CN117805396B - Kit for diagnosing endometrium inflammatory infertility and application thereof - Google Patents
Kit for diagnosing endometrium inflammatory infertility and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biological medicines, and particularly relates to a kit for diagnosing endometrium inflammatory infertility and application thereof. The invention firstly provides a biomarker for diagnosing endometrium inflammatory infertility, wherein the biomarker is at least one of ER autoantibody, PR autoantibody, CD3 autoantibody, CD16 autoantibody, CD38 autoantibody and CD138 autoantibody; the invention also provides a kit for diagnosing the endometrium inflammatory infertility, which comprises a reagent for detecting the biomarker, and the reagent detects the biomarker in a sample through enzyme-linked immunosorbent assay, protein chip, immunoblotting or microfluidic immunodetection. The invention can be used for auxiliary diagnosis of endometrium inflammatory infertility by detecting ER, PR, CD3, CD16, CD38 and CD138 autoantibody levels in human serum.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a kit for diagnosing endometrium inflammatory infertility and application thereof.
Background
Infertility is a clinical common disease of women in gestational age, the female infertility rate is 9% -18% in the global scope, and the female infertility rate is continuously increased along with the influence of factors such as the delay of the wedding age of the female, the increase of pressure and the like. Infertility is caused by various reasons, mainly including endocrine disorders, lesions of the reproductive system, immune factors and micro-ecological states (such as leucorrhea pH, vaginal cleanliness, fungi, trichomonas and clue cells), etc. Among these factors, diagnosis and treatment of infertility caused by endocrine disorders and organic lesions are relatively easy, but the etiology and pathogenesis of immune infertility are relatively complex, and it has not been completely elucidated at present, and various immune imbalances can lead to the occurrence of infertility.
Endometritis is an important risk factor for infertility, and the main influence mechanism is that the endometritis patient has an influence on the form and compatibility of endometrium, and can directly influence embryo implantation, so that infertility is caused. Inflammatory cells have cytotoxin effect, can reduce sperm motility or cause sperm death, reduce sperm quantity in oviduct, influence fertility, and cause infertility. In addition, pathogens causing inflammation can stimulate the immune response of the organism and generate antibodies, so that the release of a large amount of inflammatory cells and cytokines is stimulated, and the implantation of pregnant eggs and the implantation of placenta and embryo development are not facilitated. But because endometritis is difficult to be found, the diagnosis and treatment effects of patients on infertility are delayed.
Specific antigen refers to a specific antigen recognized by lymphocyte antigen receptor, which can provoke the immune system of human body to generate immune response, thereby triggering a large amount of autoantibodies corresponding to the specific antigen to be released into serum. The research team uses HuProt TM human proteome chips to select 6 autoantibodies of specific antigens, namely ER, PR, CD3, CD16, CD38 and CD138 on the basis of a genomics database established by technologies of whole genome association analysis, whole genome sequencing, whole genome exon sequencing and the like in the early stage, and the existence of the autoantibodies of the 6 specific antigens can be detected before the diagnosis of the endometrium inflammatory infertility, and the autoantibodies have higher specificity and sensitivity to the endometrium inflammatory infertility patients. However, the use of 6 specific antigens, ER, PR, CD3, CD16, CD38, CD138, for detecting the expression of the corresponding autoantibodies in the endometrium of patients with endometrium inflammatory infertility has not been reported. Therefore, the research provides a kit for diagnosing the endometrium inflammatory infertility, which can promote the diagnosis and treatment level of the chronic endometrium inflammatory infertility in China and also provides thinking for future further research on the chronic endometrium inflammatory infertility.
Disclosure of Invention
Aiming at the problems and the defects existing in the prior art, the invention aims to provide a kit for diagnosing endometrium inflammatory infertility and application thereof.
In order to achieve the aim of the invention, the technical scheme adopted by the invention is as follows:
In a first aspect, the invention provides a biomarker for diagnosis of endometrial inflammatory infertility, said biomarker being at least one of ER autoantibody, PR autoantibody, CD3 autoantibody, CD16 autoantibody, CD38 autoantibody, CD138 autoantibody.
In a second aspect, the invention provides the use of an agent for detecting a biomarker as described in the first aspect, in the manufacture of a product for use in the diagnosis of endometrial inflammatory infertility.
