阅读说明：本技术 Paip1自身抗体在食管鳞癌辅助诊断中的应用 (Application of PAIP1 autoantibody in auxiliary diagnosis of esophageal squamous cell carcinoma ) 是由 代丽萍 杨倩 李科明 仵金玉 王鹏 叶华 史健翔 于 2021-09-16 设计创作，主要内容包括：本发明属于医药生物技术领域,具体公开了一种用于食管鳞癌辅助诊断的生物标志物及检测试剂盒。本发明提供的用于食管鳞癌辅助诊断的生物标志物为抗肿瘤相关抗原PAIP1的自身抗体,该标志物在食管鳞癌患者血清中的表达水平高于正常人,且具有显著差异。本发明还提供一种用于食管鳞癌辅助诊断的试剂盒,该试剂盒含有用于检测上述标志物的试剂,所述试剂为通过酶联免疫吸附、蛋白芯片、免疫印迹或微流控免疫检测样本中所述生物标志物的试剂。本发明通过检测人血清中抗肿瘤相关抗原PAIP1的表达水平,可以有效区分食管鳞癌患者和正常人,可用于食管鳞癌的辅助诊断。(The invention belongs to the technical field of medical biology, and particularly discloses a biomarker and a detection kit for auxiliary diagnosis of esophageal squamous cell carcinoma. The biomarker for auxiliary diagnosis of esophageal squamous carcinoma provided by the invention is an autoantibody of an anti-tumor associated antigen PAIP1, and the expression level of the biomarker in the serum of patients with esophageal squamous carcinoma is higher than that of normal people, and has obvious difference. The invention also provides a kit for auxiliary diagnosis of esophageal squamous cell carcinoma, which contains a reagent for detecting the marker, wherein the reagent is a reagent for detecting the biomarker in a sample by enzyme-linked immunosorbent assay, a protein chip, immunoblotting or microfluidic immunoassay. The invention can effectively distinguish esophageal squamous carcinoma patients from normal people by detecting the expression level of the anti-tumor associated antigen PAIP1 in human serum, and can be used for auxiliary diagnosis of esophageal squamous carcinoma.)

1. A biomarker for the auxiliary diagnosis of esophageal squamous carcinoma, which is an autoantibody against tumor-associated antigen PAIP 1.

2. The biomarker of claim 1, wherein the autoantibody to the tumor associated antigen PAIP1 is an autoantibody to the tumor associated antigen PAIP1 in the serum, plasma, interstitial fluid or urine of the subject.

3. Use of a reagent for detecting a biomarker according to claim 1 or 2 in the manufacture of a product for use in the assisted diagnosis of esophageal squamous cell carcinoma.

4. The use of claim 3, wherein the reagent is a reagent for the detection of the biomarker in the sample by enzyme-linked immunosorbent, protein chip, immunoblot or microfluidic immunoassay.

5. The use of claim 4, wherein the sample is serum, plasma, interstitial fluid or urine.

6. The use of claim 5, wherein the reagent is an antigen for detecting the biomarker.

7. The use according to claim 6, wherein the product is a protein chip, a kit or a formulation.

8. A kit for use in the assisted diagnosis of esophageal squamous carcinoma, wherein the kit comprises reagents for detecting the biomarkers of claim 1 or 2.

9. The kit of claim 8, wherein the kit detects the biomarker in a sample by enzyme-linked immunosorbent, protein chip, immunoblot, or microfluidic immunoassay.

10. The kit of claim 9, wherein the kit is an ELISA detection kit comprising a solid support and PAIP1 protein coated on the solid support.

Technical Field

The invention belongs to the technical field of medical biology, and particularly discloses application of a PAIP1 autoantibody in auxiliary diagnosis of esophageal squamous cell carcinoma.

Background

Esophageal Cancer (EC) is one of the common digestive tract cancers, and seriously threatens human health worldwide. Global epidemiological statistics of cancer show that the incidence and mortality of esophageal cancer worldwide is on the 8 th and 6 th sites of malignancy, respectively, in 2020. Particularly, China is a country with high incidence rate of esophageal cancer worldwide, and the incidence rate and the death rate of esophageal cancer are the 6 th and the 4 th malignant tumors in China. And esophageal squamous carcinoma accounts for more than 90% of all esophageal cancers in China, and is the most common esophageal cancer in China.

