Immunochromatographic test strip for quantitative detection of CRP, preparation method thereof and coating buffer

Abstract

This application discloses a coating buffer for an immunochromatographic test strip for quantitative detection of CRP, comprising a buffer, a saccharide, and alcohol-based reduced glutathione. This application also provides a method for preparing an immunochromatographic test strip for quantitative detection of CRP and an immunochromatographic test strip for quantitative detection of CRP. The coating buffer and test strip provided herein can improve the sensitivity of C-reactive protein detection and reduce interference, resulting in enhanced precision, better product performance, and improved control of batch variability.

Description

Technical Field

[0001] The present application relates to the technical field of detection reagents, and in particular to an immunochromatographic test strip for quantitatively detecting CRP, a preparation method thereof, and a coating buffer. Background Art

[0002] C-reactive protein (CRP) is a protein (acute protein) that rises sharply in plasma when the body is infected or tissue is damaged. It activates complement and enhances phagocytosis by phagocytes, playing a role in regulating the clearance of invading pathogens and damaged, necrotic, and apoptotic tissue cells. CRP is a pentamer composed of five identical, non-glycosylated subunits, non-covalently linked. Within each subunit, a disulfide bond is formed between cysteines 36 and 97. Plasma CRP concentrations are extremely low in normal individuals. However, they rise significantly during cellular infection and tissue damage, with both a rapid rise and fall. It is one of the preferred indicators for assessing the extent of tissue damage in acute inflammation and the effectiveness of treatment, and is widely used in the differential diagnosis of clinical diseases.

[0003] Fluorescent immunochromatography is a quantitative analysis method developed in the late 20th century that combines immunochromatography and fluorescence detection technology. It has the characteristics of simple operation, rapidity, and low requirements for laboratory environment, and is suitable for some application scenarios where test results need to be obtained quickly. However, compared with new detection technologies such as chemiluminescence and molecular diagnosis, fluorescent immunochromatography has the problems of poor sensitivity and difficulty in quantification, especially for the detection of substances such as C-reactive protein, whose concentration changes very drastically. The sensitivity and accuracy of the detection have a great impact on the judgment of the results. Therefore, how to improve the sensitivity and accuracy of C-reactive protein immunochromatographic test strips is a problem that needs to be solved urgently by those skilled in the art. Summary of the Invention

[0004] To address the above technical problems, the first objective of the present invention is to provide a coating buffer for an immunochromatographic test strip for quantitatively detecting CRP; the second objective of the present invention is to provide a method for preparing an immunochromatographic test strip for quantitatively detecting CRP; and the third objective of the present invention is to provide an immunochromatographic test strip for quantitatively detecting CRP. The coating buffer and test strip provided herein can improve the detection sensitivity of CRP and reduce interference, thereby enhancing precision, improving product performance, and facilitating control of batch variation.

[0005] The technical solutions provided by the present invention are as follows:

[0006] A coating buffer for an immunochromatographic test strip for quantitatively detecting CRP comprises a buffer, sugars, and alcohol-reduced glutathione.

[0007] Preferably, the mass percentage concentration of reduced glutathione is 0.05-0.5%.

[0008] Preferably, the buffer concentration is 0.01-0.1 mol / L;

[0009] The mass percentage concentration of sugars is 0.5-5%;

[0010] The mass percentage concentration of alcohols is 0.1-2%.

[0011] Preferably, the buffer is any one of tris-hydroxyaminomethane buffer, 4-hydroxyethylpiperazineethanesulfonic acid, phosphate buffer, and borate buffer;

[0012] The sugar is any one or more of trehalose, sucrose, lactose, and dextran;

[0013] The alcohol is any one or more of isopropanol, methanol, and ethanol.

[0014] Preferably, the invention further comprises a preservative at a concentration of 0.01-0.1% by mass. Preferably, the preservative is any one or more of sodium azide, PC300, potassium sorbate, and Krovin300M.

[0015] A method for preparing an immunochromatographic test strip for quantitatively detecting CRP, wherein the test strip is formed by coating a CRP-specific antibody with any of the coating buffers described above.

[0016] Preferably, the steps include:

[0017] S1. Apply CRP-specific antibody A coupled to fluorescent microspheres and quality control antibody A coupled to fluorescent microspheres to the conjugate pad;

[0018] S2. Coating the CRP-specific antibody B on the reaction membrane using any of the coating buffers described above to form a detection zone;

[0019] Coat the quality control antibody B on the reaction membrane to form a quality control band;

[0020] S3, assembling the sample pad, the conjugate pad prepared in step S1, the reaction membrane prepared in step S2, and the absorbent pad onto the bottom plate to obtain a test strip;

[0021] There is no order restriction for steps S1 and S2.

