Non-invasive selenium level prediction method based on saliva selenoprotein P detection and application

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a non-invasive selenium level prediction method based on saliva selenoprotein P detection and application. According to the invention, a heterologous expression antigen SELENOP (40-194aa) is used for immunizing a mouse to generate an antibody, and the monoclonal antibody capable of efficiently recognizing SELENOP is obtained through screening; and establishing an ELISA detection method by using the obtained monoclonal antibody. By utilizing the detection method and an ICP-MS selenium content detection method, the selenoprotein P content in human plasma and saliva and the total selenium content in the plasma are detected and analyzed, and obvious positive correlation exists among the selenoprotein P content, the saliva selenoprotein P content and the total selenium content in the plasma, so that a correlation curve between the selenoprotein P in the saliva and the selenium content in the human plasma is established; and thus, the aim of non-invasively detecting and evaluating the selenium level of the human body through the saliva selenoprotein P is fulfilled. The technical scheme provided by the invention can be used for large-scale screening and conventional health monitoring.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and particularly relates to a non-invasive selenium level prediction method and application based on the detection of salivary selenoprotein P. Background Art

[0002] Selenium, as an essential trace element for the human body, plays an irreplaceable role in key physiological processes such as antioxidant defense, immune regulation, and thyroid hormone metabolism. Research has shown that selenium deficiency is closely related to various chronic diseases such as cardiovascular diseases, cancers, and thyroid dysfunctions, while excessive intake may also cause toxic reactions. Therefore, accurately assessing the selenium level in the human body and formulating personalized selenium supplementation strategies have become important topics in the fields of public health and clinical nutrition. However, there is currently a common problem of blindness in selenium supplementation practices, and there is an urgent need to establish scientific and convenient detection methods and guiding standards to balance the selenium nutritional needs and safety of different populations.

[0003] Currently, the detection of human selenium levels mainly relies on blood, urine, or hair samples, and techniques such as hydride generation atomic fluorescence spectrometry (AFS), fluorescence spectrophotometry, and inductively coupled plasma mass spectrometry (ICP-MS) are used to determine the total selenium content. Patent CN113030373A proposes a chemiluminescence detection method based on blood selenium, but it still relies on an invasive blood collection process. In recent years, selenoprotein P has attracted much attention due to its high correlation with selenium metabolism. Research has confirmed that its serum concentration can more directly reflect the bioavailability of selenium. Nevertheless, existing detection methods mostly focus on the total selenium content, and the sample collection is complex. For example, urine selenium requires a 24-hour collection, and hair selenium is easily interfered by detergents and growth cycles, making it difficult to meet the convenient and accurate clinical needs.

[0004] The existing selenium detection technologies have the following significant drawbacks: (1) Invasiveness and sampling limitations: Blood selenium detection requires venous puncture, resulting in poor compliance among children, pregnant women, and those requiring frequent monitoring; urine selenium detection is limited by the sampling duration (24 hours) and individual differences (such as renal function and dietary fluctuations), with an error rate as high as 30%. (2) Single biomarker selection: Total selenium content cannot distinguish between inorganic selenium and bioactive selenium forms (such as selenocysteine), leading to a disconnection between the evaluation results and physiological functions. For example, cancer patients may present a contradictory state of "normal total selenium but functional selenium deficiency" due to selenium protein synthesis disorders. (3) Lack of personalized guidance: Existing methods do not integrate selenium protein kinetic parameters (such as the half-life of SEPP1), genetic polymorphisms (such as GPx1 genotype), and pathological factors (such as the increased selenium requirement in diabetic patients), making it difficult to formulate a dynamic selenium supplementation plan. Studies have shown that the prediction error of the traditional blood selenium detection for the selenium supplementation dose in the elderly population can reach 40%, as it ignores the age-related decline in selenium absorption rate. (4) Insufficient technical cost and popularity: ICP-MS equipment is expensive (over 2 million yuan per unit), the fluorescence method has low sensitivity (detection limit > 1 μg / L), and there is a lack of a standardized detection system for non-invasive samples such as saliva, restricting its application in primary healthcare.

[0005] In summary, the existing technologies have not solved the core bottlenecks in the "precision detection - functional evaluation - personalized intervention" chain, and there is an urgent need to develop a new method to predict human selenium levels. Summary of the Invention

[0006] In view of the problems existing in the prior art, the technical problems to be solved by the present invention include:

[0007] 1) Provide a simple, low-cost, and non-invasive selenium level detection method: Detecting selenoprotein P through saliva samples, which solves the problems of complex blood sampling, high cost, and strong invasiveness in traditional methods, making the detection process more convenient and rapid, and suitable for large-scale population health monitoring.

[0008] 2) Achieve accurate prediction of selenium levels: By establishing a correlation curve between saliva selenoprotein P and total selenium content, the present invention can accurately predict the human selenium status by measuring the saliva selenoprotein P level, thereby providing data support for personalized selenium supplementation plans and avoiding the inaccurate prediction problems caused by only measuring the total selenium content in the prior art.

