Patent Application
20200182873
The present invention relates to a detection method and a detection kit for a hepatitis B virus surface antigen.
Hepatitis B is a viral hepatitis caused by infection with hepatitis B virus (hereinafter also referred to as “HBV”). A hepatitis B virus surface antigen (hereinafter also referred to as “HBsAg”) corresponding to an envelope antigen of HBV is released into the blood when HBV grows in hepatocytes. Therefore, infection with HBV can be examined by detecting an HBsAg in the blood. An HBsAg is transiently detected in the blood in the case of transient HBV infection, and is persistently detected in the blood in the case of persistent HBV infection.
In order to highly precisely examine HBV infection, it is necessary to highly sensitively detect an HBsAg. For example, PTL 1 discloses that an HBsAg in serum can be highly sensitively detected by using a combination of an antibody binding to the S region of an HBsAg and an antibody binding to the Pre-S1 region and Pre-S2 region.
PTL 1: Japanese Patent Application Laid-Open No. 2001-133460
In order to highly sensitively detect an HBsAg, while prescription of a reagent to be used may be devised as in the invention described in PTL 1, a detection method having high detection sensitivity in principle may be employed. For simply performing examination, a detection method in which not serum or plasma but blood (whole blood) can be directly used as a specimen is preferred. However, a detection method by which an HBsAg can be highly sensitively detected by using blood (whole blood) as a specimen has not been proposed until now.
The present invention was accomplished in consideration of these points, and an object is to provide a detection method and a detection kit for an HBsAg by which an HBsAg can be highly sensitively detected even when blood (whole blood) is used as a specimen.
A detection method for an HBsAg, according to one embodiment of the present invention comprises: preparing a detection chip including a metal film and a binding substance which is immobilized on the metal film and which specifically binds to an HBsAg; providing a specimen onto the metal film to cause an HBsAg contained in the specimen to bind to the binding substance; labeling, with a fluorescent substance, the HBsAg before or after binding to the binding substance; and detecting fluorescence emitted from the fluorescent substance when the metal film is irradiated with excitation light in such a manner as to generate surface plasmon resonance in the metal film with the HBsAg labeled with the fluorescent substance kept in a state binding to the binding substance.
Furthermore, a detection kit for an HBsAg, according to one embodiment of the present invention comprises: a detection chip including a metal film and a binding substance which is immobilized on the metal film and which specifically binds to an HBsAg; and a labeling reagent for labeling am HBsAg with a fluorescent substance.
According to the present invention, an HBsAg can be highly sensitively detected even when blood (whole blood) is used as a specimen.
Embodiments of the present invention will now be described in detail with reference to the accompanying drawings.
[Detection Method for HBsAg]
In a detection method for an HBsAg of the present embodiment, an HBsAg is detected by utilizing surface plasmon-field enhanced fluorescence spectroscopy (hereinafter also referred to as “SPFS”). In SPFS, a fluorescent substance is excited to emit fluorescence by an electric field enhanced by surface plasmon resonance (hereinafter also referred to as “SPR”), and therefore, as compared with general fluoroimmunoassay, a target (that is, an HBsAg in the present embodiment) can be highly sensitively detected. In SPFS, whole blood can be used as a specimen.
The detection method for an HBsAg of the present embodiment will now be specifically described.
(Preparation of Detection Chip)
First, a detection chip including a metal film and a binding substance specifically binding to an HBsAg is prepared (step S10). In SPFS, SPR is generated by causing evanescent waves, caused by irradiating a metal film with light (that is, excitation light in the present embodiment), and surface plasmon to couple to each other. As a method for generating SPR, a method in which a prism is disposed on one plane of a metal film (Kretschmann configuration), a method in which a diffraction grating is formed in a metal film, and the like are known. SPFS employing the former method is designated as prism coupling (PC)-SPFS, and SPFS employing the latter method is designated as grating coupling (GC)-SPFS. The detection method for an HBsAg of the present embodiment may employ either of PC-SPFS and GC-SPFS.
As described above, when a metal film is irradiated with excitation light, SPR is generated. The type of a metal constituting the metal film is not particularly limited as long as the metal can generate SPR. Examples of the metal constituting the metal film include gold, silver, copper, aluminum and an alloy thereof.
The binding substance can specifically bind to an HBsAg, and is immobilized on the metal film for capturing an HBsAg contained in a specimen. In general, the binding substance is uniformly immobilized in a prescribed region (reaction field) on the metal film. The type of the binding substance immobilized on the metal film is not particularly limited as long as it can specifically bind to an HBsAg. Examples of the binding substance include an antibody capable of specifically binding to an HBsAg (an anti-HBsAg antibody), a nucleic acid capable of specifically binding to an HBsAg, a lipid capable of specifically binding to an HBsAg, and a protein, excluding an antibody, capable of specifically binding to an HBsAg. When the binding substance is an anti-HBsAg antibody, the anti-HBsAg antibody may be a monoclonal antibody, a polyclonal antibody, or a fragment of an antibody. One or two or more binding substances may be immobilized on the metal film. For example, the anti-HBsAg antibody immobilized on the metal film may be one or two or more anti-hepatitis B virus surface antigen monoclonal antibodies or anti-hepatitis B virus surface antigen polyclonal antibodies.
