodies or a
fragment thereof bound to the labeled substance. Any combination of
a monoclonal antibody immobilized onto the solid support and a
labeled monoclonal antibody may be used, and the combinations that
provide high sensitivity may be selected.
[0119] Solid supports that may be used include, for example,
microtiter plates, test tubes, and capillaries made of polystyrene,
polycarbonate, polypropylene, or polyvinyl, beads (latex beads, red
blood cells, metal compounds etc.), membranes (liposome etc.),
filters, and the like.
Effects of the Invention
[0120] In accordance with the method of the present invention,
virus antigens can be conveniently released from the virus particle
in a state suitable for an immunoassay that effects detection using
antibody as a probe. Furthermore, by treating a sample containing
the virus particle in accordance with the present invention, a
simple and sensitive detection and quantitation of virus antigens
can be effected by an immunoassay in which the antigen is detected
using antibody etc. as a probe. It is also possible to create a
kit, an assay kit and a diagnostic reagent that determines the
presence or absence of viruses and quantitates viruses in the
sample using an immunoassay that employs the sample treating method
of the present invention.
EXAMPLES
[0121] The following examples illustrate the present invention, but
they should not be construed to limit the scope of the present
invention.
Example 1
Expression and Purification of a HCV-Derived Polypeptide
(A) Construction of an Expression Plasmid
[0122] A plasmid corresponding to the core region of HCV was
constructed as follows: one microgram each of DNA of plasmids
pUCC11-C21 and pUCC10-E12 obtained by integrating the C11-C21 clone
and the C10-E12 clone (Japanese Unexamined Patent Publication
(Kokai) No. 6 (1994)-38765) respectively, into pUC119 was digested
in 20 .mu.l of a restriction enzyme reaction solution [50 mM
Tris-HCl, pH 7.5, 10 mM MgCl.sub.2, 1 mM dithiothreitol, 100 mM
NaCl, 15 units of EcoRI and 15 units of ClaI enzyme] and the
restriction enzyme reaction solution [10 mM Tris-HCl, pH 7.5, 10 mM
MgCl.sub.2, 1 mM dithiothreitol, 50 mM NaCl, 15 units of ClaI and
15 units of KpnI enzyme] at 37.degree. C. for one hour each, and
then was subjected to 0.8% agarose gel electrophoresis to purify
about 380 by of EcoRI-ClaI fragment and about 920 by of ClaI-KpnI
fragment.
[0123] To the two DNA fragments and a vector obtained by digesting
pUC119 with EcoRI and KpnI was added to 5 .mu.l of 10.times.ligase
buffer solution [660 mM Tris-HCl, pH 7.5, 66 mM MgCl.sub.2, 100 mM
dithiothreitol, 1 mM ATP], 1 .mu.l of T4 lgase (350 units/.mu.l)
and water to make total volume 50 .mu.l, and then was incubated at
16.degree. C. overnight to carry out a ligation reaction. Using
this plasmid, E. coli JM109 was transformed to obtain the plasmid
pUCC21-E12.
[0124] One nanogram of the DNA of this plasmid, pUCC21-E12, was
subjected to PCR using two primers: 5'-GAATTCATGGGCACGAATCCTAAA-3'
(SEQ ID NO: 7), and 5'-TTAGTCCTCCAGAACCCGGAC-3' (SEQ ID NO: 8). PCR
was carried out using the GeneAmp.TM. (DNA Amplification Reagent
Kit, manufactured by Perkin Elmer Cetus) under the condition of DNA
denaturation at 95.degree. C. for 1.5 min, annealing at 50.degree.
C. for 2 min, and DNA synthesis at 70.degree. C. for 3 min. DNA
fragments thus obtained were separated on 0.8% agarose gel
electrophoresis and were purified by the glass powder method (Gene
Clean).
[0125] On the other hand, pUC19 was digested with SmaI, and the DNA
fragment obtained by PCR was added to 5 .mu.l of 10.times. ligase
buffer solutin [660 mM Tris-HCl, pH 7.5, 66 mM MgCl.sub.2, 100 mM
dithiothreitol, 1 mM ATP], 1 .mu.l of T4 lgase (350 units/.mu.l)
and water to make total volume 50 .mu.l, and then were incubated at
16.degree. C. overnight to carry out a ligation reaction. Using
this plasmid, E. coli JM109 was transformed to obtain the plasmid
pUCC21-E12SmaI. One microgram of this plasmid DNA was digested in
20 .mu.l of the restriction enzyme reaction solution [150 mM NaCl,
6 mM Tris-HCl, pH 7.5, 6 mM MgCl.sub.2, 15 units of EcoRI and 15
units of BamHI enzyme] and then was subjected to 0.8% agarose gel
electrophoresis to separate about 490 by of EcoRI-BamHI fragment,
which was purified by the glass powder method.
[0126] Then 1 .mu.g of the DNA of the expression vector TrpTrpE
(Japanese Unexamined Patent Publication (Kokai) No. 5(1993)-84085)
was digested in 20 .mu.l of the restriction enzyme reaction
solution [150 mM NaCl, 6 mM Tris-HCl, pH 7.5, 6 mM MgCl.sub.2, 15
units of EcoRI and 15 units of BamHI enzyme] at 37.degree. C. for 1
hour. To the reaction mixture was added 39 .mu.l of water and then
was heated at 70.degree. C. for 5 minutes. Thereafter 1 .mu.l of a
bacterial alkaline phosphatase (BAP) was added and incubated at
37.degree. C. for 1 hour.
[0127] Phenol was added to the reaction mixture for phenol
extraction. The aqueous layer thus obtained was precipitated with
ethanol and the precipitate obtained was dried. One microgram of
DNA of the EcoRI-BamHI-treated vector obtained and the above core
140 fragment were added to 5 .mu.l of 10.times.ligase buffer
solutin [660 mM Tris-HCl, pH 7.5, 66 mM MgCl.sub.2, 100 mM
dithiothreitol, 1 mM ATP], 1 .mu.l of T4 lgase (350 units/.mu.l)
and water to make total valme 50 .mu.l, and were incubated
overnight at 16.degree. C. to carry out a ligation reaction.
[0128] Using 10 .mu.l of this reaction mixture, E. coli strain
HB101 was transformed. The sensitive E. coli strain used for
transformation can be constructed by the calcium chloride method
[Mandel, M. and Higa, A., J. Mol. Biol., 53, 159-162 (1970)]. The
transformed E. coli was plated on a LB plate (1% tryptophan, 0.5%
NaCl, 1.5% agar) containing 25 .mu.g/ml ampiciliin and was
incubated overnight at 37.degree. C. Using an inoculating loop, one
loopful of a the bacterial colony that has formed on the plate was
transferred to an LB culture medium containing 25 .mu.g/ml
ampicillin and incubated overnight at 37.degree. C. One and a half
milliliters of the bacterial culture was centrifuged to collect the
cells and then the plasmid DNA was subjected to minipreparation
using the alkali method [Manniatis et al., Molceular Cloning: A
Laboratory Manual (1982)].
