CN117805372A - Application of biomarker in preparation of reagent or kit for detecting hashimoto thyroiditis spleen-kidney yang deficiency syndrome - Google Patents
Application of biomarker in preparation of reagent or kit for detecting hashimoto thyroiditis spleen-kidney yang deficiency syndrome Download PDFInfo
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
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Abstract
The invention discloses application of a biomarker in preparation of a reagent or a kit for detecting hashimoto thyroiditis spleen-kidney yang deficiency syndrome, wherein the biomarker is at least one of autoantibodies related to MAP9, IRF9, PLEKHO2, NPM3, DR1, KDM1A, PCGF, GPCPD1 and IGBP1. The biomarkers for detecting autoantibodies characteristic of the thyroiditis liver qi stagnation syndrome are MAP9 and IRF9 related autoantibodies; biomarkers for detecting autoantibodies characteristic of the hashimoto thyroiditis yin deficiency fire excess syndrome are two related autoantibodies of PLEKHO2 and NPM3; biomarkers for detecting autoantibodies characteristic of the spleen-kidney yang deficiency syndrome of hashimoto thyroiditis are five related autoantibodies of DR1, KDM1A, PCGF, GPCPD1 and IGBP1. The serum related autoantibody markers can reflect the syndrome features of the thyroiditis, so that the syndrome differentiation of traditional Chinese medicine is accurate, and the demonstration of the syndrome essence and the molecular connotation of the thyroiditis is facilitated.
Description
Technical Field
The invention relates to the technical fields of immunology and serum antibody histology, in particular to a biomarker for detecting autoantibodies with different symptoms of hashimoto thyroiditis and application thereof.
Background
Hashimoto's disease (HT), also known as Hashimoto's disease, or chronic lymphocytic thyroiditis (Chronic lymphocytic thyroiditis, CLT), is the most common organ-specific autoimmune disease at present, clinically manifested as diffuse enlargement of the thyroid gland, elevated serum levels of thyroautoantibodies, and the most common cause of hypothyroidism, with about 20% -30% of patients reported to develop hypothyroidism. The incidence of HT is rapidly increasing year by year at present, and has become a common disease which seriously threatens the health, work and life of people. The literature reports that HT has a global annual incidence of about 0.3-1.5 per 1000 people, and female prevalence is 4-10 times that of male. At present, large-scale HT epidemiological investigation data are not available in China, and the relevant reports of HT prevalence rate in different places are different by 1.03-23.08%. HT often develops with hidden disease and early stage has no specific clinical symptoms, so that the disease is easy to miss and misdiagnose, most cases are first diagnosed by thyroid nodule, thyromegaly, hypothyroidism or autoimmune diseases combined with other forms, and most patients are in an unknown state and are mostly found in the routine physical examination. In addition, HT is susceptible to canceration, and the incidence rate of the combined thyroid cancer is 0.5-38% abroad and 0.6-29.4% domestically. Currently there is an inherent lack of effective drugs for the treatment of HT, especially in the early stages of onset.
The dialectical treatment is the advantage of traditional Chinese medicine treatment, has obviously increased reports about HT treatment by traditional Chinese medicine in recent years, and has obvious curative effects on improving symptoms and reducing thyroid autoantibodies. The syndrome differentiation and treatment of traditional Chinese medicine is based on the symptoms and signs, and includes the changes of microscopic substances such as viscera, organs, tissues, cells and molecules. With the development of genome, proteome, transcriptome, metabolome and other histology, a plurality of researchers use histology technology to screen, analyze, identify and verify biological markers in recent years, research the molecular connotation of traditional Chinese medicine symptoms, and improve the accuracy of syndrome diagnosis from the molecular level, thereby precisely finding out the targets of syndrome molecular pathogenesis and treatment, and having important significance for disease diagnosis and treatment. According to the research on the traditional Chinese medicine syndrome distribution rule of HT patients, in the HT pathological process, the symptoms of liver qi stagnation are common in the early thyroid function normal stage, the symptoms of fire excess from yin deficiency are mainly common in the hyperthyroidism stage, and the symptoms of spleen-kidney yang deficiency are common in the hypothyroidism stage. However, there are no molecular mechanisms for treating HT by using histology to study the syndrome and pathogenesis of HT and the differentiation of syndromes in traditional Chinese medicine.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a biomarker for detecting autoantibodies with different symptoms of hashimoto thyroiditis and application thereof, wherein the antibody marker can reflect the symptoms of hashimoto thyroiditis.
In order to achieve the above object, one of the technical solutions of the present invention is:
the biomarker for detecting the autoantibodies with different symptoms of hashimoto thyroiditis is at least one of MAP9, IRF9, PLEKHO2, NPM3, DR1, KDM1A, PCGF2, GPCPD1 and IGBP1 related autoantibodies.
Further, the biomarker for detecting the autoantibody characteristic of the thyroiditis liver qi stagnation syndrome is at least one of two related autoantibodies of MAP9 and IRF9; preferably, a combination of two related autoantibodies.
