A tumor diagnostic marker, its diagnostic kit and application

Abstract

The present invention discloses a kit for detecting CGA protein. The kit comprises an effective amount of CGA protein, two effective amounts of monoclonal antibodies against CGA protein, and a supporting detection reagent. The nucleotide sequence of the CGA protein after codon optimization is as shown in SEQ ID NO.1. The two monoclonal antibodies against CGA protein are monoclonal antibody 1 and monoclonal antibody 2. The sequences of the heavy chain variable region and the light chain variable region encoding monoclonal antibody 1 are as shown in SEQ ID NO.2 and SEQ ID NO.3 respectively. The sequences of the heavy chain variable region and the light chain variable region encoding monoclonal antibody 2 are as shown in SEQ ID NO.4 and SEQ ID NO.5 respectively. The kit of the present invention has the advantages of high sensitivity, wide linear range, fast detection speed, and high detection accuracy.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a tumor diagnostic marker, a diagnostic kit thereof and an application. Background Art

[0002] Chromogranin A (CGA) is an acidic and hydrophilic protein composed of 439 amino acids with a molecular mass of 48,000. It is a member of the neuropeptide family and is widely distributed in the neuroendocrine system. Because of its long half-life, it has become a powerful indicator for evaluating the activity of the entire neuroendocrine system. It is a marker for diagnosing neuroendocrine tumors, and its role in cardiovascular diseases has also attracted increasing attention.

[0003] CGA has mainly two biological functions in cells: (1) selectively regulating the aggregation of target peptide hormones and neurotransmitters on the Golgi reticulum membrane; (2) promoting the permeability of the Golgi network and participating in the metabolism of Ca2+ and catecholamines. CGA has different protease cleavage sites, and its proteases are tissue-specific and are decomposed into different tissue-specific segments in different tissues. It has now been confirmed that there are 3 segments, namely vasostatin-1, -2, pancreastatin and catestatin, which play different roles respectively.

[0004] CGA is widely present in neuroendocrine cells, and elevated CGA levels occur in almost all types of neuroendocrine tumors. After surgical treatment of neuroendocrine tumors, the plasma CGA level decreases. Plasma CGA can not only be used as a tumor marker for the auxiliary diagnosis of neuroendocrine tumors, but also as a dynamic monitoring and prognostic indicator for the development process of tumors, for monitoring the development and metastasis of tumors. An elevated plasma CGA level has a predictive effect on the course of prostate cancer. There is obvious neuroendocrine differentiation in prostate cancer tissues, and CGA produced by differentiated neuroendocrine cells can be used as a serum marker for the diagnosis of prostate cancer. However, CGA is a special product secreted by neuroendocrine cells and is not unique to prostate cancer, and it will also increase in other neuroendocrine tumors. Therefore, the combined detection of CGA and prostate-specific antigen clinically has clinical significance for the diagnosis and prognosis evaluation of prostate cancer. The plasma CGA level can be used for the diagnosis of prostate cancer, especially for the monitoring and diagnosis of prostate cancer with negative prostate-specific antigen or advanced prostate cancer with distant metastasis.

[0005] In summary, CGA is one of the tumor markers for many diseases. Therefore, the detection of CGA is of great significance in the diagnosis of related diseases. At present, ELISA test kits and plate chemiluminescence test kits (described in CN102520195 B) are mainly used, and both the throughput and sensitivity are relatively low. Therefore, it is necessary to develop a new CGA diagnostic kit with high sensitivity. Summary of the Invention

[0006] To overcome the deficiencies of the prior art, one object of the present invention is to provide a CGA protein, its monoclonal antibody, and a preparation method thereof; another object of the present invention is to provide a kit for detecting CGA protein prepared using the monoclonal antibody and CGA protein prepared by the present invention.

