CN106885908A - The detection kit of blood-serum P SMD4 albumen and its detection method and application - Google Patents

Abstract

The invention belongs to the field of immunology and biotechnology, in particular to a detection kit for serum PSMD4 protein and its detection method and application. The ELISA detection kit and detection method of PSMD4 protein provided by the invention can be used for various malignant tumors ( In particular, serological diagnosis of lung, gastric and colorectal cancers) and tumor screening. The invention is simple and convenient to operate, can accurately and highly sensitively detect the PSMD4 protein content in the serum of malignant tumor patients, and provides a new means and method for clinical testing and basic research.

Description

Detection kit for serum PSMD4 protein and its detection method and application

technical field

The invention belongs to the field of immunology and biotechnology, in particular to an ELISA detection kit for PSMD4 protein and its detection method and its application in serological diagnosis and tumor screening of various malignant tumors (lung cancer, gastric cancer and colorectal cancer) .

Background technique

Cancer refers to all malignant tumors. According to the statistics of the World Health Organization, there are more than 14 million new cancer patients in the world every year, and China has become the country most seriously affected by cancer in the world. The incidence of cancer in mainland China accounts for almost half of the global cancer incidence. Cancer ranks second among various causes of death in China, and ranks first in cities. Among them, the mortality rate of lung cancer has risen the fastest, ranking first in the mortality rate of malignant tumors. Followed by liver cancer, gastric cancer and colorectal cancer. What is worrying is that in the next 10 years, the incidence and mortality of cancer in China will continue to rise. Therefore, early diagnosis of cancer is of great significance to prolong the survival time of patients and reduce the mortality of patients.

At present, the diagnosis of cancer mainly relies on the combination of laboratory examination, imaging examination and pathological examination. However, neither imaging examination nor pathological examination can screen out early cancer patients on a large scale. Laboratory examinations need to rely on the detection of multiple tumor immune markers to guide clinical diagnosis. There are many kinds of tumor markers currently used clinically, such as AFP is the most valuable indicator for the diagnosis of liver cancer, serum carcinoembryonic antigen (CEA) for colon cancer; gastric juice sulfoglycoprotein (FSA) and gastric cancer-associated antigen (GCAA) for gastric cancer , Prostate-specific antigen (PSA) of prostate cancer, etc. However, according to research reports in recent years, various tumor markers are prone to missed and misdiagnosed problems. At present, there are no tumor markers with good specificity and high sensitivity in clinical practice that can be used for large-scale screening of patients with various cancers. Therefore, there is still a need for highly sensitive malignant tumor-specific markers for primary screening of large-scale populations of cancer patients, thereby improving the early diagnosis rate of malignant tumors.

The study of serum markers of malignant tumors has always been the focus of scientists' research, but most of the research results are difficult to meet the needs of practical applications. It is particularly important to explore serological indicators with high sensitivity and specificity.

The research object in the present invention—PSMD4 protein, is a proteasome 26S non-ATPase subunit 4 (GeneID: 5710). The molecule contains two 15-amino acid ubiquitin interacting motifs (ubiquitin interacting motif, UIM), which can selectively bind ubiquitinated proteins and mediate protein degradation. It has been reported that the abnormal expression of PSMD4 is related to inflammatory bowel disease, ulcerative colitis and other diseases, but the expression and biological function of PSMD4 in malignant tumors are still not very clear. Studies have shown that PSMD4 may be related to cell differentiation and body development (see literature: Hamazaki, J., et al., Rpn10-mediated degradation of ubiquitinated proteins is essential for mouse development. Mol Cell Biol, 2007.27(19): p.6629- 38). We found that PSMD4 is an important oncoprotein and can be released into the blood. The applicant has applied for the Chinese patent CN201310005491.4 on the new use of PSMD4 as a marker for liver cancer prognosis and diagnosis.

At present, there is no literature report on the accurate detection of PSMD4 protein in serum samples of tumor patients; there is no accurate, simple and sensitive detection method for PSMD4 protein content in serum samples of tumor patients; there is no literature report on the detection of PSMD4 protein. ELISA detection kit and the application of such kit in tumor serological diagnosis and screening.

