Highly sensitive method for detecting anti-HBV antibodies

Abstract

To provide a method that improves sensitivity for detecting an anti-HBV antibody in a specimen.SOLUTION: In a method for detecting an anti-HBV antibody derived from a specimen by detecting a label in a complex including [i) anti-HBV antibody-ii) solid-phased HBV antigen-iii) labeled anti-HBV antibody] formed by reacting i) the anti-HBV antibody derived from the specimen, ii) a solid-phased HBV antigen that is an HBV antigen immobilized in a solid phase, and iii) a labeled anti-HBV antibody with the label attached to the anti-HBV antibody, i) the anti-HBV antibody is reacted with ii) the solid-phased HBV antigen and iv) an anti-IgG antibody is allowed to coexist with i) the anti-HBV antibody and reacted with i) the anti-HBV antibody, simultaneously with or before reacting ii) the solid-phased HBV antigen with iii) the labeled anti-HBV antibody.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for detecting an anti-HBV antibody. [Background technology]

[0002] It has been reported that there are 257 million Hepatitis B virus (HBV) patients worldwide (Non-Patent Document 1), and because the rate of people who can undergo testing and treatment is still low, particularly in developing countries, the number of deaths from viral hepatitis worldwide is 1.34 million, a number comparable to the number of deaths from tuberculosis and HIV. It remains a major public health problem, with an increase in chronic hepatitis caused by the European subtype in recent years and fulminant symptoms due to HBV reactivation when immunosuppressants are administered to previously infected individuals.

[0003] HBV is a spherical particle with a double structure, 42 nm in diameter, and is known to consist of an envelope and a core. When humans are infected with HBV, antibodies against the HBV core (hereinafter referred to as "anti-HBc antibodies") are produced, and it is known that anti-HBc antibodies exist in the blood from the early stage of HBV infection to a long period after infection (Non-Patent Document 2). In addition, anti-HBc antibodies appear in serum after the appearance of hepatitis B surface antigen (hereinafter "HBs antigen"), but in acute hepatitis B, they persist from the disappearance of HBs antigen until the appearance of antibodies against HBs antigen (hereinafter "anti-HBs antibody"). From these facts, anti-HBc antibodies can be said to be an important marker for detecting HBV infection at a stage when HBs antigen and anti-HBs antibodies are not found in serum (Non-Patent Document 3). Furthermore, since the antibody titer of anti-HBc antibodies increases during acute hepatitis B, they are also an important marker for distinguishing acute onset of chronic hepatitis B from acute hepatitis B (Non-Patent Document 4). However, there are cases where existing anti-HBc antibody detection reagents do not detect the antibody even though it is positive, and there is a demand for higher sensitivity. In addition, the Guidelines for Countermeasures against Hepatitis B Caused by Immunosuppression and Chemotherapy (revised in 2011) were formulated as a measure against the HBV reactivation cases that occurred in Hong Kong in 2006 due to the use of immunosuppressants. However, it is known that cases with positive anti-HBc antibodies and negative anti-HBs antibodies have a high risk of reactivation. Patients with a history of infection have high anti-HBc antibody titers, but as time passes after infection, anti-HBc antibody titers gradually decrease, so even patients with HBV-DNA levels above the lower limit of detection may show negative titers at some point. In this respect, improving the sensitivity of anti-HBc antibody detection reagents is also important. In addition, improving the measurement sensitivity is an important issue not only for anti-HBc antibodies but also for anti-HBV antibodies in general. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] WHOFact sheet N°204 Hepatitis B, Update July2015 [Non-Patent Document 2] Mikio Nishioka, Blood antigen-antibody system in hepatitis B virus infection and its interpretation, Medical Postgraduates, 24(2), 106-108, 198 [Non-Patent Document 3] H.Iisuka et al.,Correlation between anti-HBc titer and HBV DNA in blood units without detectable HBsAg,Vox Sang,63,107-111,1992 [Non-Patent Document 4] Kazuhiko Miyake and Masami Yamanaka, "Practical diagnosis of chronic hepatitis: To what extent can serum biochemistry and immunological tests be used for diagnosis?", Medical Practice, 4(7), 1080-1083, 1987. Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide a method for improving sensitivity in detecting anti-HBV antibodies in a sample. [Means for solving the problem]

[0006] As a result of intensive research aimed at solving the above problems, the present inventors have found that the sensitivity can be improved by allowing an anti-IgG antibody to coexist in the reaction system, and have thus completed the present invention.

