WO2016082445A1 - Application of peptidylarginine deiminase 2 in preparation of reagent for clinical diagnosis of tumors - Google Patents

Abstract

An application of peptidylarginine deiminase 2 in preparation of a reagent or detection object for clinical diagnosis of tumors, wherein the tumors comprise liver cancers, gastric cancers, lung cancers, breast cancers, esophageal cancers, ovarian cancers, uterine cancers and colorectal cancers. In the specific application, an antibody against peptidylarginine deiminase 2 is prepared by a conventional method, and a qualitative or quantitative method and a matching kit for detecting peptidylarginine deiminase 2 are established. A blood diagnosis reagent for diagnosing tumors comprises a peptidylarginine deiminase 2 antibody, an HRP-IgG antibody and conventional reagents in the blood diagnosis reagent, wherein the conventional reagents in the blood diagnosis reagent comprise a carbonate buffer solution, a PBST lotion, a blocking solution, a developing solution and a stop solution. Experiments prove that PAD2 can be applied as a tumor blood marker, and PAD2 has feasibility as the tumor marker.

Description

Application of Peptidyl Arginine Deiminase 2 in Preparation of Tumor Clinical Diagnostic Reagents Technical field

The invention relates to the use of peptidyl arginine deiminase 2 as a tumor blood marker for preparing tumor clinical blood diagnostic reagents.

Background technique

Peptidylarginine deiminase (PAD or PADI) is an enzyme in human tissues. Currently, five PAD enzymes (ie, PAD 1, 2, 3, 4, and 6) have been discovered. These enzymes are encoded by a cluster of genes located on the lp36 region of the human chromosome and have different tissue distributions. It can perform post-translational modification of other tissue proteins in the presence of calcium ions. This enzyme catalyzes the conversion of arginine to citrulline by catalyzing the amino group of arginine in the polypeptide chain to a carbonyl group. Citrulline is an unnatural amino acid. The process by which arginine is converted to citrulline in a PAD-catalyzed polypeptide is referred to as citrullination. After the citrullinization of the protein, the enzyme activity, metabolic activity, regulatory function and structural function are changed due to structural changes. Therefore, citrulline is an important post-translational modification of proteins as important as phosphorylation, acetylation, glycosylation, methylation, and ubiquitination.

In recent years, PAD4 (peptidyl arginine deiminase 4, or PADI4) has been shown to play an important role in the pathogenesis of human rheumatoid arthritis through immunology, cell biochemistry and molecular genetics research. Its role, and can be used as an adenocarcinoma marker, in the preparation of clinical diagnostic reagents for adenocarcinoma. PAD2 (peptidyl arginine deiminase 2, PADI2) and PAD4, although they are peptidyl arginine deiminase, have different tissue distribution, and there is currently no PAD2 as tumor blood. Relevant reports of markers.

Summary of the invention

In view of the above prior art, the present invention has obtained a new tumor blood marker, peptidyl arginine deiminase 2 (PAD2), which can be used for clinical diagnosis of tumors.

The invention is achieved by the following technical solutions:

The present invention firstly found that PAD2 is in liver cancer, gastric cancer, lung cancer, breast cancer, esophageal cancer, ovarian cancer, uterine cancer compared with serum of serum, benign tumor patients, serum of chronic inflammation patients and normal human serum after tumor resection. The expression level in serum of patients with malignant tumors such as colorectal/rectal cancer is significantly increased. PAD2 can be expressed in the serum of many malignant tumor patients at the same time, which is less in the tumor markers currently used (currently, the sensitivity and specificity of tumor blood markers are not ideal, and there are more than 20 clinical serum markers in clinical practice. However, the vast majority can only be used to detect a tumor with poor sensitivity and specificity. Usually, only joint detection can be used to provide meaningful data for clinical diagnosis. The present invention will PAD2 The expression level in the blood was compared with the known tumor marker level, and the positive rate and specificity of PAD2 tumor detection were higher than CEA, and the broad spectrum was higher than CA1250, AFP, PSA, CA199 and the like.

