WO2017008388A1 - Pancreatic protein biomarker, uses of same, detection chip therefor, and detection device - Google Patents

Abstract

A pancreatic cancer protein biomarker and applications thereof. The marker comprises detectable CRP, ICAM-1, OPG, and CA19-9 in stable existence in the human serum/plasma. The pancreatic protein biomarker is acquired by means of screening via a low-abundance difference research, is shown to participate in the development and progression of pancreatic cancer, is a credible early-stage biomarker of pancreatic cancer, and provides increased specificity and sensitivity compared with an existing detection marker. On such basis, further provided are a detection chip for the pancreatic cancer protein biomarker and a detection apparatus comprising the detection chip. The detection chip is fixed on a solid-phase substrate to form a chip on the basis of a specific CRP monoclonal antibody, ICAM-1 monoclonal antibody, OPG monoclonal antibody, and CA19-9 monoclonal antibody immunized and prepared using CRP, ICAM-1, OPG, and CA10-9, detects the biomarker proteins by means of antigen-antibody specificity combination, and provides increased accuracy and specificity.

Description

 Pancreatic cancer protein biomarker, use thereof, and detection chip and detection device thereof

Technical field

The invention belongs to the technical field of biological detection, and particularly relates to a pancreatic cancer protein biomarker, a use thereof, a detection chip thereof and a detection device.

Background technique

Pancreatic cancer is a malignant tumor of the digestive tract with a high degree of malignancy and is difficult to diagnose and treat. About 90% of ductal adenocarcinomas originate from the ductal epithelium. Usually after onset, the 5-year survival rate is <1%, which is one of the worst prognosis.

The important reason for the low prognosis of pancreatic cancer is that there is no symptom in the early stage of pancreatic cancer, and it is difficult to detect it during the incubation period or initial stage of pancreatic cancer, thus delaying the optimal treatment time. It is mainly difficult to be detected because there is no symptom at the beginning of the disease, and there is a certain positive rate when there is no detection of CA19-9, CEA, CA242 pancreatic cancer detection markers with the best detection accuracy. High specificity. According to statistics, the use of the above markers in the detection of pancreatic cancer during pregnancy (before canceration) is less sensitive, such as 1 year before the onset, 68% sensitivity, two years before the onset, the sensitivity is 53%. The use of other routine clinical techniques, such as B-ultrasound, CT, MRI, ERCP, PTCD, angiography, laparoscopic examination, etc., found that cancer has been in the advanced stage of cancer, greatly delaying the diagnosis and treatment.

Summary of the invention

The object of the present invention is to overcome the above-mentioned deficiencies of the prior art, and to provide a pancreatic cancer protein biomarker, a use thereof, a detection chip thereof and a detection device capable of better accuracy and specificity.

In order to achieve the above object, the technical solution of the embodiment of the present invention is as follows:

A pancreatic cancer protein biomarker comprising CRP, ICAM-1, OPG and CA19-9 stably present and detectable in human serum/plasma.

The above-mentioned pancreatic oncoprotein biomarker of the present invention is screened by means of "differential subtraction method", that is, the normal sample is used as a control, and the whole protein in the normal sample is deducted from the cancerous sample, and the extra part is a regulatory protein specific to cancer. Verification shows that it is involved in the development of pancreatic cancer and is a credible early biomarker for pancreatic cancer, which has higher specificity and sensitivity than existing detection markers.

Based on the above protein biomarkers, the present invention further provides the use of the above pancreatic cancer protein biomarkers in a pancreatic cancer detecting device.

The pancreatic cancer protein biomarker of the present invention is used as a basic pancreatic cancer detecting device, and the detected target is a differential analysis method to screen the above marker protein, which has higher specificity and sensitivity than the existing detection marker. .

And, the present invention further provides a detection chip for a pancreatic cancer protein biomarker, comprising a solid phase matrix, and CRP monoclonal antibody, ICAM-1 monoclonal antibody, OPG monoclonal antibody and CA19-9 immobilized on the solid phase substrate. Monoclonal antibody.

