WO2009122591A1 - Kit for detecting viral antigen and viral antibody capable of detecting viral antigen and viral antibody at a time, and method for detecting viral antigen and viral antibody - Google Patents

Abstract

It is intended to provide a kit for detecting a viral antigen and a viral antibody capable of detecting a viral antigen and a viral antibody at a time, which can detect antibodies against HIV-1 and HIV-2, and HIV-1 p24 in one step at a time, and a method for detecting a viral antigen and a viral antibody. It relates to a kit for detecting a viral antigen and a viral antibody contained in a human specimen. At least a non-human-derived capture antibody immobilized on a support and specifically binding to a viral antigen, a non-human-derived capture antigen specifically binding to a viral antibody, a non-human-derived detection antibody labeled with a labeling substance and specifically binding to a viral antigen and a non-human-derived detection antigen labeled with a labeling substance and specifically binding to a viral antibody are included, and binding between the capture antibody and the capture antigen and between the detection antigen and the detection antibody are effected by a one-step reaction.

Description

Virus antigen and virus antibody detection kit capable of detecting virus antigen and virus antibody at one time, and virus antigen and virus antibody detection method

The present invention relates to a virus antigen and virus antibody detection kit capable of detecting a virus antigen and a virus antibody at the same time, and a method for detecting a virus antigen and a virus antibody, and more particularly, to immunize a virus antigen and a virus antibody contained in a human specimen. The present invention relates to a virus antigen and virus antibody detection kit capable of detecting virus antigens and virus antibodies detected at a time, and a method for detecting virus antigens and virus antibodies.

It is known that various infectious diseases are caused by viruses that infect humans.
For example, acquired immunodeficiency syndrome (AIDS) is an opportunistic infection of other pathogens due to extreme decline in immune capacity as a result of destruction of helper T cells by infection with human immunodeficiency virus (HIV). It is a disease affected by There are two types of HIV, HIV-1 and HIV-2, depending on the origin of the virus.

Conventionally, a method for detecting HIV-1 contained in a sample such as human serum and a kit used therefor are known (see, for example, Patent Document 1).
In this method or kit, a mouse antibody (capture antibody) that recognizes and binds p24, which is the capsid protein of the HIV-1 virus, as an antigen, and a labeled mouse antibody that also recognizes and binds the HIV-1 p24 antigen (capture antibody). Detection antibody) and detection and quantification of HIV-1 virus.

This conventional HIV-1 detection method is as follows.
First, a solution containing a capture antibody is applied to a microplate for HIV-1 detection, and the capture antibody is immobilized using the microplate surface as a carrier. Subsequently, a detection antibody is added to the human specimen on this microplate and a reaction is performed by the sandwich method, and a binding reaction between the HIV-1 p24 antigen contained in the specimen, the capture antibody and the detection antibody is conducted. Next, the microplate after the reaction is washed to remove unreacted serum proteins and detection antibodies, and a substrate and a coloring solution are added to the washed microplate, and the detection is labeled by enzyme immunoassay (ELISA method). Quantify antibody. This indirectly detects and quantifies the HIV-1 p24 antigen (ie, viral antigen) bound to the detection antibody.

Some commercially available HIV-1 detection kits use a human-derived anti-p24 antibody as a p24 detection antibody and / or a capture antibody. However, since human-derived p24 capture antibodies and detection antibodies need to be separated and purified from the serum of HIV-1 infected persons, it is difficult to obtain a large amount of antibodies at a time, leading to an increase in kit manufacturing costs. There was an inconvenience.
In addition, some goat-derived detection antibodies and capture antibodies are used, but as with human-derived detection and capture antibodies, it is difficult to obtain a large amount of antibodies at a time, and the cost of kit production There was an inconvenience of incurring a rise.

Also, since the amount of serum that can be obtained from the same infected person is limited to a small amount, it is necessary to obtain antibodies from multiple infected persons. However, since the characteristics of the antibody vary from one infected person to another, the detection accuracy varies from lot to lot of the detection kit, which makes it difficult to perform accurate detection and quantification.

Therefore, in Patent Document 1, antibodies produced from non-human organisms are used as detection antibodies and capture antibodies. Specifically, a monoclonal antibody produced from a mouse-derived hybridoma is used. In this way, by obtaining non-human hybridomas and producing antibodies, it is possible to obtain a large amount of antibodies in a uniform lot compared to obtaining capture antibodies and detection antibodies from human serum. Is possible. As a result, it is possible to improve the stability of detection sensitivity and reduce the cost of manufacturing a detection kit.

International Publication No. WO2006 / 126287 Pamphlet

By the way, eradication of infectious diseases is one of the common global issues to realize world peace.
In particular, the outbreak and spread of infectious diseases in developing countries can lead to re-emerging infectious diseases in developed countries due to the internationalization of transportation and human exchange.
As recently reported, especially in developing countries, the spread of viral infections such as HIV and hepatitis viruses has become a serious problem.

