Method for measuring hemagglutinin of influenza virus

Abstract

To provide a hemagglutinin measurement system using lectin, the system enabling accurate measurement of hemagglutinin of influenza virus expressed in a baculovirus-insect cell expression system.SOLUTION: A method for measuring hemagglutinin of influenza virus expressed in a baculovirus-insect cell expression system includes: forming a sandwich immune complex including a lectin selected from pea lectin and lentil lectin; the hemagglutinin; and an anti-hemagglutinin antibody recognizing the hemagglutinin.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for measuring hemagglutinin, an antigen of influenza viruses. [Background technology]

[0002] Influenza viruses belong to the Orthomyxoviridae family and are classified into types A, B, and C based on antigenic differences in the nucleoprotein and matrix protein present inside the virus. Types A and B are the viruses that are prevalent every year, and type A viruses in particular are prone to antigenic mutations, with 16 types of hemagglutinin and 9 types of neuraminidase subtypes based on differences in glycoproteins, which are the surface antigens of the virus particles. Therefore, vaccine strains must be selected based on predictions of the epidemic each season, and reagents for measuring hemagglutinin, the main antigen in the vaccine, must also be prepared as strains change.

[0003] A highly sensitive and high-throughput method for measuring hemagglutinin is the sandwich ELISA method using monoclonal and polyclonal antibodies. However, this method requires two strain-specific antibodies, and it is difficult to use monoclonal antibodies to measure the hemagglutinin of new vaccine production strains or pandemic viruses, given the time required to produce the antibodies. In response to this, Patent Document 1 discloses that a measurement system for detecting hemagglutinin in a short period of time can be constructed by using a specific lectin that binds to influenza virus hemagglutinin but not to antibodies. Patent document 2 also discloses a method in which a sample containing influenza virus hemagglutinin is brought into contact with snowdrop lectin (GNA) immobilized on a solid support to capture it, and the captured hemagglutinin is detected by immunoassay. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 6280868 [Patent Document 2] Special Publication No. 2016-539314 Summary of the Invention [Problem to be solved by the invention]

[0005] However, it has been found that in measurement systems using lectins, the reactivity of lectins to hemagglutinins varies depending on the hemagglutinin preparation method, making it impossible to accurately measure hemagglutinins expressed in baculovirus-insect cell expression systems. Therefore, the present invention provides a lectin-based hemagglutinin measurement system that can accurately measure influenza virus hemagglutinins expressed in a baculovirus-insect cell expression system. [Means for solving the problem]

[0006] As a result of extensive research, the present inventors have found that pea lectin or lentil lectin has high reaction specificity for influenza virus hemagglutinin expressed in a baculovirus-insect cell expression system, and that the use of these lectins enables the effective detection of the hemagglutinin in a sandwich immunoassay.

[0007] That is, the present invention relates to the following 1) to 4). 1) A method for measuring influenza virus hemagglutinin expressed in a baculovirus-insect cell expression system, the method comprising a step of forming a sandwich immune complex containing a lectin selected from pea lectin and lentil lectin, the hemagglutinin, and an anti-hemagglutinin antibody that recognizes the hemagglutinin. 2) The method of 1), wherein the sandwich immune complex further comprises a labeled anti-anti-hemagglutinin antibody. 3) The method according to 2), wherein the labeled anti-anti-hemagglutinin antibody is a labeled anti-mouse IgG antibody. 4) The method of 2) or 3), wherein the lectin is immobilized on a solid phase, and the labeled anti-anti-hemagglutinin antibody bound to the solid phase is measured via the lectin, the hemagglutinin, and the anti-hemagglutinin antibody. [Effects of the Invention]

[0008] According to the present invention, influenza virus hemagglutinin expressed in a baculovirus-insect cell expression system can be quantified with high accuracy by sandwich immunoassay using a single type of anti-hemagglutinin antibody. [Brief explanation of the drawings]

[0009] [Figure 1] Absorbance measured by sandwich immunoassay. [Figure 2] Absorbance of negative control measured by sandwich immunoassay. [Figure 3] Calibration curve (four-parameter logistic curve) for sandwich immunoassay using standard antigens. DETAILED DESCRIPTION OF THE INVENTION

[0010] The method for measuring influenza virus hemagglutinin expressed in a baculovirus-insect cell expression system of the present invention comprises a step of forming a sandwich immune complex containing a lectin selected from pea lectin and lentil lectin, the hemagglutinin, and an anti-hemagglutinin antibody that recognizes the hemagglutinin, and is an immunological method for measuring influenza virus hemagglutinin, in which the hemagglutinin is measured based on the immune complex.

