CN113687076B - 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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- G—PHYSICS
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- G01N33/57488—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 involving compounds identifable in body fluids
Abstract
The invention discloses a combined detection serum marker for early diagnosis of lung adenocarcinoma, which comprises TSHR, ERBB2, survivin, PIK3CA and IgM autoantibodies of JAK2, wherein ELISA serum detection kit is prepared by applying specific proteins corresponding to the combined detection serum marker, the combined detection serum marker plays an important role in improving sensitivity, specificity and accuracy of early diagnosis of lung adenocarcinoma, and a diagnosis model is constructed by applying the combined detection serum marker and the CEA 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, it is counted that lung cancer has 220 ten thousand new cases and 180 death cases, which respectively account for 11.4% and 18.0% of all cancers, and is the cancer with the second global incidence and the first death rate. Lung adenocarcinoma is the most common histological subtype in non-small cell lung cancer, accounting for about 40% of lung malignancies, and is common in women and non-smoky. Numerous studies have shown that patients with in situ adenocarcinoma (Adenocarcinoma in Situ, AIS) and micro-invasive adenocarcinoma (Minimally Invasive Adenocarcinoma, MIA) can have nearly 100% disease-free survival rates for 5 years if undergoing radical surgery. Therefore, the early diagnosis and timely effective treatment of the lung adenocarcinoma patients are of great significance for the prevention and treatment of lung cancer. At present, lung cancer patients are screened and diagnosed clinically mainly through low-dose spiral CT (LDCT) and pathological tissue biopsy, but the former has high false positive rate and the latter has high traumatism, so that the patients bear a large burden. In recent years, serological markers which are easy to operate, have small traumatism and are easily accepted by patients are widely concerned. Clinically, carcinoembryonic antigen (CEA) is widely applied to auxiliary diagnosis of lung cancer as a traditional serum tumor marker, but single detection has the defects of low sensitivity, low specificity and the like. IgM autoantibodies, which are the first type of antibodies produced in the humoral immune response process, have great potential as early diagnosis indicators of lung cancer, but have not been studied 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, which plays an important role in improving the sensitivity, specificity and accuracy of early diagnosis of lung adenocarcinoma, and simultaneously, a diagnosis model is constructed by combining the combined detection serum marker and CEA serum marker, so that a serological detection method which has high sensitivity, strong specificity and low cost and can assist in 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;
the combined detection ELISA kit for early diagnosis of lung adenocarcinoma is characterized by comprising a solid phase carrier and a specific protein corresponding to a combined detection serum marker coated on the solid phase carrier.
Preferably, the solid phase carrier is a 96-well ELISA plate.
Further, the kit also comprises a sample diluent, a second antibody diluent, positive control serum, negative control serum, a chromogenic solution, a stop solution, a blocking solution and a washing solution.
Preferably, the second antibody carries a detectable label;
preferably, the label is horseradish peroxidase;
preferably, the second antibody is a goat anti-human IgM antibody;
the method for detecting lung adenocarcinoma serum samples 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 a coating solution, placing the combined detection serum marker protein in a 96-well plate according to 50 mu l/Kong Baobei, and placing the mixture in a refrigerator at 4 ℃ overnight;
b) Closing: discarding protein diluent in the well, adding sealing liquid into the 96-well plate, sealing 100 μl/well in a refrigerator at 4deg.C overnight;
c) Washing: the blocking solution in the wells was discarded, the ELISA plate was placed in an automatic plate washer, washed 3 times repeatedly for 10 seconds per well with 300. Mu.l of wash solution, and finally dried.
d) Incubation resistance: adding diluted serum and blank control at corresponding positions in a 96-well plate, and incubating in a water bath at 37 ℃ for 1 hour at 50 μl/well, wherein the diluted serum is diluted by an antibody diluent according to a ratio of 1:100; the blank control is antibody diluent;
e) Washing: discarding serum solution in the hole, placing the ELISA plate in an automatic plate washer, repeatedly washing for 5 times according to 300 μl of washing liquid/hole for 10 seconds/time, and finally drying;
f) Secondary antibody incubation: diluting HRP-marked goat anti-human IgM and antibody diluent according to the proportion of 1:2000, adding a 96-well ELISA plate, 50 μl/well, and incubating in a 37 ℃ water bath kettle for 1 hour;
g) Washing: the secondary antibody solution in the wells was discarded, the ELISA plate was placed in an automatic plate washer, washed 5 times repeatedly for 10 seconds per well with 300. Mu.l of wash solution, and finally dried.