The esophageal squamous carcinoma is hidden, and the prognosis is poor. The early stage is almost asymptomatic or the symptoms are not typical and can be ignored easily, most patients reach the middle and late stage of cancer when the typical symptoms appear, the prognosis of patients with esophageal squamous cell carcinoma is poor, and the 5-year survival rate is only 19.9 percent. Research proves that the survival rate of 5 years of patients with esophageal squamous cell carcinoma can reach 63.4 percent if the patients can be diagnosed and treated radically at the early stage. At present, the examination means of esophageal squamous carcinoma mainly comprises X-ray barium meal examination, CT/PET-CT scanning, electronic endoscopy and pathological tissue biopsy, but because the early diagnosis technology is limited, the cost is higher or invasive operation limits the possibility of being widely used for screening, a non-invasive early esophageal squamous carcinoma diagnosis method is urgently needed to be established. The detection of serum tumor markers is economically feasible and can be an accurate complement to the early diagnosis of certain cancers. However, until now, there is no marker with strong specificity and high sensitivity in esophageal squamous carcinoma.

It has been found that as the tumor tissue develops, specific protein products capable of activating the immune system can be produced in the serum of tumor patients, and these proteins are called tumor-associated antigens, and the body can produce corresponding antibodies against these abnormally expressed proteins through the cascade amplification effect of the immune system, and these antibodies are called tumor-associated antigen autoantibodies. These autoantibodies may appear in advance of clinical symptoms in patients with tumors, may be present in the blood of patients for a long time, and are easy to detect and less traumatic to patients during detection, thus having the potential for immunological diagnosis in early stages of tumors.

In conclusion, in order to finally reduce the death rate of the esophageal squamous cell carcinoma and improve the survival rate, the screening and identification of more sensitive and specific serological autoantibody markers and the development of a kit for detecting the esophageal squamous cell carcinoma autoantibody, which has the advantages of simple operation, low cost and wide application range, are urgently needed in the field.

Disclosure of Invention

In view of the problems and disadvantages in the prior art, an object of the present invention is to provide a biomarker for assistant diagnosis of esophageal squamous cell carcinoma, and a second object of the present invention is to provide an application of a reagent for detecting the biomarker in preparation of a product for assistant diagnosis of esophageal squamous cell carcinoma, and a third object of the present invention is to provide a kit for assistant diagnosis of esophageal squamous cell carcinoma.

Based on the above purpose, the technical scheme adopted by the invention is as follows:

the invention provides a biomarker for auxiliary diagnosis of esophageal squamous cell carcinoma, wherein the biomarker is an autoantibody against tumor associated antigen PAIP 1. The expression level of the autoantibody of the anti-tumor associated antigen PAIP1 in the serum of patients with esophageal squamous cell carcinoma is obviously higher than that of normal people, and has significant difference.

Preferably, the autoantibody against the tumor associated antigen PAIP1 is an autoantibody against the tumor associated antigen PAIP1 in the serum and plasma of a subject according to the above biomarker. More preferably, the autoantibodies to the tumor associated antigen PAIP1 are autoantibodies to the tumor associated antigen PAIP1 in the serum or plasma of a subject.

Preferably, the autoantibody to the tumor associated antigen PAIP1 is an autoantibody to the tumor associated antigen PAIP1 in the serum or plasma of a subject prior to treatment with a tumor, according to the biomarkers described above. More preferably, the tumor treatment is chemotherapy, radiation therapy or surgical resection of a tumor.

Preferably, the subject is a mammal, more preferably, the subject is a primate mammal, according to the biomarkers described above; most preferably, the subject is a human.

In a second aspect, the invention provides a use of the reagent for detecting the biomarker according to the first aspect in the preparation of a product for auxiliary diagnosis of esophageal squamous cell carcinoma.

According to the above application, preferably, the reagent is a reagent for detecting the biomarker in the sample by enzyme-linked immunosorbent, protein chip, immunoblot or microfluidic immunoassay.

Preferably, the sample is serum, plasma, interstitial fluid or urine, according to the above-mentioned use.

Preferably, the reagent is an antigen for detecting the biomarker according to the above-mentioned application. More preferably, the agent is a PAIP1 protein.

According to the above-mentioned use, preferably, the product is a protein chip, a kit or a formulation.

According to a third aspect of the present invention, there is provided a kit for assisted diagnosis of esophageal squamous cell carcinoma, the kit comprising reagents for detecting the biomarkers of the first aspect.

