CN113687076A - Combined detection serum marker for early diagnosis of lung adenocarcinoma and application thereof - Google Patents
Combined detection serum marker for early diagnosis of lung adenocarcinoma and application thereof Download PDFInfo
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Abstract
The invention discloses a combined detection serum marker for early diagnosis of lung adenocarcinoma, which comprises IgM autoantibodies of TSHR, ERBB2, Survivin, PIK3CA and JAK2, and an ELISA serum detection kit prepared by using specific protein corresponding to the combined detection serum marker.
Description
Technical Field
The invention belongs to the technical field of biomedical detection, and particularly relates to a combined detection serum marker for early diagnosis of lung adenocarcinoma and application thereof.
Background
In 2020, 220 ten thousand new cases and 180 ten thousand deaths of lung cancer are counted, which account for 11.4% and 18.0% of all cancers respectively, and are the second cancer with the global morbidity and the first cancer with the mortality. Lung adenocarcinoma, the most common histological subtype of non-small cell lung cancer, accounts for about 40% of lung malignancies and is most frequently found in women and non-smokers. Numerous studies have shown that patients with orthotopic Adenocarcinoma In Situ (AIS) and slightly Invasive Adenocarcinoma (MIA) can have a disease-free survival rate of approximately 100% in 5 years if they undergo radical surgery. Therefore, the early diagnosis and timely effective treatment of the lung adenocarcinoma patients have great significance for preventing and treating lung cancer. Currently, lung cancer patients are clinically screened and diagnosed mainly by low-dose spiral ct (ldct) and pathological tissue biopsy, but the former has higher false positive rate and the latter has larger wound, so that the patients are burdened. In recent years, serological markers which are easy to operate, less traumatic and easily accepted by patients have attracted much attention. Clinically, carcinoembryonic antigen (CEA) as a traditional serum tumor marker is widely applied to lung cancer auxiliary diagnosis, but the single detection has the defects of low sensitivity and specificity and the like. The IgM autoantibody, as a first class antibody generated in the humoral immune response process, has great potential to be used as an early diagnosis index of lung cancer, and no related research exists at present.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a combined detection serum marker for early diagnosis of lung adenocarcinoma, the combined detection serum marker plays an important role in improving the sensitivity, specificity and accuracy of early diagnosis of lung adenocarcinoma, and the combined detection serum marker and the CEA serum marker are simultaneously applied to construct a diagnosis model, so that the serological detection method which has high sensitivity, strong specificity and low cost and can assist clinical diagnosis of lung adenocarcinoma is provided.
The invention provides a combined detection serum marker for early diagnosis of lung adenocarcinoma, wherein the serum marker is five IgM autoantibodies, including autoantibodies of TSHR, ERBB2, Survivin, PIK3CA and JAK 2;
a combined detection ELISA kit for early diagnosis of lung adenocarcinoma is characterized in that the kit comprises a solid phase carrier and a specific protein corresponding to the combined detection serum marker coated on the solid phase carrier.
Preferably, the solid phase carrier is a 96-hole enzyme label plate.
Further, the kit also comprises a sample diluent, a second antibody diluent, positive control serum, negative control serum, a color development liquid, a stop solution, a blocking solution and a washing solution.
Preferably, the second antibody carries a detectable label;
preferably, the marker is horseradish peroxidase;
preferably, the second antibody is a goat anti-human IgM antibody;
the method for detecting the lung adenocarcinoma serum sample by the combined detection ELISA kit specifically comprises the following steps:
a) coating: diluting the concentration of the combined detection serum marker protein to 0.125 mu g/ml by using coating liquid, coating the combined detection serum marker protein into a 96-well plate according to 50 mu l/well, and placing the 96-well plate in a refrigerator at 4 ℃ overnight;
b) and (3) sealing: discarding the protein diluent in the hole, adding the sealing liquid into a 96-well plate, sealing the plate at 100 mu l/hole in a refrigerator at 4 ℃ overnight;
c) washing: discarding the blocking solution in the hole, placing the enzyme label plate in an automatic plate washing machine, repeatedly washing for 3 times according to 300 mul of washing solution/hole and 10 seconds/time, and finally patting to dry.