[0009] 3) Promote the wide application of saliva selenoprotein P as a biomarker: By developing an ELISA rapid detection method based on the detection of selenoprotein P, the present invention promotes the standardized application of saliva selenoprotein P in selenium level evaluation and selenium supplementation guidance.

[0010] By solving the above technical problems, the present invention can significantly improve the accuracy and efficiency of selenium level assessment, promote personalized management of selenium supplementation, and meet the health needs of different populations.

[0011] The present invention provides a truncated selenium protein P antigen, and the amino acid sequence of the truncated body is the 40-194aa of the sequence shown in SEQ ID NO.2.

[0012] The present invention also provides a gene encoding the truncated selenium protein P antigen as described in claim 1, and its nucleotide sequence is the 118-582nt of the sequence shown in SEQ ID NO.1.

[0013] The present invention also provides an expression vector containing the above encoding gene.

[0014] The present invention also provides the application of the above truncated selenium protein P antigen in the preparation of a selenium protein P antibody detection reagent.

[0015] The present invention also provides a selenium protein P antibody, and a monoclonal antibody is obtained by immunizing mice with the above truncated selenium protein P antigen.

[0016] The present invention also provides an ELISA kit for detecting selenium protein P, and the kit includes a selenium protein P antibody obtained by using the above truncated selenium protein P antigen. The kit also includes an ELISA coating buffer, a washing solution PBST, a 5% BSA blocking solution, a PBS dilution solution, a TMB solution, and a termination solution.

[0017] The present invention also provides a non-invasive selenium level prediction method based on the detection of salivary selenium protein P, using the above ELISA kit to detect the content of selenium protein P in saliva, and predicting the selenium level with reference to the correlation curve between salivary selenium protein P and plasma total selenium content.

[0018] Further, by successively establishing the correlation curve between the content of salivary selenium protein P and plasma selenium protein P, and the correlation curve between plasma selenium protein P and plasma total selenium content, a correlation curve between salivary selenium protein P and plasma total selenium content is constructed.

[0019] The present invention also provides the application of the above non-invasive selenium level prediction method based on the detection of salivary selenium protein P in the assessment of selenium level in organisms for non-diagnostic and non-therapeutic purposes, especially in the assessment of human selenium level.

[0020] In summary, the advantages and positive effects of the present invention are as follows:

[0021] The detection method provided by the present invention can rapidly and accurately determine the content of selenoprotein P through non-invasive saliva samples or conventional blood samples, thereby predicting the selenium level in the human body. Compared with traditional selenium detection methods, the present invention has the following advantages:

[0022] Simple, rapid and low-cost: The ELISA detection method is easy to operate and has a low cost, making it suitable for large-scale screening and routine health monitoring.

[0023] High precision: As a biological activity marker of selenium, selenoprotein P can more accurately reflect the selenium level in the human body.

[0024] Non-invasive: The collection of saliva samples is convenient, making it suitable for wide application in health management and the formulation of personalized selenium supplementation programs.

[0025] Through the present invention, accurate assessment of selenium levels can be achieved, and personalized selenium supplementation guidance can be provided for different populations (such as pregnant women, the elderly, children, etc.).

[0026] The present invention solves a series of problems in the prior art by proposing a method for predicting the selenium level in the human body based on detecting the content of selenoprotein P in saliva. Compared with the prior art, the present invention has the following remarkable advantages:

[0027] 1) Non-invasive and convenient sample collection method: Traditional selenium level detection methods mainly rely on the collection of blood or urine samples, which are usually invasive and may cause discomfort to patients, especially when large-scale health screenings are required. In addition, the collection of blood samples requires professional personnel and special equipment, increasing the operation difficulty and time cost. In contrast, the present invention uses saliva samples as an alternative. The collection of saliva samples is simple, non-invasive and comfortable, which not only reduces the burden on patients but also improves the efficiency of sample collection. It is suitable for large-scale population health screenings, and is particularly friendly to special populations such as children, pregnant women, and the elderly. This improvement significantly enhances the universality and convenience of detection.

[0028] 2) Customized selenoprotein P monoclonal antibody to improve detection accuracy and specificity: The present invention adopts a customized human selenoprotein P monoclonal antibody, which makes the detection of selenoprotein P have higher specificity and sensitivity. Compared with the non-specific antibodies or antibodies with strong cross-reactivity widely used in the prior art, the precisely designed antibody of the present invention can effectively avoid the interference of other proteins, thereby improving the accuracy of the detection results of selenoprotein P. This improvement provides a more reliable biomarker for the accurate assessment of selenium levels.