[0007] On the one hand, the present invention provides a kit for detecting CGA protein, which includes an effective amount of CGA protein, two effective amounts of monoclonal antibodies against CGA protein, and supporting detection reagents. The two monoclonal antibodies against CGA protein are monoclonal antibody 1 and monoclonal antibody 2. The nucleotide sequences encoding the heavy chain variable region and light chain variable region of monoclonal antibody 1 are shown in SEQ ID NO.2 and SEQ ID NO.3, and the nucleotide sequences encoding the heavy chain variable region and light chain variable region of monoclonal antibody 2 are shown in SEQ ID NO.4 and SEQ ID NO.5.

[0008] Preferably, the effective amount of CGA protein in the present invention is a calibration product prepared using CGA protein, and the concentrations of the calibration product are 3000 ng / ml, 500 ng / ml, 100 ng / ml, 25 ng / ml, 5 ng / ml, 2.5 ng / ml, and 0 ng / ml, respectively.

[0009] Preferably, the nucleotide sequence of the CGA protein after codon optimization in the present invention is shown in SEQ ID NO.1.

[0010] Preferably, monoclonal antibody 1 of the present invention is conjugated with magnetic microparticles, and the dilution factor of the conjugated magnetic microparticles of monoclonal antibody 1 during use is 50 times, and the concentration of the diluted magnetic microparticles is 0.25 mg / ml.

[0011] Preferably, monoclonal antibody 2 of the present invention is labeled with acridinium ester, and the dilution factor of the monoclonal antibody 2 labeled with acridinium ester during use is 500 times.

[0012] Preferably, the supporting detection reagents of the present invention include a washing solution and an acridinium ester luminescent solution.

[0013] Preferably, the washing solution of the present invention is a PBST buffer solution.

[0014] On the other hand, the present invention also provides the use of monoclonal antibody 1 against CGA protein and monoclonal antibody 2 against CGA protein in the preparation of CGA protein detection reagents.

[0015] On the other hand, the present invention also provides the use of the CGA protein in the preparation of CGA protein detection reagents.

[0016] Based on the prepared CGA protein, four specific monoclonal antibodies were screened in the present invention, providing a good tool for the detection of CGA protein. Among them, both monoclonal antibody 1 and monoclonal antibody 2 have good specificity, sensitivity and adaptability for paired detection, and are suitable for the detection of CGA protein.

[0017] Based on the studied CGA protein and monoclonal antibodies, a kit for the detection of CGA protein was developed in the present invention. The kit of the present invention has the advantages of high sensitivity, wide linear range, fast detection speed and high detection accuracy. Therefore, this kit has very important reference value for the clinical auxiliary diagnosis of pancreatic cancer and pheochromocytoma. Brief Description of the Drawings

[0018] Figure 1 Analysis results of the signal peptide of CGA protein.

[0019] Figure 2 Analysis results of SDS-PAGE of CGA protein. Among them, 1 is the purified CGA protein.

[0020] Figure 3 SDS-PAGE detection diagrams of four monoclonal antibodies. Among them, 1 is monoclonal antibody 2, 2 is monoclonal antibody 2, 3 is monoclonal antibody 3, and 4 is monoclonal antibody 4.

[0021] Figure 4 Standard curve of this kit. Detailed Description of the Invention

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which this invention belongs. The terms used in the description of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0023] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0024] Example 1: Preparation of CGA Protein

[0025] The protein of GenBank: AAA52017.1 was selected for analysis. First, its signal peptide was analyzed. The results showed ( Figure 1) The probability of having a signal peptide is 99.9%. Signal peptide type: SP (Sec / SPI), cleavage site: 18 - 19, probability: 96.3%. Signal peptide amino acid sequence: MRSAAVLALLLCAGQVTA. Therefore, the signal peptide sequence (the first 18 amino acids at the N-terminus) was selected to be removed, and the remaining 439 - amino acid sequence was used for prokaryotic expression.