According to the above application, preferably, the reagent is a reagent for detecting the biomarker in a sample by enzyme-linked immunosorbent, protein chip, immunoblotting or microfluidic immunodetection.
According to the above application, preferably, the reagent is an antigen or antibody that detects the biomarker. More preferably, the agent is an antigen that detects the biomarker, the antigen being at least one of ER protein, PR protein, CD3 protein, CD16 protein, CD38 protein, CD138 protein.
According to the above application, preferably, the sample is serum, plasma, interstitial fluid. More preferably, the sample is serum.
According to the above application, preferably, the product is a protein chip, a kit or a preparation.
According to the above use, preferably, the endometrium inflammatory infertility is chronic endometrium inflammatory infertility.
In a third aspect the invention provides a kit for the diagnosis of endometrial inflammatory infertility, said kit comprising reagents for detecting a biomarker as described in the first aspect above, said biomarker being at least one of ER autoantibody, PR autoantibody, CD3 autoantibody, CD16 autoantibody, CD38 autoantibody, CD138 autoantibody.
According to the kit described above, preferably, the kit detects the biomarker in the sample by enzyme-linked immunosorbent assay, protein chip, immunoblotting or microfluidic immunodetection.
According to the kit described above, preferably, the kit is an ELISA detection kit comprising a solid support and an antigen coated on the solid support; the antigen is at least one of ER protein, PR protein, CD3 protein, CD16 protein, CD38 protein and CD138 protein.
According to the above kit, preferably, the detection sample of the kit is serum, plasma, interstitial fluid.
According to the above kit, preferably, the ELISA detection kit further comprises a sample diluent, a secondary antibody, an antibody diluent, a washing solution, a color development solution and a stop solution.
According to the kit described above, preferably, the endometrium inflammatory infertility is chronic endometrium inflammatory infertility.
The basic information of the related antigens ER protein, PR protein, CD3 protein, CD16 protein, CD38 protein and CD138 protein in the invention is as follows:
Estrogen receptors (Estrogen Receptor, abbreviated ER) are present in normal mammary epithelium, endometrium, myometrium, cervical tissue and ovaries. ER is widely found in endometrial tissue cells and complexes with estrogens promote cell growth and proliferation. The ER protein in NCBI has the protein sequence number: np_001001443.
Progestin receptors (Progesterone Receptor, abbreviated PR) are present in normal mammary epithelium, endometrium, myometrium, cervical tissue and ovaries. The progestogen, through binding to its PR, inhibits mitosis, promotes cell differentiation and maturation, has an antagonistic effect on estrogens, protects the endometrium, and plays an important role in female infertility hormone dysfunction. The protein sequence number of PR protein in NCBI is: np_001192285.
CD3 is a reliable marker of T cells, is used as a T cell antigen receptor and distributed on the surfaces of all mature T cells, plays an indispensable role in T cell immunity and is mainly used for diagnosing T cell lymphomas. The protein sequence number of CD3 protein in NCBI is: np_001135555.
CD16 is a biomarker associated with monocytes and natural killer (naturalkilling, NK) cells in the lymphatic system. NK cells are the most abundant immune cells in endometrium before embryo implantation and decidua of early pregnancy, secrete various cytokines and vascular growth factors, regulate remodeling of endometrium spiral artery and invasion of trophoblast cells, and play an important role in reducing endometrial receptivity. The protein sequence number of the CD16 protein in NCBI is: np_001121068.
CD38 is a type II transmembrane glycoprotein with a molecular weight of 42KD, and is mainly expressed in plasma cells and partially activated T/B cells, and is used for diagnosis of plasma cell lymphoma. The protein sequence number of the CD38 protein in NCBI is: np_001091148.
CD138 is a transmembrane heparin sulfate protein expressed on the surface of pre-B cells and plasma cells, and is mainly used for diagnosis of plasma cell lymphoma. In recent years, CD138 is proposed as ligand glycan, is a good marker of plasma cells, can directly detect the plasma cells, can exclude the influence of morphological similar cells, and provides a reference basis for diagnosing chronic endometritis. CD138 can bind chemotactic factors, induce neutrophil to gather at an inflammation part, inhibit T cell migration, and regulate leukocyte-endothelial cell reaction of an organism, thereby participating in inflammatory reaction, and leading to migration and invasion of trophoblast. A variety of pathogenic bacteria can cause the host cell surface CD138 molecules to shed from the outer functional region, thereby enhancing self-toxic effects. CD138 molecules outside epithelial cell membranes fall off when endometrium is damaged, and are combined with bacteria or fibroblasts to participate in membrane tissue repair and inflammatory reaction in a body. The protein sequence number of CD138 protein in NCBI is: np_001006947.
