WO2024001044A1 - 一种与肺癌相关的生物标志物组合、含其的试剂盒及其用途 - Google Patents

一种与肺癌相关的生物标志物组合、含其的试剂盒及其用途 Download PDF

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WO2024001044A1
WO2024001044A1 PCT/CN2022/136007 CN2022136007W WO2024001044A1 WO 2024001044 A1 WO2024001044 A1 WO 2024001044A1 CN 2022136007 W CN2022136007 W CN 2022136007W WO 2024001044 A1 WO2024001044 A1 WO 2024001044A1
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antigen
autoantibodies
lung cancer
magnetic beads
efhd2
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PCT/CN2022/136007
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English (en)
French (fr)
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胡海
赵峰
黄朝远
黄璐
林当
周腊梅
王增松
陈嘉雯
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广州市丹蓝生物科技有限公司
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57423—Specifically defined cancers of lung
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites

Definitions

  • the present invention relates to the field of cancer diagnosis and treatment, and in particular to a biomarker combination related to lung cancer, a kit containing the same and its application.
  • Lung cancer is one of the cancers with the highest morbidity and mortality in the world. In recent years, the incidence of lung cancer has gradually increased. The latest health statistics yearbook shows that lung cancer mortality ranks first in the mortality rate of malignant tumors in my country. As of 2017, there were 320 million smokers in my country, 740 million people were exposed to second-hand smoke, and 700,000 new cases of lung cancer were diagnosed every year. Coupled with the aging of the population, rural urbanization and urban modernization, air pollution and environmental pollution are becoming increasingly serious, and the incidence and mortality of lung cancer caused by unhealthy lifestyles will further increase.
  • early tumor diagnosis is to accurately diagnose cancers that are still in their early stages of development before clinical signs appear.
  • low-dose spiral CT is widely used to detect early lung nodules and is an effective method for early screening of lung cancer.
  • the problem with low-dose spiral CT is that its test results are high in false positives, and multiple CT tests may accelerate the progression of lung cancer.
  • Other early lung cancer detection methods include PET, MRI, bronchoscopy, needle biopsy and other examinations, which have problems such as inability to screen early, invasiveness, and high price.
  • traditional conventional tumor markers such as CEA and CYFRA21-1 have very low or even no content in the blood of early-stage tumors.
  • Pathological testing is the pathological staining analysis of lesion tissue obtained through surgery or biopsy. It is currently the only gold standard for diagnosing tumors. However, this invasive examination method cannot yet be used as a detection method for early diagnosis and screening of cancer.
  • major breakthroughs have been made in the personalized treatment of lung cancer targeted drugs through the detection of gene mutations such as EGFR, KRAS, ALK, and BRAF.
  • gene mutations such as EGFR, KRAS, ALK, and BRAF.
  • these genetic mutation tests cannot be used for early diagnosis of lung cancer or screening of people at high risk for lung cancer.
  • Circulating tumor cells can break away from primary tumor lesions and enter the peripheral blood circulation spontaneously or by interference from external factors. They are a particularly promising technical field for evaluating the effects of chemotherapy, prognosis and disease monitoring of lung cancer.
  • Lung cancer autoantibody spectrum detection has more advantages.
  • Tumor-specific antigens are specific protein products that are released, shed, or secreted after cell necrosis during tumor occurrence and progression.
  • the body's immune system can recognize tumor-specific antigens expressed by tumor cells and produce autoantibodies against these antigens.
  • the level of autoantibodies is much higher than the level of antigens.
  • Autoantibodies have the characteristics of immune surveillance, immune amplification, and circulation diffusion. Compared with other tumor antigen markers and DNA markers, autoantibodies have: It appears early in serum, has a long blood detection time window, high blood signal intensity, and high early detection sensitivity. Therefore, joint detection of multiple autoantibody markers is a means to obtain highly sensitive and specific detection.
  • the product and the seven autoantibody detection kits (enzyme-linked immunoassay) of Hangzhou Kaibao Biotechnology Co., Ltd. are products that jointly detect a set of seven related autoantibody tumor markers, using the enzyme-linked immunoassay (ELISA) detection method.
  • ELISA enzyme-linked immunoassay
  • This test is used as a compensation method for CT examination of lung malignant tumors to assist diagnosis and treatment experts in making judgments.
  • the ELISA detection method does not have high-throughput characteristics, and the sensitivity or specificity of these two products for the detection of early lung cancer needs to be improved.
  • biomarker combination In order to solve the technical problem of low sensitivity and specificity of biomarker combinations or kits for early lung cancer diagnosis or prognosis in the prior art, a biomarker combination, a kit containing the same and its application are provided.
  • one of the technical solutions of the present invention is: a biomarker combination related to lung cancer, the biomarker combination includes ESO-1, TM4SF1, p53, BMI1, FXR1 and EFHD2 antigens or combines them of autoantibodies.
  • the biomarker combination further includes BRAF and/or CAGE antigens or autoantibodies that bind them.
  • the biomarker combination further includes PGP9.5 and/or ZNF573 antigens or autoantibodies that bind thereto, and preferably also includes GBU4-5 antigen or autoantibodies that bind thereto.
  • the biomarker combination further comprises MAGE A4 and/or SOX2 antigens or autoantibodies binding thereto.
  • the biomarker combination is a biomarker combination in serum, which is selected from any of the following groups:
  • ESO-1 autoantibodies ESO-1 autoantibodies, TM4SF1 autoantibodies, p53 autoantibodies, BMI1 autoantibodies, FXR1 autoantibodies and EFHD2 autoantibodies;
  • ESO-1 autoantibodies TM4SF1 autoantibodies, p53 autoantibodies, BMI1 autoantibodies, FXR1 autoantibodies, EFHD2 autoantibodies and BRAF autoantibodies;
  • ESO-1 autoantibodies TM4SF1 autoantibodies, p53 autoantibodies, BMI1 autoantibodies, FXR1 autoantibodies, EFHD2 autoantibodies, BRAF autoantibodies and CAGE autoantibodies;
  • BRAF autoantibodies ESO-1 autoantibodies, EFHD2 autoantibodies, TM4SF1 autoantibodies, ZNF573 autoantibodies, BMI1 autoantibodies, CAGE autoantibodies, FXR1 autoantibodies, PGP9.5 autoantibodies and P53 autoantibodies;
  • BRAF autoantibodies ESO-1 autoantibodies, EFHD2 autoantibodies, TM4SF1 autoantibodies, ZNF573 autoantibodies, BMI1 autoantibodies, CAGE autoantibodies, FXR1 autoantibodies, PGP9.5 autoantibodies, P53 autoantibodies and MAGEA4 autoantibodies;
  • BRAF autoantibodies ESO-1 autoantibodies, EFHD2 autoantibodies, TM4SF1 autoantibodies, ZNF573 autoantibodies, BMI1 autoantibodies, CAGE autoantibodies, FXR1 autoantibodies, PGP9.5 autoantibodies, P53 autoantibodies, GBU4-5 autoantibodies Antibodies, MAGEA4 autoantibodies and SOX2 autoantibodies.
  • the second technical solution of the present invention is: a diagnostic kit, which includes reagents for detecting biomarkers in the biomarker combination as described in one of the technical solutions, for example Detection of autoantibodies for antigen detection, or antigens for detection of autoantibodies.
  • the antigen or detection autoantibody in the biomarker combination, also contains a tag peptide.
  • the tag peptide includes: one or more of His tag, streptavidin tag, avidin tag, biotin tag, GST tag, C-myc tag, Flag tag and HA tag.
  • the biomarker can be expressed by E. coli, yeast, insect cells or animal cells; and/or, the biomarker can be expressed by Ni affinity chromatography, ion exchange chromatography, molecular sieve , dialysis, ultrafiltration or hydrophobic chromatography purification.
  • the reagent for detecting the biomarker in the biomarker combination is selected from any of the following groups:
  • ESO-1 antigen ESO-1 antigen, TM4SF1 antigen, p53 antigen, BMI1 antigen, FXR1 antigen and EFHD2 antigen;
  • ESO-1 antigen ESO-1 antigen, TM4SF1 antigen, p53 antigen, BMI1 antigen, FXR1 antigen, EFHD2 antigen and BRAF antigen;
  • ESO-1 antigen ESO-1 antigen, TM4SF1 antigen, p53 antigen, BMI1 antigen, FXR1 antigen, EFHD2 antigen, BRAF antigen and CAGE antigen;
  • BRAF antigen BRAF antigen, ESO-1 antigen, EFHD2 antigen, TM4SF1 antigen, ZNF573 antigen, BMI1 antigen, CAGE antigen, FXR1 antigen, PGP9.5 antigen and P53 antigen;
  • BRAF antigen BRAF antigen, ESO-1 antigen, EFHD2 antigen, TM4SF1 antigen, ZNF573 antigen, BMI1 antigen, CAGE antigen, FXR1 antigen, PGP9.5 antigen, P53 antigen and MAGEA4 antigen;
  • BRAF antigen BRAF antigen, ESO-1 antigen, EFHD2 antigen, TM4SF1 antigen, ZNF573 antigen, BMI1 antigen, CAGE antigen, FXR1 antigen, PGP9.5 antigen, P53 antigen, GBU4-5 antigen, MAGEA4 antigen and SOX2 antigen.
  • the detection antigen or detection autoantibody is coupled with magnetic beads to form coupled magnetic beads, and the coupled magnetic beads are mixed to form a coupled magnetic bead mixture, which is coupled with the antigen.
  • the preparation method of coupled magnetic beads includes the following steps:
  • Activation of magnetic beads Shake and mix the magnetic beads to be coupled, wash the magnetic beads with activation buffer, add NHS solution and EDC solution in sequence, shake and mix, rotate and mix and incubate to prepare activated magnetic beads.
  • the activation buffer is 0.1M sodium dihydrogen phosphate solution
  • the NHS solution is an activation buffer containing 5% (w/v) N-hydroxysuccinimide
  • the EDC solution is Activation buffer containing 5% (w/v) 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride;
  • step (2) Coupling of Biotin-BSA: After washing the magnetic beads activated in step (1) with coupling buffer, add BSA-biotin, mix and incubate, add washing solution, wash, add blocking buffer, and mix Incubate to prepare magnetic beads coupled to Biotin-BSA;
  • the coupling buffer is a 50mM 2-[N-morpholino]ethanesulfonic acid solution
  • the washing solution is a solution containing 0.05% (w /v) Tween-20, 0.03% (v/v) Proclin300 in 1 ⁇ PBS buffer
  • the blocking buffer is 1 ⁇ containing 1% (w/v) BSA, 0.03% (v/v) Proclin300 PBS buffer
  • the concentration of BSA-biotin is 2 mg/mL;
  • Antigen coating Wash the Biotin-BSA-coupled magnetic beads in step (2) with washing liquid, add the corresponding antigen, mix and incubate, then wash with washing liquid, and add magnetic bead preservation solution Save to prepare coupled magnetic beads; preferably, the washing solution is 1 ⁇ PBS buffer containing 0.05% (w/v) Tween-20 and 0.03% (v/v) Proclin300, and the magnetic beads
  • the preservation solution is 1 ⁇ PBS buffer containing 0.5% (w/v) BSA, 3% (w/v) trehalose, 5% (w/v) mannitol, and 0.03% (v/v) Proclin300; better Ground, the coating concentration of BRAF antigen is 400 ⁇ g/mL, the coating concentration of ESO-1 antigen is 100 ⁇ g/mL, the coating concentration of EFHD2 antigen is 200 ⁇ g/mL, the coating concentration of TM4SF1 antigen is 300 ⁇ g/mL, and the coating concentration of ZNF573 antigen The coating concentration of
  • the preparation method of the coupled magnetic beads further includes:
  • step (3) Mix the coupled magnetic beads in step (3), add the magnetic bead freeze-drying buffer, mix and then freeze-dry; preferably, the magnetic bead freeze-drying buffer It is 1 ⁇ PBS buffer containing 0.5% (w/v) BSA, 3% (w/v) trehalose, 5% (w/v) mannitol, and 0.03% (v/v) Proclin300.
  • the v/v is the volume percentage
  • the w/v is the mass to volume ratio
  • the diagnostic kit further includes one of sample diluent, calibrator diluent, washing liquid, analysis buffer, anti-human IgG secondary antibody, calibrator, and quality control substance.
  • the diagnostic kit preferably further includes a 96-well plate.
  • the sample diluent is a phosphate buffer containing 2% (v/v) donkey serum and 0.5% (w/v) BSA, preferably also containing a preservative;
  • the calibrator diluent is a phosphate buffer containing 1% (w/v) BSA, preferably also containing a preservative;
  • the washing liquid is PBST washing liquid containing Tween20;
  • the analysis buffer is a phosphate buffer containing 0.2% (w/v) BSA, preferably also containing a preservative;
  • the anti-human IgG secondary antibody is an RPE-labeled donkey anti-human IgG fluorescent antibody
  • the calibrator is a recombinant humanized Anti-Myc-tagged immunoglobulin containing 1% (w/v) BSA and phosphate buffer; preferably, the concentration of Anti-Myc is 3000 U/mL, and the The calibrator also contains a protective agent and/or preservative, and/or, the calibrator is freeze-dried; and/or,
  • the quality control product includes quality control product I and quality control product II.
  • the concentration of the quality control product I is a phosphate buffer containing 200 U/ml Anti-Myc and 1% (w/v) BSA.
  • the concentration of control product II is phosphate buffer containing 7.41 U/ml Anti-Myc and 1% (w/v) BSA; preferably, the quality control product I and the quality control product II also contain a protective agent and /or preservatives, and/or, the quality control product I and the quality control product II are made into freeze-dried products.
  • the protective agent contains 1% (w/v) BSA, 3% (w/v) trehalose, 5% (w/v) mannitol, 0.03% (v/v) ) Proclin300 in 1 ⁇ PBS buffer.
  • the v/v is the volume percentage
  • the w/v is the mass to volume ratio
  • the third technical solution of the present invention is: a detection method for non-diagnostic purposes.
  • the detection method uses a biomarker combination as described in one of the technical solutions or as described in the second technical solution.
  • the diagnostic kit detects the corresponding antigen or the autoantibody that binds to it.
  • the specific steps are: contacting the biomarker combination with the sample. If binding is detected, it indicates the presence of the corresponding antigen or the autoantibody that binds to it.
  • the fourth technical solution of the present invention is: the biomarker combination as described in the first technical solution or the diagnostic kit as described in the second technical solution is prepared to detect specific proteins or antibodies in human serum. Applications of molecular reagents.
  • the reagent is used for diagnosis or auxiliary diagnosis of early stage lung cancer, or for recurrence monitoring and/or prognosis monitoring after lung cancer treatment.
  • the lung cancer is squamous cell carcinoma, small cell lung cancer, adenocarcinoma, such as invasive adenocarcinoma.
  • a lung cancer diagnosis system which includes the following modules:
  • Input module which is used to input the detection value of the biomarker combination as described in one of the technical solutions contained in the sample to be tested;
  • the lung cancer diagnosis system further includes a login module, and/or the lung cancer diagnosis system also includes a printing module; the login module requires input of a user name and password, and the printing module can Print the results generated by the input module, analysis module and judgment module;
  • test sample is serum
  • the diagnosis when the regression value (p value) ⁇ cutoff value, the diagnosis is “negative”; when the regression value > cutoff value, the diagnosis is “positive”.
  • the present invention analyzes and processes the detection results of the lung cancer autoantibody detection kit and outputs them in the form of a report, which has the characteristics of convenient data analysis and intuitive result reporting.
  • a lung cancer diagnosis device including:
  • the eighth technical solution of the present invention is: the biomarker combination as described in the first technical solution, the diagnostic kit as described in the second technical solution, and the lung cancer diagnostic system as described in the fifth technical solution.
  • the computer-readable medium as described in the sixth technical solution or the lung cancer diagnostic device as described in the seventh technical solution is used for early diagnosis of lung cancer or its prognosis or uncertainty in the identification of benign and malignant pulmonary nodules, the method It includes using the diagnostic kit as described in the second technical solution to detect the biomarker combination as described in the technical solution one in the test sample, and using the detection value with the lung cancer diagnostic system as described in the fifth technical solution, as in the technical solution.