At the same time, the present invention also proposes a detecting device comprising the above-described pancreatic cancer protein biomarker detection chip.

The detection chip and the detection device of the present invention are prepared by immunizing CRP, ICAM-1, OPG and CA19-9 with specific CRP monoclonal antibody, ICAM-1 monoclonal antibody, OPG monoclonal antibody and CA19-9 monoclonal antibody, and the antigen is passed through the antigen. - Specific binding of antibodies to detect the above biomarker proteins with relatively high accuracy and specificity.

DRAWINGS

The present invention will be further described below in conjunction with the accompanying drawings and embodiments, in which:

1 is a schematic view showing the arrangement of spotting holes on a solid phase substrate in a chip according to an embodiment of the present invention;

2 is a schematic view showing the overall shape of a pancreatic cancer protein biomarker after the detection chip is packaged according to an embodiment of the present invention.

detailed description

The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It is understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The present invention provides a pancreatic cancer protein biomarker comprising CRP (C-reactive protein), ICAM-1 (intercellular adhesion molecule-1), OPG (osteoprotegerin) and CA19-9 (sugar chain antigen 19-9) .

The present invention proposes the above biomarkers based on the detection and examination of pancreatic cancer, and the markers of the present invention are more accurate than the conventional clinical analysis means and the commonly used CA19-9, CEA, CA242 pancreatic cancer detection markers. The discovery and presentation of the above-described combination of biomarkers of the present invention is based on the fact that the biomarker itself is a measurable indicator reflecting the presence and state of the disease, and any physiological changes in the course of disease, stress and recovery can ultimately It is the most intuitive means of disease research at the protein level; and multiple protein biomarker groups are closely related to the clinical and prognosis of patients with chronic diseases, indicating that simultaneous detection of multiple protein factors contributes to a more comprehensive understanding of the disease. process.

The protein in the above-mentioned biomarker combination belongs to a regulatory protein peculiar to a disease. Because tissue fluids, such as plasma, have thousands of protein species and concentration ranges up to 1010, the most advanced instruments can only measure a concentration range of 103; to overcome the interference of healthy background proteins on disease-specific protein. In the present invention, the above marker proteins were screened by differential analysis and verified. The specific screening process is as follows:

(1) A total of 210 subjects with known physical conditions were selected, including 55 healthy controls, 65 patients with initial pancreatic cancer (not metastasized), and 90 patients with metastatic pancreatic cancer. (This study has been approved by an ethics review agency and all subjects have signed informed consent.)

Take 2 ml of the blood sample of the subject, add 1% heparin, and centrifuge at 15 °C for 15 min at 4500 RPM. The plasma fractions were divided into healthy control plasma samples, initial pancreatic cancer (non-metastatic) patients, and plasma samples according to the disease. Metastasis of plasma samples from patients with pancreatic cancer;

(2) then using a healthy control plasma sample as an antigen to immunize rabbits, preparing a polyclonal antibody; and using the prepared polyclonal antibody as an affinity medium to prepare an affinity chromatography column;

(3) Samples of patients with initial pancreatic cancer and samples of patients with metastatic pancreatic cancer were sampled through the above-mentioned affinity chromatography column, and the flow-through was collected.

Due to the thousands of protein species in body fluids such as plasma, the concentration range spans up to 1010. Currently, the most advanced instruments can only measure the concentration range span 103; currently, antibodies are used to remove 20 to 100 high-abundance proteins in the sample. Although the detection rate of low-abundance proteins is increased, when compared with more than 1000 proteins in plasma, the residual relatively high-abundance protein has a ng concentration on the markers used in cancer and cancer-related developmental gestation-related markers. The analysis signal of the pg concentration still has a severe flooding effect. Based on the above reasons, the present invention adopts a subtractive subtraction technique, and uses a healthy control plasma sample as an antigen to immunize a rabbit, and prepares a polyclonal antibody against a healthy control plasma protein, and uses the antibody as an affinity adsorption medium to prepare a chromatography column. A large number of health-related proteins in patient plasma samples are specifically adsorbed and removed to obtain as much disease-related as possible, excluding interference from health-related high-abundance proteins.

