Method for improving precision and stability of beta-HCG fluorescence immunochromatography test strip, test strip and preparation method

Abstract

The invention discloses a method for improving precision and stability of a beta-HCG fluorescence immunochromatography test strip. A beta-HCG specific antibody coupled with fluorescent microspheres is used after being pre-aged. The invention also provides a preparation method of the beta-HCG fluorescence immunochromatography test strip and the prepared test strip. According to the method provided by the invention, the precision and the stability of the prepared test strip can be improved, the precision of the test strip can be within 10% within the linear range of 2.5 mIU / mL-200000mIU / mL, and the test strip can be stably stored for 18 months at the temperature of 2-30 DEG C, and has the advantages of simplicity in operation, high sensitivity, high accuracy and the like.

Description

Technical Field

[0001] The present application relates to the technical field of detection reagents, and in particular to a method for improving the precision and stability of a β-HCG fluorescent immunochromatographic test strip, a test strip, and a preparation method. Background Art

[0002] Human chorionic gonadotropin (HCG) is a glycoprotein secreted by the trophoblast cells of the placenta. It is composed of α and β dimer glycoproteins. The α subunit is similar to the follicle stimulating hormone (FSH), luteinizing hormone (LH) and thyroid stimulating hormone (TSH) secreted by the pituitary gland, so they can cross-react with each other. The last 28-30 amino acids of the β subunit are specific, so the β subunit is used to prepare HCG in specific antibody determination, named β-HCG.

[0003] β-HCG examination is of great significance for early pregnancy diagnosis, and has certain value in the diagnosis, differentiation and course observation of pregnancy-related diseases, trophoblastic tumors and other diseases. Increased β-HCG is seen in early pregnancy, hydatidiform mole, malignant hydatidiform mole, choriocarcinoma, seminoma, teratoma, ectopic HCG-secreting tumors (such as gastric cancer, pancreatic cancer, lung cancer, colon cancer, liver cancer, ovarian cancer, digestive system carcinoid, etc.); while decreased β-HCG is seen in miscarriage, etc.

[0004] As a pregnancy indicator, β-HCG increases very quickly during pregnancy, unlike other hormones in the human body that change very little. β-HCG levels can rise to 2500mIU / mL 35-50 days after pregnancy, and to 100,000mIU / mL at 2-3 months. The commonly used β-HCG detection methods in China currently include immunochromatography, enzyme immunoassay, chemiluminescence, etc., which have a wide linear range and can detect β-HCG with greatly varying concentrations, but they have poor precision and large batch differences. Summary of the invention

[0005] In order to solve the above technical problems, the first purpose of the present invention is to provide a method for improving the precision and stability of β-HCG fluorescent immunochromatography test strips; the second purpose of the present invention is to provide a method for preparing β-HCG fluorescent immunochromatography test strips; the third purpose of the present invention is to provide a β-HCG fluorescent immunochromatography test strip. The method provided in this application screens out firmly coupled microsphere antibodies through aging, thereby improving the availability of microsphere antibodies on the conjugate pad, and can improve the precision and stability of the prepared test strips. The precision of the test strips can reach within 10% within the linear range of 2.5mIU / mL-200000mIU / mL, and the test strips can be stably stored at 2-30°C for 18 months, and have the advantages of simple operation, high sensitivity, and high accuracy.

[0006] The technical solution provided by the present invention is as follows:

[0007] A method for improving the precision and stability of a beta-HCG fluorescent immunochromatographic test strip comprises pre-aging beta-HCG specific antibodies coupled to fluorescent microspheres before use.

[0008] Preferably, the β-HCG specific antibody coupled to the fluorescent microspheres is placed in an environment of 55°C±5°C for 14-18 hours for pre-aging.

[0009] A method for preparing a β-HCG fluorescent immunochromatographic test strip comprises the following steps:

[0010] S1. Pre-aging the β-HCG specific antibody A coupled to the fluorescent microspheres according to any of the above methods and then applying it to the conjugate pad;

[0011] S2, coating the β-HCG specific antibody B on the reaction membrane to form a detection zone;

[0012] The quality control antibody is coated on the reaction membrane to form a quality control band;

[0013] S3, assembling the sample pad, the binding pad prepared in step S1, the reaction membrane prepared in step S2, and the water absorbent pad onto the bottom plate to obtain a test strip;

[0014] There is no order restriction for steps S1 and S2.