  • the computer-readable medium described in the sixth technical solution or the judgment result of the lung cancer diagnostic device described in the seventh technical solution is: the biomarker combination as described in the first technical solution, the diagnostic kit as described in the second technical solution, and the lung cancer diagnostic system as described in the fifth technical solution.
  • the test sample is serum
  • the lung cancer is squamous cell carcinoma, small cell lung cancer, adenocarcinoma, such as invasive adenocarcinoma.
  • the antigens used in the present invention include 13 lung cancer-related antigen proteins expressed and purified from E. coli.
  • the 13 antigenic proteins all carry streptavidin and Myc tags, which are used for protein expression, purification, magnetic bead coupling and detection.
  • the surface of the magnetic beads is pre-coupled with biotinylated BSA (Biotin-BSA), and then 13 antigenic proteins are indirectly coupled to 13 different species through a highly specific affinity reaction between biotin and streptavidin. Encoded magnetic bead surface.
  • Mix 13 kinds of magnetic beads in a certain amount add diluted serum samples and incubate them.
  • the corresponding lung cancer autoantibodies in the serum specifically bind to the antigens coated on the 13 kinds of magnetic beads.
  • Phycoerythrin (R-Phycoerythrin, RPE) is added. ) labeled RPE-donkey anti-human IgG secondary antibody, and after incubation reaction, a "magnetic bead-lung cancer related antigen-autoantibody-secondary antibody" conjugate is formed. Detection is carried out on the Luminex multifunctional flow cytometer.
  • the fluorescently encoded magnetic beads are formed in a single row by the sheath flow system and irradiated by two laser beams in sequence. The fluorescent signal is received and analyzed by the instrument. One laser beam is used to identify the fluorescent code of the magnetic beads. To distinguish the type of autoantibodies being tested, another laser beam measures the fluorescence intensity on the magnetic beads.
  • the fluorescence signal on the magnetic beads is positively correlated with the concentration of autoantibodies in the serum.
  • the prepared calibrators of different concentrations are fitted with the fluorescence signal intensities corresponding to various magnetic beads to form a dose-response standard curve.
  • the concentration of various autoantibodies in the serum sample can be calculated through the standard curve equation.
  • the kit of the present invention 13 kinds of magnetic beads are mixed in a certain amount and then freeze-dried to prepare a freeze-dried powder of the coupled magnetic bead mixture. After optimizing each step separately, the preparation process is determined.
  • the affinity between biotin and avidin is strong, so once the two are combined, they are extremely stable and are not affected by the incubation and multiple washings in the kit method.
  • the binding reaction time is shorter than the time required for the antigen-antibody reaction, and has great advantages. Good specificity can better improve the coating effect of lung cancer-related antigens and magnetic beads in this kit.
  • kits of the present invention a group of antigens coupled to magnetic beads are expressed and purified through genetic engineering methods. Each antigen is tagged with the same Myc peptide.
  • This product uses recombinant humanized anti-Myc-tagged immunoglobulin (Anti-Myc) as a calibrator, which can specifically recognize all antigens with Myc tags.
  • Anti-Myc humanized anti-Myc-tagged immunoglobulin
  • Experimental results show that the reaction between Anti-Myc antibodies and the Myc peptide tag on the antigen surface is similar to the reaction between autoantibodies and antigens in positive serum.
  • the reaction curve of Anti-Myc antibody as calibrator with each antigen and the reaction curve of positive serum sample as serum calibrator with the same antigen are linearly related. Therefore, in this kit, the relative concentrations of 13 autoantibodies in serum are calculated using the standard curve of the reaction between 13 antigens and Anti-Myc antibodies.
  • the invention provides a biomarker combination, a kit containing the same and its application.
  • the detection kit contains a set of optimized and recombined 13 self-antigen protein combinations; each of the self-antigen combinations All kinds of autoantigens can detect the corresponding IgG type or IgM type autoantibodies.
  • the amino acid sequence of the antigen protein is full length or an ectopic spliced sequence; the antigen proteins are all connected with a tag peptide, and the tag peptide can be selected from: His tag , streptavidin tag, avidin tag, c-Myc tag, Flag tag, HA tag and biotin tag.
  • the positive quality control products are recombinant human anti-tag peptide immunoglobulin G, recombinant human anti-tag peptide immunoglobulin M, or Anti- Myc, or fragments of them.
  • the reagents and raw materials used in the present invention are all commercially available.
  • the present invention utilizes the advantages of autoantibody spectrum in early diagnosis and screening of lung cancer and in the judgment of benign and malignant uncertain pulmonary nodules, and uses liquid phase chip technology (flow fluorescence method) to detect the levels of autoantibodies to 13 lung cancer-related antigens.
  • liquid phase chip technology flow fluorescence method
  • liquid suspension chip technology is used to conduct high-throughput detection of lung cancer autoantibody profiles through flow fluorescence, which is superior to traditional detection technology, and the biomarker combination of the present invention and Its kit has the following advantages:
  • the product prepared by the present invention uses more biomarkers, has higher sensitivity and specificity, and can detect more types of tumor autoantibodies. Joint detection greatly improves detection accuracy.
  • the kit prepared by the method of the present invention has superior product performance: in terms of the linearity of the dose-response curve, the test results of three batches of kits in the pilot test showed that they were all in the range of 1.23 to 600U/ml. Good linear relationship, r ⁇ 0.9900. In terms of analytical sensitivity, the test results of the three batches of kits showed that they were no higher than 1.23U/ml. In terms of accuracy, the test results of three batches of kits showed that the recovery rates were all between 80 and 120%. In terms of intra-batch precision, the intra-batch precision (CV) of the three batches of kits was not higher than 15.0%. In terms of inter-batch precision, the inter-batch precision (CV) of the three batches of kits was not higher than 15%.
  • the kit of the present invention has good anti-interference ability for common interference samples such as hemolysis, jaundice, and high-lipid serum samples.
  • the kit of the present invention has reasonable design and feasible technology; the quality control system is stable and reliable; the product stability is good, the operation is simple, and the detection results are reliable and accurate. High clinical application value.
  • Figure 1 shows the concentration of antibodies for each marker in serum samples of lung cancer people, benign nodule people, and healthy people.
  • Figure 2 shows the distribution of each marker in the lung cancer group and benign lung disease group.
  • Figure 3 shows the ROC curve analysis of the lung cancer group vs. the control group.
  • FIG. 4 shows the purification profile of FXR1.
  • FIG. 5 shows the SDS-PAGE electrophoresis pattern of purified FXR1.
  • Figure 6 shows the interchangeability of magnetic beads before and after freeze-drying.
  • Figure 7 shows the determination of ESO-1 coating concentration.
  • Figure 8 shows the determination of FXR1 coating concentration.
  • Figure 9 shows the determination of EFHD2 coating concentration.
  • 10 ⁇ PBS concentrate containing 0.2% (w/v) potassium chloride, 0.2% (w/v) potassium dihydrogen phosphate, 8% (w/v) sodium chloride, 2.916% (w/v) ) disodium phosphate dodecahydrate in PBS buffer.
  • 1000ml weigh 2g potassium chloride, 2g potassium dihydrogen phosphate, 80g sodium chloride, 29.16g disodium hydrogen phosphate dodecahydrate, add purified water, stir and dissolve, dilute to 1000ml, filter with 0.45 ⁇ M membrane into a clean container. Label the material and store it at room temperature.
  • Activation buffer 0.1M sodium dihydrogen phosphate solution (pH 6.2). Take the preparation of 200ml as an example: weigh 2.40g of sodium dihydrogen phosphate, dissolve it in 150ml of purified water, adjust the pH to 6.20 ⁇ 0.05 with 3mol/L sodium hydroxide, dilute to 200ml, and filter with a 0.45 ⁇ m filter into a clean container , fill in and attach material labels, and store at 2-8°C until use.
  • Coupling buffer 50mM MES (2-[N-morpholino]ethanesulfonic acid) solution (pH 6.0). Take the preparation of 200ml as an example: weigh 1.96g MES, dissolve it in 150ml purified water, adjust the pH to 6.00 ⁇ 0.05 with 3mol/L sodium hydroxide, dilute to 200ml with purified water, and filter with a 0.45 ⁇ m filter into a clean container . Fill out and affix the material label, and store at 2-8°C until use.
  • Blocking buffer 1 ⁇ PBS buffer (pH 7.4) containing 1% BSA and 0.03% Proclin300.
  • 200ml 1 ⁇ PBS buffer (pH 7.4) containing 1% BSA and 0.03% Proclin300.
  • Wash buffer 1 ⁇ PBS buffer (pH 7.4) containing 0.05% Tween-20 and 0.03% Proclin300.
  • 200ml 1 ⁇ PBS buffer (pH 7.4) containing 0.05% Tween-20 and 0.03% Proclin300.
  • 100 ⁇ L of Tween-20 100 ⁇ L of Tween-20, 60 ⁇ l of Proclin300, and stir evenly.
  • Use 3 mol/L sodium hydroxide or 3 mol/L hydrochloric acid to adjust the pH to 7.40 ⁇ 0.05, dilute to 200 ml with purified water, and filter with a 0.45 ⁇ m filter into a clean container. Fill out and affix the material label, and store at 2-8°C until use.
  • Magnetic bead storage solution/magnetic bead lyophilization buffer (Storage buffer): 1 ⁇ PBS buffer (pH 7.4) containing 0.5% BSA, 3% trehalose, 5% mannitol, and 0.03% Proclin300.
  • Storage buffer 1 ⁇ PBS buffer (pH 7.4) containing 0.5% BSA, 3% trehalose, 5% mannitol, and 0.03% Proclin300.
  • Coating buffer 1 ⁇ PBS solution (pH 7.4). Containing 0.02% (w/v) potassium chloride, 0.02% (w/v) potassium dihydrogen phosphate, 0.8% (w/v) sodium chloride, 0.292% (w/v) disodium hydrogen phosphate dodecahydrate PBS buffer.
  • 200ml 1 ⁇ PBS solution (pH 7.4). Containing 0.02% (w/v) potassium chloride, 0.02% (w/v) potassium dihydrogen phosphate, 0.8% (w/v) sodium chloride, 0.292% (w/v) disodium hydrogen phosphate dodecahydrate PBS buffer.
  • 200ml as an example: measure 20ml of 10 ⁇ PBS solution, add 150ml of purified water and stir evenly, adjust the pH to 7.40 ⁇ 0.05 with 3mol/L sodium hydroxide or 3mol/L hydrochloric acid, and adjust the volume to 200mL with purified water. Filter through a 0.45 ⁇ m filter membrane into a clean container. Fill out
  • NHS solution activation buffer containing 5% (w/v) NHS (N-hydroxysuccinimide).
  • NHS N-hydroxysuccinimide
  • EDC solution activation buffer containing 5% (w/v) EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride).
  • EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
  • Calibrator freeze-drying buffer 1 ⁇ PBS buffer (pH 7.4) containing 1% BSA, 3% trehalose, 5% mannitol, and 0.03% Proclin300.
  • 200ml Take the preparation of 200ml as an example: weigh 2g BSA, 6g trehalose, and 10g mannitol, add an appropriate amount of purified water and stir to dissolve, add 20ml of 10 ⁇ PBS solution, add 60 ⁇ l Proclin300, and stir evenly.
  • the reagents or solutions used above are fed according to the production batch during the production process, and are produced and prepared separately in strict accordance with the production instructions and formulas.
  • QA strictly reviews the production instructions, formulas and materials for each solution. After preparation, the production date and validity period are noted. as a spare.
  • the development of this product is based on a kit for detecting lung cancer autoantibodies developed on the Luminex multifunctional flow cytometer.
  • the applicable instrument for the developed product is: Luminex multifunctional flow cytometer.
  • the coated magnetic beads selected during the development process of this product are magnetic beads provided by Luminex.
  • RPE-donkey anti-human IgG secondary antibody and RPE-goat anti-human IgG secondary antibody (Jackson ImmunoResearch), make the same dilution gradient respectively, and detect product 13 under the same conditions. Screen the appropriate source of RPE-labeled anti-human from the blank fluorescence background value of the magnetic beads coupled to the marker and the correlation linear R and the lowest detection limit of the standard curve. IgG secondary antibody.
  • 1.1 Glycerol bacteria inoculation Take out the frozen FXR1 glycerol bacteria from the -20°C refrigerator, thaw it in a 4°C refrigerator, and then transfer it to a clean workbench. Add 1 ⁇ kanamycin to the vial culture medium, then inoculate 1 ⁇ glycerol bacteria into the resistance-containing culture medium, and then place the culture medium in a constant-temperature oscillator, 220 rpm, and cultivate overnight at 37°C for 16 hours.
  • 1.2 Amplification culture The next day, inoculate the shaken bacterial solution into a large bottle of LB culture medium containing 1 ⁇ kanamycin (10ml of bacterial liquid per 1L of culture medium), continue to put it into a constant-temperature oscillator at 220 rpm, and culture at 37°C .
  • Inclusion body washing Wash the inclusion body precipitate with 2M inclusion body washing solution, 4M inclusion body washing solution and PBS respectively, and centrifuge to collect the precipitate.
  • Inclusion body dissolution Add inclusion body dissolution solution to the inclusion body precipitate, stir magnetically at 4°C for 4 hours, centrifuge the fully dissolved inclusion bodies to collect the supernatant, and filter with a 0.45um filter membrane for purification.
  • Mobility B 50mM PB+10mM Tris+8M Urea+400mM Imidazole+0.5M NaCl PH8.0
  • Activation of magnetic beads Vortex and mix the magnetic beads to be coupled by ultrasonic. Pipette the corresponding magnetic bead suspension into a centrifuge tube, place the centrifuge tube on a magnetic separator to magnetically absorb the beads, and then carefully remove the supernatant with a pipette. Remove the magnetic separator, take coupling 0.5ml magnetic beads as an example, add 500 ⁇ L activation buffer for every 0.5ml magnetic beads (approximately 12.5 ⁇ 10 6 /mL) into the tube, vortex and mix the magnetic beads evenly with ultrasound. Place the centrifuge tube on a magnetic separator to magnetically absorb the beads, and then carefully remove the supernatant with a pipette.
  • Preparation of magnetic bead mixture Take out the 13 kinds of intermediate magnetic beads that have been coupled to coated antigens and tested to be qualified. Mix according to the appropriate proportion of volume according to the concentration of each magnetic bead, and add magnetic bead lyophilization buffer so that the concentration of each detection magnetic bead in the magnetic bead mixture is not less than 3 ⁇ 10 4 /ml. Shake and ultrasonicate to mix the magnetic beads and prepare for dispensing.
  • the magnetic bead mixture before freeze-drying and the mixture after freeze-drying were detected and analyzed for verification.
  • Enterprise-level calibrators were used to prepare a standard curve and diluted into 7 standard points according to 600, 200, 66.67, 22.22, 7.41, 2.47, and 1.23U/ml. (S1 ⁇ S7), detect the magnetic beads before and after freeze-drying respectively, make duplicate wells for each concentration, compare the fluorescence value (MFI) of each indicator of the magnetic bead mixture before and after freeze-drying at 7 calibration points in the standard curve,
  • MFI fluorescence value
  • Magnetic beads coupled with Biotin-BSA were prepared through a process, and then 13 antigenic proteins were indirectly coupled to determine the optimal coating coupling concentration of the 13 antigenic proteins.
  • 13 antigenic proteins were indirectly coupled to determine the optimal coating coupling concentration of the 13 antigenic proteins.
  • the methods for determining the coating concentration are:
  • the ESO-1 coating concentration reaches saturation when it is higher than 50 ⁇ g/mL.
  • the ESO-1 coating concentration is set to 100 ⁇ g/mL.
  • the FXR1 coating concentration reaches saturation when it is higher than 100 ⁇ g/mL.
  • the FXR1 coating concentration is set to 200 ⁇ g/mL.
