CN106053812B - A kind of a variety of autoantibody joint-detection ELISA kits for being used for liver cancer early screening and diagnosis - Google Patents

Abstract

The invention discloses a kind of for liver cancer early screening and a variety of autoantibody joint-detection ELISA kits of diagnosis, the kit includes solid phase carrier and the tumor associated antigen being coated on solid phase carrier, and the tumor associated antigen is CyclinB1, p90, c myc, p53, NPM1, HCC1,14 3 3zeta and p62.Further, the kit also includes Sample dilution, secondary antibody, secondary antibody dilution, positive control serum, negative control sera, nitrite ion, terminate liquid, cleaning solution.The present invention ELISA kit be used for liver cancer early diagnosis there is high sensitivity, stability is good, it is easy to operate the advantages that.In addition, 11 blood samples can be detected simultaneously using the ELISA kit of the present invention, cost is reduced, improves efficiency, liver cancer patient just can be diagnosed before clinical symptoms appearance and treatment can be received.

Description

A combined detection of multiple autoantibodies for early screening and diagnosis of liver cancer ELISA kit

technical field

The invention relates to the technical field of biomedicine, in particular to an ELISA kit for combined detection of multiple autoantibodies used for early screening and diagnosis of liver cancer.

Background technique

Primary liver cancer is one of the most common malignant tumors in the world, and more than half of the new cases of liver cancer each year are in China, which seriously threatens people's lives and health. The onset of liver cancer is very insidious, and the prognosis is poor. Surgical resection and liver transplantation are considered to be the means of radical treatment for liver cancer, but most patients have already entered the advanced stage of liver cancer when they are first diagnosed, thus losing the opportunity for treatment. The main reason is that patients with early liver cancer usually have no obvious symptoms, or the size is small, and it is difficult to detect by imaging examinations. As the main serum marker, alpha-fetoprotein (AFP) has the disadvantage of low sensitivity. As for the "Updated Edition of Guidelines for Clinical Hepatocellular Carcinoma" released by the American Association for the Study of Liver Diseases (AASLD) in 2010, AFP is no longer recommended as a screening indicator for liver cancer. Researchers have conducted research on the molecular level of liver cancer, providing an important theoretical basis for the molecular diagnosis of liver cancer and the formulation of individualized treatment plans after surgery.

At present, the latest early detection methods for malignant tumors are multi-tumor marker protein chip detection system (C12 chip), tumor gene mutation detection kit, six tumor marker detection kits, human malignant tumor-specific growth factor (TSGF) enzyme-linked Immunoassay (ELISA) Kit. However, the multi-tumor marker protein chip detection system (C12 chip) has high detection false positives, and there is no specific liver cancer detection marker, and the detection efficiency is low. Tumor gene mutation detection reagents rely on complicated operations such as polymerase chain reaction (PCR), and are prone to false positives, which is an unreliable analysis method. Six tumor marker assay kits and human malignant tumor-specific growth factor (TSGF) enzyme-linked immunoassay (ELISA) kits can only detect tumors in patients, but cannot determine the specific type of tumors.

In recent years, in the research field of human oncology, many studies have found that the serum of cancer patients contains a unique set of cellular proteins that induce autoantibody responses, called tumor-associated antigens (tumor-associated antigens, TAAs), which induce the production of The antibody is called anti-TAA-antibody (autoantibody). The introduction of this concept guides a new direction for the research on the early diagnosis of liver cancer. In 1998, Dr. Zhang at the Scripps Research Institute in the United States discovered a new tumor-associated antigen p62. This TAA, called p62, can stimulate the body to generate an immune response and produce autoantibodies. Of great significance is the timing of anti-p62 autoantibodies in the circulation of liver cancer patients: using the sequence from chronic hepatitis development to cirrhosis to liver cancer In the serum, it was found that the anti-p62 autoantibody did not appear in the chronic hepatitis stage of the patient, nor did it appear in the early and middle stages of liver cirrhosis, but the content of the anti-p62 autoantibody increased sharply in the period before the occurrence of advanced liver cancer in the liver cirrhosis stage, but it did not appear at this time The clinical symptoms and signs of liver cancer, liver cancer can not be found clinically, and this phenomenon has been repeated in the serial serum of many liver cancer patients. This shows that anti-p62 autoantibody is a potential serological marker for early diagnosis of liver cancer. Subsequently, the anti-p62 autoantibodies were detected in the serum of patients with liver cancer, liver cirrhosis, chronic hepatitis and normal people in small samples. The results showed that the specificity of this autoantibody was very high, but the sensitivity could not reach 20%. Obviously, if the anti-p62 autoantibody is used alone as a marker for the diagnosis of liver cancer, the diagnostic value is low. In the next work, the researchers tried to find more sensitive and specific anti-TAA autoantibodies, but the results were not satisfactory. In a 2003 study using 7 anti-TAA autoantibodies to detect liver cancer, the sensitivity of a single index did not exceed 20%, but by combining these 7 TAA autoantibodies in parallel, the detection sensitivity could reach 56.9%, while the diagnostic specificity is still high. It shows that the combination of multiple TAA autoantibodies has a high diagnostic value and is a strategy with potential application.