Compared with the prior art, the invention has the positive beneficial effects that:
(1) The invention discovers for the first time that the expression level of ER, PR, CD3, CD16, CD38 and CD138 autoantibodies in serum of patients with endometrium inflammatory infertility is obviously higher than that of normal people, and the difference has statistical significance, and the endometrium inflammatory infertility can be effectively detected by detecting the expression level of ER, PR, CD3, CD16, CD38 and CD138 autoantibodies in the serum of the human; proved by verification, when any one marker of ER, PR, CD3, CD16, CD38 and CD138 autoantibodies is singly adopted for diagnosing the chronic endometrium inflammatory infertility, the AUC value of the ROC curve is above 0.59, wherein the AUC value of the ROC curve of the ER autoantibody for diagnosing the chronic endometrium inflammatory infertility reaches 0.747; moreover, when a plurality of markers are used in combination, the AUC value of the ROC curve is closer to 1 than that of a single index, so that the discrimination effect is good and the diagnosis effect is good. Therefore, the marker for diagnosis of endometrium inflammatory infertility of the present invention can be used for auxiliary diagnosis of endometrium inflammatory infertility.
(2) According to the invention, when six markers, namely ER autoantibody, PR autoantibody, CD3 autoantibody, CD16 autoantibody, CD38 autoantibody and CD138 autoantibody, are used as a combination for diagnosis and detection of endometrium inflammatory infertility, the AUC of the ROC curve is 0.9355 (95% CI: 0.9041-0.9670), the detection sensitivity is as high as 93.6% (namely, the ratio of the six markers to be correctly diagnosed as endometrium inflammatory infertility when the six markers are used for diagnosis in chronic endometrium inflammatory infertility patients is 93.6%), the specificity is as high as 80.7% (namely, the ratio of the six markers to be determined as healthy persons when the six markers are used for diagnosis in healthy control is 80.7%), so that the marker has higher sensitivity and specificity, greatly improves the detection rate of endometrium inflammatory infertility, is beneficial to screening and early detection of endometrium inflammatory infertility groups, and brings great good fortune to chronic endometrium inflammatory infertility patients and families.
(3) The kit detects the expression level of ER, PR, CD3, CD16, CD38 and CD138 autoantibodies in human serum by an indirect ELISA method, can accurately distinguish patients with endometrium inflammatory infertility from normal control diagnosis, and provides a new reference basis for a clinician to diagnose the endometrium inflammatory infertility.
(4) The detection sample of the kit is serum, so that invasive diagnosis can be avoided, the risk of chronic endometritis infertility can be obtained by taking the serum for detection in a minimally invasive mode, the blood quantity is less, the pain of a detected person is less, and the compliance is high; moreover, the operation is simple, the detection result time is short, and the method has wide market prospect and social benefit.
Drawings
FIG. 1 is a schematic diagram of an indirect ELISA assay;
FIG. 2 is a graph showing the results of the expression levels of six autoantibodies to specific antigens in the serum of the infertility group;
FIG. 3 is a graph showing the results of the expression levels of autoantibodies to six specific antigens in serum of a normal control group;
FIG. 4 is a graph showing the positive rate results of six autoantibodies to specific antigens in the infertility group and the control group;
FIG. 5 is a ROC curve of six autoantibodies to specific antigens alone to diagnose and distinguish infertility patients from normal individuals;
FIG. 6 is a ROC curve for the diagnosis of endometrial inflammatory infertility using autoantibody combinations of various specific antigens.
Detailed Description
The following detailed description is exemplary and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present invention. Furthermore, it should be further understood that the terms "comprises" and/or "comprising," when used in this specification, specify the presence of stated features, steps, operations, components, and/or groups thereof.
The experimental methods in the following examples, in which specific conditions are not specified, are all conventional in the art or according to the conditions suggested by the manufacturer; the reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
In order to enable those skilled in the art to more clearly understand the technical scheme of the present invention, the technical scheme of the present invention will be described in detail with reference to specific embodiments.