[0015] Preferably, in step S2, the concentration of the β-HCG specific antibody B coated on the reaction membrane is 0.8-1.2 mg / ml; the concentration of the quality control antibody coated on the reaction membrane is 0.3-0.6 mg / ml.

[0016] Preferably, the conjugate pad used in step S1 is first soaked in a conjugate pad treatment solution, and the conjugate pad treatment solution comprises the following components: a buffer, a blocking agent, a surfactant, and a sugar;

[0017] The sample pad used in step S3 is first soaked in a sample pad treatment solution, and the sample pad treatment solution includes the following components: buffer, blocking agent, surfactant, sugar, blocking agent, and red blood cell monoclonal antibody.

[0018] Preferably, in the binding pad treatment solution or the sample pad treatment solution,

[0019] The buffer concentration is 0.01-0.1 mol / L;

[0020] The mass percentage concentration of the sealant is 0.5-5%;

[0021] The mass percentage concentration of the surfactant is 0.1-5%;

[0022] The mass percentage concentration of sugars is 0.5-5%;

[0023] The blocking agent concentration is 0.1-1.0 mg / mL;

[0024] The concentration of monoclonal antibody against erythrocytes is 0.1-1.0 mg / mL.

[0025] Preferably, in the binding pad treatment solution or the sample pad treatment solution,

[0026] The buffer is any one of tris-hydroxyaminomethane buffer, 4-hydroxyethylpiperazineethanesulfonic acid, phosphate buffer, and borate buffer;

[0027] The blocking agent is any one or more of BSA, casein, and skimmed milk powder;

[0028] The surfactant is any one or more of Tween 20, S9, and CHAPS;

[0029] The sugar is any one or more of trehalose, sucrose, lactose and dextran.

[0030] Preferably, the pH of the conjugate pad treatment solution and the sample pad treatment solution is 7.40±0.05.

[0031] Preferably, in step S1, the β-HCG specific antibody A coupled to the fluorescent microspheres is sprayed onto the conjugate pad and then dried at 37±5° C. for 14-18 hours.

[0032] A β-HCG fluorescent immunochromatographic test strip prepared by any of the preparation methods described above.

[0033] The present application first provides a method for improving the precision and stability of β-HCG fluorescent immunochromatographic test strips, and the β-HCG specific antibody coupled to the fluorescent microsphere is pre-aged before use. After the pre-aging treatment, the firmly coupled microsphere antibody is screened out, thereby increasing the availability of the microsphere antibody on the conjugate pad, and the β-HCG specific antibody effectively improves the precision and stability of the test strip; the test strip prepared has a linear range of 2.5mIU / mL-200000mIU / mL, and the precision can reach less than 10%, and the test strip can be stably stored at 2-30°C for 18 months, with the advantages of simple operation, high sensitivity, and high accuracy.

[0034] The present application also provides a method for preparing a β-HCG fluorescent immunochromatographic test strip and the prepared test strip.

[0035] The applicant has verified through experiments that the β-HCG specific antibody coupled with fluorescent microspheres obtained by the pre-aging method provided in this application has the following advantages compared with conventional test strips after being made into test strips:

[0036] First, the signal-to-noise ratio and sensitivity of the test strips obtained by the method of the present application are close, indicating that the pre-aging of the microsphere markers does not affect the overall gradient and sensitivity of the test strips.

[0037] Second, the precision of the test strips obtained by the method of the present application is within 10%, while the precision of conventional test strips can only reach within 15%, indicating that the method of the present application can effectively improve the precision of the test strips.

[0038] Third, the test strip obtained by the method of the present application can be stable for 19 weeks after aging at 55°C, and the expected shelf life of the test strip is 24 months according to the Arrhenius model; while the conventional test strip can only be stable for 14 weeks after aging at 55°C, and the expected shelf life of the test strip is 18 months according to the Arrhenius model; this shows that pre-aging the microsphere marker can effectively improve the stability of the test strip.