[0022] Preferably, in step S2, the CRP-specific antibody B is diluted to 0.5-1.5 mg / L using the coating buffer according to any one of claims 1-6, coated on the reaction membrane, and then dried at 50-60° C. for 65-80 h.

[0023] An immunochromatographic test strip for quantitative detection of CRP prepared by any of the preparation methods described above.

[0024] C-reactive protein (CRP) exists in the human body as a pentamer and needs to be reduced to a monomer before it can be detected. This application improves the sensitivity of CRP detection by adding reduced glutathione to the coating buffer of the immunochromatographic test strip used for quantitative detection of CRP. The specific reasons are as follows:

[0025] First, inflammatory samples often contain higher concentrations of hydrogen peroxide. Reduced glutathione can offset some of the oxidative substances in the sample, preventing them from affecting the specific antigen-antibody binding, and helping to reduce the interference caused by high CRP samples.

[0026] Second, CRP exists in the human body in the form of pentamers, and the CRP produced in the human body needs to be reduced to monomers before it can be detected. Reduced glutathione can participate in this process, so that the CRP in the sample disaggregates into monomers when passing through the detection line, which is beneficial to signal enhancement and makes the CRP in the sample easier to detect.

[0027] Third, the characteristics of CRP chromatography test strips are short reaction time and solid phase carrier reaction. Compared with liquid phase reaction, the contact time between the test sample and the antibody is short. Therefore, adding reduced glutathione can facilitate rapid reaction in a short time and help improve sensitivity.

[0028] The coating buffer provided in this application contains a buffer, sugars, alcohols and reduced glutathione.

[0029] Preferably, the mass percentage concentration of reduced glutathione in the coating buffer is 0.05-0.5%.

[0030] The coating buffer preferably has a buffer concentration of 0.01-0.1 mol / L, a sugar concentration of 0.5-5% by weight, and an alcohol concentration of 0.1-2% by weight. The buffer may be any one of tris-hydroxyaminomethane buffer, 4-hydroxyethylpiperazineethanesulfonic acid, phosphate buffer, or borate buffer; the sugar may be any one or more of trehalose, sucrose, lactose, and dextran; and the alcohol may be any one or more of isopropanol, methanol, and ethanol.

[0031] The coating buffer may further include a preservative at a concentration of 0.01-0.1% by mass, wherein the preservative is any one or more of sodium azide, PC300, potassium sorbate, and Krovin300M.

[0032] The present application also provides a method for preparing an immunochromatographic test strip for quantitative detection of CRP. The CRP-specific antibody is coated with the above-mentioned coating buffer to form a detection strip. When the sample passes through the detection line, the reduced glutathione contained in the detection line interacts with the CRP in the sample, thereby improving the detection effect.

[0033] The preferred preparation method comprises the following steps: S1, applying CRP-specific antibody A coupled to fluorescent microspheres and quality control antibody A coupled to fluorescent microspheres to a conjugate pad; S2, coating CRP-specific antibody B on a reaction membrane using a coating buffer according to any one of claims 1 to 6 to form a detection strip; coating quality control antibody B on a reaction membrane to form a quality control strip; S3, assembling the sample pad, the conjugate pad prepared in step S1, the reaction membrane prepared in step S2, and the absorbent pad to a bottom plate to obtain a test strip; wherein there is no order restriction for steps S1 and S2.

[0034] Since the control strip is a combination of a control antibody and a control antigen, and is unrelated to the binding of the CRP antigen and antibody, the coating solution used for coating the control antibody B can be the aforementioned coating buffer or a coating solution commonly used in the art. However, a coating solution with a similar basic formula used for the same test strip is more effective, so it is more preferable to use a coating solution with a similar formula to the coating buffer used for the test strip to coat the control antibody B. For example, if the coating buffer used for the test strip is formulated with phosphate buffer, trehalose, isopropanol, sodium azide, and reduced glutathione, then the coating solution used for the control strip can be formulated with phosphate buffer, trehalose, isopropanol, and sodium azide.