  • Five groups of magnetic beads with different concentrations were used to detect the five groups of magnetic beads using Anti-Myc (0, 0.62, 1.23, 3.70, 11.11, 33.33, 100, 300U/mL), and the fluorescence value (MFI) was read. The results are shown in Table 8 and As shown in Figure 9.
  • the EFHD2 coating concentration reaches saturation at 100 ⁇ g/mL.
  • the EFHD2 coating concentration is set at 200 ⁇ g/mL.
  • BRAF 400 ⁇ g/mL
  • TM4SF1 300 ⁇ g/mL
  • ZNF573 150 ⁇ g/mL
  • BMI1 200 ⁇ g/mL
  • CAGE 200 ⁇ g/mL
  • PGP9.5 300 ⁇ g/mL
  • P53 200 ⁇ g/mL
  • GBU4-5 300 ⁇ g/mL
  • MAGE A4 300 ⁇ g/mL
  • SOX2 300 ⁇ g/mL
  • the kit consists of sample diluent, calibrator diluent, washing solution, analysis buffer, RPE-donkey anti-human IgG secondary antibody concentrate, calibrator, quality control product, coupled magnetic beads, 96-well plate and other components. Each component is formulated separately, packaged independently, and then assembled into boxes. Its preparation process is summarized as follows:
  • RPE-donkey anti-human IgG secondary antibody concentrate RPE-labeled donkey anti-human IgG fluorescent antibody concentrate.
  • calibrator Prepare recombinant humanized anti-Myc immunoglobulin containing 1% BSA, protective agent, preservative and phosphate buffer, and prepare the calibrator according to the concentration of 3000U/mL, according to 0.5 mL of each bottle was packed into freeze-drying bottles and placed in a freeze-drying machine for freeze-drying.
  • Lyophilized product of a mixture of 13 magnetic beads coupled to lung cancer-related antigens phosphate buffer solution lyophilized powder containing 0.5% BSA, protective agent, and preservative.
  • the expression levels of corresponding autoantibodies in the serum of lung cancer patients were detected.
  • Each antigen marker includes BRAF, ESO-1, EFHD2, TM4SF1, ZNF573, BMI1, CAGE, FXR1, PGP9.5, P53, GBU4-5, MAGEA4 and SOX2.
  • Three groups of serum samples were tested: 102 first-time diagnosed and untreated lung cancer patients, 52 people with benign lung lesions, and 64 healthy people undergoing physical examination.
  • the autoantibodies of each marker were found in lung cancer patients, benign lung nodule patients, and And the concentrations of healthy people are shown in Figure 1 respectively.
  • the sensitivity and specificity analysis of each marker are shown in Table 9.
  • GBU4-5 is used for the detection of Chinese races. If satisfactory results cannot be achieved, more targeted markers must be screened out for large-scale testing of the Chinese population.
  • the present invention also screened out five unreported markers for lung cancer screening: TM4SF1, FXR1, ZNF573, EFHD2, and BMI1, which have high diagnostic value.
  • TM4SF1 which has the best effect, was detected in 35% of the three groups of people tested. There were 3 positive cases of lung cancer and 3 false positive cases, with a sensitivity of 34.31% and a specificity of 96.55%.
  • Control combination 01 P53, MAGEA1, GAGE7, PGP9.5, MAGEA4, Annexin1, SOX2, GBU4-5 (reference: CN103869086B, literature reports that the sensitivity of this combination using ELISA detection method is 62.5%, and the specificity is 87.3%);
  • Control combination 02 p53, MAGEA1, GAGE7, PGP9.5, MAGEA4, NY-ESO-1, Annexin1 (reference: CN103869086B, literature reports that the sensitivity of this combination using ELISA detection method is 47.5%, and the specificity is 92.7%);
  • Control combination 03 p53, NY-ESO-1, CAGE, GBU4-5, MAGE A4, HuD, SOX2-B (Reference: CJ.Chapman, GF.Healey, A Murray, et al. test: improved clinical utility through additional autoantibody assays[J].Tumour Biol.2012Oct;33(5):1319-1326.
  • the sensitivity of this combination reported in the literature is 41% and the specificity is 91%. This combination is also used ELISA detection method).
  • Control combination 04 Combination 18 (PLG, APEX1, PARP1, PGP9.5, TP53, MAGEA1, CDKN2A, SPAG9, NY-ESO-1, MAGEA4, GAGE7, EEF2, BRAF, CAGE, GBU4-5) described in CN202011301551.3 , SOX2, GAD2, ELAVL3) (this combination is a combination of 18 biomarkers.
  • the literature reports that the detection method of this combination using liquid suspension chip technology has a sensitivity of 80.0% and a specificity of 89.6%);
  • Control combination 05 Combination 42 (PLG, APEX1, PARP1, PGP9.5, TP53, MAGEA1, CDKN2A, SPAG9, NY-ESO-1, MAGEA4, GAGE7, EEF2, BRAF, CAGE, GBU4-5) described in CN202011301551.3 , SOX2, GAD2, CRYAA) (this combination is a combination of 18 biomarkers.
  • the literature reports that the detection method of this combination using liquid suspension chip technology has a sensitivity of 81.5% and a specificity of 89.6%);
  • Control combination 06 Combination 44 (PLG, PGP9.5, TP53, SPAG9, NY-ESO-1, MAGEA4, ANXA1, BRAF, CAGE, GBU4-5, SOX2, GAD2, ELAVL3) described in CN202011301551.3 (this combination It is a combination of 13 biomarkers.
  • Combination 1 of the present invention ESO-1, TM4SF1, p53, BMI1, FXR1, EFHD2;
  • Combination 2 of the present invention ESO-1, TM4SF1, p53, BMI1, FXR1, EFHD2, BRAF;
  • Combination 3 of the present invention ESO-1, TM4SF1, p53, BMI1, FXR1, EFHD2, BRAF, CAGE;
  • Combination 4 of the present invention BRAF, ESO-1, EFHD2, TM4SF1, ZNF573, BMI1, CAGE, FXR1, PGP9.5, P53;
  • Combination 5 of the present invention BRAF, ESO-1, EFHD2, TM4SF1, ZNF573, BMI1, CAGE, FXR1, PGP9.5, P53, MAGEA4;
  • Combination 6 of the present invention BRAF, ESO-1, EFHD2, TM4SF1, ZNF573, BMI1, CAGE, FXR1, PGP9.5, P53, GBU4-5, MAGEA4, SOX2.
  • control combination 01, control combination 02 and control combination 03 are respectively antigen combinations of two similar products at home and abroad.
  • the detection method is ELISA. They all assess the risk of lung cancer in patients by detecting a set of 7-8 markers. Risk, its publicly reported sensitivity ranges from 41% to 62.5%, and its specificity ranges from 87.3% to 92.7%; Control Combination 04, Control Combination 05 and Control Combination 06 are combinations obtained by the applicant during early screening.
  • the present invention uses a smaller number of marker combinations, and the sensitivity, specificity and other inspection indicators are no lower or higher than the existing marker combinations. For example, by reducing the number of markers (combination 1, 6 markers), under the condition of the smallest number of markers, the highest specificity of all combinations was obtained, while the sensitivity was not lower than the control combination 01 (8 markers) , higher than control combination 02 and control combination 03 (both have 7 markers). Thanks to the very high specificity of the six marker combinations at the core of the present invention (combination 1 of the present invention), by further increasing the number of markers, although the specificity of the detection shows a downward trend, it is still within the acceptable range. At the same time, Sensitivity has been greatly improved.
  • Combination 6 has a detection specificity of 91.4% and a sensitivity of 85.3%.
  • Combination 5 of the present invention has a detection specificity of 81.4% and a sensitivity of 92.2%.
  • the present invention uses a liquid suspension chip detection platform to conduct high-throughput joint detection of multiple markers, which has the advantage of high-throughput detection compared with the traditional ELISA method.
  • Example 8 Determination of the positive reference value of the lung cancer autoantibody detection kit (flow fluorescence immunoassay) of the present invention
  • This kit is used to semi-quantitatively detect the concentration of 13 lung cancer-related autoantibodies in human serum. Seven standard points are set in each kit to fit the dose-response curve and used to calculate the relative concentration of each autoantibody in the serum sample.
  • the relative concentration of each autoantibody detected by this kit is a continuous variable, and the detection result is a binary variable (positive or negative).
  • the relative concentration of each autoantibody is quantitative data (dependent variable), and the sample determination result is classification data (independent variable), and classification data has only two options—positive or negative, so we use the classic binary
  • the meta-logistic regression model is used as the judgment model for this product.
  • the research method for the preclinical positive judgment value of this product is to detect lung cancer patients and patients with benign lung lesions to obtain the relative concentrations of 13 lung cancer-related autoantibodies in the serum, and simultaneously detect a certain amount of healthy people as a reference.
  • a binary logistic regression model combining 13 indicators was established and the ROC curve (receiver operating characteristic curve) was performed. ) analysis, determine the best positive reference value, and make corresponding calculations for the positive judgment value, sensitivity, and specificity.
  • Kit name Thirteen types of lung cancer autoantibodies detection kit
  • Subjects clinically diagnosed with benign lung tumors including but not limited to: pulmonary cysts, inflammatory pseudotumors, etc.;
  • lung-related diseases including but not limited to: pneumonia, tuberculosis, emphysema, etc.;
  • test sample type of this kit is serum, and the required sample volume is 10 ⁇ L.
  • sample If the sample is not tested immediately, it must be stored at 2 to 8°C (within 5 days) or below -20°C (within one year). Try to use fresh serum and avoid repeated freezing and thawing. Repeated freezing and thawing should not exceed 3 times. Serum samples should be stored at room temperature (18 ⁇ 25°C) for no more than 12 hours.
  • Hemolysis or lipemia samples may affect the results, and it is recommended to re-collect and submit them for testing.
  • Frozen serum should be allowed to thaw naturally at 2-8°C before use, and should be mixed thoroughly before testing.
  • the applicable model of this kit is the Luminex System, a multifunctional flow cytometer of the American company Luminex.
  • the model is Luminex 200.
  • the registration certificate number is National Machinery Injection 20172221166. Please refer to the instrument instruction manual for on-board operation.
  • the established model determination formula is as follows:
  • the positive determination formula of this product is as follows.
  • the positive reference value is 0.45241.
  • the detection values of 13 markers in the sample are substituted into the following formula to calculate the p value. , it is judged as positive when p>cutoff, and it is judged as negative when p ⁇ cutoff
  • Kit name Thirteen types of lung cancer autoantibodies detection kit (flow fluorescence immunoassay)
  • Kit name Thirteen types of lung cancer autoantibodies detection kit (flow fluorescence immunoassay)
  • kits to test 6 positive reference sera and 6 negative reference sera respectively. Each sample is tested once. The test results of each sample are substituted into the judgment model of this kit to calculate the regression value of each sample. , when the regression value of the sample ⁇ cutoff value, it is positive, and when the regression value of the sample ⁇ cutoff value, it is negative.
  • Embodiment 12 Stability test method of the kit according to the present invention
  • kits Take three batches of kits (batch numbers: 202107001, 202108001, 202109001) and store them in the market packaging under storage conditions, and take samples for testing every 3 months.
  • the kit is placed according to the marketed packaging at 37°C for 0, 4, 7, and 9 days, and at 2 to 8°C until the end of the validity period to detect the linearity and accuracy of the dose-response curve of the kit, and the detection of quality control product I and quality control product II. , minimum detection and repeatability, all indicators tested meet the requirements, and the validity period of this detection kit is determined to be 12 months, stored at 2-8°C, protected from light, and sealed.
  • each freeze-dried component (R6 calibrator (lyophilized powder), R7 quality control product I (lyophilized powder), R8 quality control product II (lyophilized powder) were reconstituted according to the instructions. After reconstitution, they were placed in a refrigerator at 2 to 8°C. Samples were taken at 6 hours and 12 hours to detect the performance indicators of the kit (linearity, accuracy of dose-response curve, quality control product I). , quality control product II detection, minimum detection limit, repeatability), all indicators tested met the requirements. The results showed that each freeze-dried component can be refrigerated at 2 to 8°C for 12 hours after reconstitution.
  • the kit take out the kit, take out the R5RPE-donkey anti-human IgG secondary antibody concentrated solution, and prepare the RPE-donkey anti-human IgG secondary antibody working solution according to the product instructions, and place it at 2 to 8°C.
  • Refrigerator, sampling and testing kit performance indicators linearity, accuracy of dose-response curve, detection of quality control product I and quality control product II, minimum detection limit, repeatability
  • the tests all met the requirements. The results showed that the working solution of the kit should be used within 12 hours after preparation. It is recommended that the working solution be prepared and used immediately.
  • the specific test steps and test results are as follows.

