Patent Application
20090017443
The present invention relates to a method for treatment of a sample containing hepatitis B virus (herein after, referred to as “HBV”) to detect or quantify HBV antigens in blood with high sensitivity and a method for detection or quantification of HBV antigens using said method for treatment.
HBV is the virus identified for the first time as a causative virus of post-transfusion hepatitis and HBV infection occurs through blood transfusion at the time of surgery. Accordingly, for screening of blood for transfusion, it is extremely important to make diagnosis of the presence or absence of HBV infection in blood.
As diagnostic methods for this HBV infection, there is a method for detection of antibody to HBV in a sample, a method for detection of HBV antigens, or a method for detection of HBV genes.
Among these methods, the method for detection of HBV genes includes a nucleic acid amplification test (NAT) and a DNA probe test, which are currently widely used in clinical setting. Further, attention is directed to a relation between the amount of HBV DNA and the pathos is of HBV carrier by virtue of widespread use of the NAT method, and the NAT method has come to be mainly used for monitoring after treatment with an antiviral drug.
NAT methods such as PCR method and TMA method are highly sensitive methods for detecting gene fragments. However, when HBV genomic DNA is extracted from a sample, these methods require a treatment time as long as two hours in the manual method as well as include a plural process steps, and so forth, which is complicated. In addition, the complexity of this process increases chances of contamination and increases the possibility of having false positive samples. There is also a problem that technical skills are needed to obtain consistent assay values. Although the recent development of an automated instrument has allowed measures against contamination to be taken and the processing time for DNA extraction to be shortened, a still highly expensive instrument is required, and thus, is not in common use except in institutions where a large number of samples are handled. Further, since DNA primers must match the target gene, several kinds of primers need to be used, which gives rise to a problem that the cost per test becomes high as compared with that of immunoassays.
Because of these problems associated with the test for the detection of HBV genome, attention is paid to methods for detection of viral antigens. In HBV antigen tests, a method for detection of HBs antigen has been conventionally used for blood screening and a method for measurement of HBe antigen has been widely used for a proliferation marker of HBV.
In addition to these antigen tests, a method for direct detection of HBV core antigen (HBc antigen) has also been developed. Usuda et al. (Journal of Virological Methods, 72, 95-103, 1998) developed a method for detection of HBc antigen in serum using monoclonal antibodies having specificity for HBV core (HBc) antigen and showed that the method was clinically useful similarly to the above-described NAT method for detection of viral genome. This HBc antigen detection system is relatively tolerant to contamination because amplification procedures are not included in the detection process.
However, there are several problems left behind even in the above method. The process of sample treatment for measurement is complicated and is time-consuming, which is problematic when the method is intended for use in screening, monitoring, and the like. For treatment of a sample (serum), a multi-step treatment process including treatment with an anti-HBs polyclonal antibody (37° C., 2 hours), centrifugation (10 min), removal of supernatant, treatment with surfactants, treatment with an alkali (35 min) and addition of a neutralizing agent, is necessary for concentration of virus particles and removal of serum components. Since these steps involve highly skilled work, certain experience is essential to obtain reproducibility. Further, a minimum treatment time of about three hours is required. Furthermore, automation is difficult and simultaneous mass treatment is also difficult because steps such as centrifugation and removal of supernatant are involved; thus in terms of the process as well, the above method is not appropriate for use that requires mass treatment.
Further, Oshihara et al. have developed a method in which HBc antigen is assayed by means of treatment with an alkali, treatment with pronase, and addition of Nonidet P40 (NP-40) that is a nonionic surfactant and mercaptoethanol without performing the treatment with anti-HBs polyclonal antibodies (Japanese Patent Laid-Open No. 8-50133). However, this method indicates low sensitivity and the concentration of HBc antigen in the detection limit is equivalent to 2.2 pg/ml of the concentration of HBV-DNA which is estimated at the order of 105 to 106 copies/ml.
In addition to the above-described methods for detection of HBc antigen, a method for assay of HBV core-related antigens (HBcr antigens) to allow simultaneous assay of HBe antigen and HBc antigen (International Publication WO 02/14871 A1) and a method for assay of p22 cr antigen of HBV (HBV p22cr antigen) that forms an HBV virus-like particle (International Publication WO 04/22585 A1) have been developed. These methods are more sensitive than the methods for detection of HBc antigen but indicate still an unsatisfactory sensitivity when compared with the methods of measurement of HBV genome.