[0035] The specific steps for preparing the immunochromatographic test strip for quantitative detection of CRP are as follows:

[0036] S1. Activate the fluorescent microspheres, and then couple the activated fluorescent microspheres with CRP-specific antibody A and quality control antibody A, respectively, to obtain CRP-specific antibody A-coupled fluorescent microspheres and quality control antibody A-coupled fluorescent microspheres; use the coupling storage solution to re-dissolve the CRP-specific antibody A-coupled fluorescent microspheres and the quality control antibody A-coupled fluorescent microspheres and spray them onto the conjugate pad;

[0037] S2. Use the above-mentioned coating buffer to dilute the CRP-specific antibody B and coat it on the reaction membrane to form a detection zone; use a commercially available coating solution or the above-mentioned coating buffer to coat the quality control antibody B on the reaction membrane to form a quality control zone.

[0038] S3, assembling the sample pad, the conjugate pad prepared in step S1, the reaction membrane prepared in step S2, and the absorbent pad onto the bottom plate in order from top to bottom, with the detection zone located on the side close to the conjugate pad, to obtain a test strip;

[0039] There is no order restriction for steps S1 and S2.

[0040] More preferably, the specific preparation steps of the immunochromatographic test strip for quantitative detection of CRP are as follows:

[0041] S1-1. Take 1 ml of the washed fluorescent microsphere solution with a solid content of 1%, ultrasonically treat it for 30-60 seconds, and successively add NHS solution with a concentration of 5-20 mg / mL and EDC solution with a concentration of 5-20 mg / mL, and the volume ratio of NHS solution to fluorescent microsphere solution is 1:(30-100), the volume ratio of EDC solution to fluorescent microsphere solution is 1:(50-200), and the volume ratio of EDC solution, NHS solution and fluorescent microspheres are the same or different; then incubate for 20-60 minutes, centrifuge and discard the supernatant, and then add borate buffer with a concentration of 0.01-0.05 mol / L, the volume ratio of buffer solution to fluorescent microsphere solution is 1:(0.5-2), and ultrasonically treat until there is no precipitate in the solution to obtain an activated fluorescent microsphere solution;

[0042] S1-2, add CRP-specific antibody A to the activated fluorescent microsphere solution at a ratio of 0.05-1.5 mg / mL, mix well, and incubate for 2-5 hours. After completion, add BSA solution with a mass percentage concentration of 0.1-1%, and the volume ratio of BSA solution to CRP-specific antibody A is (0.2-0.6 ml): 1 mg. Continue incubation for 0.5-2 hours. After completion, centrifuge, discard the supernatant, and wash 2-3 times. The washed microspheres are re-dissolved with the coupling storage solution to obtain a CRP-specific antibody A-coupled fluorescent microsphere solution;

[0043] Use the same method as S1-2 to prepare the quality control antibody A coupled with fluorescent microspheres solution;

[0044] Spray CRP-specific antibody A-coupled fluorescent microsphere solution and quality control antibody A-coupled fluorescent microsphere solution onto the conjugate pad;

[0045] S2. Dilute the CRP-specific antibody B to 0.5-1.5 mg / L using the above-mentioned coating buffer, coat it on the reaction membrane, and then dry it at 50-60°C for 65-80 hours to form a test strip; dilute the quality control antibody B to 0.5-1.5 mg / L using a commercially available coating solution or the above-mentioned coating buffer, coat it on the reaction membrane, and then dry it at 50-60°C for 65-80 hours to form a quality control strip;

[0046] S3-1. Prepare a sample pad treatment solution containing 0.05-0.2% bovine serum albumin and 2-8% trehalose by mass. Dissolve the sample pad treatment solution at a rate of 0.05-0.08 mL / cm 2 The ratio of 100 μg / cm2 was spread on the glass fiber and fully soaked, drained, and dried at 35-40°C for 12-20h to obtain a sample pad;

[0047] S3-2, assembling the sample pad, the conjugate pad prepared in step S1, the reaction membrane prepared in step S2, and the absorbent pad onto the bottom plate in sequence from top to bottom, with the detection band located on the side closest to the conjugate pad, to obtain a test strip;

[0048] There is a sequence between step S1-1 and step S1-2, but there is no sequence restriction for step S1 as a whole and step S2.

[0049] In the above preparation method, the coupling storage solution can be prepared by mixing 0.01 mol / L tris-hydroxyaminomethane buffer, 5% by mass trehalose, and 1% by mass BSA, with a pH of 7.4. Alternatively, a commercially available coupling storage solution can be used.

[0050] In this application, quality control antibodies can be antibodies known in the art, such as goat anti-chicken IgY. The NHS solution used is an aqueous solution of N-hydroxysuccinimide, and the EDC solution is an aqueous solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide. BSA is bovine serum albumin.

[0051] In step S1-1, the fluorescent microsphere solution is a purchased commodity. The commodity properties of different solid contents can be purchased according to needs, and the solution can also be diluted to different solid contents as needed.