Contents of the invention

The purpose of the present invention is to provide a kind of ELISA detection kit of PSMD4 albumen; Another purpose of the present invention is to provide the detection method utilizing above-mentioned kit; The third purpose of the present invention is to provide above-mentioned kit in multiple malignant tumors (such as Lung cancer, gastric cancer, colorectal cancer, etc.) serological diagnosis and screening applications.

The invention aims to provide an accurate, simple, high-sensitivity and widely-usable detection means for the PSMD4 protein content in various malignant tumor serum samples in clinic and laboratory.

After extensive and in-depth research, the inventors used enzyme-linked immunosorbent assay to detect the expression level of PSMD4 protein in the serum of various malignant tumor patients, and first confirmed the high expression of PSMD4 protein in the serum of various malignant tumor patients.

In the first aspect of the present invention, an ELISA detection kit for PSMD4 protein is provided, and the ELISA detection kit includes:

(1) ELISA microtiter plate coated with PSMD4 antibody, described coating antibody is the polyclonal antibody of rabbit anti-human PSMD4; Preferably, the polyclonal antibody of rabbit anti-human PSMD4 is purchased from PTG company; Optimum , the antibody working concentration is 2μg/ml.

(2) The antibody for detecting the PSMD4 antigen is a mouse monoclonal antibody against human PSMD4; preferably, the mouse monoclonal antibody against human PSMD4 is purchased from PTG; optimally, the working concentration of the detection antibody is 0.5 μg /ml.

(3) Enzyme-labeled secondary antibody, HRP (horseradish peroxidase)-labeled goat anti-mouse IgG; preferably, HRP-labeled goat anti-mouse IgG was purchased from PTG Company; optimally, the dilution concentration was 1 :5000.

(4) The standard protein is a recombinant human PSMD4 fusion protein; preferably, the recombinant human PSMD4 fusion protein is purchased from PTG Company.

(5) The kit also includes: conventional sample diluent, coating buffer, blocking solution, ELISA plate washing solution, antibody diluent, chromogenic solution and stop solution;

The kit, the ELISA plate is a commercially available ELISA plate, preferably the ELISA plate from Greiner, Germany (96-well single detachable plate #650180);

Coating buffer, select 1×PBS from sigam company, pH: 9.6;

Blocking solution, choose the blocking solution containing 3% bovine serum albumin + PBS solution;

ELISA plate washing solution, preferably 1×PBS solution containing 0.05% Tween-20;

Sample diluent: when the sample is serum, preferably 1×PBS, pH: 7.4;

Antibody diluent, choose 1×PBS;

Chromogenic solution, select 3,3',5,5'-tetramethylbenzidine (TMB) from sigam company;

Termination solution, preferably 2mol/L sulfuric acid solution.

The ELISA detection kit of a kind of PSMD4 protein of the present invention, used reagent, condition are further optimized:

(1) Optimization of coating antibody: the kit of the present invention uses different antibody coating respectively: polyclonal antibody of rabbit anti-human PSMD4 (Abnova company, #H00005710-AP11-1), polyclonal antibody of rabbit anti-human PSMD4 Antibody (PTG Company, #14899-1-AP), mouse anti-human PSMD4 monoclonal antibody (Abnova Company, #H00005710-AP11-2), mouse anti-human PSMD4 monoclonal antibody (PTG Company, #66179-1- Ig), the detection concentration is 7.5 μg/ml, 5 μg/ml, 2.0 μg/ml, 1 μg/ml, 0.5 μg/ml, 0.25 μg/ml. As a result, it was found that rabbit polyclonal antibody against human PSMD4 (Abnova Company, #H00005710-AP11-1), mouse monoclonal antibody against human PSMD4 (Abnova Company, #H00005710-AP11-2; PTG Company, #66179-1- Ig) as the coating antibody, there is obvious non-specific reaction, and the concentration of the standard has no correlation with the absorbance value; but there is no obvious reaction when using the polyclonal antibody of rabbit anti-human PSMD4 (PTG company, #14899-1-AP). For non-specific reactions, the concentration and absorbance of the standard have a good correlation. After repeated optimization, the antibody working concentration of 2 μg/ml is the best, and the correlation between the concentration of the standard and the absorbance value is the best.