d) Primary antibody incubation: adding diluted serum and blank control at corresponding positions in a 96-well plate, incubating for 1 hour in a water bath kettle at 37 ℃ in a way of 50 mul/well, wherein the diluted serum is obtained by diluting the serum with an antibody diluent according to the proportion of 1: 100; blank control is antibody dilution;
e) washing: discarding serum solution in the hole, placing the enzyme label plate in an automatic plate washing machine, repeatedly washing for 5 times according to 300 mul of washing liquid/hole and 10 seconds/time, and finally patting to dry;
f) and (3) secondary antibody incubation: diluting goat anti-human IgM marked by HRP and an antibody diluent according to a ratio of 1:2000, adding a 96-hole enzyme label plate, incubating at 50 mu l/hole in a water bath kettle at 37 ℃ for 1 hour;
g) washing: the secondary antibody solution in the wells was discarded, the microplate was placed in an automatic plate washer, and washing was repeated 5 times per 10 seconds per well in accordance with 300. mu.l of wash solution, and finally patted dry.
h) Color development: adding 50 μ l of developing solution into each well, placing white paper under the enzyme label plate, and developing at room temperature in dark for 20 min.
i) And (4) terminating: stop solutions were added to each well at 25. mu.l/well.
j) Measuring the absorbance: respectively measuring absorbance values corresponding to the wavelengths of 450nm and 620nm by using a microplate reader, and determining the absorbance values as OD450-OD620Relative OD values were obtained and the blank control was then deducted.
The invention also provides a construction method of the combined diagnosis model for early diagnosis of lung adenocarcinoma, which comprises the following steps:
(1) protein chip technology screening of IgM autoantibodies: serological analysis is carried out on lung adenocarcinoma patients and normal control serum samples through a protein chip technology, and potential five IgM autoantibodies which can be used for lung adenocarcinoma diagnosis are screened;
(2) verifying the selected candidate IgM autoantibody indexes in serum samples of lung adenocarcinoma patients and normal controls through an ELISA experiment to obtain combined detection serum markers for early diagnosis of lung adenocarcinoma, wherein the combined detection serum markers comprise autoantibodies of TSHR, ERBB2, Survivin, PIK3CA and JAK 2;
(3) combining a combined detection serum marker for early diagnosis of lung adenocarcinoma with a traditional tumor marker CEA by Logistic regression to construct a lung adenocarcinoma diagnosis model P ═ 1/(1+ Exp (- (1.655 OD)ERBB2+7.862*ODJAK2-10.285*ODTSHR+17.135*ODPIK3CA-11.294*ODSurvivin+0.299 × CEA-1.899)); OD in the formulaERBB2、ODJAK2、ODTSHR、ODPIK3CA、ODSurvivinRespectively subtracting the absorbance value of the blank control from the relative OD value of each IgM autoantibody index; CEA is the CEA content in the serum of a patient obtained by detecting clinically by an electrochemiluminescence method, and the unit is ng/mL.
Drawings
FIG. 1 is SNR scatter diagram of 5 IgM autoantibody serum markers screened by the protein chip in the experimental example and ROC curve chart for diagnosing lung adenocarcinoma.
FIG. 2 is an OD scatter plot of 5 IgM autoantibody serum markers and ROC curve for diagnosing lung adenocarcinoma from the results of ELISA verification in the experimental examples.
FIG. 3 is a ROC graph showing that ELISA verifies 5 IgM autoantibody serum markers, CEA serum markers and the combination of the two in the experimental examples to diagnose lung adenocarcinoma.
Advantageous effects
The five combined detection serum markers for early diagnosis of lung adenocarcinoma are used for detecting 83 lung adenocarcinoma cases and 83 normal control serum samples, and the results show that the AUC after the five combined detection serum markers are combined is 0.698, while the AUC of the traditional tumor marker CEA is 0.692, and meanwhile, the AUC after the five combined detection serum markers are combined with CEA can reach 0.827.