According to the above kit, preferably, the kit detects the biomarker in a sample by enzyme-linked immunosorbent, protein chip, immunoblot or microfluidic immunoassay. More preferably, the kit detects the biomarker in the sample by antigen-antibody reaction.

According to the above kit, preferably, the kit is an ELISA detection kit. More preferably, the ELISA detection kit comprises a solid phase carrier and PAIP1 protein coated on the solid phase carrier.

Preferably, the sample is serum, plasma, interstitial fluid or urine according to the above-mentioned kit.

According to the kit, preferably, the ELISA detection kit further comprises a sample diluent, a second antibody diluent, a washing solution, a color development solution and a stop solution.

The basic information of the tumor-associated antigen PAIP1 in the invention is as follows: PAIP1 (Polyadenylate-binding protein-interacting protein 1), a mammalian specific protein, interacts with Polyadenylate binding protein and eukaryotic translation initiation factor 4A, and participates in translation initiation and protein biosynthesis, however, the specific mechanism by which PAIP1 regulates translation is not clear. Plays a role as a helper activator in the regulation of translation initiation of poly (a) -containing mRNAs. Its stimulatory effect on translation is mediated by its effect on PABPC 1. Competes with PAIP2 for binding to PABPC 1. Its binding to EIF4A and PABPC1 may enhance contact between mRNA ends. May also be involved in translation-coupled mRNA conversion. Together with other RNA binding proteins, are involved in cytoplasmic decenyl/translation and decay interactions of FOS mRNA mediated by the primary coding region instability determinant (mCRD). The protein sequence number of the PAIP1 protein used in the research in NCBI is NP-006442.2.

Compared with the prior art, the invention has the following positive beneficial effects:

(1) the invention discovers for the first time that the expression level of the autoantibody of the anti-tumor associated antigen PAIP1 in the serum of a patient with esophageal squamous cell carcinoma is obviously higher than that of a normal person and has obvious difference, so that the autoantibody of the anti-tumor associated antigen PAIP1 can be used as a marker for auxiliary diagnosis of the esophageal squamous cell carcinoma; the AUC of the ROC curve for diagnosing esophageal squamous cell carcinoma by using the autoantibody against the tumor-associated antigen PAIP1 is 0.627 (95% CI: 0.541-0.713), and when the cutoff value is 0.3225, the sensitivity of diagnosing esophageal squamous cell carcinoma by using the autoantibody against the tumor-associated antigen PAIP1 reaches 30.9%, and the specificity reaches 90.1%.

(2) The kit disclosed by the invention detects the expression level of the autoantibody of the anti-tumor associated antigen PAIP1 in human serum by an indirect ELISA method, can accurately compare esophageal squamous cell carcinoma patients with health controls, has detection sensitivity and specificity respectively reaching 30.9% and 90.1%, is remarkably higher than the detection sensitivity and specificity of the existing clinical endoscope on esophageal squamous cell carcinoma, and is beneficial to screening and early discovery of asymptomatic high-risk groups of esophageal squamous cell carcinoma, so that the death rate of the esophageal squamous cell carcinoma patients is greatly reduced, and great welfare is brought to the esophageal squamous cell carcinoma patients and families.

(3) The detection sample of the kit is serum, so that invasive diagnosis can be avoided, the risk of esophageal squamous cell carcinoma can be obtained at an early stage by taking serum in a minimally invasive way for detection, the blood demand is low, the pain of detected personnel is low, and the compliance is high; moreover, the method is simple to operate, short in detection result time and wide in market prospect and social benefit.

Drawings

FIG. 1 is a graph showing the results of protein chip detection of the expression levels of autoantibodies against tumor associated antigen PAIP1 in esophageal squamous carcinoma serum and normal serum; wherein EC represents esophageal squamous carcinoma, NC represents normal control;

FIG. 2 is a graph showing the results of measuring the serum expression level of autoantibodies against the tumor associated antigen PAIP1 by ELISA;

FIG. 3 is a ROC plot of autoantibody diagnosis against the tumor associated antigen PAIP1 distinguishing the esophageal squamous carcinoma group from the normal control group.

Detailed Description

The following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, elements, and/or combinations thereof, unless the context clearly indicates otherwise.

The experimental methods in the following examples, which do not indicate specific conditions, all employ conventional techniques in the art, or follow the conditions suggested by the manufacturers; the reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.

