Sample diluent, sample preparation method, and rheumatoid arthritis test kit

Abstract

The present application relates to the technical field of genetic testing, and discloses a sample diluent, a sample preparation method, and a rheumatoid arthritis test kit. The sample diluent comprises the following concentrations of components: 5-40 g / L of an alkali metal salt, 1-20 g / L of a protein protectant, 5-50 mM of an alkyl glycoside surfactant, 0.01-0.1% of a Tween surfactant, 1-100 μg / mL of an immunoadsorbent, and 10-200 mM of a buffer, wherein the pH value of the buffer ranges from 6.5 to 8.0. The present application provides an actual basis for synovial fluid testing in clinical rheumatoid arthritis, and takes into account a small sample size, such as testing of a site where synovial fluid is difficult to aspirate. In addition, by using an iFlash series instrument, a result can be obtained in as little as 10 min. Compared with the operating time of at least 1 hour of conventional ELISA, the test process is greatly accelerated, and high sensitivity and specificity are achieved.

Description

Sample diluent, sample preparation method and rheumatoid arthritis detection kit TECHNICAL FIELD

[0001] The application belongs to the technical field of gene detection, and particularly relates to a sample diluent, a sample preparation method and a rheumatoid arthritis detection kit. BACKGROUND

[0002] Synovial fluid, also known as synovial fluid, is a hyaluronidase protein complex mixed with plasma exudate produced in the joint cavity. Under normal circumstances, the synovial fluid is cool, viscous, colorless and transparent or light yellow, and mainly plays a role in lubricating the joint, reducing joint vibration and synovial inflammation. In addition, the synovial fluid can provide nutrition for the articular cartilage. When the joint is inflamed due to trauma or infection, the synovial fluid will increase due to the change of vascular permeability. In clinical practice, the detection of the transparency, viscosity and clot formation of the synovial fluid can provide a basis for the diagnosis and treatment of arthritis.

[0003] Rheumatoid arthritis (RA) is a systemic disease mainly caused by inflammatory synovitis, which often involves organs other than joints and can lead to joint deformity and loss of function. At present, the diagnosis of RA is mainly based on the questionnaire and serological diagnosis, which is prone to errors. In the early stage of the disease, the pathological state of the patient cannot be accurately determined. Therefore, developing a more sensitive detection method can help accurately diagnose rheumatoid arthritis.

[0004] At present, there is no in vitro diagnostic reagent for synovial fluid. In the traditional technology, the diagnosis of rheumatoid arthritis patients mostly relies on the content of related autoimmune antibodies in the blood, which has certain limitations and cannot reflect the true situation of the joint, and the accuracy is poor.

[0005] SUMMARY

[0006] Therefore, an embodiment of the application provides a sample diluent for synovial fluid samples. The sample diluent can be used to dilute the synovial fluid sample to be tested, and the content of sCD146 in the synovial fluid can be accurately measured, thereby improving the accuracy of the diagnosis of rheumatoid arthritis patients.

[0007] One or more embodiments of the application provide a sample diluent, which comprises the following components in the following concentrations:

[0008] 5g / L-40g / L alkali metal salt, 1g / L-20g / L protein protective agent, 5mM-50mM alkyl glycoside surfactant, 0.01%-0.1% Tween surfactant, 1ug / mL-100ug / mL immunosorbent and 10mM-200mM buffer, and the pH value is 6.5-8.0.

[0009] In one embodiment, the sample diluent includes the following concentrations of each component:

[0010] 8g / L-10g / L alkali metal salt, 4g / L-6g / L protein protective agent, 18g / L-22g / L alkyl polyglycoside surfactant, 0.04%-0.06% Tween surfactant, 5μg / mL-20μg / mL immunoadsorbent, and 40mM-60mM buffer, pH 6.8-7.2.

[0011] In one embodiment, the alkali metal salt includes one or more of NaCl, KCl, and MgCl2.

