A detection reagent, kit and method for detecting plasma Staphylococcus aureus antibodies

Abstract

The invention discloses a detection reagent, a kit and a method for detecting plasma Staphylococcus aureus antibodies, belonging to the field of biological detection, wherein the detection reagent comprises one or two linear antigen polypeptides highly complementary to the target Emp antibody, and the antigen polypeptide is an antigen overlapping epitope of Emp. The detection kit is a 96-well ELISA plate coated with a KLH-coupled antigen polypeptide mixture, which is dried and vacuum-sealed with a non-metallic medical packaging material. The detection method is as follows: the first step is to couple each antigen with the KLH protein respectively; the second step is to dilute the mixture of the KLH-coupled antigen polypeptide with a coating solution to a working solution of 20-50 μg / ml; the third step is to coat a 96-well microplate with the working solution; the fourth step is to prepare a 3% BSA blocking solution with a washing solution; the fifth step is to add samples and analyze the Emp IgG antibody in steps; the beneficial effect is that it can be artificially synthesized, and the production cost is extremely low. It can be used to study the pathogenesis of ischemic cardiovascular and cerebrovascular diseases. It has high social and economic benefits.

Description

Technical Field

[0001] The invention belongs to the field of biological detection, and particularly relates to a detection reagent, a kit and a method for detecting plasma Staphylococcus aureus antibodies. Background Art

[0002] Currently, there are approximately 100 trillion microorganisms from thousands of different species living in the human intestine, collectively referred to as the "microbiome". The number of microbiota is 10 times the total number of cells in our body, and all the genes it carries are called the "microbiome", which is 150 times the number of the human genome. The human intestinal microbiome is composed of a variety of microorganisms, including bacteria, archaea, fungi, viruses and parasites, mainly represented by bacteria. On average, 150-170 species of bacteria dominate the intestine of each person and benefit from the nutrient-rich environment of the intestine. The intestinal flora plays a key role in nutrient utilization, anti-infection, regulation of immune system development and maturation, and host metabolic biological processes.

[0003] The intestinal flora coexists with the intestinal mucus layer. With the development of genome sequencing technology and bioinformatics, it has been found that the colonization, diversity and density of microorganisms vary with the location of the gastrointestinal tract. The number of bacteria in the stomach and jejunum is the lowest, with an average of only tens of thousands of bacteria per milliliter of intestinal content; the bacterial density in the ileum begins to increase, and the number of bacteria in the large intestine is the highest, with an average of tens of billions of bacteria per milliliter of intestinal content. The flora forms a rich and dense microbial biofilm on the large intestinal mucosa, and about 60% of the components in human feces are composed of intestinal microorganisms. The human microbiome has evolved with the host and is an important part of the human body. The establishment and colonization of the intestinal flora is a complex process involving many microbe-microbe and microbe-host interactions. The healthy intestinal flora ecology begins to be established in infancy and early childhood, and the mode of delivery can affect the establishment of the intestinal flora in early life. The bacterial community acquired by normal delivery is similar to the maternal vaginal microbial flora, while the intestinal flora biodiversity of infants born by caesarean section is lower than that of normal delivery babies. The abnormal intestinal microbial colonization caused by caesarean section delivery may increase the risk of suffering from a variety of geriatric diseases in later life. In addition, feeding methods also affect the establishment of intestinal flora in early life. Breastfed infants may have a healthier intestinal microbial ecology than formula-fed infants. It takes about three years after birth for children to establish an intestinal flora structure similar to that of adults, which coincides with the maturation of the immune system. In addition, the composition and structure of the intestinal flora change with age, eating habits, and overall health. There are obvious inter-individual differences in the composition of intestinal bacteria in healthy elderly subjects, with a high proportion of pathogenic bacteria and a low proportion of probiotics. The intestinal flora is also affected by the intake of antibiotics and other commonly used drugs.