[0011] The hemagglutinin to be measured in the present invention is influenza virus hemagglutinin expressed in a baculovirus-insect cell expression system (hereinafter, sometimes simply referred to as "hemagglutinin"). Here, the baculovirus-insect cell expression system refers to an expression system that combines insect cells and baculovirus, and is also called a baculovirus expression vector system (BEVS). Therefore, the influenza virus hemagglutinin expressed in the baculovirus-insect cell expression system of the present invention means a hemagglutinin expressed in insect cells infected with a recombinant baculovirus containing a gene encoding influenza virus hemagglutinin.

[0012] Specifically, the lectins used in the present invention are one or two lectins selected from pea lectin and lentil lectin. Pea lectin and lentil lectin can be isolated and prepared from pea (Pisum sativum) and lentil (Lens culimaris), respectively, by known methods, but commercially available products can also be purchased and used. As will be shown in the Examples below, the lectin of the present invention has properties suitable for forming sandwich immune complexes in sandwich immunoassays, namely, it reacts well with influenza virus hemagglutinin expressed in a baculovirus-insect cell expression system but does not show reactivity with antibodies.

[0013] In the method of the present invention, the lectin is preferably immobilized on a solid phase to form a sandwich immune complex. Any solid phase used in well-known sandwich immunoassays such as sandwich ELISA can be used, including plates, tubes, beads, membranes, and gels. Materials that can be used include polystyrene, polypropylene, nylon, latex, glass, cross-linked dextrin, agarose, cross-linked agarose, and polyacrylamide.

[0014] Methods for adsorbing lectins to these solid phases include covalent bonding, physical adsorption, ionic bonding, and biochemical specific binding (e.g., binding biotin-binding lectin to a streptavidin-binding solid phase). Physical adsorption and biochemical specific binding are particularly preferred because of their simple operation. Examples of physical adsorption methods include dissolving lectin in a pH 7-9 buffer solution (e.g., Tris-HCl buffer saline, phosphate buffer saline, carbonate buffer, etc.) containing 0.05% Tween 20 (trade name), adding the solution to a solid phase (e.g., a well of a microplate), and allowing it to stand at room temperature for 1-2 hours or overnight at about 4°C for adsorption. Biochemical specific binding methods, in which streptavidin-binding solid phases (plates or beads) are commercially available, include dissolving lectin in a pH 7-9 buffer solution (e.g., Tris-HCl buffer saline, phosphate buffer saline, carbonate buffer, etc.) containing 0.05% Tween 20 (trade name), adding the solution to a commercially available streptavidin-binding solid phase, and allowing it to stand at room temperature for 1-2 hours or overnight at about 4°C for adsorption (see Examples below). In either the physical adsorption method or the biochemical specific binding method, the concentration of the lectin reacted with the solid phase is not particularly limited, but is usually about 0.1 μg / mL to 60 μg / mL in final concentration.

[0015] On the solid phase surface to which lectins have been adsorbed, there may remain unadsorbed surface areas, and if hemagglutinin or other molecular species in the sample are adsorbed to these areas, accurate measurement results may not be obtained. Therefore, it is preferable to add a blocking substance to cover the unadsorbed areas of lectins before contacting the sample with the solid phase. Examples of such blocking substances include serum albumin, casein, milk protein, lactic acid fermentation products, collagen, and their degradation products, which can be collected from mammals, such as cows. Commercially available blocking substances for immunoassays can also be used.