The five combined detection serum markers for early diagnosis of lung adenocarcinoma show a certain diagnosis effect on lung adenocarcinoma, and can provide a simple and feasible diagnosis method with no injury, low cost and higher diagnosis efficiency for clinic after being combined with the existing clinical tumor marker CEA to construct a diagnosis model. The model can effectively distinguish lung adenocarcinoma patients from normal controls, and has good diagnostic value in different lung adenocarcinoma patient stratification. In addition, the model was statistically significant in the different groups (early and late lung adenocarcinoma patients; lung adenocarcinoma patients with and without lymph node metastasis; lung adenocarcinoma patients with and without distant metastasis). The model has certain clinical auxiliary functions for diagnosing and layering lung adenocarcinoma patients.
Detailed Description
The technical solutions of the present invention will be described in detail below with reference to the accompanying drawings and specific experimental examples, but it should be emphasized that the present invention is not limited to the specific embodiments.
Examples of the experiments
1. Serum specimen Collection and preparation
1.1 serum sample Collection
The study is included in 430 cases of study objects, and is divided into a discovery group and a verification group, wherein the discovery group is used for screening IgM autoantibody indexes with higher diagnosis value on lung adenocarcinoma. And the verification group is used for verifying the candidate IgM autoantibody index and constructing a diagnosis model by combining with CEA.
All the serum samples of the subjects were collected in a hospital in Henan province during the year 2016-. The serum CEA detection result is provided by the hospital clinical laboratory and is detected by a MODULAR 70 type full-automatic analyzer and a matched kit produced by Roche of Switzerland, and the principle of the method is an electrochemical luminescence method. The experimental procedures were performed by a skilled technician and the results were published after examination by an experienced clinical laboratory physician. CEA cutoff value was 5.0 ng/mL. Specific subject information is shown in table 1.
TABLE 1 clinical information of subjects in the serum sample discovery and validation groups of the invention
P*＜0.05
1.2 preparation of serum samples
All specimens are prepared by collecting whole blood of a research object by a red blood collection tube by 5-10mL, placing the whole blood at room temperature for 2 hours, centrifuging the whole blood for 15 minutes at 1000g, packaging the supernatant into 500 mu L/tube, and storing the tube at-80 ℃ after labeling, so that repeated freeze thawing is avoided.
2. Preparation of protein chip for screening early diagnosis of lung adenocarcinoma and jointly detecting serum protein marker
Protein chips were customized by biosciences, Inc., Bo Chong, Guangzhou, and contained 154 recombinant proteins or protein fragments, 11 of which (CIP2A/p90, c-Myc, cyclin B1, IMP1, IMP2, IMP3, RalA, RBM39, YWHAZ and two Surviin fragments) were studied before the laboratory (evaluation and identification of an immunogenic model for diagnosis of Breast Cancer. oncoimmunization 9(1) (2020)1682382. and use of diagnostic assessment of early to select tumor-associated antibodies in diagnostic imaging of Cancer genes of Cancer research, 1826. protein chip 1829, protein chip 2019, protein chip 26, protein chip, and protein chip, protein chip.
3. Protein chip technology based screening experiment
The method comprises the steps of carrying out serological analysis on 68 lung adenocarcinoma patients and 68 normal controls in a discovery group by using a protein chip technology, and screening potential combined detection serum markers which can be used for lung adenocarcinoma diagnosis.
3.1 reagents required for the experiment
Sealing liquid: 10% BSA was mixed with 1 XPBS at a ratio of 3:7 and placed on ice.
Serum incubation liquid: 10% BSA was mixed with 1 XPBST solution at a ratio of 1:9 and placed on ice.
Cleaning solution: 1 XPBST, 4 ℃ storage.
Secondary antibody incubation solution: including fluorescently labeled anti-human IgM secondary antibody (cy 5-labeled, appearing red).