After enrichment, low-abundance differentially expressed proteins were analyzed and detected by SDS-PAGE and mass spectrometry after removing a large amount of background interference from high-abundance proteins and enriching the amount.

(4) Identification and quantification of protein by mass spectrometry using the collected flowthrough

In the early screening stage, the healthy control group, the initial stage of pancreatic cancer (not metastasized), and the patients with metastatic pancreatic cancer were identified by mass spectrometry, and the protein was identified three times in each of the three replicates of each group, and 210, 320, and 378 proteins were identified. protein.

The present invention aims to find early biomarkers of pancreatic cancer, which are only found in patients with initial pancreatic cancer (untransferred) and metastatic pancreatic cancer, but there are 45 proteins that are not present in healthy controls. There were 179 proteins in healthy controls, early pancreatic cancer patients (not metastasized), and patients with metastatic pancreatic cancer. This trial is aimed at the analysis of only 45 proteins co-occurring in patients with early stage pancreatic cancer (no metastasis) and metastatic pancreatic cancer.

Through literature reports and bioinformatics analysis (Gene Ontology and KEGG Pathway analysis) did not appear in the Jiankang control, but 45 of the patients present in the plasma of pancreatic cancer were analyzed for functional annotation. It was found that CRP (C-reactive protein), ICAM-1 (intercellular adhesion molecule-1), OPG (osteoprotegerin), and CA19-9 play a very important role in the development and progression of pancreatic cancer.

(5) ELISA verification

The amount of expression of the above differential protein in the blood of the normal control and each disease group was confirmed by ELISA. Specifically, plasma was diluted 1:100 with a dilution buffer, and then the content of biomarkers in blood of different disease groups was measured using an ELISA plate for different protein biomarkers, and each sample was repeatedly measured three times. ELISA technology test results data using SPSS 14.0 software processing, expressed as mean ± standard deviation. Inter-group comparisons of normal distribution data were performed using ANOVA; the accuracy, specificity, and sensitivity of various biomarker predictions were analyzed using receiver operating characteristic curves (ROC). The results of the test are shown in Table 1. The results from Table 1 show that the expression of CRP (C-reactive protein), ICAM-1 (intercellular adhesion molecule-1), OPG (osteoprotegerin), and CA19-9 in the pancreatic cancer group. The amount is significantly higher than the healthy control and increases as the patient's condition is severe (increased proteinuria).

Table 1. ELISA validation of four early markers of pancreatic cancer

category	Healthy control	Pancreatic cancer early (no metastasis)	Late pancreatic cancer (transferred)	P value
n value	(n=55)	(n=65)	(n=90)
Gender: Male Female)	35/20	18/22	29/24	ND
age	58±11	60±12	62±5	ND
CRP	16.45mg/ml	67.87mg/ml*	120.2mg/ml*	<0.001
ICAM-1	177.1ng/ml	535.4ng/ml*	1010ng/ml*	<0.001
OPG	441.7pg/ml	719.5pg/ml*	824.1pg/ml*	<0.001
CA19-9	5.42U/ml	36U/ml*	56.04U/ml*	<0.001

Note: ND: No Difference; *p<0.001 vs. healthy control;

Further use of receiver operating characteristic curve (ROC) to analyze four biomarkers in the blood of pancreatic cancer patients and healthy controls, the results showed CRP (C-reactive protein), ICAM-1 (intercellular adhesion molecule-1), OPG ( The area under the curve of osteoprotegerin and CA19-9 (AUC) were 0.892 (p<0.01), 0.858 (p<0.01), 0.907 (p<0.01), and 0.901 (p<0.01), respectively. Sensitivity and specificity analysis showed that the sensitivity, specificity and concentration cutoff values of the four biomarkers in the blood were selected in Table 2 when the highest “youden” index was selected. The results from Table 2 indicate these four protein molecules. May be involved in the development of pancreatic cancer, a credible early biomarker for pancreatic cancer.

