WO2006126286A1

WO2006126286A1 - Hybridoma producing antibody that recognizes hiv-1

Info

Publication number: WO2006126286A1
Application number: PCT/JP2005/015856
Authority: WO
Inventors: Masami Moriyama; Yuetsu Tanaka
Original assignee: Masami Moriyama; Yuetsu Tanaka
Priority date: 2005-05-27
Filing date: 2005-08-31
Publication date: 2006-11-30

Abstract

A hybridoma producing an antibody that directly recognizes HIV-1, which can be used for the diagnosis of HIV-1 infection simply and reliably is provided. The hybridoma specifically produces an antibody (anti-HIV-1 p24 antibody) that recognizes and binds to p24 antigen (HIV-1 p24) that is expressed in HIV-1 in the growth phase, which can specifically bind to HIV-1 p24, diagnose the presence or absence of HIV-1 infection and determine the amount of virus. The hybridoma produces an antibody that is used for a kit for detecting HIV-1 in human serum, a human serum-derived blood preparation or serum for transfusion, specifically binds to HIV-1 p24 and is useful for detecting anti-HIV-1 p24 antibody and virus itself.

Description

Specification

Hyperidoma producing antibodies that recognize HIV-1

Technical field

[0001] The present invention relates to a hybridoma producing an antibody used for diagnosis of human immunodeficiency virus (HIV-1) infection.

Background art

[0002] AIDS (AIDS) is an abbreviation for Acquired Immunodeficiency Syndrome, a cell that controls immunity by infection with human immunodeficiency virus (HIV-1). A kind of opportunistic infection syndrome caused by extremely reduced immune immunity as a result of destruction of helper T cells. AIDS is a disease that affects immunity, which is the most basic function for humans to live, and overcoming this disease is considered to be an important issue that modern medicine must solve.

[0003] AIDS has spread throughout the world within a short period of time in the 1980s, and is still widely prevalent today. In modern times, the World Health Organization (WHO) estimates that approximately 5,000 to 6,000 new cases of infection will occur each day, and that the UK Department of Insurance estimates that 16,000 new Z days It is said that there are some infected people. According to UNICEF estimates, by 2010, about 40 million child-powered AIDS are even said to lose their parents. In particular, in East Africa, where AIDS treatment and HIV-1 infection prevention are inadequate, about 40% of children have a disastrous situation where one parent is lost by AIDS by the age of 15.

[0004] Various methods for diagnosing AIDS infection have been developed and put into practical use, and it is now possible to diagnose AIDS in developing countries. In addition, the development of AIDS treatments has been actively carried out, and it has become possible to delay the progression of AIDS after infection, but the cost of diagnosis is high and the price of the treatment is high. Therefore, the current situation is that only some people in the developing countries benefit from it. On the other hand, even in developed countries, the treatment of this disease with many side effects is not completely elucidated, and the current methods for diagnosing AIDS infection are quite complicated, and the infection is diagnosed at low cost. The development of diagnostic methods that can be refused is desired. [0005] By the way, AIDS is a kind of disease caused by a viral infection called retrovirus. That is, the AIDS pathogen is an RNA virus belonging to the Retroviridae family, and since HIV-1 prefers to infect lymphocytes and directly or indirectly destroys lymphocytes, the cells resulting from the reduction Sexual immune deficiency (acquired) occurs and AIDS develops.

[0006] The power of genetic dispersibility among viruses is the power of patients in the United States, Yotsuno, Haiti, Asia, and Africa To date, the various HIV-1 strains isolated Has an antigenic site. This antigenic site is retained in the major proteins, namely core protein p24, envelope glycoprotein gpl20 and transmembrane protein gp41. Thus, for example, the first retrovirus LAV strain that was confirmed to be the cause of AIDS development could be used against all HIV-1 class viruses in all carriers involved in the carrier's hometown. It can be used as a virus strain of an antigen for antibody detection. Therefore, this strain is currently used to detect HIV-1 antibodies in blood donors and patients using the immunofluorescence method, Western plot method, RIPA (immunoprecipitation assay) method, etc., which are currently known as ELISA methods. Widely used. However, serological studies conducted on HIV-1 lysates from patients from West Africa showed that these lysates showed signs of AIDS both clinically and immunologically. Serum reaction was negative or very weak and positive.

[0007] The AIDS test conducted today is an antibody test against HIV-1, and how to evaluate this antibody test is a problem. In general, antibody evaluation methods vary depending on the type of virus, and two indicators, sensitivity and specificity, are used to evaluate antibody tests. Sensitivity is the percentage of true infected persons who test positive, and specificity is the percentage of true non-infected persons who test negative. Therefore, a highly sensitive test can more reliably identify an infected person as positive, but conversely, a person who is not infected is more likely to be false positive. On the other hand, a highly specific test method can more reliably identify a non-infected person as negative, but it also increases the possibility of a false negative in which an infected person is mistakenly determined to be negative.