[0029] 3) Rapid and efficient indirect ELISA detection method: The present invention uses the indirect ELISA (enzyme-linked immunosorbent assay) technology for the quantitative detection of selenoprotein P, which only requires one capture antibody and one enzyme-labeled antibody, and has the advantages of simple operation, short detection cycle, and low cost. Compared with high-end technologies such as ICP-MS, the ELISA method not only requires less equipment, but also can be efficiently carried out in a conventional laboratory environment, and can quickly screen a large number of samples. In practical applications, the ELISA method provides an economical and efficient detection means for public health institutions, hospitals, clinical laboratories, etc.

[0030] 4) Predicting selenium levels through selenoprotein P to avoid the limitations of relying solely on total selenium content: Traditional selenium level detection usually relies on measuring the total selenium content in blood or urine. However, the total selenium content cannot fully reflect the biological activity and metabolic status of selenium in the body, and requires high-end instruments such as ICP-MS, and the operation process is cumbersome. While the present invention can more accurately evaluate the selenium level of the human body by measuring the content of selenoprotein P, because selenoprotein P is the main carrier protein of selenium and is closely related to the absorption, transport, and utilization of selenium. Therefore, the present invention can avoid the problem that the existing methods cannot accurately reflect the biological function of selenium and provide a more scientific evaluation result of selenium level.

[0031] 5) Establishing a correlation curve between selenoprotein P and total selenium content to provide personalized selenium supplementation guidance: The present invention can achieve precise prediction of selenium levels by establishing a correlation curve between the content of selenoprotein P and the total selenium content. The advantage of this technical solution is that it can scientifically predict the selenium status in the body based on the selenoprotein P level of an individual, thereby providing data support for the formulation of personalized selenium supplementation programs. By this method, problems such as overdose or deficiency caused by blind selenium supplementation can be avoided, ensuring the safety and effectiveness of selenium supplementation. This improvement provides personalized nutritional guidance for different populations (such as pregnant women, the elderly, children, etc.) and has important public health significance.

[0032] Strong adaptability and broad application prospects: The technology of the present invention can not only be used for the evaluation of selenium levels in clinical practice, but also be widely applied to various fields such as health management, disease prevention, and health monitoring of the elderly. The selenium level prediction method based on the detection of selenoprotein P can play an important role in public health projects, individual nutritional management, disease prevention, etc. In addition, this technology is also applicable to the nutritional management of special groups such as athletes, the elderly, and children, providing new technical support for personalized health management Brief Description of the Drawings

[0033] Figure 1 is the result of amino acid sequence specificity and conservation analysis;

[0034] Figure 2 is the result of antigenic epitope prediction;

[0035] Figure 3 It is the PCR verification result of expression vector construction;

[0036] Figure 4 It is the WESTERN BLOT verification result of antigen purification;

[0037] Figure 5 It is the ELISA sensitivity verification result of mouse monoclonal antibody;

[0038] Figure 6 It is the correlation analysis of plasma selenoprotein P - total plasma selenium content;

[0039] Figure 7 It is the correlation analysis of salivary selenoprotein P - plasma selenoprotein P content;

[0040] Figure 8 It is the correlation analysis of salivary selenoprotein P - total plasma selenium content;

[0041] Figure 9 It is the correlation curve of salivary selenoprotein P - total plasma selenium content;

[0042] Figure 10 It is the schematic diagram of the principle of the present invention. Detailed implementation manners

[0043] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with embodiments. The equipment and reagents used in each embodiment and test example can be obtained from commercial channels unless otherwise specified. The specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0044] Based on the information contained in this application, those skilled in the art can easily make various changes to the precise description of the present invention without departing from the spirit and scope of the appended claims. It should be understood that the scope of the present invention is not limited to the defined processes, properties or components, because these embodiments and other descriptions are only for schematically illustrating specific aspects of the present invention. In fact, various changes that can be made by those skilled in the art or related fields to the embodiments of the present invention are all covered within the scope of the appended claims.

[0045] For a better understanding of the present invention rather than limiting the scope thereof, all numbers representing amounts, percentages, and other numerical values used in this application should be understood to be modified by the word "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification and the appended claims are approximate values, which may vary depending on the desired properties to be obtained. Each numerical parameter should be considered at least as obtained by the reported significant figures and by conventional rounding methods. In the present invention, "about" means within 10% of a given value or range, preferably within 5%.

[0046] In the following embodiments of the present invention, when the temperature is not specifically limited, it is under normal temperature conditions. Normal temperature refers to the natural room temperature conditions in the four seasons without additional cooling or heating treatment. Generally, the normal temperature is controlled at 10 - 30 °C, preferably 15 - 25 °C.

[0047] In the examples, for the experimental methods without specific conditions noted, they are usually carried out under conventional conditions, such as those required by "Molecular Cloning: A Laboratory Manual" (Chinese version) (edited by J. Sambrook, M.R. Green, translated by He Fuchu. Fourth Edition, Beijing: Science Press, 2017) and the purchased reagent kits, etc.