[0026] The His tag was added to the C-terminus of the above 439 amino acids, and a total of 445 amino acids were codon-optimized. The nucleotide sequence of the codon-optimized CGA protein (as shown in SEQ ID NO.1) was subcloned into the pET-30a plasmid to construct the prokaryotic expression vector pET-30a-CGA. The recombinant clone pET-30a-CGA was transformed into Escherichia coli E.coli BL21(DE3). A single colony was picked and cultured in LB medium containing kanamycin (final concentration 50 μg / ml). When it reached the logarithmic growth phase (OD600 was 0.6 - 1.0), IPTG was added and the temperature was transferred to 16 °C for induction for 16 h to express the target protein. Nickel column purification method was used for purification, and the target protein was eluted with 400 mM imidazole. The eluate was dialyzed and exchanged into PBS. 20 μl of CGA protein was taken and examined using SDS-PAGE protein gel. There was a clear band at about 48 kDa, and the purity was greater than 90% ( Figure 2 ). The protein content was detected using the BCA kit, and the protein concentration was 1.23 mg / ml. The prepared CGA protein was aliquoted (0.5 ml / tube) and stored at -15 °C or below for later use.

[0027] Example 2: Preparation of monoclonal antibody against CGA protein

[0028] The CGA protein prepared in Example 1 was used as the antigen, and monoclonal antibodies were prepared using the conventional hybridoma cell technology. The specific method is as follows:

[0029] 2.1 Animal immunization

[0030] The CGA protein prepared in Example 1 was used to immunize 6 - 8-week-old female BALB / c mice. The immunization procedure was as follows: The CGA protein was emulsified with Freund's complete adjuvant to prepare the vaccine (200 μg / ml), and injected subcutaneously at multiple points, 0.5 ml / mouse; After two weeks, immunization was carried out with the same dose and method, but the adjuvant was changed to Freund's incomplete adjuvant; After one week, the third immunization was carried out with the same immunization dose, and the immunization method was intraperitoneal injection without adjuvant; Blood was collected 7 days later to detect the ELISA antibody titer. The spleen cells of mice with a higher ELISA antibody titer (ELISA titer ≥ 10 5 ) were selected for cell fusion.

[0031] ELISA antibody titer detection: Coat an ELISA plate with the CGA protein prepared in Example 1 at a concentration of 1 μg / ml overnight at 2 - 8°C; after washing and blocking, serially dilute mouse serum 10-fold (such as 10-fold, 100-fold, 1000-fold, etc.) as samples and add them to the ELISA plate, 0.1 ml per well, incubate at 37°C for 1 h; after washing, add a 5000-fold diluted HRP-labeled goat anti-mouse IgG secondary antibody, incubate at 37°C for 30 min; add the chromogenic solution, 0.1 ml per well, incubate at 37°C for 10 min, add 2 M sulfuric acid to terminate, 0.1 ml per well; detect the OD450nm value; use a P / N value > 2.1 as the positive index.

[0032] 2.2 Cell fusion

[0033] 2.2.1 Cell fusion: Mix 1×10 8 prepared spleen cells with 1×10 7 prepared myeloma cells SP2 / 0 in a 50 ml fusion tube, supplement incomplete medium to 30 ml, and mix well. Centrifuge at 1000 r / min for 10 minutes and aspirate as much supernatant as possible. Add 1 ml of preheated 50% PEG in 30 s using a 1 ml pipette while gently stirring. Let the pipette stand for 1 min. Add preheated incomplete culture medium to terminate the PEG action. Centrifuge at 800 rpm for 5 minutes and discard the supernatant. Add 5 ml of complete medium, resuspend the cells and mix well, then supplement complete medium to 50 ml. Aliquot into a 96-well cell culture plate, 0.1 ml per well, and culture in an incubator at 37°C and 5% CO2. After 6 h, supplement selection medium, 50 μl per well. Half-change the medium with selection medium after 3 days. Observe the growth of hybridoma cells. When they grow to more than 1 / 10 of the well bottom area, aspirate the supernatant for antibody detection. The method is shown in 2.1.

[0034] 2.2.2 Selection of hybridoma cells: Detect according to the method in 2.1, and select hybridoma cells with a higher ELISA titer (≥10 6 ) for subcloning, and screen monoclonal hybridoma cells by the limited dilution method.