[0007] (1) i) anti-HBV antibodies derived from the specimen; ii) a solid-phase HBV antigen in which an HBV antigen is immobilized on a solid phase; and iii) Labeled anti-HBV antibody obtained by binding a label to an anti-HBV antibody By reacting A method for detecting an anti-HBV antibody derived from a specimen by forming a complex having [i) an anti-HBV antibody, ii) a solid-phase HBV antigen, and iii) a labeled anti-HBV antibody, and detecting the label in the complex, comprising: ii) reacting the immobilized HBV antigen with iii) the labeled anti-HBV antibody, either simultaneously or prior to the reaction; i) reacting an anti-HBV antibody with ii) a solid-phase HBV antigen; and iv) allowing the anti-IgG antibody to coexist with i) the anti-HBV antibody and react with i) the anti-HBV antibody; A method for detecting an anti-HBV antibody, comprising:

[0008] The present invention will be described in detail below.

[0009] The present invention is a method for detecting anti-HBV antibodies derived from a sample by carrying out the above-mentioned reaction.

[0010] In the present invention, the HBV antigen is not particularly limited. Examples include hepatitis B core antigen (hereinafter referred to as "HBc antigen"), hepatitis B envelope antigen (hereinafter referred to as "HBe antigen"), and HBs antigen. HBc antigen is preferred.

[0011] Examples of anti-HBV antibodies include antibodies against the above-mentioned HBV antigens, specifically anti-HBc antibodies, anti-HBe antibodies, anti-HBs antibodies, etc., with anti-HBc antibodies being preferred.

[0012] The specimen may be, for example, a human body fluid, specifically blood, urine, etc., and preferred examples include blood, particularly whole blood, serum, plasma, etc.

[0013] As a sign, 125 I, 3 Radioactive substances such as H, enzymes such as horseradish peroxidase, β-D-galactosidase, alkaline phosphatase, fluorescent substances such as fluorescein, gold colloid, selenium colloid, luminescent or color-developing substances such as luciferin, etc. These labels may be directly bound to the anti-HBV antibody or indirectly bound via biotin-avidin or the like.

[0014] As the solid phase, beads, microparticles, the container itself, etc. can be used. Microparticles are particularly preferred, and may be inorganic materials such as glass, metal, ceramics, etc., or organic materials such as polymers. These microparticles may also contain magnetic materials. The particle size of the microparticles is preferably 0.1 to 50 μm, more preferably 1 to 10 μm.

[0015] iii) The antibody used for the labeled anti-HBV antibody may be a polyclonal antibody or a monoclonal antibody, and may be derived from virtually any animal species that produces antibodies, such as rabbits, goats, sheep, pigs, horses, mice, or rats. The antibody may be in the form of a complete antibody or an antibody fragment such as F(ab')2 or Fab' that is obtained by cleaving the complete antibody through enzymatic or chemical treatment.

[0016] The anti-IgG antibody iv) used in the present invention is not particularly limited, but when a human sample is used, an anti-human IgG antibody is preferred.

[0017] The present invention uses a competitive reaction system. Since HBV antigens are polyvalent antigens (having multiple identical epitopes), multiple identical antibodies can bind to them.

[0018] The present invention comprises reacting i) an anti-HBV antibody, ii) a solid-phase HBV antigen, and iii) a labeled anti-HBV antibody, and simultaneously with or prior to reacting ii) the solid-phase HBV antigen with iii) the labeled anti-HBV antibody, i) It is essential to react anti-HBV antibodies with ii) immobilized HBV antigens, and ·iv) It is essential that anti-IgG antibodies coexist with i) anti-HBV antibodies to react with i) anti-HBV antibodies.