[0048] Humans have 25 selenoproteins. Among them, selenoprotein P (SELENOP) is a major protein for storing and transporting selenium in mammals. It is mainly synthesized in the liver and transports selenium elements through the blood circulation, and is widely distributed in organs and body fluids such as the liver, brain, kidneys, blood, and saliva. A remarkable feature of selenoprotein P is that it contains a large number of selenocysteine (Sec) residues, usually having 10 or more Sec residues, which makes it the main storage and transport protein of selenium in the body. Selenocysteine is the key for selenoprotein P to effectively execute its biological functions, endowing it with strong antioxidant capacity and selenium transport capacity. Therefore, measuring the content of SELENOP can be used as an effective indicator for evaluating the selenium nutritional status of the human body. Considering the invasiveness and cumbersome nature of blood collection, we pioneered the use of saliva samples to detect the content of human selenoprotein P to characterize the selenium content level in the human body, and found a significant positive correlation among saliva selenoprotein, plasma selenoprotein, and plasma total selenium content.

[0049] In the present invention, the antigen SELENOP(40-194aa) was heterologously expressed in the strain E.coli Rosetta, and the screening antigen SELENOP(40-194aa) was obtained through protein purification. Then, we introduced the recombinant SELENOP protein into mice by immunization to stimulate their immune systems to produce antibodies. Subsequently, the spleen cells of the mice were collected and fused with the myeloma cell line to form hybridoma cells. Then, through screening and cloning, monoclonal antibodies capable of efficiently recognizing SELENOP were obtained. After obtaining the monoclonal antibodies, we optimized and improved an indirect ELISA detection method for selenoprotein P. This method uses the SELENOP murine monoclonal antibody as the capture antibody and goat anti-mouse F(ab’)2 as the enzyme-labeled antibody, and can quickly and sensitively detect the content of selenoprotein P in blood and saliva.

[0050] Subsequently, using this ELISA detection method and the selenium content detection method of ICP-MS, we simultaneously detected the content of selenoprotein P and the total selenium content in plasma of more than a dozen human samples, and analyzed the correlation of the three groups of data. It was found that there was a significant positive correlation between the content of salivary selenoprotein P and the content of plasma selenoprotein P and the total selenium content, and a correlation curve between salivary selenoprotein P and human plasma selenium content was established. Through the above work, we can non-invasively and quickly judge the level of blood selenium content in the human body through the ELISA detection of salivary selenoprotein P, which is used for large-scale screening and routine health monitoring. In addition, we also formulated the selenium intake that different populations need to supplement according to the level of salivary selenoprotein P, and developed a scientific selenium supplementation strategy for different populations to achieve precise and scientific selenium supplementation. At the same time, we will also study the relationship between selenium content and various chronic diseases, laying a foundation for guiding the application of organic selenium in disease treatment.

[0051] The technical solutions of the present application will be described in detail below in conjunction with specific embodiments.

[0052] Example 1 Heterologous expression of antigen selenoprotein P

[0053] 1. Selection of antigen region

[0054] In the present invention, specific and antigenic epitope predictions were respectively carried out on different antigen regions of selenoprotein P (as shown in Table 1, expression route: 40-194aa, 260-381aa; polypeptide route: 331-343aa, 28-40aa. The nucleotide sequence of the SELENOP antigen is SEQ ID NO.1, and the amino acid sequence is SEQ ID NO.2). It was found through analysis that the performance differences of different antigen regions were relatively large, and the truncated SELENOP antigen (40-194aa) was superior in all aspects. Therefore, this region was selected for heterologous expression and purification, and mouse monoclonal antibodies were produced by immunizing mice.

[0055] Table 1 Selection of antigen regions

[0056]

[0057]

[0058] Analysis of amino acid sequence specificity and conservation: As Figure 1 shown, in Homo sapiens, the specificity of the gene was analyzed and found to be good. Epitope analysis ( Figure 2 ) also indicated that this protein segment could serve as a suitable epitope.

[0059] 2. Construction of expression vector and preparation of antigen

[0060] After the full gene of SELENOP(40-194aa) was synthesized, the pET-28a-SUMO vector was double digested with BamHI and XhoI, and the PCR product of the SELENOP gene was digested with the same restriction sites. Subsequently, the digested SELENOP gene fragment was ligated to the linearized pET-28a-SUMO vector by T4 DNA ligase to construct a recombinant plasmid. The ligation product was transformed into Escherichia coli (such as DH5α), spread on an antibiotic screening plate, and positive clones were picked and verified by restriction digestion and sequencing to successfully construct the pET-28a-SUMO recombinant vector containing the SELENOP gene. The vector was transformed into the expression strain E. coli Rosetta, cultured until OD600nm reached 0.5-0.6, induced with 0.8 mM IPTG at 37 °C for 4 hours, and the bacteria were lysed and purified for protein identification. The results were as Figure 4 , and the size of the purified protein was correct and could be used for subsequent mouse immunization injection to produce monoclonal antibodies against selenoprotein P.