Follow-up studies for more than ten years have been trying to find more sensitive and specific anti-TAA autoantibodies for the diagnosis of liver cancer, and optimize the combination for the diagnosis of liver cancer. There are two commonly used methods to find valuable TAA autoantibodies: one is serological analysis of recombinant cDNA expression libraries (SEREX); the other is proteomics technology.

In this study, eight TAA combinations were screened by proteomics technology. These eight TAAs are CyclinB1, p90, c-myc, p53, NPM1, HCC1, 14-3-3zeta and p62. development is closely related. CyclinB1 is a cell cycle protein and a positive regulator of the cell cycle. It plays an important role in the process of controlling cells to enter the G2/M phase checkpoint. Its overexpression can lead to uncontrolled cell division and proliferation, thereby inducing tumorigenesis. In cancer The abnormal expression of CyclinB1 can also be recognized by the body's immune system in the early stage of cell death, and the body undergoes an immune response to produce a large number of B1 cell cyclin antibodies. p90, also known as CIP2A, is a tumor suppressor of protein phosphatase 2A, which interferes with the tumor suppressor protein phosphatase 2A, thereby promoting cell proliferation. c-myc transcription factor protein, is a transcription factor that promotes cell division and proliferation. Studies have shown that the unlimited expression of c-myc gene in tumors can make some cells never die. p53, a tumor suppressor protein, controls cell cycle initiation. The mutation of p53 gene occurs in most tumor cells, and the mutant p53 protein will be encoded after mutation. The mutant protein can interfere with the function of p53, and its overexpression and accumulation will lead to the loss of function of the normal p53 protein, which will cause the production of p53 antibody in the serum and induce the occurrence and development of tumors. NPM1 is a nucleoside phosphoprotein involved in the regulation of the ARP/p53 pathway. It is often overexpressed, mutated, rearranged and deleted in human cell carcinomas, so it is often considered as a tumor marker. HCC1/CAPERα, the mRNA inhibitory tumor protease inhibitor, is a steroid hormone receptor-mediated transcription and alternative splicing factor that plays an important role in controlling the oncogenic activity of Rel/NF-kB. 14-3-3zeta, a member of the 14-3-3 family, mediates signal transduction by binding to phosphoserine-containing proteins and interacts with target proteins during cancer initiation and progression to exert oncogenic potential effect. The mechanism is not well understood, however, in recent years, the importance of 14-3-3zeta protein in cancer has become increasingly apparent. Many studies on the relationship between hepatocellular carcinoma and 14-3-3zeta have shown that 14-3-3zeta plays an important role in the process of hepatocellular carcinoma. 14-3-3zeta can be used as a potential tumor-associated antigen, and the discovery of 14-3-3zeta antibody may help the early diagnosis of hepatocellular carcinoma. p62/IMP2, insulin-like growth factor 2 mRNA-binding protein 2, p62 gene was initially obtained by immune screening from autoantibodies in serum of patients with hepatocellular carcinoma in cDNA expression library. p62 is a cytoplasmic mRNA-binding protein that binds to messenger RNA encoding insulin-like growth factor II (IGF-II), involved in insulin signaling and insulin secretion, is a known overexpression in hepatocellular carcinoma, and in transgenic Oncogenic growth factor-binding proteins in animals. Therefore, this study will combine the screened eight TAA autoantibodies to develop an ELISA kit for early diagnosis and screening of liver cancer with high sensitivity, high specificity.