(2) Optimization of the detection antibody: the kit of the present invention selects different antibodies as the detection antibody respectively: when the coated antibody is two polyclonal antibodies of rabbit anti-human PSMD4 (Abnova company; #H00005710-AP11-1; PTG Company, #14899-1-AP) selected two kinds of monoclonal antibodies (Abnova Company, #H00005710-AP11-2; PTG Company, #66179-1-Ig) of mouse anti-human PSMD4 as detection antibodies respectively; When the antibody is two kinds of monoclonal antibodies (Abnova Company, #H00005710-AP11-2; PTG Company, #66179-1-Ig) of mouse anti-human PSMD4, two kinds of polyclonal antibodies of rabbit anti-human PSMD4 (Abnova; #H00005710 -AP11-1; PTG Corporation, #14899-1-AP). 2.0 μg/ml, 1 μg/ml, 0.5 μg/ml, 0.25 μg/ml, 0.1 μg/ml were selected as detection concentrations. As a result, it was found that the polyclonal antibody (Abnova Company; #H00005710-AP11-1) coated with antibody rabbit anti-human PSMD4 and the two monoclonal antibodies of mouse anti-human PSMD4 (Abnova Company, #H00005710-AP11-2; PTG Company, # 66179-1-Ig) showed obvious non-specific reaction when it was compatible, and there was no correlation between the concentration of the standard substance and the absorbance value; the polyclonal antibody of rabbit anti-human PSMD4 (PTG company, #14899-1-AP) and the mouse anti-human PSMD4 Monoclonal antibody (Abnova company, #H00005710-AP11-2) also has obvious non-specific reaction when it is compatible, and there is no correlation between the concentration of the standard and the absorbance value; (Abnova Company, #H00005710-AP11-2; PTG Company, #66179-1-Ig) and two polyclonal antibodies of rabbit anti-human PSMD4 (Abnova; #H00005710-AP11-1; PTG Company, #14899-1-Ig) AP) compatibility, the result also has obvious non-specific reaction, and the concentration of the standard substance has no correlation with the absorbance value. Through repeated permutation and combination verification, it was found that only when the rabbit anti-human PSMD4 polyclonal antibody (PTG Company, #14899-1-AP) was used in combination with the mouse anti-human PSMD4 monoclonal antibody (PTG Company, #66179-1-Ig) When , the concentration and absorbance of the standard have a good correlation. After repeated optimization, when the working concentration of the detection antibody is 0.5 μg/ml, the correlation between the concentration of the standard substance and the absorbance value is the best.

(3) Optimization of blocking solution: select different blocking solutions respectively, containing 1% bovine serum albumin+PBS solution, containing 2% bovine serum albumin+PBS solution, containing 3% bovine serum albumin+PBS solution, containing 4 % bovine serum albumin + PBS solution, containing 5% bovine serum albumin + PBS solution, the blocking time is 1 hour at room temperature, 2 hours at room temperature, and overnight at 4°C. The results showed that the best combination of blocking solution and blocking time was the blocking solution containing 3% bovine serum albumin+PBS solution and blocking at room temperature for 2 hours.

(4) Optimization of the enzyme-labeled secondary antibody: select different species of enzyme-labeled secondary antibodies according to the difference of the detection antibody, and the detection antibody is mouse anti-human PSMD4 monoclonal antibody (Abnova Company, #H00005710-AP11-2; PTG Company, # 66179-1-Ig) goat anti-mouse HRP-labeled IgG antibody (Jackson #715-005-150) and goat anti-mouse HRP-labeled IgG antibody (PTG #SA00001-1); the detection antibody is rabbit Anti-human PSMD4 polyclonal antibody (Abnova; #H00005710-AP11-1; PTG Company, #14899-1-AP) was used goat anti-rabbit HRP-labeled IgG antibody (PTG Company #SA00001-15). Antibody dilution factor selection 1:1000, 1:2000, 1:3000, 1:5000, 1:10000. Both groups of goat anti-mouse HRP-labeled IgG antibody (Jackson #715-005-150) and goat anti-rabbit HRP-labeled IgG antibody (PTG #SA00001-15) did not show good results in combination validation. Correlation, there is a non-specific reaction. In all the verification combinations, the mouse anti-human PSMD4 monoclonal antibody (PTG company, #66179-1-Ig) as the detection antibody and the enzyme-labeled secondary antibody were goat anti-mouse HRP-labeled IgG antibody compatibility results showed a good correlation ( PTG Company #SA00001-1), the dilution concentration of 1:5000 is the best.