[0129] Then 1 .mu.g of the DNA of the plasmid DNA thus obtained was
digested in 20 .mu.l of the restriction enzyme reaction solution
[150 mM NaCl, 6 mM Tris-HCl, pH 7.5, 6 mM MgCl.sub.2, 15 units of
EcoRI and 15 units of BamHI enzyme] at 37.degree. C. for 1 hour,
and then was subjected to agarose gel electrophoresis. The TrpTrpE
core 160 expression plasmid that produced about 490 by of
EcoRI-BamHI fragment were selected.
(B) Expression and Purification of a Polypeptide Encoded by the
Clone Core 160
[0130] E. coli strain HB101 having an expression plasmid TrpTrpE
core 160 was inoculated to 3 ml of 2YT medium (1.6% trypton, 1%
yeast extracts, 0.5% NaCl) containing 50 .mu.g/ml of ampicillin,
and was cultivated at 37.degree. C. for 9 hours. One milliliter of
the culture was passaged to 100 ml of M9-CA medium (0.6%
Na.sub.2HPO.sub.4, 0.5% KH.sub.2PO.sub.4, 0.5% NaCl, 0.1%
NH.sub.4Cl, 0.1 mM CaCl.sub.2, 2 mM MgSO.sub.4, 0.5% casamino acid,
0.2% glucose) containing 50 .mu.g/ml of ampicillin, and cultured at
37.degree. C. Indol acrylate was added to a final concentration of
40 mg/l at OD600=0.3 and was cultured for more 16 hours. The
culture was centrifuged to collect the cells.
[0131] To the cells was added 20 ml of the buffer A [50 mM
Tris-HCl, pH 8.0, 1 mM EDTA, 30 mM NaCl] to suspend them. The
suspension was again centrifuged to obtain 2.6 g of expression
cells. The cells thus obtained were suspended in 10 ml of the
buffer A. After disrupting the membrane of the E. coli with
sonication, it was centrifuged to obtain an insoluble fraction
containing a fusion polypeptide of a polypeptide encoded by HCV
cDNA and TrpE. To the fraction was added 10 ml of the buffer A
containing 6 M urea to solubilize and extract the fusion
polypeptide. The solubilized extract was subjected to ion exchange
column chromatography using S-Sepharose to purify the fusion
polypeptide.
Example 2
Method of Constructing a Hybridoma
[0132] The fusion polypeptide (TrpCll) prepared by the method
described above was dissolved in 6 M urea, and then diluted in 10
mM phosphate buffer, pH 7.3, containing 0.15 M NaCl to a final
concentration of 0.2 to 1.0 mg/ml, and mixed with an equal amount
of adjuvant (Titermax) to make a TrpCll suspension. This suspension
prepared at 0.1 to 0.5 mg/mi of TrpCll was intraperitoneally given
to 4 to 6 week old BALB/c mice. Similar immunization was conducted
every two weeks and after about two more weeks 10 .mu.g of TrpCll
dissolved in physiological saline was administered through the tail
vein.
[0133] Three days after the last booster, the spleen was
aseptically isolated from the immunized animal and was cut into
pieces using scissors, which were then crumbed into individual
cells and washed three times with the RPMI-1640 medium. After
washing, a mouse myeloma cell line SP2/0Ag14 at the logarithmic
growth phase as described above, 2.56.times.10.sup.7 of said cells
and 1.64.times.10.sup.8 spleen cells were mixed in a 50 ml
centrifuge tube. The mixture was centrifuged at 200.times.g for 5
minutes, the supernatant was removed, and 1 ml of the RPMI-1640
medium containing 50% polyethylene glycol (PEG) 4000 (manufactured
by Merck) was added to the precipitate, and 10 ml of the RPMI-1640
medium was further added to carry out cell fusion.
[0134] After PEG was removed by centrifugation (200.times.g, 5
minutes), the fused cells were cultured in a RPMI1640 medium
containing 10% bovine serum, hypoxanthine, aminopterin, and
thymidine (hereinafter referred to as HAT) in a 96-well plate for
about 10 days to grow only hybridomas. Then, the clones producing
the antibody of interest were detected by the ELISA method to
obtain the hybridomas that produce monoclonal antibody having the
desired reaction specificity of the present invention.
[0135] The hybridomas thus obtained were monocioned according to
the conventional limiting dilution method, and the hybrodomas
obtained were designated HC11-11, HC11-14, HC11-10, and HC11-3, and
HC11-7. Said four hybridomas were deposited with the National
Institute of Bioscience and Human Technology, Agency of Industrial
Science and Technology, on Jul. 4, 1997, as FERM BP-6005, FERM
BP-6006, FERM BP-6004, FERM BP-6002, AND FERM BP-6003,
respectively.
Example 3
Construction of Monoclonal Antibody
[0136] The hybridomas obtained in the method of Example 2 were
inoculated to the abdominal cavity of mice treated with pristane
etc., and the monoclonal antibodies produced in the ascites fluid
was collected. The monoclonal antibodies were purified using the
Protein A-bound Sepharose column to separate IgG fractions.
[0137] By an immunoassay using rabbit anti-mouse Ig isotype
antibody (manufactured by Zymed), the isotype of each of the
monoclonal antibodies C11-14, C11-11, C11-10, C11-7; and C11-3
produced from the above five hybridomas, respectively, was found to
be IgG2 for C11-10 and C11-7; and IgGl for CH11-11, C11-14, and
C11-3. For the five monoclonal antibodies obtained, epitope
analysis was conducted using the synthetic peptides composed of 20
amino acids synthesized according to the sequence derived from the
HCV core region. The result indicated, as shown in Table 1, that
they were the monoclonal antibodies that specifically recognize
part of the core region.
TABLE-US-00001 TABLE 1 Antibody Recognition site C11-14
.sup.41Gly-.sup.50Arg (SEQ ID NO: 4) C11-10 .sup.21Asp-.sup.40Arg
(SEQ ID NO: 3) C11-3 .sup.100Pro-.sup.120Gly (SEQ ID NO: 5) C11-7
.sup.111Asp-.sup.130Phe (SEQ ID NO: 6) C11-11
.sup.100Pro-.sup.120Gly (SEQ ID NO: 5)
Example 4
Study on the Condition of Sample Treatment
1) SDS Concentration
[0138] To 100 .mu.l of a normal human serum and HCV-RNA-positive
sera were added 100 .mu.l of the treatment solution containing a
different concentration of SDS and 0.6% CHAPS. The mixtures were
then placed in an incubator set at 56.degree. C. and were treated
for 30 minutes, and 80 .mu.l each of the treated mixtures was used
as a sample. The result obtained using the assay method described
below is shown in FIG. 1 with the SDS concentration at the time of
treatment taken as the abscissa.