Table 2: Results of ROC analysis of biomarkers in pancreatic cancer patients and healthy controls

Differential protein	Sensitivity	Specificity	Concentration cutoff
CRP (C-reactive protein)	80.8%	76.2%	65mg/ml
ICAM-1	71.2%	60.5%	456ng/ml
OPG	79.6%	55.2%	721pg/ml
CA19-9	84.7%	85.8%	39 U/ml

(6) Verification of early screening of another 100 pancreatic cancers with the above four protein markers

For the other 100 patients with early stage pancreatic cancer, the relative content of these four protein markers was determined by reference to the normal range and the cutoff values of the four protein molecules to determine the relative content of the four protein markers. The various combinations were statistically processed to compare the sensitivity and specificity of the different combinations. The results are shown in Table 3 below. Among them, in the combination indicated by the symbol "∩" in Table 3, sensitivity and specificity were calculated when all the biomarkers contained in the combination were positive (i.e., higher than the cutoff value). In the combination indicated by the symbol "∪", when one of the biomarkers contained in the combination is positive, it is judged to be positive, and the sensitivity and specificity are calculated.

Table 3 Sensitivity and specificity for the diagnosis of early pancreatic cancer protein biomarker combinations:

combination	Sensitivity	Specificity
CA19-9 ∪ OPG	0.933	0.665
CA19-9∩OPG	0.55	0.942
CA19-9 ∪ CRP	0.92	0.594
CA19-9∩CRP	0.547	0.983
CA19-9 ∪ ICAM-1	0.968	0.615
CA19-9∩ICAM-1	0.527	0.941
ICAM-1 ∪ OPG	0.923	0.505
ICAM-1∩OPG	0.541	0.969
ICAM-1 ∪ CRP ∪ OPG	0.989	0.4707
ICAM-1∩CRP∩OPG	0.532	0.983
ICAM-1 ∪ CRP ∪ OPG ∪ CA19-9	0.989	0.475
ICAM-1∩CRP∩OPG∩CA19-9	0.536	0.983

According to the results of the combined detection in Table 2 above, this indicates that the combination of biomarkers selected according to the purpose of the experiment can be used for disease diagnosis efficiently and specifically; and a combination with high sensitivity (CA19-9∪OPG; CA19-9∪CRP; CA19-9∪ICAM-1; ICAM-1∪OPG; ICAM-1∪CRP∪OPG; ICAM-1∪CRP∪OPG∪CA19-9) suitable for initial screening; on the contrary, specificity High combination (CA19-9∩OPG; CA19-9∩CRP; CA19-9∩ICAM-1; ICAM-1∩OPG; ICAM-1∩CRP∩OPG; ICAM-1∩CRP∩OPG∩CA19-9) It is suitable for secondary inspection or tertiary inspection, etc., which requires more reliable judgment. For example, if a positive occurs at the initial screening, the initial examination result is secondarily determined by a combination method with high specificity, and finally an effective and reliable early pancreatic cancer diagnosis reference result is given.

Therefore, based on the above proposals for biomarkers, the present invention proposes the above four biomarker proteins for use in a detection device for pancreatic cancer. Detecting device determined as described above The four biomarker proteins are used as the basis for the corresponding biochemical detection, and their application has higher specificity and sensitivity than the existing detection methods and methods compared with other existing detection methods and means.

The invention further provides a detection chip for a pancreatic cancer protein biomarker based on the combination of the above pancreatic cancer protein biomarker, the detection chip comprises: a solid phase carrier, and a CRP monoclonal antibody immobilized on the solid phase carrier, ICAM- 1 mAb, OPG mAb and CA19-9 mAb.

The detection chip of the pancreatic cancer protein biomarker of the present invention, by immobilizing the monoclonal antibody of the above protein biomarker on a solid phase carrier, and capturing the protein to be tested which can specifically bind thereto by the corresponding monoclonal antibody, thereby realizing Separation of the target marker protein in serum, plasma, lymph, interstitial fluid, urine, exudate, cell lysate, and secretion sample waiting for the sample; followed by washing, purification, confirmation, and biochemical analysis; The differential expression results of these biomarker proteins in the sample to be tested are obtained.