[0008] Antibody detection methods using biochemical reactions have 100% sensitivity and specificity There is no such thing as 100%. Specificity decreases with high-sensitivity testing methods, and sensitivity increases with increasing specificity. Therefore, today's AIDS tests use highly sensitive and test methods for screening, and those with high characteristics as confirmation tests, in order to ensure that infected persons are positively detected. Currently. Specifically, the particle agglutination method (Particle Agglutination: PA method) using gelatin particles is used as a screening, and if this method is determined to be positive, the Western plot (WB) method or IF method is used. Confirmation by (fluorescent antibody method), etc. (non-patent document 1 as a review)

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Therefore, the establishment of a simple diagnostic method that can detect HIV-1 infection more accurately is desired!

Non-patent document 1: "How to save AIDS-the forefront of research and treatment" by Shinji Harada, Chuko Shinsho, July 1997

Disclosure of the invention

Problems to be solved by the invention

[0009] In view of the current situation, the present invention provides a hybridoma that produces an antibody that directly recognizes HIV-1, which can be used for diagnosis of HIV-1 infection easily and reliably. And

[0010] That is, the present inventor has intensively studied to solve the above-mentioned problems! ヽ, focusing on viremia in which a large amount of virus is detected in the blood in the early stage of HIV-1 infection. _P 24 antigen in the blood at the growth phase was examined whether or not detectable (HIV-1 p24) directly. As a result, we succeeded in creating a hyperidoma that produces antibodies that specifically recognize and bind to HIV-1 p24 during the virus growth phase.

[0011] Then, using these specific antibodies produced by these cells, hybridomas, a sandwich ELISA kit capable of directly detecting and quantifying HIV-1 p24 can be prepared. As a result, it was confirmed that HIV-1 infection could be diagnosed with high sensitivity and ease, and the present invention was completed.

[0012] In diagnosing the presence or absence of HIV-1 infection, directly detecting HIV-1 p24 directly detects the HIV-1 body, ie, the virus body. Traditional diagnostic methods include HIV- The detection of HIV-1 antibody in place of the detection of 1, and its sensitivity and specificity were problematic, but the present invention diagnoses HIV-1 infection with specific and high sensitivity. It can be said that it is particularly excellent. The present invention has been completed as a result of intensive new studies.

Means for solving the problem

[0013] Therefore, the present invention for solving the difficult problem is

(1) Hyperidoma that specifically produces an antibody (anti-HIV-1 p24 antibody) that recognizes and binds to the p24 antigen (HIV-1 p24) expressed in the human immunodeficiency virus (HIV-1) in the proliferating phase;

(2) Inactivation of mice 匕 HIV-1 immunized mouse spleen cells obtained by inoculating HIV-1 and mouse myeloma cells are fused, and the resulting fused cells are screened for infected cells. The hyperpridoma according to 1 above, which specifically produces an anti-HIV-1 p24 antibody that recognizes and binds to HIV-1 p24, obtained by further cloning;

(3) The hyperidoma according to 1 or 2 above, wherein the anti-HIV-1 p24 antibody that recognizes and binds to HIV-1 p24 is a capture antibody that captures HIV-1 p24;

(4) The hyperidoma according to 1 or 2 above, wherein the anti-HIV-1 p24 antibody that recognizes and binds to HIV-1 p24 is a detection antibody that detects HIV-1 p24;

(5) Producing HIV-1 p24 capture antibody and Z or HIV-1 p24 detection antibody used to diagnose HIV-1 infection in human serum, human serum-derived blood products or blood transfusion serum 1 or 2 above High Pridoma as described in;

(6) The hyperpridoma according to any one of 3 to 5, wherein the capture antibody for capturing HIV-1 p24 is an RT-N-48 antibody;

(7) The hyperidoma according to any one of 3 to 5 above, wherein the detection antibody for detecting HIV-1 p24 is an RT-C-50 antibody;

It is.

The invention's effect

[0014] The hyperidoma provided by the present invention provides a hyperidoma that produces an antibody that can be used for more accurately detecting the presence or absence of HIV-1 infection. By using this antibody, HIV- 1 The presence or absence of infection can be easily diagnosed with higher sensitivity. In particular, the antibody produced by the hyperidoma provided by the present invention is capable of detecting HIV-1 p24, that is, the virus itself, and its diagnosis is extremely accurate. It has the advantage that it can be used for quantification.