Example 1: preparation of kit and detection of serum expression level of ER, PR, CD3, CD16, CD38, CD138 autoantibody by using kit
The inventor adopts HuProt TM human proteome chips to screen out specific antigen autoantibodies which are differentially expressed in serum of patients with endometrium inflammatory infertility and healthy control, wherein the specific antigen autoantibodies are ER autoantibodies, PR autoantibodies, CD3 autoantibodies, CD16 autoantibodies, CD38 autoantibodies and CD138 autoantibodies respectively, and the expression levels of the ER autoantibodies, PR autoantibodies, CD3 autoantibodies, CD16 autoantibodies, CD38 autoantibodies and CD138 autoantibodies in the serum of the patients with endometrium inflammatory infertility are obviously higher than that of the healthy control, and the difference has statistical significance.
To verify the difference in the expression levels of the six autoantibodies, the inventors prepared an ELISA (enzyme linked immunosorbent assay, ELISA) kit, and further detected the expression levels of the screened PR autoantibodies, CD3 autoantibodies, CD16 autoantibodies, CD38 autoantibodies, CD138 autoantibodies in the serum of a large sample population by indirect enzyme-linked immunosorbent assay using the ELISA kit. The principle of indirect enzyme-linked immunosorbent assay (shown in figure 1) is that an antigen is connected to a solid-phase carrier, an antibody to be detected in a sample is combined with the antigen to be detected to form a solid-phase antigen-detected antibody complex, then an enzyme-labeled secondary antibody is combined with an antibody in the solid-phase antigen-detected antibody complex to form a solid-phase antigen-detected antibody-enzyme-labeled secondary antibody complex, and then the color development degree after a substrate is added is measured to determine the content of the antibody to be detected.
1. Experimental samples:
The serum 109 cases of patients with chronic endometritis inflammatory infertility (infertility group) and the serum 109 cases of normal human physical examination (normal control group) of Zhengzhou city women and children health care hospital are selected. 109 cases of chronic endometrium inflammatory infertility patients were selected from women between 20 and 40 years old, and all underwent the examination by excluding factor uterine bleeding; 109 normal persons (20-40 years old females) all come from the hospital health physical examination population without any evidence of intimal related disease. The study was approved by the ethical committee of the women and child healthcare institute in zheng state, and all subjects signed informed consent.
Serum collection: 5ml of peripheral blood of the study object in early morning in a fasting state is placed in a blood collection tube without an anticoagulant, kept stand for 1h at room temperature, placed in a centrifuge, and centrifuged at 3000rpm for 10min at 4 ℃. And then sucking out serum on the upper layer of the blood collection tube, sub-packaging the serum into an EP tube with the volume of 1.5ml, marking sample numbers on the top and the side surface of the EP tube, putting the EP tube in a refrigerator with the temperature of-80 ℃ for freezing preservation, and recording the blood collection date and the storage position. Before use, the serum is taken out, placed in a refrigerator at 4 ℃ for thawing and split charging, so that repeated thawing of the serum is avoided.
2. Experimental materials and reagents:
(1) 6 specific antigen proteins: ER protein, PR protein were purchased from Gene technology (Shanghai) Co., ltd, and CD3 protein, CD16 protein, CD38 protein and CD138 protein were purchased from Beijing Zhongjingqiao Biotechnology Co., ltd;
(2) 96-well ELISA plates (8 rows by 12 columns);
(3) Coating liquid: 50mM carbonate buffer, ph=9.6;
(4) Sealing liquid: PBST buffer containing 0.2% (v/v) Tween 20 of 2% (v/v) Bovine Serum Albumin (BSA);
(5) Sample dilution: PBST buffer containing 1% (W/V) BSA;
(6) Second antibody dilution: PBST buffer containing 1% (W/V) BSA;
(7) Enzyme-labeled secondary antibody: horseradish peroxidase-labeled RecA protein (Invitrogen);
(8) Washing liquid: 0.01M PBST (phosphate Tween) buffer at pH7.4 containing 0.05% Tween 20;
(9) Positive control serum: ER positive control serum, namely, the serum of the infertility patient with positive ER antibodies is detected by using an indirect ELISA and a Westernblot method;
(10) Negative control serum: ER negative control serum, namely, serum of normal people with the ER antibody expression level being the average content of serum antibodies of normal people is detected by using an indirect ELISA and a Westernblot method;
(11) Color development liquid A:0.02% (W/V) TMB, formulated: 0.005g of methyl benzidine (TMB) was dissolved in 25ml of deionized water;
(12) Color development liquid B:0.006% (W/V) carbamide peroxide, formulated: taking 4.665g of citric acid and 418.40g of Na2HPO, fully dissolving in 400ml of deionized water, adding 3.2ml of 0.75% urea hydrogen peroxide, adjusting the pH value to 5.0-5.5, adding deionized water to a final volume of 500ml, uniformly mixing, and preserving at 4 ℃;
(13) Stop solution: 10% sulfuric acid;
(14) Enzyme-labeled instrument: star Fax 2100 (Aware. USA).