Based on the above findings, PAD2 has higher specificity as a tumor serum marker. For this reason, the present invention has developed a tumor diagnosis kit and a detection method with wide diagnostic range, high sensitivity and high specificity, which can be widely used in clinical practice. Preliminary tumor investigation and health screening provide a reliable basis for clinical diagnosis.

Specifically, an anti-peptidyl arginine deiminase 2 antibody is prepared by a conventional method, and a qualitative or quantitative method for detecting peptidyl arginine deiminase 2 and a kit are established.

A blood diagnostic reagent for diagnosing tumors, comprising a peptidyl arginine deiminase 2 antibody, an HRP-IgG antibody, and a conventional reagent in a blood diagnostic reagent.

Conventional reagents in the blood diagnostic reagent include carbonate buffer, PBST wash, blocking solution, color developing solution, and stop solution.

Further, the qualitative detection method is an indirect ELISA method, and the principle is to use an enzyme-labeled anti-antibody (anti-human immunoglobulin antibody) to detect a test antibody bound to a solid phase antigen, as follows:

(1) The blood sample to be tested is diluted 10 times with carbonate buffer (pH 9.6), added to a blank ELISA plate, and incubated at 37 ° C for 2 h;

(2) discard the liquid in the well, wash the plate three times with PBST washing solution (the PBST washing solution refers to PBS solution plus Tween-20, the pH of the PBS solution is 7.4, the concentration of Tween-20 is 0.1%, volume percentage);

(3) Each well was added blocking solution (0.5% BSA, mass percentage) 200 μL / well, and incubated at 37 ° C for 1 h;

(4) Discard the liquid in the well and wash the plate three times with PBST wash solution;

(5) Each group was added with PAD2 antibody dilution (1:4000 dilution) 100 μL / well, incubated at 37 ° C for 1 h;

(6) Discard the liquid in the well and wash the plate three times with PBST wash solution;

(7) Each well was diluted with HRP-IgG antibody (1:3000 dilution) 100 μL, incubated at 37 ° C for 1 h;

(8) Discard the liquid in the well and wash the plate with PBST wash solution five times;

(9) Add coloring solution A and B to each well [developer A (containing hydrogen peroxide) as hydrogen donor (DH2), substrate B is developer B (including TMB, 3, 3', 5, 5'-tetramethylbenzidine) 50 μL each, developed at 37 °C.

(10) Add 50 μL of stop solution (2MH ₂ SO ₄ solution) to each well to stop color development, shake well, and measure the OD _450nm value with a microplate reader;

(11) Calculation: Negative serum (normal human serum) was used as the control, and the mean value of negative serum OD _{450nm was} detected (the average value was detected by using multiple negative serums as a control); the OD _450nm value was substituted into the formula P/N value= The OD _{450nm value of} the blood sample to be tested / the mean _{value of the} negative serum OD4 _50nm , and the P/N value of the blood sample to be tested is calculated;

(12) Judging: If the P/N value of the blood sample to be tested is greater than or equal to 2.1, the detection result of the blood sample to be tested is positive, and the patient to which the blood sample to be tested belongs may have a tumor, which can be used as a basis for clinical diagnosis. One.

The antibodies referred to above are prepared by a conventional method. The various reagents referred to above are conventional reagents which are known in the art.

Similarly, when performing quantitative detection, the above detection principle is also used to prepare a series of standard standards, and then a standard curve of the standard concentration and the OD _{450 nm} value or the OD _{630 nm} value is drawn, and a linear regression equation is obtained, and then the blood sample to be detected is obtained. The OD _{450 nm} value or the OD _{630 nm} value is substituted into a standard curve to determine the concentration in the sample.