3.2 concrete operating procedure of experiment
a) Rewarming: taking out the chip from a refrigerator at-80 deg.C, re-warming in a refrigerator at 4 deg.C for half an hour, and re-warming at room temperature for 15 min.
b) And (3) sealing: and fixing the rewarming chip in 14blocks in a fence, adding sealing liquid into each block, placing the blocks on a side swing shaking bed, and sealing for 3 hours at room temperature.
c) Incubation of serum samples: after the sealing is finished, the sealing liquid is poured out, then the prepared serum incubation liquid is quickly added, 14 samples are incubated on each chip (the samples are frozen and thawed in a chromatography cabinet at 4 ℃, the samples are diluted by the serum incubation liquid according to the proportion of 1:50, the sample loading volume of each serum sample is 200 mu L, the shaking table is laterally swung at 20rpm, and the overnight incubation is carried out at 4 ℃.
d) Cleaning: taking out the chip and the chip clamp together, sucking out the sample, then quickly adding an equal volume of 1 XPBST solution, and circulating for a plurality of times to ensure that no cross contamination exists among the serum samples when the chip clamp is detached. After the chip clamp was removed, the chip was placed in a chip washing cassette containing washing solution, and washed on a horizontal shaker at room temperature at 80rpm for 3 times, each time for 10 min.
e) And (3) secondary antibody incubation: the chip was transferred to an incubation box containing 3mL of secondary antibody incubation solution, and the shaking table was shaken laterally at 40rpm, protected from light, and left at room temperature for 60 min.
f) Cleaning: the chip was removed (note that the upper surface of the chip was not touched or scratched), placed in a chip washing cassette to which a washing solution was added, and washed 3 times at 80rpm for 10min each time on a horizontal shaker. After completion with ddH2O washing for 10min 2 times.
g) And (3) drying: the chip is placed in a chip drier for centrifugal drying.
h) Scanning: operating according to the operating specifications and instructions of the scanner.
i) Data extraction: and aligning the chip image and each array of the result as a whole, pressing an automatic alignment button, and extracting and storing data.
j) And (3) data processing and analysis: and (3) carrying out statistical analysis on the data, firstly screening 31 IgM autoantibodies with potential diagnostic efficacy according to AUC >0.5 and P <0.05, and finally selecting 5 IgM autoantibodies with top five AUC as combined detection serum markers for early diagnosis of lung adenocarcinoma, wherein the IgM autoantibodies are TSHR autoantibody, ERBB2 autoantibody, Survivin autoantibody, PIK3CA autoantibody and JAK2 autoantibody. An SNR scatter diagram of 5 IgM autoantibodies combined detection serum markers screened based on the protein chip result and an ROC curve chart for diagnosing lung adenocarcinoma are shown in FIG. 1.
The adopted data statistical analysis method specifically comprises the following steps:
the method uses a Mann-WhitneyU test method to analyze the difference of autoantibody levels between a lung adenocarcinoma group and a normal group, uses SPSS26.0 software to perform ROC analysis, and uses the OD value corresponding to the maximum Yoden index when the specificity is more than 90% as a cutoff value to calculate the AUC (95% CI), sensitivity, specificity, positive predicted value, negative predicted value, positive likelihood ratio, negative likelihood ratio and accuracy of an index and a model, thereby judging the diagnosis capability of the index and the model on the lung adenocarcinoma. Scatter plots were made using GraphPadPrism 8. All statistical analyses were performed using SPSS26.0 software, with P <0.05 as the statistical criterion.
4. Verification of 5 IgM autoantibodies combined detection serum markers by ELISA method
Detecting the levels of five IgM autoantibodies, namely TSHR, ERBB2, Survivin, PIK3CA and JAK2, of 147 lung adenocarcinoma patients and 147 normal control serum in a test group by using an ELISA detection method;
4.1 reagents required for the experiment
Coating solution (1L): mixing 1.5g of Na2CO3And 2.9g NaHCO3Dissolving in 800ml deionized water, mixing, adding water to desired volume of 1L, and storing at 4 deg.C.