2) CHAPS Concentration
[0139] To 100 .mu.l of a normal human serum and HCV-RNA-positive
sera were added 100 .mu.l of the treatment solution containing a
different concentration of CHAPS and 5% SDS. The mixtures were then
placed in an incubator set at 56.degree. C. and were treated for 30
minutes, and 80 .mu.l each of the treated mixtures was used as a
sample. The result obtained using the assay method described below
is shown in FIG. 2 with the CHAPS concentration at the time of
treatment taken as the abscissa.
3) Urea Concentration
[0140] To 100 .mu.l of a normal human serum and HCV-RNA-positive
sera were added 100 .mu.l of the treatment solution (5% SDS, 0.6%
CHAPS) containing a different concentration of urea. The mixtures
were then placed in an incubator set at 56.degree. C. and were
treated for 30 minutes, and 80 .mu.l each of the treated mixtures
was used as a sample. The result obtained using the assay method
described below is shown in FIG. 3 with the urea concentration at
the time of treatment taken as the abscissa.
4) Triton X100 Concentration
[0141] To 100 .mu.l of a normal human serum and HCV-RNA-positive
sera were added 100 .mu.l of the treatment solution (5% SDS, 0.6%
CHAPS, 6 M urea) containing a different concentration of Triton
X100. The mixtures were then placed in an incubator set at
56.degree. C. and were treated for 30 minutes, and 80 .mu.l each of
the treated mixtures was used as a sample. The result obtained
using the assay method described below is shown in FIG. 4 with the
Triton X100 concentration at the time of treatment taken as the
abscissa.
5) Reaction Temperature
[0142] To 100 .mu.l of a normal human serum and HCV-RNA-positive
sera were added 100 .mu.l of the treatment solution (5% SDS, 0.6%
CHAPS, 6 M urea, 0.75% Triton X100). The mixtures were treated at
4.degree. C., room temperature (23.degree. C.), 37.degree. C.,
45.degree. C., 56.degree. C., and 70.degree. C. for 30 minutes, and
80 .mu.l each of the treated mixtures were used as a sample. The
result obtained using the assay method described below is shown in
FIG. 5.
Assay Methods
[0143] Samples obtained in the study on the condition of serum
treatment were each evaluated using the respective assay method
described below. Thus, an anti-HCV core antigen monoclonal antibody
(a mixture of equal amounts of antibody C11-3 and C11-7) was
diluted to a final total concentration of 6 .mu.g/ml in 0.1 M
carbonate buffer, pH 9.6, and 100 .mu.l each of the dilutions was
dispensed per well of a 96-well microtiter plate (manufactured by
Nunc). After the plate was incubated overnight at 4.degree. C., it
was washed twice with 0.35 ml of 10 mM sodium phosphate buffer, pH
7.3, containing 0.15 M NaCl. Then, 0.35 ml of 10 mM sodium
phosphate buffer, pH 7.35, containing 0.5% casein-Na (hereinafter
referred to as the blocking solution) was added and the plate was
further incubated at room temperature for 2 hours.
[0144] After the blocking solution was removed, 160 .mu.l of 100 mM
sodium phosphate buffer, pH 7.3, containing 0.15 M NaCl, 1% BSA,
0.5% casein-Na, and 0.05% Tween 20, and samples for measurement
obtained by the serum treating method were added into respective
wells. The plate was then incubated at room temperature for 2
hours, washed five times with 300 .mu.l of the wash solution. Then
100 .mu.l of a peroxidase (POD)-labeled monoclonal antibody (a
mixture of equal amounts of C11-10 and C11-14) was added and was
incubated at room temperature for 30 minutes. After the incubation
was over, the plate was washed five times with 300 .mu.l of the
above wash solution. One hundred microliters of the substrate
(ortho-phenylene diamine, hereinafter referred to as OPD) solution
was added to the plate and the plate was incubated at room
temperature for 30 minutes, followed by the addition of 100 .mu.l
of 2 N sulfuric acid solution. Absorbance was measured at a
wavelength of 492 nm (OD492) with the absorbance at 630 nm as a
reference.
[0145] Each treatment condition was optimized, as shown in FIGS. 1
to 4. It was difficult to detect the core antigen in the untreated
samples, but such a simple treatment enabled the detection of the
core antigen. Especially, it was shown, the core antigen can be
satisfactorily detected by employing the condition of SDS at 0.5%
or greater, CHAPS at 0.1% or greater, urea at 1M or greater, and
Triton X100 at 0.1 to 0.75%, and a temperature range of 4.degree.
C. to 70.degree. C.
Example 5
The Detection and Assay Method of the Core Antigen in the
Structural Region (1)
[0146] To 100 .mu.l of serum was added 100 .mu.l of the treatment
solution (5% SDS, 0.6% CHAPS, 6 M urea, 0.75% Triton X100). It was
then placed in an incubator set at 56.degree. C. and was treated
for 30 minutes, and 120 .mu.l of the treated mixture was used as a
sample.
[0147] An anti-HCV core antigen monoclonal antibody (a mixture of
equal amounts of C11-3 and C11-7) was diluted to a final total
concentration of 6 .mu.g/ml in 0.1 M carbonate buffer, pH 9.6, and
100 .mu.l each of the diluted mixture was dispensed per well of a
96-well microtiter plate (manufactured by Nunc). After the plate
was incubated overnight at 4.degree. C., it was washed twice with
0.35 ml of 10 nM sodium phosphate buffer, pH 7.3, containing 0.15 M
NaCl. Then, 0.35 ml of the blocking solution was added and the
plate was further incubated at room temperature for 2 hours.
[0148] After the blocking solution was removed, 120 .mu.l of the
reaction buffer and samples for measurement obtained in the above
treating method were added into respective wells, and incubated at
room temperature for 2 hours. The plate was washed five times with
300 .mu.l of the wash solution, and then 100 .mu.l of a peroxidase
(POD)-labeled monoclonal antibody (a mixture of equal amounts of
C11-10 and C11-14) was added to the plate and the plate was
incubated at room temperature for 30 minutes. The plate was washed
five times with 300 .mu.l of the wash solution and 100 .mu.l of the
substrate (OPD) solution was added, and incubated at room
temperature for 45 minutes, followed by the addition of 100 .mu.l.
of 2 N sulfuric acid solution. Absorbance was measured at a
wavelength of 492 nm (OD492) with the absorbance at 630 nm as a
reference. As a standard serum, the panel serum 50, defined as 1
U/ml, was serially diluted in 10 mM sodium phosphate buffer, pH
7.3, containing 1% BSA, which was similarly treated and
measured.