Wherein, the monoclonal antibody of the four biomarker proteins contained in the detection chip of the pancreatic cancer protein biomarker is used in the present invention to immunize a monoclonal antibody by using the rabbit as an immunogen, for example, the preparation of CRP monoclonal antibody is Examples include:

(1) Female mice of about 4 months were used for immunological injection of CRP protein. After injection, the CRP protein was used as an antigen to enter the peripheral immune organs of rabbits through blood circulation or lymphatic circulation, and the corresponding B lymphocyte clones were stimulated and activated. Proliferate and differentiate into sensitized B lymphocytes;

(2) When the immunization reaches the target level, the rabbit is sacrificed, and the spleen of the rabbit is taken; after the fine crushing, the single cell suspension is digested by enzymatic digestion, and the cell fusion hybridization is performed with the tumor cells;

(3) After cell fusion, selective culture is carried out using HAT selective medium to screen for successfully fused hybrid cells;

(4) The successful fusion cell dilution method is used for cloning and culture of hybrid cells; after culture, positive hybridoma cells capable of producing monoclonal antibodies against CRP protein are screened by immunization method; and the positive hybridoma cells are cultured in vitro. In the process of culture, the expression is induced at the appropriate time, and after the completion of the culture, the CRP monoclonal antibody can be obtained.

The above CRP monoclonal antibody, ICAM-1 monoclonal antibody, OPG monoclonal antibody and CA19-9 monoclonal antibody are separately prepared, and then fixed to a solid phase carrier by covalent crosslinking or non-covalent crosslinking.

The solid phase carrier as a protein chip in practice can generally be selected from a nitrocellulose membrane, a nylon membrane, a gold membrane, a flat plate or a glass substrate. In the present invention, a nitrocellulose membrane or a glass substrate is preferably used for the purpose of performing low-abundance differential protein-specific adsorption for the above-mentioned monoclonal antibody. The reason is that the nitrocellulose membrane itself is a permeation membrane, and the nitrocellulose cellulose microporous membrane has a large capacity for an empty structure and a strong binding force to proteins.

Among them, the use of a glass substrate is mainly convenient, inexpensive, easy to handle, and has sufficient stability and inertness, and is very suitable for large-scale commercial production of the product of the present invention. However, in the use, the glass substrate needs to be treated according to the characteristics of the present invention, because the glass substrate as the protein carrier is a glass plate, and the surface hydroxyl group is firstly used with N,N-diethoxyaminopropyltriethoxylate. The silane is surface-treated; then the antibody protein is coupled, but the glass substrate has a very strong non-specific adsorption, which is likely to cause very serious background interference, especially in the above four kinds of target proteins to be detected in the present invention. When a protein is used, a large amount of high-abundance protein interferes more strongly. Therefore, when the glass substrate is used as a solid phase carrier in the present invention, the glass substrate is surface-treated with PEG (polyethylene glycol), and the surface treated with PEG has better signal intensity than the siliconized surface, and the macromolecular analysis component can be improved ( The ability to bind to a ligand, such as a ligand, can attenuate non-specific binding; this is because PEG has a large spatial structure, which reduces the steric hindrance between proteins and can increase the specificity of affinity adsorption.

In order to facilitate the comparison of the above-mentioned protein chip detection of the present invention, a positive control and a negative control are also immobilized on the above-mentioned protein chip of the present invention. Among them, the positive control can use IgG monoclonal antibody, and the positive control IgG monoclonal antibody is preferably prepared by using the same immune body as above. For example, if the above CRP monoclonal antibody is prepared by using rabbit as an immunogen, then the IgG monoclonal antibody is also the best. It is prepared by using rabbit as an immunological body; the positive control is used to detect the binding of the target protein. Of course, corresponding to the above positive control, the negative control can use a sample-like dilution of the sample, which does not contain any bindable protein, just as a blank control.