The latest blood diagnostic kit for HIV infection is a method for simultaneously measuring antibodies to HIV-1 and HIV-2 and HIV-1 p24, which is called a fourth generation diagnostic kit.
For detection of the antibody, antigen capture method, that is, the serum is reacted with a plate coated with HIV-1 and HIV-2 antigens. If the antibody is positive, the enzyme (HRP) labeled HIV-1 and HIV to be reacted next A method using binding of a mixture of -2 antigens to a plate is used. This reaction is a one-step reaction and the inspection is completed.
However, since detection of HIV-1 p24 that enables early diagnosis currently uses a biotin-labeled antibody, it requires a two-step reaction with HRP-labeled Streptavidin, which takes time and effort.

Therefore, if the HIV-1 p24 detection method can be performed in one step by such a method as currently performed, the test reaction can be completed in one step even in the fourth generation kit.
This is a simpler and more advanced method.
Furthermore, if a new method that enables the storage of the kit's plates and reagents at room temperature is devised, it is expected to support AIDS countermeasures for developing countries without cold storage facilities.

In view of the above problems, an object of the present invention is to simultaneously detect antibodies against HIV-1 and HIV-2 and simultaneously detect viral antigens capable of detecting HIV-1 p24 in one step and viral antigens capable of detecting viral antibodies at a time. And a detection method for detecting a viral antigen and a viral antibody.

According to the virus antigen and virus antibody detection kit capable of detecting the virus antigen and virus antibody according to the present invention at the same time, the above object is a detection kit for virus antigen and virus antibody contained in a human sample, A non-human-derived capture antibody that specifically binds to the viral antigen, a non-human-derived capture antigen that specifically binds to the viral antibody, and a labeling substance that is labeled with a specific substance. A non-human-derived detection antibody that binds to the target, and a non-human-derived detection antigen that is labeled with the labeling substance and specifically binds to the viral antibody, the capture antibody and the capture antigen, and This is solved by performing the binding between the detection antigen and the detection antibody in a one-step reaction.

In addition, according to the method for detecting a virus antigen and a virus antibody according to the present invention, the above-described problem is a method for detecting a virus antigen and a virus antibody contained in a human sample, which specifically binds to the virus antigen. Immobilizing a capture antibody derived from a human and a capture antigen derived from a non-human specifically binding to the virus antibody on a carrier; a detection antibody derived from a non-human specifically binding to the virus antigen; and the virus antibody Labeling a labeling substance on a non-human-derived detection antigen that specifically binds in any order, reacting the human specimen with the capture antibody and the capture antigen, and the virus antigen and the virus antibody A step of performing a reaction for binding the capture antibody and the capture antigen in one step, and the detection antibody and the detection antibody conjugated to the viral antigen and the viral antibody. A step of detecting the labeling substance of the detection antigen, is solved by performing.

Thus, in the present invention, the reaction of binding the capture antibody and the capture antigen to the virus antigen and the virus antibody can be performed in one step.
That is, it is not necessary to perform a two-step reaction as in the prior art.
Taking human immunodeficiency virus (HIV) as an example, an antigen capture method is currently used to detect the antibody. This is because a mixture of HIV-1 and HIV-2 antigens labeled with an enzyme (HRP) that reacts with serum on a plate coated with HIV-1 and HIV-2 antigens, and then reacts if the antibody is positive. It is a method using bonding to. This reaction is a one-step reaction and the inspection is completed.
However, since detection of HIV-1 p24 that enables early diagnosis currently uses a biotin-labeled antibody, it requires a two-step reaction with HRP-labeled Streptavidin, which takes time and effort.
Therefore, with the kit of the present invention, the test reaction can be completed in a short time by making the HIV-1 p24 detection method one step.
In addition, since the number of operation steps is reduced, the inspection can be performed more easily.

At this time, if the capture antibody, the capture antigen, the detection antibody, and the detection antigen are provided separately, it is preferable because individual tests can be performed separately.
For example, a more detailed diagnosis of infection can be performed by separately performing individual tests on a sample determined to be positive in the first test.

At this time, it is preferable that the immobilized carrier is maintained in a moisturized state.
This is because the non-human-derived capture antibody that specifically binds to the viral antigen and the non-human-derived capture antigen that specifically binds to the viral antibody are immobilized on a carrier, followed by the capture antibody and This is realized by performing a step of maintaining the carrier on which the capture antigen is immobilized in a moisturized state.
Further, at this time, it is more preferable that the moisturizing state is realized by shutting off the outside air while the medium impregnated with moisture is in contact with the carrier.
In the step of maintaining the carrier on which the capture antibody and the capture antigen are immobilized in a moisturized state, the moisturized state is a state in which a medium impregnated with water is in contact with the carrier and the outside air. Realized by blocking.