[0016] The anti-hemagglutinin antibody used in the method of the present invention, which recognizes influenza virus hemagglutinin, undergoes an antigen-antibody reaction with influenza virus hemagglutinin and may be a monoclonal or polyclonal antibody. When performing type-specific measurement of influenza virus, an anti-hemagglutinin monoclonal antibody that undergoes a specific antigen-antibody reaction with each type of hemagglutinin is usually used. Such anti-hemagglutinin antibodies include, for example, rabbit anti-influenza A H1N1 (Swine Flu 2009) hemagglutinin / HA polyclonal antibody (manufactured by Sino Biological, Inc.), mouse monoclonal to influenza A virus hemagglutinin (manufactured by Abcam), Influenza B hemagglutinin / HA antibody, rabbit PAb (manufactured by Sino Biological, Inc.), and Influenza B hemagglutinin / HA antibody, mouse MAb (manufactured by Sino Biological, Inc.).

[0017] The anti-hemagglutinin antibody can be labeled appropriately to prepare a labeled anti-hemagglutinin antibody. The labeling substances used for labeling include enzymes (peroxidase, alkaline phosphatase, β-galactosidase, luciferase, acetylcholinesterase, etc.), isotopes ( 125 I, 131 I, 3 Examples of labeling substances include fluorescent dyes (luminol, fluorescein isothiocyanate, umbelliferone, 7-amino-4-methylcoumarin-3-acetic acid, etc.), chemiluminescent substances, haptens, biotin, and avidin (e.g., streptavidin, etc.), but are not particularly limited as long as they are generally usable for labeling proteins. Note that the labeling substance used here also includes substances that are not directly detected themselves, such as biotin, but are used in methods in which a detectable label is bound to a substance (e.g., avidin) that has the ability to specifically bind to the substance.

[0018] The method for labeling the antibody can be appropriately selected from known methods suitable for the labeling substance, such as the glutaraldehyde method, periodate cross-linking method, maleimide cross-linking method, carbodiimide method, activated ester method, etc. when labeling an enzyme, and the chloramine T method, lactoperoxidase method, etc. when labeling with a radioisotope. Note that labeled anti-hemagglutinin antibodies against various types are commercially available, and commercially available products can also be used.

[0019] In the method of the present invention, the sandwich immune complex is formed by reacting the above-mentioned lectin, an anti-hemagglutinin antibody, and a specimen containing hemagglutinin. The anti-hemagglutinin antibody may be the labeled anti-hemagglutinin antibody described above or an unlabeled anti-hemagglutinin antibody. When an unlabeled anti-hemagglutinin antibody is used, a sandwich immune complex is formed that further contains a labeled anti-anti-hemagglutinin antibody (e.g., a labeled anti-immunoglobulin antibody).

[0020] The specimen to which the method of the present invention is applied may be any specimen containing influenza virus hemagglutinin expressed in a baculovirus-insect cell expression system. However, it is preferable to use specimens that have been solubilized with an ionic surfactant such as sodium dodecyl sulfate (SDS), lithium dodecyl sulfate (LiDS), hexadecyltrimethylammonium bromide (CTAB), hexadecyltrimethylammonium chloride (CTAC), or hexadecylpyridinium chloride (HPC), preferably CTAB. The solubilization conditions using an ionic surfactant are preferably such that the ionic surfactant is added to the sample to a final concentration of 0.1 to 2.0%, and the sample is allowed to stand or stirred at 37°C for about 1 to 2 hours.

[0021] The lectin, hemagglutinin, and anti-hemagglutinin antibody may be reacted simultaneously. Alternatively, for example, the immobilized lectin may be reacted with the hemagglutinin first, followed by washing and then reaction with the anti-hemagglutinin antibody. Alternatively, the hemagglutinin may be reacted with the anti-hemagglutinin antibody first to form an immune complex, which may then be reacted with the immobilized lectin. Each reaction can be carried out at room temperature for approximately 30 to 120 minutes. The final concentration of the hemagglutinin in the reaction system typically ranges from about 1 ng / mL to 1 μg / mL, and the final concentration of the anti-hemagglutinin antibody typically ranges from about 0.2 to 50 μg / mL.