In order to facilitate the comparison and prevent cross-contamination, the solid phase carrier of the above-mentioned protein chip of the present invention is provided with spotting holes, and the spotting holes may be disposed on the surface of the solid phase carrier in a matrix or array manner. The corresponding monoclonal antibodies used to detect the biomarker protein are then immobilized in these spotted wells. The structure of the sampled hole is used for operation detection. On the one hand, the protein in the sample hole can be well separated, and the cross-contamination is minimized; on the other hand, the concave structure of the hole can facilitate the addition and preservation of the spotting buffer. Etc., to avoid problems such as drying after incubation, and thirdly, the rules and sizes of the spotting holes can be standardized, which facilitates horizontal comparison of the amount of spotting. Controlling the amount of sample is also more conducive to lateral comparison and calculation of results.

The present invention also provides a detection kit for a pancreatic cancer protein biomarker for the purpose of specifically detecting the above-mentioned pancreatic cancer protein organism for the convenience of the detection chip of the pancreatic cancer protein biomarker described above. Marker CRP monoclonal antibody, ICAM-1 mAb, OPG mAb and CA19-9 mAb. Specific detection was carried out using a monoclonal antibody preparation kit corresponding to the above protein biomarker. The detection by the kit can be carried out in various ways. For example, the above four monoclonal antibodies are respectively used as a chromatographic medium to form a chromatography column, and then the sample to be tested is subjected to a column, and then assisted by electrophoresis, mass spectrometry and the like. Alternatively, the enzyme-labeled antigen may be used, and the sample protein to be tested is subjected to an enzyme labeling, and then subjected to an affinity reaction with the above-mentioned four monoclonal antibodies, and then the signal is detected by the enzyme-labeled antigen bound to the respective monoclonal antibody.

However, the above two detection methods are inconvenient because the convenience and accuracy of the detection are further improved. The kit of the present invention refers to the double-anti-sandwich method to assist the auxiliary reagent, and further, the auxiliary reagent includes the sample dilution buffer. In the present invention, a 30-70 mM PBS phosphate buffer containing 1.5% to 2% BSA (bovine serum albumin) stabilizer is used as the sample dilution buffer; the sample dilution buffer is used for sample dilution and washing, wherein BSA It is a protein stabilizer that prevents protein breakdown and non-specific adsorption; and BSA also mitigates some adverse environmental factors such as heat, surface tension and chemical factors, thereby reducing the denaturation of some proteins during the test.

In order to improve the detection and sensitivity analysis in the reagent reagent detection, the above kit of the present invention further needs to cooperate with the enzyme-labeled secondary antibody according to the principle of the double-anti-sandwich method; after the sample to be tested, each protein of interest will be It is captured by the corresponding monoclonal antibody and affinity-adsorbed. At this time, an excess of the enzyme-labeled secondary antibody is added to bind to the target protein to form a sandwich structure; then the amount of the enzyme-labeled secondary antibody can be reacted in the sample to be tested. The expression of the target protein can be obtained by calculating the signal carried by the enzyme-labeled secondary antibody. In the practice of the present invention, the above-mentioned enzyme-labeled secondary antibody can be carried out by using a relatively common HRP (horseradish peroxidase)-containing enzyme-labeled secondary antibody, and in the case of satisfying the accuracy, the self-designed and manufactured product is also omitted. trouble.

At the same time, in order to facilitate the detection of the enzyme-labeled secondary antibody signal, the kit may further comprise a concentrated washing solution containing 1~2%. BSA, 0.03~0.07% Tween-20 PBS, used to concentrate the enzyme-labeled secondary antibody and signal detection color components. Of course, it is necessary to add a developer, TMB (tetramethylbenzidine) substrate solution afterwards.

Of course, in order to facilitate the detection by the double-sandwich method, a solid phase matrix can also be provided in the kit, which in practice is a matrix similar to the above-mentioned chip, functioning as a carrier and a reaction medium. The specific shape and morphology can also be referred to in the above description of the chip. During the process of use, the corresponding monoclonal antibody is fixed to the spotting hole of the solid phase matrix according to the instructions of the kit manual, and then the detection of the sample to be tested is performed; Of course, if the solid phase matrix carrier is not provided in the kit of the present invention, it can also be carried out by using a spotting plate of the ELISA kit.