h) Color development: 50 μl of color development liquid is added to each well, white paper is required to be filled under the ELISA plate, and the plate is placed at room temperature and developed for 20min in dark place.
i) And (3) terminating: stop solution was added to each well at 25. Mu.l/well.
j) Absorbance was measured: measuring absorbance values corresponding to wavelengths of 450nm and 620nm respectively by using an enzyme-labeled instrument to obtain OD 450 -OD 620 Relative OD values were obtained and the blank was then snapped off.
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 for IgM autoantibodies: serologic analysis is carried out on lung adenocarcinoma patients and normal control serum samples by a protein chip technology, and five potential 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 ELISA experiments to obtain combined detection serum markers which can be used for early diagnosis of lung adenocarcinoma, wherein the combined detection serum markers comprise autoantibodies of TSHR, ERBB2, survivin, PIK3CA and JAK 2;
(3) The combined detection serum marker for early diagnosis of lung adenocarcinoma is combined with the traditional tumor marker CEA by using Logistic regression, so that a lung adenocarcinoma diagnosis model P=1/(1+exp (1.655 times OD) is constructed ERBB2 +7.862*OD JAK2 -10.285*OD TSHR +17.135*OD PIK3CA -11.294*OD Survivin +0.299 cea-1.899))); OD in ERBB2 、OD JAK2 、OD TSHR 、OD PIK3CA 、OD Survivin Subtracting the absorbance value after blank control from the relative OD value of each IgM autoantibody index; CEA is the content of CEA in serum of a patient detected by using an electrochemiluminescence method clinically, and the unit is ng/mL.
Drawings
FIG. 1 is a graph of the SNR scatter of 5 IgM autoantibody serum markers screened by a protein chip in experimental example and a ROC graph for diagnosing lung adenocarcinoma.
FIG. 2 is an OD scatter plot of 5 IgM autoantibody serum markers and a ROC graph for diagnosing lung adenocarcinoma as a result of ELISA verification in experimental examples.
FIG. 3 is a ROC graph of ELISA verification of 5 IgM autoantibody serum markers, CEA serum markers and a combination of both for lung adenocarcinoma in experimental examples.
Advantageous effects
The five combined detection serum markers for early diagnosis of lung adenocarcinoma are used for detecting serum samples of 83 lung adenocarcinomas and 83 normal controls, and the results show that the AUC after the combination of the five combined detection serum markers is 0.698, the AUC of the traditional tumor marker CEA is 0.692, and the AUC after the combination of the five combined detection serum markers and CEA can reach 0.827.
The five combined detection serum markers for early diagnosis of lung adenocarcinoma of the invention show a certain diagnosis effect on lung adenocarcinoma, and the five combined detection serum markers are combined with the clinically existing tumor marker CEA to construct a diagnosis model, so that a diagnosis method with simplicity, feasibility, no wound, low cost and higher diagnosis efficiency can be provided for clinic. The model can effectively distinguish lung adenocarcinoma patients from normal control, and has good diagnosis value in layering of different lung adenocarcinoma patients. Furthermore, the model is statistically significant for differences in different groupings (early and late stage lung adenocarcinoma patients; lung adenocarcinoma patients with and without lymph node metastasis; lung adenocarcinoma patients with and without distant metastasis). The model has a certain clinical auxiliary function for diagnosing and layering lung adenocarcinoma patients.
Detailed Description
The following describes the technical scheme of the present invention in detail with reference to the drawings and specific experimental examples, but it should be emphasized that the present invention is not limited to the specific embodiments described.
Experimental example
1. Serum specimen collection and preparation
1.1 collection of serum samples
The study was included in 430 subjects, and was divided into a discovery group and a validation group, the discovery group was used to screen for IgM autoantibody indicators with higher diagnostic value for lung adenocarcinoma. The validation set was used to validate the candidate IgM autoantibody indicators and construct a diagnostic model in combination with CEA.