Contents of the invention

The object of the present invention is to provide a multiple autoantibody combined detection ELISA kit for early screening and diagnosis of liver cancer. The ELISA kit has high sensitivity and specificity, and is easy to use, cheap, and low in detection cost. It can be popularized and used in ordinary laboratories.

The technical scheme adopted in the present invention is:

An ELISA kit for combined detection of multiple autoantibodies for early screening and diagnosis of liver cancer, the kit includes a solid phase carrier and a tumor-associated antigen coated on the solid phase carrier, the tumor-associated antigen is CyclinB1, p90, c-myc, p53, NPM1, HCC1, 14-3-3zeta and p62.

According to the above ELISA kit for joint detection of multiple autoantibodies, the kit also includes sample diluent, secondary antibody, secondary antibody diluent, positive control serum, negative control serum, chromogenic solution, stop solution, washing solution . The sample diluent is PBST (phosphate Tween) buffer containing 1% (W/V) BSA; the second antibody diluent is PBST (phosphate Tween) containing 1% (W/V) BSA ) buffer solution; the chromogenic solution is composed of chromogenic solution A and chromogenic solution B, and the chromogenic solution A is 0.02% (W/V) TMB (3,3',5,5'-tetramethyl benzidine), the chromogenic solution B is 0.006% (W/V) urea peroxide; the stop solution is 10% vitriol oil; the washing solution is pH7.4 containing 0.05% Tween-20 0.01 M PBST (Phosphate Tween) buffer.

According to the ELISA kit for combined detection of multiple autoantibodies above, the second antibody has a detectable label; the detectable label is preferably horseradish peroxidase; further, the second antibody Preferable is the RecA protein (recombinant protein A).

According to the ELISA kit for the combined detection of multiple autoantibodies above, the positive control serum is preferably a p53 positive control serum, and the negative control serum is preferably a p53 negative control serum; wherein, the p53 positive control serum is obtained using indirect ELISA and Western blot method detects p53 antibody positive sera of liver cancer patients, and the p53 negative control serum is the serum of normal people whose expression level of p53 antibody is detected by indirect ELISA method and is the average serum antibody content of normal population. Because studies have clearly shown that p53 antigen is involved in the occurrence and development of liver cancer, a large number of current literatures have also reported that anti-p53 antibodies have a high positive rate in the serum of patients with liver cancer. Therefore, in order to improve work efficiency, the present invention selects p53 antibody-positive serum as a positive control. Because the positive control serum and negative control serum are carefully screened serum, the strength of other antigen-antibody reactions in the same ELISA kit can be used as a reference to achieve the purpose of quality control.

According to the above-mentioned multiple autoantibody combined detection ELISA kit, the solid phase carrier is preferably a 96-well microtiter plate; the 96-well microtiter plate is coated with CyclinB1, p90, Eight tumor-associated antigens, c-myc, p53, NPM1, HCC1, 14-3-3zeta, and p62, were coated with one antigen in each row, and each antigen was coated in 11 spotting wells. Serum samples of the same test subject are diluted and added to a column of the 96-well microtiter plate of the ELISA kit of the present invention, so as to simultaneously detect the expression levels of eight TAA antibodies in the serum samples. Further, the 96-well ELISA plate is also provided with blank control wells, positive control wells and negative control wells, the blank control wells are coated with an antigen-free coating solution, and the positive control wells and negative control wells are All wells were coated with p53 antigen.

According to the ELISA kit for combined detection of multiple autoantibodies, the detection object of the ELISA kit for combined detection of multiple autoantibodies is human serum. The same serum sample is diluted and added to a column of a 96-well microtiter plate to simultaneously detect the expression levels of eight TAA antibodies in the serum sample.

The positive beneficial effect that the present invention obtains:

(1) The present invention uses CyclinB1, p90, c-myc, p53, NPM1, HCC1, 14-3-3zeta and p62 as a combination for the early screening and diagnosis of liver cancer for the first time, with high accuracy.