Abstract

提供了一种与肺癌相关的生物标志物组合、含其的试剂盒及其用途。所述生物标志物组合包含ESO-1、TM4SF1、p53、BMI1、FXR1和EFHD2抗原或结合其的自身抗体，所述试剂盒包括用于检测所述的生物标志物组合中生物标志物的试剂。还提供了使用所述生物标志物组合或试剂盒的非诊断目的的检测方法、肺癌诊断系统、可实现所述的肺癌诊断系统功能的计算机可读介质以及肺癌诊断装置。所述生物标志物组合具有更高的敏感性和特异性，将更多种类的肿瘤自身抗体联合进行检测，从而大大提高了检测准确性。所述试剂盒，设计合理，技术可行；质量控制体系稳定可靠；产品稳定性能良好，操作简单，检测结果可靠准确，具有较高的临床应用价值。

Description

一种与肺癌相关的生物标志物组合、含其的试剂盒及其用途
本申请要求申请日为2022/7/1的中国专利申请202210775248X的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明涉及癌症诊断和治疗领域，尤其是涉及一种与肺癌相关的生物标志物组合、含其的试剂盒及其应用。
背景技术
肺癌是全球发病率和死亡率最高的癌症之一，近年来肺癌发病率逐步升高，最新的卫生统计年鉴显示，肺癌死亡率占我国恶性肿瘤死亡率的第一位。截至2017年，我国有3.2亿烟民，7.4亿人遭受二手烟暴露，每年新增70万例肺癌患者。加之人口老龄化进程、农村城镇化进程及城市现代化进程加剧，大气污染与环境污染日趋严重，不健康生活方式等导致的肺癌发病与死亡率还将进一步攀升。
尽管肺癌的治疗手段日新月异，其5年生存率仅14.1％，60％的患者在诊断后1年内死亡。统计资料显示肺癌的预后与诊断时的临床分期密切相关。0期肺癌患者术后的5年生存率可达90％以上，Ia期患者术后5年生存率为60％，而Ⅱ-Ⅳ期患者总的5年生存率则从40％下降到5％以下。由此可见肺癌早期诊断直接影响着患者的预后。然而令人遗憾的是，目前肺癌早期诊断率仅15％，0期患者一般没有任何症状，确诊时还不到肺癌患者总数的0.6％。故提高肺癌早期诊断率，争取“早期发现、早期诊断、早期治疗”，是降低患者死亡率的重要措施。
肿瘤早期诊断目的在于能够在临床体征出现之前准确的诊断出尚处于发展早期的癌症。随着医学影像技术的发展，低剂量螺旋CT被广泛用于检测肺部早期结节，是肺癌早期筛查的有效手段。但低剂量螺旋CT的问题在于其检测结果的高假阳性，而且多次CT检测还有可能加速肺癌的恶化。其他的早期肺癌检查方法包括PET、核磁共振、支气管内镜，针吸活检等检查存在无法早期筛查、有创伤、价格昂贵等问题。而CEA、CYFRA21-1等传统常规肿瘤标志物，在肿瘤早期血液中含量非常低甚至没有，目前主要用于恶性肿瘤患者病情的动态监测，不能作为早期诊断或确诊依据。病理检测是手术或活检获得病灶组织进行病理学染色分析，是目前唯一能确诊肿瘤的金标准。但这种侵入性检查手段还无法作为癌症早期诊断与筛查的检测手段。在肺癌的临床研究中，通过EGFR、KRAS、 ALK、BRAF等基因突变检测，对肺癌靶向治疗药物的个性化治疗取得了重大突破。然而，这些基因突变检测无法用于肺癌早期诊断或肺癌高危人群的筛查。使用DNA甲基化检测来进行结直肠癌的早期诊断已经有一些商业化的产品，但用于肺癌早期诊断的产品还没有上市。循环肿瘤细胞(CTC)可由肿瘤细胞自发或受外界因素干扰而从肿瘤原发病灶脱离进入外周血循环，是评价化学疗法效果，肺癌预后、病情监测的一个特别有希望的技术领域。
肺癌自身抗体谱检测具有更多优势，肿瘤特异性抗原是肿瘤发生、进展过程中细胞坏死后释放、脱落或者外分泌的特异蛋白产物。在癌症初期，机体免疫系统可识别肿瘤细胞表达的肿瘤特异性抗原，产生针对这些抗原的自身抗体。在早期肺癌及癌前患者血液中，自身抗体水平远高于抗原水平，自身抗体具有免疫监视、免疫放大、循环扩散的特点，并且自身抗体相对于其他肿瘤抗原标志物及DNA标志物具有：在血清中出现的时间早、血液检测时间窗长、血液信号强度高、早期检测灵敏度高等特点。因此将多个自身抗体标志物联合检测是获取高敏感性和高特异性检测的手段。
目前市场上，Oncimmune公司的
Figure PCTCN2022136007-appb-000001
产品及杭州凯保罗生物科技有限公司的七种自身抗体检测试剂盒(酶联免疫法)是由一组7个相关自身抗体肿瘤标志物联合检测的产品，采用酶联免疫(ELISA)检测方法，用该检测作为肺部恶性肿瘤CT检查的一种补偿手段，辅助诊疗专家进行判定。其采用ELISA检测方法不具有高通量的特点，而且此2个产品用于早期肺癌的检测时的敏感性或特异性有待提高。
发明内容
为解决现有技术中早期肺癌诊断或其预后的生物标志物组合或试剂盒敏感性和特异性低的技术问题，提供一种生物标志物组合、含其的试剂盒及其应用。
为解决上述技术问题，本发明的技术方案之一为：一种与肺癌相关的生物标志物组合，所述生物标志物组合包含ESO-1、TM4SF1、p53、BMI1、FXR1和EFHD2抗原或结合其的自身抗体。
如技术方案之一所述的生物标志物组合，所述生物标志物组合还包含BRAF和/或CAGE抗原或结合其的自身抗体。
在本发明的较佳实施方案中，所述生物标志物组合还包含PGP9.5和/或ZNF573抗原或结合其的自身抗体，优选还包含GBU4-5抗原或结合其的自身抗体。
在本发明的更佳实施方案中，所述生物标志物组合还包含MAGE A4和/或SOX2抗原或结合其的自身抗体。
在本发明的进一步更佳实施方案中，所述生物标志物组合为血清中的生物标志物组合，其选自以下任一组：
1)ESO-1自身抗体、TM4SF1自身抗体、p53自身抗体、BMI1自身抗体、FXR1自身抗体和EFHD2自身抗体；
2)ESO-1自身抗体、TM4SF1自身抗体、p53自身抗体、BMI1自身抗体、FXR1自身抗体、EFHD2自身抗体和BRAF自身抗体；
3)ESO-1自身抗体、TM4SF1自身抗体、p53自身抗体、BMI1自身抗体、FXR1自身抗体、EFHD2自身抗体、BRAF自身抗体和CAGE自身抗体；
4)BRAF自身抗体、ESO-1自身抗体、EFHD2自身抗体、TM4SF1自身抗体、ZNF573自身抗体、BMI1自身抗体、CAGE自身抗体、FXR1自身抗体、PGP9.5自身抗体和P53自身抗体；
5)BRAF自身抗体、ESO-1自身抗体、EFHD2自身抗体、TM4SF1自身抗体、ZNF573自身抗体、BMI1自身抗体、CAGE自身抗体、FXR1自身抗体、PGP9.5自身抗体、P53自身抗体和MAGEA4自身抗体；
6)BRAF自身抗体、ESO-1自身抗体、EFHD2自身抗体、TM4SF1自身抗体、ZNF573自身抗体、BMI1自身抗体、CAGE自身抗体、FXR1自身抗体、PGP9.5自身抗体、P53自身抗体、GBU4-5自身抗体、MAGEA4自身抗体和SOX2自身抗体。
为解决上述技术问题，本发明的技术方案之二为：一种诊断试剂盒，所述诊断试剂盒包括用于检测如技术方案之一所述的生物标志物组合中生物标志物的试剂，例如用于抗原检测的检测自身抗体，或用于自身抗体检测的抗原。
在本发明的较佳实施方案中，所述生物标志物组合中，所述抗原或检测自身抗体还含有标签肽。
在本发明的优选实施方案中，所述标签肽包括：His标签、streptavidin标签、avidin标签、生物素标签、GST标签、C-myc标签、Flag标签和HA标签中的一种或多种。
在本发明的更优选实施方案中，所述生物标志物可由大肠杆菌、酵母、昆虫细胞或动物细胞表达；和/或，所述生物标志物通过Ni亲和层析、离子交换层析、分子筛、透析、超滤或疏水层析纯化。
在本发明的更佳实施方案中，所述用于检测所述生物标志物组合中生物标志物的试剂选自以下任一组：
1)ESO-1抗原、TM4SF1抗原、p53抗原、BMI1抗原、FXR1抗原和EFHD2抗原；
2)ESO-1抗原、TM4SF1抗原、p53抗原、BMI1抗原、FXR1抗原、EFHD2抗原和 BRAF抗原；
3)ESO-1抗原、TM4SF1抗原、p53抗原、BMI1抗原、FXR1抗原、EFHD2抗原、BRAF抗原和CAGE抗原；
4)BRAF抗原、ESO-1抗原、EFHD2抗原、TM4SF1抗原、ZNF573抗原、BMI1抗原、CAGE抗原、FXR1抗原、PGP9.5抗原和P53抗原；
5)BRAF抗原、ESO-1抗原、EFHD2抗原、TM4SF1抗原、ZNF573抗原、BMI1抗原、CAGE抗原、FXR1抗原、PGP9.5抗原、P53抗原和MAGEA4抗原；
6)BRAF抗原、ESO-1抗原、EFHD2抗原、TM4SF1抗原、ZNF573抗原、BMI1抗原、CAGE抗原、FXR1抗原、PGP9.5抗原、P53抗原、GBU4-5抗原、MAGEA4抗原和SOX2抗原。
如技术方案之二所述的诊断试剂盒，所述检测抗原或检测自身抗体与磁珠偶联，形成偶联磁珠，所述偶联磁珠混合后形成偶联磁珠混合物，以抗原偶联磁珠为例，偶联磁珠的制备方法包含以下步骤：
(1)磁珠的激活：将待偶联的磁珠震荡混匀，使用激活缓冲液洗涤磁珠，并依次加入NHS溶液和EDC溶液，震荡混匀后，旋转混合孵育，制得激活的磁珠；较佳地，所述激活缓冲液为0.1M磷酸二氢钠溶液，所述NHS溶液为含5％(w/v)N-羟基琥珀酰亚胺的激活缓冲液，所述EDC溶液为含5％(w/v)1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐的激活缓冲液；
(2)Biotin-BSA的偶联：将步骤(1)中激活的磁珠用偶联缓冲液洗涤后，加入BSA-biotin混匀并孵育，加入洗涤液洗涤后，加入封闭缓冲液，混匀孵育，制得偶联Biotin-BSA的磁珠；较佳地，所述偶联缓冲液为50mM 2-[N-吗啉代]乙烷磺酸溶液，所述洗涤液为含0.05％(w/v)吐温-20、0.03％(v/v)Proclin300的1×PBS缓冲液，所述封闭缓冲液为含1％(w/v)BSA、0.03％(v/v)Proclin300的1×PBS缓冲液；所述BSA-biotin的浓度为2mg/mL；
(3)抗原的包被：将步骤(2)中偶联Biotin-BSA的磁珠用洗涤液洗涤，加入相应的所述抗原，混匀并孵育，再用洗涤液洗涤，加入磁珠保存液保存，制得偶联磁珠；较佳地，所述洗涤液为含0.05％(w/v)吐温-20、0.03％(v/v)Proclin300的1×PBS缓冲液，所述磁珠保存液为含0.5％(w/v)BSA、3％(w/v)海藻糖、5％(w/v)甘露醇、0.03％(v/v)Proclin300的1×PBS缓冲液；更佳地，BRAF抗原的包被浓度为400μg/mL、ESO-1抗原的包被浓度为100μg/mL、EFHD2抗原的包被浓度为200μg/mL、TM4SF1抗原的包被浓度为300μg/mL、ZNF573抗原的包被浓度为150μg/mL、BMI1抗原的包被浓度为 200μg/mL、CAGE抗原的包被浓度为200μg/mL、FXR1抗原的包被浓度为200μg/mL、PGP9.5抗原的包被浓度为300μg/mL、P53抗原的包被浓度为200μg/mL、GBU4-5抗原的包被浓度为300μg/mL、MAGEA4抗原的包被浓度为300μg/mL和/或SOX2抗原的包被浓度为300μg/mL。
在本发明的优选实施方案中，所述偶联磁珠的制备方法还包含：
(4)偶联磁珠混合物的冻干：将步骤(3)中偶联磁珠混合，加入磁珠冻干缓冲液，混匀后冻干；较佳地，所述磁珠冻干缓冲液为含0.5％(w/v)BSA、3％(w/v)海藻糖、5％(w/v)甘露醇、0.03％(v/v)Proclin300的1×PBS缓冲液。
其中，所述v/v为体积百分比，所述w/v为质量体积比。
如技术方案之二所述的诊断试剂盒，所述诊断试剂盒还包括样品稀释液、校准品稀释液、洗涤液、分析缓冲液、抗人IgG二抗、校准品、质控品中的一种或多种；所述诊断试剂盒优选还包括96孔板。
在本发明的较佳实施方案中：
所述样品稀释液为含2％(v/v)驴血清、0.5％(w/v)BSA的磷酸盐缓冲液，优选还含有防腐剂；
所述校准品稀释液为含1％(w/v)BSA的磷酸盐缓冲液，优选还含有防腐剂；
所述洗涤液为含Tween20的PBST洗涤液；
所述分析缓冲液为含0.2％(w/v)BSA的磷酸盐缓冲液，优选还含有防腐剂；
所述抗人IgG二抗为RPE标记的驴抗人IgG荧光抗体；
所述校准品为含1％(w/v)BSA及磷酸盐缓冲液的重组人源化Anti-Myc标签的免疫球蛋白；优选地，所述Anti-Myc的浓度为3000U/mL，所述校准品还含有保护剂和/或防腐剂，和/或，所述校准品被制成冷冻干燥品；和/或，
所述质控品包括质控品I和质控品II，所述质控品I浓度为含200U/ml Anti-Myc、含1％(w/v)BSA的磷酸盐缓冲液，所述质控品II浓度为含7.41U/ml Anti-Myc、含1％(w/v)BSA的磷酸盐缓冲液；优选地，所述质控品I和所述质控品II还含有保护剂和/或防腐剂，和/或，所述质控品I和所述质控品II被制成冷冻干燥品。
在本发明的优选实施方案中，所述保护剂为含1％(w/v)BSA、3％(w/v)海藻糖、5％(w/v)甘露醇、0.03％(v/v)Proclin300的1×PBS缓冲液。
其中，所述v/v为体积百分比，所述w/v为质量体积比。
为解决上述技术问题，本发明的技术方案之三为：一种非诊断目的的检测方法，所述检测方法使用如技术方案之一所述的生物标志物组合或如技术方案之二所述的诊断试 剂盒对相应的抗原或结合其的自身抗体进行检测，具体步骤是：使所述生物标志物组合接触样品，若检测到结合，则说明相应的抗原或结合其的自身抗体的存在。
为解决上述技术问题，本发明的技术方案之四为：如技术方案之一所述的生物标志物组合或如技术方案之二所述的诊断试剂盒在制备检测人血清中特定的蛋白或抗体分子的试剂中的应用。
在本发明的较佳实施方案中，所述试剂用于诊断或辅助诊断早期肺癌，或用于经肺癌治疗后的复发监测和/或预后监测。
在本发明的更佳实施方案中，所述肺癌为鳞癌、小细胞肺癌、腺癌例如浸润性腺癌。
为解决上述技术问题，本发明的技术方案之五为：一种肺癌诊断系统，所述肺癌诊断系统包括以下模块：
(1)输入模块，其用于输入待测样本中所含的如技术方案之一所述生物标志物组合的检测值；
(2)分析模块，其用于计算p，其中
Figure PCTCN2022136007-appb-000002
X＝－4.672652+0.000868*TM4SF1+0.000273*p53+0.001453*PGP9.5+0.00033*SO X2+0.000075*BMI1+0.000101*FXR1+0.000835*MAGE A4+0.002701*ESO1+0.001060*ZNF573+0.001213*BRAF+0.000082*GBU4-5－0.000002*EFHD2+0.000141*CAGE；
(3)判断模块，cutoff为0.45241；当p＞cutoff时判定为阳性，p≤cutoff时判定为阴性；
在本发明的较佳实施方案中，所述肺癌诊断系统还包括登录模块，和/或所述肺癌诊断系统还包括打印模块；所述登录模块需要输入用户名称和用户密码，所述打印模块可打印输入模块、分析模块和判断模块产生的结果；
在本发明的更佳实施方案中，所述测试样本为血清。