Thus, by maintaining the carrier on which the capture antibody and the capture antigen are immobilized in a moisturized state, it can be stored at room temperature while maintaining the virus antigen detection capability and the virus antibody detection capability.
Such a kit and method used as a blood test method for viral infections are very beneficial because they can simultaneously screen multiple specimens in a simple facility and are excellent in economy and convenience. All known antigen-antibody test kits and methods require refrigeration for transport and storage for reasons of accuracy control.
In other words, exposure of the kit to room temperature for a long time is contraindicated.
For this reason, it was difficult to store and use in regions of the world where there is no power supply or refrigeration facilities. However, as shown in the present invention, the carrier on which the capture antibody and the capture antigen are immobilized must be kept moisturized. Thus, it can be stored at room temperature for a long time.
Therefore, the use area is not limited, and it can be used effectively worldwide.

In addition, the detection kit for the virus antigen and virus antibody is more convenient if it further comprises a test reagent for detecting the detection antibody bound to the virus antigen and the virus antibody and the labeling substance of the detection antigen. Therefore, it is preferable.

Further, the viral antigen is human immunodeficiency virus type 1 (HIV-1),
The capture antibody and the detection antibody are both antibodies having HIV-1 p24 as an antigen, and the viral antibodies are human immunodeficiency virus type 1 (HIV-1) and human immunodeficiency virus type 2 (HIV−). It is preferable that the capture antigen and the detection antigen are HIV-1 gp41 antigen, HIV-1 gp120 antigen, and HIV-2 gp36 antigen.

The virus antigen detection method and virus antigen detection kit according to the present invention can detect HIV-1 p24 antigen in one step simultaneously with antibodies against HIV-1 and HIV-2.
For this reason, while being able to shorten test | inspection time, an operation step reduces and it can test | inspect an infectious disease easily.
In addition, the virus antigen detection method and virus antigen detection kit according to the present invention can maintain the performance even at room temperature (about 10 ° C. to 40 ° C.).
Therefore, refrigeration is not an essential condition for transport and storage.
For this reason, it can be used effectively in a wide area around the world.

It is a calibration curve showing the detection sensitivity of HIV-1 p24 antigen.

Hereinafter, an embodiment of the present invention will be described.
It should be noted that members, arrangements, and the like described below do not limit the present invention, and it goes without saying that various modifications can be made in accordance with the spirit of the present invention.
In the present specification, “normal temperature” means a temperature at which the kit according to the present invention is likely to be naturally exposed in a normal distribution route and storage environment, that is, a temperature of about 10 ° C. to about 40 ° C. Is assumed.
However, this temperature is not strictly limited to this range, but it means a temperature that is assumed to be common sense and sensible at the time of distribution and storage, and the range numbers are assumed to be common sense and sensible. It is only an example of the temperature to be applied.
Of course, extremely low temperatures and extremely higher temperatures naturally exist on the earth, but if the temperature is much lower than this, the conditions are the same as in the refrigerated state, and if the temperature is much higher, This is a condition with low possibility of actual use and distribution.

Hereinafter, as an example of viral antigens and antibodies, detection kit and detection method of viral antigens using human immunodeficiency virus type 1 (HIV-1) p24, human immunodeficiency virus type 1 (HIV-1), A virus antibody detection kit and detection method using Type 2 (HIV-2) will be described.
The virus in the present invention is not limited to HIV-1 and HIV-2 as long as it infects humans and is contained in a specimen.
For example, other infectious viruses such as hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), and parvovirus may be used.

Further, as a human specimen, serum is described as an example.
However, the human specimen only needs to include the above-mentioned viral antigens or anti-heterologous immunoglobulin antibodies (hereinafter referred to as anti-heterologous Ig antibodies). For example, lymph, tissue fluid, bone marrow fluid, joint fluid, urine, saliva, Other bodily fluids such as vaginal secretions and semen, and those obtained by subjecting these bodily fluids to predetermined treatment may be mentioned.

The virus antigen detection kit of the present invention comprises a non-human-derived capture antibody and capture antigen immobilized on a carrier, a non-human-derived detection antibody and detection antigen labeled with a labeling substance, and a human specimen. And a blocking antibody having an epitope recognized by the anti-heterologous Ig antibody.
This epitope is identical to the epitope common to the capture and detection antibodies recognized by the anti-heterologous Ig antibody.

As an antigen / antibody measurement system, the target antigen and target antibody are reacted with both the capture antibody and capture antigen, the detection antibody and detection antigen, and detected by sandwich ELISA.
By using a blocking antibody, a false positive reaction due to binding of the anti-heterologous Ig antibody to the capture antibody / blocking antibody can be prevented, and the virus antigen can be detected with high accuracy.

