KR100671825B1 - Kit for diagnosis of cervical cancer by human papilloma virus - Google Patents

Abstract

A kit for diagnosing cervical cancer is provided to be able to diagnose the cervical cancer at the initial stage, thereby being usefully used for significantly decreasing death rate caused by the cervical cancer. The kit for diagnosing cervical cancer comprises: a monoclonal antibody of human papilloma virus(HPV) 16th E7 protein or HPV 18th E7 protein which is fixed on a fixable solid support of the protein; and a solution for crushing an antibody-signal material complex coupled to a signal coloring material of the antibody and cervical cancer cells. In the kit, the solid support is in the form of a strip or a plate, the signal coloring material consists of an antibody-gold particle zygote where each of the monoclonal antibody of HPV 16th E7 protein or HPV 18th E7 protein is conjugated to respectively gold particles and the solution for crushing the cell includes a surfactant such as 0.1-5 vol.% of triton.

Description

Kit for Diagnosis of Cervical Cancer by Human Papilloma Virus

1 is a photograph showing the components of the diagnostic kit developed through the present invention.

①: antibody fixing strip

②: container containing antibody-signal material conjugate

③: antibody-signal material conjugate mixed with cell disruption solution

Figure 2 is a photograph and analysis table showing the antibody combination test for detection of HPV No. 16 E7 recombinant protein.

Figure 3 is a photograph and analysis table showing the antibody combination test for detection of HPV 18 E7 recombinant protein.

Figure 4 is a photograph showing the detection limit test results for HPV No. 16 E7 recombinant protein, wherein the fixed antibody is 16-2, gold conjugated antibody 16-5, HPV No. 16 E7 protein amount of the strip 1 5 ng respectively , Strip 2 is 1 ng, strip 3 is 0.5 ng, strip 4 is 0.2 ng, strip 5 is 0.1 ng, strip 6 is 0.05 ng.

Figure 5 is a photograph showing the detection limit test results for HPV 18 E7 recombinant protein, wherein the fixed antibody is 18-3, gold-conjugated antibody 18-2, HPV 18 E7 protein amount of sample 1 strip 5 ng, strip 2 is 1 ng, strip 3 is 0.5 ng, strip 4 is 0.2 ng, strip 5 is 0.1 ng, strip 6 is 0.05 ng.

6 is a graph showing the correlation between HeLa cell number and protein concentration of HeLa cell lysate.

Figure 7 is a photograph showing the specificity test results for cancer cells of HPV 16 E7 protein diagnostic kit, wherein the fixed antibody is 16-2, gold-conjugated antibody is 16-5, the type of cancer cells used as samples 1 times HeLa cells, strip 2 are SiHa cells, strip 3 are PC3M cells.

Figure 8 is a photograph showing the specificity test results for cancer cells of HPV E7 protein diagnostic kit, wherein the fixed antibody is 18-3, gold-conjugated antibody is 18-2, the type of cancer cells used as samples 1 times HeLa cells, strip 2 are SiHa cells, strip 3 are PC3M cells.

The present invention relates to a diagnostic kit for cervical cancer caused by human papillomavirus, and more particularly, to a diagnostic kit for cervical cancer using monoclonal antibodies against HPV 16 E7 protein or HPV 18 E7 protein, which are the main causes of cervical cancer. It is about.

Human papilloma virus (hereinafter referred to as HPV) has been found to be a major cause of cervical cancer. Cervical cancer ranks fourth with 10.1% of cancers occurring in women and 22.3% with epithelial carcinoma. More than 7,000 new patients occur each year, and the incidence rate is 26.5 out of 100,000 female population, the most important cancer of Korean women. Early diagnosis is essential to reduce mortality from cervical cancer. Cervical cancer screening methods, such as Pap smear (Pap smear), cervical magnification test, etc. There are classical methods, and despite the various disadvantages because there is no alternative diagnostic method has been mainly used until now. In recent years, HPV DNA testing has been developed, with reports that HPV is the main source of cervical cancer. Initially, nucleic acid amplification reaction (hereinafter, referred to as PCR) method was mainly used, but HPV is classified into a high-risk group that has about 120 types of cancers and a low-risk group that has no significant correlation with cervical cancer. As a result, DNA chips have been developed to distinguish between high-risk and low-risk groups of HPV.