10 XPBST wash (1L): mixing 81.8g NaCl, 28.8g Na2HPO4·12H2O and 3.1gNaH2PO4·2H2Dissolving O in 800ml deionized water, adding 5ml Tween 20, mixing, adding water to constant volume of 1L, and storing at room temperature.
1 XPBST wash (1L): 100ml of 10 XPBST lotion was put into a container, and water was added to the container to a constant volume of 1L and mixed well, and then stored at room temperature.
Blocking solution (2% BSA, 100 ml): 2g BSA (BIOSHARP Co.) was dissolved in 100ml1 XPBST solution, mixed well and stored at 4 ℃.
Antibody dilutions (1% BSA, 100 ml): 1g BSA (BIOSHARP Co.) was dissolved in 100ml1 XPBST solution, mixed well and stored at 4 ℃.
Stop solution (200 ml): 100ml of deionized water is taken in a container, 20ml of concentrated sulfuric acid is slowly injected into the container along a glass rod, stirring is carried out while adding, finally, water is added to a constant volume of 200ml, and the mixture is stored at room temperature.
Substrate solution a (100 ml): 20mg of TMB was dissolved in 100ml of deionized water, mixed well, and stored at 4 ℃ in the dark.
0.75％H2O2Solution (10 ml): 75mg of urea hydrogen peroxide is dissolved in 10ml of deionized water, mixed evenly and stored at 4 ℃ in the dark.
Substrate solution B (100 ml): mixing 3.7g of Na2HPO4·12H2O and 0.92g citric acid were dissolved in 80ml deionized water and 800. mu.l 0.75% H was added2O2The solution is added with water to be constant volume to 100ml and is stored in dark at 4 ℃.
Color development liquid: mixing the substrate solution A and the substrate solution B according to the ratio of 1:1, and storing at 4 ℃ in a dark place. It is prepared as before use.
4.2 concrete operation procedure of experiment
a) Coating: diluting protein (Wuhanyun clone) concentration to 0.125 μ g/ml with coating solution, coating in 96-well plate at 50 μ l/well, and placing in 4 deg.C refrigerator overnight;
b) and (3) sealing: the protein dilutions in the wells were discarded, and blocking solution was added to 96-well plates at 100. mu.l/well and blocked overnight in a refrigerator at 4 ℃.
c) Washing: the blocking solution in the wells was discarded, the microplate was placed in an automatic plate washer, washed according to the set program (300. mu.l wash solution/well, 10 seconds/time, 3 repetitions), and finally patted dry.
d) Primary antibody incubation: diluted serum and blank control (antibody dilution) were added to the corresponding positions in the 96-well plate, 50. mu.l/well, and incubated in a 37 ℃ water bath for 1 hour, wherein the diluted serum was diluted with the antibody dilution (1 XPBST solution) at a ratio of 1: 100.
e) Washing: the serum solution in the wells was discarded, the microplate was placed in an automatic plate washer, washed according to the set program (300. mu.l wash/well, 10 seconds/time, 5 repetitions), and finally patted dry.
f) And (3) secondary antibody incubation: diluting HRP-labeled goat anti-human IgM (SIGMA) and an antibody diluent according to a ratio of 1:2000, adding the diluted solution into a 96-well enzyme label plate, incubating the diluted solution at 50 mu l/well in a water bath kettle at 37 ℃ for 1 hour.
g) Washing: the secondary antibody solution in the wells was discarded, the microplate was placed in an automatic plate washer, washed according to the set program (300. mu.l wash/well, 10 seconds/time, 5 repetitions), and finally patted dry.
h) Color development: 50 mul of color development liquid (prepared as before) is added into each hole respectively, white paper needs to be padded under the enzyme label plate to ensure that the color change is easy to detect, and the plate is placed at room temperature and is protected from light to develop color for 20 min.
i) And (4) terminating: add stop solution, 25. mu.l/well.
j) Measuring the absorbance: and (3) respectively measuring absorbance values corresponding to the wavelengths of 450nm and 620nm by using a microplate reader, taking OD450-OD620 as a relative OD value, and deducting a blank control.