(Capture antibody)
The capture antibody is a non-human-derived monoclonal antibody (anti-HIV-1 p24), which recognizes a virus contained in a human specimen as an antigen and specifically binds to it.
The capture antibody of this embodiment uses the core protein p24 as an antigen site common to HIV-1 of various infected persons as an antigen, and specifically recognizes and binds to a partial region as an epitope. It is an antibody.

(Capture antigen)
Capture antigens include recombinant HIV-1 gp41, gp120 antigen and recombinant HIV-2
It is a gp36 antigen and recognizes and specifically binds a viral antibody contained in a human specimen.
The capture antigen of this embodiment binds to HIV-1 gp41, gp120 and HIV-2 gp36 antibodies of various infected persons.

The capture antibody and the capture antigen are used by being immobilized on a carrier such as a plastic plate, and this is immobilized on the carrier by binding to the virus antigen and the virus antibody.
Examples of the material of the plastic plate include polypropylene, polystyrene, polyethylene, and the like that exhibit binding properties to the capture antibody.
In the present embodiment, the capture antibody and the capture antigen are immobilized by adsorption between the plastic plate and the antibody molecule of the capture antibody, and adsorption between the plastic plate and the antigen molecule of the capture antigen.

The immobilization of the capture antibody or capture antigen and the carrier is not limited to the above-described adsorption, and may be immobilized by, for example, covalent bond or electrostatic interaction.
Further, the carrier for immobilizing the capture antibody and the capture antigen is not limited to a plastic plate, but may be a plastic tube, a slide glass, a resin bead, or the like, and a material exhibiting binding properties to the capture antibody and the capture antigen.

(Detection antibody)
Like the capture antibody, the detection antibody is an antibody derived from non-human, and is a primary antibody that recognizes and binds to a virus contained in a human specimen as an antigen.
The detection antibody of the present embodiment is an HIV-1 p24 antibody that specifically binds to the HIV-1 p24 antigen, similar to the capture antibody described above, but recognizes an epitope different from the epitope recognized by the capture antibody. Join.
That is, the capture antibody and the detection antibody recognize different regions of the same antigen (in this case, p24) as antigens.

The capture antibody and the detection antibody are antibodies derived from organisms other than humans (non-human organisms) and have the same class / subclass.
The capture antibody and detection antibody used in this embodiment are both mouse-derived monoclonal antibodies, and the class / subclass is IgG1.
In addition, if these antibodies are non-human-derived antibodies, the biological species derived from them is not limited to mice. For example, hamsters, rats, guinea pigs, rabbits, goats, sheep, chickens, horses, cows, etc. Good. Further, the subclass is not limited to IgG1, and may be another subclass such as IgG2a, IgG2b, IgG3.

The capture antibody and the detection antibody in this embodiment are both antibodies that use HIV-1 p24 as an antigen, but may be antibodies that recognize other regions of HIV-1 as antigens.
For example, an antibody that recognizes HIV-1 envelope glycoprotein gp120 or transmembrane protein gp41 may be used.

The capture antibody and detection antibody in the present invention can be obtained from, for example, a non-human hybridoma. For example, when a mouse-derived hybridoma is used as the non-human hybridoma, it can be obtained by the following procedure.
First, purified HIV-1 p24 is inoculated into BALB / c mice and immunized, and a tissue producing anti-HIV-1 p24 antibody, for example, spleen cells is collected. Next, the obtained spleen cells and mouse myeloma cells (for example, SP-2 / 0) are subjected to cell fusion using a known cell fusion method using polyethylene glycol to establish a plurality of types of hybridomas.

Subsequently, among the antibodies produced from the obtained hybridomas, antibodies that react with HIV-1 p24 are selected by an enzyme immunoreaction method (ELISA method) using purified HIV-1 p24 as an antigen.
Furthermore, after the selected hybridoma is transplanted into a BALB / c mouse, the obtained ascites is obtained and treated by an ammonium sulfate salting-out method and a gel filtration method. Thereby, an antibody specific for the p24 antigen can be obtained.
The fusion of spleen cells and myeloma cells is not limited to the method using polyethylene glycol as described above, and may be a method using Sendai virus or a method using electrical treatment.

The detection antibody of this embodiment is labeled with horseradish peroxidase (HRP) as a labeling substance. In the labeled detection antibody, HRP is covalently bound to an antibody molecule (mouse IgG molecule). The detection antibody can be labeled by a known method such as the periodic acid method, the maleimide method, the glutaraldehyde method, or the like.

In this embodiment, peroxidase is used as the labeling molecule. However, the labeling molecule that binds to the detection antibody is not limited to the peroxidase described above, and other molecules may be used depending on the detection system. For example, other enzymes such as alkaline phosphatase, FITC, biotin, gold colloid, etc. may be labeled.