[0149] FIG. 6 shows a dilution line of the panel serum 50 used as a
standard serum. The core antigen in the sample was determined in a
dose-dependent manner and could be detected to a level of about 0.5
mU/ml. It was demonstrated, therefore, that by combining a very
simple method of sample treatment and the monoclonal antibody of
the present invention, the HCV core antigen can be detected or
quantitated.
Example 6
Detection and Quantitation of the HCV Core Antigen (2)
[0150] A Method using an Alkaline Phosphatase-Labeled Monoclonal
Antibody
[0151] A 96-well black microtiter plate (Nunc) as the solid
carrier, an alkaline phosphatase-labeled monoclonal antibody as the
labeled antibody, and CDPstar (Emerald II as the sensitizer) as the
substrate were used. A dilution line of the panel serum 50 used as
a standard serum is shown in FIG. 7, in which the core antigen in
the sample was determined in a dose-dependent manner and could be
detected to a level of about 0.5 mU/ml. It was demonstrated,
therefore, that the method using an alkaline phosphatase-labeled
monoclonal antibody can also detect or quantitate the HCV core
antigen.
Example 7
Study on Additives for Suppressing Sensitivity Reduction in the
Hemolyzed Serum
[0152] When serum components were tested on the effect on
sensitivity, it was found that the addition of hemoglobin
drastically reduced sensitivity. It was thought that the reduction
was caused by the heme released from the denatured hemoglobin
produced by pretreatment using a pretreatment agent containing SDS,
CHAPS, or Triton X100. Thus, additives that could reduce the effect
of the denatured hemoglobin were tested by adding them to the
pretreatment agent.
[0153] The effect of urea addition was studied by adding urea to
the model samples that were created by adding a high concentration
hemoglobin (manufactured by Kokusai Shiyaku: Kansho Check) to a HCV
core antigen positive serum (panel serum No. 3), and by determining
the core antigen according to Example 6. The level of activity of
the core antigen in the 430 mg/dl hemoglobin addition group
relative to 100% of the no-hemoglobin addition group used as the
control is shown in Table 2. It was confirmed that when no urea is
added, the level of activity of the core antigen in the hemoglobin
addition group decreased by 30%, but by increasing the amount of
added urea the level of activity of the core antigen in the
hemoglobin addition group increased and interference by hemoglobin
decreased.
TABLE-US-00002 TABLE 2 Suppressive effect of urea on interference
by hemoglobin Additive % Relative to control No addition 30.0 0.5M
urea 36.3 1M urea 39.7 2M urea 43.0 3M urea 48.8 4M urea 53.7
[0154] On the other hand, since there is a possibility of the
interaction of each of amino acids with the heme and the buffering
effect by the amino group and the carboxyl group, various amino
acids were added and the degree of the effect was examined. The
result is shown in Table 3.
TABLE-US-00003 TABLE 3 Suppressive effect of various amino acids on
interference by hemoglobin Additive % Relative to control No
addition 22.7 0.1M histidine 53.7 0.1M tryptophan 70.8 0.1M
phenylalanine 45.8 0.1M leucine 25.9 0.1M glutamine 36.1 0.1M
lysine 42.1 0.1M arginine 31.4 0.1M glutamic acid 49.8 0.1M glycine
39.1 0.1M proline 31.2 0.1M serine 32.5
[0155] Tryptophan and histidine exhibited the most potent
suppressive effect on interference. The dose-dependency of the
suppressive effect on interference was studied and the result is
shown in Table 4.
TABLE-US-00004 TABLE 4 Suppressive effect of histidine and
tryptophan on interference by hemoglobin Additive % Relative to
control No addition 24.2 0.05M histidine 49.3 0.1M histidine 59.4
0.15M histidine 74.5 0.2M histidine 77.0 0.05M tryptophan 58.7 0.1M
tryptophan 71.5 0.15M tryptophan 77.9 0.2M tryptophan 89.0
[0156] Since the heme is coordinated by a side chain in hemoglobin
and retained in hemoglobin, the effect was suggested to be
attributable to the side chain. Accordingly, the effect of
imidazole, a side chain in histidine, and indoleacrylic acid
containing an indole ring, a side chain in tryptophan, were studied
and the result is shown in Table 5.
TABLE-US-00005 TABLE 5 Suppressive effect of imidazole and
indoleacrylic acid on interference by hemoglobin Additive %
Relative to control No addition 22.1 0.05 M imidazole 35.2 0.1 M
imidazole 42.0 0.15 M imidazole 58.8 0.2 M imidazole 70.7 5 mM
indoleacrylic acid 50.4 10 mM indoleacrylic acid 69.0 20 mM
indoleacrylic acid 90.3 30 mM indoleacrylic acid 96.8
[0157] When indole or indoleacrylic acid was added to the reaction,
a dose-dependent suppressive effect of interference by hemoglobin
was observed as with the addition of amino acids. This indicated
that by adding to the reaction a substance that contains an
imidazole ring, for example histidine, or an indole ring, for
example tryptophan, the sensitive detection of the core antigen can
be attained even for the samples that contains hemoglobin.
[0158] The effect of combination of the above additives was
studied. The result is shown in Table 8. By combining histidine and
tryptophan, recovery of 90% or greater was obtained, and the
addition of urea further increased detection sensitivity.
TABLE-US-00006 TABLE 6 Additive % Relative to control 0.1M
histidine/0.1M tryptophan 91.1 4M urea/0.1M Tris/0.1M histidine
112.6
Example 8
Analysis the Molecular Form Recognized in the Serum Treatment and
in the Assay Method
[0159] Each method of serum treatment was used to treat 0.25 ml of
the panel serum 13. The treated serum was fractionated on a gel
filtration column (Superdex 200HR, 1.times.30), and anti-core
immunological activity in the fractions was measured. The result is
shown in Table 8. The figure suggested that the molecules having a
molecular weight of about 20 to 30 kDa are being recognized and
that the core antigen in the virus has been released through the
disruption of the virus and the inactivation of the anti-core
antibody in the serum by the above-mentioned pretreatment.
Example 9
Assay Method of the Core Antigen in the HCV Structural Region in
the Serum
[0160] Sera determined to have 10.sup.3 to 10.sup.7 copies/ml of
HCV-RNA using AmpliCore HCV Monitor kit (Roche), a PCR method, and
normal human sera were used to quantitate the HCV core antigen in
the sera using the method described above.