From the viewpoint of improving detection sensitivity for an HBsAg, an antibody specifically binding to a specific region of the HBsAg may be used as the binding substance immobilized on the metal film. For example, the anti-HBsAg antibody immobilized on the metal film may be an antibody binding to the S region of an HBsAg, or may be a combination of an antibody binding to the S region of an HBsAg and an antibody binding to the Pre-S2 region of an HBsAg.
A method for immobilizing the binding substance is not particularly limited. For example, a self-assembled monolayer (hereinafter referred to as “SAM”) or a polymer film to which the binding substance (such as an anti-HBsAg antibody) is caused to bind may be formed on the metal film. An example of the SAM includes a film made of a substituted aliphatic thiol such as HOOC—(CH2)11—SH. Examples of a material constituting the polymer film include polyethylene glycol and MPC polymer. A polymer having a reactive group (or a functional group that can be converted into a reactive group) capable of binding to the binding substance (such as an anti-HBsAg antibody) may be immobilized on the metal film, and the binding substance (such as an anti-HBsAg antibody) may be caused to bind to the polymer.
A detection chip is a structure having each side with a length preferably of several mm to several cm, and may be a more compact structure or a larger structure not belonging to the category of “chip”.
As illustrated in
As illustrated in
As illustrated in
As illustrated in
Passage cover 310 is made of a material transparent to fluorescence L3. It is noted that a part of passage cover 310 may be made of a material not transparent to fluorescence L3 as long as outcoupling of fluorescence L3 cannot be prevented. An example of the material transparent to fluorescence L3 includes a resin. Passage cover 310 may be connected, without using adhesive layer 350, to metal film 120 (or prism 110) through laser welding, ultrasonic welding, pressure bonding with a clamp member or the like. In this case, the shape of the side surface of passage 320 is defined by passage cover 310.
A pipette tip is inserted into liquid injection port 330. At this point, an opening of liquid injection port 330 (that is, a through hole provided in liquid injection port covering film 331) comes into tight contact with the outer circumference of the pipette tip. Therefore, a liquid can be introduced into passage 320 by injecting the liquid into liquid injection port 330 from the pipette tip, and a liquid held in passage 320 can be removed by sucking the liquid held in liquid injection port 330 into the pipette tip. When injection and suction of a liquid are alternately performed, the liquid can be fed to reciprocate in passage 320.
When a liquid in an amount exceeding the volume of passage 320 is introduced from liquid injection port 330 into passage 320, the liquid flows from passage 320 into reservoir 340. Also when a liquid is fed to reciprocate in passage 320, the liquid flows into reservoir 340. The liquid thus flown into reservoir 340 is stirred within reservoir 340. When the liquid is stirred in reservoir 340, a concentration of a component (such as an HBsAg or a cleaning component) of the liquid (such as a specimen or a cleaning liquid) passing through passage 320 is made uniform, and hence various reactions can be easily caused in passage 320, or a cleaning effect is improved.
(Primary Reaction)
Next, a specimen is provided onto the metal film of the detection chip to cause an HBsAg contained in the specimen to bind to the binding substance (primary reaction; step S20). A method for providing a specimen is not particularly limited. For example, a specimen may be provided onto the metal film using a pipette having a pipette tip attached to a tip thereof. In general, after completing the primary reaction, the surface of the metal film is cleaned with a buffer or the like to remove a component not binding to the binding substance.
The type of the specimen is not particularly limited. Examples of the specimen include blood, serum, plasma, and a diluted solution thereof. In the detection method for an HBsAg of the present embodiment, an HBsAg is detected by employing SPFS, and hence, whole blood can be used as the specimen.
(Secondary Reaction)
Next, a labeling reagent is provided onto the metal film of the detection chip to label, with a fluorescent substance, the HBsAg having bound to the binding substance (secondary reaction; step S30). A method for providing a labeling reagent is not particularly limited. For example, a labeling reagent may be provided onto the metal film using a pipette having a pipette tip attached to a tip thereof. In general, after completing the secondary reaction, the surface of the metal film is cleaned with a buffer or the like to remove the fluorescent substance not labeling the HBsAg.