Further, in the quantitative detection, the double antibody sandwich ELISA is used to detect PAD2, and the steps are as follows:

(1) Coating: PAD2 monoclonal antibody 1 was diluted to 1 μg/mL with a coating solution (pH 9.6 carbonate buffer), and added to a blank plate, 100 μL/well, at 4 ° C saturated humidity. Place overnight;

(2) Washing the plate: discard the liquid in the well, and add 300 μL of PBST washing solution to each well (the PBST washing solution refers to PBS solution plus Tween-20, the pH of the PBS solution is 7.4, and the concentration of Tween-20 is 0.1%. Volume percent), soak for 15 seconds, discard the liquid. Wash the plate three times in a row;

(3) blocking: each well was added blocking solution (0.5% BSA, mass percentage) 200 μL / well, incubated at 37 ° C for 1 h;

(4) Washing the plate three times, the same step (2);

(5) Setting: leave one hole as blank control, no liquid is added temporarily; set 7 holes of PAD2 standard, add 100μL standard each;

(6) Loading: The blood sample to be tested is diluted 10 times with PBST, and then added to the reaction well in order, and incubated at room temperature for 1.5 h;

(7) Washing the plate three times, the same step (2);

(8) Add enzyme: add 100 μL of HRP-labeled PAD2 monoclonal antibody dilution (1/3000) to each well (excluding blank control), incubate for 45 min at room temperature;

(9) Washing the plate three times, the same step (2);

(10) Color development: Add coloring solution A and B sequentially to each well [developer A (containing hydrogen peroxide) as hydrogen donor (DH2), substrate B is developer B (including TMB, 3, 3) ',5,5'-tetramethylbenzidine)] 50 μL each (including blank control), shake and mix, and color at room temperature for 10 minutes;

(11) Termination: 50 μL of each stop solution (including blank control) was added to each well, and the reaction was stopped by shaking and shaking;

(12) Determination: zero control with blank control wells, and the OD value of each well was measured by a microplate reader at a wavelength of 450 nm within 30 minutes; the OD value of each well was also measured by dual wavelengths of 450 nm / 630 nm;

(13) Calculation: The logarithm of the concentration value of the series standard is the abscissa (X axis), and the logarithm of the OD value of the standard is the ordinate (Y axis), and the standard curve is established (log-log), and the calculation is to be tested. The PAD2 content of the sample.

In addition, the qualitative detection method can also be colloidal gold method, the detection principle is: using double antibody sandwich method and colloidal gold immunotechnology, pre-adding anti-PAD2 antibody colloidal gold conjugate on colloidal gold pad, detecting in nitrocellulose membrane Line and control lines were coated with anti-PAD2 antibodies, respectively. When the test is performed, if it is a positive sample, the PAD2 in the sample can form a complex with the gold standard anti-PAD2 antibody, and the complex is moved forward along the test paper, and then combined with the antibody coated with the test line to form a "" The gold-labeled antibody-PAD2-antibody" complex is agglutinated for color development. The nitrocellulose membrane was coated with a quality control line as a control at the same time, so it was positive when a red quality control line and a red reaction line appeared. When there is no PAD2 antigen in the specimen to be examined, only one red quality control line is judged to be negative. As a quality control, a red quality control line will appear regardless of whether the result is positive or negative. If no red line (or only reaction line) appears, the test is invalid. The specific detection method is:

(1) Collect a small amount of sample in a clean container (serum samples are collected intravenously according to the conventional method; the collected samples are tested within five days, and should be stored at 4 ° C, and stored at -20 ° C for more than five days to avoid repeated freezing and thawing; if the sample If turbidity or sedimentation occurs, it should be centrifuged or filtered and clarified before testing); open the inner packaging and take out the test strip.

(2) According to the product model:

1 Test strip: Immerse the end of the test strip marked with MAX in the sample for 30 seconds (note: the liquid surface should not pass the MAX line) and take it out on the table.

2 Test card: In the sample hole marked with “S” on the test card, add 4 drops of sample (about 100 μl) with a dropper.

(3) The time after the appearance of the quality control line (C line) of the test strip is observed, the observation result is within 20 minutes, and the observation result is invalid after 20 minutes.

Explanation of test results:

(1) Negative result: only the quality control line (C line) appears.

(2) Positive result: Two lines appear, namely the quality control line (C line) and the detection line (T line).

(3) Invalid result: Wireless appears, or only the detection line (T line) appears, and must be re-detected.