[0061] Example 2 Expression of monoclonal mouse antibody against human selenoprotein P

[0062] First, the recombinant SELENOP protein prepared in Example 1 was introduced into mice by immunization to stimulate their immune systems to produce antibodies (Table 2). Then, referring to the method of Ma Jingchang et al. (Ma Jingchang, Wu Shuwen, Wang Yuling, et al. Preparation and application of monoclonal antibody against human tumor suppressor 2 (ST2) in mice [J]. Chinese Journal of Cellular and Molecular Immunology, 2021, 37(11): 1026-1031. DOI: 10.13423 / j.cnki.cjcmi.009298.), spleen cells of the mice were collected and fused with the antibody-producing cell line myeloma cells SP2 / 0 to form hybridoma cells. Subsequently, through screening and cloning, monoclonal antibodies capable of efficiently recognizing SELENOP were obtained. Finally, antibodies with high specificity and high affinity were selected for large-scale culture and purification to obtain the mouse monoclonal antibody CMC0619-01-A against selenoprotein P for various ELISA assays, with an antibody concentration of 1.32 mg / mL. Then, through the following ELISA experiment, it was demonstrated that this antibody had a good titer and could meet the experimental requirements such as ELISA assays ( Figure 5 ).

[0063] Experimental protocol:

[0064] Coating: SELENOP(40-194aa)-HIS-SUMO, 1 μg / mL, 25 μl / well, coating (384-well plate) overnight at 4 °C

[0065] Blocking: ELISA blocking solution 50 μl / well, incubate at room temperature for 1 h.

[0066] Primary antibody: Starting concentration of the antibody is 1 μg / ml, diluted 3-fold in gradient, 8 gradients, 25 μl / well, incubate at room temperature for 1 h.

[0067] Secondary antibody: Peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG(H+L) [Jackson ImmunoResearch, 115-035-003], 1:10000, 25 μl / well, incubate at room temperature for 1 h.

[0068] Color development: TMB [Thermo Fisher, 34029] (1:5), 25 μl / well, develop color at room temperature in the dark for 3 min.

[0069] Note: NC is dilution buffer

[0070] Table 2: Immunization record of mouse antigen injection

[0071] Number of immunizations Immunization cycle Immunization time Immunization dose (μg) Immunoadjuvant Status of immunized animals First immunization Day 0 2022 / 9 / 20 100 μg / animal Complete Freund's adjuvant Good Second immunization Day 14 2022 / 10 / 4 100 μg / animal Incomplete Freund's adjuvant Good Third immunization Day 28 2022 / 10 / 18 100 μg / animal Incomplete Freund's adjuvant Good Fourth immunization Day 42 2022 / 11 / 1 100 μg / animal Incomplete Freund's adjuvant Good Fifth immunization Day 56 2022 / 11 / 15 100 μg / animal Incomplete Freund's adjuvant Good Sixth immunization Day 70 2022 / 11 / 29 100 μg / animal Incomplete Freund's adjuvant Good Seventh immunization Day 388 2023 / 10 / 13 100 μg / animal Incomplete Freund's adjuvant Good Boost immunization Day 402 2023 / 10 / 27 100 μg / animal / Good Blood collection from immunized animals Day 407 2023 / 11 / 01 / / /

[0072] Establishment of Example 3 ELISA Rapid Detection Method

[0073] 1. Detection Principle and Steps

[0074] First, fix the mouse monoclonal capture antibody on the surface of the ELISA well plate. The F(ab’)2 region of the capture antibody can specifically bind to the target antigen - selenoprotein P. Then, add the saliva samples to be tested (containing selenoprotein P in the samples) and standards to the well plate respectively. After incubation, the selenoprotein P antigen in the samples binds to the F(ab’)2 region of the capture antibody. At the same time, the concentration of selenoprotein P in the standards is also fixed through a similar binding reaction. Next, add the HRP-labeled anti-mouse F(ab’)2 secondary antibody. This labeled secondary antibody can bind to the remaining F(ab’)2 region of the capture antibody. The labeled secondary antibody brings horseradish peroxidase (HRP) by binding to the capture antibody, thus providing signal amplification in subsequent reactions. In the reaction system, HRP catalyzes the substrate reaction of the TMB substrate chromogenic solution. TMB substrate produces a blue soluble substance under the catalysis of HRP. The color depth is proportional to the amount of HRP in the reaction system, and the amount of HRP is inversely proportional to the concentration of selenoprotein P in the sample. Finally, add sulfuric acid to terminate the reaction, and the chromogenic solution turns from blue to yellow. By detecting the absorbance value of the reaction system at 450 nm, the content of selenoprotein P in the sample can be quantitatively determined. The absorbance value is inversely proportional to the concentration of selenoprotein P in the sample. A higher absorbance value represents a lower antigen concentration, and a lower absorbance value represents a higher antigen concentration. This process can achieve accurate quantitative detection of selenoprotein P.