However, even if a DNA chip is used to easily distinguish DNA types of HPV, this is the evidence that cervical cancer is developed or there is a limit to predicting cervical cancer at some point. Recent studies have shown that the E7 protein produced by HPV plays a key role in the development and progression of cervical cancer caused by HPV. In other words, the E7 protein binds to pRB, which is most important for cell cycle regulation, to separate E2F from pRB-E2F conjugates, to produce the proteins necessary for cell division, and to allow cells to progress from cancer to control and control. It is. Therefore, it is important to test for the presence of HPV through DNA testing of HPV, but it is important to diagnose whether high-risk HPV is in the current stage of activation and it is necessary to confirm the expression of E7 protein, a key substance.

Conventional techniques related to cervical cancer screening are as follows.

First, the diagnostic method currently used to detect cervical cancer early is Pap smear. Compared to normal cells, HPV infection causes morphological variation. Cells collected from the cervix are stained by H & E methods and examined by a pathologist or a cytologist to find cancer cells. This method is simple and economical, and now hundreds of thousands of tests are performed annually in Korea alone, thus contributing to a significant reduction in cervical cancer mortality over the last 50 years. However, this method has a high probability of inaccuracy due to errors in cell sampling methods, and the accuracy of diagnosis becomes a problem (false negative rate of 5-45%) because the diagnosis of cancer depends only on the subjective microscopic observation and judgment of the examiner. Although the method of improving the existing Pap smear method such as the thin prep method has been introduced, the cell test result is still inaccurate, and the number of experts who can determine abnormal morphology of cells is limited in Korea. Even in Western countries where national cervical cancer screening programs are well established, the limitation of cytology is no longer reducing cancer incidence. Therefore, there is a need for developing a new diagnostic method for screening for HPV, the cause of cancer.

Second, there is HPV DNA test by PCR. The PCR method was developed in 1983 by Dr. B. Mullis of Cetus. Even if there is a very small amount of DNA of 103 or less in the sample to be tested, the DNA can be amplified several million times or more, and thus the amplified DNA can be confirmed by a simple detection method. The result of the amplification mainly confirmed the DNA amplified by the electrophoresis method and is still widely used. Electrophoresis method has the advantage that can be easily learned while using carcinogens such as ethidium bromide, there is a risk that you need to use ultraviolet rays to confirm this, there is a problem that can not preserve the result. In order to preserve the result, it is inconvenient to store the photograph using expensive equipment such as a CCD camera.

Third, there is a hybrid capture method of Digene of the United States. Hybrid capture is a method of extracting DNA from a patient sample, hybridizing with an RNA probe, and then confirming it by immunoassay (ELISA) using an antibody that recognizes a DNA-RNA hybrid. This method requires a great deal of attention because it uses unstable RNA, which is difficult to test.

Fourth, recently developed method is HPV DNA chip test. Immobilized probes for β-globin with oligonucleotide probes specific to 19 HPV types at appropriate concentrations and controls on modified slides to which aldehyde groups are attached. Each probe is a high risk group (16, 18, 31, 33, 35, 39, 45, 51, 56, 58, 59, 66) and low risk groups (6, 11, 34, 40, 42, 44). In order to indicate the position of each probe, oligonucleotides bound to biotin that do not react with HPV DNA at all are fixed as a label and used as markers. Target DNA to react with the probe is PCR amplified using a primer combination and then titrated. After the hybridization under the conditions, the state bound to each type of probe is detected using streptavidin-R-phycoerythrin staining reagent. The target DNA, which is a PCR product, is randomly inserted into the biotin during PCR amplification, and the biotin binds to streptavidin, which is bound to the dye reagent, to visually indicate the hybridization reaction.