3. Preparing an antigen coated ELISA plate:
respectively preparing an ELISA plate coated by a specific antigen ER, an ELISA plate coated by PR, an ELISA plate coated by CD3, an ELISA plate coated by CD16, an ELISA plate coated by CD38 and an ELISA plate coated by CD 138.
Taking an ELISA plate coated with antigen ER as an example, the specific operation steps are as follows:
(1) Preparing a specific antigen solution: the specific antigen ER protein is prepared into ER protein solution with the concentration of 0.125 mu g/mL by adopting coating liquid.
(2) And (3) coating an ELISA plate: adding the specific antigen solution (ER protein solution) prepared in the step 1) into each reaction well of a 96-well ELISA plate respectively, wherein the sample adding amount is 50 mu L/well; ER antigen solution (the loading amount is 50 mu L/hole) is added into the positive control hole and the negative control hole, coating solution (the loading amount is 50 mu L/hole) is added into the blank control hole, the mixture is incubated for 1h in a constant temperature incubator at 37 ℃, the coating solution is removed after overnight at 4 ℃, and then the mixture is washed 3 times by using washing solution for 3min each time.
(3) Closing: adding a sealing liquid into the reaction holes of the coated 96-hole ELISA plate, sealing for 2 hours in a water bath at 37 ℃ with the sample adding amount of 100 mu L/hole, removing the sealing liquid, washing with a washing liquid (with the sample adding amount of 300 mu L/hole) for 3 times, and performing beating drying to obtain the ELISA plate coated with the specific related antigen ER.
The preparation steps of the antigen PR coated ELISA plate, the antigen CD3 coated ELISA plate, the antigen CD16 coated ELISA plate, the antigen CD38 coated ELISA plate and the antigen CD138 coated ELISA plate are basically the same as those of the ER coated ELISA plate, and the coating concentration is the same. The difference is that: the specific related antigens adopted in the step 1) are different; the specific antigen solutions added into the 96-well ELISA plate reaction wells in step 2) are different. Wherein, when preparing the antigen PR coated ELISA plate, the specific antigen adopted in the step 1) is PR protein; in the step 2), the specific antigen solution added into the 96-well ELISA plate reaction well is PR protein solution. When preparing an antigen CD3 coated ELISA plate, the specific antigen adopted in the step 1) is CD3 protein; in the step 2), the specific antigen solution added into the reaction well of the 96-well ELISA plate is a CD3 protein solution. When preparing an antigen CD16 coated ELISA plate, the specific antigen adopted in the step 1) is CD16 protein, and in the step 2), the specific antigen solution added into the 96-well ELISA plate reaction well is CD16 protein solution. When preparing an antigen CD38 coated ELISA plate, the specific antigen adopted in the step 1) is CD38 protein, and in the step 2), the specific antigen solution added into the 96-well ELISA plate reaction well is CD38 protein solution. When preparing the antigen CD138 coated ELISA plate, the specific antigen adopted in the step 1) is CD138 protein, and in the step 2), the specific antigen solution added into the 96-well ELISA plate reaction well is CD138 protein solution.
4. The composition of the kit of the invention is as follows:
(1) The antigen coated 96-well elisa plate prepared in step 3;
(2) Sample dilution: PBST buffer containing 1% (W/V) BSA;
(3) Second antibody dilution: PBST buffer containing 1% (W/V) BSA;
(4) Enzyme-labeled secondary antibody: horseradish peroxidase-labeled RecA protein (Invitrogen);
(5) Color development liquid: the color development liquid consists of color development liquid A and color development liquid B, wherein the color development liquid A is 0.02% (W/V) TMB, and the color development liquid B is 0.006% (W/V) carbamide peroxide; when in use, the color development liquid A and the color development liquid B are uniformly mixed according to the equal volume of 1:1;
(6) Stop solution: 10% sulfuric acid;
(7) Washing liquid: 0.01M PBST (phosphate Tween) buffer at pH7.4 containing 0.05% Tween 20;
(8) Positive control serum: ER positive control serum;
(9) Negative control serum: ER negative control serum.