The type of the labeling reagent is not particularly limited as long as the HBsAg having bound to the binding substance can be labeled with a fluorescent substance. For example, the labeling reagent is a binding substance, labeled with a fluorescent substance, specifically binding to an HBsAg. The type of the binding substance contained in the labeling reagent is not particularly limited as long as it can specifically bind to an HBsAg. Examples of the binding substance include an antibody capable of specifically binding to an HBsAg (an anti-HBsAg antibody), a nucleic acid capable of specifically binding to an HBsAg, a lipid capable of specifically binding to an HBsAg, and a protein, excluding an antibody, capable of specifically binding to an HBsAg. The binding substance contained in the labeling reagent may be the same type as or different type from the binding substance immobilized on the metal film. When the binding substance is an anti-HBsAg antibody, the anti-HBsAg antibody may be a monoclonal antibody, a polyclonal antibody, or a fragment of an antibody. One or two or more binding substances may be immobilized on the metal film. For example, the anti-HBsAg antibody labeled with a fluorescent substance may be one or two or more anti-HBsAg monoclonal antibodies or anti-HBsAg polyclonal antibodies. In this case, the anti-HBsAg monoclonal antibodies and the anti-HBsAg polyclonal antibodies labeled with a fluorescent substance are preferably different from one or two or more anti-HBsAg monoclonal antibodies immobilized on the metal film.
From the viewpoint of improving detection sensitivity for an HBsAg, an antibody binding to a specific region of an HBsAg may be used as the anti-HBsAg antibody labeled with a fluorescent substance. For example, the anti-HBsAg antibody labeled with a fluorescent substance may be an antibody binding to the S region of an HBsAg, or may be a combination of an antibody binding to the S region of an HBsAg and an antibody binding to the Pre-S2 region of an HBsAg.
The type of the fluorescent substance is not particularly limited as long as it can be used in SPFS. Examples of the fluorescent substance include cyanine-based dyes, Alex Fluor(R) dye of Thermo Scientific, and CF dye of Biotium. Alexa Fluor dye and CF dye have high quantum efficiency for the wavelength of excitation light used in SPFS as compared with other commercially available fluorescent dyes. CF dye is not largely discolored in fluorescence detection, and hence the fluorescence detection can be stably performed. A method for labeling the binding substance with a fluorescent substance is not particularly limited, and can be appropriately selected from known methods. For example, a fluorescent substance may be caused to bind to an amino group or a sulfhydryl group of the binding substance (such as an anti-HBsAg antibody).
Although an HBsAg is labeled with the fluorescent substance after causing the HBsAg to bind to the binding substance immobilized on the metal film in the above description, an HBsAg may be labeled with the fluorescent substance before causing the HBsAg to bind to the binding substance immobilized on the metal film. In this case, the specimen and the labeling reagent may be mixed before providing the specimen onto the metal film. Alternatively, a step of causing the binding substance immobilized on the metal film to bind to an HBsAg and a step of labeling the HBsAg with the fluorescent substance may be simultaneously performed. In this case, the specimen and the labeling reagent may be simultaneously provided onto the metal film.
(Fluorescence Detection)
Next, fluorescence corresponding to the presence or amount of the HBsAg is detected by SPFS (step S40). Specifically, with the HBsAg labeled with the fluorescent substance and binding to the binding substance immobilized on the metal film, the metal film is irradiated with the excitation light so as to generate SPR, and fluorescence thus emitted from the fluorescent substance is detected. In general, a precedently measured optical blank value is subtracted from a measured fluorescent value to calculate a signal value in correlation with the amount of the HBsAg. If necessary, the signal value may be converted into the amount or concentration of the HBsAg by using a calibration curve or the like precedently created.
When detection chip 100 for use in PC-SPFS is used, metal film 120 is irraciated with excitation light L1 through prism 110 as illustrated in
When detection chip 200 for use in GC-SPFS is used, metal film 210 (diffraction grating 211) is directly irradiated with excitation light L1 as illustrated in
The type of the excitation light is not particularly limited, and is generally laser light. For example, the excitation light is laser light emitted from a laser light source having an output power of 15 to 30 mW. When the output power is 15 mW or more, fluorescence intensity can be increased to appropriately detect the fluorescence. When the output power is 30 mW or less, the binding substance immobilized on the metal film and the like can be prevented from being harmfully affected. The wavelength of the excitation light is appropriately set in accordance with the excitation wavelength of the fluorescent substance to be used.
A detector for the fluorescence is preferably disposed, with respect to the detection chip, in a direction where the fluorescence intensity is the highest. For example, when detection chip 100 for use in PC-SPFS is used, the direction where the intensity of fluorescence L3 is the highest is a normal direction of metal film 120 as illustrated in
Through the above-described procedures, the presence or amount of an HBsAg contained in a specimen can be detected.
[Detection Kit for HBsAg]
A detection kit for an HBsAg according to the present embodiment is a set of the aforementioned detection chip and the aforementioned labeling reagent. When the detection chip and the labeling reagent are thus precedently prepared as a set, a user (such as a health care provider) can more simply perform the detection method for an HBsAg.