While HPV DNA testing using PCR or DNA chips has the advantage of distinguishing HPV DNA by type, the detection system requires expensive equipment, and even if HPV DNA is identified, it will directly cause cervical cancer. There is a limit to show the correlation, but it is used as a test that can be used as a reference for making various clinical findings. In addition, this DNA test is time-consuming because it is a multi-processing process, prone to errors in the test, and abortive or latent infection that does not produce protein after infection may be positive. In addition, since patients have to pay a high cost, there is a disadvantage in that efficiency and economical efficiency are inadequate for use in diagnosing HPV infection, which shows an infection rate of 0.5-2.5% of the entire female population.

As described above, the cervical cancer screening method, as described above, has been used in the Pap smear, cervical augmentation, HPV DNA, and the like. However, cytology and Pap smears can only be tested if the disease progresses to some extent and changes in the tissues. HPV DNA is an early diagnosis, but it is difficult to directly diagnose the prognosis of the disease. There is. Therefore, there is a need for early testing, which is an intermediate step between HPV DNA testing, cytology, and cervical augmentation testing, which requires the ability to test for specific proteins needed for metastasis to cervical cancer. Of the many types of HPV produced by the high-risk HPV, type 16 HPV and type 18 HPV, one of the specific proteins that causes cervical cancer is the E7 protein, which allows for accurate prognostic diagnosis. No specific method has been developed to test for E7 proteins produced in 16 HPV and 18 HPV.

Therefore, the present inventors synthesize monoclonal antibodies that specifically recognize E7 proteins of HPV of form 16 and HPV of form 18, and use the diagnostic kit having high sensitivity and specificity, and use the same early to The present invention has been completed by developing a method for diagnosing the development of cervical cancer.

It is an object of the present invention to provide a diagnostic kit for cervical cancer caused by HPV.

Still another object of the present invention is to provide a method for diagnosing cervical cancer using the diagnostic kit.

In order to achieve the above object, the present invention provides a diagnostic kit for cervical cancer using monoclonal antibodies against HPV No. 16 E7 protein and HPV No. 18 E7 protein which is the main cause of cervical cancer.

The present invention also provides a method for diagnosing cervical cancer using the diagnostic kit.

Hereinafter, the present invention will be described in detail.

The present invention provides a diagnostic kit for cervical cancer using monoclonal antibodies against HPV No. 16 E7 protein or HPV No. 18 E7 protein, the main cause of cervical cancer.

In the present invention, the diagnostic kit is a monoclonal antibody to the HPV No. 16 E7 protein or HPV No. 18 E7 protein is fixed to a solid support capable of fixing the protein, the antibody-signal combined with the signaling material of the antibody It is preferably composed of a substance conjugate and a solution for disrupting cells of cervical cancer cells.

At this time, the form of the solid support is preferably a strip or plate, more preferably plastic or glass, most preferably a nitrocellulose membrane. In addition, it is preferable that only one of the monoclonal antibodies against the HPV No. 16 E7 protein and the monoclonal antibodies against the HPV No. 18 E7 protein is fixed to each of the solid supports or both species.

In addition, the signal chromophore is preferably composed of an antibody-gold particle conjugate conjugated to gold particles to monoclonal antibodies to the HPV 16 E7 protein or HPV 18 E7 protein, respectively, in addition to the gold particles, respectively, additional magnetic material, stain More preferably, the antibody-signal material conjugate selected from the group consisting of a substance, an enzyme and a fluorescent material is bound to the antibody, and the antibody-signal material conjugate is preferably lyophilized or kept in solution.

In addition, the solution for crushing the cells preferably contains a surfactant such as 0.1 to 5% (v / v) Triton.

In addition, the diagnostic kit preferably further includes a detection device for measuring absorbance, magnetic field or fluorescence, respectively, with a spectrometer, a magnetic field meter or a fluorescence analyzer to measure signal color development.

In the present invention, a monoclonal antibody that specifically recognizes the E7 protein produced by HPV 16 and HPV 18 is fixed to a specific position on the surface of a specific support surface in the form of a strip. The antibody-signal material conjugate combined with the main component is composed of a cell disruption solution containing a surfactant that breaks down cervical cancer cells and exposes E7 protein in cervical cancer cells for testing. More specifically, at least two high titer monoclonal antibodies that specifically recognize different epitopes (specific regions of the E7 protein to which the antibody binds) of the E7 protein produced by HPV 16 were tested. A pair of antibody combination systems were constructed by immobilizing HPV No. 16 E7 protein epitope specific monoclonal antibodies on the support surface and binding different types of HPV No. 16 E7 protein epitope specific monoclonal antibodies with signal material. In addition, at least two high titer monoclonal antibodies that specifically recognize different epitopes of the E7 protein produced by HPV 18 (specific regions of the E7 protein to which the antibody binds) were tested. A protein epitope specific monoclonal antibody was immobilized on the support surface and a pair of antibody combination systems were constructed by combining different types of HPV No. 18 E7 protein epitope specific monoclonal antibodies with signal material. The cell disruption solution for crushing cervical cancer cells contains 0.5 to 3% protein such as bovine serum albumin in a phosphate buffer solution containing 0.5 to 3% (v / v) of surfactant. More preferably, the cell disruption solution contains Triton X-100 (final concentration 1%), bovine serum albumin protein (final concentration 0.5%), and PMSF (final 10 mM) in phosphate buffer solution (pH 7.4). .

The present invention also provides a method for diagnosing cervical cancer using the diagnostic kit.

More specifically, the cervical cancer cell sample is placed in a cell disruption solution and the cells are disrupted to expose the HPV E7 protein in the cell disruption solution, and the cell disruption solution thus treated is placed in a container or pad containing the antibody-signal conjugate. The HPV E7 protein exposed to the cell disruption solution is reacted with the antibody-signal conjugate. In a solution in which the HPV E7 protein and the antibody-signal complex reacted, a strip containing a monoclonal antibody that specifically recognizes the HPV E7 protein was added.In the case of the sample containing the HPV E7 protein, the E7-antibody-signal complex was stripped. In response to the monoclonal antibody immobilized on the E7-antibody-signal conjugate, signal color appears at the site of the strip where the monoclonal antibody is immobilized (see FIG. 1). If the sample does not contain the HPV E7 protein, the antibody-signal complex no longer reacts with the monoclonal antibody immobilized on the strip, so no signal development occurs at that site.

The present invention provides cell lines producing monoclonal antibodies specific for HPV No. 16 E7 protein and cell lines producing monoclonal antibodies specific for HPV No. 18 E7 protein (SP2 / 0 AG-14 cells (Invitrogen) and splenocytes of mice). Cell cultures), and these were injected into Balb / c rats and purified from the ascites by affinity chromatography. The monoclonal antibody against the E7 protein of HPV 16 prepared as described above and the monoclonal antibody against the E7 protein of HPV 18 were immobilized on a nitrocellulose membrane, reacted with gold particles, and then dissolved with bovine serum albumin. Prepare a gold particle conjugate. In addition, the monoclonal antibody against the E7 protein of HPV 16 and the monoclonal antibody against the E7 protein of HPV 18 are selected through various combination tests to determine the combination conditions showing the optimal sensitivity (see FIGS. 2 and 3).

The detection limit for HPV 16 E7 protein and HPV 18 E7 protein was determined by the optimal combination obtained above (FIGS. 4 and 5). Reference).

In addition, the specificity was compared using SiHa cervical cancer cells infected with HPV type 16 virus, HeLa cervical cancer cells infected with HPV type 18 virus, and PC3M prostate cancer cells, which are not related to cervical cancer. As a result, the diagnostic system for HPV 16 E7 protein shows high specificity for HPV 16 E7 protein, which responds only to SiHa cells and does not respond to HeLa cells and PC3M cells (FIGS. 6 and 7). Reference). In addition, the diagnostic system for HPV 18 E7 protein only responds to HeLa cells (FIG. 8). Reference).

The present invention enables to overcome the limitation that the prognosis of the development of cervical cancer can not be overcome by HPV DNA test as well as early diagnosis of cervical cancer. To date, the diagnosis of cervical cancer has been widely used for cytology or cervical augmentation examination of tissue deformation through histologic staining. However, cytology and cervical augmentation examination are used to detect the progress of cervical cancer. It also had the disadvantage that it could only be confirmed by a low sensitivity and a highly trained expert. As the cause of cervical cancer is mainly caused by human papillomavirus, HPV DNA test has been developed and used for early diagnosis of cervical cancer. However, HPV DNA testing provides information that HPV is present, but has the limitation that HPV is present and the prognosis of when cervical cancer develops cannot be diagnosed. Therefore, there was an urgent need for a method for diagnosing the prognosis of cervical cancer caused by HPV, which is an intermediate step between HPV DNA test, cytology and cervical augmentation test. It is expected that the mortality rate of cervical cancer in women can be drastically reduced.

Hereinafter, the present invention will be described in more detail based on the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited by the examples.

< Example  1> Isolation and Purification of Monoclonal Antibodies

Cell lines producing monoclonal antibodies specific for HPV No. 16 E7 protein selected by cell fusion and marginal dilution (fusion of SP2 / 0 AG-14 cells (Invitrogen) and mouse splenocytes) and HPV No. 18 Cell lines producing monoclonal antibodies specific for the E7 protein (prepared by fusing SP2 / 0 AG-14 cells (Invitrogen) and spleen cells of mice) were cultured respectively. The cell lines were purchased from Metaball Lab, Inc. There are six cell lines that produce monoclonal antibodies specific for the HPV 16 E7 protein, and each of the cell lines 16-1, 16-2, 16-3, 16-4, 16-5, 16-6 It was named burn. Three cell lines producing monoclonal antibodies specific for the HPV 18 E7 protein were named 18-1, 18-2, and 18-3. Each cell line propagated by cell culture was injected into Balb / c rats bred in an animal room, and when the ascites grew, the ascites was collected and purified by affinity chromatography.

In more detail, the fused cells producing the selected antibodies were cultured in DMEM-HT medium in a 25 ml flask, and then injected into the abdominal cavity of Balb / c rats 10 weeks or older. Prior to this, in order to secure a large amount of ascites fluid, prestain was injected into rats one week before injection of fusion cells. After 10-14 days, ascites was collected from the abdominal cavity of the rats and centrifuged to remove cell fragments, red blood cells, and lipids. Separation and purification was performed using affinity chromatography. Protein agarose column (Pierce) was used for affinity chromatography, and antibody was isolated and purified by pH elution and neutralization.

< Example  2> HPV  16th E7  Manufacture of diagnostic system for protein

<2-1> Antibody Fixation Strip Preparation

Monoclonal antibodies against E7 protein of HPV No. 16 prepared in Example 1 were dispensed into nitrocellulose membranes at 1 mg / ml concentration and dried at 37 ° C. for 10 minutes to fix.

<2-2> antibody- Gold particles  Conjugate manufacturing

Monoclonal antibody against E7 protein of HPV 16 was reacted with gold particles for 30 minutes at room temperature at a concentration of 0.1 mg / ml. Bovine serum albumin was added to 1% of the reaction solution, and the supernatant was discarded by centrifugation. The precipitate was dissolved in a phosphate buffer solution containing 1% of bovine serum albumin to prepare an antibody-gold particle conjugate.