根据本发明的实施例，本发明中，当回归值(p值)≤cutoff值，则诊断为“阴性”；当回归值＞cutoff值，则诊断为“阳性”。本发明将所述的肺癌自身抗体检测试剂盒的检测结果进行分析处理并以报告的形式输出，具有数据分析便捷、结果报告直观等特点。
为解决上述技术问题，本发明的技术方案之六为：一种计算机可读介质，所述的计算机可读介质存储了计算机程序，所述计算机程序被处理器执行时可实现如技术方案之五所述的肺癌诊断系统的功能。
为解决上述技术问题，本发明的技术方案之七为：一种肺癌诊断装置，包括：
(1)如技术方案之六所述的计算机可读介质；
(2)处理器，用于执行计算机程序以实现所述肺癌诊断系统的功能。
为解决上述技术问题，本发明的技术方案之八为：如技术方案之一所述的生物标志物组合、如技术方案之二所述的诊断试剂盒、如技术方案之五所述肺癌诊断系统、如技术方案之六所述的计算机可读介质或如技术方案之七所述的肺癌诊断装置用于早期肺癌诊断或其预后或不确定性肺小结节良恶性鉴别的方法，所述方法包含使用如技术方案之二所述的诊断试剂盒检测测试样本中如技术方案之一所述的生物标志物组合，并将检测值用如技术方案之五所述肺癌诊断系统、如技术方案之六所述的计算机可读介质或如技术方案之七所述的肺癌诊断装置判断结果。
在本发明的更佳实施方案中，所述测试样本为血清，和/或，所述肺癌为鳞癌、小细胞肺癌、腺癌例如浸润性腺癌。
本研究对100多种肿瘤相关标志物抗原进行了E.coli基因工程菌重组表达，将BRAF、ESO-1、EFHD2、TM4SF1、ZNF573、BMI1、CAGE、FXR1、PGP9.5、P53、GBU4-5、MAGEA4、SOX2这13个抗原蛋白，按照CN202011301551.3所述的在E.coli表达方法，SDS-PAGE分析结果说明各个抗原进行表达，可以获得稳定表达的基因工程菌种，经进一步纯化获得可用于分析检测的目的蛋白。
本发明使用的抗原包括13个从E.coli表达纯化的肺癌相关抗原蛋白。13个抗原蛋白均带有链霉亲和素和Myc标签，用于蛋白的表达、纯化、磁珠偶联和检测。磁珠表面预先偶联有生物素化的BSA(Biotin-BSA)，然后通过生物素与链霉亲和素之间的高特异性亲和反应将13个抗原蛋白分别间接偶联到13种不同编码的磁珠表面。将13种磁珠以一定数量混合，加入稀释好的血清样本孵育后，血清中对应的肺癌自身抗体与13种磁珠上分别包被的抗原特异结合，加入藻红蛋白(R-Phycoerythrin，RPE)标记的RPE-驴抗人IgG二抗后，经孵育反应，形成“磁珠-肺癌相关抗原-自身抗体-二抗”结合物。在Luminex多功能流式点阵仪上进行检测，荧光编码的磁珠由鞘流系统形成单列依次通过两束激光照射，荧光信号被仪器接收分析，一束激光用于识别磁珠的荧光编码从而区分被测自身抗体的类别，另一束激光测定磁珠上的的荧光强度，磁珠上所带的荧光信号与血清中的自身抗体浓度正相关。经配制的不同浓度的校准品分别与各种磁珠对应的荧光信号强度拟合成剂量-反应标准曲线，即可通过标准曲线方程计算得出血清样本中各种自身抗体的浓度。
本发明的试剂盒中，将13种磁珠以一定数量混合后，通过冷冻干燥，制备偶联磁珠混合物的冻干粉。分别对各个步骤进行优化后，确定制备工艺。生物素和亲和素间亲和力强，故二者一旦结合，就极为稳定，不受试剂盒方法中的孵育及多次洗涤影响，而且这 种结合反应时间比抗原抗体反应所需时间短，具有很好的特异性，能更好的提高本试剂盒中肺癌相关抗原与磁珠的包被效果。
本发明的试剂盒中，磁珠偶联的一组抗原是通过基因工程方法表达并纯化。每个抗原都带有同一个Myc肽段标签。本产品采用重组人源化抗Myc标签的免疫球蛋白(Anti-Myc)作为校准品，可特异性的识别所有带有Myc标签的抗原。实验结果表明，Anti-Myc抗体与抗原表面的Myc肽段标签反应和阳性血清中自身抗体与抗原反应特点相似。而且，以Anti-Myc抗体为校准品与每一种抗原的反应曲线和阳性血清样本作为血清校准品与同一个抗原的反应曲线是线性相关的。因此本试剂盒中，用13种抗原分别与Anti-Myc抗体反应的标准曲线，分别计算血清中13种自身抗体的相对浓度。
本发明提供了一种生物标志物组合、含其的试剂盒及其应用，所述检测试剂盒包含一组经过优化重组的13个自身抗原蛋白的组合；所述的自身抗原组合中的每一种自身抗原均可检测相应IgG型或IgM型自身抗体，抗原蛋白的氨基酸序列为全长或经异位剪切的序列；抗原蛋白均接有标签肽，所述标签肽可以选自：His标签、streptavidin标签、avidin标签，c-Myc标签、Flag标签、HA标签和生物素标签，阳性质控品为重组人抗标签肽免疫球蛋白G、重组人抗标签肽免疫球蛋白M、或Anti-Myc，或者是它们的片段。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明利用自身抗体谱在肺癌早诊及筛查中和不确定性肺结节良恶性判断中的优势，采用液相芯片技术(流式荧光法)检测13个肺癌相关抗原自身抗体的水平，利用抗原-抗体反应的高特异性和高敏感性特点，采用液态悬浮芯片技术，通过流式荧光进行肺癌自身抗体谱高通量检测，优于传统检测技术，且本发明的生物标志物组合及其试剂盒，具有以下优势：
(1)高敏感性和/或高特异性。具体地，本发明所制备得到的产品与国内外ELISA法检测7个自身抗体相比，采用了更多的生物标志物，具有更高的敏感性和特异性，将更多种类的肿瘤自身抗体联合进行检测，从而大大提高了检测准确性。
(2)利用本发明所述的方法制备得到的试剂盒，其产品性能优越：在剂量-反应曲线的线性方面，中试三批试剂盒检测结果显示，均在1.23～600U/ml范围内呈良好线性关系，r≥0.9900。在分析灵敏度方面，三批试剂盒检测结果显示，均不高于1.23U/ml。在准确度方面，三批试剂盒检测结果显示，回收率均在80～120％之间。在批内精密度方面，三批试剂盒批内精密度(CV)均不高于15.0％。在批间精密度方面，三批试剂盒批间精 密度(CV)均不高于15％。在阴性参考品符合率方面，检测6例阴性参考品，三批试剂盒结果均为阴性。在阳性参考品符合率方面，检测6例阳性参考品，三批试剂盒结果均为阳性。在稳定性方面，37℃放置3天或者按规定条件放置至有效期末，三批试剂盒检验结果均符合质量标准规定。
(3)本发明所述的试剂盒，对常见的干扰样本如溶血、黄疸、及高脂血清样本，具有良好的抗干扰能力。
(4)与目前市场上常用的同类试剂盒相比，本发明所述的试剂盒，其设计合理，技术可行；质量控制体系稳定可靠；产品稳定性能良好，操作简单，检测结果可靠准确，具有较高的临床应用价值。
附图说明
图1为各标志物的抗体在肺癌人群、良性结节人群以及健康人群血清标本中的浓度。
图2为各标志物在肺癌组和良性肺病组中的分布。
图3为肺癌组vs对照组的ROC曲线分析。
图4为FXR1的纯化图谱。
图5为纯化后FXR1的SDS-PAGE电泳图。
图6为磁珠冻干前、后的互换性。
图7为ESO-1包被浓度的确定。
图8为FXR1包被浓度的确定。
图9为EFHD2包被浓度的确定。
具体实施方式
为了便于理解本发明，下面将参照相关附图对本发明进行更全面的描述。附图中给出了本发明的较佳实施例。但是，本发明可以以许多不同的形式来实现，并不限于本文所描述的实施例。相反地，提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。
除非另有定义，本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本发明。本文所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
本发明所用试剂或溶液的配制：
(1)10×PBS浓缩液：含0.2％(w/v)氯化钾、0.2％(w/v)磷酸二氢钾，8％(w/v)氯化钠、2.916％(w/v)十二水合磷酸氢二钠的pbs缓冲液。以配制1000ml为例：称取2g氯化钾、2g磷酸二氢钾，80g氯化钠、29.16g十二水合磷酸氢二钠，加入纯化水搅拌溶解，定容至1000ml，0.45μM滤膜过滤至洁净容器中。并贴上物料标签，常温保存。
(2)激活缓冲液(Activation buffer)：0.1M磷酸二氢钠溶液(pH6.2)。以配制200ml为例：称取2.40g磷酸二氢钠，溶解于150ml纯化水中，用3mol/L的氢氧化钠调pH至6.20±0.05，定容至200ml，0.45μm滤膜过滤至洁净容器中，填写并贴上物料标签，2-8℃保存待用。
(3)偶联缓冲液(Coupling buffer)：50mM MES(2-[N-吗啉代]乙烷磺酸)溶液(pH6.0)。以配制200ml为例：称取1.96g MES，溶解于150ml纯化水中，用3mol/L的氢氧化钠调pH至6.00±0.05，用纯化水定容至200ml，0.45μm滤膜过滤至洁净容器中。填写并贴上物料标签，2-8℃保存待用。
(4)封闭缓冲液(Blocking buffer)：含1％BSA、0.03％Proclin300的1×PBS缓冲液(pH 7.4)。以配制200ml为例：称取BSA 2g，加入150ml纯化水搅拌溶解，加入10×PBS溶液20ml，加入60μl Proclin300，搅拌均匀。用3mol/L的氢氧化钠或3mol/L的盐酸调pH至7.40±0.05，用纯化水定容至200ml，0.45μm滤膜过滤至洁净容器中。填写并贴上物料标签，2-8℃保存待用。
(5)洗涤液(Wash buffer)：含0.05％吐温-20、0.03％Proclin300的1×PBS缓冲液(pH 7.4)。以配制200ml为例：量取10×PBS溶液20ml，加入150ml纯化水，加入100μL吐温-20，60μl Proclin300，搅拌均匀。用3mol/L的氢氧化钠或3mol/L的盐酸调pH至7.40±0.05，用纯化水定容至200ml，0.45μm滤膜过滤至洁净容器中。填写并贴上物料标签，2-8℃保存待用。
(6)磁珠保存液/磁珠冻干缓冲液(Storage buffer)：含0.5％BSA、3％海藻糖、5％甘露醇、0.03％Proclin300的1×PBS缓冲液(pH 7.4)。以配制200ml为例：称取BSA 1g，加入150ml纯化水搅拌溶解，加入10×PBS溶液20ml，搅拌均匀。加入6g海藻糖，10g甘露醇，搅拌溶解。加入60μl Proclin300，搅拌均匀。用纯化水定容至200ml，0.45μm滤膜过滤至洁净容器中。填写并贴上物料标签，2-8℃保存待用。
(7)包被缓冲液：1×PBS溶液(pH 7.4)。含0.02％(w/v)氯化钾、0.02％(w/v)磷酸二氢钾，0.8％(w/v)氯化钠、0.292％(w/v)十二水合磷酸氢二钠的pbs缓冲液。以配制200ml为例：量取10×PBS溶液20ml，加入150ml纯化水搅拌均匀，用3mol/L的氢氧化钠或3mol/L的盐酸调pH至7.40±0.05，用纯化水定容至200mL，0.45μm滤膜 过滤至洁净容器中。填写并贴上物料标签，2-8℃保存待用。
(8)50mg/mL NHS溶液：含5％(w/v)NHS(N-羟基琥珀酰亚胺)的激活缓冲液。以配制5ml为例：称取NHS 0.25g，溶解于5ml的激活缓冲液中，分装成500μl/支，填写并贴上物料标签，2-8℃保存待用。
(9)50mg/mL EDC溶液：含5％(w/v)EDC(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)的激活缓冲液。以配制5ml为例：称取EDC 0.25g，溶解于5ml的激活缓冲液中，该试剂为一次性使用，现配现用。
(10)校准品冻干缓冲液：含1％BSA、3％海藻糖、5％甘露醇、0.03％Proclin300的1×PBS缓冲液(pH 7.4)。以配制200ml为例：称取2g BSA、6g海藻糖，10g甘露醇，加入适量纯化水搅拌溶解，加入10×PBS溶液20ml，加入60μl Proclin300，搅拌均匀。用3mol/L的氢氧化钠或3mol/L的盐酸调pH至7.40±0.05。用纯化水定容至200ml，0.45μm滤膜过滤。
以上使用的试剂或溶液在生产过程中按照生产批量投料，严格按照生产指令配方单独生产配制，每个溶液在配制过程中QA严格审核生产指令及配方与投料，配制后并注明生产日期与有效期以备用。
包被用磁珠的筛选：
本产品的开发是基于在Luminex多功能流式点阵仪上开发的检测肺癌自身抗体的试剂盒。开发的产品适用仪器为：Luminex多功能流式点阵仪。为适用配套仪器，本产品开发过程中选用的包被磁珠为Luminex公司提供的磁珠。
实施例1 RPE标记的抗人IgG二抗的筛选
1、实验方案
选择2个不同来源的RPE标记的抗人IgG二抗：RPE-驴抗人IgG二抗及RPE-山羊抗人IgG二抗(Jackson ImmunoResearch)，分别做同样的稀释梯度，同一条件下检测产品13种标志物偶联的磁珠的空白荧光背景值及标曲的相关线性R与最低检测限，从空白荧光背景值及标曲的相关线性R与最低检测限筛选合适来源的RPE标记的抗人IgG二抗。
2、实验结果
检测结果参见表1和表2。
表1.RPE驴抗人IgG二抗筛选试验检测结果
检测标志物名称 标曲相关线性R 最低检测限(U/mL) 空白荧光背景值
p53 0.992 1.08 17
PGP9.5 0.993 1.04 20
TM4SF1 0.991 1.15 20
SOX2 0.997 1.18 52
MAGE A4 0.997 1.08 17
ESO1 0.994 1.09 21
FXR1 0.993 1.14 15
EFHD2 0.992 1.12 19
BRAF 0.993 1.08 16
GBU4-5 0.996 1.10 17
ZNF573 0.997 1.20 33
BMI1 0.993 1.03 21
CAGE 0.995 1.12 16
表2.RPE-山羊抗人IgG二抗筛选试验检测结果
Figure PCTCN2022136007-appb-000003
3、分析及结论
结果提示，从Jackson筛选的2个不同种属来源的二抗，驴来源及山羊来源的二抗经检测标曲都具有较好的线性相关性，R都≥0.990，最低检测限都不高于1.50U/ml，但是RPE-驴抗人IgG二抗相比RPE-山羊抗人IgG二抗在检测时具有更好的荧光背景(整体荧光背景值更低)，故本试剂盒选用RPE-驴抗人IgG二抗。
实施例2抗原蛋白的生产操作过程
1、菌体培养
1.1甘油菌接种：从-20℃冰箱取出冻存的FXR1甘油菌，放在4℃冰箱融化，再转移 到洁净工作台。往小瓶培养基加入1‰卡那霉素，然后取1‰甘油菌接种于含抗性培养基中，再把培养基放到恒温振荡器中，220rpm，37℃过夜培养16h。
1.2放大培养：次日将摇好的菌液接种到含1‰卡那霉素的大瓶LB培养基中(每1L培养基接入10ml菌液)，继续放入恒温振荡器220rpm，37℃培养。
1.3诱导表达：培养2h后，在洁净工作台内取样1ml在紫外分光光度计测量OD600，OD600值达到0.6～1.2之间就可以往菌液加入1‰IPTG，然后继续放进恒温振荡器中37℃诱导培养4h。
2、菌体处理
2.1收集菌体：将菌液进行离心，离心机设置参数5000rpm，15min，弃上清，收集菌体。