Therefore, it becomes possible to directly detect HIV-1 infection in human serum, human serum-derived blood products or blood transfusion serum, and can effectively prevent the spread of HIV-1 infection.

[0015] That is, since the conventional diagnostic methods have detected HIV-1 antibody, the sensitivity and specificity of the diagnosis are not necessarily sufficient. By using the antibody produced by the provided hyperidoma, the virus itself can be detected, and HIV-1 infection can be diagnosed more reliably.

[0016] Further, the antibody produced by the hyperidoma provided by the present invention is used in a sandwich ELISA kit for diagnosing the presence or absence of HIV-1 infection, so that the HIV-1 infection diagnostic kit can be produced at low cost and in a large amount. It can be manufactured. Therefore, even in developing countries or countries where the incidence of AIDS is constant, there is an advantage that it is possible to more easily diagnose AIDS infection, which is an intractable disease, and to help with treatment.

BEST MODE FOR CARRYING OUT THE INVENTION

[0017] As described above, the hyperidoma provided by the present invention produces an antibody that recognizes the p24 antigen (HIV-1 p24) expressed in HIV-1 in the proliferating phase, that is, an anti-HIV-1 p24 antibody It is a hybridoma. The p24 antigen (HIV-1 p24) expressed during the growth phase of HIV-1 in the early stage of HIV-1 infection is an infectious antigen, and in general, antibodies against this antigen were supposed to interfere with the detection system. However, the hybridoma provided by the present invention is a hybridoma that produces an antibody capable of effectively recognizing HIV-1 p24 itself without interfering with the detection system.

[0018] In general, infection with HIV-1 reveals viremia in the early stages of infection, that is, a large amount of virus is detected in the blood. Therefore, a large amount of p24 antigen (HIV-1 p24) is present in the blood. If this HIV-1 p24 is reliably detected, HIV-1 infection can be diagnosed more effectively and with high sensitivity.

[0019] The present invention is a hyperidoma that produces an antibody that directly recognizes the p24 antigen at the time when viremia after the initial infection of HIV-1 is observed. This makes it easy to diagnose HIV-1 infection directly and with higher sensitivity.

[0020] Therefore, the conventional diagnosis of HIV-1 infection includes, for example, the core protein p25, or envelope glycoprotein gpl10 and transmembrane protein gp41-43 as antigen sites common to HIV-1 in various infected persons. The hybridoma provided by the present invention produces an antibody capable of recognizing HIV-1 p 24 in the proliferative phase after HIV-1 infection. By using this method, it can be said that detecting the virus itself directly is superior in diagnosing HIV-1 infection.

[0021] Such a hyperidoma is basically produced by inoculating HIV-1 with, for example, a mouse so that the mouse functions immune to HIV-1 and, as a result, produces anti-HIV-1 antibody. Tissue cells, preferably spleen cells, are collected and fused with commonly used mouse myeloma cells (mouse melanoma cells), and the resulting fused cells can be used for normal hybridoma cloning. By performing screening in a culture using a HAT selection medium, a hybridoma that effectively recognizes and binds to HIV-1 p24 can be obtained.

Operations such as powerful cell fusion, high-pridoma cloning, and screening can be performed by appropriately applying generally used techniques (see, for example, Examples described later).

[0022] Neubridoma obtained by force is an antibody that recognizes HIV-1 p24 extremely effectively and with high sensitivity, that is, a hybridoma that produces an anti-HIV-1 p24 antibody. Among them, one of the antibodies produced by hyperidoma is a capture antibody that captures HIV-1 p2 4, and another antibody functions as a detection antibody that detects HIV-1 p24. be able to.

[0023] Specifically, a capture antibody that captures HIV-1 p24, named RT-N-48 antibody by the present inventors, and an HIV-1 p24 named RT-C-50 antibody. Functions as a detection antibody to detect.

Therefore, the present invention specifically provides an RT-N-48 antibody that captures HIV-1 p24 and a hybridoma that produces an RT-C-50 antibody that detects HIV-1 p24. It is.

[0024] The hybridoma provided by the present invention produces an antibody that directly recognizes HIV-1 p24, and the produced antibody functions as an antibody that captures and detects HIV-1 p24. . Accordingly, by using these antibodies, HIV-1 infection can be directly diagnosed with high sensitivity by ELISA kit for detecting HIV-1 antigen 'antibody, that is, sandwich method.

[0025] Specifically, RT-N-48 antibody as a capture antibody against HIV-1 p24 is applied to, for example, a well microphone mouth plate for HIV-1 detection. On the other hand, for example, RT-C-50 antibody as a detection antibody to which peroxidase (POD) as a detection dye is bound is prepared, and the detection antibody is added together with the detection sample on the above plate, and the reaction by the sandwich method is performed. The color can be developed with a color former and diagnosed for HIV-1 infection.