Patent Document 1: Japanese Patent Laid-Open No. 8-50133
Patent Document 2: International Publication WO 02/14871 A1
Patent Document 3: International Publication WO 04/22585 A1
Non-Patent Document 1: Journal of Virological Methods, 72, 95-103, 1998
Immunoassays can be performed easily and at a low cost; however, the current method for assay of HBe antigen that is used as a proliferation marker cannot measure HBe antigen occurring as immune complexes in the presence of anti-HBe antibodies. Further, the methods for assay of HBc antigen are not applied in clinical studies owing to the complexity of pretreatment as described above and insufficiency of sensitivity, although the amounts of HBc antigen correlate with the amounts of HBV DNA.
On the other hand, in the assay of HBV core-related antigens and in the assay of HBV p22cr antigen, pretreatment of a sample is carried out using a surfactant and heat (from 56 to 70° C.) to disrupt antibodies and virus particles, and then HBV core-related antigens or HBV p22cr antigens are measured.
However, these methods also need to treat a sample off-board and the adaptation to the full automation is difficult.
Accordingly, the object of the present invention is to provide a pretreatment method for assay of HBV core-related antigens (HBe and HBc antigens), HBV p22cr antigen, and the like even in the presence of anti-HBV antibodies for screening of hepatitis B, monitoring in the treatment of patients with chronic hepatitis B, and so forth, and an assay method with the use thereof. In other words, the object is to provide a system for detection of HBV antigens that can be easily applied to a mass treatment system such as automation by simple pretreatment in shorter time.
As a result of assiduous research intended to solve the above problems, the present inventors focused attention on (a) a method for treatment of a sample containing HBV that allows HBV antigens in the sample to be converted into a state suitable for detection with a probe only by a simple procedure in a short time and (b) a method for treatment that allows antibodies against HBV antigens originating from a host that compete with a probe for capture or detection to be simultaneously inactivated by the simple procedure in a short time in order to detect HBV antigens in the sample. Further, the present inventors found that, for assay of HBV antigens, not only can HBV antigens present in a sample be released from virus particles or immune complexes but also human antibodies against HBV present in the sample are inactivated by (c) treatment of the sample with an acidifying agent and (d) treatment with a surfactant, a protein denaturant, and a reducing agent in addition to the former treatment, and that (e) a sample most suitable for an immunoassay with a probe such as antibody can be provided by the use of the treatment method. Furthermore, the present inventors found it possible to provide (f) a step of treating a sample with a treatment agent that releases HBV antigens present in the sample containing HBV antigens from the virus particles and that also simultaneously inactivates the human antibodies against HBV present in the sample, a method for detection and quantification of HBV antigens by an immunoassay involving the treatment step, and (g) an HBV antigen assay kit containing the treatment agent, and achieved the present invention based on these findings.
Thus, the following items 1 to 3 shown below are provided according to the present invention:
1. A method for treatment of a sample containing HBV characterized in that release of HBV antigens and inactivation of antibodies that bind to HBV antigens are performed by treating a sample containing HBV with a treatment agent containing
(1) an acidifying agent and
(2) a surfactant and/or a protein denaturant.
2. A method for immunological detection of HBV antigens including
(1) a step of conducting the treatment of a sample containing HBV according to the preceding item 1 and
(2) a step of detecting HBV antigens with the use of a probe that binds to the HBV antigens.
3. A diagnostic reagent or a diagnostic kit containing the acidifying agent (1) and at least one substance selected from the group (2) that are described below in the treatment agent for treating a sample to detect HBV antigens:
(1) an acidifying agent; and
(2) an amphoteric surfactant having an alkyl group and a tertiary amine or a quaternary ammonium salt within the same molecule, a cationic surfactant having an alkyl group and a tertiary amine or a quaternary ammonium salt within the same molecule, a nonionic surfactant, and a protein denaturant.