At the same time, in order to facilitate comparative analysis of the results, standards, positive controls and negative controls can be provided in the kit. For the setting of the comparison, refer to the description in the above chip, and the usage is the same as the sample to be tested, and the operation is performed synchronously with the sample to be tested, and then the results are compared to provide a comparative reference.

Based on the above detection chip, the present invention also proposes a pancreatic cancer detecting device apparatus including the above detection chip. In a specific implementation, the above chip and the device for detecting the chip signal, such as a CCD detector, may be combined with similar detection signal devices; or the chip may be directly fixed in the detection port of the detection to form a whole.

The detection device device of the invention comprising the detection chip, the detection chip is made into a whole device capable of directly detecting the biomarker protein completely, and the biomarker protein is detected by the specific binding of the antigen-antibody, which is relatively high. Accuracy and specificity.

To illustrate the accuracy of the biomarkers employed in the present invention and the authenticity of the test results of the application kits, and to enable the above-described technical details and process methods to be more easily understood and implemented by those skilled in the art, the following The examples and actual analytical data are exemplified.

Example 1

Example 1 is used to demonstrate the accuracy of pancreatic cancer detection using the above-described biomarkers of the present invention and the authenticity of the test results using the kit.

S11, preparing a solid phase matrix carrier (taking a nitrocellulose membrane as an example):

Obtain the matrix nitrocellulose membrane and follow the design (5*5 array in lattice form, as shown in Figure 1, labeled 1 as the anchor point in Figure 1, 2 as the positive control spotted well, and 7 as the negative control spot) Hole, 3 is CRP spotting hole, 4 is ICAM-1 spotting hole, 5 is OPG spotting hole, 6 is CA19-9 spotting hole) arrangement and spotting position is prepared on the film with detection index coating Spotted hole, closed; finally dried, stored at 4 ° C after packaging, the shape is shown in Figure 2;

S12, immobilizing CRP monoclonal antibody, ICAM-1 monoclonal antibody, OPG monoclonal antibody and CA19-9 monoclonal antibody on a solid substrate, wherein CRP monoclonal antibody is taken as an example for description (wherein the preparation of CRP monoclonal antibody is as described in the above embodiment) The preparation process of rabbit immunization will not be repeated here; the remaining ICAM-1 monoclonal antibody, OPG monoclonal antibody and CA19-9 monoclonal antibody are fixed according to the process):

First, 0.01 to 0.5 mg/mL of CRP monoclonal antibody to 50 mM Preparing a solution of the monoclonal antibody for immobilization in a PBS buffer of pH 7.2;

After stimulating the spotted well on the nitrocellulose membrane slide with glutaraldehyde, use 100 μl The previously prepared monoclonal antibody solution was spotted on the spotting hole, and then the cover piece was covered and incubated for about 5 hours. Then, after the incubation is completed, use 50 mM Wash PBS buffer pH 7.2 three to four times, remove unbound antibody and impurities, and dry for use;

In order to block the portion of the glutaraldehyde-activated spotted well to which the CRP mAb was to be immobilized (the portion which does not react with the target protein CRP on the surface), a 50 mM pH 7 containing 2% BSA was added dropwise to the spotted well. .2 The PBS buffer solution was then incubated with a cover slip for 2 h; the front and back surfaces of the slides blocked with BSA were washed three to four times with 50 mM pH PBS buffer solution and allowed to air dry.

S13, obtaining a urine sample to be detected, and then diluted with the negative control and the positive control sample dilution (30-70 mM PBS buffer containing 1.5% to 2% BSA stabilizer) for 3 times, respectively, and adding the detected points respectively. In the sample well, 0.1 mL was added to each well, and each sample was subjected to 3 parallel tests, and the mixture was incubated at 37 ° C for 1 hour to fully react, and then the solution in the well was discarded, and washed with a concentrated washing solution 3 times;

S14, 100 μl of enzyme-labeled working solution (HRP-containing enzyme-labeled secondary antibody) was added to each well, incubated at 37 ° C for 30 minutes, and shaken and washed 3 times for 1 minute each time;

In S15, a developer TMB (tetramethylbenzidine) substrate liquid matched with the enzyme standard working solution is added to the reaction area of the chip, and 20 ul per well is added for chemiluminescence.