The capture antibody may be immobilized by directly binding to p24 as in the above-described embodiment, or indirectly bound and immobilized via another molecule (for example, an antibody immobilized on a carrier). It may be a thing to become.
Similarly, the detection antibody may be labeled by directly binding to p24, and indirectly bound and labeled through another molecule (for example, a labeled secondary antibody). It may be a thing.

(Detection antigen)
Further, recombinant HIV-1 antigen and recombinant HIV-2 antigen are used as detection antigens, and horseradish peroxidase (HRP) is labeled as a labeling substance on these antigens.
In the labeled detection antigen, HRP is covalently bound to the antigen molecule.
The detection antigen can be labeled by a known method such as the periodic acid method, the maleimide method, or the glutaraldehyde method.

In this embodiment, peroxidase is used as the label molecule, but the label molecule that binds to the detection antigen is not limited to the peroxidase described above, and other molecules may be used depending on the detection system. The same as in the case of the detection antibody.

(Blocking antibody)
Next, the blocking antibody of the present invention and the anti-heterologous Ig antibody that binds to the blocking antibody will be described. A blocking antibody is an antibody that binds to and absorbs an anti-heterologous Ig antibody contained in a human specimen.
An anti-heterologous Ig antibody is an antibody that recognizes and binds to an epitope common to both the capture antibody and the detection antibody described above. This anti-heterologous Ig antibody binds to both the capture antibody and the detection antibody and exhibits an HIV-1 false positive reaction.

The blocking antibody is an antibody having the same epitope in the molecule as the common epitope, and is recognized and bound by the anti-heterologous Ig antibody. For this reason, by adding a blocking antibody to the human specimen, the anti-heterologous Ig antibody specifically binds to the blocking antibody, and as a result, the anti-heterologous Ig antibody is absorbed. That is, the anti-heterologous Ig antibody cannot bind to the capture antibody or the detection antibody by binding to the blocking antibody. For this reason, bridge formation between the capture antibody and the detection antibody by the anti-heterologous Ig antibody can be prevented.

The blocking antibody can be obtained from the same species as the species from which the capture antibody and the detection antibody are derived. The class and subclass of blocking antibody is the same as the capture antibody and detection antibody. For example, if the capture antibody and the detection antibody are mouse IgG1, the blocking antibody is also mouse IgG1.
It is preferable that the blocking antibody does not bind to the virus antigen to be detected at all. This is because when the blocking antibody recognizes and binds to the virus antigen, the binding of the capture antibody or detection antibody to the virus antigen may be inhibited, and in this case, accurate detection and quantification of the virus antigen becomes difficult.

The blocking antibody can be obtained by a method obtained by a hybridoma method or a method obtained directly from mouse plasma.
In the method obtained by the hybridoma method, a hybridoma that produces a blocking antibody is established to produce the blocking antibody.
For example, when a mouse IgG1 antibody is used as a blocking antibody, a mouse IgG1 antibody is obtained by immunizing a mouse with an appropriate antigen unrelated to the virus antigen to be detected and fusing the obtained mouse spleen cells with mouse myeloma cells. Is produced.

Moreover, in the method of directly obtaining from mouse plasma, an antigen unrelated to the virus antigen to be detected is appropriately selected to immunize the mouse, and mouse IgG1 is obtained from the ascites or serum of this immunized mouse using a chromatography method or the like. Purify the antibody. Examples of the chromatography method include a method in which one or more known chromatography methods such as ion exchange chromatography, molecular adsorption chromatography, gel filtration chromatography, and affinity chromatography are combined.

The amount of the blocking antibody is preferably added so as to be excessive with respect to the amount of the anti-heterologous Ig antibody (anti-mouse IgG antibody). This is because the amount of the blocking antibody is excessive with respect to the anti-mouse IgG antibody, so that most of the anti-mouse IgG antibody binds to the blocking antibody and is absorbed.
Specifically, the amount of the blocking antibody is preferably 5 times or more, preferably 10 times or more, more preferably 50 times or more of one anti-heterologous Ig antibody molecule. . On the other hand, the upper limit of the amount of blocking antibody is not particularly limited, but if it is too much, the burden is increased in terms of cost. Therefore, the amount is preferably 10,000 times or less, more preferably 1000 times or less, and particularly preferably 100 times or less.