The reagents (2) - (9) are respectively packaged and then form a kit with the antigen coated 96-well ELISA plate.
5. The prepared kit is adopted to detect the autoantibody expression level of 6 specific antigens in a serum sample:
The prepared kit is adopted to detect the expression level of the specific antigen autoantibody in the serum sample 6 of the infertility group and the normal control group. The same serum sample is respectively used for detecting the expression levels of ER autoantibodies, PR autoantibodies, CD3 autoantibodies, CD16 autoantibodies, CD38 autoantibodies and CD138 autoantibodies in the serum sample by adopting the ELISA method by adopting the 6 specific antigen coated ELISA plates prepared above.
Taking the example of detecting the expression level of ER autoantibodies, the specific operation steps are as follows:
(1) Incubation of serum samples:
And diluting the serum sample to be detected by using a serum sample diluent according to the volume ratio of 1:100. Adding the diluted serum sample into the reaction wells of the 1 st-11 th row of the ER protein coated 96-well ELISA plate prepared in the step (1), wherein the sample adding amount is 50 μl/well; adding positive serum diluted according to a volume ratio of 1:100 into a1 st-3 rd reaction well of a 12 th row of a 96-well ELISA plate coated with ER protein, wherein the sample adding amount is 50 μl/well, and adding negative serum diluted according to a volume ratio of 1:100 into a 4 th-6 th row of reaction space, wherein the sample adding amount is 50 μl/well; adding serum-free antibody diluent (the loading amount is 50 μl/hole) into the 7 th-8 th reaction hole of the 12 th row of the 96-well ELISA plate coated with ER protein as a blank control; the 96-well enzyme label was then incubated in a 37℃water bath for 1h, after which the reaction wells were discarded, washed 5 times with wash solution (300. Mu.l/well) and dried by pipetting.
(2) Secondary antibody incubation:
diluting the horseradish peroxidase-labeled RecA protein with a secondary antibody diluent according to the proportion of 1:40000, adding the diluted horseradish peroxidase-labeled RecA protein into a reaction hole of a 96-hole ELISA plate, placing the reaction hole in a water bath at 37 ℃ for incubation for 1h, discarding the liquid in the reaction hole, washing the reaction hole for 5 times with a washing solution (the sample adding amount is 300 μl/hole), and beating to dryness.
(3) Color development and termination reaction:
Uniformly mixing the color development liquid A and the color development liquid B according to the equal volume of 1:1, then rapidly adding the mixed color development liquid into the reaction holes of the 96-hole ELISA plate, wherein the sample adding amount is 50 μl/hole, performing light-proof color development reaction at room temperature for 5-15min, and then adding 25 μl of stop solution into each reaction hole to stop the color development reaction; and respectively reading the absorbance OD450 and the absorbance OD620 at the wavelengths of 450nm and 620nm by using a microplate reader, wherein the absorbance OD620 at the wavelength of 620nm is used as a background value, and the difference value between the absorbance OD450 and the absorbance OD620 is used as a final result of the detected absorbance value.
The specific procedure for detecting the expression levels of PR autoantibodies, CD3 autoantibodies, CD16 autoantibodies, CD38 autoantibodies, CD138 autoantibodies in serum samples is essentially the same as that described above for detecting ER autoantibodies, except that: in the step 1), the ELISA plates used in the detection are PR, CD3, CD16, CD38 and CD138 protein coated ELISA plates respectively.
6. And (3) data processing:
The absorbance values (OD values) of the serum samples of the infertility group and the normal control group are subjected to Kolmogorov-Smirnova test, and as a result, the expression levels of the 6 specific related antigen autoantibodies in the serum sample of the study object are not consistent with the normal distribution (P < 0.05), so that the 25 th percentile (P25), the median (P50) and the 75 th percentile (P75) are used for describing the expression level distribution of the 6 specific antigen autoantibodies; a nonparametric test (Mann-WhitneyU) was then used to compare whether there was a difference in the expression level of autoantibodies in the infertility group and the normal control group.