Example 1: screening of markers for esophageal squamous carcinoma diagnosis by using human proteome chips

1. Experimental samples:

30 cases of esophageal squamous carcinoma patient serum (esophageal squamous carcinoma group) and 24 cases of normal human serum (normal control group) from a specimen bank of an important tumor epidemiology laboratory in Henan province are collected; wherein, 30 cases of the serum of the esophageal squamous cell carcinoma patients are from esophageal squamous cell carcinoma patients which are diagnosed by pathology and are not treated; the 24 normal human sera were from healthy subjects who entered the cohort criteria: no cardiovascular, respiratory, liver, kidney, gastrointestinal, endocrine, hematological, psychiatric, or neurological disease, and history of the above, no acute or chronic disease, no autoimmune disease, no evidence of any tumor association; furthermore, the difference between sex and age between 30 patients with esophageal squamous carcinoma and 24 healthy subjects was not statistically significant. The study was approved by the ethical committee of zheng state university, and all subjects had signed informed consent.

Mixing every 3 cases of serum of 30 cases of esophageal squamous carcinoma patient serum into 1 case of mixed esophageal squamous carcinoma serum sample to obtain 10 cases of mixed esophageal squamous carcinoma serum samples; each 3 of 21 normal human sera were mixed into 1 mixed normal serum sample to obtain 7 normal serum samples.

Collecting serum: collecting peripheral blood 5 ml of the subject in fasting state, placing in a blood collecting tube without anticoagulant, standing at room temperature for 1h, placing in a centrifuge, setting to 4 deg.C, and centrifuging at 3000 rpm for 10 min. Then sucking out the serum on the upper layer of the blood collection tube, subpackaging into 1.5 ml EP tubes, marking sample numbers on the top and the side of the EP tube, placing the EP tubes in a refrigerator at minus 80 ℃ for freezing storage, and recording the blood collection date and the storage position. Before use, the serum is taken out and put in a refrigerator at 4 ℃ for unfreezing and subpackaging, and repeated freeze thawing of the serum is avoided.

2. Human proteome chip detection

The expression levels of autoantibodies in 10 mixed esophageal squamous carcinoma serum samples, 7 mixed normal serum samples and 3 normal human serum samples were tested using the HuProt (TM) human proteome chip (available from Biotech, Inc., Bo Chong, Guangzhou). Each chip can detect 14 serum samples at the same time, and the protein fixed on the chip interacts with the specific antibody in the serum to be combined.

(1) The experimental method comprises the following steps:

1) rewarming: the human whole protein histone chip is taken out from a freezing refrigerator with the temperature of minus 80 ℃, placed in a refrigerator with the temperature of 4 ℃ for 30 min, and then placed in the room temperature for 15 min.

2) And (3) sealing: each rewarmed protein chip was immobilized on 14 pens, blocking solution (3 ml 10% BSA, added to 7ml 1 XPBS solution, mixed well on ice) was added to each pen, and then the protein chips were placed on a side-shaking table, shaken gently, and blocked at room temperature for 3 h.

3) Incubation of serum samples: after blocking is completed, the blocking solution is discarded, and then the diluted serum sample (1 ml 10% BSA and 9ml 1 XPBST solution) is quickly added into the pen with the sample amount of 200 ul/pen by using the diluent to dilute the serum sample according to the ratio of 1: 50; then placing the protein chip in a refrigerator at 4 ℃, and incubating overnight in a side-swinging shaker (the rotating speed is 20 rpm); each protein chip can incubate 14 serum samples.

4) Cleaning: the protein chip and chip holder were removed from the rocking platform, the serum sample was discarded, and PBST buffer (sample size 200 ul/pen) was added quickly, and the cycle was repeated several times. The chip holder was removed with tweezers, and the protein chip was placed in a box containing a washing solution (1 XPBST, stored in a refrigerator at 4 ℃), and washed on a horizontal shaker (80 rpm) for 10min each time at room temperature for 3 times.

5) And (3) secondary antibody incubation: the protein chip was placed in a cassette containing 3ml of secondary antibody incubation solution (secondary antibody was diluted with diluent at a ratio of 1:100 to obtain secondary antibody incubation solution, secondary antibody was fluorescently labeled anti-human IgM, IgG antibody, and the diluent was 1ml of 10% BSA added to 9ml of 1 XPBST solution), and then transferred to a side-shaking table (rotation speed 40 rpm) and incubated for 60min at room temperature in the dark.