(5) Optimization of sample diluents: different sample diluents were selected, and 1×PBS (pH: 7.4) or 1×PBS solution containing 0.1% BSA and 0.5% Tween-20 was selected as the sample diluent for serum sample detection. It was found that the best serum sample dilution solution was 1×PBS (pH:7.4) which was better than 1×TBS solution containing 0.1% BSA and 0.5% Tween-20.

The second aspect of the present invention provides a detection method using the above PSMD4 protein ELISA detection kit.

Before detection, first prepare the detection ELISA plate: Dilute the antibody (rabbit anti-human PSMD4 polyclonal antibody) to 2.0ug/ml in the coating buffer, add 100 μl to each well of the ELISA plate, seal the plate and place Incubate overnight at 4°C and set aside.

The detection method of the present invention comprises the following steps: the serum sample is diluted 1:1 with a sample diluent, added with a volume of 100 μl/hole, and incubated at room temperature for 2 hours; PSMD4 recombinant protein is diluted into different concentration gradients with a sample diluent, Add sample per well volume, incubate at room temperature for 2 hours; shake off the liquid in each well, add ELISA plate washing solution, 300 μl per well, wash 3-5 times, and dry; Dilute PSMD4 detection antibody to 0.5 μg/well ml, 100 μl per well, incubate at room temperature for 1 hour; shake off the liquid in each well, add ELISA plate washing solution, 300 μl per well, wash 3-5 times, and dry; add HRP-labeled goat anti-mouse IgG, 100 μl per well, incubate at room temperature for 40 minutes; shake off the liquid in each well, add ELISA plate washing solution, 300 μl per well, wash 6-8 times, and dry; add TMB, 100 μl per well, incubate at room temperature for 10 -20 minutes, add 2mol/L sulfuric acid solution to terminate the reaction, 50 μl per well; measure the OD value on a microplate reader (450nm).

PSMD4 recombinant protein is diluted into different concentration gradients with sample diluent, and the concentration gradient can be set by itself according to the detection needs. In a preferred embodiment of the present invention, the concentration gradient is: 10ng/ml, 3ng/ml, 1ng/ml, 0.3 ng/ml, 0.1ng/ml, 0.03ng/ml, 0.01ng/ml.

The third aspect of the present invention provides the application of the above PSMD4 protein ELISA detection kit in the serological diagnosis of various malignant tumors.

The invention provides an application of an ELISA detection kit for PSMD4 protein in the preparation of malignant tumor serological diagnosis or screening kits.

The serum PSMD4 protein detection kit of the present invention can specifically, accurately, qualitatively and quantitatively detect, diagnose and screen various malignant tumors at the same time.

Said malignant tumor, in a preferred embodiment of the present invention, is lung cancer, gastric cancer, and/or colorectal cancer, etc.

The application of the PSMD4 protein ELISA detection kit of the present invention in the preparation of malignant tumor serological diagnosis or screening kits, the PSMD4 protein ELISA detection kit comprises the above kit composition.

The application of the PSMD4 protein ELISA detection kit of the present invention in the preparation of a malignant tumor serological diagnosis or screening kit, the method for the serological diagnosis or screening of the kit includes the above detection method steps.

Beneficial effects of the present invention:

1) Accurate: There is currently no commercial ELISA kit for human PSMD4 protein on the market, and there is no method for quantitative detection of human PSMD4 protein content after literature search. Preliminary work found that PSMD4 is an oncoprotein, but it is currently impossible to identify multiple Determination of PSMD4 in the serum of patients with malignant tumors. This ELISA kit can accurately detect the content of PSMD4 protein in the serum of various malignant tumor patients, and the results are quantitatively analyzed by a microplate reader, eliminating the subjectivity of semi-quantitative methods such as immunohistochemistry and immunofluorescence.

2) High sensitivity: the PSMD4 protein detected by this method can be as low as 10pg/ml, and the sensitivity is significantly higher than that of semi-quantitative methods such as ordinary Western blot and immunohistochemistry.