2.2菌体超声破碎：往收集到的菌体中加入Lysis buffer(1g菌体加入10ml Lysis buffer)，将菌体混匀。300W冰浴超声破碎15min。超声结束后，用离心机4℃、12000rpm、离心15min，弃上清，收集包涵体沉淀。
2.2包涵体洗涤：将包涵体沉淀依次分别采用2M包涵体洗涤液、4M包涵体洗涤液及PBS进行洗涤，并离心收集沉淀，
2.3包涵体溶解：往包涵体沉淀加入包涵体溶解液，4℃条件磁力搅拌4h，将充分溶解好的包涵体离心收集上清，并使用0.45um滤膜过滤，待纯化。
3、纯化
3.1设备：蛋白纯化仪(AKTA prime plus)、层析柱(φ＝25mm，H＝20cm)、层析填料(GE Ni Sepharose TM 6Fast Flow)
3.2流动相配制：
流动性A：50mM PB+10mM Tris+8M尿素+10mM咪唑+0.15M NaCl PH8.0
流动性B：50mM PB+10mM Tris+8M尿素+400mM咪唑+0.5M NaCl PH8.0
3.3层析纯化：用A液平衡层析柱。开始上样，上样流速设置5.0ml/min，上样完成后梯度洗脱收集目的蛋白。关于FXR1的纯化色谱图见图4。纯化后目的蛋白的SDS-PAGE电泳图见图5中A所示。
4、蛋白复性
将纯化收集的目的蛋白加入到透析袋，采用梯度透析复性，先放入4M尿素PBS溶液中，4℃搅拌透析8h。之后再把蛋白液放入2M尿素PBS溶液中4℃搅拌透析12h。之后把透析的蛋白溶液放入PBS缓冲液搅拌透析复性8h。复性后目的蛋白的SDS-PAGE电泳图见图5中B所示。
同理，用本发明类似的方法，可制备其余12种生物标志物BRAF、ESO-1、EFHD2、TM4SF1、ZNF573、BMI1、CAGE、PGP9.5、P53、GBU4-5、MAGEA4、SOX2的抗原蛋白。
实施例3偶联磁珠混合物的制备工艺
1、磁珠的激活：将待偶联的磁珠，经涡旋震荡并超声混匀。吸取相应的磁珠混悬液至离心管，将离心管置于磁力分离器上磁性吸附磁珠，然后用移液器小心移除上清。移走磁力分离器，以偶联0.5ml磁珠为例，往管内按每0.5ml磁珠(约12.5×10 6个/mL)加入500μL激活缓冲液，涡旋震荡并超声混匀磁珠。将离心管置于磁力分离器上磁性吸附磁珠，然后用移液器小心移除上清。重复此步骤，将磁珠用激活缓冲液洗涤共两次。移走磁力分离器，往管内加入400μL激活缓冲液，涡旋震荡并超声混匀磁珠。往管内加入50μL的50mg/mL的NHS溶液，涡旋震荡混匀磁珠。往管内加入50μL的50mg/mL的EDC溶液，立即涡旋震荡混匀磁珠；将离心管置于旋转混合仪，室温、避光、20rpm旋转混合孵育20分钟。
2、Biotin-BSA的偶联：
(1)将上步骤激活后磁珠的离心管置于磁力分离器上吸附磁珠，然后用移液器小心移除上清。往管内加入0.5mL的偶联缓冲液，涡旋震荡并超声混匀磁珠。重复此步骤两次。将磁珠用偶联缓冲液洗涤共三次。将含有偶联缓冲液的磁珠置于磁力分离器上吸附磁珠，然后用移液器小心移除上清。
(2)往管内加入970μL的偶联缓冲液，涡旋震荡并超声混匀磁珠。往管内加入30μl的BSA-biotin(2mg/mL)，涡旋震荡混匀磁珠。将离心管置于旋转混合仪，室温、避光、20rpm旋转混合孵育2小时。
(3)将孵育后磁珠的离心管放在磁力分离器上吸附磁珠，然后用移液器小心移除上清。移走磁力分离器，加入1mL的洗涤液，涡旋震荡并超声混匀磁珠。将离心管放在磁力分离器上吸附磁珠，然后用移液器小心移除上清。移走磁力分离器并加入1ml的封闭缓冲液，涡旋震荡并超声混匀磁珠。将离心管置于旋转混合仪，室温、避光、20rpm旋转混合孵育1小时。
3、抗原的包被
(1)将上步骤的磁珠离心管，放在磁力分离器上吸附磁珠，然后用移液器小心移除上清。移开磁力分离器，往管内加入1ml的洗涤液，涡旋震荡并超声混匀磁珠。将离心管短暂离心，以甩下管壁残留液体后，将离心管置于磁力分离器上吸附磁珠，然后用移 液器小心移除上清。
(2)管移开磁力分离器，按照表1所述的磁珠编码与包被抗原表，往管内加入相应的抗原。涡旋震荡并超声混匀磁珠。将离心管置于旋转混合仪，室温、避光、20rpm旋转混合孵育1小时。
(3)将离心管置于磁力分离器上吸附磁珠，然后用移液器小心移除上清。移开磁力分离器，往离心管中加入2ml洗涤液，涡旋震荡并超声混匀磁珠。将离心管置于磁力分离器上吸附磁珠，然后用移液器小心移除上清。重复此步骤，将磁珠洗涤共三次。
(4)移开磁力分离器，往离心管中加入2ml磁珠保存液，涡旋震荡并超声混匀磁珠，将离心管避光暂存于2-8℃。吸取少量磁珠进行计数后，根据计数结果，使用磁珠保存液将磁珠浓度调整至2.0×10 6个/ml。置于-20℃冰箱暂存。
4、偶联磁珠混合物的冻干
(1)磁珠混合液配制：取出偶联包被抗原并检验合格的13种磁珠中间品。根据每种磁珠的浓度按合适的比例体积混合，加入磁珠冻干缓冲液，使磁珠混合液中每种检测磁珠的浓度不低于3×10 4个/ml。震荡并超声混匀磁珠后准备分装。
(2)磁珠混合液分装与冻干：将磁珠混合液混匀后，按标示量分装至冻干瓶，将冻干瓶转移至冻干机箱体进行冻干，冻干完成后，进行压塞、旋盖、贴签。
5、偶联磁珠混合物冻干效果验证
将冻干前磁珠混合物与冻干后混合物进行检测分析进行验证，采用企业级校准品准备标准曲线，按照600、200、66.67、22.22、7.41、2.47、1.23U/ml稀释成7个标准点(S1～S7)，分别对冻干前后磁珠进行检测，每个浓度作复孔，比较标准曲线中的7个校准点的冻干前后磁珠混合物每个指标的荧光值(MFI)检测，检测结果如下表3和表4：
表3冻干后磁珠检测结果
Figure PCTCN2022136007-appb-000004
Figure PCTCN2022136007-appb-000005
表4冻干前磁珠检测结果
Figure PCTCN2022136007-appb-000006
计算冻干后磁珠与冻干前磁珠对7个校准点检测的荧光值(MFI)的变异系数(CV)，比较两组磁珠荧光信号的偏差，结果如下表5：
表5冻干前后磁珠检测的变异系数
Figure PCTCN2022136007-appb-000007
对冻干磁珠与冻干前留样磁珠的7个校准点共91个荧光值(MFI)进行线性回归分析如图6所示。
通过以上结果可以看出，冻干前后磁珠分别检测的7个校准点荧光值MFI的CV值均＜10％，表明两组磁珠检测的荧光值差异较小。对冻干磁珠与冻干前留样磁珠的7个校准点共91个荧光值(MFI)进行线性回归分析，回归方程为Y＝1.0008X+6.1681，R^2＝0.9972，说明两组数据具有互换性，综上，表明磁珠按照现制备工艺进行冻干，其功 能性不受影响，该冻干工艺可行。
实施例4抗原蛋白包被浓度的确定
通过工艺制备偶联有Biotin-BSA的磁珠，然后分别间接偶联13种抗原蛋白，确定13种抗原蛋白的最佳包被偶联浓度。例如针对ESO-1、FXR1、EFHD2，其包被浓度的确定方法分别为：
1、ESO-1包被浓度的确定
使用包被缓冲液将ESO-1配制成10μg/mL、25μg/mL、50μg/mL、100μg/mL、200μg/mL五组不同浓度，分别包被已经偶联有Biotin-BSA的磁珠，制备对应不同ESO-1浓度的五组磁珠，使用Anti-Myc(0、0.62、1.23、3.70、11.11、33.33、100、300U/mL)分别检测五组磁珠，读取荧光值(MFI)，结果如表6和图7所示。
表6 ESO-1包被浓度的确定
Figure PCTCN2022136007-appb-000008
由结果可知，ESO-1包被浓度在高于50μg/mL时达到饱和，为确保生产中磁珠能充分饱和偶联抗原蛋白，将ESO-1包被浓度定为100μg/mL。
2、FXR1包被浓度的确定
使用包被缓冲液将FXR1配制成5μg/mL、15μg/mL、50μg/mL、100μg/mL、200μg/mL五组浓度，分别包被已经偶联有Biotin-BSA的磁珠，制备对应不同FXR1浓度的五组磁珠，使用Anti-Myc(0、0.62、1.23、3.70、11.11、33.33、100、300U/mL)分别检测五组磁珠，读取荧光值(MFI)，结果如表7和图8所示。
表7 FXR1包被浓度的确定
Figure PCTCN2022136007-appb-000009
Figure PCTCN2022136007-appb-000010
由结果可知，FXR1包被浓度在高于100μg/mL时达到饱和，为确保生产中磁珠能饱和偶联抗原蛋白，将FXR1包被浓度定为200μg/mL。
3、EFHD2包被浓度的确定
使用包被缓冲液将EFHD2配制成10μg/mL、30μg/mL、100μg/mL、200μg/mL、270μg/mL五组浓度，分别包被已经偶联有Biotin-BSA的磁珠，制备对应不同EFHD2浓度的五组磁珠，使用Anti-Myc(0、0.62、1.23、3.70、11.11、33.33、100、300U/mL)分别检测五组磁珠，读取荧光值(MFI)，结果如表8和图9所示。
表8 EFHD2包被浓度的确定
Figure PCTCN2022136007-appb-000011
由结果可知，EFHD2包被浓度在100μg/mL时达到饱和，为确保生产中磁珠能饱和偶联抗原蛋白，将EFHD2包被浓度定为200μg/mL。
同理，用本发明类似的方法，可确定其余10种生物标志物的包被浓度：BRAF(400μg/mL)、TM4SF1(300μg/mL)、ZNF573(150μg/mL)、BMI1(200μg/mL)、CAGE(200μg/mL)、PGP9.5(300μg/mL)、P53(200μg/mL)、GBU4-5(300μg/mL)、MAGE A4(300μg/mL)、SOX2(300μg/mL)。
实施例5试剂盒生产工艺过程
试剂盒由样品稀释液、校准品稀释液、洗涤液、分析缓冲液、RPE-驴抗人IgG二抗浓缩液、校准品、质控品、偶联磁珠、96孔板等组分构成。各组分分别配制，独立分装，再组装成盒。其制备工艺概括如下：
(1)样品稀释液、校准品稀释液、洗涤液、分析缓冲液、RPE-驴抗人IgG二抗浓缩液的配制及分装：
1)样品稀释液：含2％驴血清、0.5％BSA及防腐剂的磷酸盐缓冲液，pH＝7.4。
2)校准品稀释液：含1％BSA、防腐剂的磷酸盐缓冲液，pH＝7.4。
3)洗涤液：含Tween20的PBST洗涤液，pH＝7.1。
4)分析缓冲液：含0.2％BSA、防腐剂的磷酸盐缓冲液，pH＝7.4。
5)RPE-驴抗人IgG二抗浓缩液：RPE标记的驴抗人IgG荧光抗体浓缩液。
(2)校准品、质控品的配制分装与冷冻干燥：
1)校准品的配制及冻干：配制含1％BSA、保护剂、防腐剂及磷酸盐缓冲液的重组人源化抗Myc的免疫球蛋白，按照浓度配制成3000U/mL校准品，按0.5mL每瓶分装至冻干瓶中，放入冻干机进行冷冻干燥。
2)质控品Ⅰ的配制及冻干：配制高浓度Anti-Myc；含1％BSA、保护剂、防腐剂的磷酸盐缓冲液的冷冻干燥品。将Anti-Myc配置成200U/ml，按0.5mL每瓶分装至冻干小瓶中，放入冻干机进行冷冻干燥，冻干后的质控品Ⅰ进行质量检测合格后使用。
3)质控品Ⅱ的配制及冻干：配制低浓度Anti-Myc，含1％BSA、保护剂、防腐剂的磷酸盐缓冲液的冷冻干燥品。将使用企业级校准品标定的Anti-Myc原料，使用校准品稀释液稀释成7.41U/ml，每瓶0.5mL，分装至小瓶中，放入冻干机进行冷冻干燥，冻干后的质控品Ⅰ进行质量检测合格后使用。
(3)13种相关抗原蛋白的磁珠偶联与分装及冻干：
偶联肺癌相关抗原的13种磁珠混合物冻干品，含0.5％BSA、保护剂、防腐剂的磷酸盐缓冲溶液冻干粉。
(4)96孔板(空板)装袋贴签。
(5)试剂盒组装。
实施例6各抗原标志物对应自身抗体在肺癌人群中表达水平的检测
按照微球的流式免疫荧光检测方法，检测肺癌患者血清中相应自身抗体表达水平。
各抗原标志物包括BRAF、ESO-1、EFHD2、TM4SF1、ZNF573、BMI1、CAGE、FXR1、PGP9.5、P53、GBU4-5、MAGEA4和SOX2。检测102例首次确诊、未经治疗的肺癌患者人群、52例肺部良性病变人群及64例健康体检人群共三组血清标本，各标志物自身抗体在肺癌人群、肺部良性病变结节人群、以及健康人群的浓度分别如图1所示，各标志物敏感性和特异性分析如表9所示。
表9.各抗原标志物的临床诊断价值敏感性特异性分析
排序 抗原名称 敏感性 特异性
1 BRAF 15.69 95.69
2 ESO-1 13.73 94.83
3 EFHD2 11.76 94.83
4 TM4SF1 34.31 96.55
5 ZNF573 9.8 93.97
6 BMI1 12.75 95.69
7 CAGE 10.78 92.24
8 FXR1 14.71 96.55
9 PGP9.5 15.69 96.55
10 P53 14.71 95.69
11 GBU4-5 5.88 95.69
12 MAGEA4 14.71 97.41
13 SOX2 13.73 96.55
从图1各标志物自身抗体在不同人群中的浓度分布可知，其中ESO1、TM4SF1、FXR1、BMI1、p53五个标志物，肺癌组平均值与对照组(肺部良性病变+健康人群)平均值的比值显著高于其他标志物，进一步地，从表12可知ESO1、TM4SF1、FXR1、BMI1和p53等5个标志物的敏感性在13个标志物中排名前列，提示这5个标志物在13个标志物中具有最高的诊断性能。其中MAGEA4、SOX2、pGP9.5三个标志物在肺癌组中常常出现强阳性信号，提示这三种标志物具有较高的特异性。
GBU4-5在肺癌中的检出敏感性只有5.88％，与国外的研究报道有较大出入，考虑到国内、外研究所采用的患者人种不同，提示GBU4-5用于中国人种的检测不能取得满意效果，必须针对性得对于中国人种群的大规模检测，才能筛选出更适合的标志物。
ZNF573、MAGEA4、SOX2、在肺癌组中的分布水平显著高于健康组，但与结节组相比，两组间差异小于健康组，提示这七个标志物对于健康人群具有更高的鉴别能力。
本发明还筛选到TM4SF1、FXR1、ZNF573、EFHD2、BMI1五个未经报道用于肺癌筛查的标志物，具有校高的诊断价值，其中效果最佳的TM4SF1在三组检测人群中，检测35例肺癌阳性，3例假阳性，灵敏度34.31％，特异性96.55％。
实施例7抗原组合及临床意义
1.选取的不同抗原标志物的组合如下：
对照组合01：P53、MAGEA1、GAGE7、PGP9.5、MAGEA4、Annexin1、SOX2、GBU4-5(参考文献：CN103869086B，文献报道该组合采用ELISA检测方法的敏感性62.5％、特异性87.3％)；
对照组合02：p53、MAGEA1、GAGE7、PGP9.5、MAGEA4、NY-ESO-1、Annexin1(参考文献：CN103869086B，文献报道该组合采用ELISA检测方法的敏感性47.5％、特异性92.7％)；
对照组合03：p53,NY-ESO-1,CAGE,GBU4-5,MAGE A4,HuD,SOX2-B(参考文献:CJ.Chapman,GF.Healey,A Murray,et al.