[0012] In one embodiment, the protein protective agent includes one or more of BSA, fish gelatin, hydrolyzed casein, human serum albumin, glycine, arginine, and polylysine.

[0013] In one embodiment, the alkyl polyglycoside surfactant includes one or more of APG0810, APG1214, APG0814, APG0816, and APG1216.

[0014] Optionally, the Tween surfactant includes one or more of Tween-80, Tween-40, and Tween-20.

[0015] In one embodiment, the immunoadsorbent includes one or more of goat anti-human IgG antibody, mouse anti-human IgG antibody, and rabbit anti-human IgG antibody.

[0016] Optionally, the buffer includes one or more of Tris buffer, PBS buffer, MES buffer, and HEPES buffer.

[0017] In one embodiment, the sample diluent further includes a preservative.

[0018] Optionally, the preservative includes one or more of Proclin 300, sodium azide, sodium benzoate, and potassium sorbate.

[0019] One or more embodiments of the present application provide a method for preparing a sample for detection, comprising:

[0020] Mixing synovial fluid with the sample diluent described above to prepare a sample for detection of rheumatoid arthritis.

[0021] The volume ratio of the synovial fluid to the sample diluent is 1:(3-5).

[0022] A rheumatoid arthritis detection kit, the kit comprising the sample diluent as described above.

[0023] In one of the embodiments, the kit further comprises one or more of a magnetic bead component, an acridinium component, and a SA component;

[0024] The magnetic bead component comprises magnetic beads coated with a first antibody specifically recognizing sCD146;

[0025] The acridinium component comprises acridinium-labeled bovine serum albumin and biotin;

[0026] The SA component comprises a second antibody specifically recognizing sCD146 labeled with streptavidin;

[0027] Optionally, the magnetic bead component, the acridinium component, and the SA component each comprises a diluent;

[0028] Optionally, the diluent comprises a 10 mM-100 mM Tris buffer pair, 1 g / L-50 g / L NaCl, 1 g / L-50 g / L BSA, 0.1 %-5% Tween-20, and 0.01 %-0.5% Proclin 300 in water. BRIEF DESCRIPTION OF DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the present application, more completely understand the present application and its beneficial effects, the drawings needed to be used in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some of the embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative effort based on these drawings.

[0030] Figure 1 is a ROC curve for distinguishing normal joint fluid from rheumatoid arthritis patient joint fluid for clinical application;

[0031] Sensitivity is sensitivity; Specificity is specificity; Associated criterion is associated criterion. DETAILED DESCRIPTION

[0032] The application will be described in further detail below with reference to the embodiments and examples. It should be understood that these embodiments and examples are only used to explain the present application and not intended to limit the scope of the present application, and the purpose of providing these embodiments and examples is to make the disclosure of the present application more thoroughly and comprehensively understood. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein, and those skilled in the art can make various modifications or changes without departing from the spirit of the present application, and the equivalent forms obtained thereby also fall within the protection scope of the present application. In addition, in the following description, a large number of specific details are given in order to provide a more complete understanding of the present application, and it should be understood that the present application can be implemented without one or more of these details.

[0033] Unless otherwise defined, 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 application belongs.

[0034] The term

[0035] All documents referred to in this application are incorporated by reference in this application as if each document were individually incorporated by reference. Unless otherwise conflicting or inappropriate, the disclosures in the documents referred to in this application are incorporated by reference in their entirety. When the present application refers to the documents, the definitions of the relevant technical features, terms, names, phrases, etc. in the documents are also incorporated by reference. When the present application refers to the documents, the examples and preferred modes of the relevant technical features are also incorporated by reference into the present application, but are limited to the implementation of the present application. It should be understood that when the content of the reference conflicts with the description in the present application, the present application is used as the reference or is modified according to the description in the present application.

[0036] In the present application, the technical features described in an open manner include both the closed technical solutions consisting of the listed features and the open technical solutions containing the listed features.

[0037] The term "and / or" or "and / or" means any and all combinations of one or more relevant listed items.