[0161] As a standard serum the panel serum 50 (defined as 1 U/ml)
was serially diluted in 10 mM sodium phosphate buffer, pH 7.3,
containing 1% BSA, and treated in a similar manner. The result is
shown in Table 7. Of the samples tested, the core antigen in all
the normal human sera was below the detection limit and could be
detected in all of the PCR-positive samples. The correlation is
shown in FIG. 9, which revealed that the correlation with the PCR
method was also as high as 0.8 or greater.
TABLE-US-00007 TABLE 7 Levels of HCV-RNA and the core antigen RNA
core antigen Sample # (K copies/ml) (mU/ml) Normal human serum 1 --
N.D. 2 -- N.D. 3 -- N.D. 4 -- N.D. 5 -- N.D. Panel serum 81 1.6 2.1
80 8 2.1 82 8 8.5 33 16 3.7 31 30 37.0 26 87 266.7 39 97 63.8 41
170 116.1 16 400 133.7 50 1000 1000 45 1300 277.3 13 1600 1806
N.D.: Not detected
Example 10
Study on the Condition of Sample Treatment Study on Treatment
Conditions
1) Guanidine Hydrochloride Concentration
[0162] To 100 .mu.l of a normal human serum and HCV-RNA-positive
sera were added 100 .mu.l of the treatment solution containing a
different concentration of guanidine hydrochloride and 0.5 N HCl.
The mixtures were treated at room temperature for 30 minutes, and
80 .mu.l each of the treated mixtures was used as a sample. The
result obtained using the assay method described below is shown in
FIG. 10 with the guanidine hydrochloride concentration at the time
of treatment taken as the abscissa.
2) Triton X100 Concentration
[0163] To 100 .mu.l of a normal human serum and HCV-RNA-positive
sera were added 100 .mu.l of the treatment solution containing a
different concentration of Triton X100 (6 M guanidine
hydrochloride, 0.5 N HCl). The mixtures were treated at room
temperature for 30 minutes, and 80 .mu.l each of the treated
mixtures was used as a sample. The result obtained using the assay
method described below is shown in FIG. 11 with the Triton X100
concentration at the time of treatment taken as the abscissa.
3) Tween 20 Concentration
[0164] To 100 .mu.l of a normal human serum and HCV-RNA-positive
sera were added 100 .mu.l of the treatment solution containing a
different concentration of Triton X100 (6 M guanidine
hydrochloride, 0.5 N HCl, 12.5% Triton X100). The mixtures were
treated at room temperature for 30 minutes, and 80 .mu.l each of
the treated mixtures was used as a sample. The result obtained
using the assay method described below is shown in FIG. 12 with the
Tween 20 concentration at the time of treatment taken as the
abscissa.
4) Reaction Temperature
[0165] To 100 .mu.l of a normal human serum and HCV-RNA-positive
sera were added 100 .mu.l of the treatment solution (6 M guanidine
hydrochloride, 0.5 N HCl, 12.5% Triton X, 0.75% Tween 20). The
mixtures were treated at 4.degree. C., room temperature (23.degree.
C.), 37.degree. C., and 45.degree. C. for 30 minutes, and 80 .mu.l
each of the treated mixtures were used as a sample. The result
obtained using the assay method described below is shown in FIG.
13.
Assay Methods
[0166] Samples obtained in the study on the condition of serum
treatment were each evaluated using the respective assay method
described below. Thus, an anti-HCV core antigen monoclonal antibody
(a mixture of equal amounts of antibody C11-14 and C11-11) was
diluted to a final total concentration of 6 .mu.l g/ml in 0.1 M
carbonate buffer, pH 9.6, and 100 .mu.l each of the dilutions was
dispensed per well of a 96-well microtiter plate (manufactured by
Nunc). After the plate was incubated overnight at 4.degree. C., it
was washed twice with 0.35 ml of 10 mM sodium phosphate buffer, pH
7.3, containing 0.15 M NaCl. Then, 0.35 ml of 10 mM sodium
phosphate buffer, pH 7.35, containing 0.5% casein-Na (hereinafter
referred to as the blocking solution) was added and the plate was
further incubated at room temperature for 2 hours.
[0167] After the blocking solution was removed, 160 .mu.l of the
mixture of 140 .mu.l of 100 mM sodium phosphate buffer, pH 7.3,
containing 0.15 M NaCl, 1% BSA, 0.5% casein-Na, and 0.05% Tween 20,
and 20 .mu.l of 1 M Tris (hereinafter referred to as the reaction
buffer), and samples for measurement obtained by the
above-mentioned serum treating method were added into respective
wells, incubated at room temperature for 2 hours, washed five times
with 300 .mu.l of the wash solution, and then 100 .mu.l of the
peroxidase (POD)-labeled monoclonal antibody (C11-10) was added and
was incubated at room temperature for 30 minutes. After the
incubation was over, the plate was washed five times with 300 .mu.l
of the above wash solution. One hundred microliters of the
substrate (ortho-phenylene diamine, hereinafter referred to as OPD)
solution was added to the plate and the plate was incubated at room
temperature for 30 minutes, followed by the addition of 100 .mu.l
of 2 N sulfuric acid solution. Absorbance was measured at a
wavelength of 492 nm (OD492) with the absorbance at 630 nm as a
reference.
[0168] Each treatment condition was optimized as shown in FIGS. 10
to 13. It was difficult to detect the core antigen in the untreated
samples, but such a simple treatment drastically enabled the
detection of the core antigen. In any case no enhancement in
signals was observed in the healthy humans. It was also shown that
the core antigen can be satisfactorily detected by employing the
condition of guanidine hydrochloride at 2 M or greater and Triton
X100 at 0.2% or greater, and a temperature range of 4.degree. C. to
45.degree. C.
Example 11
The Detection and Assay Method of the Core Antigen
[0169] To 100 .mu.l of serum was added 100 .mu.l of a treatment
solution (6 M guanidine hydrochloride, 0.5 N HCl, 12.5% Triton
X100, 0.75% Tween 20). It was treated at room temperature for 30
minutes, and 100 .mu.l of the treated mixture was used as a
sample.
[0170] An anti-HCV core antigen monoclonal antibody (a mixture of
equal amounts of C11-14 and C11-11) was diluted to a final total
concentration of 6 .mu.g/ml in 0.1 M carbonate buffer, pH 9.6, and
100 .mu.l each of the diluted mixture was dispensed per well of a
96-well microtiter plate (manufactured by Nunc).
[0171] After the plate was incubated overnight at 4.degree. C., it
was washed twice with 0.35 ml of 10 nM sodium phosphate buffer, pH
7.3, containing 0.15 M NaCl. Then, 0.35 ml of the blocking solution
was added and the plate was further allowed to stand at room
temperature for 2 hours. After the blocking solution was removed,
150 .mu.l of the reaction buffer and samples for measurement
obtained in the above treating method were added into respective
wells, and incubated at room temperature for 2 hours.