[0022] The formed sandwich immune complex is then detected and measured. Specifically, after the reaction, the solid phase is washed, and the labeled substance in the sandwich immune complex bound to the solid phase is detected and measured. Examples of the washing solution include buffer solutions containing surfactants such as Tween (trade name) surfactants (e.g., phosphate buffer, phosphate buffer saline, Tris-HCl buffer, Tris-HCl buffer saline). The method for detecting a labeling substance varies depending on the labeling substance used, but examples include a method in which, when biotin is used as the labeling substance, an enzyme such as peroxidase is bound to a complex containing biotin as a labeling substance via streptavidin or the like, a color-developing substance such as tetramethylbenzidine and aqueous hydrogen peroxide are added as substrates for the enzyme, and the degree of color development of the product by the enzyme reaction is measured by the change in absorbance.Furthermore, when a fluorescent substance or chemiluminescent substance is used as the labeling substance, examples include a method in which the fluorescence or luminescence of the solution after the reaction is measured.

[0023] In the measurement method of the present invention, the hemagglutinin concentration in a sample can be quantified by preparing a calibration curve showing the relationship between the detection results of hemagglutinin and the labeled substance using a hemagglutinin standard solution of known concentration in advance, and then using the detection results for a sample of unknown concentration and the calibration curve.

[0024] A preferred embodiment of the measurement method of the present invention will be described below. 1) First, the lectin is adsorbed (coated) onto the solid phase. The preferred method of adsorption is as described above. After the adsorption, it is preferable to add a buffer solution containing a blocking substance such as skim milk and leave it at room temperature for about 30 to 2 hours to coat the areas where the lectin has not been adsorbed. 2) Next, a specimen is added to the solid phase to which the lectin has been adsorbed, and the specimen is left standing or stirred for an appropriate time, for example, 30 to 120 minutes at room temperature, to allow the hemagglutinin to bind to the lectin. 3) Thereafter, the solid phase to which this complex is bound is washed with a washing solution such as a buffer solution containing a Tween surfactant or the like (for example, Tris-HCl buffered saline, phosphate buffered saline, etc.). 4) Furthermore, a labeled anti-hemagglutinin antibody, or an anti-hemagglutinin antibody and a labeled anti-anti-hemagglutinin antibody (e.g., a labeled anti-immunoglobulin antibody) is added to the solid phase, and the mixture is left standing or stirred at room temperature for 30 to 120 minutes, for example, to bind the labeled anti-hemagglutinin antibody or the anti-hemagglutinin antibody-labeled anti-anti-hemagglutinin antibody to the hemagglutinin. This procedure forms a lectin-hemagglutinin-labeled anti-anti-hemagglutinin antibody complex or a lectin-hemagglutinin-anti-hemagglutinin antibody-labeled anti-anti-hemagglutinin antibody complex on the solid phase. 5) Next, the labeled substance in the complex is detected and measured.

[0025] Separately, a calibration curve is prepared showing the relationship between the concentration of the hemagglutinin standard and the detection result (e.g., absorbance) of the labeled substance, and the detection result for the unknown sample and the calibration curve are used to quantify the hemagglutinin in the unknown sample. [Example]

[0026] The present invention will be specifically explained below with reference to examples, but the present invention is not limited to these examples. Example 1 Lectin Screening (1) Hemagglutinin (HA) antigen i) The HA antigen expressed in the baculovirus-insect cell expression system (BEVS) was obtained by infecting insect cells (Sf9) with a recombinant baculovirus incorporating HA derived from B / Phuket / 3073 / 2013 (Thermo Fisher Scientific, Bac-to-Bac system). Two days after infection, the cells were harvested and a 1% Triton X-100 solution was added to extract the HA antigen. The resulting extract was clarified by centrifugation and purified using a cation exchange resin, Cellufine S-500 (JNC). In addition, to confirm the reaction with HA derived from influenza virus prepared in chicken eggs, a standard antigen manufactured by NIBSC was used as a control.

[0027] ii) These standard antigens, HA and HA prepared using BEVS, were diluted 10-fold or more with 1% hexadecyltrimethylammonium bromide (CTAB) solution and incubated at 37°C for 2 hours. Subsequently, they were diluted 2-fold with 1% CTAB solution, further incubated at 37°C for 2 hours, and then diluted 10-fold or more with 1% Triton X-100 solution to prepare solubilized samples. The solubilized standard antigen HA was adjusted to an HA concentration of 31.25 ng / mL for measurement.