[0038] The term "synovial fluid" is a liquid formed by the filtration of plasma in the synovial capillary, and can also be a liquid formed by the secretion of hyaluronic acid, collagen and proteoglycan by synovial cells. The amount of synovial fluid in a normal joint is small, and its function is to help lubricate the joint and nourish the joint cartilage, intra-articular ligaments and other tissues. In addition, synovial cells have phagocytic function and can remove debris or foreign bodies in the joint, and synovial fluid can also remove metabolic products through lymphatic fluid or blood.

[0039] When the joint produces pathological changes or certain systemic diseases, the joint fluid increases, i.e. arthrohydrops is formed, causing joint pain and discomfort. The normal amount of joint fluid in the knee joint is only a small amount, which is used to nourish the articular cartilage, lubricate the joint, and reduce friction during joint movement. The synovial fluid is secreted by the synovial membrane, and the synovial fluid is constantly updated during joint movement. When the joint produces pathological changes or certain systemic diseases, arthrohydrops can be formed.

[0040] Soluble CD146 is CD146 protein cut by metalloproteinase or directly secreted into body fluid, containing 552 amino acids. sCD146 in serum is related to various diseases, including inflammation, cancer. The level of this marker is closely related to the occurrence of inflammation.

[0041] In addition to the correlation between sCD146 in blood and inflammation, studies have found that the content of sCD146 in the synovial fluid of RA patients increases significantly, and the increased level of sCD146 is higher in the early stage of the disease, which can be used as an early diagnostic tool.

[0042] In actual practice, the synovial fluid often contains a small amount, which is difficult to extract, and cannot be directly used for detection, so it is necessary to supplement the volume of the sample to be tested, which increases the requirement for the sensitivity of the detection reagent; The synovial fluid contains a large amount of hyaluronic acid and mucin, which causes the viscosity of the synovial fluid to be very large, and direct testing is not possible, and the interference of rheumatoid factor antibody in the synovial fluid of rheumatoid arthritis patients needs to be excluded.

[0043] At the same time, in the synovial fluid of rheumatoid arthritis patients, turbidity and cell proliferation often occur due to inflammation, and the protein content is also higher than that of normal people, which has a greater impact on direct measurement of synovial fluid. Therefore, a special diluent needs to be developed for this sample type to ensure accurate measurement of the test substance.

[0044] The present application provides a sample diluent, which comprises the following components in the following concentrations:

[0045] 5g / L-40g / L alkali metal salt, 1g / L-20g / L protein protective agent, 5mM-50mM alkyl glycoside surfactant, 0.01%-0.1% Tween surfactant, 1μg / mL-100μg / mL immunosorbent, and 10mM-200mM buffer, pH 6.5-8.0.

[0046] In a specific example, the sample diluent comprises the following components in the following concentrations:

[0047] 8g / L~10g / L alkali metal salt, 4g / L~6g / L protein protective agent, 18g / L~22g / L alkyl polyglucoside surfactant, 0.04%~0.06% Tween surfactant, 5μg / mL~20μg / mL immunoadsorbent, and 40mM~60mM buffer, pH 6.8~7.2.

[0048] Optionally, the alkali metal salt includes one or more of NaCl, KCl, and MgCl2.

[0049] In one specific example, the protein protective agent includes one or more of BSA, fish gelatin, hydrolyzed casein, human serum albumin, glycine, arginine, and polylysine. There are many types of protein protective agents, including mainly sugars, polyols, amino acids and their derivatives, inorganic salts, glycerol, polymers, clear albumin, antioxidants, metal chelators, surfactants, and protease inhibitors, etc. There are different theories about the mechanism of action of protein protective agents, mainly including water molecule repulsion theory, preferential solvent binding theory, and hydration layer theory, etc. Protein protective agents refer to compounds or biological agents that can protect proteins from denaturation, degradation, and damage.