All subjects serum specimens were collected in a three-phase hospital in Henan province during 2016-2019, and all subjects' basic information, pathological stage, serum CEA levels, and the like were consolidated by the hospital internal system with patient consent and approval by the institutional review board and the ethical committee of the hospital. The serum CEA detection result is provided by the clinical laboratory of the hospital and is obtained by adopting a MODULARE70 type full-automatic analyzer and a kit, which are produced by Swiss Rogowski company, and the principle is an electrochemiluminescence method. The experimental procedure was performed by a skilled artisan and the results were published after examination by an experienced clinical laboratory physician. The CEA threshold was 5.0ng/mL. Specific subject information is shown in table 1.
TABLE 1 clinical information of subjects in the serum sample discovery group and validation group of the invention
P * ＜0.05
1.2 preparation of serum samples
All specimens are prepared by collecting 5-10mL of whole blood of a study object by using a red head blood collection tube, standing at room temperature for 2 hours, centrifuging at 1000g for 15 minutes, sub-packaging the supernatant into 500 mu L/tube, labeling, and preserving at-80 ℃ to avoid repeated freeze thawing.
2. Preparation of protein chip for screening combined detection serum protein marker for early diagnosis of lung adenocarcinoma
Protein chips were custom-made by Guangzhou Bo biotechnology Co., ltd, which contains 154 recombinant proteins or protein fragments, of which 11 (CIP 2A/p90, c-Myc, cyclinB1, IMP1, IMP2, IMP3, ralA, RBM39, YWHAZ and two Survivin fragments) were found in studies prior to the present laboratory (Establishment and validation of an immunodiagnostic model for prediction of breast cancer.Oncoimmunogy 9 (1) (2020) 1682382. And Using recursive partitioning approach to select tumor-associated antigens in immunodiagnosis of gastric adenocarcinoma, cancer Sci 110 (6) (2019) 1829-1841.) to code for proteins or protein fragments with potential diagnostic value, the other 143 being based on 138 Cancer driver-encoded proteins (Cancer genome landscapes, science (New York, N.Y.) 339 (6127) (2013) 1546-58.).
3. Screening experiment based on protein chip technology
Serological analysis is carried out on 68 lung adenocarcinoma patients found in the group and 68 normal controls through a protein chip technology, and potential combined detection serum markers which can be used for lung adenocarcinoma diagnosis are screened.
3.1 reagents required for experiments
Sealing liquid: 10% BSA was mixed with 1 XPBS solution at a ratio of 3:7 and placed on ice.
Serum incubation: 10% BSA was mixed with 1 XPBST solution at a ratio of 1:9 and placed on ice.
Cleaning liquid: 1 XPBST, stored at 4 ℃.
Secondary antibody incubation liquid: including fluorescent-labeled anti-human IgM secondary antibodies (cy 5 label, red).
3.2 Experimental specific procedure
a) And (3) rewarming: taking out the chip from the refrigerator at-80 ℃, rewarming for half an hour at the refrigerator at 4 ℃, and rewarming for 15min at room temperature.
b) Closing: the chips after the rewarming were fixed in 14blocks with a blocking solution added to each block and placed on a side shaking table and blocked for 3 hours at room temperature.
c) Incubation of serum samples: after the sealing is completed, the sealing liquid is poured out, and then a prepared serum incubation liquid is rapidly added, 14 samples are incubated for each chip (the samples are firstly placed in a chromatographic cabinet at 4 ℃ for freeze thawing, the samples are diluted by the serum incubation liquid in a ratio of 1:50, the loading volume of each serum sample is 200 mu L, and a side swinging table is 20rpm for overnight incubation at 4 ℃.
d) Cleaning: the chip and the chip clamp are taken out together, the sample is sucked, then the equal volume of 1 XPBST solution is added rapidly, and the cycle is performed for a plurality of times, so that no cross contamination exists between serum samples when the chip clamp is removed. After the chip clamp is removed, the chip is placed in a chip cleaning box with cleaning liquid, a horizontal shaking table and a room temperature of 80rpm, and the chip is cleaned for 3 times for 10 minutes each time.
e) Secondary antibody incubation: the chip was transferred to an incubation box with 3mL of secondary antibody incubation, side shaking table 40rpm, protected from light, room temperature for 60min.
f) Cleaning: the chip was removed (taking care not to touch or scratch the upper surface of the chip), placed in a chip cleaning cartridge with cleaning solution added, and cleaned 3 times at 80rpm for 10min each on a horizontal shaker. After completion use of ddH 2 O was washed 2 times for 10min each.