6) Cleaning: cleaning according to the step 4), and cleaning with double distilled water for 2 times (10 min each time) after the cleaning is finished.

7) And (3) drying: and (3) placing the protein chip treated in the step 6) into a chip dryer for centrifugal drying.

8) Scanning: and (3) performing normative scanning on the dried protein chip according to the operation instruction of a scanning instrument and recording a fluorescence signal, wherein the strength of the fluorescence signal has a positive correlation with the affinity and the quantity of a corresponding antibody.

9) Data extraction: and opening a corresponding GAL file, aligning each array on the GAL file with the whole chip image, clicking an automatic alignment button, extracting data of each serum sample, and storing the data as a GPR.

(2) Data processing:

f532 Median refers to the Median of the foreground values of the signal points under the 532nm channel, and B532 Median refers to the Median of the background values of the signal points under the 532nm channel. In order to eliminate the sample-to-sample variation caused by the variance of background values, background normalization processing is performed on the data extracted from each serum sample, that is, signal-to-noise ratio (SNR) = F532 media/B532 media is defined, and the SNR values of 10 mixed esophageal squamous carcinoma serum samples, 7 mixed normal serum samples and 3 normal serum samples are obtained by calculating according to an SNR calculation formula. The SNR values of 10 mixed esophageal squamous carcinoma serum samples, 7 mixed normal serum samples and 3 normal serum samples were subjected to z-score normalization so as to be subjected to N (0, 1) standard normal distribution. For any one of the autoantibodies, calculating the difference multiple of the esophageal squamous carcinoma group and the normal control group (difference multiple = SNR mean value after the esophageal squamous carcinoma group is normalized by z-score/SNR mean value after the normal control group is normalized by z-score) to represent the degree that the esophageal squamous carcinoma group is higher than the normal control group, and further setting the screening condition: the difference multiple is more than 2, the sensitivity is more than or equal to 60 percent, the specificity is more than or equal to 90 percent, and the anti-tumor associated antigen autoantibodies meeting the conditions are screened out.

(3) The experimental results are as follows:

through screening, the difference multiple (fold change) of the autoantibody of the anti-tumor associated antigen PAIP1 is 2.242, and the sensitivity and the specificity are 60% and 90% respectively. The SNR values of the autoantibodies against the tumor associated antigen PAIP1 in the esophageal squamous cell carcinoma group and the normal control group were further plotted in a bar graph, and the results are shown in FIG. 1. As shown in FIG. 1, the level of the autoantibody against the tumor associated antigen PAIP1 in the serum of the esophageal squamous cell carcinoma group is significantly higher than that of the serum of the normal control group, and the difference has statistical significance.

Example 2: ELISA for detecting serum expression level of autoantibody against tumor-associated antigen PAIP1

The expression level of anti-PAIP 1 autoantibodies was further detected in the plasma of a large panel of persons using enzyme linked immunosorbent assay (ELISA).

1. Experimental samples:

160 primary esophageal squamous carcinoma patients (esophageal squamous carcinoma group) and 229 normal control serum (normal control group) samples included in the study are both from a specimen bank of an important laboratory in tumor epidemiology in Henan province; wherein, the 160 cases of the serum of the esophageal squamous cell carcinoma patients are from esophageal squamous cell carcinoma patients which are diagnosed by pathology and are not treated; 160 normal human sera were from healthy subjects who entered the cohort criteria: no cardiovascular, respiratory, liver, kidney, gastrointestinal, endocrine, hematological, psychiatric, or neurological disease, and history of the above, no acute or chronic disease, no autoimmune disease, no evidence of any tumor association; furthermore, there was no statistical difference between gender and age between 160 patients with esophageal squamous carcinoma and 160 healthy subjects. The study was approved by the ethical committee of zheng state university, and all subjects had signed informed consent.

Collecting serum: collecting peripheral blood 5 ml of the subject in fasting state, placing in a blood collecting tube without anticoagulant, standing at room temperature for 1h, placing in a centrifuge, setting to 4 deg.C, and centrifuging at 3000 rpm for 10 min. Then sucking out the serum on the upper layer of the blood collection tube, subpackaging into 1.5 ml EP tubes, marking sample numbers on the top and the side of the EP tube, placing the EP tubes in a refrigerator at minus 80 ℃ for freezing storage, and recording the blood collection date and the storage position. Before use, the serum is taken out and put in a refrigerator at 4 ℃ for unfreezing and subpackaging, and repeated freeze thawing of the serum is avoided.