[0004] The gastrointestinal tract is the largest pool of immune cells in the human body, and the number of intestinal lymphocytes accounts for 70% of the total number of cells in the human immune system. The intestinal microbial ecology is dynamic, which means that not all bacterial members can permanently settle in the intestine. The intestinal flora regulates the host's immune system, which in turn affects the composition of the intestinal flora. The host immune system ensures the establishment of beneficial flora, controls the growth of specific bacteria, and prevents pathogenic bacteria from damaging the intestine. In a healthy state, the intestinal microbiota plays a beneficial role in the human body by digesting food, producing metabolites, promoting nutrient absorption and drug metabolism. The intestinal flora also helps maintain the integrity of the intestinal mucosal barrier structure, immune regulation, and resistance to pathogen invasion. Therefore, it is believed that the intestinal flora is a key "organ" that affects a variety of physiological and biochemical functions of the host. If the intestinal flora is damaged, the whole body will be affected. Studies have found that dysbiosis of the intestinal flora is associated with the occurrence and development of a variety of human diseases, including ischemic cardiovascular and cerebrovascular diseases. The intestinal flora has been used as a potential target for nutritional and medical intervention.

[0005] As research deepens, people have found that intestinal flora testing may have potential in the risk prediction and prevention of ischemic cardiovascular and cerebrovascular events. A relatively authoritative study performed carotid endarterectomy on patients with severe carotid stenosis under sterile conditions, and used whole genome sequencing technology to identify bacteria obtained from atherosclerotic plaque tissue culture in living subjects. The results showed that each sample contained its own unique microbial flora, such as Bacteroides, Parabacteroides, Bacteroides ovatus, Ruminococcus, and Staphylococcus. Among them, Staphylococcus dominated all samples, especially Staphylococcus pasteurianus. These bacteria may be dormant, but they can be activated in in vitro studies, and after activation, they all have virulence, intracellular invasion, and drug resistance. Clinical routine antibiotics (such as penicillin, tetracycline, ampicillin, streptomycin, and gentamicin) were used for drug sensitivity testing of Staphylococcus pasteurianus isolates, and the results showed that all strains showed a certain degree of sensitivity and resistance to different antibiotics. It is inferred that Staphylococcus pasteurianus is involved in the formation and development of carotid atherosclerotic plaques. Sampling studies in some regions have also found that there are multiple strains in carotid plaques, including Staphylococcus. Another study reported that a study of 15 patients with clinical atherosclerosis and 15 age- and sex-matched healthy controls found that there were multiple strains from the intestines and oral cavity in carotid plaques, including Staphylococcus. Certain intestinal flora strains have also been found in coronary artery atherosclerotic plaques. A large sample study from relevant departments found that coronary atherosclerosis was associated with the transitional proliferation of oral streptococci. These observations support the view that there is a "core" microbiome composed of lineages at the same genus level in atherosclerotic plaques. The microbiome of atherosclerotic plaques comes at least in part from the mouth and intestines.

[0006] Streptococcus and Staphylococcus are the two most common bacteria in the human body, living in large numbers in the mouth, intestines, respiratory tract and skin. Under normal circumstances, streptococci and staphylococci do not infect the body and cause disease. One of the main reasons is the presence of a large number of anti-streptococcal and anti-staphylococcal antibodies in the human body. A study reported in 2021 used phage immunoprecipitation sequencing technology to detect anti-microbial flora IgG antibodies in 997 normal human serum samples. The results showed that more than 99% of people carried anti-streptococcal and anti-staphylococcal IgG antibodies. It can be considered that the presence of these IgG antibodies can ensure the long-term coexistence of humans with streptococci and staphylococci, preventing bacteria from entering the body and causing disease. Summary of the invention

[0007] The purpose of the present invention is to determine the level of Staphylococcus aureus Emp IgG antibodies, and to provide a detection reagent, a kit and a method for detecting plasma Staphylococcus aureus antibodies.

[0008] The detection reagent for detecting plasma Staphylococcus aureus antibodies provided by the present invention comprises one or two linear antigen polypeptides highly complementary to the target Emp antibody, the antigen polypeptides are antigen overlapping epitopes of Emp, and the antigen overlapping epitopes can specifically bind to IgG antibodies against Emp in human plasma.

