JP7622350B2 - Highly sensitive method for detecting anti-HBV antibodies - Google Patents

Description

The present invention relates to a method for detecting anti-HBV antibodies.

It has been reported that there are 257 million Hepatitis B virus (HBV) patients worldwide (Non-Patent Document 1), and because the percentage of people who can receive 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. HBV 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.

HBV is a spherical particle with a diameter of 42 nm and a double structure, which 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 antibody 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 high sensitivity. In addition, the Guidelines for Countermeasures against Hepatitis B Caused by Immunosuppression and Chemotherapy (revised in 2011) have been formulated as a measure against the case of HBV reactivation caused by the use of immunosuppressants that occurred in Hong Kong in 2006. It is known that cases in which anti-HBc antibody is positive and anti-HBs antibody is negative have a high risk of reactivation. Patients with a history of infection have high anti-HBc antibody titers, but as time passes after infection, the anti-HBc antibody titer gradually decreases, so that even patients with HBV-DNA above the lower limit of detection may show negative titers at a certain point, and improving the sensitivity of anti-HBc antibody detection reagents is also important from this point of view. In addition, improving the measurement sensitivity is an important issue not only for anti-HBc antibodies but also for anti-HBV antibodies in general.

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

The present invention aims to provide a method that improves sensitivity when detecting anti-HBV antibodies in a sample.

As a result of intensive research aimed at solving the above problems, the inventors discovered that the sensitivity can be improved by allowing anti-IgG antibodies to coexist in the reaction system, and thus completed the present invention. That is, the present invention is as follows.

(1)
i) anti-HBV antibodies derived from the sample;
ii) a solid-phase HBV antigen obtained by immobilizing an HBV antigen on a solid phase, and iii) a labeled anti-HBV antibody obtained by binding a label to an anti-HBV antibody,
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) simultaneously with or prior to reacting the immobilized HBV antigen with iii) the labeled anti-HBV antibody,
i) reacting an anti-HBV antibody with ii) a solid-phase HBV antigen, and iv) allowing an 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:

The present invention is described in detail below.

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

In the present invention, the HBV antigen is not particularly limited. Examples include hepatitis B core antigen (hereinafter "HBc antigen"), hepatitis B envelope antigen (hereinafter "HBe antigen"), HBs antigen, etc. Preferred is HBc antigen.

Examples of anti-HBV antibodies include antibodies against the aforementioned HBV antigens, specifically anti-HBc antibodies, anti-HBe antibodies, anti-HBs antibodies, etc., with anti-HBc antibodies being preferred.

Examples of the specimen include human body fluids, specifically blood and urine, and preferred examples include blood, particularly whole blood, serum, and plasma.

Examples of labels that can be used include radioactive substances such as ¹²⁵ I and ³ H, enzymes such as horseradish peroxidase, β-D-galactosidase, and alkaline phosphatase, fluorescent substances such as fluorescein, gold colloids, selenium colloids, and luminescent or color-developing substances such as luciferin, etc. These labels may be directly bound to the anti-HBV antibody, or may be indirectly bound via biotin-avidin or the like.

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, and more preferably 1 to 10 μm.

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.

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

The present invention uses a competitive reaction system. Note that since HBV antigens are polyvalent antigens (having multiple identical epitopes), multiple identical antibodies can bind to them.

The present invention involves 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,
- It is essential that i) anti-HBV antibody and ii) solid-phase HBV antigen are reacted with each other, and - iv) it is essential that anti-IgG antibody is allowed to coexist with i) anti-HBV antibody, thereby allowing i) reaction with anti-HBV antibody.

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

Because 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. These are also included in the above-mentioned complexes, which are expressed 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].

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].

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

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 the labeled anti-HBV antibody to ii) the solid-phase HBV antigen, and as a result, it is possible to determine 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.

FIG. 1 is a diagram showing the measurement results of Example 1 and Comparative Example 1.

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

[Example 1]
The immunoassay was performed by a two-step competitive method using a fully automated enzyme immunoassay device (AIA-CL2400, manufactured by 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.

(1) Preparation of solid-phase suspension HBc antigen immobilized magnetic microparticles prepared by immobilizing HBc antigen on magnetic microparticles were diluted and mixed with MOPSO buffer solution 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.

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

(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 anti-HBc antibody immunoreaction reagent (reagent A).

(4) Measurement Measurement was performed by the two-step competitive method using the above-mentioned (3) Reagent A. Specifically, of the two holes of Reagent A, a dissolving solution was poured into the hole containing the lyophilized solid suspension to dissolve it, and a specimen (patient serum) was dispensed therein and reacted at 37°C for 6 minutes (first reaction), and unreacted matter was removed by B/F separation and washing three times with a 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). Thereafter, unreacted substances were removed by B/F separation and washing three times with the washing solution, after which 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 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 whether it was positive or negative.

Since this measurement is a competitive method, the negative control, i.e., the measured value when anti-HBc antibody is not present in the specimen, is used as the standard. The positive control refers to the concentration at which no change in the measured value is observed when anti-HBc antibody is serially diluted with specimen diluent and measured by this measurement method, and the measured value at this point is defined as 100 INH%. On the other hand, when anti-HBc antibody is present in the specimen, the amount of anti-HBc antibody is expressed from the ratio (INH (%)) at which the labeled anti-HBc antibody is inhibited from binding to the solid-phase HBc antigen. 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)] x 100
Values equal to or greater than 50% in this calculation were considered positive (+), otherwise negative (-).

[Comparative Example 1]
Measurement was performed in the same manner as in Example 1, except that the 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 the anti-IgG antibody was not used.

[Measurement results]
Eight positive specimens and seven negative specimens were diluted with negative serum to the vicinity of 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.

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.

As is clear from Table 1 and Figure 1, Reagent A, which is a reaction system using anti-IgG antibodies, was able to measure positive samples with greater sensitivity than Reagent B.

Claims (1)

i) anti-HBV antibodies derived from the sample;
ii) a solid-phase HBV antigen obtained by immobilizing an HBV antigen on a solid phase, and iii) a labeled anti-HBV antibody obtained by binding a label to an anti-HBV antibody,
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:
- before reacting ii ) the solid-phased HBV antigen with iii) the labeled anti-HBV antibody , i ) reacting the anti-HBV antibody with ii) the solid-phased HBV antigen, and - simultaneously with reacting ii) the solid-phased HBV antigen with iii) the labeled anti-HBV antibody, iv) allowing an 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: