CN112114145A - Kit for detecting cervical cancer caused by HPV (human papillomavirus) virus infection - Google Patents
Kit for detecting cervical cancer caused by HPV (human papillomavirus) virus infection Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57411—Specifically defined cancers of cervix
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
- G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4703—Regulators; Modulating activity
- G01N2333/4704—Inhibitors; Supressors
Abstract
The invention provides a kit for detecting cervical cancer caused by HPV virus infection, which comprises reagent subgroups 1 and 2, wherein the reagent subgroups 1 and 2 respectively use reagents containing levels of detecting Spondin1 and p16INK4a as markers for screening and diagnosing cervical cancer caused by HPV virus infection, and the reagents are mainly divided into a capture antibody, a detection antibody, a chemiluminescence substrate AMPPD, a diluent and a washing concentrated solution, the detection antibody is prepared by adding alkaline phosphatase into PBS and adding glutaraldehyde, and then respectively mixing with a monoclonal antibody for detecting mouse anti-human Spondin1 or a monoclonal antibody for detecting mouse anti-human p16INK4a, and detectable samples are blood, serum, plasma, lymph, cerebrospinal fluid, ascites, urine and tissue biopsy from a subject. The cervical cancer diagnosis method of the kit and the sensitivity and specificity of the kit provided by the invention are both obviously improved, the false positive rate is greatly reduced, and the kit can be effectively used for early screening of cervical cancer.
Description
Technical Field
The invention relates to the technical field of immunodetection, in particular to a kit for detecting cervical cancer caused by HPV virus infection.
Background
Cervical cancer is the second most malignant tumor harmful to women in China, the risk degree of the cervical cancer is second to that of breast cancer, the morbidity of the cervical cancer is ranked fourth to that of female tumors, and the mortality of the cervical cancer is the second, so that the cervical cancer is the main cause of death of diseases related to female tumors. Cervical cancer severely reduces the quality of life of women and threatens the life health of women. Therefore, early diagnosis and early treatment of cervical cancer and effective control of cervical cancer incidence and mortality become urgent.
HPV infection is one of the main causes of cervical cancer occurrence, and the correlation rate is as high as 99.7%. Two requirements for inducing cervical cancer by HPV infection are high risk HPV (hrHPV) infection and persistent HPV infection. The HPV is divided into low-risk type, medium-risk type and high-risk type, the high-risk type HPV comprises 16, 18, 31, 33, 39, 45, 52 subtypes and the like, wherein the HPV16 and HPV18 are the most common high-risk type subtypes and account for 75% of the pathogenesis of cervical cancer.
The p16INK4a gene is an anti-cancer gene directly involved in negative feedback regulation of the cell cycle, and inactivation of the p16INK4a gene can cause over-proliferation of cells, so that cells which are not fully developed in the G1 stage enter the S stage in advance, and tumors are generated. p16INK4a was almost overexpressed in 100% of cervical cancers and precancerous lesions resulting from HR-HPV infection, but not in HPV-negative cervical cancers and normal tissues. It can be seen that p 16. sup. INK4a gene is abnormally expressed in HPV-infected cervical diseases.
The Spondin1 protein (accession number UniProtKB-Q9HCB 6in uniprot. org), also known as F-Spondin, encoded by the SPON1 gene in humans, is 807 amino acids in length, is a cell adhesion protein that promotes spinal cord and sensory neuron cell attachment and neurite growth in vitro. By similarity analysis, it is thought that it may contribute to the growth and guidance of axons in the spinal cord and peripheral nervous system. However, the combination of p 16. sup. INK4a and Spondin1 for biomarkers for early diagnosis of cervical cancer has never been disclosed.
Disclosure of Invention
The invention provides a method and application for predicting, evaluating and diagnosing the cervical cancer caused by HPV infection by measuring the levels of biomarker combinations Spondin1 and p16INK4a, and also provides a kit for predicting, evaluating and diagnosing the cervical cancer caused by HPV infection, thereby providing a method and a kit which are convenient to operate, high in sensitivity and specificity and can be effectively used for screening the cervical cancer caused by HPV infection.