[0009] "Emp antibody" is a polyclonal antibody that can recognize the polypeptide sequences of SEQ ID NO: 1 and SEQ ID NO: 2, as shown in the following table:

[0010] abbreviation Antigen peptide sequence Emp 1 H- cdfvvpesginkiiptydefkkapkvnv -OH (SEQ ID NO:1) Emp 2 H-cevdkqivakdpevnrfitqnkvnh -OH (SEQ ID NO:2)

[0011] The full-length amino acid sequence of the Staphylococcus aureus Emp molecule, 341 amino acids, and the linear antigen epitope polypeptide region are as follows:

[0012] mkkkllvltmstlfatqfmnsnhanastesvdknfvvpesginkiiptydefkkapkvnvsnladnknfvasedklnkiadpsaaskivdknfavpesklgiivpeykeinnrvnvttnnpaskqvdkqivakdpevnrfitqnkvnhrfittqthykkvitsyksthvhk hvnhatssihhhftikpseaprythpsqsqsliinhhfavpgyhghkvvtpgqasirihhfcavpqinsfkvipsyghnshrmhvpsfqnnttathqnakvnktynykyfytykvvkgvkkhfsfskshgckivkpalniknvnyqyavpsnspthvvpefqgilpaprv.

[0013] The mass or volume concentration ratio of SEQ ID NO: 1 to SEQ ID NO: 2 in the detection reagent is 1:1.

[0014] The two antigen polypeptides are respectively coupled to a carrier protein, and then the two antigen polypeptides coupled to the carrier protein are mixed at a mass concentration ratio of 1:1. The carrier protein is keyhole limpet hemocyanin KLH.

[0015] The detection kit for detecting plasma Staphylococcus aureus antibodies provided by the present invention comprises coating a KLH-coupled antigen polypeptide mixture on a 96-well ELISA plate, and then vacuum-sealing and packaging the mixture with a non-metallic medical packaging material after drying.

[0016] Non-metallic medical packaging materials are medical plastics.

[0017] The kit may also include a positive control and / or a negative control.

[0018] The present invention provides a method for detecting plasma Staphylococcus aureus antibodies, which comprises the following steps:

[0019] Step 1: Before operation, couple each antigen with KLH protein separately, then mix equal volumes and store in a -20℃ refrigerator with a temperature error within ±2℃.

[0020] Step 2: At the beginning of the operation, the mixed solution of KLH-coupled antigen peptide is first diluted with coating solution to a working solution of 20-50 μg / ml. The coating solution is 0.1 M carbonate buffer, and the pH value is measured to be 9.6;

[0021] Step 3: Coat the 96-well microplate with the working solution, incubate overnight at 4°C, and then wash the plate three times with a washing solution containing 0.15 M sodium chloride and 0.1% TWEEN-20 in 0.1 M phosphate buffer with a pH between 7.0 and 7.4.

[0022] Step 4: Use the washing solution to prepare a 3% BSA blocking solution, block the antigen-coated 96-well microplate, incubate at room temperature for 1 hour, wash the plate twice with the washing solution, dry it in a 40°C oven, and vacuum seal it with non-metallic packaging materials;

[0023] Step 5: Add samples and analyze Emp IgG antibodies according to the following steps:

[0024] Step 1. Open the vacuum-sealed package of non-metallic packaging material, take out the 96-well microplate, wash the plate three times with a 0.1 M phosphate buffer solution containing 0.15 M sodium chloride and 0.1% TWEEN-20, and measure the pH value to be 7.2, and then detect the plasma antibody according to the steps;

[0025] Step 2, set up duplicate wells for the plasma samples to be tested, and set up 2 negative control wells, the reference is a negative control solution without Emp IgG antibody, thereby reflecting the experimental index values ​​of the two antigen polypeptides in the Emp IgG antibody negative reaction system, and 2 positive control wells, the reference is a mixture of human Emp IgG antibodies, thereby reflecting the experimental index values ​​of the two antigen polypeptides in the Emp IgG antibody positive reaction system;

[0026] Step 3, dilute the plasma sample to be tested 1:100 with an analytical solution, which is 0.1 M phosphate buffer containing 0.15 M sodium chloride, 10 mM EDTA and 0.5% BSA, with a pH between 7.0 and 7.4, add 100 μl to each well, incubate at 25°C for 1-2 hours, and then wash the plate 3 times;