The biomarker for detecting the autoantibodies characteristic of the hashimoto thyroiditis yin deficiency fire excess syndrome is at least one of two related autoantibodies PLEKHO2 and NPM3; preferably, a combination of two related autoantibodies.
The biomarker for detecting autoantibodies characteristic of the spleen-kidney yang deficiency syndrome of hashimoto thyroiditis is at least one of five related autoantibodies, namely DR1, KDM1A, PCGF, GPCPD1 and IGBP 1; preferably, a combination of five related autoantibodies.
One of the technical schemes of the invention is as follows: the application of the biomarker in preparing a hashimoto thyroiditis treatment drug.
One of the technical schemes of the invention is as follows: the application of the biomarker in preparing reagents and kits related to detection of autoantibodies characteristic of hashimoto thyroiditis syndrome.
One of the technical schemes of the invention is as follows: a kit for detecting autoantibodies characteristic of hashimoto thyroiditis liver qi stagnation syndrome comprises at least one of MAP9 and IRF9 human recombinant proteins, and the kit is used for detecting at least one of MAP9 and IRF9 related autoantibodies in serum.
One of the technical schemes of the invention is as follows: a kit for detecting autoantibodies characteristic of hashimoto thyroiditis yin deficiency fire excess syndrome comprises at least one of PLEKHO2 and NPM3 human recombinant proteins, and the kit is used for detecting at least one of PLEKHO2 and NPM3 related autoantibodies in serum.
One of the technical schemes of the invention is as follows: a kit for detecting autoantibodies characteristic of hashimoto thyroiditis spleen-kidney yang deficiency syndrome comprises at least one of five human recombinant proteins DR1, KDM1A, PCGF, GPCPD1 and IGBP1, and the kit is used for detecting at least one of five related autoantibodies DR1, KDM1A, PCGF, GPCPD1 and IGBP1 in serum.
Further, the kit also comprises a human IgG standard, goat anti-human IgG marked by horseradish peroxidase and a reagent commonly used in ELISA technology.
The invention has the beneficial effects that:
in order to study the change of autoantibodies related to different symptoms of HT and characteristic protein markers and molecular pathogenesis of the symptoms, the invention respectively selects typical symptoms of liver qi stagnation, yin deficiency fire excess and spleen-kidney yang deficiency in patients with different thyroid functional states of normal, hyperfunction and hypofunction of HT, and applies HuProt TM Human proteome chip technology (comprising>20000 newly sequenced recombinant human proteins, corresponding to 16152 uniqueProtein coding genes, the coverage rate is up to 81%, and the highest flux human recombinant protein group chip in the world so far) performs serogroup analysis on HT different syndrome patients and healthy control serum, screens HT syndrome related autoantibodies, and then adopts an indirect ELISA method to verify and detect potential characteristic autoantibodies in serum of the thyroiditis different syndrome patients in a larger sample so as to obtain characteristic proteins capable of reflecting thyroiditis syndrome, so that the molecular connotation of the syndrome is revealed more clearly, the traditional Chinese medicine syndrome differentiation is accurate, the traditional Chinese medicine syndrome differentiation is guided from the molecular level, and a new reference basis is provided for the basic research of thyroiditis syndrome.
Specifically, the technical scheme of the invention comprises the following steps: (1) establishing a unified standard specimen library and database: the system collects complete basic information of the thyroiditis patient, four diagnosis information of traditional Chinese medicine symptoms and clinical case data, and collects blood samples meeting the standard according to a standard operation procedure. (2) Serum samples of patients with typical symptoms of liver qi stagnation, yin deficiency fire hyperactivity and spleen-kidney yang deficiency are respectively selected from patients with different thyroid functional states of hashimoto thyroiditis with normal thyroid function, hyperthyroidism and hypothyroidism. (3) Using high-throughput HuProt TM The human proteome chip technology analyzes the serum related autoantibodies which are differentially expressed in different patients with thyroiditis and normal control groups, and screens the differentially expressed significant syndrome characteristic related autoantibodies by using a statistical and bioinformatics method. (4) Further carrying out large sample verification in different patients with hashimoto thyroiditis, other patients with thyroiditis and normal control patients, and determining the sensitivity and specificity of the autoantibodies related to the characteristics of the hashimoto thyroiditis in distinguishing different syndrome proteins. (5) Development of a syndrome characteristic related autoantibody detection kit: according to the kit for detecting the characteristic protein of the characteristic related autoantibody of the differential expression in serum of the thyroiditis and normal people with different symptoms, the characteristic protein can reflect the characteristics of the thyroiditis, the traditional Chinese medicine differentiation is accurate, and a new reference basis is provided for the substantial research of the thyroiditis.
The application of the kit can detect serum related autoantibodies with traditional Chinese medicine syndrome characteristic of thyroiditis, and the serum related autoantibody markers can reflect the syndrome characteristics of thyroiditis, so that the traditional Chinese medicine syndrome differentiation is accurate, and the composition and the molecular connotation of thyroiditis are favorable for revealing.
Drawings
FIG. 1 shows the principle of protein chip detection.