[0050] Optionally, the alkyl polyglucoside surfactant includes but is not limited to one or more of APG0810, APG1214, APG0814, APG0816, and APG1216. Alkyl polyglucoside refers to alkyl polyglucoside (APG for short) synthesized from glucose and fatty alcohol, which refers to a glycoside with more than or equal to 2 sugar units in a complex glycoside compound, collectively referred to as alkyl polyglucoside (or alkyl polyglycoside).

[0051] Generally, the degree of polymerization n of alkyl polyglycoside is in the range of 1.1~3, and R is C8~C16 alkyl. APG is a white solid powder or light yellow oily liquid at room temperature, has high solubility in water, and is relatively difficult to dissolve in commonly used organic solvents.

[0052] Compared with other surfactants, alkyl polyglucoside has good compatibility, low skin irritation, low toxicity, and good biodegradability. Alkyl polyglucoside is synthesized from starch as the main raw material. Not only the cost is low, but also there is no pollution, which meets the requirements of modern environmental protection.

[0053] Optionally, the Tween surfactant comprises one or more of Tween-80, Tween-40 and Tween-20. Since Tween is an ester of sorbitol with different higher fatty acids, Tween is actually a series of products of the same type, such as Tween-20, Tween-40, Tween-60, Tween-80. Among them, Tween-60 is stearate; Tween-80 is oleate; Tween-20 is laurate, which is a mixture of polyoxyethylene sorbitan monolaurate and part of polyoxyethylene sorbitan monolaurate.

[0054] In one specific example, the immunoadsorbent comprises one or more of goat anti-human IgG antibody, mouse anti-human IgG antibody and rabbit anti-human IgG antibody.

[0055] Optionally, the sample diluent further comprises a preservative.

[0056] Further optionally, the preservative comprises one or more of Proclin 300, sodium azide, sodium benzoate and potassium sorbate.

[0057] In one specific example, the present application also provides a sample preparation method, wherein the sample comprises synovial fluid; the volume ratio of the synovial fluid to the sample diluent is 1:(3-5). Diluting the synovial fluid to be tested with the sample diluent can accurately measure sCD146 in the synovial fluid, and can also directly use a fully automated instrument to measure the marker of the synovial fluid. It is also suitable for cases where the synovial fluid of some patients is difficult to extract and the amount of synovial fluid obtained is small.

[0058] The present application also provides a rheumatoid arthritis detection kit, characterized in that the kit comprises the sample diluent.

[0059] In one specific example, the kit further comprises a magnetic bead component, an acridine component and an SA component; the magnetic bead component comprises magnetic beads coated with a first antibody specifically recognizing sCD146; the acridine component comprises bovine serum albumin labeled with acridinium ester and biotin; and the SA component comprises a second antibody of sCD146 labeled with streptavidin.

[0060] The present application provides an actual basis for testing synovial fluid for clinical rheumatoid arthritis, and takes into account the case of small sample amount (such as determination of joints where synovial fluid is difficult to extract). At the same time, combined with the iFlash series of instruments, the result can be obtained in 10 minutes at the fastest, which is at least 1 hour faster than the operation time of traditional ELISA, and has high sensitivity and specificity.

[0061] The embodiments of the present application will be described in detail below with examples. It should be understood that these examples are only used to illustrate the present application and not used to limit the scope of the present application. The experimental methods in the following examples without specific conditions are preferred to refer to the instructions given in the present application, and can also be performed according to the experimental manuals or conventional conditions in the art, or according to the conditions suggested by the manufacturers, or according to the experimental methods known in the art.

[0062] In the following specific examples, the measurement parameters of the raw material components may have slight deviations within the weighing accuracy range if not otherwise specified. The temperature and time parameters allow for acceptable deviations caused by the instrument testing accuracy or the operation accuracy.

[0063] It should be understood that the size of the sequence number of each process described above in various embodiments of the present application does not mean the order of execution, and the execution order of each process should be determined according to its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0064] Example 1

[0065] The synovial fluid of rheumatoid arthritis patients and other types of arthritis patients will increase in cell content and protein content due to factors such as increased white blood cells in the joint cavity and infection, and the synovial fluid will become mixed. At the same time, because the synovial fluid often contains a large amount of hyaluronic acid, the synovial fluid of some patients is difficult to extract, and the amount of synovial fluid obtained is small, etc. Therefore, it is not possible to directly use full-automatic instruments to measure the markers in the synovial fluid.