<2-3> Optimal Monoclonal Antibody Combination Screening

Six monoclonal antibodies against the EV protein of HPV 16 were tested in combination with each other to select the optimal combination conditions. That is, six kinds of monoclonal antibodies were fixed to nitrocellulose membranes, and cellulose papers were superimposed on top of nitrocellulose membranes to prepare antibody fixing strips. Of the six types of monoclonal antibodies, three types of monoclonal antibodies selected through preliminary experiments were conjugated to gold particles to prepare antibody-gold particle conjugates. The most sensitive combinations for recombinant HPV No. 16 E7 protein were selected by combining six antibody-fixed strips and three antibody-gold particle conjugates, each immobilized with one of six antibodies. That is, the combination having the best detection ability against the concentration of 100 ng / ml of HPV No. 16 E7 protein was selected. As a result, the antibody immobilized on the nitrocellulose membrane was the best 16-2 antibody, and the combination antibody-gold particle conjugate showed the best detection ability 16-16 antibody (Fig. 2) .

< Example  3> HPV  18th E7  Manufacture of diagnostic system for protein

<3-1> Antibody Fixation Strip Preparation

Monoclonal antibody against E7 protein of HPV No. 18 was dropped at 1 mg / ml onto nitrocellulose membrane at 1 mg / ml, and then fixed by drying at 37 ° C. for 10 minutes.

<3-2> Antibody Gold particles  Conjugate manufacturing

Monoclonal antibody against E7 protein of HPV 18 was reacted with gold particles for 30 minutes at room temperature at 0.1 mg / ml. Bovine serum albumin was added to 1% of the reaction solution, and the supernatant was discarded by centrifugation. The precipitate was dissolved in a phosphate buffer solution containing 1% of bovine serum albumin to prepare an antibody-gold particle conjugate.

<3-3> Optimal Monoclonal Antibody Combination Screening

Three monoclonal antibodies against EV protein of HPV 18 were tested in combination with each other to select the optimal combination conditions. That is, three types of monoclonal antibodies were fixed to nitrocellulose membranes, and cellulose papers were superimposed on top of nitrocellulose membranes to prepare antibody fixing strips. Each of the three types of monoclonal antibodies was conjugated to gold particles to prepare antibody-gold particle conjugates. The most sensitive combinations were selected for recombinant HPV No. 18 E7 protein by combining three types of antibody fixation strips and three types of antibody-gold particle conjugates, each immobilized with three types of antibodies. That is, the combination having the best detection ability for the concentration of 100 ng / ml of HPV No. 18 E7 protein was selected. As a result, the antibody immobilized on the nitrocellulose membrane showed the best 18-3 antibody, and the antibody-gold particle conjugate, which was combined, showed the best detection capability of the 18-2 antibody (FIG. 3). .

< Example  4> E7  Detection limit test for protein

<4-1> HPV16 E7  Detection limit test for protein

The monoclonal antibody No. 16-2 was fixed at 1 mg / ml to the nitrocellulose membrane using an antigen dispenser. In addition, the antibody-fixing strip was prepared by immobilizing anti-mouse IgG at a concentration of 1 mg / ml at the upper portion of the 16-2 monoclonal antibody. Using the antibody-fixing conjugate prepared by conjugating the antibody-fixed strip thus prepared and the monoclonal antibody No. 16-5 to the gold particle, the detection limit of HPV No. 16 E7 protein was tested and detected up to about 0.1 ng. Showed good sensitivity (FIG. 4).

<4-2> HPV18 E7  Detection limit test for protein

Monoclonal antibody No. 18-3 was fixed at 1 mg / mL to the nitrocellulose membrane using an antigen dispenser. An antibody-fixed strip was prepared by immobilizing anti-mouse IgG at a concentration of 1 mg / ml at the upper portion of the monoclonal antibody 18-3, using an antigen divider. Using the antibody-fixing conjugate prepared by conjugating the antibody-fixed strip prepared above and the monoclonal antibody No. 18-2 to the gold particle, the detection limit of HPV No. 18 E7 protein was tested. Showed good sensitivity (FIG. 5).