[0026] It should be noted that the HIV-1 p24 capture antibody and detection antibody produced by the hyperidoma provided by the present invention can be sandwiched by appropriately stepwise diluting the concentration applied to the well microphone mouth plate, for example. It is possible to qualitatively and quantify HIV-1 p24 or antibodies against it by measuring the absorbance at a wavelength of 450 nm, for example, by measuring the absorbance at 450 nm and comparing the standard curve with the standard. .

Example

[0027] In the following, the present invention will be described in more detail by explaining the production of specific high-pridoma. The present invention is not limited to these, but various modifications can be made without departing from the purpose. These modifications are also included in the technical scope of the present invention.

[0028] Test Example 1: Preparation of hybridoma

In order to obtain the hyperidoma of the present invention, as a first step, mice were immunized with HIV-1 and immune tissue cells (spleen cells) were excised.

[Method]

[0029] (a) Concentrated HIV-1 was obtained by the following method.

Clinical isolate virus (HIV-1) strain was grown on human peripheral blood mononuclear cells (PBMC) stimulated with anti-CD3 antibody and interleukin-2 (IL-2) and inactivated with AT-2 drug Thereafter, concentrated HIV-1 was obtained as a fraction concentrated by ultracentrifugation. [0030] (b) Immunization to mice:

Concentrated HIV-1 obtained in (a) above is solubilized with a surfactant, mixed with an equal amount of Freund Adjuvant (CFA), and inoculated subcutaneously in mice in the state of emulsions. , Sensitized.

Two weeks later, the same amount of concentrated HIV-1 soluble yeast was mixed with Freund's complete adjuvant (CFA) in an equal amount and immunized subcutaneously in mice. This immunization was repeated 3 times every 2 weeks.

Two weeks after this final immunization, the same amount of concentrated HIV-1 soluble yeast was inoculated into the peritoneal cavity of the immunized mouse, and three days later, the immune spleen was removed from the mouse.

[0031] (c) Cell fusion:

Mouse myeloma (mouse myeloma) cells SP2 / 0 were grown logarithmically and washed in RPMI-1 medium supplemented with 10% FCS (fetal urine serum).

On the other hand, the mouse spleen excised above was lined with a stainless mesh to obtain a cell suspension, and this cell suspension and the proliferated mouse myeloma cells SP2 / 0 cells at a ratio of 4: 1 polyethylene glycol 4000 ( Cell fusion was performed using PEG4000).

[0032] (d) Selection of hybridoma

The fused cells (fused cells) were seeded on a 96-well microphone mouthplate, and selection of high-pridoma was performed.

The selection of the cells was performed using a commercially available HAT selection medium. That is, the medium was changed every 3 days, and the culture supernatant of the fused cells that appeared during the culture 14 to 21 days after the fusion was screened by the following fluorescent antibody method.

[0033] (e) Screening (positive):

HIV-1 infected cell line Molt4 / IIIB and non-infected cell Molt4 cells are smeared on separate 15-well slide glasses (Dainippon Pharmaceutical Co., Ltd.), air-dried, solidified with 100% ethanol at 20 ° CZ for 10 minutes, then E .

Place an Atsey sample on each slide glass well, react at room temperature for 30 minutes, wash, and then commercially available FITC label (note: fluorescein isothiocyanate: antibody fluorescent labeling reagent) goat anti-mouse IgG ( 1/100 dilution) was further reacted at room temperature for 30 minutes. After washing, using a fluorescence microscope, MoltVlIIB cells (Note: infected cells) were stained, and Molt4 cells (Note: non-infected cells) were not stained.

The reactivity to HIV-1 p24 was confirmed by Western plot (WB) method using Molt4 / IIIB and a soluble product of Molt4 cells as antigens.

[0034] (f) Screening (secondary):

HIV-1 infected activated human PBMC and non-infected control cells are smeared on separate 15-well slide glasses (Dainippon Pharmaceutical Co., Ltd.), air-dried, and then solidified with 100% ethanol for 10 minutes at 20 ° CZ for 7 minutes.

Place the Atsy sample on each slide glass well, react at room temperature for 30 minutes, wash, and then commercially available FITC label (note: fluorescein isothiocyanate: antibody fluorescent labeling reagent) goat anti-mouse IgG ( 1/100 dilution) was further reacted at room temperature for 30 minutes.

After washing, the infected cells were stained using an fluorescence microscope, and antibodies that did not stain uninfected cells were screened.