(Virus antigen and antibody detection kit)
Hereinafter, one embodiment of the detection kit for viral antigens and antibodies of the present invention is shown.
The virus antigen and antibody detection kit of the present embodiment is composed of the following materials.
• Microplate • Antigen / antibody dilution • Washing solution • HIV-1 p24 capture antibody (capture antibody)
HIV-1 gp41, gp120 antigen (capture antigen)
HIV-2 gp36 antigen (capture antigen)
・ HRP labeled p24 detection antibody (detection antibody)
-HRP labeled HIV-1 gp41, gp120 antigen (detection antigen)
HRP standardized HIV-2 gp36 antigen (detection antigen)
・ Mouse IgG (blocking antibody)
・ Hydrogen peroxide solution ・ Coloring agent (eg tetramethylbenzidine)
・ Stop solution (sulfuric acid)

(Virus antigen detection method)
Next, a method for detecting a virus antigen and an antibody using the above-described virus antigen detection kit will be described.
First, the capture antibody and capture antigen mixed solution is placed in the well of the microplate, and the capture antibody and capture antigen are adsorbed and immobilized on the microplate surface.
A region where the capture antibody is not adsorbed on the microplate surface may be blocked with a blocking solution such as skim milk.
In addition, a detection antibody and a detection antigen labeled with HRP are prepared.
HRP can be labeled by a known technique such as the periodic acid method described above.
The capture antibody and capture antigen can be immobilized and the detection antibody and detection antigen can be labeled with HRP in any order.

On the other hand, human specimens (eg, serum) contain anti-mouse IgG antibody (anti-heterologous Ig antibody) in addition to HIV-1 p24.
When this human specimen is mixed with an excessive amount of blocking antibody and reacted, anti-mouse IgG antibody binds to the blocking antibody and is absorbed.
It is preferable to add a blocking antibody so that it is contained in an excessive amount relative to the anti-mouse IgG antibody, since most of the anti-mouse IgG antibody contained in the human sample reacts with the blocking antibody and is absorbed.

The absorbed anti-mouse IgG antibody does not react with the capture antibody or the detection antibody even if contained in the human sample, so that the human sample after the reaction can be used as it is for the detection reaction of the virus antigen.

Next, the human specimen after the blocking antibody reaction is placed in the well of the microplate, and the capture antibody immobilized on the surface of the microplate by the antigen-antibody reaction is bound to p24, and the capture antigen, the HIV-1 antibody, and the HIV- Two antibodies are bound.
At the same time or subsequently, the HRP-labeled p24 detection antibody, the HRP-labeled HIV-1 antigen, and the HRP-labeled HIV-2 antigen are placed in a well, and p24 and the detection antibody are reacted by an antigen-antibody reaction, The HIV-1 antibody and the HIV-2 antibody are reacted with the detection antigen.
After the reaction, the microplate is washed to remove serum proteins, unreacted substances and detection antibodies.

After washing, a color former (TMB) is added and reacted with peroxidase to develop blue color.
Finally, a stop agent (sulfuric acid) is mixed to stop the color reaction.
Color development by the color former is measured by absorbance at a wavelength of 450 nm. This absorbance is proportional to the concentration of HIV-1 p24, HIV-1 antibody, HIV-2 antibody.
At the same time, a calibration curve is prepared based on the concentration and absorbance of each standard solution, and the concentrations of antibodies and antigens contained in the human sample can be quantified from the calibration curve and the absorbance of the human sample.

Viral antigens and viral antibodies can be detected by the above series of steps. The blocking antibody may be added to the human specimen either before or after the human specimen is placed on the microplate. By setting the timing of adding the blocking antibody to the human sample before mounting, the anti-heterologous Ig antibody in the human sample can be absorbed in advance by the blocking antibody before mounting on the microplate. On the other hand, by adding the blocking antibody simultaneously with placing the human specimen on the microplate, the operation of adding the blocking antibody in advance becomes unnecessary, and the operation becomes simple.
The detection kit of the present invention contains not only a human sample containing an anti-heterologous Ig antibody but also an anti-heterologous Ig antibody, since the blocking antibody has little influence on the detection system even if it is excessively contained in the human sample. It goes without saying that the virus antigen can be detected even in a human sample.

The virus antigen detection method and virus antigen detection kit of the present invention will be described in detail below by showing specific experimental examples.

1. Production of capture antibody and antigen / detection antibody and antigen Capture antibody and capture antigen (HIV-1 p24 capture antibody, HIV-1 gp41, gp120 antigen, HIV-2 gp36 antigen), detection antibody and detection antigen (HRP-labeled p24 detection) Antibody, HRP-labeled HIV-1 gp41, gp120 detection antigen, HRP standardized HIV-2 gp24 detection antigen) are prepared by known methods.