K) And (3) data processing and analysis: the expression levels of the five IgM autoantibodies in lung adenocarcinoma serum and normal control serum were significantly different (P <0.05) (shown in FIG. 2), revealing the consistency of the ELISA detection result and the protein chip result. However, as in many similar marker studies, a single index shows low diagnostic value, and the OD value corresponding to the maximum john index is selected as a cutoff value under the condition that the specificity is greater than 90%, and the sensitivity of a single IgM autoantibody is analyzed to be in the range of 9.52% to 17.01%, and the diagnostic accuracy is only 51.70% to 54.76% (table 2).
TABLE 2 diagnostic efficacy of 5 IgM autoantibody serum detection markers in the Experimental examples in ELISA verification
The OD value corresponding to the maximum john index is defined as the cutoff value when the specificity is greater than 90%.
5. Construction of combined detection serum marker and CEA combined diagnosis model for early diagnosis of lung adenocarcinoma
83 lung adenocarcinoma patients and 83 normal controls with serum CEA detection results were matched from all subjects in the validation group based on five IgMELISA result of antibody and clinical CEA detection information, index joint analysis is carried out by using Logistic regression analysis, candidate IgM autoantibody index is combined with traditional tumor marker CEA by using Logistic regression, and lung adenocarcinoma diagnosis model P is constructed by 1/(1+ Exp (- (1.655 OD)ERBB2+7.862*ODJAK2-10.285*ODTSHR+17.135*ODPIK3CA-11.294*ODSurvivin+0.299 × CEA-1.899)); OD in the formulaERBB2、ODJAK2、ODTSHR、ODPIK3CA、ODSurvivinRespectively subtracting the absorbance value of the blank control from the relative OD value of each IgM autoantibody index; CEA is the CEA content in the serum of a patient obtained by detecting clinically by an electrochemiluminescence method, and the unit is ng/mL.
The diagnosis result is shown in FIG. 3, the AUC of a single CEA is only 0.692, the AUC after the combination of the five IgM autoantibodies is 0.698, and the AUC after the combination of the five IgM autoantibodies and the CEA can reach 0.827; the result shows that the diagnostic model constructed by combining the two different types of biomarkers can effectively improve the diagnostic efficacy in the lung adenocarcinoma.
6. Application of combined detection serum marker and CEA combined diagnosis model for early diagnosis of lung adenocarcinoma in diagnosis of lung adenocarcinoma
1) Diagnostic efficacy of diagnostic models in lung adenocarcinoma and normal controls
According to the invention, 5 candidate IgM autoantibody indexes are combined with the traditional tumor marker CEA through Logistic regression to construct a lung adenocarcinoma diagnosis model. The AUC (95% CI) of the model in 83 lung adenocarcinoma samples and 83 normal control samples was 0.827(0.765-0.890), and the sensitivity and specificity were 56.63% and 93.98% (FIG. 3 and Table 3). The results show that the model has good diagnostic capability for lung adenocarcinoma patients.
2) Diagnostic efficacy of diagnostic models in patients with early and late stage lung adenocarcinoma
Lung adenocarcinoma patients were classified according to their TNM stage into 23 early stage lung adenocarcinoma patients, 55 late stage lung adenocarcinoma patients and 5 ill-staged lung adenocarcinoma patients. The ROC results of the diagnostic model in different stages show that the AUC (95% CI) of 23 cases of early lung adenocarcinoma to 83 cases of normal control has the sensitivity, specificity and accuracy of 0.744(0.625-0.864), 39.13%, 95.18% and 83.02% respectively; in addition, the sensitivity, specificity and accuracy were 0.861(0.793-0.928), 65.45%, 92.77%, 81.88% for 55 advanced lung adenocarcinomas versus 83 normal controls (95% CI), respectively (table 3). The result shows that the model has higher accuracy for diagnosing early lung adenocarcinoma.