7. Judging the detection result of the kit:
And taking the average number of OD values measured by the negative control holes plus two standard deviations (mean+2SD) as cut-off values, wherein the judgment that the OD value reading in the reaction holes is larger than or equal to the cut-off value is positive, and the judgment that the OD value reading in the reaction holes is smaller than the cut-off value is negative.
8. Experimental results:
(1) Expression level of autoantibodies to 6 specific antigens in infertility group, normal control group:
The statistics of the expression level of the autoantibodies of the 6 specific antigens in the infertility group and the normal control group are shown in fig. 2 and 3.
As can be seen from FIG. 2, the average expression level of the 6 specific antigen autoantibodies was high in the infertility group, and the average OD value was floating around 0.35. As can be seen from FIG. 3, the average expression level of the 6 specific related antigen autoantibodies in the normal control group is lower, and the average OD value is less than 0.25. As can be seen from fig. 2 and 3, the average level of the 6 specific antigen autoantibodies in the serum of the patients with infertility is significantly higher than that of the normal control group, which indicates that the 6 specific related antigen autoantibodies can be used for diagnosing endometrium inflammatory infertility.
(2) Positive rate of autoantibodies to 6 specific antigens in infertility group, normal control group:
And respectively counting the positive rates of the specific antigen autoantibodies in the infertility group and the normal control group (the positive rate is obtained by dividing the number of positive objects detected in each group by the total number of objects detected in the group) by taking the detection result of the kit as a standard, and drawing a bar graph of the positive rates of the 4 tumor-associated antigen autoantibodies in the infertility group and the normal control group by using Excel software (shown in figure 4). The SPSS22.0 software is used for statistical test, the two independent sample chi-square test method is adopted to compare the antibody positive rate of the infertility group and the normal control group, the test level alpha=0.05, and when P is less than 0.05, the result has statistical significance.
As can be seen from FIG. 4, the positive rate of 6 specific antigen autoantibodies in the serum of the patients in the infertility group is in the range of 30% -38.5%, while the positive rate in the control group is not more than 11%. Moreover, the positive rate of the 6 specific antigen autoantibodies in the infertility group is higher than that of the control group through statistical test. The 6 specific antigen autoantibodies can be used as detection indexes of endometrium inflammatory infertility and used for early diagnosis of endometrium inflammatory infertility.
(3) Value assessment of 6 specific antigen autoantibodies alone for diagnosis of endometrial inflammatory infertility:
further, according to the levels of the 6 specific antigen autoantibodies in the infertility group and the normal control group detected by the kit, the individual diagnosis of the autoantibodies of the 6 anti-tumor related antigens is respectively drawn by GRAPHPAD PRISM 8.0.0 to distinguish ROC curves of the patients with endometrium inflammatory infertility and normal persons (as shown in figure 5), the value of the diagnosis of the autoantibodies of the 6 specific antigens to distinguish the patients with endometrium inflammatory infertility and normal persons is verified, and the corresponding sensitivity and specificity are calculated (as shown in table 1).
As can be seen from fig. 5, the AUC of ER autoantibodies when used to diagnose infertility patients from normal individuals was 0.747; the AUC of PR autoantibody diagnosis is 0.652 when the PR autoantibody diagnosis is adopted to distinguish infertility patients from normal people; the AUC of CD3 autoantibodies when used to diagnose and distinguish infertility patients from normal humans was 0.639; the AUC of CD16 autoantibodies when used to diagnose and distinguish infertility patients from normal individuals was 0.617; the AUC of CD38 autoantibodies when used to diagnose and distinguish infertility patients from normal humans was 0.614; the AUC of CD138 autoantibody diagnosis when used to differentiate infertility patients from normal individuals was 0.594. From this, it is clear that the AUC of each of the 6 autoantibodies against the tumor-associated antigen alone for diagnosis and distinction of infertility patients is greater than 0.5, and therefore, ER autoantibodies, PR autoantibodies, CD3 autoantibodies, CD16 autoantibodies, CD38 autoantibodies, CD138 autoantibodies can be used for the auxiliary diagnosis of endometrial inflammatory infertility.