[0052] In the present application, the reaction membrane may be a membrane known in the art, such as a nitrocellulose membrane. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments recorded in this application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0054] Figure 1 This is a linear relationship diagram of the test results of the biochemical reactor in Example 3 of the present invention (Example 2, all sample values);

[0055] Figure 2 This is a linear relationship diagram of the test results of the biochemical reactor in Example 3 of the present invention (Comparative Example 2, all sample values);

[0056] Figure 3 This is a linear relationship diagram of the test results of the biochemical reactor in Example 3 of the present invention (Example 2, sample value below 10 mg / L);

[0057] Figure 4This is a linear relationship diagram of the test results of the biochemical reactor in Example 3 of the present invention (Comparative Example 2, sample value below 10 mg / L). DETAILED DESCRIPTION

[0058] In order to help those skilled in the art better understand the technical solutions in this application, the technical solutions in the embodiments of this application will be clearly and completely described below. Obviously, the embodiments described are only part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without making any creative efforts shall fall within the scope of protection of this application.

[0059] Example 1 Coating Buffer

[0060] Buffer: phosphate buffer, 0.02 mol / L;

[0061] Carbohydrate: trehalose, mass fraction 5%;

[0062] Alcohols: isopropyl alcohol, mass fraction 1%;

[0063] Preservative: sodium azide, mass fraction 0.05%;

[0064] Reduced glutathione, mass fraction 0.1%;

[0065] Mix the above raw materials evenly.

[0066] Comparative Example 1

[0067] The reagents and concentrations were the same as in Example 1, except that reduced glutathione was not added.

[0068] Example 2 Immunochromatographic test strips for quantitative detection of CRP

[0069] S1-1. Take 1 ml of the washed 1% solid content fluorescent microsphere solution, sonicate for 30 seconds, add 10 μL of a 10 mg / L NHS solution and 10 mg / L EDC solution, incubate on a rotary mixer for 30 minutes, centrifuge and discard the supernatant, then add 1 ml of a 0.05 mol / L borate buffer, and sonicate until no precipitate remains in the solution to obtain an activated fluorescent microsphere solution;

[0070] S1-2. Add 0.1 mg of CRP-specific antibody A to the activated fluorescent microsphere solution, mix well, and incubate for 3 h. After completion, add 50 μL of 0.5% BSA solution and continue incubation for 1 h. After completion, centrifuge, discard the supernatant, and wash twice with ultrapure water. The washed microspheres are re-dissolved in 1 ml of coupling storage solution to obtain CRP-specific antibody A-coupled fluorescent microsphere solution, which is stored at 4°C.

[0071] The coupling storage solution includes 0.01 mol / L tris-hydroxyaminomethane buffer, 5% trehalose and 1% BSA, and the pH value of the coupling storage solution is 7.4;

[0072] Using the same method as in S1-2, the quality control antibody was prepared into a quality control antibody A (goat anti-chicken IgY)-conjugated fluorescent microsphere solution. The CRP-specific antibody A-conjugated fluorescent microsphere solution and the quality control antibody A-conjugated fluorescent microsphere solution were then sprayed onto the conjugate pad. S2: Using the coating buffer from Example 1, the CRP-specific antibody B and quality control antibody B (chicken IgY) were each diluted to 1.0 mg / L. The membranes were then coated onto nitrocellulose membranes using a film sprayer to form a test band (T line) and a quality control band (C line). The membranes were then dried at 55°C for 72 hours and stored dry.

[0073] S3-1. Prepare a sample pad treatment solution containing 0.1% bovine serum albumin and 5% trehalose by mass. Dissolve the sample pad treatment solution at 0.05 mL / cm 2 The ratio of 1% to 2% was spread on the glass fiber and fully soaked, drained, and dried at 37℃ for 16h. After drying, it was cut into 16mm*300mm size with a chopper to obtain the sample pad, and stored in an aluminum foil bag with desiccant and sealed.

[0074] S3-2. Stick the sample pad, conjugate pad, nitrocellulose membrane, and absorbent paper onto the PVC base plate in sequence from top to bottom, with each other connected. The detection strip is located on the side close to the conjugate pad. Cut the strips into strips of appropriate width according to the experimental requirements. After encapsulation, they can be used for clinical sample testing.

[0075] Comparative Example 2

[0076] The process and parameters are the same as those in Example 2, except that the coating buffer prepared in Comparative Example 1 is used in step S2.