Further, a preferred embodiment of the method for treatment of a sample containing HBV includes the following 1) or 2):
1) a method for treatment of a sample containing HBV in which release of HBV antigens and inactivation of antibodies that bind to HBV antigens are carried out by treating a sample containing HBV with a treatment agent containing
(1) an acidifying agent and
(2) any one of a protein denaturant, an amphoteric surfactant having an alkyl group and a tertiary amine or a quaternary ammonium salt within the same molecule, a cationic surfactant having an alkyl group and a tertiary amine or a quaternary ammonium salt within the same molecule, and a nonionic surfactant.
2) a method for treatment of a sample containing HBV characterized in that release of HBV antigens and inactivation of antibodies against HBV antigens are carried out by treating a sample containing HBV with a treatment agent containing
(1) an acidifying agent and
(2) combination of any two or more kinds of an amphoteric surfactant having an alkyl group and a tertiary amine or a quaternary ammonium salt within the same molecule, a cationic surfactant having an alkyl group and a tertiary amine or a quaternary ammonium salt within the same molecule, a protein denaturant, a nonionic surfactant, and a reducing agent.
According to the present invention, it becomes possible to release HBV antigens easily in a short time from the virus particles in a state suitable for an immunoassay method in which an antigen is detected with a probe such as antibody as well as to inactivate antibodies against HBV antigens. Further, it becomes possible to detect and quantify HBV antigens easily in a short time with high sensitivity by treating a sample containing HBV according to the method described in the present invention and subjecting to the immuno assay method in which an antigen is detected with a probe such as antibody. Furthermore, according to the present invention, it is possible to solve the problem of precipitation caused by acid treatment by the use of the surfactant and the like in addition to the acidifying agent, disrupt protein efficiently, release viral antigens with ease in a short time, and bring about a remarkably excellent sensitivity-enhancing effect.
Samples used in the method for treatment of a sample containing HBV antigens according to the present invention include biological fluids such as whole blood, plasma, serum, urine, saliva, and cerebrospinal fluid, liver tissues, and the like.
The infectious particle of HBV is thought to be Dane particle having a structure with a diameter of 42 nm. The envelope lipoproteins are HBs antigens, and HBc antigens form an inner nucleocapsid (core particle) with a diameter of 27 nm. In addition, there is HBV p22cr antigen that forms an HBV nucleocapsid-like particle, and this molecule is thought to form a core-like particle and have HBs antigen on its outside.
The diagnosis of hepatitis B is generally performed by detecting HBs antigen or HBe antigen. However, the measurement of these antigens do not accurately reflect the time of the infection and the amount of infectious particles. For this reason, it is necessary to determine HBc antigen, HBV core-related antigens, and HBV p22cr antigen that make up the virus particle or the virus-like particle.
In samples, HBc antigen and p22cr antigen form the virus particle and HBe antigen and the like forms immune complexes with anti-HBV antibodies. In order to detect HBc antigen, HBe antigen, and HBV p22cr among these antigens, it is necessary to I) allow HBc antigen and HBV p22cr antigen not only to be released from HBV particles by disrupting the HBV particles but also to be converted to their monomer forms as much as possible, II) inactivate or remove antibodies, originating from a host, against HBc antigen and HBe antigen of HBV, and III) release HBc antigen, HBe antigen, and HBV p22cr antigen from interactions with blood components other than the antibodies against the HBV antigens. Although the antibodies against the HBV antigens can be removed by centrifugation and affinity chromatography, treatment steps increase, and therefore, it seems desirable to carry out the inactivation.
A maximum release of HBc antigen, HBe antigen, and HBV p22cr antigen, contained in a limited amount of sample in a detection system, in their monomer states from HBV particles, antibodies against HBV antigens, and other blood components results in an increase of the number of the antigen molecules that can react with a probe. It is important to maximally release the antigens in their monomer states by a short-time and simple sample treatment, thereby enhancing their reactivity with a probe.
As the conditions to inactivate the activities of antibodies present in a sample, an alkaline treatment, an acid treatment, and the like are known. When serum and the like are subjected to an acid treatment, certain serum-derived proteins and the like are irreversibly denatured and precipitation or cloudiness occurs in certain cases. Therefore, when a sample after treated with an acid is pipetted, trouble such as clogging often occurs. Further, in measurement, precipitates entangling denatured proteins and the like may adsorb to a carrier or solid phase linked with a probe such as antibody to capture a target antigen, resulting in a false positive. In addition, the target antigen is entangled in those precipitates and the amount of the antigen that can be bound to the probe is decreased, thereby presenting a problem of sensitivity reduction.