3) Simple and convenient: the reagents and experimental consumables used in this method are all commercially available products, which are easy to obtain; in the detection, only a pipette and a microplate reader are required for sample addition and reading, which can be carried out in ordinary laboratories and hospitals This test.

The ELISA kit provided by the invention is simple and convenient to operate, and can accurately and highly sensitively detect the content of PSMD4 protein in the serum of patients with various malignant tumors; it provides a new method and method for screening early malignant tumor patients and basic research. method.

The kit of the present invention is mainly used for quantitative detection of PSMD4 protein in serum samples of various malignant tumor patients, and can be applied to PSMD4 protein in various biological samples (such as cell culture supernatant, cell lysate) in basic research. detection.

The present invention does not require complex instruments for detection, is easy to be popularized and applied in scientific research institutions and medical institutions, can detect clinical specimens on a large scale, and quickly obtain massive data and information related to human PSMD4 protein, providing early diagnosis and treatment for patients with various malignant tumors. Screening provides clinical reference value and has a broad market prospect and great economic and social benefits.

Description of drawings

Fig. 1 is the result of detecting PSMD4 fusion protein by double-antibody sandwich ELISA method, wherein A is a histogram, and B is a scatter plot (linear correlation).

Fig. 2 is the result of detecting PSMD4 protein in serum of normal population and lung cancer patients by double antibody sandwich ELISA method, normal (healthy control); lung cancer (lung cancer);

Figure 3 is the result of detecting PSMD4 protein in normal population and gastric cancer patient serum by double antibody sandwich ELISA method, normal (healthy control); gastric cancer (gastric cancer);

Figure 4 is the result of detecting PSMD4 protein in serum of normal population and colorectal cancer patients by double antibody sandwich ELISA method, normal (healthy control); colorectal cancer (colorectal cancer).

detailed description

Now, the present invention will be further described in conjunction with the embodiments and accompanying drawings, but the implementation of the present invention is not limited thereto.

Rabbit polyclonal antibody against human PSMD4, Abnova; H00005710-AP11-1

Mouse monoclonal antibody against human PSMD4, Abnova; H00005710-AP11-2

Rabbit polyclonal antibody against human PSMD4, PTG; 14899-1-AP

Mouse monoclonal antibody against human PSMD4, PTG Company; 66179-1-Ig

HRP-labeled goat anti-mouse IgG, PTG; SA00001-1

HRP-labeled goat anti-rabbit IgG, PTG; SA00001-15

HRP-conjugated goat anti-mouse IgG, Jackson; 715-005-150

Recombinant human PSMD4 fusion protein, PTG company; ag6691

The chromogenic solution was 3,3′,5,5′-tetramethylbenzidine (TMB) from Sigam Company.

Example 1:

Preparation of detection ELISA plate: Coating buffer 1×PBS, pH: 9.6 Dilute the antibody (rabbit anti-human PSMD4 polyclonal antibody) to 2.0ug/ml, add 100 μl to each well of the ELISA plate, After sealing the plate, incubate overnight at 4°C for later use.

Detection of serum samples and recombinant proteins: Shake off the liquid in each well, add ELISA microplate washing solution (1×PBS solution containing 0.05% Tween-20), 300 μl per well, wash 2-3 times, and dry; Block with a blocking solution containing 3% bovine serum albumin in PBS solution, add a sample at a volume of 200ul/well, and block for 2 hours at room temperature: shake off the liquid in each well, add ELISA plate washing solution, 300μl per well , washed 2-3 times, dried, serum samples were diluted 1:1 with sample diluent 1×PBS (pH:7.4), added at a volume of 100 μl/well, incubated at room temperature for 2 hours; PSMD4 recombinant protein was diluted with samples Dilute the solution into different concentration gradients, add samples at a volume of 100 μl/well, and incubate at room temperature for 2 hours; shake off the liquid in each well, add ELISA plate washing solution, 300 μl per well, wash 3-5 times, and dry; Dilute the PSMD4 detection antibody to 0.5μg/ml, 100μl per well, incubate at room temperature for 1 hour; shake off the liquid in each well, add ELISA microplate washing solution, 300μl per hole, wash 3-5 times, dry; add HRP-labeled goat anti-mouse IgG, 100 μl per well, incubate at room temperature for 40 minutes; shake off the liquid in each well, add ELISA plate washing solution, 300 μl per well, wash 6-8 times, and dry; add TMB, 100 μl per well, incubate at room temperature in the dark for 10-20 minutes, add 2 mol/L sulfuric acid solution to terminate the reaction, 50 μl per well; measure the OD value on a microplate reader (450 nm).