FIG. 2 shows the results of the chip scan quality evaluation (protein spotting).
Note that: fig. a is a chip scan: shown as a whole scan (left) and a partial magnified image (right), respectively, wherein the 1-4 st arrow from left to right indicates a positive control (GST, concentration from left to right is 10 ng/. Mu.L, 50 ng/. Mu.L, 100 ng/. Mu.L, 200 ng/. Mu.L, respectively), and the rightmost arrow indicates a negative control (BSA protein); panel B shows the results of the chip preparation reproducibility evaluation.
Fig. 3 is a SNR cluster analysis of 59 related autoantibodies with significant differences in the hashimoto thyroiditis liver qi depression group.
Note that: the transverse direction is a sample, and the longitudinal direction is the protein (IgG or IgM response type) corresponding to the sample.
Fig. 4 is a SNR cluster analysis of 147 related autoantibodies with significant differences in hashimoto thyroiditis yin deficiency fire hyperactivity syndrome group.
Note that: the transverse direction is a sample, and the longitudinal direction is the protein (IgG or IgM response type) corresponding to the sample.
Fig. 5 is an SNR cluster analysis of 152 related autoantibodies with significant differences in the hashimoto thyroiditis spleen-kidney yang deficiency group.
Note that: the transverse direction is a sample, and the longitudinal direction is the protein (IgG or IgM response type) corresponding to the sample.
FIG. 6 is a graph of the analysis of Venn profile of autoantibodies characteristic of different syndromes of Hashimoto thyroiditis.
Note that: left lower: HT0105: bridge thyroiditis liver qi stagnation syndrome (thyroid gland normal), upper left: HT0610: deficiency of yin and hyperactivity of fire (hyperthyroidism), thyroiditis, upper right: HT1115: hashimoto thyroiditis spleen-kidney yang deficiency syndrome (hypothyroidism), right side down: HT: hashimoto thyroiditis whole group.
FIG. 7 is a scatter plot and ROC plot of specific autoantibodies associated with the symptoms of Hashimoto thyroiditis liver depression and qi stagnation.
Note that: HT: group of hashimoto thyroiditis liver depression and qi stagnation syndrome, C: normal control group, OTD: other thyroid disorders.
FIG. 8 is a graph showing the separate detection and combined detection of ROC for two related autoantibodies MAP9 and IRF 9.
FIG. 9 is a scatter plot and ROC plot of the specific autoantibodies associated with the hashimoto thyroiditis yin deficiency fire hyperactivity syndrome.
Note that: HT: group of hashimoto thyroiditis yin deficiency fire excess, C: normal control group, OTD: other thyroid disorders.
FIG. 10 is a graph showing the detection of two related autoantibodies PLEKHO2 and NPM3 alone and in combination with ROC.
FIG. 11 is a scatter plot and ROC plot of specific autoantibodies associated with the spleen-kidney yang deficiency syndrome of hashimoto thyroiditis.
Note that: HT: group of hashimoto thyroiditis spleen-kidney yang deficiency syndrome, C: normal control group, OTD: other thyroid disorders.
FIG. 12 is a graph showing the individual detection and combined detection of ROC for five related autoantibodies DR1, KDM1A, PCGF, GPCPD1, IGBP1.
Detailed Description
The following describes the embodiments of the present invention in further detail with reference to examples.
Example 1 protein chip screening for autoantibodies characteristic of different symptoms of Hashimoto thyroiditis
1. Serum specimen source
Venous serum samples of untreated hashimoto thyroiditis patients at outpatient and hospitalized primordial were collected from the first affiliated hospital of the university of chinese medicine, henna, third affiliated hospital, university of zheng, from 1 st 2017 to 12 th 2019. From the above, 15 cases of patients with different hashimoto thyroiditis, namely 5 cases of patients with liver depression and qi stagnation, thyroid gland normal function, yin deficiency and fire excess, hyperthyroidism, spleen and kidney yang deficiency and hypothyroidism, were selected as HuProt TM Proteome chip test samples, each group suffering fromThere were no statistical differences in gender and age comparisons among individuals.
Venous serum samples of 5 normal (healthy) controls were taken from the national people's free radical 988 hospital health physical examination center healthy population from 1 st 2017 to 12 nd 2019. The selection requirements of normal contrasters meet the following criteria: any one of the diagnostic standards of the hashimoto thyroiditis patients is not met, and the laboratory examination of thyroiditis ultrasonic, TPOAb, TGAb, FT, FT4 and TSH is normal; no history of major disease; the person or the direct relatives have no autoimmune diseases. The normal control group was gender and age matched with hashimoto thyroiditis cases.
2. Main instrument and equipment for protein chip experiment
120090 crystal core slideshrer TM Chip drier (Beijing Boao Crystal Biotechnology Co., ltd.), crystal core LuxScan TM 10K-A microarray chip scanner (Beijing Boao Crystal Biotechnology Co., ltd.), cyTM3-conjugated AffiniPure Goat Anti-Human IgG (Jackson Co., USA), alexA Fluor 647-conjugated AffiniPure Donkey Anti-Human IgM (Jackson Co., USA), multifure X1R refrigerated high-speed centrifuge (Thermo Co., USA).