[0172] The plate was washed five times with 300 .mu.l of the wash
solution, and then 100 .mu.l of a peroxidase (POD)-labeled
monoclonal antibody (C11-10) was added to the plate. The plate was
incubated at room temperature for 30 minutes. Then the plate was
washed five times with 300 .mu.l of the wash solution, and 100
.mu.l of the substrate (OPD) solution was added. After incubating
the plate at room temperature for 45 minutes, 100 .mu.l of 2 N
sulfuric acid solution was added. Absorbance was measured at a
wavelength of 492 nm (OD492) with the absorbance at 630 nm as a
reference. As a standard serum, the panel serum 50, defined as 1
U/ml, was serially diluted in 10 mM sodium phosphate buffer, pH
7.3, containing 1% BSA, which was similarly treated and
measured.
[0173] FIG. 14 shows a dilution line of the panel serum 50 used as
a standard serum. The core antigen in the sample was determined in
a dose-dependent manner and could be detected to a level as low as
about 0.5 mU/ml. It was demonstrated, therefore, that by combining
a very simple method of sample treatment and the monoclonal
antibody of the present invention, the HCV core antigen can be
detected or quantitated.
Example 12
Analysis the Molecular Form Recognized in the Serum Treatment and
in the Assay Method
[0174] Each method of serum treatment was used to treat 0.25 ml of
the panel serum 13. The treated serum was fractionated by a gel
filtration column (Superdex 200HR, 1.times.30), and anti-core
immunological activity in the fractions was measured. The result is
shown in Table 15. The figure suggested that molecules having a
molecular weight of about 20 to 30 kDa are being recognized and
that the core antigen in the virus has been released from various
interactions through the disruption of the virus and the
inactivation of the anti-core antibody in the serum by the
above-mentioned pretreatment.
Example 13
Assay Method of the Core Antigen in the Serum
[0175] Sera determined to have 10.sup.3 to 10.sup.7 copies/ml of
HCV-RNA using AmpliCore HCV Monitor kit (Roche), a PCR method, and
normal human sera were used to quantitate the HCV core antigen in
the sera using the method described above.
[0176] As a standard serum the panel serum 50 (sefined as 1 U/ml)
was serially diluted in 10 mM sodium phosphate buffer, pH 7.3,
containing 1% BSA, and treated in a similar manner. The result is
shown in Table 8. Of the samples tested, the core antigen in all
the normal human sera was below the detection limit and could be
detected in all of the PCR-positive samples. The correlation is
shown in FIG. 16, which revealed that the correlation with the PCR
method was also as high as 0.8 or greater.
TABLE-US-00008 TABLE 8 Levels of HCV-RNA and the core antigen RNA
Core antigen Sample # (K copies/ml) (mU/ml) Normal human serum 1 --
N.D. 2 -- N.D. 3 -- N.D. 4 -- N.D. 5 -- N.D. 6 -- N.D. 7 -- N.D.
Panel serum 1 50 166.4 7 830 471.1 8 26 61.5 11 240 107.4 13 1600
1426 15 25 40.1 16 400 240.3 19 840 1369 26 87 1093 31 30 45.8 33
16 58.5 39 97 89.0 41 170 43.9 44 180 57.5 49 33 47.7 50 1000 1005
84 8.7 63.5 N.D.: Not detected
Example 14
Detection of the Hepatitis B Virus (HBV) Core Antigen
[0177] We have so far explained the detection of the HCV core
antigen. We have investigated whether this treating method is
applicable to the detection of structural proteins in other
viruses.
[0178] A monoclonal antibody (Tokushu Menneki Kenkyuusho [Special
Immunology Research Institute]) against HBV core antigen was
diluted to a concentration of 3 .mu.g/ml in 0.1 M carbonate buffer,
pH 9,6, and was dispensed in an aliquot of 100 .mu.l. After
incubating overnight at 4.degree. C., the plate was washed with a
phosphate buffer, and a 350 .mu.l aliquot of 1% BSA solution was
dispensed to the plate. After allowing to stand at room temperature
for 2 hours, the 1% BSA solution was aspirated off, and 200 .mu.l
of the reaction solution was added.
[0179] A recombinant HBV core antigen was used as a standard, and
five patient sera that tested positive for HBe antigen and negative
for anti-HBe antibody and ten normal human sera were used as
samples. To 100 .mu.l of a sample, 50 .mu.l of a treatment reagent
(7.5% SDS, 0.75% CHAPS, 0.15% Triton X100, 2 M urea, 0.1 M
histidine, 0.1 M tryptophan) was added and treated at 56.degree. C.
for 30 minutes. After the treatment, 50 .mu.l thereof was added to
a well filled with the reaction solution, and was incubated at room
temperature for 90 minutes.
[0180] As a comparison (without pretreatment), 100 .mu.l of each
sample was diluted with 50 .mu.l of purified water and 50 .mu.l of
the diluted sample was used for the reaction. After washing five
times with the wash solution, a biotin-labeled anti-HBV core
monoclonal antibody (a mixture of equal amounts of HBc-2, HBc-5,
HBc-14) was added, and incubated at room temperature for 30
minutes. After washing five times with the wash solution, the
avidin-labeled alkaline phosphatase was added and the mixture was
reacted at room temperature for 30 minutes.
[0181] After washing five times with the wash solution, CDPstar
(Emerald II as the sensitizer) was added, reacted at room
temperature for 15 minutes, and relative chemiluminescence thereof
was measured. A standard curve for a serially diluted recombinant
HBV core antigen is shown in FIG. 17, and the amount of the core
antigen in the measured samples is shown in Table 9. The detection
limit was 21 ng/ml. When a cut-off value that distinguishes the
core antigen-positive from the negative was defined at 60 ng/ml,
all 10 normal human sera, with or without pretreatment, tested
negative for the core antigen, and in the sera of patients with
hepatitis B virus, the core antigen could not be detected in the
case of no pretreatment, but with pretreatment, all the sera tested
positive for the core antigen.
[0182] It is thought that in the sera of patients with the
hepatitis B virus, pretreatment disrupted the virus particle and
inactivated the anti-HBc antibody, thereby enabling the detection
of the core antigen. From the foregoing, it was confirmed that this
method of sample treatment is useful for the detection of the
structural proteins of viruses other than HCV, such as HBV, that
have DNA as the genome. Needless to say, this holds true for
HCV-related viruses such as flaviviruses and retroviruses, for
example HIV.