2. Experimental materials and reagents:

(1) the PAIP1 recombinant protein, purchased from Wuhan Huamei bioengineering GmbH;

(2) 96-well enzyme-linked plate (8 rows × 12 columns);

(3) coating liquid: contains 0.15% sodium carbonate (Na)₂ CO₃) And 0.29% sodium bicarbonate (NaHCO)₃) An aqueous solution of (a);

(4) sealing liquid: PBST buffer containing 2% (v/v) Bovine Serum Albumin (BSA) and 0.2% (v/v) Tween 20;

(5) serum sample diluent: PBST buffer containing 1% (W/V) BSA;

(6) enzyme-labeled secondary antibody: horse Radish Peroxidase (HRP) labeled mouse anti-human immunoglobulin antibody (hereinafter, HRP labeled mouse anti-human IgG antibody);

(7) antibody dilution: PBST buffer containing 1% (W/V) BSA;

(8) washing liquid: PBST buffer containing 0.2% (v/v) Tween 20;

(9) color development liquid: the color developing solution comprises a color developing solution A and a color developing solution B, wherein the color developing solution A is 20% of aqueous solution of tetramethylbenzidine dihydrochloride, and the color developing solution B is 3.7% of Na₂HPO₄ •12H₂O, 0.92% citric acid and 0.75% (V/V) aqueous urea hydrogen peroxide solution; when in use, the color development liquid A and the color development liquid B are uniformly mixed in equal volume according to the ratio of 1: 1;

(10) stopping liquid: 10% sulfuric acid.

3. The experimental method comprises the following steps:

(1) preparing an ELISA plate coated by tumor-associated antigen PAIP 1:

the specific operation steps for preparing the ELISA plate coated by the tumor-associated antigen PAIP1 are as follows:

1) preparation of tumor-associated antigen PAIP1 solution: the PAIP1 recombinant protein was dissolved in the coating solution to prepare a PAIP1 protein solution at a concentration of 0.25. mu.g/mL.

2) Coating an enzyme label plate: adding the PAIP1 protein solution prepared in the step 1) into reaction holes of the 1 st to 11 th columns of the 96-well enzyme label plate, wherein the sample adding amount is 50 mu l/hole; adding a human IgG standard substance (the human IgG standard substance with a series of concentration gradients is coated in different reaction holes in the 12 th column, the coated human IgG standard substance with the series of concentration gradients can be used for making a standard curve, and simultaneously the stability of experimental operation can be ensured), wherein the sample adding amount is 50 mu l/hole, the preservative film is sealed to prevent volatilization, the coating is carried out overnight at 4 ℃, and then the coating liquid in the reaction holes is discarded.

3) And (3) sealing: adding a sealing solution into the reaction holes of the coated 96-hole ELISA plate, wherein the loading amount is 100 mu l/hole, sealing in water bath at 37 ℃ for 2h, then removing the sealing solution, washing for 3 times by using a washing solution and drying by beating to obtain the tumor-associated antigen PAIP 1-coated ELISA plate.

(2) Measurement of the level of autoantibody expression against the tumor-associated antigen PAIP1 in a serum sample:

the autoantibody expression level of the tumor-associated antigen PAIP1 in a serum sample is detected by an ELISA method by using the ELISA plate coated with the tumor-associated antigen PAIP1 prepared above. The specific operation steps are as follows:

1) incubation of serum samples:

diluting a serum sample to be detected by using a serum sample diluent according to the volume ratio of 1:100, and then adding the diluted serum sample into reaction holes of the 1 st to 10 th columns of a 96-hole enzyme label plate coated with PAIP1 recombinant protein, wherein the sample adding amount is 50 mu l/hole; adding 50 mul/well of quality control serum (the quality control serum is used as quality control to carry out normalization among different enzyme-labeled plates) into the reaction wells 1-5 of the 96-well enzyme-labeled plate coated with PAIP1 recombinant protein, and adding 50 mul/well of antibody diluent without serum (used as blank control) into the reaction wells 6-8 of the 11 th column; adding serum-free antibody diluent to each reaction well in column 12 in an amount of 50. mu.l/well; the 96-well enzyme-labeled reagent was incubated in a water bath at 37 ℃ for 1 hour, and then the liquid in the reaction well was discarded, washed 5 times with a washing solution (sample amount: 300. mu.l/well) and dried.