3. Protein chip detection principle
1 HuProt was used for each sample TM Protein chips were tested, specific antibodies (including IgG, igM or other types of antibodies) were bound to proteins immobilized on the chips, unbound antibodies and other proteins were removed by washing, and signals were visualized by fluorescent labeling of secondary Anti-human IgM (cy 5 label, wavelength 635 nm) and Anti-human IgG fluorescent secondary antibody (cy 3 label, wavelength 532 nm) and by means of a fluorescent scanner. The intensity of the signal is positively correlated with the affinity and quantity of the antibodies. The chip detection schematic diagram is shown in fig. 1.
4. Protein chip quality control
The reliability of marker screening is ensured by carrying out quality detection on the chip before the high-throughput chip experiment. The protein N-terminal of the protein chip is provided with GST (glutathione S-transferase) label for purifying the protein, so that the quality of the high-density proteome chip can be evaluated by utilizing the hybridization of the anti-GST antibody and the protein chip. The protein fusion GST was subjected to expression purification, and after purification, a chip was prepared, and each protein was repeated at 2 spots. After the chip preparation is completed, chip quality detection is carried out through Anti-GST, and the protein sample application result is shown in FIG. 2. As can be seen from FIG. 2, the prospect value generated by the hybridization of the repeated protein Anti-GST is taken as an object, and R2 after linear fitting is 0.98 through the analysis of a scatter diagram, which indicates that the overall repeatability of the chip preparation is good; the detection rate of the protein spots is evaluated by negative control by taking the signal-to-noise ratio generated by Anti-GST hybridization as a calculation object, and the result shows that the protein detection rate is more than 99 percent, which indicates that the chip is qualified in preparation.
5. Protein chip detection experiment procedure
(1) Closing: taking out the preserved protein chip from the temperature of minus 80 ℃, placing the protein chip on a side swinging table, adding a sealing liquid, and sealing for 3 hours at room temperature;
(2) Incubation of serum samples: discarding the sealing solution, rapidly adding a serum incubation solution (the serum is diluted by 200 times by the incubation solution), placing the mixture on a side swinging table, and incubating at 4 ℃ overnight;
(3) Cleaning: taking out the chip (taking care that the upper surface of the chip cannot be touched or scratched) by using tweezers, placing the chip on a horizontal shaking table, and cleaning the chip for 3 times at room temperature for 10 min/time;
(4) Secondary antibody incubation: placing the mixture on a side swing table, and incubating a secondary antibody incubation liquid (the secondary antibody is diluted by 1000 times by the incubation liquid) for 1h at room temperature (taking care of light-shielding operation from the beginning of the step);
(5) Cleaning: placing in a horizontal shaking table, cleaning the liquid at room temperature for 3 times and 10 min/time, and cleaning the liquid with ultrapure water at room temperature for 2 times and 10 min/time after the cleaning is completed;
(6) And (3) drying: placing the chip in a chip dryer for centrifugal drying;
(7) Scanning: operating in accordance with scanner operating specifications and instructions. The setting parameters are as follows: power 90%, PMT value650, if a flare exists, adjust PMT or Power to scan for a no flare.
6. Protein chip experimental data processing and statistical analysis
The chip scan results were read by GenePix Pro v6.0 software to obtain the raw data. And processing the chip data by using the R language limma program package. In order to eliminate the condition of uneven signals caused by inconsistent background values among different protein points in the same chip, the background correction method is adopted for processing. The implementation is the ratio of the foreground to background value, i.e. F/B, for each protein, and on this basis the SNR (Signal Noise Ratio, signal to noise ratio), i.e. the mean of the F/B of the two repeated proteins, is defined. In order to reduce errors caused by systematic differences among different samples, different chips, experimental operation and the like, sample SNR is normalized before data comparison, and statistical analysis is performed based on normalized data to screen out specific response antibodies of an experimental group, which are distinguished from a control group.
SPSS22.0 statistical software was used for data processing and statistical analysis. The orthonormally test is carried out on the continuous variable by adopting a Kolomogorov-Smirnov test, and the continuous variable is used for the variable conforming to normal distributionA representation; for continuous variables conforming to normal distribution, adopting t test or single factor analysis of variance to perform group comparison, and adopting Dunnett test for pairwise comparison among multiple groups; for continuous variables with non-normal distribution, mann-Whitney U rank sum test is adopted for comparison between two groups, and χ is adopted for comparison between classified variables 2 Inspection does not meet χ 2 The data of the test conditions are precisely tested by Fisher. The test level alpha=0.05, and the difference of P <0.05 is statistically significant.
7. Antibody marker screening
The protein sites 23034 of each chip are removed from ND and Control points on the chip, and 23032 protein sites are reserved. Taking the normalized SNR value as a calculation object, screening potential specific high-response antibody markers based on a statistical method, and specifically analyzing as follows:
(1) For either protein, it is assumed that the samples to be aligned are from two identical populations and pass the Mann-Whitney U rank sum test, single tail, test, and characterize as p-value. Definition, when p-value <0.05, the original hypothesis is rejected, i.e. there is a significant difference between the two.