[0066] I. Detection steps

[0067] (1) After the synovial fluid is extracted, 50 μL is added to 200 μL of sample diluent, mixed, centrifuged to obtain the supernatant, and then the iFlash series instrument can be used for testing.

[0068] The sample diluent includes 9 g / L NaCl, 5 g / L BSA, 20 mM APG0810, 0.05% Tween-80, 0.1% Proclin 300, 10 μg / mL goat anti-human IgG antibody, and 50 mM Tris buffer.

[0069] (2) The detection of the sample is performed by one-step reaction. After dilution, the sample is mixed with R1 magnetic bead component, R2 acridinium ester and R3 SA component in the instrument to form a magnetic bead antibody complex-antigen-antibody-streptavidin complex-biotin-acridinium ester complex immunocomplex. After sufficient reaction, the instrument performs the steps of cleaning and luminescence.

[0070] 1) R1 magnetic bead component: buffer solution (pH 7.0) containing magnetic beads coated with a first antibody specifically recognizing sCD146, the buffer solution containing diluent, mainly an aqueous solution containing a buffer pair (10-100 mM Tris), a metal salt (1-50 g / L NaCl), a protein protective agent (1-50 g / L BSA), a surfactant (0.1%-5% Tween-20), and a preservative (0.01-0.5% Proclin 300).

[0071] 2) R2 acridinium ester component: bovine serum albumin labeled with acridinium ester and biotin and the corresponding buffer solution (pH 7.0), the buffer solution containing diluent, mainly an aqueous solution containing a buffer pair (10-100 mM Tris), a metal salt (1-50 g / L NaCl), a protein protective agent (1-50 g / L BSA), a surfactant (0.1%-5% Tween-20), and a preservative (0.01-0.5% Proclin 300).

[0072] 3) R3 SA component: sCD146-specific second antibody labeled with streptavidin and the corresponding buffer solution (pH 7.0), the buffer solution containing diluent, mainly an aqueous solution containing a buffer pair (10-100 mM Tris), a metal salt (1-50 g / L NaCl), a protein protective agent (1-50 g / L BSA), a surfactant (0.1%-5% Tween-20), and a preservative (0.01-0.5% Proclin 300).

[0073] The present application selects the following clinically representative samples: 3 samples of ordinary people, 3 samples of rheumatoid arthritis, and 3 samples of joint fluid caused by infection.

[0074] The average concentration and the percentage deviation of the standard concentration are important parameters for evaluating the accuracy of concentration measurement. It can reflect the reliability and accuracy of the measurement results. When the percentage deviation is small (deviation less than or equal to ±10%), the measurement result is close to the theoretical result, and the measurement accuracy is high. On the contrary, when the percentage deviation is large (deviation greater than ±10%), there may be measurement errors or uncertainties, which need to be further analyzed and improved.

[0075] The present application finds that when physiological saline is used to dilute the sample, the correlation between the measured concentration of the sample and the dilution ratio is maintained at 0.9 or higher only when the dilution ratio is greater than 20 times. Therefore, we use physiological saline to dilute the sample by 20 times, 30 times, 40 times, and 50 times. The back-calculated concentration is obtained by multiplying the measured concentration by the dilution ratio. The average of the back-calculated concentrations of the samples at each dilution ratio is taken as the theoretical concentration of the sample.

[0076] The detection results of the optimal diluent for diluting the sample are shown in Table 1. When the dilution ratio is less than or equal to 5, the measured concentration of each sample deviates from the theoretical concentration by less than or equal to 10%, which is considered to be able to dilute the sample well and will not have a significant impact on the measured value.