[0035] 2.2.3 Subcloning of hybridoma cells (limited dilution method): Dilute the hybridoma cell suspension to be cloned with HT medium containing 20% serum to three different dilutions of 5, 10, and 20 cells per milliliter. Aliquot each type of hybridoma cells into a 96-well plate, 0.1 ml per well. Culture at 37°C and 5% CO2 for 6 days. When visible clones appear, the antibodies can be detected; observe under an inverted microscope, mark the wells with only single clone growth, and take the supernatant for antibody detection. Expand the cells from the wells with positive antibody detection and cryopreserve them. After detection, a total of 4 better hybridoma cells were screened, named hybridoma cells 1 - 4 respectively, and they were cryopreserved in liquid nitrogen for future use.

[0036] 2.3 Production and purification of monoclonal antibodies

[0037] 2.3.1 Production of monoclonal antibodies: The 4 hybridoma cell strains obtained in 2.2 were expanded in culture, counted after suspension, and reserved for use. BALB / c mice aged 6 - 8 weeks were intraperitoneally inoculated with liquid paraffin at 0.5 ml per mouse. 7 - 10 days later, the hybridoma cells diluted with PBS were intraperitoneally inoculated at 1 - 4 per mouse, 5×10 5 / 0.2 ml per mouse. After an interval of 5 - 7 days, the ascites production in mice was observed daily. If the abdomen was significantly enlarged and the skin felt tense when touched by hand, the ascites could be collected. Usually, 3 ml of ascites could be collected from each mouse; the ascites was centrifuged (2000 rpm for 5 minutes) to remove cell components and other precipitates, the supernatant was collected, the antibody titer was measured, aliquoted, and stored at -70°C for future use.

[0038] 2.3.2 Purification of monoclonal antibodies: The ascites was centrifuged at 12000 rpm for 15 min at 4°C to remove impurities. Take 1 part of ascites and add 2 parts of 0.06 mol / L acetate buffer at pH 5.0. Octanoic acid was added dropwise with stirring at room temperature according to the ratio of 33 μl of octanoic acid per ml of diluted ascites, mixed at room temperature for 30 min, left to stand at 4°C for 2 hours, then taken out and centrifuged at 12000 rpm for 30 minutes, and the precipitate was discarded; the supernatant was filtered through a nylon sieve and dialyzed against 50 volumes of PBS at 4°C for 6 h. An equal volume of saturated ammonium sulfate solution was added to the dialyzed supernatant. Left to stand at 4°C for more than 1 h, centrifuged at 10000g for 30 minutes, and the supernatant was discarded. The precipitate was dissolved in an appropriate amount of PBS and dialyzed against 50 - 100 volumes of PBS overnight. Take a small amount of the dialyzed sample, dilute it appropriately, aliquot it, and store it at -70°C or below for future use.

[0039] 2.3.3 According to the above method, 4 monoclonal antibodies were obtained in total (named monoclonal antibodies 1 - 4, corresponding to hybridoma cell strains 1 - 4).

[0040] Example 3: Detection of monoclonal antibodies against CGA protein

[0041] 3.1 Purity test: Take 20 μl of the 4 monoclonal antibodies prepared in Example 2, add 5 μl of 5*loading buffer, then heat in a boiling water bath for 10 min, and store at -20°C for future use; prepare a 10% SDS-PAGE gel, add the treated sample into the sample well, and detect the SDS-PAGE purity of the monoclonal antibodies. The results showed ( Figure 3 ) that the SDS-PAGE purities of the 4 monoclonal antibodies were all greater than 90%, which were 95%, 95%, 98%, and 96% respectively.

[0042] 3.2 Concentration test: Take 20 μl of the 4 monoclonal antibodies prepared in Example 2, detect their concentrations according to the instructions of the BCA kit, and calculate the actual concentration of the monoclonal antibodies based on the results of purity detection (total protein concentration detected by BCA * purity of 3.1). After detection, the concentrations of monoclonal antibodies 1 - 4 are 1.78 mg / ml, 1.65 mg / ml, 1.81 mg / ml, and 1.79 mg / ml respectively.