[0019] This forms a complex having [iv) anti-IgG antibody-i) anti-HBV antibody-ii) solid-phase HBV antigen-iii) labeled anti-HBV antibody], and the label in the complex is detected to detect the anti-HBV antibody derived from the sample. At this time, the anti-IgG antibody iv) binds to the anti-HBV antibody i) and modifies the anti-HBV antibody i) to make it bulky, which can efficiently inhibit the binding of the labeled anti-HBV antibody iii) to the solid-phase HBV antigen ii), and as a result, it is considered that the amount of anti-HBV antibody derived from the sample can be determined with high sensitivity.

[0020] Since HBV antigen is a polyvalent antigen, multiple i) anti-HBV antibodies and multiple iii) labeled anti-HBV antibodies may bind to ii) solid-phase HBV antigen in the above-mentioned complex. However, these are all included and are referred to as a complex having [i) anti-HBV antibody-ii) solid-phase HBV antigen-iii) labeled anti-HBV antibody] or a complex having [iv) anti-IgG antibody-i) anti-HBV antibody-ii) solid-phase HBV antigen-iii) labeled anti-HBV antibody], as described above.

[0021] On the other hand, the reaction order in the present invention is as described above, but the following embodiment is preferable. That is, first, ii) solid-phased HBV antigen and i) anti-HBV antibody derived from the specimen are mixed, so that ii) solid-phased HBV antigen and i) HBV antibody derived from the specimen react with each other. After this, it is preferable to remove unreacted substances by B / F separation or washing. Next, iii) labeled anti-HBV antibody and iv) anti-IgG antibody are simultaneously added to the reaction product and reacted. After this, it is preferable to remove unreacted substances by B / F separation or washing. Finally, the amount of anti-HBV antibody derived from the specimen can be determined by measuring the label in the complex having the generated [iv) anti-IgG antibody-i) anti-HBV antibody-ii) solid-phased HBV antigen-iii) labeled anti-HBV antibody].

[0022] In the present invention, proteins, sugars, buffer solutions, and salts may be present during the reaction, and examples of proteins that can be used include bovine serum albumin, collagen peptide, and milk protein. The protein concentration is preferably in the range of 0.1 to 20% (weight / volume), and more preferably in the range of 1 to 10% (weight / volume). For sugars, for example, sucrose, mannitol, trehalose, and inositol can be used. For buffer solutions, for example, Tris, MOPSO, MOPS, and MES can be used, and for salts, for example, sodium chloride, potassium chloride, magnesium chloride, zinc chloride, and disodium ethylenediaminetetraacetate can be used. In addition to these, other reagent components can also be present during the reaction, if necessary. Effect of the Invention

[0023] According to the present invention, it is possible to use an anti-IgG antibody to i) modify an anti-HBV antibody to make it bulky, thereby iii) efficiently inhibiting the binding of a labeled anti-HBV antibody to ii) a solid-phase HBV antigen, and as a result, it is possible to measure the amount of anti-HBV antibody in a sample with high sensitivity. Furthermore, although the present invention is a competitive method, it is possible to obtain a measurement sensitivity equivalent to that of the sandwich method. [Brief description of the drawings]

[0024] [Figure 1] FIG. 1 is a diagram showing the measurement results of Example 1 and Comparative Example 1. EXAMPLES

[0025] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0026] [Example 1] The immunoassay was performed by a two-step competitive assay using a fully automated enzyme immunoassay device (AIA-CL2400, Tosoh Corporation) and an anti-HBc antibody immunoreaction reagent (reagent A) for the device. The anti-HBc antibody immunoreaction reagent (reagent A) was prepared as follows.

[0027] (1) Preparation of solid suspension The HBc antigen-immobilized magnetic microparticles prepared by immobilizing HBc antigen on magnetic microparticles were diluted and mixed with MOPSO buffer containing collagen peptide, animal serum, sugar, and salts to prepare a solid-phase suspension, which was then dispensed into one of the holes of a two-hole resin container.

[0028] (2) Preparation of a solution containing labeled antibody and anti-IgG antibody Alkaline phosphatase-labeled anti-HBc antibody, prepared by binding alkaline phosphatase to anti-HBc antibody, and anti-human IgG antibody were diluted and mixed with MOPSO buffer containing collagen peptide, animal serum, sugar, and salts to prepare a solution containing the labeled antibody and anti-human IgG antibody, which was then dispensed into the other hole of the aforementioned two-hole resin container.