[Effects]
As described so far, since SPFS is employed in the detection method or the detection kit for an HBsAg of the present embodiment, an HBsAg can be highly sensitively detected in a short period of time even when blood (whole blood) is used as a specimen.
Now, the present invention will be described in detail with reference to Examples, and it is noted that the present invention is not limited to these Examples.
Detection chip 300 having the structure illustrated in
Blood was collected from four healthy volunteers. 10 μL of purified HBsAg (Institute of Immunology Co., Ltd.) was added to 1990 μL of the blood (whole blood) of each of these four volunteers. The thus obtained blood to which the antigen had been added was divided into two portions, one of which was directly used as a whole blood sample, and the other of which was subjected to centrifugation to obtain a plasma sample. Each of the whole blood sample and the plasma sample was diluted three times before use. A concentration of the HBsAg in each of these diluted samples was set to 0.40 IU/mL (Low) or 40.00 IU/mL (Mid).
Each sample was introduced into passage 320 through liquid injection port 330 with a pipette tip, and fed to reciprocate therein (primary reaction). After removing the sample from passage 320 though liquid injection port 330, passage 320 was cleaned once with a cleaning solution. Subsequently, a labeling reagent (a mouse anti-HBsAg monoclonal antibody (Institute of Immunology Co., Ltd.)) labeled, via an amino group, with CF dye (Biotium, Inc.) was introduced into passage 320 through liquid injection port 330 and fed to reciprocate therein (secondary reaction). After removing the labeling reagent from passage 320 through liquid injection port 330, passage 320 was cleaned three times with a cleaning solution. Subsequently, a measurement liquid was introduced into passage 320 through liquid injection port 330. In this state, a fluorescent value was measured by SPFS. Specifically, metal film 120 was irradiated with excitation light (laser light) from the side of prism 110 with the incident angle of the excitation light against metal film 120 set to a reinforcement angle, and fluorescence emitted at this point was detected. A precedently measured optical blank value was subtracted from the thus obtained fluorescent value to calculate a signal value in correlation with the amount of the HBsAg. A precedently prepared calibration curve was used to calculate a concentration (IU/mL) (quantitative value) of the HBsAg based on the signal value. The concentration of the HBsAg in the whole blood sample was corrected by using a hematocrit value measured by a micro-hematocrit method.
The measurement results of the respective samples are shown in Table 1.
It is understood from Table 1 that a concentration of an HBsAg can be measured, even when whole blood is used as a specimen, in the same manner as in using plasma. The measured values for blood No. 3 or No. 4 were slightly lower than the concentration of the HBsAg added to the sample probably because of fluctuation caused in preparation of the sample, specimen characteristics or the like.
Detection chip 300 was prepared in the same manner as in Experiment 1. Four commercially available HBsAg-positive plasma (ProMedEx) samples were prepared as specimens. In the same manner as in Experiment 1, each specimen was measured for a signal value twice a day for 3 days to calculate a concentration (IU/mL) (quantitative value) of the HBsAg.
The measurement results of the respective specimens are shown in Table 2.
It is understood from Table 2 that a variation coefficient CV is as low as 7.8% or less, and that the detection method for an HBsAg of the present embodiment has high daily reproducibility.
Detection chip 300 was prepared in the same manner as in Experiment 1. Three commercially available HBsAg-positive plasma (ProMedEx) samples were prepared as specimens. Each plasma was diluted by once, 4 times, 16 times, 64 times, 256 times, 1024 times, 4096 times and 16384 times to prepare diluted samples. Each diluted sample was measured for a signal value to calculate a concentration (IU/mL) (quantitative value) of the HBsAg in the same manner as in Experiment 1.
The measurement results of the respective specimens are illustrated in
Detection chip 300 was prepared in the same manner as in Experiment 1. Nineteen commercially available HBsAg-positive plasma (ProMedEx) samples were prepared as specimens. Each specimen was measured for a signal value to calculate a concentration (IU/mL) (quantitative value) of the HBsAg in the same manner as in Experiment 1.
In each of the same nineteen specimens, the HBsAg was detected by using a commercially available automatic chemiluminescent immunoassay apparatus (AD VIA Centaur; Siemens).
The measurement results of the respective specimens are illustrated in
The present application is based upon and claims the benefit of priority of Japanese Patent Application No. 2017-167618, filed on Aug. 31, 2017. The entire contents of the specification and drawings thereof are incorporated herein by reference.
When a detection method or a detection kit for an HBsAg of the present embodiment is employed, infection with HBV can be highly precisely examined in a short period of time. Accordingly, the detection method and the detection kit for an HBsAg of the present invention are useful for, for example, laboratory examinations and the like.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2017-167618 | Aug 2017 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2018/026280 | 7/12/2018 | WO | 00 |