The present invention finds that PAD2 can be highly expressed in cells of liver cancer, gastric cancer, lung cancer, breast cancer, esophageal cancer, ovarian cancer, uterine cancer, colorectal cancer and many other malignant tumors as well as blood. Therefore, PAD2 can be used as a tumor blood marker. Application, experimentally proved that its specificity is higher than CEA, broad-spectrum is higher than CA125, AFP, PSA, CA199 and so on. Although PAD2 is expressed in a variety of tumors, it does not achieve absolute specificity, but it is well known to those skilled in the art that the specificity of clinical detection indicators is not absolutely unique and cannot be denied as one of the means for diagnosing related diseases. Sex, for example, doctors often make a final diagnosis by combining several test indicators with clinical manifestations. Therefore, PAD2 is feasible as a tumor marker; although PAD2 has non-unique specificity, PAD2 as a tumor blood marker combined with other detection indicators and clinical manifestations is sufficient for detecting tumors (this method is also currently clinical) Common way). In addition, the present invention also found that PAD2 can also be used as a blood marker for the detection of other diseases such as inflammation, such as hepatitis B, general inflammation. (with white blood cells, neutrophils, symptoms of decreased lymphocyte ratio), kidney disease (including nephrotic syndrome, renal failure, nephritis), can also be used as one of the clinical diagnosis of other diseases such as inflammation.

detailed description

The present invention will be further described below in conjunction with the embodiments.

The reagents, methods and the like referred to in the following examples are conventional reagents and methods in the prior art unless otherwise specified.

Example 1 PAD2 as an example of detection of tumor blood markers

PAD2 was used as a tumor blood marker to detect the blood of tumor patients. The number of tumor patients and the types of tumors are shown in Table 1.

The detection method is:

(1) The blood sample to be tested is diluted 10 times with carbonate buffer (pH 9.6), added to a blank ELISA plate, and incubated at 37 ° C for 2 h;

(2) discard the liquid in the well, wash the plate three times with PBST washing solution (the PBST washing solution refers to PBS solution plus Tween-20, the pH of the PBS solution is 7.4, the concentration of Tween-20 is 0.1%, volume percentage);

(3) Each well was added blocking solution (0.5% BSA, mass percentage) 200 μL / well, and incubated at 37 ° C for 1 h;

(4) Discard the liquid in the well and wash the plate three times with PBST wash solution;

(5) Each group was added with PAD2 antibody dilution (1:4000 dilution) 100 μL / well, incubated at 37 ° C for 1 h;

(6) Discard the liquid in the well and wash the plate three times with PBST wash solution;

(7) Each well was diluted with HRP-IgG antibody (1:3000 dilution) 100 μL, incubated at 37 ° C for 1 h;

(8) Discard the liquid in the well and wash the plate with PBST wash solution five times;

(9) Add coloring solution A and B to each well [developer A (containing hydrogen peroxide) as hydrogen donor (DH2), substrate B is developer B (including TMB, 3, 3', 5, 5'-tetramethylbenzidine) each 50 μL, developed at 37 ° C for 10 min.

(10) Add 50 μL of stop solution (2M H ₂ SO ₄ solution) to each well to stop color development, shake well, and measure the OD _450nm value with a microplate reader;

(11) Calculation: Negative serum (normal human serum) as control, detection of negative serum OD _450nm mean value; OD _450nm value substituted into formula P / N value = blood sample OD _{450nm value} / negative serum OD4 _50nm mean value, calculated to be Detecting the P/N value of the blood sample;

(12) Judging: If the P/N value of the blood sample to be tested is greater than or equal to 2.1, the detection result of the blood sample to be tested is positive, and the patient to which the blood sample to be tested belongs may have a tumor, which can be used as a basis for clinical diagnosis. One.

Results: The number of positive cases and positive rate are shown in Table 1. The positive rate of other tumor markers is shown in Table 2 (can be disclosed in the prior art) The data can be seen from Table 1 and Table 2. As a tumor blood marker, PAD2 has good specificity and broad spectrum.