(2) For any one protein, calculating the differential expression multiple of the disease group and the healthy control group, namely fold change = disease group mean/healthy control group mean, wherein the fold change = disease group mean/healthy control group mean is used for indicating the degree of the disease group higher than the healthy control group; the definition is that the fold change is larger than or equal to 1.5, namely the potential difference, and the larger the difference multiple is, the more obvious the difference between the two groups is.
(3) For different sample groups, in order to avoid the comparison between negative proteins, before sample comparison, a positive protein judgment threshold is set according to the distribution of protein spot signal SNR values on a chip after normalization, and the protein is judged to be positive protein when IgG-SNR >4 and IgM-SNR >5 are defined. On the basis, setting a cutoff threshold (cutoff-IgG is more than or equal to 4 and cutoff-IgM is more than or equal to 5) by taking SNR of a control group on the protein as a calculation object, respectively calculating positive rates of a disease group and a healthy control group, and defining the lowest positive rate of the disease group as 50%, namely, the number of positive samples of the disease group on the protein is not less than 3/5 or 8/15; the highest positive rate of the healthy control group is 0%, namely, the number of the healthy control group positive samples on the protein is 0/5, and the positive rate (sensitivity) of the disease group and the positive rate (1-specificity) of the healthy control group are respectively calculated.
8. Results
8.1 Hashimoto thyroiditis liver qi stagnation syndrome related autoantibodies
By comparing the hashimoto thyroiditis liver qi depression syndrome (thyroid gland normal function, HT 0105) with the normal healthy control group (NC), 59 related autoantibodies with obvious differences are screened out altogether, wherein 43 IgG response types and 16 IgM response types (fold change is more than or equal to 1.5, and p-value is less than 0.05). Wherein 33 relevant autoantibodies with fold change.gtoreq.2 are shown in Table 1.
TABLE 1 related autoantibodies with significant differences in desmotothyroiditis liver qi stagnation syndrome (HT 0105) compared to healthy control (NC)
SNR cluster analysis of 59 related autoantibodies with significant differences were screened as shown in fig. 3, and HT0105 group samples and NC group samples could be distinguished by one-step clustering from unsupervised learning clustering results. Therefore, the classification accuracy of the screened proteins in the whole group for distinguishing the thyroiditis liver qi stagnation syndrome and the healthy control group is 100%.
8.2 Equipped thyroiditis yin deficiency fire excess syndrome related autoantibodies
By comparing the hashimoto thyroiditis yin deficiency fire excess syndrome (hyperthyroidism, HT 0610) with the normal healthy control group (NC), 147 related autoantibodies with obvious differences are screened out, wherein 53 IgG response types and 94 IgM response types (fold change is more than or equal to 1.5, and p-value is less than 0.05). Wherein 78 related autoantibodies with fold change.gtoreq.2 are shown in Table 2.
TABLE 2 relevant autoantibodies with significant differences in the hashimoto thyroiditis yin deficiency fire hyperactivity syndrome (HT 0610) compared to healthy controls (NC)
SNR cluster analysis of 147 related autoantibodies with significant differences screened is shown in fig. 4, and from the result of unsupervised learning clustering, HT0610 group samples and NC group samples can be distinguished through one-step clustering. Therefore, the classification accuracy of the screened protein in the whole group for distinguishing the hashimoto thyroiditis yin deficiency fire hyperactivity from the healthy control group is 100%.
8.3 Hashimoto thyroiditis spleen-kidney yang deficiency syndrome related autoantibodies
By comparing the hashimoto thyroiditis spleen-kidney yang deficiency syndrome (hypothyroidism, HT 1115) with the normal healthy control group (NC), 152 related autoantibodies with obvious differences are screened out altogether, wherein 91 IgG response types and 61 IgM response types (fold change is more than or equal to 1.5, and p-value is less than 0.05). Wherein 73 related autoantibodies with fold change not less than 2 are shown in Table 3.
TABLE 3 related autoantibodies with significant differences in the hashimoto thyroiditis, spleen and kidney yang deficiency syndrome (HT 1115) compared to healthy control group (NC)
SNR cluster analysis of the 152 related autoantibodies with significant differences screened was as shown in fig. 5, and from the unsupervised learning clustering result, HT1115 group samples and NC group samples could be distinguished by one-step clustering. Therefore, the classification accuracy of the screened protein in the whole group for distinguishing the hashimoto thyroiditis spleen-kidney yang deficiency syndrome and the healthy control group is 100%.