[0027] Step 4: Dilute the goat anti-human IgG antibody labeled with horseradish peroxidase with analytical solution to verify whether the substance detected in the plasma is a specific antibody. The antibody dilution ratio is 1:25000-1:50000, add 100 μl to each well, and incubate at 25°C for 1-2 hours;

[0028] Step 5: Wash the plate three times with 0.15 M sodium chloride and 0.1% TWEEN-20 in 0.1 M phosphate buffer with a pH value between 7.0 and 7.4, then add 100 μl of 3,3',5,5'-tetramethylbenzidine TMB and peroxidase mixture to each well and incubate at room temperature in the dark for 20-30 minutes.

[0029] Step 6. Add 50 µl of 12% sulfuric acid solution to each well as the stop solution, and then use a microplate reader to detect the optical density OD value. The detection wavelength is 450 nm and the reference wavelength is 630 nm. The detection process is completed within 10 minutes after adding the stop solution, thereby quantitatively analyzing the Emp IgG antibody level in individual plasma.

[0030] Beneficial effects of the present invention:

[0031] Compared with the prior art, the detection reagent, kit and method for detecting plasma Staphylococcus aureus antibodies provided by the present invention have the following beneficial effects:

[0032] (1) The composition for detecting Emp IgG antibodies of the present invention uses two antigenic polypeptides, each of which has a sequence length of no more than 30 amino acids and can be artificially synthesized with extremely low production costs.

[0033] (2) The two antigenic polypeptides of the present invention are antigenic epitopes derived from the Emp protein of Staphylococcus aureus. These antigenic epitopes can specifically bind to the Emp IgG antibodies in human blood and can be used to study the pathogenesis of ischemic cardiovascular and cerebrovascular diseases.

[0034] (3) The method for detecting Emp IgG antibodies of the present invention has high sensitivity, good specificity, and a low false negative rate. It can be conveniently and inexpensively used to predict the risk of ischemic cardiovascular and cerebrovascular events, and can be further extended to the field of pharmaceutical or biological preparation production, with high social and economic benefits. DETAILED DESCRIPTION

[0035] The detection reagent for detecting plasma Staphylococcus aureus antibodies provided by the present invention comprises one or two linear antigen polypeptides highly complementary to the target Emp antibody, the antigen polypeptides are antigen overlapping epitopes of Emp, and the antigen overlapping epitopes can specifically bind to IgG antibodies against Emp in human plasma.

[0036] "Emp antibody" is a polyclonal antibody that can recognize the polypeptide sequences of SEQ ID NO: 1 and SEQ ID NO: 2, as shown in Table 1:

[0037] Table 1. Two linear antigen peptide sequences for detecting Emp IgG antibodies in human plasma

[0038] abbreviation Antigen peptide sequence Emp 1 H- cdfvvpesginkiiptydefkkapkvnv -OH (SEQ ID NO:1) Emp 2 H-cevdkqivakdpevnrfitqnkvnh -OH (SEQ ID NO:2)

[0039] The full-length amino acid sequence of the Staphylococcus aureus Emp molecule, 341 amino acids, and the linear antigen epitope polypeptide region are as follows:

[0040] mkkkllvltmstlfatqfmnsnhanastesvdknfvvpesginkiiptydefkkapkvnvsnladnknfvasedklnkiadpsaaskivdknfavpesklgiivpeykeinnrvnvttnnpaskqvdkqivakdpevnrfitqnkvnhrfittqthykkvitsyksthvhk hvnhatssihhhftikpseaprythpsqsqsliinhhfavpgyhghkvvtpgqasirihhfcavpqinsfkvipsyghnshrmhvpsfqnnttathqnakvnktynykyfytykvvkgvkkhfsfskshgckivkpalniknvnyqyavpsnspthvvpefqgilpaprv.

[0041] The mass or volume concentration ratio of SEQ ID NO: 1 to SEQ ID NO: 2 in the detection reagent is 1:1.

[0042] The two antigen polypeptides are respectively coupled to a carrier protein, and then the two antigen polypeptides coupled to the carrier protein are mixed at a mass concentration ratio of 1:1. The carrier protein is keyhole limpet hemocyanin KLH.