g) And (3) drying: and (5) placing the chip in a chip dryer for centrifugal drying.
h) Scanning: operating in accordance with scanner operating specifications and instructions.
i) And (3) data extraction: and (3) aligning the chip image with each array as a whole, pressing an automatic alignment button, extracting data and storing the data.
j) Data processing analysis: the data were statistically analyzed by first screening 31 IgM autoantibodies with potential diagnostic efficacy according to AUC >0.5 and P <0.05, and finally selecting the five 5 IgM autoantibodies with top AUC rank as combined detection serum markers for early diagnosis of lung adenocarcinoma, respectively TSHR, ERBB2, survivin, PIK3CA and JAK2 autoantibodies. SNR scatter plots of the combined detection of serum markers based on the 5 IgM autoantibodies screened by the protein chip results and ROC graphs for diagnosing lung adenocarcinoma are shown in FIG. 1.
The adopted data statistical analysis method is specifically as follows:
according to the invention, the difference of autoantibody levels between a lung adenocarcinoma group and a normal group is analyzed by using a Mann-WhitneyU test method, ROC analysis is performed by using SPSS26.0 software, and the AUC (95% CI), sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio, negative likelihood ratio and accuracy of an index and a model are calculated by taking an OD value corresponding to the maximum about a Density index when the specificity is more than 90% as a cut-off value, so that the diagnostic capability of the index and the model on lung adenocarcinoma is judged. The scatter plot was completed using GraphPadPrism 8. All statistical analyses were performed using SPSS26.0 software, P <0.05 being the statistical criterion.
4. ELISA method is used for verifying serum markers for joint detection of 5 IgM autoantibodies
Using ELISA detection method to detect the levels of five IgM autoantibodies of TSHR, ERBB2, survivin, PIK3CA and JAK2 in 147 lung adenocarcinoma patients and 147 normal control serum in the verification group;
4.1 reagents required for experiments
Coating liquid (1L): will be 1.5g Na 2 CO 3 And 2.9g NaHCO 3 Dissolving in 800ml deionized water, mixing, adding water to 1L, and storing at 4deg.C.
10 XPBST wash (1L): 81.8g NaCl,28.8g Na 2 HPO 4 ·12H 2 O and 3.1g NaH 2 PO 4 ·2H 2 O is dissolved in 800ml deionized water, 5ml Tween 20 is added, the mixture is uniformly mixed, finally water is added to fix the volume to 1L, and the mixture is preserved at room temperature.
1 XPBST wash (1L): 100ml of 10 XPBST lotion was taken into a container, water was added to a volume of 1L and mixed well, and stored at room temperature.
Blocking solution (2% bsa,100 ml): 2g of BSA (BIOSHARP) was dissolved in 100ml of 1 XPBST solution, and the mixture was stirred and stored at 4 ℃.
Antibody dilutions (1% bsa,100 ml): 1g of BSA (BIOSHARP) was dissolved in 100ml of 1 XPBST solution, and the mixture was stirred and stored at 4 ℃.
Stop solution (200 ml): taking 100ml of deionized water in a container, slowly injecting 20ml of concentrated sulfuric acid into the container along a glass rod, stirring while adding water, and finally adding water to a volume of 200ml, and preserving at room temperature.
Substrate solution A (100 ml): 20mgTMB was dissolved in 100ml deionized water, mixed well and stored at 4deg.C in the dark.
0.75％H 2 O 2 Solution (10 ml): 75mg of urea hydrogen peroxide is dissolved in 10ml of deionized water, evenly mixed and stored at 4 ℃ in a dark place.
Substrate solution B (100 ml): will 3.7gNa 2 HPO 4 ·12H 2 O and 0.92g of citric acid were dissolved in 80ml of deionized water, and 800. Mu.l of 0.75% H was added thereto 2 O 2 The solution was then added with water to a volume of 100ml and stored at 4℃in the absence of light.
Color development liquid: mixing the substrate solution A and the substrate solution B according to the ratio of 1:1, and storing in a dark place at 4 ℃. Is prepared in the prior art.