The present invention makes it possible to achieve prevention of precipitation and cloudiness caused by the acid treatment, prevention of false positive, and enhancement of sensitivity by adding another substance to an acidifying agent.
Here, as the acidifying agent, hydrochloric acid, sulfuric acid, acetic acid, trifluoroacetic acid, trichloroacetic acid, and the like are appropriate. In particular, the concentration of the acidifying agent at the treatment is preferably 0.05 N or higher and 1 N or lower, and further preferably from 0.25 N to 1 N. In this case, a sample added acidifying agent is treated at pH 2.5 or lower, and in most samples at pH 2.0 or lower.
One of the substances added to the acidifying agent in the treatment agent includes a surfactant. Various surfactants are known to have an activity to disrupt a higher structure of protein and exert effects such as disruption of viral particle membrane, denaturation of antibodies, and solubilization of insoluble proteins. However, in the presence of such a surfactant, a conformational epitope of a target antigen is also disrupted, resulting in weakening of binding to a probe such as antibody to capture the antigen, which presents a serious problem of sensitivity reduction.
On the other hand, the denaturing activity of the surfactant may often be reversible, and a temporarily denatured structure is sometimes returned to the original structure by reducing the concentration of the surfactant by means of dilution or dialysis. Therefore, the antibodies originating from a sample may compete with a probe for measurement, and as the result, it is apparent that sensitivity may be reduced. Thus, the addition of the surfactant has such an ambivalent nature described above. Surfactants are classified into various groups according to their structures and properties. For example, there are ionic and nonionic surfactant, and the ionic surfactant further include anionic, cationic, amphoteric surfactants, and the like.
The present inventors have found that the problem associated with the acid treatment such as occurrence of precipitates and the problem associated with the surfactant treatment such as reactivation of antibodies in a sample can be solved by combining the acidifying agent with the surfactant, and that the combination shows a significant enhancement effect in sensitivity with respect to the detection of HBV antigens.
Particularly among surfactants, the present inventors have found that a striking effect is obtained by using an amphoteric surfactant having an alkyl group and a tertiary amine or a quaternary ammonium salt within the same molecule or a cationic surfactant having an alkyl group and a tertiary amine or a quaternary ammonium salt within the same molecule.
Further, a remarkably striking effect is obtained by combining an acidifying agent with an amphoteric surfactant having a straight chain alkyl group of 12 or more carbon atoms and a tertiary amine or a quaternary ammonium salt within the same molecule or a cationic surfactant having a straight chain alkyl group of 12 or more carbon atoms and a tertiary amine or a quaternary ammonium salt within the same molecule.
Furthermore, it has been found that the addition of a nonionic surfactant, e.g. polyoxyethylene iso-octylphenyl ethers such as Triton X-100 or polyoxyethylene sorbitan alkyl esters such as Tween 20, the addition of a protein denaturant such as urea or thiourea, and the addition of a reducing agent such as cysteine, cysteamine, dimethyl-aminoethanethiol, diethylaminoethanethiol, diisopropyl-aminoethanethiol, or dithiothreitol to the treatment agent containing the acidifying agent and the surfactant are more preferable.
The present invention provides the method for treatment of a sample containing HBV characterized in that release of HBV antigens and inactivation of antibodies bound to HBV antigens are carried out by treating a sample containing HBV with a treatment agent containing (1) an acidifying agent, (2) an amphoteric surfactant having an alkyl group and a tertiary amine or a quaternary ammonium salt within the same molecule or a cationic surfactant having an alkyl group and a tertiary amine or a quaternary ammonium salt within the same molecule, a nonionic surfactant, and further a protein denaturant, and (3) a reducing agent.
As the amphoteric surfactant having an alkyl group and a tertiary amine or a quaternary ammonium salt within the same molecule, N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, N-octadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, and the like are appropriate.
Further, as the cationic surfactant having an alkyl group and a tertiary amine or a quaternary ammonium salt within the same molecule, decyltrimethylammonium chloride, dodecyltrimethylammonium chloride, tetradecyltrimethyl-ammonium chloride, hexadecyltrimethylammonium chloride, decyltrimethylammonium bromide, dodecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide, hexadecyl-trimethylammonium bromide, lauryl pyridinium chloride, tetradecyl pyridinium chloride, cetyl pyridinium chloride, and the like are appropriate.