Figure PCTCN2022136007-appb-000012
test:improved clinical utility through additional autoantibody assays[J].Tumour Biol.2012Oct；33(5):1319-1326，该文献报道的此组合的敏感性为41％，特异性为91％，该组合也是采用ELISA检测方法)。
对照组合04：CN202011301551.3所述的组合18(PLG、APEX1、PARP1、PGP9.5、TP53、MAGEA1、CDKN2A、SPAG9、NY-ESO-1、MAGEA4、GAGE7、EEF2、BRAF、CAGE、GBU4-5、SOX2、GAD2、ELAVL3)(该组合为含18种生物标志物的组合，该文献报道该组合采用液态悬浮芯片技术的检测方法的敏感性80.0％、特异性89.6％)；
对照组合05：CN202011301551.3所述的组合42(PLG、APEX1、PARP1、PGP9.5、TP53、MAGEA1、CDKN2A、SPAG9、NY-ESO-1、MAGEA4、GAGE7、EEF2、BRAF、CAGE、GBU4-5、SOX2、GAD2、CRYAA)(该组合为含18种生物标志物的组合，该文献报道该组合采用液态悬浮芯片技术的检测方法的敏感性81.5％、特异性89.6％)；
对照组合06：CN202011301551.3所述的组合44(PLG、PGP9.5、TP53、SPAG9、NY-ESO-1、MAGEA4、ANXA1、BRAF、CAGE、GBU4-5、SOX2、GAD2、ELAVL3)(该组合为含13种生物标志物的组合，该文献报道该组合采用液态悬浮芯片技术的检测方法的敏感性73.3％、特异性91.7％)；
本发明组合1：ESO-1、TM4SF1、p53、BMI1、FXR1、EFHD2；
本发明组合2：ESO-1、TM4SF1、p53、BMI1、FXR1、EFHD2、BRAF；
本发明组合3：ESO-1、TM4SF1、p53、BMI1、FXR1、EFHD2、BRAF、CAGE；
本发明组合4：BRAF、ESO-1、EFHD2、TM4SF1、ZNF573、BMI1、CAGE、FXR1、PGP9.5、P53；
本发明组合5：BRAF、ESO-1、EFHD2、TM4SF1、ZNF573、BMI1、CAGE、FXR1、PGP9.5、P53、MAGEA4；
本发明组合6：BRAF、ESO-1、EFHD2、TM4SF1、ZNF573、BMI1、CAGE、FXR1、PGP9.5、P53、GBU4-5、MAGEA4、SOX2。
2.选取的不同标志物抗原组合的敏感性(真阳性率)与特异性(真阴性率)，其测试结果具体见表10：
表10.各生物标志物组合的检测效果
Figure PCTCN2022136007-appb-000013
Figure PCTCN2022136007-appb-000014
利用本发明的自身抗体谱液态芯片检测平台，本发明所述的6种不同的组合，其敏感性介于61.8％～85.3％，特异性介于91.4％～96.6％之间。对照组合01、对照组合02以及对照组合03，其分别为国内外两个类似产品的抗原组合，其检测方法为ELISA，均为通过检测一组7-8个标志物，来评估患者患肺癌的风险，其公开报道的敏感性介于41％～62.5％，特异性介于87.3％～92.7％；对照组合04、对照组合05和对照组合06是本申请人在前期筛选时获得的组合。本发明通过不断优化标志物组合，在使用更少数量的标志物数组合下，在敏感性及特异性等考察指标上不低于或高于现有的标志物组合。例如通过减少标志物数量(组合1，6个标志物)，在标志物数量最少的条件下，获得了所有组合中最高的特异性，同时敏感性不低于对照组合01(8个标志物)，高于对照组合02和对照组合03(均为7个标志物)。得益于本发明核心的6个标志物组合(本发明组合1)具有非常高的特异性，因此进一步增加标志物数量，检测的特异性虽呈现下降趋势，但仍在可接受范围内，同时敏感性有了较大的提高。本发明所述的组合6，其检测的特异性达到91.4％，敏感性达到85.3％。本发明所述的组合5，其检测的特异性达到81.4％，敏感性达到92.2％。另外本发明采用液态悬浮芯片检测平台可以高通量的对多标志物进行联检，比传统的ELISA方法具有高通量检测的优势。
实施例8本发明所述的肺癌自身抗体检测试剂盒(流式荧光免疫法)阳性参考值确定
1概述
本试剂盒用于半定量检测人血清中13种肺癌相关自身抗体的浓度。每个试剂盒中设置7个标准点来进行剂量-反应曲线的拟合，用于计算血清样本中各自身抗体的相对浓度。本试剂盒检测的各个自身抗体的相对浓度为连续型变量，检测结果为二分类变量(阳性或阴性)。在本研究中，各自身抗体的相对浓度为定量数据(因变量)，样本判定结果为定类数据(自变量)，且定类数据只有两个选项—阳性或阴性，因此我们采用经典的二元Logistic回归模型作为本产品的判定模型。
本产品临床前阳性判断值研究方法为：对肺癌患者和肺部良性病变患者进行检测，得到血清中13种肺癌相关自身抗体的相对浓度，同时检测一定量的健康人群作为参考。将肺癌患者检测数据作为阳性参考，肺部良性病变患者和健康人群检测数据作为阴性参考，建立13种指标联合的二元Logistic回归模型并进行ROC曲线(receiver operating characteristic curve,受试者特征工作曲线)分析，确定最佳的阳性参考值，对阳性判断值和敏感性、特异性作出相应的计算。
2统计模型及阳性参考值的确定
2.1实验材料
2.1.1试剂盒信息
试剂盒名称：十三种肺癌自身抗体检测试剂盒
试剂盒批号：202107001
规格：12人份/盒
生产厂家：广州市丹蓝生物科技有限公司
2.1.2样本信息
2.1.2.1样本来源与数量
1)样本来源：本研究所用的样本来源于广州等三甲医院
2)样本数量：肺癌282例，良性肺病217例，体检人群122例。
2.1.2.2肺癌组入组标准
1)经临床诊断或组织学病理确诊为肺癌但尚未经过抗肿瘤治疗的患者；
2)未诊断出其它组织或器官的癌症，无肺癌手术史，三月内亦未接受抗肿瘤治疗；
2.1.2.3良性肺病组入组标准
1)临床诊断为肺部良性肿瘤的受试者，包括但不限于：肺囊肿、炎性假瘤等；
2)临床诊断为肺部相关疾病，包括但不限于：肺炎、肺结核、肺气肿等；
3)无肺癌手术史，三月内亦未经局部或全身治疗。
2.1.2.4健康人群入组标准
体格检查和相关的实验室检查均无异常的表面健康人群，无自身免疫疾病史，最近无感染史。
2.1.2.5样本要求
1)本试剂盒测试样本类型为血清，样本量所需量为10μL。
2)采集空腹静脉血不少于1mL，不抗凝，离心后常规分离得到血清。
3)如果样本不立即检测，需2～8℃(5天内)或-20℃以下(一年内)保存。尽量采 用新鲜血清，避免反复冻融，反复冻融不应超过3次，室温(18～25℃)下血清标本存放不超过12小时。
4)溶血或脂血标本可能会对结果造成影响，建议重新采集送检。
5)冷冻血清使用前应放2-8℃环境自然融解，检测前充分混匀。
2.2仪器设备
主要仪器：本试剂盒适用机型为美国Luminex公司多功能流式点阵仪Luminex System，型号为Luminex 200，注册证编号为国械注进20172221166，上机操作参阅仪器使用说明书。
其它设备：单道及多道移液器及适配枪头、储液槽、磁力分离板(型号：Magnetic Plate Separator/CN-0269-01，Luminex)、涡旋振荡器、具标准96孔板卡座转速可调式涡旋混匀器(精骐VM-02U，适配夹具VMA06)、37℃恒温箱。
2.3实验要求
实验人员应熟悉检测方法与仪器操作并保持仪器处于正常状态；用于实验的试剂应为检验合格的产品，且在有效期内。
2.4统计分析结果
2.4.1各标志物的统计描述
本研究共检测了282例肺癌，217例良性肺病，122例健康人群，各组样本、各标志物的统计描述如表11～表15所示：
表11
Figure PCTCN2022136007-appb-000015
表12
Figure PCTCN2022136007-appb-000016
Figure PCTCN2022136007-appb-000017
表13
Figure PCTCN2022136007-appb-000018
表14
Figure PCTCN2022136007-appb-000019
表15
Figure PCTCN2022136007-appb-000020
各标志物在肺癌组和良性肺病组中的分布如图2所示，由结果可知，各标志物的浓度水平在肺癌组和阴性对照组中均存在显著性差异。
2.4.2统计模型的建立
将肺癌组和良性肺病组的检测结果输入IBM SPSS Statistics 25统计软件，将样本的定类数据按以下进行转换：肺癌组＝1，良性肺病组＝0，建立二元Logistic回归模型并进行ROC曲线分析，以敏感性和1-特异性为纵坐标和横坐标作图，结果如图3所示，ROC曲线下的AUC＝0.906，95％CI为0.881～0.931，最大约登指数＝0.751，cutoff(阳性参考值)＝0.45241，特异性为90.3％，敏感性为84.8％。
所建立的模型判定公式如下：
Figure PCTCN2022136007-appb-000021
其中：
X＝－4.672652+0.000868*TM4SF1+0.000273*p53+0.001453*PGP9.5+0.00033*SO X2+0.000075*BMI1+0.000101*FXR1+0.000835*MAGE A4+0.002701*ESO1+0.001060*ZNF573+0.001213*BRAF+0.000082*GBU4-5－0.000002*EFHD2+0.000141*CAGE。
将样本的13种标志物的检测值代入上述公式，计算样本的p值(回归值)，当p≥cutoff时，判定为阳性；当p＜cutoff时，判定为阴性。
3结论
经过临床前大样本研究，确定了本产品的判定模型及阳性参考值，本产品的阳性判定公式如下，阳性参考值为0.45241，将样本的13种标志物的检测值代入下述公式计算p值，当p＞cutoff时判定为阳性，p≤cutoff时判定为阴性
Figure PCTCN2022136007-appb-000022
其中：
X＝－4.672652+0.000868*TM4SF1+0.000273*p53+0.001453*PGP9.5+0.00033*SO X2+0.000075*BMI1+0.000101*FXR1+0.000835*MAGE A4+0.002701*ESO1+0.001060*ZNF573+0.001213*BRAF+0.000082*GBU4-5－0.000002*EFHD2+0.000141*CAGE。
实施例10对本发明所述肺癌自身抗体检测试剂盒的准确度评估
1试验材料
试剂盒名称：十三种肺癌自身抗体检测试剂盒(流式荧光免疫法)
批号：202107001、202108001、202109001
2试验方法
将企业校准品使用校准品稀释液配制成2000U/ml及74.1U/ml的标准液，分别将标准液按照1：9加入到校准品稀释液中，分别制备回收样本1及回收样本2；
取以上3批试剂盒，分别检测2个回收样本，每个样本分别做2个复孔，取平均值按以下公式(1)分别计算2个稀释浓度的回收率。
计算公式(1)
Figure PCTCN2022136007-appb-000023
R---------回收率；
V---------样品A液的体积；
V0-------样品B液的体积；
C---------样品B液加入A液后的检测浓度；
c0--------样品B液的浓度；
c s---------样品A液的浓度。
3试验结果，参见表16～表18。
表16.批号202107001准确度检测结果
Figure PCTCN2022136007-appb-000024
表17.批号202108001准确度检测结果
Figure PCTCN2022136007-appb-000025
表18.批号202109001准确度检测结果
Figure PCTCN2022136007-appb-000026
4试验结论
上述实验结果表明，使用3批试剂盒分别对2个回收样本进行检测，每个样本重复检测2次，取平均值按公式(1)分别计算回收率，2个回收样本检测的回收率都在80～120％之间。
实施例11对本发明所述肺癌自身抗体检测试剂盒的阴阳性样本符合率评估
1试验材料
试剂盒名称：十三种肺癌自身抗体检测试剂盒(流式荧光免疫法)
阳性参考血清(肺癌)与阴性参考血清(良性病变)信息如表19：
表19
Figure PCTCN2022136007-appb-000027
2试验方法
使用3批试剂盒，分别对6例阳性参考血清和6例阴性参考血清进行检测，每个样本检测1次，将各个样本的检测结果代入本试剂盒的判定模型中，计算各个样本的回归值，当样本的回归值≥cutoff值时为阳性，样本的回归值＜cutoff值时为阴性。
3试验结果
1)3批试剂盒对6例阳性参考血清和6例阴性参考血清的检测结果如表20～表25：
表20.批号202107001阳性参考血清检测结果
Figure PCTCN2022136007-appb-000028
表21.批号202107001阴性参考血清检测结果
Figure PCTCN2022136007-appb-000029
表22.批号202108001阳性参考血清检测结果
Figure PCTCN2022136007-appb-000030
Figure PCTCN2022136007-appb-000031
表23.批号202108001阴性参考血清检测结果
Figure PCTCN2022136007-appb-000032
表24.批号202109001阳性参考血清检测结果
Figure PCTCN2022136007-appb-000033
表25.批号202109001阴性参考血清检测结果
Figure PCTCN2022136007-appb-000034
2)将3批试剂盒对6例阳性参考血清和6例阴性参考血清的检测结果代入本试剂盒的判定模型中，各样本的回归值及判定结果如表26～表28：
表26.批号202107001试剂盒
Figure PCTCN2022136007-appb-000035
表27.批号202108001试剂盒
Figure PCTCN2022136007-appb-000036
表28.批号202109001试剂盒
Figure PCTCN2022136007-appb-000037
Figure PCTCN2022136007-appb-000038
从上述实验结果可知，3批试剂盒对6例阳性参考血清均判定为阳性，阳性符合率为100％，3批试剂盒对6例阴性参考血清均判定为阴性，阴性符合率均为100％。
4试验结论
由上述3批试剂盒的检测结果可知，3批试剂盒对30000U/mL、22500U/mL样本的检测结果均不能准确报告，多个标志物测量的准确度低于80％，出现明显的高剂量钩状效应，3批试剂盒对15000U/mL、10000U/mL、5000U/mL、2500U/mL样本测量的准确度均位于80～120％之间，未观察到高剂量钩状效应，表明本试剂盒在1.23～600U/mL的检测范围(对应报告范围1.23～15000U/mL，25倍稀释)内不产生高剂量钩状效应，对高于检测范围的样本应将浓度稀释至检测范围内才能准确测量。
实施例12本发明所述试剂盒的稳定性试验方法
1加速稳定性
分别取三批试剂盒(批号：202107001、202108001、202109001)，置于37℃的恒温箱中，分别在第0天、4天、7天、9天取样检测。
2开瓶稳定性
2.1偶联磁珠混合物(冻干粉)、校准品(冻干粉)、质控品Ⅰ(冻干粉)、质控品Ⅱ(冻干粉)的开瓶稳定性
取本品(批号：202107001、202108001、202109001)，分别将3批次试剂盒内的R9偶联磁珠混合物(冻干粉)、R6校准品(冻干粉)、R7质控品Ⅰ(冻干粉)、R8质控品Ⅱ(冻干粉)取出按标识复溶，置于2～8℃避光保存，分别在放置的第6小时、第12小时取出进行检测。
2.2 RPE-驴抗人IgG二抗浓缩液的开瓶稳定性
取本品(批号：202107001、202108001、202109001)，分别将3批次试剂盒内的R5RPE-驴抗人IgG二抗浓缩液取出并配制成二抗工作液，置于2～8℃避光保存，分别在放置的第6小时、第12小时取出进行检测。
3长期稳定性
分别取三批试剂盒(批号：202107001、202108001、202109001)在储存条件下按上市包装放置保存，每隔3个月取样进行检测。
稳定性研究结论：
试剂盒按上市包装37℃放置0、4、7、9天，2～8℃放置至效期末检测试剂盒的剂量-反应曲线的线性、准确度、质控品Ⅰ、质控品Ⅱ的检测、最低检测以及重复性，各项指标检测都符合要求，确定了本检测试剂盒的有效期12个月，2～8℃，避光、密封保存。