[0075] Using the above ELISA technology, establish a standard curve based on the absorbance values of the selenoprotein P standards, and then calculate the concentration of selenoprotein P in the saliva samples according to the absorbance values generated after incubation of the saliva samples, and obtain the actual content of selenoprotein P in the saliva samples. Through this process, we can accurately calculate the concentration of the target antigen (selenoprotein P) in the samples, ensuring the reliability and accuracy of the detection results.

[0076] The specific steps are as follows:

[0077] Coating with capture antibody: Fix the selenoprotein P monoclonal antibody prepared in Example 2 on the surface of the wells of the ELISA plate.

[0078] Blocking the well plate: Add 5% BSA and block for 2 h.

[0079] Sample treatment: Dilute the saliva or blood samples and add them to the well plate, and make selenoprotein P bind to the capture antibody through incubation.

[0080] Washing and detection: Binding was carried out using HRP-labeled goat anti-mouse F(ab’)2 antibody. After adding the substrate, an enzymatic reaction was performed, and finally, the concentration of selenoprotein P was quantitatively detected by measuring the color change or optical density (OD value).

[0081] The content of selenoprotein P in the sample was calculated through a standard curve, and then the selenium level in the body was predicted.

[0082] 2. Establishment of ELISA detection method for the content of selenoprotein P in saliva

[0083] 1) Collection of saliva samples: The saliva was collected at 9:00 am on the same day. The subjects were asked to avoid drinking alcohol and consuming greasy, spicy, and other foods that may change the pH value of saliva within 12 hours before collection. Before collection, the subjects should brush their teeth carefully, gargle, and clean the oral cavity thoroughly, and wait for at least 10 minutes before saliva collection. Under no stimulating conditions, the tip of the tongue was pressed against the palate to let the saliva flow naturally into the saliva collector. Any behavior that stimulates saliva secretion, such as licking the tongue, was prohibited during the collection process. The collection should be completed within 5 minutes. After collecting the saliva, the whole saliva was centrifuged (4°C, 2000g, 30 minutes). The supernatant saliva was taken, the corresponding volume of protease inhibitor was added, and the protein was quantified by the BCA method and stored at -20°C.

[0084] 2) Coating of capture antibody: The primary antibody was diluted to a concentration of 0.6 μg / ml with ELISA coating buffer (50 mM sodium bicarbonate, pH 9.6), and 100 μL of the diluted antibody was added to each well of the enzyme-linked immunosorbent assay (ELISA) plate and incubated overnight at 4°C.

[0085] 3) Washing: The liquid in the wells was discarded, 350 μL of washing solution PBST (PBS containing 1% Tween) was added to each well, allowed to stand for 30 s, washed 3 times, and patted dry.

[0086] 4) Blocking of the plate: 200 μL of 5% BSA blocking solution (dissolved in PBST) was added to each well and blocked at 37°C for 2 h; Step 3) was repeated.

[0087] 5) Incubation of saliva samples and standard protein: The standard was serially diluted with PBS (pH 7.2) to 0, 1, 5, 10, 20, 40, 60, 80, 100 ng / μL, 100 μL of each dilution was added to each well, and 100 μL of saliva diluent was added to the blank well. Incubate at 37°C for 1.5 h; Step 3) was repeated.

[0088] 6) Incubation of enzyme-labeled antibody: The HRP-enzyme-labeled antibody was diluted at a ratio of 1:20000 with antibody diluent, 100 μL of the diluted enzyme-labeled antibody was added to each well, incubated at 37°C for 1 h, and the TMB was preheated at 37°C in advance; Step 3) was repeated.

[0089] 7) Color reaction: Add 90 μL of TMB solution to each well and incubate at 37 °C in the dark for 15 minutes;

[0090] 8) Termination reaction: Immediately add 50 μL of termination solution (2 mol / L sulfuric acid) to each well to terminate the reaction;

[0091] 9) Absorbance measurement: Measure the absorbance of each well at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader within 5 minutes;

[0092] Calculation of results: Prepare a standard curve and calculate the concentration of selenoprotein P in the sample.

[0093] Example 4 Correlation analysis between salivary selenoprotein P and total selenium content

[0094] 1. Quantitative detection of total selenium content in blood

[0095] To establish the correlation between salivary selenoprotein P and total selenium content, the present invention also uses inductively coupled plasma mass spectrometry (ICP-MS) to quantitatively detect the total selenium content in blood. The specific steps include:

[0096] Pretreat the blood sample to remove cell precipitation and obtain plasma, and ionize the sample using a microwave digestion instrument.

[0097] High-precision determination of the total selenium content in the plasma sample by ICP-MS.