2) And incubation of enzyme-labeled secondary antibody:

diluting an HRP-labeled mouse anti-human IgG antibody with an antibody diluent according to the proportion of 1:10000 (v/v), adding the diluted HRP-labeled mouse anti-human IgG antibody into a reaction hole of a 96-hole enzyme label plate, placing the diluted HRP-labeled mouse anti-human IgG antibody into a water bath at 37 ℃ for incubation for 1h, then discarding the liquid in the reaction hole, washing with a washing solution (the sample addition is 300 mu l/hole) for 5 times, and patting to dry.

3) Color development and termination reaction:

uniformly mixing the color development liquid A and the color development liquid B in an equal volume according to a ratio of 1:1, then quickly adding the mixed color development liquid into each reaction hole of a 96-hole enzyme label plate, wherein the sample addition amount is 50 mu l/hole, carrying out light-shielding color development reaction at room temperature for 10min, then adding 25 mu l of stop solution into each reaction hole, and stopping the color development reaction; then, the absorbance (OD) at the wavelengths of 450nm and 620 nm was read using a microplate reader, wherein the absorbance value at the wavelength of 620 nm was taken as a background value, the difference between the absorbances at the wavelengths of 450nm and 620 nm was taken as an absorbance value, and the absorbance was zeroed using a blank control well.

4. Data processing

(ii) performing Kolmogorov-Smirnova test on the absorbance values of the serum samples of the esophageal squamous carcinoma group and the normal control group, and finding that the expression level of autoantibodies against the tumor-associated antigen PAIP1 in the serum sample of the subject does not conform to the normal distribution by the Kolmogorov-Smirnova test: (P<0.05), therefore the 25 th percentile (P25), the median (P50) and the 75 th percentile (P75) were used to describe the distribution of the expression levels of autoantibodies against the tumor-associated antigen PAIP 1; a nonparametric test (Mann-Whitney U) was then used to compare the differences in the expression levels of autoantibodies in the esophageal squamous carcinoma group and the normal control group. Plotting a ROC curve for autoantibody diagnosis against the tumor-associated antigen PAIP1 based on measured expression levels of autoantibodies against the tumor-associated antigen PAIP1 in the esophageal squamous carcinoma group and the normal control group using GraphPad prism5.0, and area under the curve according to the ROC curve (AUC), analyzing the diagnostic value of autoantibodies of the anti-tumor associated antigen PAIP1 on esophageal squamous carcinoma; when the specificity is more than 90%, the OD value with the maximum Yoden index is a cut-off value, and the value above which the test piece is positive, and the value below which the test piece is negative. All statistical analyses were performed using SPSS 26.0 software,P<0.05 is a statistical judgment standard.

5. Results of the experiment

The expression levels of autoantibodies against the tumor associated antigen PAIP1 in the serum samples of the esophageal squamous carcinoma group and the normal control group are shown in FIG. 2. As shown in FIG. 2, the level of autoantibodies against the tumor-associated antigen PAIP1 in the serum samples of the esophageal squamous carcinoma group was significantly higher than that in the normal control group (P < 0.05). Therefore, the autoantibody of the anti-tumor associated antigen PAIP1 can be used for auxiliary diagnosis of esophageal squamous cell carcinoma.

FIG. 3 is a ROC plot of autoantibody diagnosis against the tumor associated antigen PAIP1 distinguishing the esophageal squamous carcinoma group from the normal control group. As can be seen from FIG. 3, the AUC of esophageal squamous carcinoma diagnosed with the autoantibodies against the tumor-associated antigen PAIP1 was 0.627 (95% CI: 0.541-0.713), and the corresponding sensitivity and specificity were 30.9% and 90.1% respectively when the cut-off value was 0.3225. Therefore, the autoantibody of the anti-tumor associated antigen PAIP1 can be used for diagnosing and distinguishing patients with esophageal squamous carcinoma from normal people.

In conclusion, the present invention effectively overcomes the disadvantages of the prior art and has high industrial utilization value. The above-described embodiments are intended to illustrate the substance of the present invention, but are not intended to limit the scope of the present invention. It will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the true spirit and scope of the invention.