Table 1

Tumor category	Total number of cases	Number of positive cases	Positive rate
				Liver cancer	488	392	80.3%
Knot, rectal cancer	352	248	70.4%
				Esophageal cancer	240	160	66.7%
Gastric cancer	384	248	64.6%
				Female breast malignancy	144	88	61.1%
Lung cancer	400	232	58.0%
				Ovarian cancer	384	216	56.2%
Breast cancer	390	216	55.4%
				Uterine cancer	680	376	55.3%
total	3462	2176	62.8%

Table 2

Example 2 PAD2 as a test for tumor blood markers 2

The patient's blood was detected by using PAD2 as a tumor blood marker, and the detection method was the same as in Example 1. At the same time, the blood of the tumor patient is detected by using CEA, CA199, F/PSA, CA125, PSA, CA242 in the prior art as a tumor blood marker (the detection method is a conventional method in the prior art; these prior art) The blood sample test data of the tumor blood marker in the hospital is the patient outpatient data of a hospital, and the data can be used as one of the basis for diagnosing whether the patient has a tumor; the invention also takes a blood sample of some patients to detect PAD2). The positive rate of each tumor blood marker was compared, and the cross comparison of PAD2 with each tumor marker was compared.

RESULTS: The number of patients (906 cases) and the number of positive cases were shown in Table 3. As can be seen from Table 3, PAD2 can be used as a tumor blood marker with good specificity.

Table 3: Comparison of PAD2 and each tumor marker

Example 3 Test Example 3 of PAD2 as Blood Marker

Using PAD2 as a blood marker to detect blood in patients with hepatitis B, general inflammation (white blood cells, neutrophils, lymphocyte ratio reduction), kidney disease (including nephrotic syndrome, renal failure, nephritis), patient cases The number and the types of diseases are shown in Table 4.

The detection method is the same as in the first embodiment.

Results: The number of positive cases and the positive rate are shown in Table 4. As can be seen from Table 4, PAD2 can be used as a blood marker to detect hepatitis B and common inflammation (with white blood cells, neutrophils, and decreased lymphocyte ratio). ), kidney disease (including nephrotic syndrome, renal failure, nephritis) and other diseases, the test results can be used as one of the clinical diagnosis.

Table 4

Disease category	Total number of cases	Number of positive cases	Positive rate
				Hepatitis B	394	33	8.4%
General inflammation	225	6	2.7%
				Kidney disease	133	2	1.5%

Claims (9)

1. The use of peptidyl arginine deiminase 2 in the preparation of clinical diagnostic reagents or assays for diagnosing tumors.
2. The use according to claim 1, wherein the tumor is selected from the group consisting of liver cancer, colorectal cancer, esophageal cancer, gastric cancer, female breast cancer, lung cancer, ovarian cancer, breast cancer, and uterine cancer.
3. The use of peptidyl arginine deiminase 2 in the preparation of a clinical diagnostic reagent or test for diagnosing inflammation selected from the group consisting of hepatitis B, general inflammation, and kidney disease.
4. The use according to claim 1 or 2 or 3, characterized in that the anti-peptidyl arginine deiminase 2 antibody is prepared by a conventional method to establish a qualitative or quantitative assay for detecting peptidyl arginine deiminase 2 Methods and kits.
5. A blood diagnostic reagent for diagnosing a tumor, comprising: a peptide-based arginine deiminase 2 antibody, an HRP-IgG antibody, and a conventional reagent in a blood diagnostic reagent.
6. The blood diagnostic reagent for diagnosing a tumor according to claim 5, wherein the tumor is selected from the group consisting of liver cancer, colorectal cancer, esophageal cancer, gastric cancer, female breast cancer, lung cancer, ovarian cancer, breast cancer, and uterine cancer.
7. The blood diagnostic reagent for diagnosing a tumor according to claim 5 or 6, wherein the conventional reagent in the blood diagnostic reagent comprises a carbonate buffer, a PBST washing solution, a blocking solution, a color developing solution, and a stopping solution.
8. A blood diagnostic reagent for diagnosing inflammation, comprising: a peptide-based arginine deiminase 2 antibody, an HRP-IgG antibody, and a conventional reagent in a blood diagnostic reagent; the inflammation is selected from the group consisting of hepatitis B, general inflammation, Kidney disease.
9. The blood diagnostic reagent for diagnosing inflammation according to claim 8, wherein the conventional reagent in the blood diagnostic reagent comprises a carbonate buffer, a PBST wash, a blocking solution, a color developing solution, and a stop solution.