3) Diagnostic efficacy of diagnostic models in lung adenocarcinoma patients with different lymph node metastasis conditions
The lung adenocarcinoma patients were classified into 32 lung adenocarcinoma patients who did not develop lymph node metastasis, 47 lung adenocarcinoma patients with lymph node metastasis and 4 lung adenocarcinoma patients with unknown lymph node metastasis. The ROC results of this diagnostic model in lung adenocarcinoma patients with different lymph node metastasis showed that the AUC (95% CI), sensitivity, specificity and accuracy of the model were 0.805(0.713-0.897), 43.75%, 92.77%, 79.13% for 32 lung adenocarcinoma patients without lymph node metastasis and 83 normal controls, respectively, while the AUC (95% CI), sensitivity, specificity and accuracy of the model were 0.843(0.764-0.921), 63.83%, 96.39%, 84.62% when 47 lung adenocarcinoma patients with lymph node metastasis and 83 normal controls were distinguished (Table 3). Also, the diagnostic model was statistically significant (P <0.05) for differences between patients with lung adenocarcinoma with and without lymph node metastasis. Therefore, the results of the invention suggest that the model can be used for diagnosing the lung adenocarcinoma patients under different lymph node metastasis conditions, and has higher diagnostic value particularly for patients with lymph node metastasis.
4) Diagnostic efficacy of diagnostic models in lung adenocarcinoma patients with different distant metastasis conditions
The lung adenocarcinoma patients were classified into 43 lung adenocarcinoma patients without distal metastasis, 34 lung adenocarcinoma patients with distal metastasis and 6 lung adenocarcinoma patients with unknown distal metastasis. The analysis results of the model in lung adenocarcinoma patients with different distant metastasis cases show that when the patients do not have distant metastasis, the AUC (95% CI), the sensitivity, the specificity and the accuracy are respectively 0.769(0.679-0.859), 44.19%, 93.98% and 76.98%; in the presence of distant metastasis, AUC (95% CI), sensitivity, specificity and accuracy were 0.892(0.817-0.967), 67.65%, 96.39%, 88.03%, respectively (table 3). In addition, this model also showed significant differences between lung adenocarcinoma patients with and without distant metastasis (P < 0.05). The results of the invention suggest that the model can be used for diagnosing lung adenocarcinoma patients under different far-end metastasis conditions, and has higher diagnostic value particularly for patients with far-end metastasis.
TABLE 3 diagnostic efficacy of the diagnostic models constructed from 5 IgM autoantibody serum detection markers and CEA in different lung adenocarcinoma patient stratification in Experimental examples
The P values represent the differences between early and late stage, lymph node metastasis (-) and lymph node metastasis (+), distant metastasis (-) and distant metastasis (+).
The above experimental examples are only preferred embodiments of the present invention, and the scope of the present invention is not limited thereto, and any simple changes or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the technical scope of the present invention are within the scope of the present invention.
Claims (9)
1. A combined detection serum marker for early diagnosis of lung adenocarcinoma, wherein the serum marker is five IgM autoantibodies including autoantibodies of TSHR, ERBB2, Survivin, PIK3CA and JAK 2.
2. A combined detection ELISA kit for early diagnosis of lung adenocarcinoma is characterized in that the kit comprises a solid phase carrier and a specific protein corresponding to the combined detection serum marker coated on the solid phase carrier.
3. The ELISA kit for combined detection of early diagnosis of lung adenocarcinoma as claimed in claim 2, wherein said solid carrier is a 96-well ELISA plate.
4. The combined detection ELISA kit for early diagnosis of lung adenocarcinoma as claimed in claim 2, wherein the kit further comprises a sample diluent, a second antibody diluent, a positive control serum, a negative control serum, a developing solution, a stop solution, a blocking solution and a washing solution.
5. The combined detection ELISA kit for early diagnosis of lung adenocarcinoma according to claim 2, wherein said second antibody is provided with a detectable label.
6. The ELISA kit for combined detection of early diagnosis of lung adenocarcinoma according to claim 2, wherein said marker is horseradish peroxidase.