(2) The present invention has prepared multiple autoantibody combined detection ELISA kits according to the indirect enzyme-linked immunosorbent method. Clinical detection efficiency; in addition, the detection cost is low, and it can be popularized and used in ordinary laboratories.

(3) The ELISA kit for joint detection of multiple autoantibodies of the present invention uses RecA protein as the second antibody, which can specifically bind to the Fc segment in the antibody structure. Compared with traditional horseradish peroxidase-labeled IgG, Using RecA protein as the second antibody has the characteristics of strong specificity and low background value, which can improve the accuracy of antibody detection and reduce the false positive rate.

(4) adopting multiple autoantibody joint detection ELISA kits of the present invention can realize the expression level of 8 kinds of TAA antibodies in 11 serum samples to be tested simultaneously, can be used for large-scale sample detection, has greatly improved detection and Diagnostic efficiency.

Description of drawings

Figure 1 Schematic diagram of indirect enzyme-linked immunoassay.

Figure 2 The antigen coating layout of the 96-well microtiter plate in the multiple autoantibody joint detection ELISA kit of the present invention (wherein, the antigen name represents that the antigen is coated, and the final detection is the corresponding antibody in the serum sample to be tested Expression level; "+" means the positive control well, which means positive control serum was added to the serum sample incubation reaction; "-" means negative control well, means the negative control serum was added to the serum sample incubation reaction; "blank" means blank In the control well, that is, the sample diluent without serum was added when the serum sample was incubated for the reaction, and the other operations were the same, and the blank control reflected the background value during the experiment.

Figure 3 Distribution diagram of 8 tumor-associated antigen autoantibodies in the serum of the control group; it can be seen from the figure that the average expression level of these 8 tumor-associated antigen autoantibodies in the control group is relatively low, and the average OD values are far less than 0.25, while the OD values There are also relatively few serums greater than 0.5.

Figure 4 Distribution of 8 tumor-associated antigen autoantibodies in the serum of the liver cancer group; it can be seen from the figure that the average expression level of these 8 tumor-associated antigen autoantibodies in the liver cancer group is relatively high, and the average OD value fluctuates around 0.25, and the OD value There are also more serums greater than 0.5, and the OD value of some serums even exceeds 1.0.

Figure 5 The results of the positive rates of autoantibodies to 8 tumor-associated antigens in the liver cancer group and the control group. (The results show that the positive rate of 8 kinds of tumor-associated antigen autoantibodies in the liver cancer group is 20.2%-41.4%, and the positive rate of these 8 kinds of tumor-associated antigen autoantibodies in the control group is not more than 10%. After statistical testing, this The positive rates of 8 tumor-associated antigen autoantibodies in liver cancer were higher than those in the control group.)

Figure 6 ROC curve (receiver operating characteristic curve) for detection of liver cancer by autoantibodies to 8 tumor-associated antigens; the ordinate is the true positive rate (sensitivity), and the abscissa is the false positive rate.

detailed description

The present invention will be further described below in conjunction with specific examples, but the protection scope of the present invention will not be limited.

Example 1: Preparation of eight tumor-associated antigens

By using a prokaryotic expression system, 8 tumor-associated antigens (CyclinB1, p90, c-myc, p53, NPM1, HCC1, 14-3-3zeta, and p62) were prokaryotically expressed and purified in order to prepare for the next experiment. The specific antigen preparation process is as follows:

1) The recombinant prokaryotic expression plasmids of eight tumor-related proteins (CyclinB1, p90, c-myc, p53, NPM1, HCC1, 14-3-3zeta and p62) were constructed using gene cloning technology.

2) Expression of the target protein: the constructed recombinant prokaryotic expression plasmids were respectively transformed into Escherichia coli Bl21(DE3), and IPTG (isopropylthiogalactoside) was used to induce the expression of the target protein.

3) Purification of the target protein: according to the tag carried by the target protein, the target protein is purified using a traditional corresponding purification scheme; wherein, for c-myc, p53, p62, 14-3-3zeta, NPM1 and HCC1 proteins were purified using Ni-NTA columns, while GST-tagged Cyclin B1 and p90 proteins were purified using glutathione S-transferase.