Similarly, the reactivity to HIV-1 p24 was confirmed by the Western plot (WB) method using Molt4 / IIIB and a soluble product of Molt4 cells as an antigen.

[0035] (g) Determination of clawing and Ig class:

Hypridoma in antibody-producing wells was cloned twice by the limiting dilution method using mouse thymocytes as feeder cells according to a conventional method. The determination of the Ig class and subclass of the antibody contained in the culture supernatant after the final cloning was determined by ELISA using a commercially available goat anti-mouse Ig kit.

[0036] (h) Large-scale preparation and purification of antibodies:

Commercially available SCID mice (CB-17) were administered 0.5 mL of pristane intraperitoneally, and one week later, 5 million hyperpridoma obtained above were intraperitoneally transplanted. Ascites was obtained after about 10 days. The ascites is pooled, the cells are precipitated by centrifugation at 15,000 rpm for 10 minutes, the resulting supernatant is further filtered through a 10-m filter, and the filtrate is precipitated with 50% aqueous ammonium sulfate solution. It was. The obtained sediment was dissolved in phosphate buffered saline (PBS), and an IgG fraction was prepared by gel filtration using Sephadex G200. The antibody concentration was estimated by OD280. [0037] By the above method, nobridoma producing 20 or more kinds of anti-HIV-lp24 antibodies was obtained. Among them, the following combination made it possible to measure HIV-1 p24 with high sensitivity by sandwich ELISA.

Capture antibody: RT-N-48

Detection antibody: RT-C-50

In both cases, the IgG class was mouse IgG.

[0038] Test Example 2: Preparation of HIV-1 p24 antigen standard solution for sandwich ELISA

(a) (Correct) Triton Χ-100 with a final concentration of 10% was added to the culture supernatant of Molt4 / IIIB cells and allowed to react in ice for 30 minutes. Aliquot into Eppendorf type tubes and store at -20 ° C.

(b) Add 10% final concentration of Triton Χ-100 to the culture supernatant grown in peripheral blood mononuclear cells P BMC of humans who were stimulated with (CD) antibody and IL-2. The mixture was reacted for 30 minutes in ice. Aliquot into Eppendorf type tubes and store at -20 ° C.

Industrial applicability

[0039] As described above, as a method for conveniently and reliably diagnose HIV-1 infection by the present invention, a hybridoma producing a direct antibody recognizing HIV-1 _P 24 is provided. The hyperidoma provided by the present invention produces an anti-HIV-1 antibody for detecting HIV-1 p24 for diagnosing HIV-1 infection and produces an antibody capable of detecting the virus body. Yes.

[0040] That is, the hybridoma provided by the present invention produces HIV-1 p24, that is, an antibody that can detect the virus body. By using a strong antibody, the presence or absence of HIV-1 infection is detected. Diagnosis is extremely accurate and can be used to quantify viral load in addition to the presence or absence of infection.

Therefore, it becomes possible to directly detect HIV-1 infection in human serum, human serum-derived blood products or blood transfusion serum, and can effectively prevent the spread of HIV-1 infection. Industrial contribution is tremendous.

Claims

The scope of the claims

[1] A hyperidoma that specifically produces an antibody (anti-HIV-1 p24 antibody) that recognizes and binds to the p24 antigen (HIV-1 p24) expressed in the proliferating human immunodeficiency virus (HIV-1).

[2] Inactive mice マウス Spleen cells that have been immunized against HIV-1 obtained by inoculating HIV-1 and mouse myeloma cells are fused, and infected cells are detected from the resulting fused cells. Anti-HI that recognizes and binds to HIV-1 p24, obtained by cleaning and further cloning

The hyperidoma according to claim 1, which specifically produces V-l p24 antibody.

[3] The hyperpridoma according to claim 1 or 2, wherein the anti-HIV-1 p24 antibody that recognizes and binds to HIV-1 p24 is a capture antibody that captures HIV-1 p24.

[4] The hyperpridoma according to claim 1 or 2, wherein the anti-HIV-1 p24 antibody that recognizes and binds to HIV-1 p24 is a detection antibody that detects HIV-1 p24.

[5] Claims to produce HIV-1 p24 capture antibody and Z or HIV-1 p24 detection antibody for use in diagnosing HIV-1 infection in human serum, human serum-derived blood products or blood transfusion serum High Pridoma according to 1 or 2.

[6] The hyperidoma according to any one of claims 3 to 5, wherein the capture antibody that captures HIV-1 p24 is an RT-N-48 antibody.

[7] The detection antibody for detecting HIV-1 p24: The hyperidoma according to any one of claims 3 to 5, which produces RT-C-50.