9. Screening of related autoantibodies with characteristics of different symptoms of hashimoto thyroiditis
From the above results, it can be seen that the thyroiditis liver qi stagnation syndrome group (thyroid gland function is normal) screens 59 related autoantibodies, the yin deficiency fire excess syndrome group (hyperthyroidism) screens 147 related autoantibodies, the spleen and kidney yang deficiency syndrome group (hypothyroidism) screens 152 related autoantibodies, and analysis of Venn diagram is utilized to obtain the characteristic related autoantibodies of different thyroiditis syndrome groups, four groups take intersection sets, wherein the present syndrome group has, the other syndrome groups do not have the characteristic related autoantibodies of the present syndrome group, and the result is shown in fig. 6. As can be seen from fig. 6, 29 characteristic autoantibodies in the group of thyroiditis liver qi depression, 103 characteristic autoantibodies in the group of thyroiditis yin deficiency and fire hyperactivity, and 104 characteristic autoantibodies in the group of thyroiditis spleen and kidney yang deficiency. Characteristic autoantibodies with the syndrome groups fold change more than or equal to 5 are selected and are shown in tables 4-6.
TABLE 4 autoantibodies characteristic of Hashimoto thyroiditis liver qi stagnation syndrome (fold change. Gtoreq.5)
TABLE 5 autoantibodies characteristic of the Qiaobu thyroiditis with fire excess from yin deficiency (fold change. Gtoreq.5)
TABLE 6 autoantibodies characteristic of the spleen-kidney yang deficiency syndrome of hashimoto thyroiditis (fold change. Gtoreq.5)
And further selecting fold change difference multiple of more than 5 times from the selected related autoantibodies which are characteristic of different groups of hashimoto thyroiditis, searching related autoantibodies which are highly expressed in thyroid tissues and cells through a GenBank database, and verifying in more serum samples. The relevant autoantibody to be verified in the liver qi stagnation syndrome group is TNRC6C, MAP9 and IRF9; the relevant autoantibodies to be verified in the yin deficiency fire excess syndrome group are PLEKHO2 and NPM3; the related autoantibodies to be verified in spleen-kidney yang deficiency syndrome groups are DR1, CTDP1, KDM1A, BRMS1L, PCGF2, GPCPD1 and IGBP1.
Example 2 indirect ELISA experiments verify that the screened antibodies are characteristic of the related autoantibodies of different symptoms of hashimoto thyroiditis
Selecting an autoantibody TNRC6C, MAP9 and IRF9 related to a liver qi stagnation syndrome group to be verified; autoantibodies PLEKHO2 and NPM3 related to yin deficiency and fire excess syndrome groups; the content of the related autoantibodies DR1, CTDP1, KDM1A, BRMS1L, PCGF2, GPCPD1 and IGBP1 in a large sample is detected by an indirect ELISA experiment, and the specificity and the sensitivity of the related autoantibodies serving as biomarkers for detecting different symptoms of thyroiditis are verified.
2.1 serum sample Source
The PASS15.0 software was used to estimate the required sample size. According to the requirement of the study, the sample size of the hashimoto thyroiditis patient group is 2 times that of the normal control group, the minimum sensitivity p1=70% of the patient group is calculated according to alpha= 0.05,1-beta=0.90, the minimum sensitivity p2=10% of the normal control group is calculated according to the standard deviation value, the average value +2SD of the normal control group is taken as a cut-off value, and the positive rate of the study factors in the hashimoto thyroiditis group and the normal control group is calculated according to the cut-off value, wherein the difference value between the positive rate and the positive rate is more than 10%. About 216 patients are calculated in the hashimoto thyroiditis group, about 108 patients are calculated in the normal control group, about 10% of cases are added in the hashimoto thyroiditis group considering the falling and losing visit cases, about 240 patients are calculated in the final hashimoto thyroiditis group, and about 120 patients are calculated in the normal control group.
The selected cases are patients with hashimoto thyroiditis without medicines or other treatments at the first affiliated hospital endocrinology of Henan traditional Chinese medicine university, third affiliated hospital endocrinology, the first affiliated hospital thyroid surgery clinic of Zhengzhou university and the initial hospitalization from 1 month in 2017 to 12 months in 2019. From the collected patients with the hashimoto thyroiditis, 95 patients with the liver qi stagnation with normal thyroid gland function, 60 patients with the yin deficiency and fire excess with hyperthyroidism and 85 patients with the spleen-kidney yang deficiency with hypothyroidism are selected, and the gender and the age of the patients with the hashimoto thyroiditis are not statistically different. 60 subjects with thyroid disease other than hashimoto thyroiditis were also collected. Normal controls were from healthy people in the national release force 988 hospital health physical examination center in the same period. Each group of cases was matched by gender, age.
2.2 Experimental procedure
(1) TNRC6C, MAP, IRF9, PLEKHO2, NPM3, DR1, CTDP1, KDM1A, BRMS1L, PCGF, GPCPD1, IGBP1 commercial human recombinant proteins purchased from Bio-Inc. were diluted to 250. Mu.g/ml with protein dilutions, respectively, each recombinant protein was diluted to the appropriate concentrations as shown in Table 7 with coating solutions, and added to 96-well ELISA plates with a pipette gun at 100. Mu.l/well, taking care to avoid air bubbles and to avoid adhesion of liquid to the walls of the wells during the addition. The human IgG standards were diluted 8 gradient concentrations (640 ng/ml, 320ng/ml, 160ng/ml, 80ng/ml, 40ng/ml, 20ng/ml, 10ng/ml, 0 ng/ml) with coating solution as quality controls to adjust plate-to-plate differences, 100. Mu.l/well, sealed with sealing plate film, placed in a refrigerator at 4℃and coated overnight.