[0077] Table 1

[0078] Comparative Example 1

[0079] The sample diluent includes 9 g / L NaCl, 5 g / L BSA, 20 mM APG0810, goat anti-human IgG antibody, and 50 mM Tris buffer. Compared with Example 1, the sample diluent of Comparative Example 1 does not contain Tween-80, and the remaining steps are the same as those of Example 1.

[0080] The detection results of Comparative Example 1 as the diluent are shown in Table 2. When the dilution ratio is less than or equal to 5, the measured concentration of each sample deviates from the theoretical concentration by less than or equal to 10% after removing Tween-80, which is considered to be able to dilute the sample well and will not have a significant impact on the measured value.

[0081] Table 2

[0082] However, when testing more samples that are relatively viscous in nature (n=50), it is found that some samples are still relatively viscous when diluted with the diluent without Tween-80, which poses a certain risk. After adding an additional surfactant, the flowability of the sample is enhanced, as shown in Table 3.

[0083] Table 3

[0084] Comparative Example 2

[0085] The sample diluent includes 9 g / L NaCl, 5 g / L BSA, goat anti-human IgG antibody, and 50 mM Tris buffer. Comparative Example 2 does not contain APG0810 and Tween-80 in the sample diluent relative to Example 1, and the remaining steps are the same as Example 1.

[0086] The detection results of Comparative Example 2 as a diluent are shown in Table 4, and the sample is diluted using a surfactant without diluent, and the dilution effect is similar to that of physiological saline. When the dilution ratio is small, the measured concentration after dilution is much lower than the theoretical concentration.

[0087] Table 4

[0088] Comparative Example 3

[0089] TBST is used as the sample diluent, and the remaining steps are the same as Example 1. The specific detection results are shown in Table 5:

[0090] Table 5

[0091] Comparative Example 4

[0092] PBST is used as the sample diluent, and the remaining steps are the same as Example 1. The specific detection results are shown in Table 6:

[0093] Table 6

[0094] As shown in Comparative Examples 3 and 4, when TBST or PBST is used as the diluent, the measured value increases when the dilution ratio is large. It is subsequently proven that the high concentration of Tween-20 in TBST causes partial lysis of cells, bacteria, viruses, etc., resulting in an increase in the content of sCD146 in the sample. Reducing the concentration of Tween-20 in TBST reduces the lysis ability, but the sample becomes sticky.

[0095] Comparative Example 5

[0096] Triton-X100 (5 g / L) and Tween-80 (0.05%) are used for compounding, and the remaining components are the same as Example 1. The specific detection results are shown in Table 7.

[0097] Table 7

[0098] From the above data, it can be seen that after replacing APG0810 with other surfactants, similar situations as TBST and PBST may occur due to the increased lysis ability of the solution, and the measured values of some samples increase after dilution.

[0099] Comparative Example 6

[0100] Using physiological saline as the sample diluent, the remaining steps are the same as Example 1, and the specific test results are shown in Table 8:

[0101] Table 8

[0102] From the above data, it can be seen that when only physiological saline is used for dilution, only when the dilution ratio is large, the concentration of the diluted sample is close to the theoretical concentration, but at this time the concentration of the diluted sample is too low, and it is difficult to measure some low value samples.

[0103] When PBST or TBST is used as the diluent, with the increase of the dilution ratio, individual samples show a high measured value, which is mainly due to the lysis of cells in the synovial fluid with the increase of the dilution ratio, resulting in the release of sCD146 on the cells into the sample, which leads to the phenomenon of measured value increase.

[0104] Therefore, it is necessary to select some surfactants with slightly weaker lysis ability. When APG0810 is selected, it can be seen that various samples do not show a large deviation after dilution, but according to observation, the viscosity of some samples is still high when the dilution ratio is low, and direct use of the instrument will cause failure. When an additional surfactant is added to the diluent, the viscosity of the sample decreases, and the activity of APG0810 may be enhanced due to the additional surfactant, which further increases the solubility of proteins in the sample. At low dilution multiples, the instrument can be directly used for testing.