The concentration at the treatment of such an amphoteric surfactant or cationic surfactant having an alkyl group and a tertiary amine or a quaternary ammonium salt within the same molecule is preferably 0.1% or higher and 15% or lower, further preferably from 0.5% to 10%.
As the nonionic surfactant added to the acidifying agent and the amphoteric surfactant or cationic surfactant having an alkyl group and a tertiary amine or a quaternary ammonium salt within the same molecule, polyoxyethylene isooctylphenyl ethers such as Triton X-100, polyoxyethylene nonylphenyl ethers such as NP40 or polyoxyethylene sorbitan alkyl esters such as Tween 80 are appropriate, and their concentrations at the treatment are preferably 1% or higher and 7.5% or lower, further preferably 1% or higher and 5% or lower.
As the protein denaturant added to the acidifying agent and the amphoteric surfactant or cationic surfactant having an alkyl group and a tertiary amine or a quaternary ammonium salt within the same molecule, urea, thiourea, and the like are appropriate, and their concentrations at the treatment are preferably 0.5 M or higher, further preferably 1 M or higher and 8 M or lower. However, in the case of no problem of solubility, for example, when urea is added in powder form in advance in a tube for treating a sample, it is possible to use at concentrations up to 10 M.
As the reducing agent added to the acidifying agent and the amphoteric surfactant or cationic surfactant having an alkyl group and a tertiary amine or a quaternary ammonium salt within the same molecule, cysteine, cysteamine, dimethylaminoethanethiol, diethylaminoethanethiol, diisopropylaminoethanethiol, dithiothreitol, and the like are appropriate, and their concentrations at the treatment are preferably 0.25 mM or higher and 1000 mM or lower, further preferably 1.5 mM or higher and 200 mM or lower.
As described above, an additional substance added to the acidifying agent includes a protein denaturant such as urea. Such a protein denaturant is known to have an activity to partially disrupt protein conformation by weakening hydrogen bonds, and it can disrupt viral particle membrane and denature antibodies against a target antigen in a sample. It has also an effect of solubilizing insoluble precipitates, for example, solubilizing a recombinant protein expressed in E. coli from its inclusion body that is an insoluble fraction. In the presence of a protein denaturant such as urea, however, a conformational epitope of the target antigen is also disrupted, resulting in weakening of binding to a probe such as antibody to capture the antigen, which presents a problem of sensitivity reduction.
On the other hand, the denaturing activity of the protein denaturant such as urea may often be reversible, and a temporarily denatured structure is sometimes returned to the original structure by reducing the concentration of the protein denaturant by means of dilution or dialysis. This results in a state in which antibodies originating from a sample may compete with a probe for measurement, and as the result, it is apparent that sensitivity may be reduced. Thus the addition of the protein denaturant such as urea has such an ambivalent nature described above.
The present inventors have perfected another invention of the present inventions by finding that the problem associated with the acid treatment such as occurrence of precipitates and the problem associated with the protein denaturant treatment such as reactivation of antibodies in a sample can be solved by combining the acid treatment with the protein denaturant treatment.
The present inventors have found that the formation of precipitates by the acid treatment can be significantly decreased by adding urea, one of protein denaturants, at 1 M or higher concentration at the treatment. For this protein denaturant, urea, thiourea, and the like are appropriate. Further, the concentration of the protein denaturant at the treatment is preferably 1 M or higher, further preferably 1.5 M or higher and 8 M or lower. Furthermore, the present inventors have found that the addition of a nonionic surfactant, e.g. polyoxyethylene isooctylphenyl ethers such as Triton X100 and polyoxyethylene sorbitan alkyl esters such as Tween 20, to the treatment agent containing the acidifying agent and the protein denaturant exerts an effect on enhancement of sensitivity. In addition, it is possible to add a reducing agent to the treatment agent containing the acidifying agent and the protein denaturant.