(2) Closing: the coating liquid in the ELISA plate is discarded, the ELISA plate is dried by beating on water absorbing paper, 200 mu l of 2% BSA is added into each hole for sealing, and the mixture is placed in a refrigerator at 4 ℃ for overnight sealing after sealing by a sealing plate film.
(3) Washing the plate: taking out the sealed overnight ELISA plate, removing sealing liquid in the plate, beating to dry, washing the plate 3 times by using 1 XPBST solution on a 96-hole full-automatic plate washer, and beating to dry.
(4) Incubation of an anti-antibody (serum): 1 XPBST antibody dilutions with 1% BSA at 1:100 (v/v) ratio the serum samples were diluted in 96-well deep well plates (as-is diluted) and the diluted serum samples were added to the elisa plate with a 12-channel pipette, 100 μl/well, blank wells were filled with 100 μl serum-free antibody dilution, sealed with a sealing plate membrane, and incubated in a half-water bath at 37 ℃ in a thermostat water bath for 1h.
(5) Washing the plate: discarding serum diluent in the ELISA plate, beating to dry, washing the plate 5 times by using 1 XPBST solution on a 96-hole full-automatic plate washer, and beating the ELISA plate on absorbent paper.
(6) Secondary antibody incubation: horseradish peroxidase-labeled goat anti-human IgG secondary antibody (as prepared) was diluted 1 XPBST antibody dilution containing 1% BSA at a ratio of 1:10000 (v/v), diluted secondary antibody was added to the ELISA plate with a 12-channel pipette, 100. Mu.l/well, and after sealing with a plate membrane, incubated in a half-water bath at 37℃for 1h.
(7) Washing the plate: discarding the secondary anti-diluent in each hole of the ELISA plate, drying by beating, washing the plate 5 times by using 1 XPBST solution on a 96-hole full-automatic plate washer, and drying the ELISA plate on absorbent paper.
(8) Color reaction: the microplate reader was turned on 30min in advance for preheating, and the specimen layout and measurement of dual wavelengths (450 nm and 620 nm) were set. Taking out substrate solution A and substrate solution B in a refrigerator at 4 ℃ and uniformly mixing according to the proportion of 1:1 (v/v) (in-situ preparation), adding a color development liquid into an ELISA plate by using a 12-channel pipetting gun, reacting at room temperature for 5-10min in a dark place, wherein the color development liquid is 100 mu l/hole.
(9) Terminating the reaction: after the color change is observed by naked eyes, a 12-channel pipetting gun is used for adding a stop solution into the ELISA plate in time, 50 mu l/hole is used for stopping the reaction, and the ELISA plate is placed into an ELISA instrument for detection after the reaction is stopped completely, so that experimental data are stored.
TABLE 7 coating concentration of proteins
2.3 data processing and statistical analysis
Statistical analysis was performed on the experimental data obtained using SPSS22.0, graphPad Prism 8.0. AUC, sensitivity, specificity and 95% confidence interval (95% ci) for each relevant autoantibody were calculated by ROC curve, screening criteria defined as AUC >0.5 and P <0.05. The maximum approximate dengue index (sensitivity + specificity-1) at a specificity greater than 90% was chosen and defined as the cutoff. All assays were performed on a two-sided assay, and differences were considered statistically significant when the P-value was less than 0.05.
2.4 results
2.4.1 related autoantibodies characteristic of Hashimoto thyroiditis liver qi stagnation syndrome
The content of autoantibodies related to the thyroiditis liver qi stagnation syndrome (thyroid gland normal) in a group of serum samples (95 cases of the thyroiditis liver qi stagnation syndrome, normal control 120 cases and 60 cases of other thyroiditis other than thyroiditis) is detected through an indirect ELISA experiment. In the group of liver qi stagnation syndrome, as shown in table 8, TNRC6C was eliminated according to AUC >0.5 and P <0.05, the concentration difference between serum of hashimoto thyroiditis patient and normal control serum was not statistically significant, the antibody concentrations of the remaining 2 related autoantibodies MAP9, IRF9 were significantly higher than that in serum of hashimoto thyroiditis patient, and the difference was statistically significant (P < 0.05).
Detection of 8 3 autoantibodies related to hashimoto thyroiditis liver qi stagnation syndrome
The scatter plots and ROC graphs of expression of two characteristic related autoantibodies of MAP9 and IRF9 in patients with hashimoto thyroiditis, other thyroiditis than hashimoto thyroiditis and healthy controls with liver qi stagnation syndrome are shown in fig. 7. As can be seen from fig. 7, the expression level of autoantibodies associated with the HT liver qi stagnation group was significantly higher than that of the healthy control group and other thyroid disease patient groups.