In order to achieve the above purpose of the present invention, the present invention adopts the following technical scheme:
in one aspect, the invention provides a kit for detecting cervical cancer caused by HPV virus infection, which takes a reagent for detecting the levels of Spondin1 and p16INK4a as a marker for screening and diagnosing the cervical cancer caused by HPV virus infection.
Further, the reagents for detecting the levels of Spondin1 and p16INK4a are an antibody for detecting Spondin1 and an antibody for detecting p16INK4a, respectively.
Preferably, the antibody for detecting Spondin1 and the antibody for detecting p16INK4a are monoclonal antibodies for detecting Spondin1 and p16INK4a, respectively.
Further, the kit comprises 2 reagent subgroups, wherein reagent subgroup 1 and reagent subgroup 2 comprise: the kit comprises a capture antibody, a detection antibody, a chemiluminescence substrate AMPPD, a diluent and a washing concentrated solution, wherein the capture antibody and the detection antibody in a reagent subgroup 1 respectively contain 2.5 mu g/mL of monoclonal antibody for detecting Spondin1, and the capture antibody and the detection antibody in a reagent subgroup 2 respectively contain 2.5 mu g/mL of monoclonal antibody for detecting p16INK4 a.
Further, the diluent consists of 1-5% of BSA, 10-20mM of PBS with pH6-8 and 0.01-0.1% of Tween 80 by mass fraction, the washing concentrate consists of 10-30mM of PBS and 0.01-0.1% of Tween 80 by mass fraction, and preferably the diluent consists of 1% of BSA, 10mM of PBS with pH7.2 and 0.05% of Tween 80 by mass fraction, and the washing concentrate consists of 25mM of PBS and 0.05% of Tween 80 by mass fraction.
Further, the capture antibody is prepared as follows: the carboxyl magnetic particles were coated with a monoclonal antibody for detecting murine anti-human Spondin1 for 2 hours.
Still further, the monoclonal antibody for detecting mouse anti-human Spondin1 is 1-5 μ g/mL, preferably the monoclonal antibody for detecting mouse anti-human Spondin1 is 2-3 μ g/mL, and more preferably the monoclonal antibody for detecting mouse anti-human Spondin1 is 2.5 μ g/mL.
Further, the coating temperature is 20-40 ℃ and the coating time is 1-5 hours, preferably, the coating temperature is 30-40 ℃ and the coating time is 1-3 hours, more preferably, the coating temperature is 37 ℃ and the coating time is 2 hours
Further, the capture antibody can also be prepared by the following preparation process: the magnetic particles coated with the monoclonal antibody for detecting mouse anti-human Spondin1 were prepared overnight.
Still further, the coating temperature is 1-5 ℃, preferably, the coating temperature is 2-5 ℃, more preferably, the coating temperature is 4 ℃.
Still further, the monoclonal antibody for detecting mouse anti-human Spondin1 is 1-5 μ g/mL, preferably the monoclonal antibody for detecting mouse anti-human Spondin1 is 2-3 μ g/mL, and more preferably the monoclonal antibody for detecting mouse anti-human Spondin1 is 2.5 μ g/mL.
Further, the detection antibody in the reagent subgroup 1 is prepared by the following steps:
1) adding Spondin1 protein standard substances into reaction cups respectively for reaction;
2) adding the alkaline phosphatase-labeled monoclonal antibody for detecting mouse anti-human Spondin1 into the solution obtained in the step 1), thus obtaining the detection antibody.
Still further, the content of the Spondin1 protein standard is 0-8000pg/ml, preferably, the content of the Spondin1 protein standard is 8000, 4000, 2000, 1000, 500, 0 pg/ml.
Further, the concentration of the alkaline phosphatase-labeled mouse anti-human Spondin1 detection monoclonal antibody is 0.1-0.5. mu.g/mL, and preferably, the concentration of the alkaline phosphatase-labeled mouse anti-human Spondin1 detection monoclonal antibody is 0.25. mu.g/mL.
Further, the preparation process of the alkaline phosphatase-labeled monoclonal antibody for detecting mouse anti-human Spondin1 is as follows:
1) adding alkaline phosphatase into PBS, adding glutaraldehyde, mixing, and activating;
2) dialyzing the solution in the step 1) to PBS, and changing the solution for activation;
3) preparing a solution by using PBS (phosphate buffer solution) for detecting a mouse anti-human Spondin1 monoclonal antibody;
4) adding the monoclonal antibody solution obtained in the step 3) into the alkaline phosphatase obtained in the step 2), uniformly mixing, and reacting to obtain the monoclonal antibody solution for detecting the mouse anti-human Spondin1 marked by the alkaline phosphatase.
Further, the PBS is 1-30mM, pH6-8, preferably the PBS is 5-20mM, pH7-8, more preferably the PBS is 10mM, pH 7.2.
Still further, the glutaraldehyde is 10-50%, preferably, the glutaraldehyde is 10-30%, more preferably, the glutaraldehyde is 25%.
Further, the volume ratio of the alkaline phosphatase to the PBS is 1: (1-5), preferably, the volume ratio of the alkaline phosphatase to the PBS is 1: (2-4), more preferably, the volume ratio of alkaline phosphatase to PBS is 1: 3.
further, the mixing temperature in the step 1) or the step 2) is 1-5 ℃, and preferably, the mixing temperature in the step 1) or the step 2) is 4 ℃.
Further, the final concentration of the alkaline phosphatase-labeled detection mouse anti-human Spondin1 monoclonal antibody in the step 4) is 2000-8000U AP/mg protein, preferably, the final concentration of the alkaline phosphatase-labeled detection mouse anti-human Spondin1 monoclonal antibody in the step 4) is 4000-6000U AP/mg protein, and more preferably, the final concentration of the alkaline phosphatase-labeled detection mouse anti-human Spondin1 monoclonal antibody in the step 4) is 5000U AP/mg protein.
Further, the capture antibody in the reagent subgroup 2 was prepared as follows: the carboxyl magnetic particles were coated with a test murine anti-human p16INK4a monoclonal antibody at a concentration of 10. mu.g/mL for 2 hours at 37 ℃.
Further, the capture antibody in the reagent subgroup 2 can also be prepared by the following preparation process: magnetic microparticles coated with a mouse monoclonal antibody against human p 16. sup. INK4a were prepared by coating the magnetic microparticles with a mouse monoclonal antibody against human p 16. sup. INK4a at a concentration of 10. mu.g/mL overnight at 4 ℃.
Further, the detection antibody in the reagent subgroup 2 is prepared as follows:
1) adding a p16INK4a protein standard into a reaction cup for reaction;
2) adding the monoclonal antibody for detecting p16INK4a marked by alkaline phosphatase into the solution obtained in the step 2), and reacting for a period of time to obtain the detection antibody.
Further, the concentration of the p16INK4a protein standard in the step 1) is 0-650U/mL, and preferably, the concentration of the p16INK4a protein standard in the step 1) is 0U/mL, 20U/mL, 40U/mL, 80U/mL, 160U/mL, 320U/mL, 640U/mL.
Further, the reaction temperature in the step 1) or the step 2) is 33-38 ℃, and the reaction temperature in the step 1) or the step 2) is 37 ℃.
Further, the concentration of the monoclonal antibody for detecting p16INK4a marked by alkaline phosphatase is 5-20 μ g/mL, and preferably, the concentration of the monoclonal antibody for detecting p16INK4a marked by alkaline phosphatase is 10 μ g/mL.
Further, the preparation process of the alkaline phosphatase labeled monoclonal antibody for detecting p 16. sup. INK4a is as follows:
1) adding alkaline phosphatase into PBS (pH7.2), adding glutaraldehyde, mixing, and activating;
2) dialyzing the solution obtained in the step 1) to PBS, and changing the solution for activation;
3) preparing a monoclonal antibody solution by using PBS (phosphate buffer solution) for detecting the monoclonal antibody of p16INK4 a;
4) adding the monoclonal antibody obtained in the step 3) into the solution containing alkaline phosphatase obtained in the step 2), mixing uniformly, reacting to obtain the monoclonal antibody for detecting p16INK4a marked by the alkaline phosphatase.
Further, the AMPPD substrate concentration is 0.1-0.5mg/ml, preferably, the AMPPD substrate concentration is 0.1-0.3mg/ml, and more preferably, the AMPPD substrate concentration is 0.1 mg/ml.
Further, the sample is a biological fluid sample from the subject. Preferably, the sample is blood, serum, plasma, lymph, cerebrospinal fluid, ascites, urine and tissue biopsy from the subject. More preferably, the sample is blood, serum and plasma from the subject.
Further, the cervical cancer is an early stage of cervical cancer and/or cervical intraepithelial neoplasia caused by HPV persistent infection.
The invention has the beneficial effects that:
the invention uses Spondin1 and p16INK4a in combination as a novel marker for cervical cancer screening and diagnosis. By detecting Spondin1 and p16INK4a of inspectors, the method can preliminarily screen HPV positive cervical cancer and CIN and can also be used as an early diagnosis basis. The method has the advantages of convenient and fast extraction of blood samples, low cost, easier operation compared with the traditional detection, and capability of being used as a main technical means for preliminary screening of cervical cancer and CIN in the future.
By jointly using the Spondin1 and p16INK4a detection reagents as markers for diagnosing cervical cancer, compared with the method for singly detecting one of the markers, the sensitivity and specificity of the cervical cancer diagnosis method and the kit provided by the invention are remarkably improved, the false positive rate is greatly reduced, and the kit can be effectively used for early screening of cervical cancer.
Detailed Description
In order to make the objects, technical solutions and technical effects of the present invention more clear, the present invention will be described in further detail with reference to specific embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.
The experimental materials and reagents used are, unless otherwise specified, all consumables and reagents which are conventionally available from commercial sources.
EXAMPLE 1 preparation of the kit
Preparation of reagent subgroup 1 for detecting Spondin1
1. Alkaline phosphatase-labeled Spondin1 monoclonal antibody
1) Adding 100 μ L of alkaline phosphatase into 300 μ L of 10mM PBS (pH7.2), adding 40 μ L of 25% glutaraldehyde, mixing, and activating at 4 deg.C for 2 hr;
2) dialyzed to 10mM PBS (pH7.2), and changed the solution 4 times for 24 hours;
3) the Spondin1 monoclonal antibody was made up to 4mg/ml with 10mM PBS (pH7.2);
4) the 125 mu LSpondin 1 monoclonal antibody is added into activated alkaline phosphatase (the final concentration is 5000U AP/mg protein), mixed evenly and reacted for 24 hours at 4 ℃ to obtain the Spondin1 monoclonal antibody marked by the alkaline phosphatase.
2. Capture antibody
Coating the carboxyl magnetic particles with a mouse anti-human Spondin1 monoclonal antibody with the concentration of 2.5 mu g/mL for 2 hours at 37 ℃ or coating the magnetic particles overnight at 4 ℃ to prepare the magnetic particles coated by the mouse anti-human Spondin1 monoclonal antibody, namely the capture antibody.
3. Detection of antibodies
1) Adding Spondin1 protein standard substances with the concentrations of 8000, 4000, 2000, 1000, 500 and 0pg/ml into different reaction cups respectively, and reacting for 30 minutes at 37 ℃;
2) adding 0.25 mug/mL alkaline phosphatase labeled Spondin1 monoclonal antibody to obtain the detection antibody.
Spondin1 reagent subgroup kit subgroup 1 components:
capture antibody: magnetic particles coated by a mouse anti-human Spondin1 monoclonal antibody, wherein the concentration of the monoclonal antibody is 2.5 mu g/mL;
detecting an antibody: alkaline phosphatase-labeled Spondin1 monoclonal antibody, wherein the concentration of the monoclonal antibody is 2.5 mu g/mL;
calibration products: the concentration of the Spondin1 protein standard substance is 8000, 4000, 2000, 1000, 500 and 0 pg/ml;
diluting liquid: BSA with the mass fraction of 1%, 10mM PBS (pH7.2), and Tween 80 with the mass fraction of 0.05%;
washing the concentrated solution: 25 × PBS (containing 0.05% tween 80);
AMPPD substrate concentration: 0.1 mg/ml;
quality control product: spondin1 protein at a concentration of 1000 pg/ml.
II, detecting p16INK4a reagent subgroup 2
1. Alkaline phosphatase-labeled p16INK4a monoclonal antibody
1) Adding 100. mu.L of alkaline phosphatase into 300. mu.L of 10mM PBS (pH7.2), adding 40. mu.L of 25% glutaraldehyde, mixing, and activating at 4 ℃ for 2 hours;
2) dialyzed to 10mM PBS (pH7.2), and changed the solution 4 times for 24 hours; the p16INK4a monoclonal antibody was made up to 4mg/ml with 10mM PBS (pH7.2);
3) the 125 mu L p16INK4a monoclonal antibody is added into activated alkaline phosphatase (the final concentration is 5000U AP/mg protein), mixed evenly and reacted for 24 hours at 4 ℃ to obtain the p16INK4a monoclonal antibody marked by the alkaline phosphatase.
2. Capture antibody
The magnetic fine particles coated with the mouse anti-human p 16. sup. INK4a monoclonal antibody, i.e., the capture antibody, were prepared by coating the above mouse anti-human p 16. sup. INK4a monoclonal antibody at 37 ℃ for 2 hours or at 4 ℃ overnight.
3. Detection of antibodies
1) Adding p16INK4a protein standard substances with the concentrations of 0U/mL, 20U/mL, 40U/mL, 80U/mL, 160U/mL, 320U/mL and 640U/mL into different reaction cups respectively, and reacting for 30 minutes at 37 ℃;
2) then adding the p16INK4a monoclonal antibody with the concentration of 10 mug/mL marked by alkaline phosphatase, and reacting for 30 minutes at 37 ℃ to obtain the detection antibody.
Kit subgroup 2 components:
capture antibody: mouse anti-human p16INK4a monoclonal antibody coated magnetic particles, wherein the concentration of the monoclonal antibody is 2.5 mug/mL;
detecting an antibody: the p16INK4a monoclonal antibody is marked by alkaline phosphatase, wherein the concentration of the monoclonal antibody is 2.5 mu g/mL;
calibration products: p16INK4a protein standard with concentration of 8000, 4000, 2000, 1000, 500, 0pg/ml
Diluting liquid: BSA with a mass fraction of 1%, 10mM PBS (pH7.2), 0.05% Tween 80
Washing the concentrated solution: 25 × PBS (containing 0.05% tween 80);
AMPPD substrate concentration: 0.1 mg/ml;
quality control product: p16INK4a protein at a concentration of 1000 pg/ml.
Example 2Spondin 1-p16INK4a Combined detection kit diagnosis and prediction of early cervical cancer
100 clinical samples were collected, 20 samples from the normal population and 80 samples from early stage cervical cancer patients, each with 1mL serum. Stage I was observed in 80 cervical cancer patients. Respectively detecting the concentrations of Spondin1 and p16INK4a markers in blood serum of a cervical cancer patient and a healthy normal person, analyzing the obtained data by using SPASS statistical software according to the detection result, and if the difference between different groups has statistical significance, performing independent sample t test, wherein the result is expressed by x +/-s; the association analysis of Spondin1 and P16INK4a was performed for each group, and P <0.05 was statistically significant. Finally, the specificity and sensitivity of the Spondin1 and p16INK4a markers were tested individually or in combination according to data statistics.
The results show that the serum p16INK4a (309.21 + -143.8) U/mL and Spondin 1(2999.96 + -105.49) pg/mL of the cervical cancer group are both higher than those of the healthy control p16INK4a (15.34 + -9.78) U/mL and Spondin 1(1867.89 + -57.26) pg/mL. Statistical treatment was performed and the differences were statistically significant (p < 0.01).
Through sensitivity and specificity analysis of cervical cancer diagnosis cases, the following methods are known: the sensitivity of detecting the Spondin1 marker alone is 54.9%, and the specificity is 79%; the sensitivity of detecting the p16INK4a marker alone was 68.9%, and the specificity was 65.2%. The sensitivity was 89.6% and the specificity was 95.3% when the two markers Spondin1 and p16INK4a were tested in combination. The detection is obviously superior to the detection of a single cervical cancer marker in terms of sensitivity and specificity.
Therefore, the combined detection result of the two markers, namely Spondin1 and p16INK4a, is remarkably superior to the detection of a single cervical cancer marker. In conclusion, Spondin1 and p16INK4a can be used as markers for diagnosing and predicting early cervical cancer, can be used for early diagnosis of cervical cancer, and improves accuracy of early prediction.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A kit for detecting cervical cancer caused by HPV virus infection, which is characterized by comprising a reagent subgroup 1 and a reagent subgroup 2, wherein the reagent subgroup 1 and the reagent subgroup 2 comprise: the kit comprises a capture antibody, a detection antibody, a chemiluminescence substrate AMPPD, a diluent and a washing concentrated solution, wherein the capture antibody and the detection antibody in a reagent subgroup 1 respectively contain 2.5 mu g/mL of monoclonal antibody for detecting Spondin1 as a marker for prediction, diagnosis and evaluation, and the capture antibody and the detection antibody in a reagent subgroup 2 respectively contain 2.5 mu g/mL of monoclonal antibody for detecting p16INK4a as a marker for prediction, diagnosis and evaluation.
2. The kit of claim 1, wherein the diluent consists of 1-5% BSA, 10-20mM PBS pH6-8, and 0.01-0.1% Tween 80 by mass, and the washing concentrate is 10-30mM PBS and 0.01-0.1% Tween 80 by mass.
3. The kit according to claim 1, wherein the capture antibody in reagent subgroup 1 is prepared as follows: the carboxyl magnetic particles were coated with a monoclonal antibody for detecting murine anti-human Spondin1 for 2 hours.
4. The kit according to claim 3, wherein the monoclonal antibody for detecting mouse anti-human Spondin1 in reagent subgroup 1 is 1-5 μ g/mL, the coating temperature is 20-40 ℃, the coating time is 1-5 hours, and the coating temperature is 1-5 ℃.
5. The kit according to claim 1, wherein the detection antibody in reagent subgroup 1 is prepared by the following steps:
1) adding Spondin1 protein standard substances into reaction cups respectively for reaction;
2) adding the alkaline phosphatase-labeled monoclonal antibody for detecting mouse anti-human Spondin1 into the solution obtained in the step 1), thus obtaining the detection antibody.
6. The kit according to claim 5, wherein the alkaline phosphatase-labeled monoclonal antibody for detecting mouse anti-human Spondin1 is prepared by the following steps:
adding alkaline phosphatase into PBS, adding glutaraldehyde, mixing, and activating;
dialyzing the solution in the step 1) to PBS, and changing the solution for activation;
preparing a solution by using PBS (phosphate buffer solution) for detecting a mouse anti-human Spondin1 monoclonal antibody;
adding the monoclonal antibody solution obtained in the step 3) into the alkaline phosphatase obtained in the step 2), uniformly mixing, and reacting to obtain the monoclonal antibody solution for detecting the mouse anti-human Spondin1 marked by the alkaline phosphatase.
7. The kit according to claim 6, wherein the final concentration of the alkaline phosphatase-labeled monoclonal antibody for detecting mouse anti-human Spondin1 in step 4) is 2000-8000U AP/mg protein.
8. The kit according to claim 1, characterized in that the capture antibodies in reagent subgroup 2 are prepared as follows: the carboxyl magnetic particles were coated with a test murine anti-human p16INK4a monoclonal antibody at a concentration of 10. mu.g/mL for 2 hours at 37 ℃.
9. The kit according to claim 1, characterized in that the detection antibodies in the reagent subgroup 2 are prepared as follows:
1) adding a p16INK4a protein standard into a reaction cup for reaction;
2) adding the monoclonal antibody for detecting p16INK4a marked by alkaline phosphatase into the solution obtained in the step 2), and reacting for a period of time to obtain the detection antibody.
10. The kit according to any one of claims 1 to 9, which is applicable to a biological fluid sample for detecting an early stage of cervical cancer and/or cervical intraepithelial neoplasia caused by HPV infection.
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| CN116449003A (en) * | 2023-05-06 | 2023-07-18 | 郑州市妇幼保健院 | Application of cervical cancer related antibody protein combination in colloidal gold test strip |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101178405A (en) * | 2006-11-10 | 2008-05-14 | 北京科美东雅生物技术有限公司 | Tumor-associated antigen 50 chemiluminescence immune analyze quantitative determination reagent box and method of producing the same |
| CN101446591A (en) * | 2008-11-15 | 2009-06-03 | 邹先进 | P16<INK4a> double antibody sandwich ELISA kit |
| CN101452001A (en) * | 2007-12-06 | 2009-06-10 | 王颖 | Quantitative determination RBP4 kit by chemiluminescence magnetic enzymoimmune method |
| CN107202888A (en) * | 2017-07-11 | 2017-09-26 | 广州江元医疗科技有限公司 | A kind of p16 for cervical liquid-based cellsINK4aImmune labeled colour reagent box |
| CN109856383A (en) * | 2019-03-05 | 2019-06-07 | 湖北泰康医疗设备有限公司 | A kind of immunochemistry staining kit for cervical carcinoma auxiliary diagnosis |
| CN109932510A (en) * | 2017-12-18 | 2019-06-25 | 天津云检医学检验所有限公司 | A kind of cervical cancer marker and its detection kit |
| CN111077302A (en) * | 2019-12-26 | 2020-04-28 | 江苏美克医学技术有限公司 | Glucan signal amplification-based ki67 and p16INK4aDetection kit |
| CN111089958A (en) * | 2019-12-26 | 2020-05-01 | 江苏美克医学技术有限公司 | P16 based on glucan signal amplificationINK4aChemiluminescence kit |
| CN111521807A (en) * | 2020-07-02 | 2020-08-11 | 北京信诺卫康科技有限公司 | Spondin1 and CA125 combined used as early ovarian cancer biomarker and kit |
-
2020
- 2020-09-21 CN CN202010992308.4A patent/CN112114145A/en active Pending
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101178405A (en) * | 2006-11-10 | 2008-05-14 | 北京科美东雅生物技术有限公司 | Tumor-associated antigen 50 chemiluminescence immune analyze quantitative determination reagent box and method of producing the same |
| CN101452001A (en) * | 2007-12-06 | 2009-06-10 | 王颖 | Quantitative determination RBP4 kit by chemiluminescence magnetic enzymoimmune method |
| CN101446591A (en) * | 2008-11-15 | 2009-06-03 | 邹先进 | P16<INK4a> double antibody sandwich ELISA kit |
| CN107202888A (en) * | 2017-07-11 | 2017-09-26 | 广州江元医疗科技有限公司 | A kind of p16 for cervical liquid-based cellsINK4aImmune labeled colour reagent box |
| CN109932510A (en) * | 2017-12-18 | 2019-06-25 | 天津云检医学检验所有限公司 | A kind of cervical cancer marker and its detection kit |
| CN109856383A (en) * | 2019-03-05 | 2019-06-07 | 湖北泰康医疗设备有限公司 | A kind of immunochemistry staining kit for cervical carcinoma auxiliary diagnosis |
| CN111077302A (en) * | 2019-12-26 | 2020-04-28 | 江苏美克医学技术有限公司 | Glucan signal amplification-based ki67 and p16INK4aDetection kit |
| CN111089958A (en) * | 2019-12-26 | 2020-05-01 | 江苏美克医学技术有限公司 | P16 based on glucan signal amplificationINK4aChemiluminescence kit |
| CN111521807A (en) * | 2020-07-02 | 2020-08-11 | 北京信诺卫康科技有限公司 | Spondin1 and CA125 combined used as early ovarian cancer biomarker and kit |
Non-Patent Citations (2)
| Title |
|---|
| 王自正: "《现代医学标记免疫学》", 31 August 2000, 人民军医出版社 * |
| 王锋 和 张艳丽: "《动物繁殖学实验教程（动物科学专业用）（第2版）》", 31 July 2007, 中国农业大学出版社 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116449003A (en) * | 2023-05-06 | 2023-07-18 | 郑州市妇幼保健院 | Application of cervical cancer related antibody protein combination in colloidal gold test strip |
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