[0098] 2. Correlation analysis between salivary selenoprotein P and total selenium content and establishment of a prediction model

[0099] After obtaining the detection results of the content of selenoprotein P and total selenium content in multiple groups of samples, the present invention establishes a correlation curve between the content of selenoprotein P and total selenium level by statistical methods (regression analysis). This curve can be used to predict the total selenium level in blood based on the content of salivary selenoprotein P. This correlation curve provides a scientific basis for selenium supplementation, can predict individual selenium levels based on the detected content of selenoprotein P, and provides support for personalized selenium supplementation programs. This technology is applicable to fields such as health screening, nutritional assessment, and clinical intervention.

[0100] Advantages: Customized human selenoprotein P monoclonal antibody: The use of a customized human monoclonal antibody ensures high-specificity detection of selenoprotein P and avoids interference from other proteins.

[0101] Dual detection of saliva and blood samples: By combining saliva and blood samples, more diverse and flexible means of selenium level assessment are provided.

[0102] Combined detection of ELISA and ICP-MS: ELISA is used to detect salivary selenoprotein P, and ICP-MS is used to detect the total selenium content. Through the correlation analysis of the two, accurate data support is provided for selenium level prediction.

[0103] Standard curve and personalized selenium supplementation plan: By establishing a correlation curve between selenoprotein P and total selenium content, personalized prediction of selenium level is achieved, and a scientific basis is provided for the personalized management of selenium supplementation.

[0104] 3. Establishment of the correlation curve between salivary selenoprotein P and human plasma selenium content

[0105] The ELISA detection method for salivary selenoprotein P has been established through Example 3. At the same time, in this application, saliva samples and plasma samples of the same subject are collected. The detection of selenoprotein P in saliva samples is shown in Example 3.

[0106] The detection of the total selenium content in plasma samples uses a microwave digestion system to perform high-temperature and high-pressure digestion on the samples, decompose the organic components in the tissues, and convert selenium into a soluble form. After digestion, the sample solution is cooled and diluted to an appropriate volume with 2% dilute nitric acid, and then the selenium content is quantitatively analyzed by inductively coupled plasma mass spectrometry (ICP-MS). The basic steps are as follows:

[0107] 1) Place the microwave digestion reaction vessel, lid, and shrapnel in 10-20% nitric acid and soak overnight, wash and dry for later use;

[0108] 2) Sample pre-acidification: Add 200 μL of saliva sample to each tube, add 8 mL of concentrated nitric acid, and pre-acidify at 120 °C for 20 min;

[0109] 3) Microwave digestion: Tighten the tube cap, load it into the turntable, and turn on the acidification program: Microwave digestion at 140 °C for 5 min and 180 °C for 25 min;

[0110] 4) Sample acidification: In the fume hood, slowly open the lid and acidify at 160 °C until it reaches 0.5-1 mL;

[0111] 5) Volume determination and sample analysis: After acidification in step 4), it is volume-determined to 10 mL, and the selenium content in the sample is quantitatively analyzed by inductively coupled plasma mass spectrometry (ICP-MS).

[0112] Through the above method, the present invention realizes the determination of the content of salivary selenoprotein P and the determination of the total plasma selenium content. Then, based on these experimental data, a linear relationship between the content of salivary selenoprotein P and the total plasma selenium content is established. The specific steps are as follows:

[0113] Data collection: By detecting the saliva samples and plasma samples of different subjects in the experiment, the concentrations of selenoprotein P in the saliva samples and the total selenium in the plasma of 10 subjects were collected and measured.

[0114] Data arrangement: The ELISA test results of saliva selenoprotein P (unit: ng / mL) for each subject were paired with the ICP-MS test results of total selenium in their plasma (unit: μg / L), and organized into a set of comparative data to form a corresponding table of the content of saliva selenoprotein P and the content of total selenium in plasma.

[0115] Table 3: Detection statistics of the content of total selenium in plasma, plasma selenoprotein P, and saliva selenoprotein P of subjects

[0116]

[0117] Statistical analysis: Use statistical software for regression analysis, and select an appropriate regression model (such as a linear regression model) to fit the relationship between the content of saliva selenoprotein P and the content of total selenium in plasma. The specific steps are as follows:

[0118] Taking the concentration of selenoprotein P in plasma as the independent variable (X-axis) and the total selenium content in plasma as the dependent variable (Y-axis), the analysis shows a positive correlation between the two ( Figure 6 ), and a best regression line is fitted by the least squares method, the regression equation is calculated, and the formula for linear regression is obtained: y = 0.0596x - 30.647 (R 2 = 0.9795).