In summary of the above, the present invention provides a method for treatment of a sample containing hepatitis B virus (HBV) characterized in that release of HBV antigens and inactivation of antibodies bound to HBV antigens are carried out by treating a sample containing HBV with a treatment agent containing (1) an acidifying agent and (2) an amphoteric surfactant or cationic surfactant having an alkyl group and a tertiary amine or a quaternary ammonium salt within the same molecule, or a protein denaturant.
Further, the treatment temperature in the method for treatment of a sample containing HBV according to the present invention may be high, but preferably from 20° C. to 50° C., further preferably from 25° C. to 42° C.
Among the treatment agents combined with the acidifying agent in the present invention, the most preferred surfactant is the amphoteric surfactant having an alkyl group and a tertiary amine or a quaternary ammonium salt within the same molecule or the cationic surfactant having an alkyl group and a tertiary amine or a quaternary ammonium salt within the same molecule, and another treatment agent is the protein denaturant. To these two treatment agents, the nonionic surfactant is added, and in addition, the reducing agent is added, by which an enhancement of treatment effect was found (refer to Example 4). This indicates that the combination of treatment agents leads to the enhancement of treatment effect. As a treatment agent combined with the acidifying agent, there is, for example, the amphoteric surfactant having an alkyl group and a tertiary amine or a quaternary ammonium salt within the same molecule, the cationic surfactant having an alkyl group and a tertiary amine or a quaternary ammonium salt within the same molecule, the protein denaturant, the nonionic surfactant, the reducing agent, or an anionic surfactant. By combining two or more of these treatment agents and treating simultaneously with the acidifying agent, it is possible to efficiently treat a sample containing HBV.
The method for immunological detection of HBV antigens according to the present invention comprises the steps of releasing HBV antigens and inactivating antibodies that are binding to HBV antigens by allowing HBV-containing samples to come in contact with the treatment agent containing the acidifying agent and the surfactant and/or the protein denaturant (step 1) and detecting the HBV antigens with the use of a probe that binds to the HBV antigens (step 2).
In the step 2, as the probe used for the detection, for example, an antibody that specifically binds to an HBV antigen, any molecule that exhibits a high affinity for HBV antign can be used. It is desirable that one of the probes to capture HBV core-related antigens in a sample that has been treated in the step 1 is, for example, a monoclonal antibody such as HB44, HB114, or HB61.
The probe referred herein is, for example, a polyclonal antibody obtained by immunizing an experimental animal such as mouse, rat, guinea pig, rabbit, chicken, goat, sheep, or bovine, a monoclonal antibody produced by a hybridoma that is obtained by fusing the spleen cells and the like isolated from an immunized individual and myeloma cells or a monoclonal antibody produced by a cell line that is obtained by immortalizing spleen cells from an immunized individual or leukocytes in the blood using EB virus, a polyclonal antibody produced by human, chimpanzee, or the like that is infected with HBV, or a molecule exhibiting high specificity and affinity to HBV antigen that is produced by recombinant technology from a variable region gene fragment obtained from a cDNA or a chromosomal DNA of immunoglobulin of mouse, human, or the like, or a variable region gene fragment constructed by combining part of cDNA or chromosomal DNA of immunoglobulin, with an artificially prepared sequence.
In the method for immunological detection of HBV antigens according to the present invention, an HBV antigen forms an immune complex with the monoclonal antibody as described above by an antigen-antibody reaction. This immune complex is formed by a sandwich immunoassay system using two or more kinds of antibodies. The presence of the HBV antigen can be detected as a signal by a color development method or a chemiluminescence method using a labeling enzyme present in this immune complex. In addition, by directly binding an antibody with a fluorescent substance and so forth and allowing the fluorescent substance to be incorporated into an immune complex, HBV antigen can also be detected as a signal of the fluorescence.
Furthermore, the present invention provides a kit for diagnosis of HBV infection using the above immunological detection method. This diagnosis kit contains the acidifying agent and the protein denaturant and/or the surfactant in the treatment agent to treat a sample containing HBV. It is preferred that the kit contains a probe such as antibody that binds to an HBV antigen.
Hereinafter, the present invention is more specifically explained by means of the following Examples. However, it should be understood that these Examples do not limit the scope of the present invention in any way.
Concentration of acidifying agent: To 100 μl of an HBV antigen-negative sample or each of HBV antigen-positive samples (#990277, #990544), 100 μl each of aqueous hydrochloric acid at various concentrations was added and the mixture was incubated for 10 min at room temperature. Then 100 μl of the mixtureas samples for the assay was examined in the measurement method described below.