[0077] Test strip performance testing

[0078] 1. Preparation of sample diluent

[0079] Weigh 8g NaCl, 0.2g KCl, 1.44g Na2HPO4, and 0.24g KH2PO4 powder, dissolve them in 800mL water, adjust the pH to 7.4, add 2g surfactant S9, make up to 1000mL with ultrapure water, filter, and store at room temperature.

[0080] 2. C-reactive protein reference test

[0081] A C-reactive protein reference product was tested using the test strips of Example 2 and Comparative Example 2 to compare the difference in linear range between the coating buffers of Example 1 and Comparative Example 1.

[0082] The test values ​​were 0.5, 1.0, 9.8, 30.6, 50.7, 71.9, 90.5, and 104.3 mg / L. Prepared test strips were tested five times at each concentration on both systems. The sample was diluted 200-fold with sample diluent, and 80 μL was immediately dripped onto the test strip. The reaction lasted for 3 minutes before the result was read.

[0083] The results of the embodiment are shown in Table 1. The results of the comparative example are shown in Table 2:

[0084] Table 1 Test results of Example 2

[0085]

[0086] Table 2 Test results of comparative example 2

[0087]

[0088] The above experimental results demonstrate that, when testing the same batch of reference samples, the high-value linear range of the modified coating buffer of Example 1 is comparable to that of the unmodified coating buffer of Comparative Example 1, but the precision is superior. However, when the sample concentration is less than 1 mg / L, the sensitivity of the test results using the unmodified sample dilution of Comparative Example 1 is insufficient, while the modified coating buffer system of Example 1 exhibits a high sensitivity that meets detection requirements.

[0089] 3. Biochemical reactor test

[0090] 5,000 test strips were prepared for each of Example 2 and Comparative Example 2. 35 clinical samples with concentration values ​​evenly distributed within the linear range were collected and tested on a Hitachi biochemical reactor. Each sample was tested twice to verify the clinical relevance of the two systems. The test method used was the same as in the second item. The results are shown in Table 3:

[0091] Table 3 Biochemical reactor test results

[0092]

[0093] According to the results in Table 3, since 10 mg / L is the reference value of CRP for determining whether a patient has inflammation, which is of great clinical significance, the test results of all sample values ​​and the test results of sample values ​​below 10 mg / L are plotted separately, as shown in the following figure: Figures 1 to 4 shown.

[0094] From Table 3 and Figures 1 to 4The results show that, when compared with a large number of test strips, the correlation between Comparative Example 2 and Example 2 is better at low values ​​below 10 mg / L, and the accuracy of tests below 1 mg / L is higher. For high-value samples with severe inflammation, the improved Example 2 effectively reduces sample interference, has a smaller degree of discreteness, and has better overall performance.

[0095] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing an immunochromatographic test strip for quantitative detection of CRP, characterized in that: The detection strip is formed by coating the CRP-specific antibody with coating buffer; The coating buffer comprises: buffer, sugars, alcohols, and reduced glutathione; Among them, the mass percentage concentration of reduced glutathione is 0.05-0.5%; the buffer concentration is 0.01-0.1 mol / L; the mass percentage concentration of sugars is 0.5-5%; the mass percentage concentration of alcohols is 0.1-2%; The buffer is any one of tris-hydroxyaminomethane buffer, 4-hydroxyethylpiperazineethanesulfonic acid, phosphate buffer, and borate buffer; the sugar is any one or more of trehalose, sucrose, lactose, and dextran; the alcohol is any one or more of isopropanol, methanol, and ethanol; It also includes a preservative with a mass percentage concentration of 0.01-0.1%.

2. The preparation method according to claim 1, characterized in that The preservative is any one or more of sodium azide, PC300, potassium sorbate, and Krovin300M.

3. The preparation method according to claim 1, characterized in that The following steps are involved: S1. Apply CRP-specific antibody A coupled to fluorescent microspheres and quality control antibody A coupled to fluorescent microspheres to the conjugate pad; S2. Coat the CRP-specific antibody B on the reaction membrane using a coating buffer to form a detection zone; Coat the quality control antibody B on the reaction membrane to form a quality control band; S3, assembling the sample pad, the conjugate pad prepared in step S1, the reaction membrane prepared in step S2, and the absorbent pad onto the bottom plate to obtain a test strip; There is no order restriction for steps S1 and S2.

4. The preparation method according to claim 3, characterized in that In step S2, the CRP-specific antibody B is diluted to 0.5-1.5 mg / L using a coating buffer, coated on a reaction membrane, and then dried at 50-60° C. for 65-80 h.

5. An immunochromatographic test strip for quantitative detection of CRP prepared by the preparation method according to any one of claims 1 to 4.

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