(4) Value assessment of 6 specific antigen autoantibody combinations for diagnosis of endometrial inflammatory infertility:
in order to improve the diagnosis sensitivity, the invention combines ER autoantibodies, PR autoantibodies, CD3 autoantibodies, CD16 autoantibodies, CD38 autoantibodies and CD138 autoantibodies in parallel, judges the serum of 109 infertility patients and 109 normal control serum which are included in the examples, and analyzes the value of different specific antigen combinations for diagnosing the endometrium inflammatory infertility.
The method for judging the diagnosis of the endometrium inflammatory infertility by using the combination of the antigen autoantibodies with different specificities comprises the following steps: when the parallel combination of two, three, four, five or six specific antigen autoantibodies is used for diagnosing and distinguishing the endometrium inflammatory infertility, the diagnosis result of any specific antigen autoantibody in the combination is positive, and the diagnosis result of the specific antigen autoantibody combination is positive. Based on the determination results, ROC curves were generated, and the sensitivity and specificity were counted, and the results are shown in fig. 6 and table 1.
Table 1 shows statistics of results of combinations of autoantibodies to antigens of different specificities for diagnosis of endometrium inflammatory infertility
As can be seen from fig. 6 and table 1, AUC and sensitivity of the combination of a plurality of specific antigen autoantibodies for diagnosis of endometrium inflammatory infertility are significantly improved as compared to the single specific antigen autoantibody; in addition, as the number of specific antigen autoantibodies in the combination increases, the sensitivity of diagnosis of endometrial inflammatory infertility also increases, and when 6 specific antigen autoantibodies are combined, the AUC of the ROC curve reaches the maximum of 0.936, the sensitivity of detection reaches 93.6%, and the diagnostic value is higher. Although the detection specificity gradually decreases with increasing number of specific antigen autoantibodies in the combination, the detection specificity can still reach 80.7% when 6 specific antigen autoantibodies are combined. Therefore, when the combination of a plurality of specific antigen autoantibodies is used for diagnosing endometrium inflammatory infertility, the diagnosis sensitivity can be greatly improved on the premise of ensuring the diagnosis specificity. In addition, as the number of specific antigen autoantibodies in the combination increases, the about log index is continuously increased and gradually tends to be 1, which indicates that the method for diagnosing the endometrium inflammatory infertility by using the combination of a plurality of specific antigen autoantibodies has better diagnosis value.
The experimental result shows that the ELISA kit has higher diagnostic value for the endometrium inflammatory infertility, and further proves that the ELISA kit is an ideal means for early diagnosis and screening of infertility.
The embodiments described above are specific embodiments of the present invention, but the embodiments of the present invention are not limited to the embodiments described above, and any other combinations, changes, modifications, substitutions, and simplifications that do not exceed the design concept of the present invention fall within the scope of the present invention.
Claims (10)
1. Use of an agent for detecting a biomarker, said biomarker being a combination of ER autoantibodies, PR autoantibodies, CD3 autoantibodies, CD16 autoantibodies, CD38 autoantibodies, CD138 autoantibodies, for the manufacture of a product for the diagnosis of endometrial inflammatory infertility.
2. The use according to claim 1, wherein the reagent is a reagent for detecting the biomarker in a sample by enzyme-linked immunosorbent, protein chip, immunoblotting or microfluidic immunodetection.
3. The use according to claim 2, wherein the agent is an antigen or antibody that detects the biomarker.
4. The use according to claim 3, wherein the antigen is a combination of ER protein, PR protein, CD3 protein, CD16 protein, CD38 protein, CD138 protein.
5. The use according to claim 2, wherein the sample is serum, plasma, interstitial fluid.
6. The use according to any one of claims 1 to 5, wherein the product is a protein chip, a kit or a preparation.
7. A kit for diagnosis of endometrial inflammatory infertility, characterized in that the kit comprises reagents for detection of a biomarker being a combination of ER autoantibodies, PR autoantibodies, CD3 autoantibodies, CD16 autoantibodies, CD38 autoantibodies, CD138 autoantibodies.
8. The kit of claim 7, wherein the kit detects the biomarker in the sample by enzyme-linked immunosorbent, protein chip, immunoblot, or microfluidic immunodetection.
9. The kit of claim 8, wherein the kit is an ELISA detection kit comprising a solid support and an antigen coated on the solid support; the antigen is a combination of ER protein, PR protein, CD3 protein, CD16 protein, CD38 protein and CD138 protein.
10. The kit according to any one of claims 7 to 9, wherein the test sample of the kit is serum, plasma, interstitial fluid.
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