[0028] (2) Lectin i) As lectins, seven types of lectins were evaluated: lentil lectin (LCA), pea lectin (PSA), and, as comparative controls, Cordyceps cristata lectin (ECL), castor bean lectin (RCA120), and Datura stramonium lectin (DSL) described in Patent Document 1, as well as banded bean lectin (GSLII) and snowdrop lectin (GNL) described in Patent Document 2.

[0029] (3) Sandwich immunoassay Seven biotinylated lectins (Vector Laboratories) were diluted to 20 μg / mL in 0.05% Tween 20 / Tris-HCl buffered saline (TBST) and added at 100 μL / well to a streptavidin-coated microplate for overnight incubation at 4°C. After the lectin incubation, each well was washed five times with 300 μL / well of TBST. Next, 300 μL / well of 2.5% skim milk / TBST was added to each well for blocking at 25°C for 1 hour, and then each well was washed five times with 300 μL / well of TBST. Subsequently, 100 μL / well of each solubilized sample and a solution without HA (negative control) were added and incubated at 25°C for 2 hours. After the reaction, each well was washed five times with 300 μL / well of TBST, and 100 μL / well of anti-HA antibody (Influenza B Hemagglutinin / HA Antibody, Rabbit PAb, manufactured by Sino Biological, Inc.) diluted with 0.5% skim milk / TBST was added to each well, followed by incubation at 25°C for 1.5 hours.

[0030] After the reaction, each well was washed five times with 300 μL / well of TBST. 100 μL / well of HRP-labeled anti-rabbit IgG antibody (Rabbit IgG Heavy and Light Chain Antibody, Bethyl Laboratories) diluted with 0.5% skim milk / TBST was added to each well and incubated at 25°C for 1 hour. After the reaction, each well was washed five times with 300 μL / well of TBST. 125 μL / well of TMB solution (TMB ELISA Substrate, Abcam) was added and incubated at 25°C for 20 minutes. The reaction was then stopped by adding 125 μL / well of 0.6N sulfuric acid to each well. After the reaction was stopped, the absorbance at 450 nm was measured.

[0031] As shown in Figure 1, ECL, RCA120, and DSL, which were confirmed to react with hen egg-derived HA, showed low absorbance values ​​with BEVS-prepared HA, suggesting that they do not react with insect cell-derived HA. GSLII reacted with both hen egg-derived and BEVS-prepared HA, but the absorbance was low, suggesting low reactivity. Furthermore, GNL showed high absorbance with both hen egg and BEVS-prepared HA. However, as shown in Figure 2, the blank value for the HA-free sample (negative control) was also high. This suggests that GNL may react with the antibody used for detection and therefore cannot be used in this analysis. On the other hand, LCA and PSA reacted with both insect cell and hen egg-derived HA and also showed low blank values. Therefore, these two lectins, LCA and PSA, were considered usable.

[0032] Example 2 Performance evaluation of HA-ELISA using LCA An ELISA method for measuring HA content (HA-ELISA) was established using LCA and anti-HA antibodies, which were found to have good reactivity with insect cell-derived HA in Example 1. This HA-ELISA was used to measure the HA content of various samples during the purification process of HA expressed in BEVS. (1) Sample preparation Insect cells (Sf9) were infected with a recombinant baculovirus incorporating HA from B / Phuket / 3073 / 2013, and the culture supernatant was collected two days after infection. The culture supernatant was concentrated by ultrafiltration and replaced with 25 mM Tris-HCl buffer (pH 7.4) containing 10 mM NaCl. After replacement, 25 mL of the replacement solution (sample name: Load sample) was loaded onto the affinity chromatography resin Cellufine DexS HbP (JNC), and the solution that passed through the resin was collected (sample name: Flow Through). The sample was then washed with 5 mL of 25 mM Tris-HCl buffer (pH 7.4) containing 10 mM NaCl, and this wash solution was also collected (sample name: Wash). After washing, HA was eluted from the resin with 5 mL of 25 mM Tris-HCl buffer (pH 7.4) containing 0.05% polysorbate 80 and 1 w / w% NaCl, each containing 50, 100, 200, 600, 1000, or 3000 mM NaCl (sample names: Elutions 1 to 6).