S16, using a CCD detector to perform chemiluminescence scanning and collecting signals, and displaying the analysis results according to the scanning results to determine whether the expression levels of these biomarker proteins are normal.

Using the above-mentioned implementation process, the above biomarker proteins of 500 subjects (age level 58-70) were detected. The results of the test showed that 24 subjects were treated according to the combination category of Table 3 above. The patients were judged to be suspected of pancreatic cancer; the 24 abnormal subjects were further followed up and re-clinical and comprehensive symptom analysis were performed within the next six months. The results showed that 22 of 24 people were diagnosed with pancreatic cancer (of which 6 were of type that had spread, 16 of which were of initial non-proliferation type), and the remaining 2 were not considered to be pancreatic cancer patients, but Other interfering conditions.

From the final statistical results, the detection kit for the above-mentioned markers of the present invention is based on the double-anti-sandwich method, and the detection of the affinity antibody for detecting the above biomarker is carried out, and the detection accuracy has high accuracy and sensitivity. . Moreover, compared with the current CA19-9, CEA, CA242 pancreatic cancer detection markers, it can cover the early stage of pancreatic cancer and not miss the treatment.

The above is only the preferred embodiment of the present invention, and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection of the present invention. Within the scope.

Claims (12)

1. A pancreatic cancer protein biomarker comprising CRP, ICAM-1, OPG and CA19-9 which are stably present and detectable in human serum/plasma.
2. The use of the pancreatic oncoprotein biomarker according to claim 1 in a pancreatic cancer detecting device.
3. A detection chip for a pancreatic oncoprotein biomarker according to claim 1, comprising a solid phase matrix, and CRP monoclonal antibody, ICAM-1 monoclonal antibody, OPG monoclonal antibody, and immobilized on the solid phase substrate CA19-9 mAb.
4. The detection chip for pancreatic oncoprotein biomarkers according to claim 3, wherein the solid phase substrate is provided with a matrix-arranged spotting hole, the CRP monoclonal antibody, ICAM-1 monoclonal antibody, and OPG single The anti-and CA19-9 mAb was immobilized in the spotted wells, respectively.
5. The detection chip for pancreatic oncoprotein biomarkers according to claim 3, wherein a positive control and/or a negative control are further disposed on the solid phase substrate.
6. The detection chip for a pancreatic cancer protein biomarker according to claim 3, wherein the solid phase substrate is one of a nitrocellulose membrane, a nylon membrane, a gold membrane, a flat plate or a glass substrate.
7. The detection chip for a pancreatic cancer protein biomarker according to claim 6, wherein the solid phase substrate is provided with a matrix-arranged spotting hole, the CRP monoclonal antibody, ICAM-1 monoclonal antibody, OPG single The anti-and CA19-9 mAb was immobilized in the spotted wells, respectively.
8. The detection chip for pancreatic oncoprotein biomarkers according to claim 6, wherein a positive control and/or a negative control are further disposed on the solid phase substrate.
9. The test chip for a pancreatic cancer protein biomarker according to claim 6, wherein the glass substrate is a glass substrate surface-treated with PEG.
10. The detection chip for a pancreatic cancer protein biomarker according to claim 9, wherein the solid phase substrate is provided with a matrix-arranged spotting hole, the CRP monoclonal antibody, ICAM-1 monoclonal antibody, and OPG single The anti-and CA19-9 mAb was immobilized in the spotted wells, respectively.
11. The detection chip for pancreatic oncoprotein biomarkers according to claim 9, wherein a positive control and/or a negative control are further disposed on the solid phase substrate.
12.  A pancreatic cancer detecting device comprising the detection chip of the pancreatic cancer protein biomarker according to claim 11.