7. The ELISA kit for combined detection of early diagnosis of lung adenocarcinoma as claimed in claim 2, wherein said second antibody is goat anti-human IgM antibody.
8. A method for detecting a lung adenocarcinoma serum sample by using the combined detection ELISA kit according to any one of claims 2-7, which comprises the following steps:
a) coating: diluting the concentration of the combined detection serum marker protein to 0.125 mu g/ml by using coating liquid, coating the combined detection serum marker protein into a 96-well plate according to 50 mu l/well, and placing the 96-well plate in a refrigerator at 4 ℃ overnight;
b) and (3) sealing: discarding the protein diluent in the hole, adding the sealing liquid into a 96-well plate, sealing the plate at 100 mu l/hole in a refrigerator at 4 ℃ overnight;
c) washing: discarding the blocking solution in the hole, placing the enzyme label plate in an automatic plate washing machine, repeatedly washing for 3 times according to 300 mul of washing liquid/hole and 10 seconds/time, and finally patting to dry;
d) primary antibody incubation: adding diluted serum and blank control at corresponding positions in a 96-well plate, incubating for 1 hour in a water bath kettle at 37 ℃ in a way of 50 mul/well, wherein the diluted serum is obtained by diluting the serum with an antibody diluent according to the proportion of 1: 100; blank control is antibody dilution;
e) washing: discarding serum solution in the hole, placing the enzyme label plate in an automatic plate washing machine, repeatedly washing for 5 times according to 300 mul of washing liquid/hole and 10 seconds/time, and finally patting to dry;
f) and (3) secondary antibody incubation: diluting goat anti-human IgM marked by HRP and an antibody diluent according to a ratio of 1:2000, adding a 96-hole enzyme label plate, incubating at 50 mu l/hole in a water bath kettle at 37 ℃ for 1 hour;
g) washing: discarding the secondary antibody solution in the hole, placing the enzyme label plate in an automatic plate washing machine, repeatedly washing for 5 times according to 300 mul of washing liquor/hole and 10 seconds/time, and finally patting to dry;
h) color development: respectively adding 50 μ l of color development liquid into each hole, padding white paper below the enzyme label plate, and placing at room temperature in dark for color development for 20 min;
i) and (4) terminating: adding stop solution into each hole at 25 mu l/hole;
j) measuring the absorbance: respectively measuring absorbance values corresponding to the wavelengths of 450nm and 620nm by using a microplate reader, and determining the absorbance values as OD450-OD620Relative OD values were obtained and the blank control was then deducted.
9. A method for constructing a combined diagnosis model for early diagnosis of lung adenocarcinoma is characterized by comprising the following steps:
(1) protein chip technology screening of IgM autoantibodies: serological analysis is carried out on lung adenocarcinoma patients and normal control serum samples through a protein chip technology, and potential five IgM autoantibodies which can be used for lung adenocarcinoma diagnosis are screened;
(2) verifying the selected candidate IgM autoantibody indexes in serum samples of lung adenocarcinoma patients and normal controls through an ELISA experiment to obtain combined detection serum markers for early diagnosis of lung adenocarcinoma, wherein the combined detection serum markers comprise autoantibodies of TSHR, ERBB2, Survivin, PIK3CA and JAK 2;
(3) combined detection serum marker for early diagnosis of lung adenocarcinoma and traditional tumor marker by Logistic regressionThe combination of the stoke CEA, and further construction of a lung adenocarcinoma diagnosis model P =1/(1 + Exp (- (1.655 OD)ERBB2 + 7.862*ODJAK2- 10.285*ODTSHR + 17.135*ODPIK3CA - 11.294*ODSurvivin+0.299 × CEA-1.899)); OD in the formulaERBB2、ODJAK2、ODTSHR、ODPIK3CA、ODSurvivinRespectively subtracting the absorbance value of the blank control from the relative OD value of each IgM autoantibody index; CEA is the CEA content in the serum of a patient obtained by detecting clinically by an electrochemiluminescence method, and the unit is ng/mL.
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