[0039] In the present application, the blocker can be any blocker known in the art, such as Roche MAK33, scantibody HBR1, HBR9, HBR Plus, etc. DETAILED DESCRIPTION

[0040] In order to enable those skilled in the art to 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 described embodiments are only part of the embodiments of this application, not all of them. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0041] Example 1 Preparation of β-HCG Fluorescent Immunochromatographic Test Strips

[0042] 1. Processing of conjugate pad:

[0043] Prepare a binding pad treatment solution: 0.01M PBS buffer, 0.2% casein by mass, 0.5% Tween20 by mass, 2.0% trehalose by mass and 0.5% BSA by mass; the pH of the binding pad treatment solution is 7.40±0.05, soak the glass fiber membrane to obtain a binding pad.

[0044] 2. Prepare fluorescent microspheres coupled to proteins and apply to the conjugate pad:

[0045] 2.1 Take the fluorescent latex microspheres, accurately measure 0.2 mL of fluorescent microspheres (solid content 1.0%) into a 2 mL EP tube with a pipette, add 0.8 mL of 0.05 M BB 7.0 buffer to wash, centrifuge (16000 rpm, 15 min), remove the supernatant. Add 1 mL of 0.05 M BB 7.0 buffer again, and use a cell disruptor to break up for 3 rounds in an ice water bath until the EP tube is left to stand for 1 min and no tiny particles are seen when it is slowly tilted.

[0046] 10 μL each of EDC solution (10 mg / mL) and NHS solution (10 mg / mL) were added to the fluorescent microspheres and mixed using a vortex mixer.

[0047] Place the fluorescent microspheres in a 25°C thermostat and rotate and mix for 15 minutes (30 rpm) to complete the activation. Centrifuge the activated fluorescent microspheres (16000 rpm, 20 minutes) and remove the supernatant. Add 1 mL of 0.05M BB 7.5 buffer and resuspend by ultrasound. Repeat the washing step twice. The buffer used in this step is 0.05M BB 7.5.

[0048] 2.2 Take 1 mL of 0.05 M BB 7.5 solution and add it to the microspheres after removing the supernatant. Use a cell disruptor in an ice-water bath to break up the microspheres until the EP tube is left to stand for 1 min and no tiny particles are seen when it is slowly tilted.

[0049] Add 0.2 mg mouse anti-human β-HCG monoclonal antibody A to the EP tube, place in a 25°C constant temperature box, and rotate and mix for 3.0 h (30 rpm), and the coupling is completed.

[0050] 2.3 Add 0.1 mL of 20% BSA solution and rotate to mix for 1.5 h (30 rpm) to complete the blocking process.

[0051] The blocked particles were centrifuged (16000 rpm, 15 min) and the supernatant was removed.

[0052] 2.4 Add 1 mL of 0.05 M BB8.0 to the particles after removing the supernatant, and break them up with a cell disruptor in an ice-water bath until the EP tube is left to stand for 1 min and no tiny particles are seen when it is slowly tilted. Repeat the washing step twice. The buffer used in this step is 0.05 M BB 8.0.

[0053] Add 1.0 mL of particle protection solution (0.05 M Tris HCl 8.0, 0.1% casein, 3% trehalose, 0.05% Tween 20, 0.5% BSA) to the centrifuged particles, and use a cell disruptor in an ice-water bath to break them up until the EP tube is left standing for 1 min and no tiny particles are seen when it is slowly tilted. A total of 1.0 mL of labeled particles is obtained and placed in a 50°C environment for 18 h to complete pre-aging.

[0054] 2.5 Take the pre-aged particles, dilute them with the particle protection liquid, and spray them on the treated binding pad at a spraying amount of 6 μL / cm, and then bake them in a forced air drying oven at 37±5°C for 16 hours to obtain the binding pad.

[0055] 3. Preparation of reaction membrane

[0056] Mouse anti-human β-HCG monoclonal antibody B was diluted to 1.0 mg / mL with T-line coating buffer (0.01M PBS7.4+2% trehalose+0.1% methanol), and goat anti-mouse IgG was diluted to 0.5 mg / mL with C-line coating buffer (0.01M PBS7.4+2% trehalose+0.1% methanol). The usage amount was 0.8 μL / cm. The membranes were coated on Sartorius nitrocellulose membranes with a film sprayer. T and C lines were drawn respectively. The membranes were dried at 55°C for 72 hours and then stored dry.