2. Preparation of detection kit for virus antigen and antibody The capture antibody and capture antigen (HIV-1 p24 capture antibody, HIV-1 gp41, gp120 capture antigen, HIV-2 gp36 capture antigen), detection antibody and detection antigen (HRP labeled p24 A detection kit of a virus antigen was prepared using a detection antibody, HRP-labeled HIV-1 gp41, gp120 detection antigen, HRP standardized HIV-2 gp36 detection antigen).
A virus antigen and antibody detection kit comprises the following reagents and the like.
(1) 96-well microplate (cassette type)
(2) HIV-1 p24 capture antibody (3) HIV-1 gp41, gp120 capture antigen, HIV-2 gp36 capture antigen mixture.
(4) HRP-labeled HIV-1 p24 detection antibody (5) HRP-labeled HIV-1 gp41, gp120 detection antigen, HRP-labeled HIV-2 gp36 detection antigen mixture.
(6) Antigen / antibody dilution solution PBS containing 0.5% Trion X-100, 0.1% bovine serum albumin (hereinafter referred to as “BSA”), 0.02% Tween20.
It also contains a blocking antibody (a proper amount of mouse IgG1 (final concentration 2.5 to 5.0 ug / ml) that blocks non-specific reactions to be prevented when detecting HIV-1 p24.
(7) 0.01% hydrogen peroxide solution (8) Plate cleaner PBS containing 0.02% Tween 20, provided at a 20-fold concentration, diluted at the time of use.
(9) Color former (dimethylformamide containing 1 mg / ml TMB)
(10) Stop solution (2N sulfuric acid)
(11) Plate seal (polyethylene adhesive seal)
(12) Sodium hypochlorite solution Used for HIV inactivation in blood after use.
In addition, it does not necessarily need to be attached.

3. Comparison experiment of detection kit of virus antigen and antibody Preparation of plates (1) The HIV-1 p24 capture antibody, HIV-1 gp41, gp120 capture antigen, and HIV-2 gp36 capture antigen mixture are smeared on the plate.
These mixed solutions are diluted to an appropriate concentration. At this time, PBS containing 2% trehalose is used as the smearing buffer.
This solution is then added to a 96 well plate at 50ul per well.
The plate is covered with a dedicated plate seal to prevent evaporation.
Incubate for 1 hour at room temperature or overnight at 4 ° C.
Trehalose is necessary to allow the solution to diffuse evenly into the wells.
(2) After incubation, the plate is washed with a plate washing solution and used immediately or stored in a refrigerator in a moisturizing bag (dedicated polyethylene bag) and sealed with a plate seal.
In addition, for moisturizing, a filter paper impregnated with purified water added with 0.001% thimerosal is enclosed.

B. Performance comparison test (1) Put 50 ul of serum as a specimen, negative control, positive control, and p24 positive control solution in each well.
Do nothing in the blank well.
(2) Further, 50 ul of detection reagents (HRP labeled HIV-1 gp41, gp120 detection antigen, HRP labeled HIV-2 gp36 detection antigen and HRP labeled HIV-1 p24 detection antibody) diluted to an appropriate concentration in each well Insert.
The mixed detection solution is diluted with an antigen-antibody dilution solution.
(3) After incubating at room temperature for 1 hour, wash with a plate washing solution 3-5 times.
(4) Add 100 ul of a coloring solution containing hydrogen peroxide as a substrate and TMB as a coloring dye to each well and react for 15 to 20 minutes. Then, 2N sulfuric acid is added to 50 ul / well to stop the reaction.
(5) OD ₄₅₀ is measured with a plate absorptiometer (Dainippon Pharmaceutical plate reader).
In some cases, the blue intensity is determined with the naked eye after the reaction of (4).

C. Sample Evaluated with a seroconverted serum panel and HIV-2 infected serum panel sold by US BBI for performance evaluation of HIV infection diagnostic kit.

D. Results a. Specificity and sensitivity test results A comparative test was performed using the kit according to this embodiment and the kit manufactured by Abbott.
In this test, a method of observing the reaction with sera before and after the HIV test is used by using a kit of a seroconversion panel sold by BBI of the United States for performance evaluation of an HIV infection diagnostic kit.
Tables 1 and 2 show the results of the kit specificity and sensitivity tests.

(Table 1)

(Table 2)

From this result, it was found that serodiagnosis of HIV-1 and HIV-2 infection can be performed with the same specificity and sensitivity as a kit commercially available from Abbott.
In other words, as shown in Table 1, “Panel BG” shows that the kit according to the present embodiment was able to detect serum that had already been positive for the antigen 5 days before the antibody was seroconverted.
Similarly, “Panel BH” shows that the kit according to the present embodiment was able to detect serum that had already been positive for the antigen 6 days before the antibody seroconverted.
In both cases, the antigen and the antibody did not react at all in the negative period, indicating the specificity.
Furthermore, as shown in Table 2, it was shown that HIV-2 infected persons can be reliably detected.
BLD was below the detection limit, and the cut-off value this time was 5 times the absorbance exhibited by normal human serum.
The S / C value is determined to be positive when 1.0 or more.

b. Detection Sensitivity Test Table 3 and FIG. 1 show the detection sensitivity of HIV-1 p24 antigen of the kit according to this embodiment.
A calibration curve was prepared with HIV-1 p24 standard solution.
The calibration curve is a log-log graph.