Example 2: Optimization

1. Optimization of coating antibody concentration:

Select different coating antibodies rabbit anti-human PSMD4 polyclonal antibody (PTG company and Abnova company), choose different concentrations (7.5μg/ml, 5μg/ml, 2.0μg/ml, 1μg/ml, 0.5μg/ml, 0.25 μg/ml) coated ELISA plate, detected according to the operating steps in Example 1, adding known PSMD4 protein standards of different concentrations. According to the obtained OD value, select the blank group with the smallest OD value and the closest linear relationship between the OD value and the standard protein concentration as the best antibody (PTG company). The coating antibody concentration is 2.0 μg/ml.

2. Optimization of blocking solution:

Choose different blocking solutions, containing 1% bovine serum albumin + PBS solution, containing 2% bovine serum albumin + PBS solution, containing 3% bovine serum albumin + PBS solution, containing 4% bovine serum albumin + PBS solution , containing 5% bovine serum albumin + PBS solution, the blocking time was selected for 1 hour at room temperature, 2 hours at room temperature, and overnight at 4°C. Detect according to the operation steps in Example 1, add known PSMD4 protein standards of different concentrations, select the blank group with the smallest OD value, and the linear relationship between the OD value and the standard protein concentration is the closest as the best blocking solution and closed time. The best combination is the blocking solution containing 3% bovine serum albumin + PBS solution and blocking at room temperature for 2 hours.

3. Optimization of detection antibody:

The detection antibody is selected from two kinds of monoclonal antibodies (Abnova Company, #H00005710-AP11-2; PTG Company, #66179-1-Ig) of mouse anti-human PSMD4, and the detection concentration is selected (2.0 μg/ml, 1 μg/ml, 0.5 μg/ml) ml, 0.25 μg/ml, 0.1 μg/ml). Detect according to the operation steps in Example 1, add known PSMD4 protein standards of different concentrations, select the blank group with the smallest OD value, and the linear relationship between the OD value and the standard protein concentration is the closest as the best detection antibody and working concentration. The working concentration of PSMD4 (PTG Company) is 0.5 μg/ml, which is the best compatibility.

4. Optimization of the enzyme-labeled secondary antibody:

When the detection antibody is mouse anti-human PSMD4 monoclonal antibody (Abnova Company, #H00005710-AP11-2; PTG Company, #66179-1-Ig), the enzyme-labeled secondary antibody is selected as goat anti-mouse HRP-labeled IgG (Jackson Company# 715-005-150; PTG Company #SA00001-1); Antibody dilution factor selection 1:1000, 1:2000, 1:3000, 1:5000, 1:10000. Perform detection according to the operation steps in Example 1, add known PSMD4 protein standards of different concentrations, select the blank group with the smallest OD value, and the linear relationship between the OD value and the standard protein concentration is the closest as the best enzyme label 2 Antibody and dilution concentration. The goat anti-mouse HRP-labeled IgG antibody has the best compatibility (PTG Company #SA00001-1), and the best dilution is 1:5000.

5. Optimization of sample diluent:

When the patient's serum sample is detected, the sample diluent is selected from 1 × PBS (pH: 7.4) or 1 × TBS solution containing 0.1% BSA, 0.5% Tween-20, and detects according to the operation steps in Example 1, adding known different Concentration PSMD4 protein standard, select the linear relationship between the OD value and the standard protein concentration is the closest as the best serum sample diluent. The best serum sample diluent is 1×PBS (pH:7.4) solution.

Embodiment 3: ELISA kit detects PSMD4 fusion protein

Dilute the PSMD4 recombinant protein (PTG#ag6691) doubly, starting from 10ng/ml, serially dilute to 7 concentrations, 10ng/ml, 3ng/ml, 1ng/ml, 0.3ng/ml, 0.1ng/ml, 0.03ng /ml, 0.01ng/ml each sample set up 2 duplicate wells, 100 μ l/hole, repeat the detection 5 times according to the step of embodiment 1, the result is similar, as shown in Figure 1, the reaction of PSMD4 recombinant protein and antibody has good Concentration-dependent, and a clear linear relationship.