[0043] 3.3 Pairing test of monoclonal antibodies

[0044] 3.3.1 HRP labeling: Use the HRP conjugation kit (abcam, ab102890) to label the 4 monoclonal antibodies with HRP respectively, and detect the labeled monoclonal antibodies using the direct ELISA method.

[0045] 3.3.2 Detection of HRP labeling effect: Coat the ELISA plate with the CGA protein prepared in Example 1, dilute it to 1 μg / ml with the coating solution, add 100 μl / well to the ELISA plate, and coat overnight at 2 - 8 °C; after washing, add 5% skim milk (prepared with PBST) for blocking, 200 μl / well, incubate at 37 °C for 2 h; after washing, gradient dilute the HRP-labeled monoclonal antibodies with PBST, and the dilution factors are 5000, 10000, 20000, 30000, etc., and add them to the ELISA plate, 100 μl / well, incubate at 37 °C for 30 min; after washing, add the TMB substrate solution, 100 μl / well, incubate at 37 °C for 10 min; detect the OD450nm value, and use OD450nm value ≥ 1.1 as the judgment standard. After detection, the dilution factors of the 4 monoclonal antibodies are 20000 times, 30000 times, 5000 times, and 10000 times respectively.

[0046] 3.3.3 Double sandwich ELISA assay: Four monoclonal antibodies were used to coat the ELISA plates, adding 100 μl per well to the ELISA plates and coating overnight at 2-8 °C; after washing, 5% skim milk (prepared with PBST) was added for blocking, 200 μl per well, and incubated at 37 °C for 2 h; after washing, the CGA protein of Example 1 was used as the sample (diluted to 10 ng / ml with PBST), 100 μl per well, incubated at 37 °C for 1 h, and then the monoclonal antibodies labeled in 3.3.1 were added crosswise. Before adding, they were diluted according to the dilution multiples in 3.3.2 with PBST respectively, 100 μl per well, and incubated at 37 °C for 30 min; after washing, TMB substrate was added, 100 μl per well, and incubated at 37 °C for 10 min; the OD450nm value was measured, and the pairing effect was determined by comparing the OD450nm values (the higher the OD450nm value, the better the pairing effect). The results are shown in Table 1. When monoclonal antibody 1 was used for coating and monoclonal antibody 2 was used for detection, the OD value was the highest; the final paired antibodies were determined to be monoclonal antibody 1 and monoclonal antibody 2, where monoclonal antibody 1 was used as the coating antibody and monoclonal antibody 2 was used as the detection antibody.

[0047] Table 1 Detection results of monoclonal antibody pairing (OD450nm value)

[0048]

[0049] 3.4 Sequence analysis of monoclonal antibodies

[0050] Monoclonal antibodies are highly homogeneous immunoglobulins produced by cloning a single B lymphocyte and specifically recognizing and binding only one antigen. Like ordinary antibodies, they are composed of two shorter and smaller molecular weight light chains (L chains) and two longer and larger molecular weight heavy chains (H chains). Both the L chain and the H chain contain a variable region (V region) with a relatively large amino acid sequence variation and a constant region (C region) with a relatively stable amino acid sequence. There are some regions in the V region where the amino acid sequence is very prone to variation, called hypervariable regions (HVRs) or complementarity-determining regions (CDRs). CDR is the core sequence of the entire antibody and the basis for the antibody to exert its biological function. It determines the specificity of the antibody to bind the corresponding antigen. Therefore, sequence analysis of it is of great significance.

[0051] We used a sequencing method based on PCR amplification (indirectly achieved by determining the DNA sequences encoding the CDR regions of monoclonal antibodies. First, total mRNA of hybridoma cells was extracted and reverse-transcribed to obtain cDNA. Then, degenerate primers for the CDR sequences were designed for PCR amplification to obtain the DNA sequences encoding the CDR sequences. Finally, DNA sequence analysis was performed. For specific details, please refer to the literature: "Chen Sumin, Yang Ping. Cloning and sequencing of the heavy chain variable region gene of a monoclonal antibody against human liver cancer. Chinese Journal of Immunology, 1995, 011(001)-10~12") to analyze and determine the heavy chain variable region and light chain variable region of the prepared monoclonal antibody 1 and monoclonal antibody 2.