TABLE-US-00009 TABLE 9 non-treated pre-treated HBV core Ag HBV core
Ag Sample # (ng/ml) Judged (ng/ml) Judged Normal human 1 <21
Neg. <21 Neg. sample 2 <21 Neg. <21 Neg. 3 <21 Neg.
<21 Neg. 4 <21 Neg. <21 Neg. 5 <21 Neg. 46 Neg. 6
<21 Neg. <21 Neg. 7 <21 Neg. 47 Neg. 8 <21 Neg. <21
Neg. 9 <21 Neg. 26 Neg. 10 <21 Neg. 56 Neg. HBV sample 11
<21 Neg. 98 Pos. 15 <21 Neg. 94 Pos. 20 <21 Neg. 780 Pos.
21 <21 Neg. 270 Pos. 46 <21 Neg. 630 Pos.
Example 15
Method for Effective Detection without Pretreatment of the
Antigen
[0183] HCV particle-containing samples were diluted in a
surfactant-added reaction solution, and the efficiency of detecting
the HCV antigen was investigated.
[0184] The detection of the HCV core antigen was carried out by a
sandwich enzymeimmunoassay (EIA) using monoclonal antibody against
the HCV core antigen. Among the monoclonal antibodies obtained in
Example 3, C11-3 and C11-7 were used as the antibody for capturing
the core antigen and C11-10 and C11-14 were used as the antibody
for detecting the captured core antigen.
[0185] EIA was essentially carried out using the following
conditions. Solutions of monoclonal antibodies C11-3 and C11-7,
each of which was diluted to 4 .mu.g/ml in an acetate buffer, were
added to a microtiter plate and were incubated overnight at
4.degree. C. After washing with the phosphate buffer, a phosphate
buffer containing 1% BSA, was added to effect blocking. To the
plate were added 100 .mu.l of the reaction solution and 100 .mu.l
of the sample. The plate was then stirred and incubated at room
temperature for 1.5 hour. Unreacted substances were removed by
washing with the phosphate buffer to which a low concentration of a
surfactant had been added. Then the alkaline phosphatase-labeled
monoclonal antibodies C11-10 and C11-14 were added and reacted at
room temperature for 30 minutes. After the reaction is over,
unreacted substances were removed by washing with the phosphate
buffer to which a low concentration of a surfactant had been added.
Then a substrate solution (CDP-Star/Emeraldll) was added and
reacted at room temperature for 20 minutes. The amount of
luminescence was measured.
[0186] To the above reaction, various surfactants were added to
investigate their effects. By using HCV-positive sera in which the
titer of antibody to HCV is below the detection limit and virtually
no antibody to HCV is contained, the activity of the core antigen
based on the amount of luminescence was expressed in terms of a
reaction ratio relative to the amount of luminescence of the normal
human serum that was defined as 1.0. The results are shown in
Tables 10 and 11.
TABLE-US-00010 TABLE 10 Reactivity relative to normal human serum
(S/N ratio) HLB (%) NO 45 NO 46 NO 3 NO 7 NO 19 No addition 15.67
1.00 1.15 1.34 1.19 Judgement criteria >30.0 >2.0 >2.0
>2.0 >2.0 Additive Anionic surfactant sodium dodecyl sulfate
40.0 0.5 5.42 2.0 5.73 sodium dodecyl-N-sarcosinate 0.5 12.79 2.70
2.0 125.43 7.27 3.83 3.70 6.71 perfluoroalkylcarboxylic acid 0.5
10.55 1.27 S-113 2.0 6.72 0.91 Cationic surfactant
cetyltrimethylammonium bromide 0.5 72.97 7.42 3.09 3.52 5.43 2.0
44.55 5.35 dodecylpyridinium chloride 0.5 53.43 4.70 2.05 1.52 2.33
2.0 12.44 2.49 n-dodecyltrimethylammonium 0.5 66.84 4.43 2.41 1.63
2.67 2.0 27.98 3.77 tetradecylammonium bromide 0.05 14.69
n-octyltrimethylammonium chloride 0.5 12.57 1.00 0.74 0.99 2.0
11.46 n-decyltrimethylammonium chloride 0.5 17.50 0.88 0.80 0.72
2.0 45.21 1.12 1.08 1.41 Amphoteric surfactant CHAPS 0.5 29.57 2.0
25.32 1.63 1.82 2.42 perfluoroalkylbetaine S-132 0.5 11.07 1.61
(from ASAHI GLASS) 2.0 10.77 1.49 3-(dodecyldimethylammonio)- 0.5
57.69 1-propane-sulfonic acid 2.0 113.19 4.57 3.44 5.26
TABLE-US-00011 TABLE 11 Reactivity relative to normal human serum
(S/N ratio) HLB (%) NO 45 NO 46 NO 3 NO 7 NO 19 No addition 15.67
1.00 1.15 1.34 1.19 Judgement >30.0 >2.0 >2.0 >2.0
>2.0 criteria Additive nonionic surfactant MEGA-10 0.5 32.11
3.38 2.0 38.49 3.53 1.97 1.87 2.84 Tween 20 16.7 0.5 16.88 2.0
12.36 Tween 40 15.6 0.5 14.96 1.02 0.99 1.41 2.0 19.10 1.32 1.25
1.64 Tween 80 15.0 0.5 12.45 1.33 1.23 1.10 2.0 17.47 Nonidet P-40
13.1 0.5 43.14 3.09 2.95 4.58 octyl glucoside 0.5 12.48 0.90 0.60
0.97 2.0 25.07 1.92 1.20 2.63 Triton N101 13.4 0.5 26.50 1.85 1.62
2.70 2.0 60.84 2.23 2.28 3.81 Triton X100 13.5 0.5 27.72 2.0 71.08
2.90 2.34 3.86 Triton X114 12.4 0.5 31.49 2.04 1.65 2.77 2.0 58.62
1.92 2.11 2.51 Triton X305 17.3 0.5 10.50 0.94 0.97 1.08 2.0 25.91
1.30 1.24 1.87 Triton X405 17.9 0.5 12.54 0.86 0.78 1.04 2.0 24.92
1.21 1.24 1.25 Others benzyl- 0.5 5.45 1.00 dimethyl- ammonium
chloride 2.0 7.01 1.12 triethylamine 0.5 3.89 0.97 Surfactant
mixture 2% sodium 244.13 6.11 5.50 12.71 dodecyl-N- sarcosinate +
2% Triton X100
[0187] The results revealed that the addition of a nonionic
surfactant having an HLB of 12 to 14, as represented by Triton
X100, causes an increase in the amount of luminescence thereby
enhancing detection sensitivity in HCV-positive sera compared to
the normal human sera. It was also clarified that, similarly, as
represented by sodium dodecyl-N-sarcosinate and dodecyl
trimethylammonium, the addition of a surfactant having in its
structure a straight-chain alkyl group having at the same time 10
or more carbon atoms and a secondary, tertiary, or quaternary amine
causes an increase in detection sensitivity in HCV-positive sera.