Embodiment 4: Specificity and sensitivity evaluation of the ELISA kit of PSMD4 protein

1. Specificity test

Detect 50 normal people, 26 lung cancer patient serum samples, 41 gastric cancer patient serum samples and 39 colorectal cancer patient serum samples with the ELISA method established in Example 1 (healthy people are selected from the staff physical examination samples of our hospital; lung cancer, gastric cancer and colorectal cancer samples were obtained from the Laboratory Department of Shanghai Changzheng Hospital, which were pathologically diagnosed as primary lung cancer, gastric cancer and colorectal cancer), PSMD4 recombinant protein was used as a positive control, and the OD value of the obtained standard was drawn with the corresponding concentration The standard curve was used to calculate the serum level of PSMD4 protein in each population.

As shown in Figure 2, the serum level of PSMD4 protein in the lung cancer group (Mean178.99pg/mL, Range0-645.95pg/ml) was significantly higher than that of the normal population (Mean98.84pg/mL, Range0-164.14pg/ml). There is statistical significance (p=0.002). It suggested that PSMD4 has certain specificity in the serological diagnosis of lung cancer.

As shown in Figure 3, the serum level of PSMD4 protein in the gastric cancer group (Mean193.99pg/mL, Range20.82-586.43pg/ml) was significantly higher than that of the normal population (Mean98.84pg/mL, Range0-164.14pg/ml) , the difference was statistically significant (p﹤0.001). It suggested that PSMD4 has certain specificity in the serological diagnosis of gastric cancer.

As shown in Figure 4, the serum level of PSMD4 protein in the colorectal cancer group (Mean258.01pg/mL, Range20.76-1010.38pg/ml) was significantly higher than that of the normal population (Mean98.84pg/mL, Range0-164.14pg/ml) ml), the difference was statistically significant (p﹤0.001). It is suggested that PSMD4 has certain specificity in the serological diagnosis of colorectal cancer. (Mean-mean; Range-extreme value range)

2. Sensitivity test

The PSMD4 recombinant protein (1ug/ul) was serially diluted with 1×PBS, and two replicate holes were set up to obtain the highest dilution factor at which the average OD value of each dilution point of the standard protein was statistically different from the OD value of the blank group.

The OD value of the ^PSMD4 fusion protein diluted 108 times is still statistically different from the OD value of the blank group, indicating that the minimum detectable concentration of PSMD4 protein is 10 pg/ml.

The basic principles, main features and advantages of the present invention have been shown and described above. Those skilled in the industry should understand that the present invention is not limited by the above-mentioned embodiments, and that described in the above-mentioned embodiments and the description only illustrates the principles of the present invention, and the present invention also has various aspects without departing from the spirit and scope of the present invention. Variations and improvements all fall within the scope of the claimed invention. The protection scope of the present invention is defined by the appended claims and their equivalents.

Claims (8)