[0052] The results showed that after detection and analysis, the sequences of the heavy chain variable region and light chain variable region encoding monoclonal antibody 1 were shown in SEQ ID NO.2 and SEQ ID NO.3 respectively; the sequences of the heavy chain variable region and light chain variable region encoding monoclonal antibody 2 were shown in SEQ ID NO.4 and SEQ ID NO.5 respectively.

[0053] Example 4: Establishment of a CGA protein detection method and preparation of a kit

[0054] 4.1 Preparation of calibrators

[0055] The calibrators were prepared using the CGA protein prepared in Example 1. The CGA protein was diluted to 7 gradients with PBST buffer containing 1% BSA, which were 3000 ng / ml, 500 ng / ml, 100 ng / ml, 25 ng / ml, 5 ng / ml, 2.5 ng / ml, and 0 ng / ml respectively.

[0056] 4.2 Magnetic particle-coupled monoclonal antibody 1 (reagent R1)

[0057] Take 5 mg of carboxyl magnetic particles (Shanghai Huizhi) in a 0.5 ml centrifuge tube, add 1 ml of 0.1 M MES buffer, vortex and mix well, place it on a magnetic stand, let it stand for 5 min to separate the magnetic particles from the liquid, discard the supernatant, wash 3 times, then add 0.5 ml of MES buffer with a pH of 6.0, and vortex. Add 0.1 mg of monoclonal antibody 1, vortex, rotate the reaction tube, and incubate at room temperature for 20 min. Add 10 μl of coupling reagent EDC with a concentration of 10 mg / ml, vortex, rotate the reaction tube, and incubate at room temperature for 2 h. After centrifugation, discard the supernatant, add 1 ml of PBST buffer, and wash 3 times. Add 1 ml of PBST buffer containing 1% BSA for blocking, and block at room temperature for 2 h. After centrifugation, discard the supernatant, add 1 ml of PBST buffer, and wash 3 times. Add 0.5 ml of PBST buffer containing 1% BSA to resuspend the magnetic particles, and store at 2 - 8 °C for later use. When in use, dilute the monoclonal antibody 1 conjugated with magnetic particles 50 times with PBST buffer containing 1% BSA + 0.1% NaN3. At this time, the concentration of magnetic particles is 0.25 mg / ml, and store at 2 - 8 °C for later use.

[0058] 4.3 Acridinium ester-labeled monoclonal antibody 2 (reagent R2)

[0059] Weigh 1.6 mg of acridinium ester NSP-DMAE-NHS (purchased from Macklin), dissolve it in anhydrous DMSO to prepare a stock solution of 6.5 mM. Under a light-shielded environment, mix 5 μl of monoclonal antibody 2 (containing 5 μg) with 195 μl of the acridinium ester stock solution, react at room temperature for 1 h, add 100 μl of 10 g / L lysine, and continue to react for 10 min to terminate the labeling reaction. Inject the labeled solution into a Sephadex G-25 desalting column and wash it with PBS. Detect the protein with a chromatograph and detect the protein concentration at 280 nm. Detect the labeled protein. Take 10 μl of the acridine-labeled protein and place it in a black 96-well plate. Inject 50 μl - 150 μl of acridinium ester luminescent solution ((0.01 - 0.1 M) NaOH + 0.05% H2O2, freshly prepared and used immediately) into each well, and detect immediately. If the luminescence is too strong, the labeled protein can be diluted several times to make it within the luminescence detection limit of the instrument; when an obvious protein peak appears on the chromatograph, collect the eluate. Add 1% BSA + 0.1% NaN3 to the eluate and store it frozen in the dark. A total of about 0.5 ml of eluate is obtained. When in use, dilute the monoclonal antibody 2 labeled with acridinium ester 500 times with PBST buffer containing 1% BSA + 0.1% NaN3, and store at 2 - 8 °C for later use.