To a 96-well microplate (FluoroNunc Module, Maxisoap surface), 100 μl of a mixture of monoclonal antibodies against HBV core-related antigens (HB44, HB114, and HB61 were mixed in a ratio of two to one to one) at a concentration of 4 μg/ml were added to each well and the plate was incubated overnight at 4° C.
After washing twice with 10 mM phosphate buffer, pH 7.3, containing 0.15 M NaCl, 350 μl of 10 mM phosphate buffer, pH 7.1, containing 0.5% casein sodium was added to each well and the plate was incubated for two hours. After removing the blocking solution, 100 μl of a reaction buffer containing a neutralizing agent and the each test sample obtained by sample treatment methods were added to each well and the plate was incubated for two hours at room temperature with shaking, washed six times with 350 μl of 10 mM phosphate buffer, pH 7.3, containing 0.05% Tween 20 (washing solution), and then 100 μl of alkaline phosphatase (ALP)-labeled monoclonal antibodies (HB91 and HB110 were mixed in equal amounts) was added to each well, and the plate was incubated for 30 min at room temperature. After washing was conducted six times with the washing solution, 100 μl of a substrate solution (TROPIX, CDP-star with Emerald II) was added and the plate was incubated for 20 min.
Luminescence intensity was measured with a luminometer (DIA-IATRON, Luminous CT-9000D) and the result is shown in
Immunoreactivity of HBV core-related antigens could hardly be detected in HBV antigen-positive samples (#990277, #990544) incubated in a solution not containing hydrochloric acid for 10 min at room temperature. However, the immuno-reactivity of HBV core-related antigens started to be observed when the concentration of hydrochloric acid at the treatment was 0.05 N or higher and reached a peak at from 0.25 to 1.0 N. Further, when the study was carried out using sulfuric acid in place of hydrochloric acid, almost the same result was obtained.
Concentrations of various surfactants in the presence of acidifying agent: To 100 μl of the HBV antigen-negative sample or each of the HBV antigen-positive sample (#990277, #990544, #990768), 100 μl of various surfactants dissolved in 1.0N aqueous hydrochloric acid was added and the mixture was incubated for 10 min at room temperature. 100 μl of the treated sample was used for the assay, and was subjected to examination in the method described in Example 1. The results are shown in Tables 1 to 5. In each table, the underlined portions of the measurement values indicate cases exceeding each judgment criterion.
According to Tables 1 to 5, a surfactant that showed reactivity higher than the criterion for each sample in at least one sample of the three samples was judged to have an effect to detect HBV core-related antigen sensitively. As the result, when various surfactants were added together with an acidifying agent such as hydrochloric acid or sulfuric acid, surfactants that greatly enhanced immunoreactivity of HBV core-related antigens in the HBV antigen-positive samples were found. The surfactants for which the effects of addition were observed were amphoteric surfactants having an alkyl group and a tertiary amine or a quaternary ammonium salt within the same molecule and cationic surfactants having an alkyl group and a tertiary amine or a quaternary ammonium salt within the same molecule.
Further, the effects of addition were also found in nonionic surfactants such as Triton X100 and Tween 20. Although anionic surfactants, sodium dodecyl sulfate (SDS) and lithium dodecyl sulfate (LDS), at a concentration equal to or higher than 0.5% produced cloudiness during reaction with the samples, their effects could be confirmed by dissolving after addition of the reaction buffer containing the neutralizing agent. A surfactant having a steroid skeleton such as CHAPS did not indicate an enhancement in reactivity. In addition, sodium N-lauroyl sarcosine, deoxycholic acid, and the like were examined, but their solubility was not sufficient in the presence of the acidifying agent.
An increase in the sensitivity was observed by adding to the acidifying agent an amphoteric surfactant having an alkyl group and a tertiary amine or a quaternary ammonium salt within the same molecule or a cationic surfactant having an alkyl group and a tertiary amine or a quaternary ammonium salt within the same molecule. When the acidifying agent was removed from this treatment agent consisting of the acidifying agent and the surfactant and the samples was treated only with the surfactant that was found to be effective, the sensitivity was significantly reduced. Hence, it was considered that enhancement in the sensitivity was based on the acidifying agent, and that the sensitivity was significantly increased by adding surfactants to the acidifying agent.