1. The application of an ELISA detection kit of PSMD4 protein in the preparation of malignant tumor serological diagnosis or screening kit. 2. the application of the ELISA detection kit of a kind of PSMD4 protein according to claim 1 in the preparation of malignant tumor serological diagnosis or screening kit, it is characterized in that, described malignant tumor is lung cancer, gastric cancer, or tumor rectal cancer. 3. the application of the ELISA detection kit of a kind of PSMD4 protein according to claim 1 and 2 in the preparation of malignant tumor serological diagnosis or screening kit, it is characterized in that, the ELISA detection kit of described PSMD4 protein include: (A) ELISA plate coated with PSMD4 antibody, and described coating antibody is the polyclonal antibody of rabbit anti-human PSMD4; (B) the antibody that detects PSMD4 antigen is the monoclonal antibody of mouse anti-human PSMD4; (C) Enzyme-labeled secondary antibody is HRP-labeled goat anti-mouse IgG; (D) a standard protein, which is a recombinant human PSMD4 fusion protein; (E) Sample diluent, coating buffer, blocking solution, ELISA plate washing solution, antibody diluent, chromogenic solution and stop solution. 4. the application of the ELISA detection kit of a kind of PSMD4 protein according to claim 3 in the preparation of malignant tumor serological diagnosis or screening kit, it is characterized in that, described ELISA microtiter plate is the ELISA of 96 holes ELISA plate. 5. the application of the ELISA detection kit of a kind of PSMD4 protein according to claim 3 in the preparation of malignant tumor serological diagnosis or screening kit, is characterized in that, in described ELISA detection kit, Coating buffer, 1×PBS, pH:7.4; The blocking solution is a blocking solution containing 3% bovine serum albumin+PBS solution; ELISA microplate washing solution is 1×PBS solution containing 0.05% Tween-20; Antibody diluent, 1×PBS; Chromogenic solution, which is 3,3′,5,5′-tetramethylbenzidine; The stop solution is 2mol/L sulfuric acid solution. 6. the application of the ELISA detection kit of a kind of PSMD4 protein according to claim 3 in the preparation of malignant tumor serological diagnosis or screening kit, is characterized in that, in described ELISA detection kit, when sample is For serum, the sample diluent is 1×PBS, pH:7.4. 7. the application of the ELISA detection kit of a kind of PSMD4 albumen according to claim 3 in preparing malignant tumor serological diagnosis or screening kit, it is characterized in that, described kit is used for serological diagnosis or screening The checking method includes the following steps: Prepare detection ELISA microtiter plate: Dilute rabbit polyclonal antibody against human PSMD4 to 2.0ug/ml in coating buffer; The sample was diluted 1:1 with the sample diluent, added to the well of the ELISA plate, and incubated at room temperature for 1-2 hours; Dilute the recombinant human PSMD4 fusion protein with sample diluent into different concentration gradients, add the sample to the wells of the ELISA plate, and incubate at room temperature for 1-2 hours; shake off the liquid in each well, add the ELISA plate washing solution, and wash 3-5 times, shake dry; Dilute the antibody for detecting PSMD4 antigen to 0.5 μg/ml, add it to the wells of the ELISA plate, and incubate at room temperature for 1-2 hours; shake off the liquid in each well, add the ELISA plate washing solution, and wash for 3-5 times, shake dry; Add HRP-labeled goat anti-mouse IgG, incubate at room temperature for 1-2 hours; shake off the liquid in each well, add ELISA plate washing solution, wash 6-8 times, and dry; Add chromogenic solution, incubate at room temperature in the dark for 10-20 minutes, add stop solution to terminate the reaction; measure OD value at 450nm on a microplate reader. 8. the application of the ELISA detection kit of a kind of PSMD4 protein according to claim 3 in the preparation of malignant tumor serological diagnosis or screening kit, it is characterized in that, described kit is used for serological diagnosis or screening Check methods include: Before detection, first prepare the detection ELISA plate: Dilute the rabbit anti-human PSMD4 polyclonal antibody to 2.0ug/ml in the coating buffer, add 100μl to each well of the ELISA plate, seal the plate and place it at 4°C Incubate overnight and set aside; Serum samples were diluted 1:1 with sample diluent 1×PBS, pH:7.4, added at a volume of 100 μl/well, and incubated at room temperature for 2 hours; PSMD4 recombinant protein was diluted with sample diluent into different concentration gradients, at 100 μl/well Add the volume of the sample and incubate at room temperature for 2 hours; shake off the liquid in each well, add ELISA plate washing solution, 300 μl per well, wash 3-5 times, and dry; dilute the PSMD4 detection antibody to 0.5 μg/ml, 100 μl per well, incubate at room temperature for 1 hour and 30 minutes; shake off the liquid in each well, add ELISA plate washing solution, 300 μl per well, wash 3-5 times, and dry; add HRP-labeled goat anti-mouse IgG, 100 μl per well, incubate at room temperature for 1 hour; shake off the liquid in each well, add ELISA plate washing solution, 300 μl per well, wash 6-8 times, and dry; add TMB, 100 μl per well, incubate at room temperature for 10 -20 minutes, add 2 mol/L sulfuric acid solution to terminate the reaction, 50 μl per well; measure the OD value on a microplate reader at 450 nm.