[0060] 4.4 Detection steps of the kit

[0061] 1) 2 μl of sample + 50 μl of reagent R1 + 50 μl of reagent R2 + 248 μl of washing solution;

[0062] 2) Incubate at 37°C for 10 min (37°C, 1000 rpm);

[0063] 3) Wash 3 times with 500 μl of washing solution each time;

[0064] 4) Add 200 μl of acridinium ester luminescent solution, and immediately detect the RLU value with a chemiluminescence analyzer.

[0065] The washing solution is PBST buffer, and the sample can be serum, whole blood, tissue fluid, and cell culture medium.

[0066] Example 5: Performance Evaluation of the Kit

[0067] 5.1 Linear Range of the Kit: Use the prepared kit to detect the calibrator prepared in 4.1 according to the test steps in 4.4. The results show ( Figure 4 and Table 2) that the kit has good linearity and uniformity in the concentration range of 2.5 ng / ml to 3000 ng / ml, indicating that the kit has a wide linear range.

[0068] Table 2 Calibration Results

[0069]

[0070]

[0071] 5.2 Sensitivity of the Kit: Perform 20 repeated tests on S0, and take the average value of the S0 measurement plus 3 times the standard deviation as the detection limit of this kit. After detection and calculation, the detection limit of this kit is 0.5 ng / ml, indicating that the kit has high sensitivity.

[0072] 5.3 Comparison of Sample Detection Results: We used this kit to measure serum specimens of a batch of patients with pancreatic cancer and pheochromocytoma diagnosed by the hospital and normal human serum specimens. The results show (Table 3) that the positive detection rates of this kit for pancreatic cancer and pheochromocytoma can reach 91.1% and 93.0% respectively, while the false positive rate for normal human detection is 0. Therefore, this kit has very important reference value for the clinical auxiliary diagnosis of pancreatic cancer and pheochromocytoma.

[0073] Table 3 Comparison of Sample Detection Results

[0074] Group Number of Specimens Number of Positive Cases Detected Positive Detection Rate Pancreatic Cancer Group 45 41 91.1％ Pheochromocytoma Group 43 40 93.0％ Normal Group 95 0 0

[0075] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A kit for detecting CGA protein, characterized in that, The described kit includes an effective amount of CGA protein, two effective amounts of monoclonal antibodies against CGA protein, and supporting detection reagents. The two monoclonal antibodies against CGA protein are monoclonal antibody 1 and monoclonal antibody 2. Among them, the sequences of the heavy chain variable region and the light chain variable region encoding monoclonal antibody 1 are shown as SEQ ID NO.2 and SEQ ID NO.3, and the sequences of the heavy chain variable region and the light chain variable region encoding monoclonal antibody 2 are shown as SEQ ID NO.4 and SEQ ID NO.

5.

2. The kit according to claim 1, wherein, The described effective amount of CGA protein is a calibrator prepared using CGA protein, and the concentrations of the calibrator are 3000 ng / ml, 500 ng / ml, 100 ng / ml, 25 ng / ml, 5 ng / ml, 2.5 ng / ml, and 0 ng / ml respectively.

3. The kit according to claim 1, characterized in that, The nucleotide sequence of the CGA protein after codon optimization is shown as SEQ ID NO.

1.

4. The kit according to claim 1, characterized in that, The monoclonal antibody 1 is conjugated with magnetic microparticles. The dilution factor of the conjugated magnetic microparticles of monoclonal antibody 1 during use is 50 times, and the concentration of the diluted magnetic microparticles is 0.25 mg / ml.

5. The kit according to claim 1, wherein The monoclonal antibody 2 is labeled with acridinium ester. The dilution factor of the monoclonal antibody 2 labeled with acridinium ester during use is 500 times.

6. The kit according to claim 1, wherein The described supporting detection reagents include a washing solution and an acridinium ester luminescent solution.

7. The kit according to claim 6, characterized in that, The described washing solution is a PBST buffer solution.

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