(Table 3)

The correlation equation of the calibration curve is shown by the following equation.
Log (y) =-2.95 + 0.926 * Log (x)
The detection limit was 4 pg / ml.

c. Reactivity test against various clade HIV-1 p24 antigens The reactivity of various HIV-1 strains was examined with the kit according to this embodiment.
The results are shown in Table 4.
The absorbance cutoff value of HIV-1 p24 antigen was 0.02, and an S / C value of 1.0 or higher was positive.

(Table 4)

As shown in Table 4, all tested HIV-1 strains could be detected.
From this result, it was shown that the kit according to the present embodiment can cope with various HIV-1 strains.

As described above, the kit according to the present embodiment can complete the test in one step, so that it is possible to test the infectious disease in a shorter time than the conventional method.

Claims (10)

1. A detection kit for a viral antigen and a viral antibody contained in a human specimen,
   A non-human-derived capture antibody that is immobilized on a carrier and specifically binds to the viral antigen; and a non-human-derived capture antigen that specifically binds to the viral antibody;
   A non-human-derived detection antibody that is labeled with a labeling substance and specifically binds to the viral antigen; and a non-human-derived detection antigen that is labeled with the labeling substance and specifically binds to the viral antibody. ,
   Detection of virus antigen and virus antibody capable of detecting virus antigen and virus antibody at a time, wherein the capture antibody and the capture antigen are combined with the detection antigen and the detection antibody in a one-step reaction. kit.
2. The virus antigen and virus antibody capable of detecting the virus antigen and virus antibody at the same time according to claim 1, wherein the capture antibody, the capture antigen, the detection antibody, and the detection antigen are provided separately. Detection kit.
3. The virus antigen and virus antibody detection kit according to claim 1, wherein the immobilized carrier is maintained in a moisturized state.
4. 4. The virus antigen and virus antibody according to claim 3, wherein the moisturizing state is realized by shutting off the outside air in a state where a medium impregnated with water is in contact with the carrier. Possible viral antigen and viral antibody detection kits.
5. The detection kit for the viral antigen and viral antibody comprises:
   The virus antigen and the virus antibody according to claim 1, further comprising a test reagent for detecting the detection antibody bound to the virus antigen and the virus antibody, and the labeling substance of the detection antigen. Detection kit for detectable viral antigens and viral antibodies.
6. The viral antigen is human immunodeficiency virus type 1 (HIV-1),
   The capture antibody and the detection antibody are both antibodies having HIV-1 p24 as an antigen,
   The viral antibody is an antibody of human immunodeficiency virus type 1 (HIV-1) and human immunodeficiency virus type 2 (HIV-2),
   The virus according to any one of claims 1 to 5, wherein each of the capture antigen and the detection antigen is an HIV-1 gp41 antigen, an HIV-1 gp120 antigen, or an HIV-2 gp36 antigen. A virus antigen and virus antibody detection kit capable of detecting antigen and virus antibody at a time.
7. A method for detecting a viral antigen and a viral antibody contained in a human specimen,
   Immobilizing on a carrier a non-human-derived capture antibody that specifically binds to the viral antigen and a non-human-derived capture antigen that specifically binds to the viral antibody;
   A non-human-derived detection antibody that specifically binds to the viral antigen and a step of labeling a labeling substance on the non-human-derived detection antigen that specifically binds to the viral antibody are performed in any order,
   A step of reacting the human specimen with the capture antibody and the capture antigen to bind the capture antibody and the capture antigen to the viral antigen and the viral antibody in one step;
   Detecting the virus antigen, the detection antibody bound to the virus antibody, and the labeling substance of the detection antigen, and a method for detecting the virus antigen and the virus antibody.
8. Following the step of immobilizing the capture antibody derived from non-human specifically binding to the viral antigen and the capture antigen derived from non-human specifically binding to the viral antibody to the carrier,
   The method for detecting a viral antigen and a viral antibody according to claim 7, wherein a step of maintaining the capture antibody and the carrier on which the capture antigen is immobilized in a moisturized state is performed.
9. In the step of keeping the carrier on which the capture antibody and the capture antigen are immobilized in a moisturized state, the moisturized state is a state in which a medium impregnated with water is in contact with the carrier and is blocked from outside air. It implement | achieves by these, The detection method of the virus antigen and virus antibody of Claim 8 characterized by the above-mentioned.
10. The viral antigen is human immunodeficiency virus type 1 (HIV-1),
    The capture antibody and the detection antibody are both antibodies having HIV-1 p24 as an antigen,
    The viral antibody is an antibody of human immunodeficiency virus type 1 (HIV-1) and human immunodeficiency virus type 2 (HIV-2),
    The virus according to any one of claims 7 to 9, wherein each of the capture antigen and the detection antigen is an HIV-1 gp41 antigen, an HIV-1 gp120 antigen, or an HIV-2 gp36 antigen. Methods for detecting antigens and viral antibodies.

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