[0043] The detection kit for detecting plasma Staphylococcus aureus antibodies provided by the present invention comprises coating a KLH-coupled antigen polypeptide mixture on a well ELISA plate, and then vacuum-sealing and packaging the mixture with a non-metallic medical packaging material after drying.

[0044] Non-metallic medical packaging materials are medical plastics.

[0045] The kit may also include a positive control and / or a negative control.

[0046] The present invention provides a method for detecting plasma Staphylococcus aureus antibodies, which comprises the following steps:

[0047] Step 1: Before operation, couple each antigen with KLH protein separately, then mix equal volumes and store in a -20℃ refrigerator with a temperature error within ±2℃.

[0048] Step 2: At the beginning of the operation, the mixture of KLH-coupled antigen peptides was first diluted with coating solution to a working solution of 20-50 μg / ml. The coating solution was 0.1 M carbonate buffer, and the pH value was measured to be 9.6;

[0049] Step 3: Coat the 96-well microplate with the working solution, incubate overnight at 4°C, and then wash the plate three times with a washing solution containing 0.15 M sodium chloride and 0.1% TWEEN-20 in 0.1 M phosphate buffer with a pH between 7.0 and 7.4.

[0050] Step 4: Use the washing solution to prepare a 3% BSA blocking solution, block the antigen-coated 96-well microplate, incubate at room temperature for 1 hour, wash the plate twice with the washing solution, dry it in a 40°C oven, and vacuum seal it with non-metallic packaging materials;

[0051] Step 5: Add samples and analyze Emp IgG antibodies according to the following steps:

[0052] Step 1. Open the vacuum-sealed package of non-metallic packaging material, take out the 96-well microplate, wash the plate three times with a 0.1 M phosphate buffer solution containing 0.15 M sodium chloride and 0.1% TWEEN-20, and measure the pH value to be 7.2, and then detect the plasma antibody according to the steps;

[0053] Step 2, set up duplicate wells for the plasma samples to be tested, and set up 2 negative control wells, the reference is a negative control solution without Emp IgG antibody, thereby reflecting the experimental index values ​​of the two antigen polypeptides in the Emp IgG antibody negative reaction system, and 2 positive control wells, the reference is a mixture of human Emp IgG antibodies, thereby reflecting the experimental index values ​​of the two antigen polypeptides in the Emp IgG antibody positive reaction system;

[0054] Step 3, dilute the plasma sample to be tested 1:100 with an analytical solution, which is 0.1 M phosphate buffer containing 0.15 M sodium chloride, 10 mM EDTA and 0.5% BSA, with a pH between 7.0 and 7.4, add 100 μl to each well, incubate at 25°C for 1-2 hours, and then wash the plate 3 times;

[0055] Step 4: Dilute the goat anti-human IgG antibody labeled with horseradish peroxidase with analytical solution to verify whether the substance detected in the plasma is a specific antibody. The antibody dilution ratio is 1:25000-1:50000, add 100 μl to each well, and incubate at 25°C for 1-2 hours;

[0056] Step 5: Wash the plate three times with 0.15 M sodium chloride and 0.1% TWEEN-20 in 0.1 M phosphate buffer with a pH value between 7.0 and 7.4, then add 100 μl of 3,3',5,5'-tetramethylbenzidine TMB and peroxidase mixture to each well and incubate at room temperature in the dark for 20-30 minutes.

[0057] Step 6. Add 50 µl of 12% sulfuric acid solution to each well as the stop solution, and then use a microplate reader to detect the optical density OD value. The detection wavelength is 450 nm and the reference wavelength is 630 nm. The detection process is completed within 10 minutes after adding the stop solution, thereby quantitatively analyzing the Emp IgG antibody level in individual plasma.

[0058] The specific test methods are as follows:

[0059] 1. Sample collection:

[0060] A total of 218 patients with ischemic stroke who had recently been treated and 198 healthy subjects who were matched with the same age were included. In addition, 76 patients with acute myocardial infarction coronary heart disease, 216 patients with non-acute myocardial infarction coronary heart disease, and 206 age-matched healthy control subjects who were treated at the same time were included. Before the detection of plasma Emp IgG antibody levels, all plasma samples were stored at -80℃, and the storage time was verified to be no more than two years, and there was no repeated freezing and thawing (no more than 3 times).