4.2 Experimental specific procedure
a) Coating: protein (Wuhan cloud clone) concentration was diluted to 0.125. Mu.g/ml with coating solution, and placed in 96-well plates at 50. Mu.l/Kong Baobei in a refrigerator at 4℃overnight;
b) Closing: the protein dilutions were discarded from the wells, blocking solution was added to the 96-well plates, 100 μl/well, and the wells were blocked overnight in a refrigerator at 4deg.C.
c) Washing: the blocking solution in the wells was discarded, the ELISA plate was placed in an automatic plate washer, washed according to the set procedure (300. Mu.l wash/well, 10 seconds/time, 3 repetitions) and finally dried.
d) Incubation resistance: diluted serum and blank (antibody diluent) were added to corresponding positions in 96-well plates and incubated in a water bath at 37℃for 1 hour at 50. Mu.l/well, wherein the diluted serum was diluted with antibody diluent (1 XPBST solution) at a ratio of 1:100.
e) Washing: the serum solution in the wells was discarded, the ELISA plate was placed in an automatic plate washer, washed according to the set procedure (300. Mu.l wash/well, 10 seconds/repeat 5 times), and finally dried.
f) Secondary antibody incubation: HRP-labeled goat anti-human IgM (SIGMA) was diluted with antibody dilution at a ratio of 1:2000, and then 96-well ELISA plates, 50. Mu.l/well, were added and incubated in a 37℃water bath for 1 hour.
g) Washing: the secondary antibody solution in the wells was discarded, the ELISA plate was placed in an automatic plate washer, washed according to the set procedure (300. Mu.l wash/well, 10 seconds/repeat 5 times), and finally patted dry.
h) Color development: 50 mu l of color development liquid (in-situ preparation) is added into each hole, white paper is required to be filled under the ELISA plate, so that color change is easy to be perceived, and the ELISA plate is placed at room temperature to develop color for 20min in a dark place.
i) And (3) terminating: stop solution, 25. Mu.l/well, was added.
j) Absorbance was measured: and (3) measuring absorbance values corresponding to wavelengths of 450nm and 620nm respectively by using an enzyme-labeled instrument, taking OD450-OD620 as a relative OD value, and then deducting a blank control.
K) Data processing analysis: the expression levels of the five IgM autoantibodies in lung adenocarcinoma and normal control serum were significantly different (P < 0.05) (shown in fig. 2), revealing the consistency of the ELISA detection results with the protein chip results. However, as in many similar marker studies in the past, single index showed lower diagnostic value, with the OD value corresponding to the maximum approximate log index selected as cut-off value at a specificity greater than 90%, and single IgM autoantibody sensitivity was analyzed to be 9.52% to 17.01% with diagnostic accuracy ranging from 51.70% to 54.76% (table 2).
Table 2 diagnostic efficacy of 5 IgM autoantibody serum detection markers in experimental example in ELISA validation
* At a specificity greater than 90%, the OD value corresponding to the maximum about log index is defined as the cutoff.
5. Construction of combined diagnosis model of combined detection serum marker and CEA (CEA) for early diagnosis of lung adenocarcinoma
83 lung adenocarcinoma patients and 83 normal controls matched with serum CEA detection results from all subjects in the validation groupBased on ELISA results and clinical CEA detection information of five IgM autoantibodies, performing joint analysis on indexes by using Logistic regression analysis, and combining candidate IgM autoantibody indexes with a traditional tumor marker CEA by using Logistic regression to construct a lung adenocarcinoma diagnosis model P=1/(1+exp (1.655X OD) ERBB2 +7.862*OD JAK2 -10.285*OD TSHR +17.135*OD PIK3CA -11.294*OD Survivin +0.299 cea-1.899))); OD in ERBB2 、OD JAK2 、OD TSHR 、OD PIK3CA 、OD Survivin Subtracting the absorbance value after blank control from the relative OD value of each IgM autoantibody index; CEA is the content of CEA in serum of a patient detected by using an electrochemiluminescence method clinically, and the unit is ng/mL.
The diagnosis results are shown in FIG. 3, the AUC of single CEA is only 0.692, the AUC after the combination of five IgM autoantibodies is 0.698, and the AUC after the combination of the five IgM autoantibodies and CEA can reach 0.827; the result shows that the combined construction of two different types of biomarkers into a diagnosis model can effectively improve the diagnosis efficiency in 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, so as to construct a lung adenocarcinoma diagnosis model. The model achieves an AUC (95% ci) of 0.827 (0.765-0.890), sensitivity and specificity of 56.63%,93.98% in the samples of 83 lung adenocarcinomas and 83 normal controls (fig. 3 and table 3). The results show that the model has good diagnosis capability for lung adenocarcinoma patients.