[0029] (3) Preparation of anti-HBc antibody immunoreaction reagent (reagent A) The two-well containers prepared in (1) and (2) above were freeze-dried to prepare an anti-HBc antibody immunoreaction reagent (reagent A).

[0030] (4) Measurement Measurement was performed by the two-step competitive assay using the above-mentioned (3) Reagent A. Specifically, of the two holes in Reagent A, a dissolving solution was poured into the hole containing the lyophilized solid phase suspension to dissolve it, and a specimen (patient serum) was dispensed therein and reacted at 37°C for 6 minutes (first reaction). Unreacted substances were removed by B / F separation and washing three times with washing solution. Next, a dissolving solution was poured into the hole containing the lyophilized labeled antibody solution for detection to dissolve it, and this was dispensed into the other hole and reacted at 37°C for 3 minutes (second reaction). After that, unreacted substances were removed by B / F separation and washing three times with washing solution, and then a luminescent substrate (compound name DIFURAT: 3-(5-tert-butyl-4,4-dimethyl-2,6,7-trioxabicyclo[3.2.0]hept-1-yl)phenyl phosphate ester disodium salt) was dispensed and reacted for 4 minutes, and the alkaline phosphatase activity bound to the solid phase was measured from the amount of luminescence to determine positive / negative.

[0031] Since this measurement is a competitive method, the negative control, i.e., the measurement value when anti-HBc antibodies are not present in the sample, is used as the standard. The positive control refers to the concentration at which no change in the measurement value is observed when anti-HBc antibodies are serially diluted with sample dilution solution and measured using this measurement method, and the measurement value at this point is defined as 100INH%. On the other hand, when anti-HBc antibodies are present in the sample, the amount of anti-HBc antibodies is expressed from the percentage (INH(%)) at which the binding of the labeled anti-HBc antibody to the immobilized HBc antigen is inhibited by the antibody. These were calculated according to the following formula. INH(%)=[(measurement value of negative control - measurement value of sample) / (measurement value of negative control - measurement value of positive control)]×100 In this calculation, values ​​equal to or greater than 50% were considered positive (+), and anything else was considered negative (-).

[0032] [Comparative Example 1] Measurement was performed in the same manner as in Example 1, except that an anti-HBc antibody immunoreaction reagent (reagent B) was used instead of the anti-HBc antibody immunoreaction reagent (reagent A). The anti-HBc antibody immunoreaction reagent (reagent B) was prepared in the same manner as the anti-HBc antibody immunoreaction reagent (reagent A), except that it was prepared without using anti-IgG antibody.

[0033] [Measurement results] Eight positive samples and seven negative samples were diluted with negative serum to the detection sensitivity of Reagent B, and the results were compared. The results are shown in Table 1. In the table, values ​​in parentheses indicate negative values.

[0034] [Table 1]

[0035] In addition, 137 patient samples (47 positive samples, 90 negative samples) were measured and the results were compared. The results are shown in Figure 1.

[0036] As is clear from Table 1 and FIG. 1, Reagent A, which is a reaction system using an anti-IgG antibody, was able to measure positive samples with higher sensitivity than Reagent B.

Claims

[Claim 1] i) anti-HBV antibodies derived from the sample; ii) a solid-phase HBV antigen in which an HBV antigen is immobilized on a solid phase; and iii) A labeled anti-HBV antibody obtained by binding a label to the anti-HBV antibody. By reacting A method for detecting an anti-HBV antibody derived from a specimen by forming a complex having [i) an anti-HBV antibody, ii) a solid-phase HBV antigen, and iii) a labeled anti-HBV antibody, and detecting the label in the complex, comprising: - i) reacting an anti-HBV antibody with ii) a solid-phase HBV antigen prior to reacting ii) a solid-phase HBV antigen with iii) a labeled anti-HBV antibody; and - ii) reacting the solid-phase HBV antigen with iii) the labeled anti-HBV antibody, and at the same time, iv) allowing an anti-IgG antibody to coexist with i) the anti-HBV antibody and reacting it with i) the anti-HBV antibody; A method for detecting an anti-HBV antibody, comprising:

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