[0061] 2. Sample testing:

[0062] Plasma samples were thawed at 4°C. The two peptide antigens used in this experiment (see Table 1) were synthesized by Gill Biochemical Co., Ltd. (Shanghai) with a purity of 95%. The specific steps are as follows:

[0063] (1) Before operation, each antigen peptide was coupled to KLH protein, then mixed in equal volumes and stored in a -20°C refrigerator.

[0064] (2) At the beginning of the operation, the mixture of the two antigen peptides and the KLH-coupled antigen was first diluted to 30 μg / ml with coating solution containing 0.1 M carbonate buffer with a measured pH value of 9.6.

[0065] (3) Coat a 96-well assay plate (Shanghai Sangon Biotechnology Co., Ltd.), incubate overnight at 4°C for 17.5 hours, and then wash the plate three times with a washing solution consisting of 0.1 M phosphate buffer containing 0.15 M sodium chloride and 0.1% TWEEN-20. The pH value was measured to be 7.2.

[0066] (4) Use the washing solution to prepare 3% BSA blocking solution, block the antigen-coated 96-well microtiter plate, incubate at room temperature for 1 hour, wash the plate twice with the washing solution, then dry it in a 40°C oven and vacuum seal it with non-metallic packaging material.

[0067] (5) The steps for plasma antibody analysis are as follows:

[0068] a) Open the vacuum-sealed package of non-metallic packaging material, take out the 96-well microplate, wash the plate three times with the aforementioned washing solution (i.e., 0.1 M phosphate buffer containing 0.15 M sodium chloride and 0.1% TWEEN-20, with a measured pH of 7.2), and then detect plasma antibodies according to the steps.

[0069] b) Duplicate wells were set for the plasma samples to be tested, and two negative control wells (NC, the reference substance was bovine serum albumin, provided by Sigma-Aldrich) and two positive control wells (PC, the reference substance was a mixture of anti-Staphylococcus aureus IgG antibodies, provided by Sigma-Aldrich) were set up.

[0070] c) Dilute the plasma sample to be tested 1:100 with the analytical solution, which is 0.1 M phosphate buffer containing 0.15 M sodium chloride, 10 mM EDTA and 0.5% BSA, with a pH between 7.0 and 7.4. Add 100 μl to each well, incubate at 25°C for 1-2 hours, and then wash the plate 3 times with the washing solution.

[0071] d) Dilute the horseradish peroxidase-labeled goat anti-human IgG antibody (supplied by Sigma-Aldrich) with analytical solution at a working concentration of 1:50,000, add 100 μl to each well, and incubate at 25°C for 1.5 hours.

[0072] e) After washing the plate three times with the aforementioned washing solution (i.e., 0.1 M phosphate buffer containing 0.15 M sodium chloride and 0.1% TWEEN-20, pH value measured to be 7.2), add 100 μl of 3,3',5,5'-tetramethylbenzidine (TMB) and peroxidase mixture (provided by Life Technologies) to each well and incubate at room temperature in the dark for 25 minutes.

[0073] f) Add 50 μl of stop solution (12% sulfuric acid solution or 12% H2SO4) to each well, and then use a microplate reader to detect the optical density (OD) value. The detection wavelength is 450nm and the reference wavelength is 630nm. The detection should be completed within 10 minutes after adding the stop solution. The subsequent steps will be based on this result for quantitative comparative analysis of Emp IgG antibodies for each individual.

[0074] 3. Data Analysis:

[0075] When analyzing the data obtained from the above tests, the positive sample ratio (PSR) is used to determine the Emp IgG antibody level in plasma; the PSR calculation formula is:

[0076] PSR = [OD value of the sample to be tested – ODNC value] / [OD value of the positive standard – ODNC value], NC is the negative control. The threshold of this method is determined by the 5th percentile PSR value of the healthy control group. Antibody levels below this threshold are considered to be negative for Emp IgG antibodies.