根据试剂盒冻干组分复溶稳定性，取本批试剂盒，将各冻干组分(R6校准品(冻干粉)、R7质控品Ⅰ(冻干粉)、R8质控品Ⅱ(冻干粉)分别按照说明书复溶，复溶后至于2～8℃冰箱，分别于6小时、12小时取样检测试剂盒各性能指标(剂量-反应曲线的线性、准确度、质控品Ⅰ、质控品Ⅱ的检测、最低检测限、重复性)，各项指标检测都符合要求，结果表明各冻干组份复溶后可于2～8℃冷藏放置12小时。
根据试剂盒工作液配制使用稳定性，取试剂盒，将R5RPE-驴抗人IgG二抗浓缩液取出，按照产品说明书配制成至RPE-驴抗人IgG二抗工作液，放置于2～8℃冰箱，分别于6小时、12小时取样检测试剂盒各性能指标(剂量-反应曲线的线性、准确度、质控品Ⅰ、质控品Ⅱ的检测、最低检测限、重复性)，各项指标检测都符合要求，结果表明试剂盒工作液配制后于12小时内使用，建议现配现用。具体试验步骤、试验结果见下述内容。
其中，批号202107001在37℃加速第7天实验结果如表29和表30：
表29.批号202107001 13个标志物的原始中位荧光值(MFI)记录表
Figure PCTCN2022136007-appb-000039
表30.批号202107001 13个标志物的原始中位荧光值(MFI)记录表
Figure PCTCN2022136007-appb-000040
Figure PCTCN2022136007-appb-000041
分别将3个批次试剂盒的13个标志物各样本测得的MFI与对应的样本浓度使用5p-Logistics数学模型进行拟合，3批试剂盒13个标志物的剂量-反应曲线相关系数R如表31所示：
表31
Figure PCTCN2022136007-appb-000042
结论：实验结果表明，3个批次的试剂盒在37℃放置7天，在600～1.23U/mL的范围内，3批次试剂盒的剂量-反应曲线的相关系数R均大于0.990，符合产品技术要求。
准确度，结果见表32。
表32.批号202107001准确度实验结果
Figure PCTCN2022136007-appb-000043
Figure PCTCN2022136007-appb-000044
结论：3批试剂盒在37℃放置7天，3批试剂盒对样品1、样品2检测的回收率均在80％～120％之间，符合产品技术要求。
质控品Ⅰ、质控品Ⅱ的检测，结果见表33。
表33.批号202107001试剂盒质控品Ⅰ、质控品Ⅱ检测结果
Figure PCTCN2022136007-appb-000045
结论：3批试剂盒在37℃放置7天，分别对各自试剂盒内的质控品Ⅰ、质控品Ⅱ进行检测，3批试剂盒的13种抗原标志物的质控品Ⅰ、质控品Ⅱ检测结果均在标示范围，符合产品技术要求。
重复性，结果见表34和表35。
表34.批号202107001重复性实验结果
Figure PCTCN2022136007-appb-000046
Figure PCTCN2022136007-appb-000047
表35.批号202107001重复性实验结果
Figure PCTCN2022136007-appb-000048
分别计算企业质控品Ⅰ、企业质控品Ⅱ重复测量的平均值(AV)，标准差(SD)，计算10次重复测量的变异系数(CV)，结果如表36：
表36
Figure PCTCN2022136007-appb-000049
Figure PCTCN2022136007-appb-000050
由上述结果可知，批号202107001试剂盒检测的重复性CV均小于15％，符合产品技术要求。
结论：由上述实验结果可知，3批试剂盒在37℃放置7天，3批试剂盒的重复性CV％均小于15％，符合产品技术要求。
最低检测限
将企业级校准品稀释至标曲S7的浓度(1.23U/mL)，使用校准品稀释液将S7稀释至1.00U/mL，重复测定10次，计算测定的平均值(AV)和变异系数(CV)，以测定的平均值(AV)作为最低检测限，结果CV应不大于15％且最低检测限不高于1.23U/mL。结果见表37。
表37.批号202107001最低检测限实验数据
Figure PCTCN2022136007-appb-000051
分别计算3批试剂盒10次重复检测的平均值(AV)和标准差(SD)，结果如表38：
表38. 3批试剂盒最低检测限计算结果
Figure PCTCN2022136007-appb-000052
Figure PCTCN2022136007-appb-000053
结论：由上述实验结果可知，3批试剂盒在37℃放置7天。3批试剂盒的最低检测限均不高于1.23U/mL，符合产品技术要求。
实验结论
上述实验结果表明，3批试剂盒在37℃放置7天，3批试剂盒的各项性能指标均满足产品技术要求中的规定。
实施例12溶血、黄疸及血脂样品对本试剂检测结果的干扰
常见的干扰样本有溶血、黄疸、及高脂血清样本，为评估本试剂的抗干扰能力，我们分别将血红蛋白、胆红素及甘油三酯以一定的浓度(浓度参考其他厂家或临床信息：血红蛋白10mg/mL，胆红素1.5mM，甘油三酯10mM)添加至3例血清样本中(样本1、样本2、样本3)，使用本试剂进行检测，观察以上三种干扰物质在血清样本中添加前、后对检测结果的影响，如果干扰物质添加前后同一样本检测偏差在±20％之内，则视为不存在干扰，实验结果表39～表41：
表39血红蛋白(10mg/mL)干扰检测结果
Figure PCTCN2022136007-appb-000054
表40胆红素(1.5mM)干扰检测结果
Figure PCTCN2022136007-appb-000055
表41甘油三脂(10mM)干扰检测结果
Figure PCTCN2022136007-appb-000056
上述实验结果表明，血清样本血红蛋白10mg/mL，胆红素1.5mM，甘油三酯10mM)三种干扰物质添加前后同一样本检测偏差在±20％之内。

Claims (10)

  1. 一种与肺癌相关的生物标志物组合，其特征在于，所述生物标志物组合包含ESO-1、TM4SF1、p53、BMI1、FXR1和EFHD2抗原或结合其的自身抗体。
  2. 如权利要求1所述的生物标志物组合，其特征在于，所述生物标志物组合还包含BRAF和/或CAGE抗原或结合其的自身抗体；
    较佳地，所述生物标志物组合还包含PGP9.5和/或ZNF573抗原或结合其的自身抗体，优选还包含GBU4-5抗原或结合其的自身抗体；
    更佳地，所述生物标志物组合还包含MAGE A4和/或SOX2抗原或结合其的自身抗体；
    进一步更佳地，所述生物标志物组合为血清中的生物标志物组合，其选自以下任一组：
    1)ESO-1自身抗体、TM4SF1自身抗体、p53自身抗体、BMI1自身抗体、FXR1自身抗体和EFHD2自身抗体；
    2)ESO-1自身抗体、TM4SF1自身抗体、p53自身抗体、BMI1自身抗体、FXR1自身抗体、EFHD2自身抗体和BRAF自身抗体；
    3)ESO-1自身抗体、TM4SF1自身抗体、p53自身抗体、BMI1自身抗体、FXR1自身抗体、EFHD2自身抗体、BRAF自身抗体和CAGE自身抗体；
    4)BRAF自身抗体、ESO-1自身抗体、EFHD2自身抗体、TM4SF1自身抗体、ZNF573自身抗体、BMI1自身抗体、CAGE自身抗体、FXR1自身抗体、PGP9.5自身抗体和P53自身抗体；
    5)BRAF自身抗体、ESO-1自身抗体、EFHD2自身抗体、TM4SF1自身抗体、ZNF573自身抗体、BMI1自身抗体、CAGE自身抗体、FXR1自身抗体、PGP9.5自身抗体、P53自身抗体和MAGEA4自身抗体；
    6)BRAF自身抗体、ESO-1自身抗体、EFHD2自身抗体、TM4SF1自身抗体、ZNF573自身抗体、BMI1自身抗体、CAGE自身抗体、FXR1自身抗体、PGP9.5自身抗体、P53自身抗体、GBU4-5自身抗体、MAGEA4自身抗体和SOX2自身抗体。
  3. 一种诊断试剂盒，其特征在于，所述诊断试剂盒包括用于检测如权利要求1或2所述的生物标志物组合中生物标志物的试剂，例如用于抗原检测的自身抗体，或用于自身抗体检测的抗原；
    较佳地，所述生物标志物组合中，所述抗原或自身抗体还含有标签肽；所述标签肽 优选包括：His标签、streptavidin标签、avidin标签、生物素标签、GST标签、C-myc标签、Flag标签和HA标签中的一种或多种；所述生物标志物更优选由大肠杆菌、酵母、昆虫细胞或动物细胞表达，和/或，所述生物标志物通过Ni亲和层析、离子交换层析、分子筛、透析、超滤或疏水层析纯化；
    更佳地，所述用于检测所述生物标志物组合中生物标志物的试剂选自以下任一组：
    1)ESO-1抗原、TM4SF1抗原、p53抗原、BMI1抗原、FXR1抗原和EFHD2抗原；
    2)ESO-1抗原、TM4SF1抗原、p53抗原、BMI1抗原、FXR1抗原、EFHD2抗原和BRAF抗原；
    3)ESO-1抗原、TM4SF1抗原、p53抗原、BMI1抗原、FXR1抗原、EFHD2抗原、BRAF抗原和CAGE抗原；
    4)BRAF抗原、ESO-1抗原、EFHD2抗原、TM4SF1抗原、ZNF573抗原、BMI1抗原、CAGE抗原、FXR1抗原、PGP9.5抗原和P53抗原；
    5)BRAF抗原、ESO-1抗原、EFHD2抗原、TM4SF1抗原、ZNF573抗原、BMI1抗原、CAGE抗原、FXR1抗原、PGP9.5抗原、P53抗原和MAGEA4抗原；
    6)BRAF抗原、ESO-1抗原、EFHD2抗原、TM4SF1抗原、ZNF573抗原、BMI1抗原、CAGE抗原、FXR1抗原、PGP9.5抗原、P53抗原、GBU4-5抗原、MAGEA4抗原和SOX2抗原。
  4. 如权利要求3所述的诊断试剂盒，其特征在于，所述检测抗原或检测自身抗体与磁珠偶联，形成偶联磁珠，所述偶联磁珠混合后形成偶联磁珠混合物，以抗原偶联磁珠为例，偶联磁珠的制备方法包含以下步骤：
    (1)磁珠的激活：将待偶联的磁珠震荡混匀，使用激活缓冲液洗涤磁珠，并依次加入NHS溶液和EDC溶液，震荡混匀后，旋转混合孵育，制得激活的磁珠；较佳地，所述激活缓冲液为0.1M磷酸二氢钠溶液，所述NHS溶液为含5％(w/v)N-羟基琥珀酰亚胺的激活缓冲液，所述EDC溶液为含5％(w/v)1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐的激活缓冲液；
    (2)Biotin-BSA的偶联：将步骤(1)中激活的磁珠用偶联缓冲液洗涤后，加入BSA-biotin混匀并孵育，加入洗涤液洗涤后，加入封闭缓冲液，混匀孵育，制得偶联Biotin-BSA的磁珠；较佳地，所述偶联缓冲液为50mM 2-[N-吗啉代]乙烷磺酸溶液，所述洗涤液为含0.05％(w/v)吐温-20、0.03％(v/v)Proclin300的1×PBS缓冲液，所述封闭缓冲液为含1％(w/v)BSA、0.03％(v/v)Proclin300的1×PBS缓冲液；所述BSA-biotin的浓度为2mg/mL；
    (3)抗原的包被：将步骤(2)中偶联Biotin-BSA的磁珠用洗涤液洗涤，加入相应的所述抗原，混匀并孵育，再用洗涤液洗涤，加入磁珠保存液保存，制得偶联磁珠；较佳地，所述洗涤液为含0.05％(w/v)吐温-20、0.03％(v/v)Proclin300的1×PBS缓冲液，所述磁珠保存液为含0.5％(w/v)BSA、3％(w/v)海藻糖、5％(w/v)甘露醇、0.03％(v/v)Proclin300的1×PBS缓冲液；更佳地，BRAF抗原的包被浓度为400μg/mL、ESO-1抗原的包被浓度为100μg/mL、EFHD2抗原的包被浓度为200μg/mL、TM4SF1抗原的包被浓度为300μg/mL、ZNF573抗原的包被浓度为150μg/mL、BMI1抗原的包被浓度为200μg/mL、CAGE抗原的包被浓度为200μg/mL、FXR1抗原的包被浓度为200μg/mL、PGP9.5抗原的包被浓度为300μg/mL、P53抗原的包被浓度为200μg/mL、GBU4-5抗原的包被浓度为300μg/mL、MAGEA4抗原的包被浓度为300μg/mL和/或SOX2抗原的包被浓度为300μg/mL；
    优选地，所述偶联磁珠的制备方法还包含：
    (4)偶联磁珠混合物的冻干：将步骤(3)中偶联磁珠混合，加入磁珠冻干缓冲液，混匀后冻干；较佳地，所述磁珠冻干缓冲液为含0.5％(w/v)BSA、3％(w/v)海藻糖、5％(w/v)甘露醇、0.03％(v/v)Proclin300的1×PBS缓冲液；
    其中，所述v/v为体积百分比，所述w/v为质量体积比。
  5. 如权利要求4所述的诊断试剂盒，其特征在于，所述诊断试剂盒还包括样品稀释液、校准品稀释液、洗涤液、分析缓冲液、抗人IgG二抗、校准品、质控品中的一种或多种；所述诊断试剂盒优选还包括96孔板；
    较佳地，所述样品稀释液为含2％(v/v)驴血清、0.5％(w/v)BSA的磷酸盐缓冲液，优选还含有防腐剂；
    所述校准品稀释液为含1％(w/v)BSA的磷酸盐缓冲液，优选还含有防腐剂；
    所述洗涤液为含Tween20的PBST洗涤液；
    所述分析缓冲液为含0.2％(w/v)BSA的磷酸盐缓冲液，优选还含有防腐剂；
    所述抗人IgG二抗为RPE标记的驴抗人IgG荧光抗体；
    所述校准品为含1％(w/v)BSA及磷酸盐缓冲液的重组人源化Anti-Myc标签的免疫球蛋白；优选地，所述Anti-Myc的浓度为3000U/mL，所述校准品还含有保护剂和/或防腐剂，和/或，所述校准品被制成冷冻干燥品；和/或，
    所述质控品包括质控品I和质控品II，所述质控品I浓度为含200U/ml Anti-Myc、含1％(w/v)BSA的磷酸盐缓冲液，所述质控品II浓度为含7.41U/ml Anti-Myc、含1％(w/v)BSA的磷酸盐缓冲液；优选地，所述质控品I和所述质控品II还含有保护剂和/或防腐 剂，和/或，所述质控品I和所述质控品II被制成冷冻干燥品；
    优选地，所述保护剂为含1％(w/v)BSA、3％(w/v)海藻糖、5％(w/v)甘露醇、0.03％(v/v)Proclin300的1×PBS缓冲液；
    其中，所述v/v为体积百分比，所述w/v为质量体积比。
  6. 一种非诊断目的的检测方法，其特征在于，所述检测方法使用如权利要求1或2所述的生物标志物组合或如权利要求3～5任一项所述的诊断试剂盒对相应的抗原或结合其的自身抗体进行检测，具体步骤是：使所述生物标志物组合接触样品，若检测到结合，则说明相应的抗原或结合其的自身抗体的存在。
  7. 如权利要求1或2所述的生物标志物组合或如权利要求3～5任一项所述的诊断试剂盒在制备检测人血清中特定的蛋白或抗体分子的试剂中的应用；
    较佳地，所述试剂用于诊断或辅助诊断早期肺癌，或用于经肺癌治疗后的复发监测和/或预后监测；
    更佳地，所述肺癌为鳞癌、小细胞肺癌、腺癌例如浸润性腺癌。
  8. 一种肺癌诊断系统，其特征在于，所述肺癌诊断系统包括以下模块：
    (1)输入模块，其用于输入待测样本中所含的如权利要求1或2所述生物标志物组合的检测值；
    (2)分析模块，其用于计算p，其中
    Figure PCTCN2022136007-appb-100001
    X＝－4.672652+0.000868*TM4SF1+0.000273*p53+0.001453*PGP9.5+0.00033*SOX2+0.000075*BMI1+0.000101*FXR1+0.000835*MAGE A4+0.002701*ESO1+0.001060*ZNF573+0.001213*BRAF+0.000082*GBU4-5－0.000002*EFHD2+0.000141*CAGE；
    (3)判断模块，cutoff为0.45241；当p＞cutoff时判定为阳性，p≤cutoff时判定为阴性；
    较佳地，所述肺癌诊断系统还包括登录模块，和/或所述肺癌诊断系统还包括打印模块；所述登录模块需要输入用户名称和用户密码，所述打印模块可打印输入模块、分析模块和判断模块产生的结果；
    更佳地，所述测试样本为血清。
  9. 一种计算机可读介质，其特征在于，所述的计算机可读介质存储了计算机程序，所述计算机程序被处理器执行时可实现如权利要求8所述的肺癌诊断系统的功能。
  10. 一种肺癌诊断装置，其特征在于，包括：
    (1)如权利要求9所述的计算机可读介质；
    (2)处理器，用于执行计算机程序以实现所述肺癌诊断系统的功能。
PCT/CN2022/136007 2022-07-01 2022-12-01 一种与肺癌相关的生物标志物组合、含其的试剂盒及其用途 WO2024001044A1 (zh)

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