4) The Bradford method was used to measure the protein concentration (Solebo kit), and the immunological activity of the purified protein was identified by Western blot.

Since the prokaryotic expression and purification of recombinant protein technology is relatively mature, the specific process will not be described in detail. Through the above preparation steps, 8 kinds of active target proteins (ie, 8 kinds of tumor-associated antigens) were successfully obtained for subsequent experiments.

Embodiment 2: the preparation of kit

According to the principle of indirect ELISA, the invention prepares an ELISA kit for combined detection of various autoantibodies that can be used for early screening and diagnosis of liver cancer. The principle of indirect enzyme-linked immunoassay is to connect the antigen to a solid-phase carrier, and the antibody to be tested in the sample combines with it to form a solid-phase antigen-test antibody complex, and then use the enzyme-labeled secondary antibody and the solid-phase antigen-test antibody to The antibodies in the complex combine to form a solid-phase antigen-test antibody-enzyme-labeled secondary antibody complex, and then measure the degree of color development after adding the substrate to determine the content of the antibody to be tested.

1. Reagents and materials

(1) Eight tumor-associated proteins (CyclinB1, p90, c-myc, p53, NPM1, HCC1, 14-3-3zeta and p62 proteins);

(2) Antigen coating solution: 50mM carbonate buffer solution, pH=9.6;

(3) Blocking solution: PBST buffer containing 2% (W/V) BSA;

(4) Enzyme-labeled secondary antibody: RecA protein labeled with horseradish peroxidase, purchased from Invitrogen;

(5) 96-well ELISA plate: purchased from Jet Biofil;

(6) Chromogenic solution A: 0.02% (W/V) TMB (3,3',5,5'-tetramethylbenzidine), preparation: weigh 0.005g of methylbenzidine (TMB), dissolve in 25ml deionized water.

(7) Chromogenic solution B: 0.006% (W/V) urea peroxide, preparation: weigh 4.665g of citric acid, 18.40g of Na ₂ HPO ₄ 12H ₂ O, fully dissolve in 400ml deionized water, add 0.75% urea hydrogen peroxide 3.2ml, adjust the pH value to 5.0-5.4, add deionized water to the final volume of 500ml, mix well and store at 4°C.

2. Preparation of antigen-coated microtiter plates

(1) Prepare eight kinds of tumor-associated antigen solutions:

Eight kinds of tumor-associated proteins were dissolved in the coating solution, mixed thoroughly, and prepared into 8 kinds of antigen solutions with different concentrations, among which, the concentration of NPM1 solution was 0.125 μg/ml, and the concentration of p53 solution was 0.25 μg/ml. The concentration of p90 solution is 0.25 μg/ml; the concentration of p62 solution is 0.5 μg/ml, the concentration of HCC1 solution is 0.5 μg/ml; the concentration of CyclinB1 solution is 1.0 μg/ml, and the concentration of 14-3-3zeta solution is 1.0 μg/ml, the concentration of c-myc solution is 1.0 μg/ml.

(2) Coated microtiter plate: Spotting according to the layout shown in Figure 2. Add the prepared eight tumor-associated antigen solutions to the spotting wells of the 96-well microtiter plate, and the sample volume is 100 μl/well. ; Add p53 antigen solution to positive control wells and negative control wells, add coating solution to blank control wells, and remove the coating solution after incubating overnight at 4°C.

(3) Blocking: add blocking solution to the sample wells of the coated 96-well ELISA plate, incubate at room temperature for 2 hours, and then remove the blocking agent.

(4) Place the 96-well microplate plate treated in step (3) in a 37°C oven for drying, pack it, and store it at 4°C for later use.