In patients with thyroiditis caused by liver qi stagnation syndrome due to two characteristic related autoantibodies of MAP9 and IRF9 obtained through ELISA verification, a ROC curve chart of single antibody detection and combined detection of the two antibodies is shown in fig. 8, and the highest AUC value (0.941) of combined detection of the two related autoantibodies can be seen, so that the combined detection of the two related autoantibodies of MAP9 and IRF9 has higher syndrome characteristic identification.
2.4.2 Equipped's thyroiditis yin deficiency fire excess syndrome characteristic related autoantibodies
The content of autoantibodies related to the deficiency of the heat of hashimoto thyroiditis (hyperthyroidism) in a group of serum samples (60 cases of the deficiency of the heat of hashimoto thyroiditis, normal control 120 cases and 60 cases of other thyroiditis other than hashimoto thyroiditis) is detected through an indirect ELISA experiment. In the yin deficiency fire hyperactivity group, as shown in table 9, the levels of antibodies in serum of hashimoto thyroiditis patients were significantly higher for both PLEKHO2 and NPM 32 related autoantibodies than in normal control serum, and the differences were statistically significant (P < 0.05).
Detection of Tab 9 2 autoantibodies related to Hashimoto thyroiditis yin deficiency fire excess syndrome
The scatter plots and ROC plots of the expression of two characteristic related autoantibodies of PLEKHO2, NPM3 in patients with hashimoto thyroiditis due to hyperactivity of fire due to yin deficiency, other thyroiditis patients than hashimoto thyroiditis, and healthy controls are shown in fig. 9. As can be seen from fig. 9, the expression level of autoantibodies associated with the HT yin deficiency fire hyperactivity group was significantly higher than that of the healthy control group and other thyroid disease patient groups.
In patients with thyroiditis caused by fire excess from yin deficiency, the ROC curve graph of single antibody detection and combined detection of two antibodies is shown in figure 10, and the highest AUC value (0.884) of the combined detection of the two related autoantibodies can be seen, which indicates that the combined detection of the two related autoantibodies of PLEKHO2 and NPM3 has higher syndrome feature recognition.
2.4.3 related autoantibodies characteristic of the Hashimoto thyroiditis spleen-kidney yang deficiency syndrome
The content of autoantibodies related to the spleen-kidney yang deficiency syndrome (hypothyroidism) of hashimoto thyroiditis in a group of serum samples (85 cases of spleen-kidney yang deficiency syndrome of hashimoto thyroiditis, normal control 120 cases and 60 cases of other thyroiditis not hashimoto thyroiditis) is detected through an indirect ELISA experiment. In the spleen-kidney yang deficiency syndrome group, as shown in table 10, CTDP1 and BRMS1L 2 autoantibodies were deleted according to AUC >0.5 and P <0.05, the concentration difference between the serum of hashimoto thyroiditis patient and normal control serum was not statistically significant, the concentration of antibodies of the other 5 related autoantibodies DR1, KDM1A, PCGF, GPCPD1 and IGBP1 in serum of hashimoto thyroiditis patient was significantly higher than that in normal control serum, and the difference was statistically significant (P < 0.05).
TABLE 10 detection of 7 autoantibodies related to hashimoto thyroiditis spleen-kidney yang deficiency syndrome
The scatter diagrams and ROC graphs of the expressions of five characteristic related autoantibodies of DR1, KDM1A, PCGF, GPCPD1 and IGBP1 in patients with hashimoto thyroiditis with spleen-kidney yang deficiency syndrome, other thyroiditis patients without hashimoto thyroiditis and healthy controls are shown in FIG. 11. As can be seen from fig. 11, the expression level of autoantibodies associated with the HT spleen-kidney yang deficiency group was significantly higher than that of the healthy control group and other thyroid disease patient groups.
The ROC curve graphs of single antibody detection and five-antibody combined detection of five characteristic related autoantibodies of DR1, KDM1A, PCGF, GPCPD1 and IGBP1 obtained by ELISA verification in patients with spleen-kidney yang deficiency type thyroiditis are shown in figure 12, and the highest AUC value (0.852) of the five related autoantibodies combined detection can be seen, which indicates that the five related autoantibodies of DR1, KDM1A, PCGF2, GPCPD1 and IGBP1 have higher syndrome feature identification.
Claims (3)
1. The application of a biomarker in preparing a reagent or a kit for detecting hashimoto thyroiditis spleen-kidney yang deficiency syndrome is characterized in that the biomarker is five autoantibodies of DR1, KDM1A, PCGF2, GPCPD1 and IGBP1.
2. The use according to claim 1, wherein the kit comprises five human recombinant proteins DR1, KDM1A, PCGF, GPCPD1 and IGBP1, and the kit is for detecting five autoantibodies DR1, KDM1A, PCGF2, GPCPD1 and IGBP1 in serum.
3. The use according to claim 2, wherein the kit further comprises a human IgG standard, horseradish peroxidase-labelled goat anti-human IgG and reagents commonly used in ELISA techniques.
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