[0077] As shown in Table 2, the rank sum test compared the difference in Emp IgG antibody levels between the ischemic stroke group and the healthy control group. Compared with the healthy control group, the Emp IgG level in the ischemic stroke group was significantly reduced, and it was statistically significant (P < 0.001).

[0078] Table 2. Comparison of plasma IgG antibody levels between the ischemic stroke group and the healthy control group

[0079] IgG Ischemic stroke (n) Healthy controls (n) Z P＊ Emp1-Emp2 0.843±0.489（218） 1.215±0.603（198） -6.650 ＜0.001

[0080] Note: Mean ± standard deviation, n = number of cases.

[0081] As shown in Table 3, Kruskal-Wallis nonparametric analysis of variance (H test) compared the differences in Emp IgG antibody levels between the acute myocardial infarction coronary heart disease group, the non-acute myocardial infarction coronary heart disease group and the healthy control group. The results showed that compared with the healthy control group, the Emp IgG antibody levels in the acute myocardial infarction coronary heart disease group and the non-acute myocardial infarction coronary heart disease group were significantly reduced, and there was statistical significance (P < 0.001).

[0082] Table 3. Comparison of plasma IgG antibody levels of patients with acute myocardial infarction, non-acute myocardial infarction and healthy controls

[0083] IgG Antibody level (n) H P Emp1-Emp2 30.22 ＜0.001 Acute myocardial infarction coronary heart disease 0.557±0.512（76） Non-acute myocardial infarction coronary heart disease 0.709±0.579（216） Healthy controls 0.936±0.672（206）

[0084] Note: Mean ± standard deviation, n = number of cases.

[0085] Experimental technology evaluation:

[0086] The above two linear antigen polypeptides can specifically bind to Staphylococcus Emp IgG in plasma. That is, the amino acid sequences of these two linear antigen polypeptides are completely complementary to the Emp IgG antibody molecules in terms of spatial structure and configuration.

Claims

1. A detection reagent for detecting plasma Staphylococcus aureus antibodies, characterized in that: It contains two linear antigenic polypeptides that are highly complementary to the target Emp antibody. The antigenic polypeptides are overlapping antigenic epitopes of Emp. The overlapping antigenic epitopes can specifically bind to the Emp IgG antibody in human plasma. The "Emp antibody" is a polyclonal antibody that can recognize the polypeptide sequences of SEQ ID NO: 1 and SEQ ID NO: 2, as follows: SEQ ID NO:

1. The polypeptide sequence of Emp 1 antigen is: H-cdfvvpesginkiiptydefkkapkvnv-OH; SEQ ID NO:

2. The Emp 2 antigen polypeptide sequence is: H-cevdkqivakdpevnrfitqnkvnh-OH.

2. A detection reagent for detecting plasma Staphylococcus aureus antibodies according to claim 1, characterized in that: The full-length amino acid sequence of the Staphylococcus aureus Emp molecule, 341 amino acids, and the linear antigen epitope polypeptide region are as follows: mkkkllvltmstlfatqfmnsnhanastesvdknfvvpesginkiiptydefkkapkvnvsnladnknfvasedklnkiadpsaaskivdknfavpesklgiivpeykeinnrvnvttnnpaskqvdkqivakdpevnrfitqnkvnhrfittqthykkvitsyksthvhk hvnhatssihhhftikpseaprythpsqsqsliinhhfavpgyhghkvvtpgqasirihhfcavpqinsfkvipsyghnshrmhvpsfqnnttathqnakvnktynykyfytykvvkgvkkhfsfskshgckivkpalniknvnyqyavpsnspthvvpefqgilpaprv.

3. A detection reagent for detecting plasma Staphylococcus aureus antibodies according to claim 1, characterized in that: The mass or volume concentration ratio of SEQ ID NO: 1 to SEQ ID NO: 2 in the detection reagent is 1:

1.

4. A detection reagent for detecting plasma Staphylococcus aureus antibodies according to claim 1, characterized in that: The two antigen polypeptides are respectively coupled with a carrier protein, and then the two antigen polypeptides coupled with the carrier protein are mixed at a mass concentration ratio of 1:1, and the carrier protein is keyhole limpet hemocyanin KLH.