[0057] 4. Sample Pad Preparation

[0058] Prepare a sample pad treatment solution: 0.01M PBS buffer, 0.1% Tween20 by mass, 0.1% casein by mass, 1.0% sucrose by mass, 0.5% BSA by mass, 0.2mg / mL blocking agent and 0.2mg / mL erythrocyte monoclonal antibody; the pH of the sample pad treatment solution is 7.40±0.05, soak the glass fiber membrane to obtain a sample pad.

[0059] 5. Test strip assembly

[0060] The sample pad, conjugate pad, reaction membrane, and absorbent paper are sequentially attached to the PVC base plate, and the membrane strips are cut into appropriate widths according to the experimental requirements and can be used for blood testing.

[0061] During the detection process, the mouse anti-human β-HCG specific antibody A coupled to the fluorescent microspheres in the binding pad combines with the β-HCG in the sample to form a fluorescent particle-antibody-antigen complex. The immune complex then chromatographs along the reaction membrane to the detection zone (T), combines with the coated mouse anti-human β-HCG specific antibody B, and is fixed on the detection zone. Its fluorescence intensity is positively correlated with the β-HCG content in the sample.

[0062] During the chromatography process, the remaining mouse anti-human β-HCG specific antibody A coupled to the fluorescent microspheres in the conjugate pad that is not bound to the β-HCG in the sample is chromatographed to the quality control band (C), combined with the pre-coated goat anti-mouse IgG, and emits fluorescence in the quality control band.

[0063] Example 2

[0064] The same as Example 1, except that in step 2.4, the particles are placed in an environment of 55° C. for 18 hours to complete pre-aging.

[0065] Example 3

[0066] The same as Example 1, except that in step 2.4, the particles are placed in a 60° C. environment for 18 hours to complete pre-aging.

[0067] Comparative Example 1

[0068] The same as Example 1, except that the particles prepared in step 2.4 are not pre-aged and are directly used in step 2.5.

[0069] Performance Evaluation

[0070] The sensitivity, signal-to-noise ratio, precision and stability of the test strips of Examples 1-3 and Comparative Example 1 were compared for a series of gradient clinical samples, wherein the sensitivity was the ratio of the test signal of the sample with a concentration near the linear lower limit (2.50mIU / mL) to the test signal of the sample with a value of 0; the signal-to-noise ratio was the ratio of the test signal of the sample with a concentration near the linear upper limit (200000.00mIU / mL) to the test signal of the sample with a value of 0; the precision was to test samples of different concentrations 10 times each, and calculate the mean value (M) and standard deviation (SD) of the measured values, and calculate the coefficient of variation (CV) according to the formula of CV=SD / M×100%, which was the precision; the stability was investigated by placing the test strips in a 55°C accelerated aging method, and introducing the Arrhenius model,

[0071] TIMETI=TIMERT / Q10 [(T1-TRT) / 10]

[0072] Where:

[0073] Time required for accelerated aging (weeks): TIMETI

[0074] Expected shelf life at room temperature (weeks): TIMERT

[0075] Accelerated aging temperature (℃): T1=55

[0076] Storage temperature (℃): TRT=30

[0077] Reaction rate coefficient: Q10 = 2

[0078] When the expected shelf life of the test strip is 18 months (78 weeks), the storage time at 55°C is calculated according to the Arrhenius model as TIMETI = 78 / 22.5 = 13.8 weeks. Therefore, the test strip is aged at 55°C for at least 14 weeks to test its accuracy.

[0079] Among them, clinical samples were all quantified using Roche electrochemiluminescence instrument Cobas e601, and the value of 0 was the sample diluent.

[0080] Table 1 T / C signal test results

[0081]

[0082] From the results in Table 1, it can be seen that the test results of the test strips of Examples 1-3 (pre-aged at 55±5°C) and the conventional test strips without pre-aging (Comparative Example 1) are similar in signal-to-noise ratio and sensitivity, indicating that pre-aging of the microsphere markers does not affect the overall gradient and sensitivity of the test strips.

[0083] Table 2 Precision test results

[0084]

[0085] It can be seen from Table 2 that the precision of the test strips of Examples 1-3 (pre-aging at 55±5°C) is within 10%, while the precision of the conventional test strips without pre-aging treatment (Comparative Example 1) can only reach within 15%, indicating that the method of pre-aging the microsphere marker provided in the present application can effectively improve the precision of the test strips.