2) Diagnostic efficacy of diagnostic models in early and late stage lung adenocarcinoma patients
The lung adenocarcinoma patients were classified into 23 early-stage lung adenocarcinoma patients, 55 late-stage lung adenocarcinoma patients and 5 unidentified lung adenocarcinoma patients according to TNM stage of the lung adenocarcinoma patients. ROC results of the diagnostic model in different phases show that AUC (95% ci) of 23 cases of early lung adenocarcinoma to 83 cases of normal control, sensitivity, specificity and accuracy are respectively 0.744 (0.625-0.864), 39.13%,95.18% and 83.02%; in addition, the AUC (95% ci), sensitivity, specificity and accuracy were 0.861 (0.793-0.928), 65.45%,92.77%,81.88%, respectively, for 55 cases of advanced lung adenocarcinoma versus 83 normal controls (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
The cases of lymph node metastasis of lung adenocarcinoma patients are classified into 32 lung adenocarcinoma patients in which lymph node metastasis does not occur, 47 lung adenocarcinoma patients in which lymph node metastasis occurs, and 4 lung adenocarcinoma patients in which lymph node metastasis is unknown. ROC results of this diagnostic model in lung adenocarcinoma patients with different lymph node metastasis showed that AUC (95% ci), sensitivity, specificity and accuracy of this model were 0.805 (0.713-0.897), 43.75%,92.77%,79.13%, respectively, for 32 lung adenocarcinoma patients without lymph node metastasis and 83 normal controls, respectively, whereas AUC (95% ci), sensitivity, specificity and accuracy of this model were 0.843 (0.764-0.921), 63.83%,96.39%,84.62%, respectively (table 3), when distinguishing 47 lung adenocarcinoma patients with lymph node metastasis and 83 normal controls. Moreover, the diagnostic model also has statistical significance (P < 0.05) for differences between lung adenocarcinoma patients with and without lymph node metastasis. Therefore, the result of the invention suggests that the model can be used for diagnosing lung adenocarcinoma patients under different lymph node metastasis conditions, and has higher diagnostic value especially for patients with lymph node metastasis.
4) Diagnostic efficacy of diagnostic models in lung adenocarcinoma patients with different distant metastasis conditions
The cases of distant metastasis of lung adenocarcinoma patients are classified into 43 cases of lung adenocarcinoma patients without distant metastasis, 34 cases of lung adenocarcinoma patients with distant metastasis and 6 cases of lung adenocarcinoma patients with unknown distant metastasis. Analysis of the model in lung adenocarcinoma patients with different distant metastasis conditions shows that when the patients do not undergo distant metastasis, the AUC (95% ci), sensitivity, specificity and accuracy are 0.769 (0.679-0.859), 44.19%,93.98%,76.98% respectively; in the presence of distant metastasis in patients, their 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, the model also has significant differences between lung adenocarcinoma patients with and without distant metastasis (P < 0.05). The result of the invention suggests that the model can be used for diagnosing lung adenocarcinoma patients under different remote metastasis conditions, and has higher diagnostic value especially for patients with remote metastasis.
Table 3 diagnostic efficacy of diagnostic models constructed from 5 IgM autoantibody serum detection markers and CEA in stratification of patients with different lung adenocarcinomas in experimental examples
P-values represent differences between early and late stage, lymph node metastasis (-) and lymph node metastasis (+), distal metastasis (-) and distal metastasis (+).
The above experimental examples are only preferred embodiments of the present invention, the protection scope of the present invention is not limited thereto, and any simple changes or equivalent substitutions of the technical solutions that are obvious to those skilled in the art within the technical scope of the present invention disclosed herein fall within the protection scope of the present invention.
Claims (8)
1. The combined detection ELISA kit for early diagnosis of lung adenocarcinoma is characterized by comprising a solid phase carrier and a specific protein corresponding to a combined detection serum marker coated on the solid phase carrier; the serum markers are five IgM autoantibodies including autoantibodies to TSHR, ERBB2, survivin, PIK3CA and JAK 2.
2. The combined detection ELISA kit of claim 1 for early diagnosis of lung adenocarcinoma, characterized in that the solid phase carrier is a 96-well ELISA plate.