[0119] Taking the concentration of selenoprotein P in saliva as the independent variable (X-axis) and the content of selenoprotein P in plasma as the dependent variable (Y-axis), the analysis shows a positive correlation between the two ( Figure 7 ), and a best regression line is fitted by the least squares method, the regression equation is calculated, and the formula for linear regression is obtained: y = 0.6631x - 142.91 (R 2 = 0.9809)

[0120] Taking the concentration of selenoprotein P in saliva as the independent variable (X-axis) and the total selenium content in plasma as the dependent variable (Y-axis), the analysis shows a positive correlation between the two ( Figure 8 ), and a best regression line is fitted by the least squares method, the regression equation is calculated, and the formula for linear regression is obtained: y = 0.0876x - 14.722 (R 2 = 0.9506)

[0121] Verification of linear relationship: After establishing the linear regression model, the following verification steps are carried out: By randomly sampling the saliva and plasma samples of a subject XXX, after gradient dilution of the samples, the content of selenoprotein P in saliva is detected by ELISA, and the total selenium content in plasma is detected by ICP-MS. According to the detection data, the regression standard curve of the two is established (y = 0.086x - 3.2795R 2 = 0.9909). And taking this as the standard curve, the total plasma selenium content of samples 1 to 12 is predicted. The predicted data is compared with the actual detection data, and the reliability analysis of the predicted data is carried out.

[0122] Establishment of selenium level prediction model:

[0123] The regression model established through the above steps ( Figure 9 ) can be used to predict the selenium level in human plasma. By measuring the content of selenoprotein P (selenoprotein P) in saliva samples and combining with the regression equation, the plasma selenium level of the subject can be accurately predicted (Table 4). This model can provide a basis for the health management of selenium, the assessment of supplementation needs, and the formulation of personalized nutrition plans.

[0124] Table 4: The content of selenoprotein P in saliva of the subjects, the total selenium content in plasma, and the plasma selenium content predicted according to the correlation curve of the two

[0125]

[0126] The prediction result, through the detection of selenoprotein P in saliva, can significantly reflect the blood selenium content level of the human body, and its error rate is between 3% - 15%, and the average error rate is about 8%. This result indicates that by measuring the content of selenoprotein P (selenoprotein P) in saliva samples and combining with the regression equation, the plasma selenium level of the subject can be basically predicted. This model can provide a basis for the health management of selenium, the assessment of supplementation needs, and the formulation of personalized nutrition plans.

[0127] Experimental data verification and model optimization:

[0128] In order to further verify the applicability and accuracy of the established linear relationship, cross-validation can be carried out using samples of different batches to verify the prediction accuracy of the model. If there are large deviations or errors, the prediction ability of the model can be further improved by optimizing the regression model (such as using methods such as non-linear regression and weighted regression).

[0129] The present invention provides a method for detecting selenoprotein P based on saliva samples, and by establishing a correlation model between saliva selenoprotein P and human blood selenium content, the schematic diagram of the principle is as Figure 10As shown, it provides an efficient and non-invasive detection method for selenium health assessment. Through a simple and convenient saliva collection process and using highly sensitive ELISA technology, this method can accurately detect the content of selenoprotein P in saliva. Based on the established blood selenium prediction model between the saliva selenoprotein P content and blood selenium level, it can effectively predict the selenium content in blood, providing a scientific basis for selenium supplementation and health management, and is widely applicable to personal nutritional health management, public health monitoring, and the formulation of personalized selenium nutrition intervention programs. At the same time, this technology has the advantages of simple operation, low cost, and large-scale application potential, with huge market potential and application prospects.

[0130] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A selenoprotein P antigen truncated form, characterized in that: The amino acid sequence of the truncated form is 40-194aa of the sequence shown in SEQ ID NO.

2.

2. A gene encoding the selenoprotein P antigen truncate as claimed in claim 1, wherein the nucleotide sequence thereof is nt 118-582 of the sequence shown in SEQ ID NO.

1.

3. An expression vector, characterized in that: Contains the coding gene as described in claim 2.

4. Use of the selenoprotein P antigen truncate according to claim 1 in the preparation of a selenoprotein P antibody detection reagent.

5. A selenoprotein P antibody, characterized in that: The monoclonal antibody is obtained by immunizing mice with the selenoprotein P antigen truncation as claimed in claim 1.

6. An ELISA kit for detecting selenoprotein P, characterized in that: The kit comprises a selenoprotein P antibody obtained using the selenoprotein P antigen truncation as described in claim 1.

7. An ELISA kit for detecting selenoprotein P according to claim 6, characterized in that: The kit also includes ELISA coating buffer, washing solution PBST, 5% BSA blocking solution, PBS diluent, TMB solution and stop solution.

8. A non-invasive method for predicting selenium levels based on salivary selenoprotein P detection, characterized in that: The ELISA kit described in claim 6 is used to detect the selenoprotein P content in saliva, and the selenium level is predicted by referring to the correlation curve between salivary selenoprotein P and plasma total selenium content.

9. The non-invasive selenium level prediction method based on salivary selenoprotein P detection according to claim 8, characterized in that: The correlation curve between salivary selenoprotein P content and plasma selenoprotein P content, and the correlation curve between plasma selenoprotein P content and plasma total selenium content were established in sequence to construct the correlation curve between salivary selenoprotein P and plasma total selenium content.

10. Use of the non-invasive selenium level prediction method based on salivary selenoprotein P detection as claimed in claim 8 or 9 in the assessment of selenium level in an organism.

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