< Example  5> Specificity test for cervical cancer cells

The specificity was compared using SiHa cervical cancer cells infected with HPV type 16 virus, HeLa cervical cancer cells infected with HPV type 18 virus, and PC3M prostate cancer cells, which are not related to cervical cancer. After culturing each cancer cell in the culture solution, the cell number was calculated using hematocytosis, and then the cell disruption solution (Prison X-100 (final concentration 1%) and bovine serum albumin protein in phosphate buffer solution (pH 7.4) Final concentration 0.5%), PMSF (including the final 10 mM)) and the cell lysate (cell lysate) was obtained by centrifugation and cell lysates were protein quantified to determine the protein concentration of the cell lysate relative to the cell number (Fig. 6). ). After crushing about 2 X 10 ⁶ cells with the cell disruption solution, the protein in the supernatant was recovered by centrifugation, and the protein was quantified to obtain a protein having a concentration of about 1 mg / ml. SiHa cells (cervical cancer cells infected with HPV 16) were used as a positive standard sample for HPV 16 E7 protein, and HeLa cells (HPV18 type infected as a positive standard sample for E7 protein of HPV18). Cervical cancer cells) and PC3M cells, which are prostate cancer cells unrelated to cervical cancer, were used as negative standard specimens. After culturing SiHa cells, HeLa cells, PC3M cells, each cell was recovered, and each cell was disrupted with a cell disruption solution. Each cell lysate thus obtained was diluted by protein concentration, first reacted with the antibody-gold particle conjugate, and then the antibody fixing strip was added thereto. The diagnostic system for the HPV 16 E7 protein only responded to SiHa cells. It showed high specificity for HPV No. 16 E7 protein, which did not respond to HeLa cells and PC3M cells (FIG. 7). In addition, the diagnostic system for HPV 18 E7 protein responded only to HeLa cells and showed high specificity for HPV 18 E7 protein that did not respond to SiHa cells and PC3M cells (FIG. 8).

As described above, the diagnostic kit of the present invention enables women to overcome the limitation that prognosis of cervical cancer cannot be diagnosed by HPV DNA test as well as early diagnosis of cervical cancer alone. It can be useful to rapidly reduce the mortality rate due to cervical cancer.

Claims (12)

Monoclonal antibodies against HPV No. 16 E7 protein or HPV No. 18 E7 protein, a major cause of cervical cancer, are immobilized on a solid support capable of immobilizing the protein, and the antibody-signal conjugate and uterus bound to the signal chromophore of the antibody Diagnosis kit for cervical cancer comprising a solution for disrupting cells of cervical cancer cells. delete The diagnostic kit according to claim 1, wherein the solid support is in the form of a strip or a plate. The diagnostic kit of claim 3, wherein the solid support is plastic or glass. The diagnostic kit of claim 4, wherein the solid support is a nitrocellulose membrane. The diagnostic kit according to claim 1, wherein only one of the monoclonal antibodies against HPV No. 16 E7 protein and the monoclonal antibodies against HPV No. 18 E7 protein is immobilized or both species are immobilized on each of the solid supports. . The diagnostic kit according to claim 1, wherein the signal generating substance comprises an antibody-gold particle conjugate conjugated to gold particles to monoclonal antibodies to the HPV 16 E7 protein and HPV 18 E7 protein, respectively. 8. The diagnostic kit according to claim 7, wherein the signal chromophore is an antibody-signal material conjugate combined with the antibody selected from the group consisting of magnetic material, dye, enzyme and fluorescent material in addition to gold particles. 9. The diagnostic kit according to claim 8, wherein the antibody-signal material conjugate is kept in lyophilized or solution state. The diagnostic kit of claim 1, wherein the solution for disrupting cells comprises 0.1 to 5% (v / v) of surfactant. The diagnostic kit of claim 1, wherein the diagnostic kit further includes a detection device for measuring absorbance, magnetic field, or fluorescence, respectively, using a spectrometer, a magnetic field meter, or a fluorescence analyzer to measure signal color development. delete

Images