3, the composition of kit of the present invention is as follows:

(1) Antigen-coated 96-well microtiter plate;

(2) Sample diluent: PBST buffer containing 1% (W/V) BSA;

(3) Secondary antibody diluent: PBST buffer containing 1% (W/V) BSA;

(4) Enzyme-labeled secondary antibody: RecA protein labeled with horseradish peroxidase, purchased from Invitrogen;

(5) Color-developing solution: The color-developing solution is composed of color-developing solution A and color-developing solution B, wherein, color-developing solution A is 0.02% (W/V) TMB (3,3',5,5'-tetramethyl Base benzidine), chromogenic solution B is 0.006% (W/V) urea peroxide;

(6) Termination solution: 10% concentrated sulfuric acid;

(7) Washing solution: 0.01 M PBST (phosphate Tween) buffer at pH 7.4 containing 0.05% Tween-20.

Embodiment 3: the detection method of kit of the present invention

1. Serum sample incubation:

Dilute the serum sample to be tested with 1% BSA solution at a ratio of 1:100, and then add the diluted serum sample to the spotting well of the antigen-coated 96-well ELISA plate according to the layout shown in Figure 2 In this method, the sample volume was 100 μl/well, and incubated in a constant temperature incubator at 37°C for 1 hour, then the liquid in the sample well was discarded, and washed 5 times with washing solution.

2. Enzyme-labeled secondary antibody incubation:

Dilute the horseradish peroxidase-labeled RecA protein with 1% BSA solution at a ratio of 1:8000, and then add the diluted horseradish peroxidase-labeled RecA protein to the wells of the 96-well microtiter plate In this method, the sample volume was 100 μl/well, and incubated in a constant temperature incubator at 37°C for 1 hour, then the liquid in the sample well was discarded, and washed 5 times with washing solution.

3. Color development and termination reaction

Mix the chromogenic solution A and the chromogenic solution B according to the equal volume of 1:1, and then quickly add the mixed chromogenic solution into the sample well of the 96-well microplate plate, the sample volume is 100 μl/well, and place in Color was developed in a water bath at 37°C in the dark for 15 minutes, and then 50 μl of stop solution was added to each well to terminate the color reaction, and then the OD value was read at 450 nm and 595 nm with an automatic microplate reader, and zeroed with a blank well.

4. Result judgment:

The mean of the OD values of all normal control groups plus two standard deviations (Mean+2SD) was used as the cut-off value (Cut-off value), which was judged positive if it was higher than this value, and negative if it was lower than this value.

Embodiment 4: Serum samples of patients with liver cancer and normal persons detected by the kit of Example 2 of the present invention

Sample preparation: According to the epidemiological analysis, the serum of 84 patients with primary liver cancer (liver cancer group) and the serum of 89 normal people (control group) were collected in this study. Among the 84 patients with primary liver cancer, there were 56 males and 28 females, with an average age of 57.0±11.3 years, and the age range was 23-77 years old; among the 89 normal subjects, there were 59 males and 30 females, with an average age of 57.2±10.9, the age range was 25-79 years old. All the sera of liver cancer patients were collected when the patients were initially diagnosed as liver cancer and had not received any radiotherapy and chemotherapy, and the time of diagnosis was from October 2011 to October 2013. Normal human serum was obtained from those who participated in the annual physical examination and had no malignant tumor-related diseases.

The kits described in Example 2 of the present invention and the detection method described in Example 3 were used to detect the autoantibodies of 8 tumor-associated antigens in 84 cases of liver cancer patients' serum (liver cancer group) and 89 cases of normal human serum (control group). content was tested. Taking the mean value plus two standard deviations of the autoantibody levels of the eight tumor-associated antigens in the serum of the control group as the cut-off value, the positive rates of the autoantibodies to the eight tumor-associated antigens in the liver cancer group and the control group were calculated, respectively. Use MedCalc software to draw the average expression level distribution map of 8 kinds of tumor-associated antigen autoantibodies in the liver cancer group and the control group (results are shown in Figure 3 and Figure 4), and the histograms of their positive rates in the two groups (results are shown in Fig. 5). In this study, SPSS21.0 software was also used for statistical testing. Two independent sample chi-square test methods were used to compare the positive rates of antibodies in the liver cancer group and the control group. The test level α was set at 0.05. The diagnostic value (results in Table 1 and Figure 6).

Table 1 Authenticity evaluation of parallel detection result sets of different TAAs (tumor-associated antigens) combinations

Note: Youden index: Youden index is the sum of sensitivity and specificity minus 1, which comprehensively evaluates the diagnostic value of this method for liver cancer. The value is greater than 0 and less than 1, and the closer it is to 1, the greater the diagnostic value.