4413
5355
16478
7048
14941
5985
15517
6313
16628
4997
15908
8062
14148
6269
15228
4302
4602
4301
4581
5578
5402
16926
6645
16655
6869
6827
18706
8451
56923
19937
7813
4128
17118
5251
43825
26990
7945
50648
28246
7316
55167
32885
9698
47512
17307
5976
48591
30527
9273
62242
36256
8276
66746
42918
16172
64794
26139
9224
73370
25348
7818
85287
41969
9925
75342
35782
11916
21151
11844
67221
20731
9871
57204
24829
13125
73931
34152
11351
77719
17943
18025
35451
8842
95495
4234
17189
4315
64879
18620
10542
137707
25964
7551
144107
20826
7126
98056
43646
15778
173578
16694
4328
20245
10125
85021
12516
53253
18948
45240
13482
168004
31229
16960
168734
14409
20956
29248
10044
37765
23853
199795
To 100 μl of the HBV antigen-negative sample or each of the HBV antigen-positive samples (#990277, #990544, #990768), 100 μl of one of protein denaturants, urea, dissolved in 1.0N aqueous hydrochloric acid was added and the mixture was incubated for 10 min at room temperature. 100 μl of the treated samples was used as for the assay, and was subjected to examination in the method described in Example 1. The ratio of the immunoreactivity of each HBV antigen-positive sample to the immunoreactivity of the HBV antigen-negative sample (Luminescence intensity of HBV antigen-positive sample/Luminescence intensity of HBV antigen-negative sample expressed by S/N ratio) was determined and shown in Table 6.
It was confirmed that there were samples showing approximately 1.5 to 3-fold higher S/N ratio in the addition of urea compared with in the treatment agent containing only the acidifying agent. At the treatment only with the acidifying agent, precipitation or cloudiness occurs in certain cases because of denaturation of serum proteins and the like, which often gives rise to pipetting trouble and a significant cause of false positive due to precipitates. Further, it seems possible that a target antigen is entangled in these precipitates, resulting in a reduction in sensitivity. It was found that the formation of these precipitates could be greatly reduced by adding urea at 1 M or higher at the treatment, and particularly, its addition at 1.5 M or higher and 8M or lower at the treatment was found to be more effective. Although urea dissolved up to about 10 M, the precipitation may be occurred dependent on storage conditions and the like. Therefore, in a solution, the concentration of urea at the treatment depends on the volume ratio of a treatment solution and a sample.
To 100 μl of the HBV antigen-negative sample (normal serum) or each of the three HBV antigen-positive samples (#990277, #990544, #990768), 100 μl of a solution in which dithiothreitol, cysteamine hydrochloride, or diethylaminoethanethiol hydrochloride that is a reducing agent was mixed with a solution containing 1.0 N hydrochloric acid, 1.5 M urea, 5.0% Triton X100, and 1.5% C16APS was added and the mixture was incubated for 10 min at room temperature. 100 μl of the treated sample was used for the assay, and was subjected to examination in the method described in Example 1 (Table 7).
The concentrations of the reducing agents were expressed by the concentrations at the treatment of the samples, respectively. Even though the reducing agent was added to the HBV antigen-negative sample (normal serum), change in the sample signal was hardly observed, whereas signal rises were observed for the HBV antigen-positive sample #990544 at 5 mM or higher concentrations of reducing agents at the sample treatment, and signal rises higher than 30% were observed for two samples (#990544, #990768) at 10 mM concentration of diethylaminoethanethiol hydrochloride.
13611
138
26295
267
15193
154
15063
153
15370
156
135
The present invention provides a simple and highly user-friendly sample treatment method for detection or quantification of HBV antigens in blood with high sensitivity and a method for detection or quantification of HBV with the use thereof and allows diagnosis of the presence or absence of HBV infection in blood and fast and accurate screening of blood for transfusion. The present invention can also provide a diagnostic kit and greatly contributes to efficiency enhancement of HBV antigen detection.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2004-149682 | May 2004 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP05/09158 | 5/19/2005 | WO | 00 | 11/16/2006 |