5. A detection kit for detecting plasma Staphylococcus aureus antibodies, characterized in that: The invention comprises a detection reagent for detecting plasma Staphylococcus aureus antibodies as described in any one of claims 1 to 4; the KLH-coupled antigen polypeptide mixture is coated on a 96-well ELISA plate, and then vacuum-sealed and packaged with non-metallic medical packaging materials after drying.

6. A detection kit for detecting plasma Staphylococcus aureus antibodies according to claim 5, characterized in that: The non-metallic medical packaging material is medical plastic.

7. A detection kit for detecting plasma Staphylococcus aureus antibodies according to claim 5, characterized in that: The kit also includes a positive control and / or a negative control.

8. A method for detecting plasma Staphylococcus aureus antibodies, characterized in that: The method comprises the following steps: The first step is to couple each antigen Emp 1 and Emp 2 as described in claim 1 with KLH protein respectively before operation, and then mix them in equal volumes and store them in a -20°C refrigerator with a temperature error within the range of ±2°C; Step 2: At the beginning of the operation, the mixed solution of KLH-coupled antigen polypeptide is first diluted with coating solution to a working solution of 20-50 μg / ml. The coating solution is 0.1 M carbonate buffer, and the pH value is measured to be 9.6; Step 3: Coat a 96-well microplate with the working solution, incubate overnight at 4°C, and then wash the plate three times with a washing solution containing 0.15M sodium chloride and 0.1% TWEEN-20 in 0.1M phosphate buffer at a pH between 7.0 and 7.

4. Step 4: Use the washing solution to prepare a 3% BSA blocking solution, block the antigen-coated 96-well microplate, incubate at room temperature for 1 hour, wash the plate twice with the washing solution, then dry it in a 40°C oven, and vacuum seal it with non-metallic packaging materials; Step 5: Add samples and detect plasma Emp antibody IgG step by step according to the following steps: Step 1, open the vacuum-sealed package of non-metallic packaging material, take out the 96-well microplate, wash the plate three times with a 0.1 M phosphate buffer solution containing 0.15 M sodium chloride and 0.1% TWEEN-20, and the pH value is measured to be 7.2, and then detect the plasma Emp antibody IgG according to the steps; Step 2, set up duplicate wells for the plasma samples to be tested, and set up two negative control wells, and the reference substance is a negative control solution without Emp IgG antibody, so as to reflect the experimental index values ​​of the two antigen polypeptides Emp 1 and Emp 2 in the Emp IgG antibody negative reaction system; and 2 positive control wells, the reference substance is a mixture of human Emp IgG antibodies, thereby reflecting the experimental index values ​​of the two antigenic peptides Emp 1 and Emp 2 in the Emp IgG antibody positive reaction system; Step 3, dilute the plasma sample to be tested 1:100 with an analytical solution, which is 0.1M phosphate buffer containing 0.15M sodium chloride, 10mM EDTA and 0.5% BSA, with a pH between 7.0-7.4, add 100μl to each well, incubate at 25°C for 1-2 hours, and then wash the plate 3 times; Step 4: Dilute the goat anti-human IgG antibody labeled with horseradish peroxidase with analytical solution to verify whether the substance detected in the plasma is a specific antibody. The antibody dilution ratio is 1:25000-1:50000, add 100 μl to each well, and incubate at 25°C for 1-2 hours; Step 5, wash the plate three times with a 0.1 M phosphate buffer solution containing 0.15 M sodium chloride and 0.1% TWEEN-20, with a pH value between 7.0 and 7.4, then add 100 μl of 3,3',5,5'-tetramethylbenzidine TMB and peroxidase mixture to each well and incubate at room temperature in the dark for 20-30 minutes; Step 6, add 50 μl of 12% sulfuric acid solution to each well as the stop solution, and then use a microplate reader to detect the optical density OD value, the detection wavelength is 450 nm, and the reference wavelength is 630 nm. The detection process is completed within 10 minutes after adding the stop solution, thereby quantitatively analyzing the Emp antibody IgG level in individual plasma.