It can be seen from Figure 3 and Figure 4 that in the control group, the average expression levels of autoantibodies to the eight tumor-associated antigens were low, and the average OD values were far less than 0.25, and there were few sera with OD values greater than 0.5; while in the liver cancer group, serum The average levels of the autoantibodies to the eight tumor-associated antigens were high, and the average OD value fluctuated around 0.25, and there were many sera with an OD value greater than 0.5, and some sera detected the OD value of the autoantibodies to the tumor-associated antigens even exceeded 1.0; thus It shows that these 8 tumor-associated antigen autoantibodies can be used in the diagnosis of liver cancer. It can be seen from Figure 5 that the positive rates of autoantibodies to 8 tumor-associated antigens in the liver cancer group ranged from 20.2% to 41.4%, while the positive rates of autoantibodies to these 8 tumor-associated antigens in the control group did not exceed 10%. The positive rates of 8 tumor-associated antigen autoantibodies in liver cancer were significantly higher than those in the control group, which proved that these 8 tumor-associated antigen autoantibodies can be used as detection indicators for early diagnosis of liver cancer.

It can be seen from Table 1 that as the number of antigen combinations increases, the diagnostic sensitivity also increases. When 8 tumor-associated antigens are combined, the diagnostic sensitivity finally reaches 86.9%, that is, the application of this method in patients with liver cancer is correct. The percentage of people diagnosed with liver cancer was 86.9%. Although the specificity is lowered, it can reach 73%, which shows that the percentage of non-liver cancer patients who are correctly diagnosed as not suffering from liver cancer by using this method is 73%. Using these 8 tumor-associated antigen combinations to diagnose liver cancer can greatly improve the diagnostic sensitivity under the premise of ensuring diagnostic specificity. The correct rate of judging whether the test object is a liver cancer patient is very high, which is 79.8%. Therefore, it can also be proved that the combination of these 8 tumor-associated antigens is an ideal method and means for the early diagnosis and screening of liver cancer.

It can be seen from Figure 6 that the area under the ROC curve of this method is 0.800, indicating that the combined detection of multiple autoantibodies using the ELISA kit has high judgment accuracy and high diagnostic value for liver cancer, which further confirms that we have developed The multiple autoantibody combined detection ELISA kit is an ideal method and means for early diagnosis and screening of liver cancer.

Claims (6)

1. It is 1. a kind of for liver cancer early screening and a variety of autoantibody joint-detection ELISA kits of diagnosis, it is characterised in that The kit includes solid phase carrier and the tumor associated antigen being coated on solid phase carrier, and the tumor associated antigen is CyclinB1, p90, c-myc, p53, NPM1, HCC1,14-3-3zeta and p62；The kit also include Sample dilution, Secondary antibody, secondary antibody dilution, positive control serum, negative control sera, nitrite ion, terminate liquid, cleaning solution；Described Two antibody are RecA albumen.
2. 2. a variety of autoantibody joint-detection ELISA kits according to claim 1, it is characterised in that the solid phase Carrier is 96 hole elisa Plates.
3. 3. a variety of autoantibody joint-detection ELISA kits according to claim 1, it is characterised in that described second Antibody carries detectable label.
4. 4. a variety of autoantibody joint-detection ELISA kits according to claim 3, it is characterised in that described to examine The label of survey is horseradish peroxidase.
5. 5. a variety of autoantibody joint-detection ELISA kits according to claim 1, it is characterised in that the positive Control serum is p53 positive control serums, and the negative control sera is p53 negative control seras；Wherein, the p53 is positive The liver cancer patient blood serum that it is the positive using indirect ELISA and Western blot methods detection p53 antibody that control serum, which is, institute It is to be averaged using indirect ELISA method detection p53 antibody expressions for normal population serum antibody to state p53 negative control seras The serum of the normal person of content.
6. 6. a variety of autoantibody joint-detection ELISA kits according to claim 1, it is characterised in that described a variety of The detection object of autoantibody joint-detection ELISA kit is human serum.