[0033] To solubilize the HA contained in these samples, they were diluted 20-fold or more with 1% CTAB solution and incubated at 37°C for 2 hours. Subsequently, they were diluted 2-fold with 1% CTAB solution, further incubated at 37°C for 2 hours, and then diluted 10-fold or more with 1% Triton X-100 solution to obtain solubilized samples. The same standard antigen from NIBSC as in Example 1 was used as a standard for the calibration curve. This standard antigen was also subjected to the same solubilization treatment, and the HA concentrations were adjusted to 7.81, 15.63, 31.25, 62.5, 125, 250, and 500 ng / mL.

[0034] (2) Sandwich immunoassay Biotinylated LCA was diluted with TBST to 20 μg / mL and added to a streptavidin-coated microplate at 100 μL / well for overnight incubation at 4°C. After the lectin reaction, each well was washed five times with 300 μL / well of TBST. Next, 300 μL / well of 2.5% skim milk / TBST was added to each well for blocking at 25°C for 1 hour, and then each well was washed five times with 300 μL / well of TBST. Next, 100 μL / well of each solubilized sample was added and incubated at 25°C for 2 hours. After the reaction, each well was washed five times with 300 μL / well of TBST. Anti-HA antibody (Influenza B Hemagglutinin / HA Antibody, Mouse MAb, Sino Biological, Inc.) diluted in 0.5% skim milk / TBST was added at 100 μL / well and incubated at 25°C for 1.5 hours. After the reaction, each well was washed five times with 300 μL / well of TBST. HRP-labeled anti-mouse IgG antibody diluted in 0.5% skim milk / TBST was added at 100 μL / well and incubated at 25°C for 1 hour. After the reaction, each well was washed five times with 300 μL / well of TBST. TMB solution was added at 125 μL / well and incubated at 25°C for 20 minutes. The reaction was then stopped by adding 125 μL / well of 0.6 N sulfuric acid. After the reaction was stopped, the absorbance at 450 nm was measured.

[0035] The four-parameter logistic curve obtained from the analysis software of the measuring device is shown in Figure 3, and the inverse regression concentration and relative standard deviation of the calibration curve are shown in Table 1.

[0036] [Table 1]

[0037] As shown in Figure 3, the calibration curve created was confirmed to be a good fit with the measured values. Furthermore, as shown in Table 1, the inverse regression concentrations calculated from each absorbance were roughly equivalent to the theoretical concentrations, and the relative standard deviation of each inverse regression concentration was less than 10%. Therefore, this analytical method is capable of creating an accurate calibration curve that reflects the theoretical concentration, and is considered to have good precision. Therefore, the HA content of each sample was calculated using this calibration curve.

[0038] As shown in Table 2, the HA content of the load sample, which is the input specimen to the affinity chromatography purification step, was set at 100%, and the percentage (yield) of each output sample was calculated.

[0039] [Table 2]

[0040] As a result, the total yield of the output sample was 98%, and the balance between the input and output to the purification process was roughly consistent. Therefore, it was considered that the HA-ELISA using LCA used in this example can accurately quantify HA even in samples with different solvents.

Claims

1. A method for measuring influenza virus hemagglutinin expressed in a baculovirus-insect cell expression system, the method comprising a step of forming a sandwich immune complex containing a lectin selected from pea lectin and lentil lectin, the hemagglutinin, and an anti-hemagglutinin antibody that recognizes the hemagglutinin.

2. The method of claim 1, wherein the sandwich immune complex further comprises a labeled anti-anti-hemagglutinin antibody.

3. The method of claim 2, wherein the labeled anti-anti-hemagglutinin antibody is a labeled anti-mouse IgG antibody.

4. The method according to claim 2 or 3, wherein the lectin is immobilized on a solid phase, and the labeled anti-anti-hemagglutinin antibody bound to the solid phase is measured via the lectin, the hemagglutinin, and the anti-hemagglutinin antibody.

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