3. The joint detection ELISA kit of claim 1 useful for early diagnosis of lung adenocarcinoma, further comprising a sample diluent, a secondary antibody diluent, a positive control serum, a negative control serum, a chromogenic solution, a stop solution, a blocking solution, and a wash solution.
4. The combined detection ELISA kit of claim 3 useful for early diagnosis of lung adenocarcinoma wherein the secondary antibody is provided with a detectable label.
5. The combined detection ELISA kit for early diagnosis of lung adenocarcinoma according to claim 4, wherein the marker is horseradish peroxidase.
6. The combined detection ELISA kit of claim 5 for early diagnosis of lung adenocarcinoma, wherein the second antibody is a goat anti-human IgM antibody.
7. A method for detecting lung adenocarcinoma serum samples for non-disease diagnostic and therapeutic purposes using a combined detection ELISA kit according to any of claims 1-6, characterized in that it comprises in particular the following steps:
a) Coating: diluting the concentration of the combined detection serum marker protein to 0.125 mu g/ml by using a coating solution, placing the combined detection serum marker protein in a 96-well plate according to 50 mu l/Kong Baobei, and placing the mixture in a refrigerator at 4 ℃ overnight;
b) Closing: discarding protein diluent in the well, adding sealing liquid into the 96-well plate, sealing 100 μl/well in a refrigerator at 4deg.C overnight;
c) Washing: discarding the sealing liquid in the hole, placing the ELISA plate in an automatic plate washer, repeatedly washing for 3 times according to 300 μl of washing liquid/hole for 10 seconds/time, and finally drying;
d) Incubation resistance: adding diluted serum and blank control at corresponding positions in a 96-well plate, and incubating in a water bath at 37 ℃ for 1 hour at 50 μl/well, wherein the diluted serum is diluted by an antibody diluent according to a ratio of 1:100; the blank control is antibody diluent;
e) Washing: discarding serum solution in the hole, placing the ELISA plate in an automatic plate washer, repeatedly washing for 5 times according to 300 μl of washing liquid/hole for 10 seconds/time, and finally drying;
f) Secondary antibody incubation: diluting HRP-marked goat anti-human IgM and antibody diluent according to the proportion of 1:2000, adding a 96-well ELISA plate, 50 μl/well, and incubating in a 37 ℃ water bath kettle for 1 hour;
g) Washing: discarding the secondary antibody solution in the hole, placing the ELISA plate in an automatic plate washer, repeatedly washing for 5 times according to 300 μl of washing liquid/hole for 10 seconds/time, and finally drying;
h) Color development: adding 50 mu l of color development liquid into each hole, filling white paper under the ELISA plate, and placing at room temperature for color development in a dark place for 20min;
i) And (3) terminating: adding a stop solution to the wells at 25. Mu.l/well, respectively;
j) Absorbance was measured: measuring absorbance values corresponding to wavelengths of 450nm and 620nm respectively by using an enzyme-labeled instrument to obtain OD 450 -OD 620 Relative OD values were obtained and the blank was then snapped off.
8. The construction method of the combined diagnosis model for early diagnosis of lung adenocarcinoma is characterized by comprising the following steps of:
(1) Protein chip technology screening for IgM autoantibodies: serologic analysis is carried out on lung adenocarcinoma patients and normal control serum samples by a protein chip technology, and five potential 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 ELISA experiments to obtain combined detection serum markers which can be used for early diagnosis of lung adenocarcinoma, wherein the combined detection serum markers comprise autoantibodies of TSHR, ERBB2, survivin, PIK3CA and JAK 2;
(3) The combined detection serum marker for early diagnosis of lung adenocarcinoma is combined with the traditional tumor marker CEA by using Logistic regression, so that a lung adenocarcinoma diagnosis model P=1/(1+exp (1.655 times OD) is constructed ERBB2 +7.862*OD JAK2 -10.285*OD TSHR +17.135*OD PIK3CA -11.294*OD Survivin +0.299 cea-1.899))); OD in ERBB2 、OD JAK2 、OD TSHR 、OD PIK3CA 、OD Survivin Subtracting the absorbance value after blank control from the relative OD value of each IgM autoantibody index; CEA is the content of CEA in serum of a patient detected by using an electrochemiluminescence method clinically, and the unit is ng/mL.
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