No such increase in sensitivity was observed with the above
surfactant with an alkyl group having not more than 8 carbons
(n-octyl trimethylammonium chloride). It was also found that by
mixing and adding these two surfactants (in Table 11, 2% sodium
dodecyl-N-sarcosinate and 2% Triton X100 were mixed), detection
sensitivity in HCV-positive sera can be further enhanced.
Example 16
Detection of the Core Antigen in the Samples During a Period
between after HCV Infection and before the Appearance of Anti-HCV
Antibody (Window Period)
[0188] By adding 2% Triton X100 and 2% sodium dodecyl-N-sarcosinate
to the primary reaction solution, a commercially available
seroconversion panel PHV905 (B.B.I. inc.) was measured according to
Example 15. The PHV905 panel used turned positive on day 21 after
the start of observation (serum No. PHV905-7) when measured by the
anti-HCV antibody test (Ortho EIA 3.0). In the test, the antibody
titer is expressed in a cut-off index (S/CO) with a value of 1.0 or
greater being judged as positive. The activity of the HCV core
antigen (the amount of luminescence) was expressed in the
reactivity (S/N) relative to that of the normal human serum that
was defined as 1.0.
[0189] As shown in FIG. 12, the activity of the core antigen is
observed before the anti-HCV antibody appears, the addition of a
surfactant exposed the core antigen from the virus particle, which
reacted with the immobilized monoclonal antibody, thereby
confirming the detection of the core antigen.
TABLE-US-00012 TABLE 12 Days after start HCV core Ag activity
Anti-HCV Ab titer Serum No. of observation (S/N) (S/CO) PHV905-1 0
5.32 0.000 905-2 4 8.30 0.000 905-3 7 15.63 0.000 905-4 11 4.37
0.300 905-5 14 14.75 0.700 905-6 18 7.57 0.700 905-7 21 4.82 2.500
905-8 25 3.31 5.000 905-9 28 1.61 5.000
Reference to Microorganisms Defined in Rule 13-2 of the Rule Based
on Patent Cooperation Treaty
[0190] Name of depository: the National Institute of Bioscience and
Human Technology, Agency of Industrial Science and Technology
[0191] Address of depolsitory: 1-3, Higashi 1-chome, Tsukuba city,
Ibalaki pref., Japan (Zip code 305)
[0192] (1) Indication of Microorganism: HC11-3 [0193] Date
deposited: Jul. 4, 1997 [0194] Deposit number: FERM BP-6002
[0195] (2) Indication of Microorganism: HC11-7 [0196] Date
deposited: Jul. 4, 1997 [0197] Deposit number: FERM BP-6003
[0198] (3) Indication of Microorganism: HC11-10 [0199] Date
deposited: Jul. 4, 1997 [0200] Deposit number: FERM BP-6004
[0201] (4) Indication of Microorganism: HC11-11 [0202] Date
deposited: Jul. 4, 1997 [0203] Deposit number: FERM BP-6005
[0204] (5) Indication of Microorganism: HC11-14 [0205] Date
deposited: Jul. 4, 1997
Sequence CWU 1
1
81177PRTHepatitis C virus 1Met Lys Ala Ile Phe Val Leu Lys Gly Ser
Leu Asp Arg Asp Pro Glu1 5 10 15Phe Met Gly Thr Asn Pro Lys Pro Gln
Arg Lys Thr Lys Arg Asn Thr 20 25 30Asn Arg Arg Pro Gln Asp Val Lys
Phe Pro Gly Gly Gly Gln Ile Val 35 40 45Gly Gly Val Tyr Leu Leu Pro
Arg Arg Gly Pro Arg Leu Gly Val Arg 50 55 60Ala Thr Arg Lys Thr Ser
Lys Arg Ser Gln Pro Arg Gly Gly Arg Arg65 70 75 80Pro Ile Pro Lys
Asp Arg Arg Ser Thr Gly Lys Ser Trp Gly Lys Pro 85 90 95Gly Tyr Pro
Trp Pro Leu Tyr Gly Asn Glu Gly Leu Gly Trp Ala Gly 100 105 110Trp
Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp 115 120
125Pro Arg His Arg Ser Arg Asn Val Gly Lys Val Ile Asp Thr Leu Thr
130 135 140Cys Gly Phe Ala Asp Leu Met Gly Tyr Ile Phe Arg Val Gly
Ala Phe145 150 155 160Leu Gly Gly Ala Ala Arg Ala Leu Ala His Gly
Val Arg Val Leu Glu 165 170 175Asp2160PRTHepatitis C virus 2Met Gly
Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn1 5 10 15Arg
Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly 20 25
30Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala
35 40 45Thr Arg Lys Thr Ser Lys Arg Ser Gln Pro Arg Gly Gly Arg Arg
Pro 50 55 60Ile Pro Lys Asp Arg Arg Ser Thr Gly Lys Ser Trp Gly Lys
Pro Gly65 70 75 80Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Leu Gly
Trp Ala Gly Trp 85 90 95Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp
Gly Pro Thr Asp Pro 100 105 110Arg His Arg Ser Arg Asn Val Gly Lys
Val Ile Asp Thr Leu Thr Cys 115 120 125Gly Phe Ala Asp Leu Met Gly
Tyr Ile Phe Arg Val Gly Ala Phe Leu 130 135 140Gly Gly Ala Ala Arg
Ala Leu Ala His Gly Val Arg Val Leu Glu Asp145 150 155
160320PRTArtificial SequenceProbe for Hepatitis C virus 3Asp Val
Lys Phe Pro Gly Gly Gly Gln Ile Val Gly Gly Val Tyr Leu1 5 10 15Leu
Pro Arg Arg 20410PRTArtificial SequenceProbe for detecting
Hepatitis C virus 4Gly Pro Arg Leu Gly Val Arg Ala Thr Arg1 5
10521PRTArtificial SequenceProbe for detecting Hepatitis C virus
5Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro Arg His Arg1 5
10 15Ser Arg Asn Val Gly 20620PRTArtificial SequenceProbe for
detecting Hepatitis C virus 6Asp Pro Arg His Arg Ser Arg Asn Val
Gly Lys Val Ile Asp Thr Leu1 5 10 15Thr Cys Gly Phe
20724DNAArtificial SequenceProbe for detecting Hepatitis C virus
7gaattcatgg gcacgaatcc taaa 24821DNAArtificial SequenceProbe for
detecting Hepatitis C virus 8ttagtcctcc agaacccgga c 21
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