[0086] Table 3 Test results of aging stability of test strips in Example 1

[0087]

[0088]

[0089]

[0090] Table 4 Example 2 Test strip aging stability test results

[0091]

[0092]

[0093]

[0094] Table 5 Example 3 Test strip aging stability test results

[0095]

[0096]

[0097]

[0098] Table 6 Comparative Example 1 Test Bar Aging Stability Test Results

[0099]

[0100]

[0101] From the results in Tables 3-6, it can be seen that the test accuracy of the test strips of Examples 1-3 (pre-aged at 55±5°C) aged at 55°C for 19 weeks is within ±10%, and the expected shelf life of the test strips calculated based on the Arrhenius model is 24 months; while the conventional test strips (Comparative Example 1) without pre-aging treatment can only be stable for 14 weeks after aging at 55°C, and the expected shelf life of the test strips calculated based on the Arrhenius model is 18 months; This indicates that pre-aging of the microsphere marker can effectively improve the stability of the test strips.

[0102] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be 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 will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for improving the precision and stability of β-HCG fluorescent immunochromatographic test strips, characterized in that: The β-HCG specific antibody coupled to the fluorescent microspheres was pre-aged before use.

2. The method according to claim 1, characterized in that The β-HCG specific antibody coupled to the fluorescent microspheres was placed in an environment of 55°C±5°C for 14-18 hours for pre-aging.

3. A method for preparing a β-HCG fluorescent immunochromatographic test strip, characterized in that: The following steps are involved: S1. Pre-aging the β-HCG specific antibody A coupled to the fluorescent microspheres according to the method of any one of claims 1 to 2, and then applying it to the conjugate pad; S2, coating the β-HCG specific antibody B on the reaction membrane to form a detection zone; The quality control antibody is coated on the reaction membrane to form a quality control band; S3, assembling the sample pad, the binding pad prepared in step S1, the reaction membrane prepared in step S2, and the water 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 concentration of the β-HCG specific antibody B coated on the reaction membrane is 0.8-1.2 mg / ml; the concentration of the quality control antibody coated on the reaction membrane is 0.3-0.6 mg / ml.

5. The preparation method according to claim 3, characterized in that: The conjugate pad used in step S1 is first soaked in a conjugate pad treatment solution, wherein the conjugate pad treatment solution comprises the following components: a buffer, a blocking agent, a surfactant, and a sugar; The sample pad used in step S3 is first soaked in a sample pad treatment solution, and the sample pad treatment solution includes the following components: buffer, blocking agent, surfactant, sugar, blocking agent, and red blood cell monoclonal antibody.

6. The preparation method according to claim 5, characterized in that: In the conjugate pad treatment solution or sample pad treatment solution, The buffer concentration is 0.01-0.1 mol / L; The mass percentage concentration of the sealant is 0.5-5%; The mass percentage concentration of the surfactant is 0.1-5%; The mass percentage concentration of sugars is 0.5-5%; The blocking agent concentration is 0.1-1.0 mg / mL; The concentration of monoclonal antibody against erythrocytes is 0.1-1.0 mg / mL.

7. The preparation method according to any one of claims 5-6, characterized in that: In the conjugate pad treatment solution or sample pad treatment solution, The buffer is any one of tris-hydroxyaminomethane buffer, 4-hydroxyethylpiperazineethanesulfonic acid, phosphate buffer, and borate buffer; The blocking agent is any one or more of BSA, casein, and skimmed milk powder; The surfactant is any one or more of Tween 20, S9, and CHAPS; The sugar is any one or more of trehalose, sucrose, lactose and dextran.

8. The preparation method according to claim 5, characterized in that: The pH of the conjugate pad treatment solution and the sample pad treatment solution was 7.40±0.

05.

9. The preparation method according to claim 3, characterized in that: In step S1, β-HCG specific antibody A coupled to fluorescent microspheres is sprayed onto the conjugate pad and then dried at 37±5° C. for 14-18 hours.

10. The β-HCG fluorescent immunochromatographic test strip prepared by the preparation method according to any one of claims 3 to 9.

Images