[0105] At the same time, according to the data shown above, using a surfactant and two surfactants in combination can effectively dilute the sample, and the measured value will not increase due to cell lysis.

[0106] By the above comparison, the synovial fluid is only diluted with physiological saline, and the content of sCD146 in the synovial fluid cannot be accurately measured. With the increase of the amount of physiological saline, the content of sCD146 decreases disproportionately, but the use of surfactant buffer containing a conventional concentration (such as TBST, PBST, etc.) can cause certain cell lysis, resulting in a higher test value of some turbid samples. However, the use of surfactants with low lysis ability cannot guarantee that the sample can obtain good dilution effect at low dilution multiple. Therefore, the present application develops a diluent that can ensure the accurate measurement of sCD146 in the synovial fluid according to different synovial fluid conditions.

[0107] Comparative Example 7

[0108] In order to confirm whether the rheumatoid antibody in the synovial fluid of the rheumatoid arthritis patient will affect the detection reagent, the present application selects two different sample diluents, one of which is the basic sample diluent, and the other is added with goat anti-human IgG antibody.

[0109] The diluent of Comparative Example 7 includes 9 g / L NaCl, 5 g / L BSA, 20 mM APG0810, 0.05% Tween-80, and 50 mM Tris buffer, and does not contain immunosuppressants compared with Example 1.

[0110] The obtained 20 synovial fluid samples of the control group and 20 synovial fluid samples of the rheumatoid arthritis patients are mixed with the diluent, and the state of the diluted sample is observed and recorded. It is found that the diluted sample added with goat anti-human IgG antibody is turbid, which indicates that the goat anti-human IgG antibody in the sample reacts with the human IgG antibody in the sample, and can eliminate the interference of part of the human IgG, as shown in Table 9:

[0111] Table 9

[0112] The above samples are detected, and the diluent added with goat anti-human IgG antibody is obviously different from the test results of the diluent without addition, and the specific results are shown in Table 10:

[0113] Table 10

[0114] From the detection results, it can be seen that after adding the goat anti-human IgG antibody, the change of the sCD146 concentration test deviation of the control group is not large, but the test concentration of most rheumatoid arthritis samples is greatly improved, which shows that after adding the goat anti-human IgG antibody, the possible interference can be eliminated.

[0115] The present application also selects other adsorbents such as mouse anti-human IgG antibody, rabbit anti-human IgG antibody, etc., which can achieve similar levels.

[0116] II. Effect verification

[0117] Further, the application also verifies the repeatability of the sample diluent test results.

[0118] In order to test the accuracy of the detection reagent in detecting synovial fluid, the application uses 5 cases of control population and 5 cases of RA population to test the synovial fluid samples, each sample is measured 10 times, and the coefficient of variation (CV) of each sample test result is calculated, and the specific results are shown in Table 11:

[0119] Table 11

[0120] From the above data, it can be seen that when different samples are diluted and tested using the sample diluent of the application, the coefficient of variation of each sample test concentration is within 2%, and the detection reagent has good repeatability in test results.

[0121] The application further verifies the linearity of the sample diluent.

[0122] In order to verify the relationship between the measured concentration and the sample dilution ratio of the detection reagent when testing the synovial fluid, the application uses the sample diluent to pre-dilute different synovial fluid samples according to the sample:diluent ratio of 8:2, 6:4, 4:6, 8:2, and 9:1, and then measures the samples according to the measurement procedure. Compare the deviation between the concentration of the diluted sample and the theoretical concentration of the diluted sample.

[0123] The specific verification is shown in Table 12, and the concentration unit is ng / mL:

[0124] Table 12

[0125] The application also explores the reference interval of synovial fluid

[0126] In order to determine the difference between the sample diluent and the synovial fluid of normal people and rheumatoid arthritis patients in clinical application, 128 cases of rheumatoid arthritis patient synovial fluid samples and 103 cases of control samples are used for testing, and the ROC curve is drawn, and the test data of the curve is shown in Table 13, and the ROC curve is shown in Figure 1.

[0127] Table 13

[0128] As can be seen from Figure 1, when the measured concentration of sCD146 is greater than 12.84 ng / mL, the specificity of diagnosing rheumatoid arthritis patients can reach 98.6%, and the sensitivity can reach more than 93.75% when using the sample diluent of the application to diagnose rheumatoid arthritis patients.

[0129] The above-described embodiments only express several implementation manners of the present application, facilitate specific and detailed understanding of the technical solutions of the present application, but cannot be understood as a limitation on the patent protection scope of the present application. It should be noted that, for ordinary skilled persons in the art, several modifications and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. In addition, it should be understood that, after reading the above teaching content of the present application, the skilled person in the art can make various modifications or modifications to the present application, and the equivalent forms obtained are also within the protection scope of the present application. It should also be understood that, on the basis of the technical solutions provided by the present application, the skilled person in the art obtains the technical solutions through logical analysis, reasoning or limited experiments, and all of them are within the protection scope of the appended claims of the present application. Therefore, the protection scope of the patent of the present application should be subject to the content of the appended claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. A sample diluent, characterized in that, The sample diluent comprises the following components at the following concentrations: 5g / L-40g / L alkali metal salt, 1g / L-20g / L protein protective agent, 5mM-50mM alkyl glycoside surfactant, 0.01%-0.1% Tween surfactant, 1μg / mL-100μg / mL immunoadsorbent, and 10mM-200mM buffer, with a pH of 6.5-8.

0.

2. The sample diluent of claim 1, wherein, The sample diluent comprises the following components at the following concentrations: 8g / L-10g / L alkali metal salt, 4g / L-6g / L protein protective agent, 18g / L-22g / L alkyl glycoside surfactant, 0.04%-0.06% Tween surfactant, 5μg / mL-20μg / mL immunoadsorbent, and 40mM-60mM buffer, with a pH of 6.8-7.

2.

3. The sample diluent of claim 1, wherein, The alkali metal salt comprises one or more of NaCl, KCl, and MgCl2.

4. The sample diluent of claim 1, wherein, The protein protective agent comprises one or more of BSA, fish skin gelatin, hydrolyzed casein, human serum albumin, glycine, arginine, and polylysine.

5. The sample diluent of any one of claims 1-4, wherein, The alkyl glycoside surfactant comprises one or more of APG0810, APG1214, APG0814, APG0816, and APG1216. Optionally, the Tween surfactant comprises one or more of Tween-80, Tween-40, and Tween-20.

6. The sample diluent of any one of claims 1-4, wherein, The immunoadsorbent comprises one or more of goat anti-human IgG antibody, mouse anti-human IgG antibody, and rabbit anti-human IgG antibody. Optionally, the buffer comprises one or more of Tris buffer, PBS buffer, MES buffer, and HEPES buffer.

7. The sample diluent of any one of claims 1 to 4, wherein, The sample diluent further comprises a preservative. Optionally, the preservative comprises one or more of Proclin 300, sodium azide, sodium benzoate, and potassium sorbate.

8. A method of preparing a sample for detection, characterized by, The sample diluent comprises the following components at the following concentrations: The sample diluent comprises the following components at the following concentrations: The sample diluent comprises the following components at the following concentrations:

9. A rheumatoid arthritis test kit, characterized by, The sample diluent comprises the following components at the following concentrations:

10. The rheumatoid arthritis detection kit according to claim 9, characterized by, The sample diluent comprises the following components at the following concentrations: The sample diluent comprises the following components at the following concentrations: The sample diluent comprises the following components at the following concentrations: The sample diluent comprises the following components at the following concentrations: The sample diluent comprises the following components at the following concentrations: The sample diluent comprises the following components at the following concentrations: The sample diluent comprises the following components at the following concentrations: The sample diluent comprises the following components at the following concentrations: The sample diluent comprises the following components at the following concentrations: The sample diluent comprises the following components at the following concentrations: The sample diluent comprises the following 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