CN112557661A - Magnetic immunochromatographic test strip and method for rapidly detecting pre-S2 antigen of hepatitis B virus - Google Patents
Magnetic immunochromatographic test strip and method for rapidly detecting pre-S2 antigen of hepatitis B virus Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
- G01N33/5761—Hepatitis B
- G01N33/5764—Hepatitis B surface antigen
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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- G01—MEASURING; TESTING
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/01—DNA viruses
- G01N2333/02—Hepadnaviridae, e.g. hepatitis B virus
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Abstract
The invention particularly relates to a magnetic immunochromatographic test strip capable of quickly, qualitatively and quantitatively detecting a pre-S2 antigen of hepatitis B virus and a detection method. The test line of the test strip is pre-hepatitis B S2 antigen-bovine serum albumin conjugate, and the quality control line is mouse IgG. Firstly, pre-hepatitis B S2 antigen in a sample reacts with antibody and immunomagnetic beads coupled with goat-anti-mouse secondary antibody through pre-incubation treatment, so that the steric hindrance effect of immobilized antibody is avoided, and the combination between the antigen and the antibody is more sufficient; then, a magnetic immunochromatographic test strip is adopted for detection. According to the invention, the specific monoclonal antibody modified on the surface of the magnetic nano material is changed into the secondary antibody which is strong in universality and easy to obtain, so that the detection cost is reduced, and the detection sensitivity is improved by utilizing the signal amplification effect of the secondary antibody; in addition, the invention utilizes the characteristics of stability and accurate quantification of the magnetic signal, and realizes accurate and sensitive quantitative detection while rapidly and qualitatively detecting the pre-S2 antigen of the hepatitis B virus by naked eyes.
Description
Technical Field
The invention belongs to the field of biotechnology and detection, and particularly relates to a preparation method and an application method of a magnetic immunochromatographic test strip suitable for quickly detecting a pre-hepatitis B S2 antigen at low cost.
Background
Hepatitis B is a liver infection caused by Hepatitis B Virus (HBV), and is an infectious disease that seriously harms human health. According to WHO statistics, about 20 hundred million people all over the world are infected by hepatitis B, about 3.78 hundred million chronic HBV patients exist, about 450 million new HBV infections occur every year, wherein about 1/4 will develop into various liver diseases and finally die from liver failure, liver cirrhosis and primary hepatocellular carcinoma caused by HBV infection. However, China is a middle and high epidemic area infected by HBV, the carrying rate of hepatitis B surface antigen (HBsAg) in hepatitis B people in China is as high as 8% -10%, and almost reaches one third of the total carrying amount of HBsAg in the world, and the infection rate, the morbidity and the like of HBV are ranked in the front of the world for a long time.
Chronic hepatitis b infection often does not show any specific symptoms, and the condition of the patient often develops to a moderate or severe stage when the patient feels obviously uncomfortable to visit the hospital, so that the early detection of hepatitis b is significant for the patient and risk groups. The most important serological index for the early detection of hepatitis B is hepatitis B surface antigen (HBsAg), which is the main component of HBV envelope and contains three forms of large, medium and small proteins. The fragment of the smaller protein of the middle protein is called pre-hepatitis B S2 antigen, and the fragment of the larger protein is called pre-hepatitis B S1 antigen. The pre-hepatitis B S2 antigen is completely exposed to the outer layer of HBV, has a polymerized human serum albumin receptor, is beneficial to the adsorption of HBV on the surface of liver cells, is considered to be more sensitive to reflect the active replication of HBV in liver cells than pre-hepatitis B S1 antigen and HBsAg, can appear in acute hepatitis B earlier than 11 weeks before the ALT (acute hepatitis B) is increased in serum, is a marker of HBV early infection, and is also an index for judging the chronic degree and the severity of hepatitis B.
With the continuous development of clinical medicine, immunology and molecular biology in recent years, a plurality of high-sensitivity novel detection means such as chemiluminescence, time-resolved fluorescence, surface enhanced raman spectroscopy and other technologies are applied to hepatitis B detection, but compared with other hepatitis B serum markers, the current clinical diagnosis technology of the pre-hepatitis B S2 antigen is less researched, the method is immature, and only qualitative judgment of negative and positive results can be achieved basically, so that the development of related quantitative products is urgently needed. The prior patent CN203337669U only proposes a construction method for simultaneously and qualitatively detecting pre-hepatitis B pre-S1 and pre-S2 antigen joint inspection cards based on gold-labeled immunochromatography (GICA), but the construction method is not perfect and the detection performance is not further analyzed. Similar problems exist in CN104297483A, CN105652001B and CN 207396502U. CN103293295A discloses a magnetic immunochromatographic test strip for detecting hepatitis B pre-S1 antigen and a preparation method thereof, but only a T-line signal value is used as a quantitative basis, and clinical true serum samples are not used for verification. CN104730237A discloses a test strip method for detecting alpha fetoprotein, hepatitis B surface antigen and HIV antibody, which combines chemiluminescence and immunochromatography techniques, has high detection sensitivity but complex operation, and only realizes semi-quantitative analysis of hepatitis B surface antigen by applying luminescent liquid on the test strip during detection. CN109884306A discloses a test strip for detecting small molecules, a kit and a detection method thereof, wherein the test strip adopts a pre-incubation mode, but still adopts a mode of marking monoclonal antibodies, and compared with the test strip and the kit, the use amount of expensive monoclonal antibodies can not be effectively reduced.
The traditional test strip generally adopts a strategy that a specific monoclonal antibody (monoclonal antibody) coupled to a corresponding detection substance on a marker is directly combined with a substance to be detected, the using amount of the antibody in a single preparation process is about 10-200 mu g, the cost is high, and the early-stage product development is not facilitated. And because the combination of the target antigen and the antibody is completed on the combination pad of the test strip, the problems of reduced antibody activity, short reaction time and insufficient combination exist. Therefore, there is a need to improve the prior art to provide a low cost detection method and reagent that can rapidly qualitatively/quantitatively detect hepatitis b virus pre-S2 antigen.
Disclosure of Invention
The invention aims to provide a method, a test strip and a kit for rapidly detecting a pre-S2 antigen of hepatitis B virus. The problems of large using amount and high cost of the antibody in the single preparation process of the traditional test strip in the prior art are solved, and the problems of reduced antibody activity, short reaction time and insufficient combination caused by direct combination of the target antigen and the antibody on the combination pad of the test strip can be solved. In addition, the invention utilizes the characteristics of stability and accurate quantification of the magnetic signal, realizes accurate and sensitive quantitative detection by utilizing a magnetic signal analyzer while rapidly and qualitatively detecting the pre-S2 antigen of the hepatitis B virus by naked eyes.
In order to achieve the purpose, the invention is realized by the following technical scheme:
a magnetic immunochromatographic test strip for rapidly detecting a pre-S2 antigen of hepatitis B virus comprises a sticky bottom plate, and a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad which are sequentially adhered and lapped on the sticky bottom plate;
the nitrocellulose membrane is coated with a detection line (T line) and a quality control line (C line); the detection line (T line) is a detection line of pre-hepatitis B S2 antigen-bovine serum albumin conjugate (pre-hepatitis B S2 antigen-BSA), and the quality control line (C line) is mouse IgG.
Preferably, the selected sticky bottom plate is a transparent PVC plastic thin plate, the sample pad is a glass fiber pad, the combination pad is a polyester fiber pad, and the water absorption pad is a cellulose membrane.
Preferably, the surfaces of the sample pad, the combination pad, the nitrocellulose membrane and the absorbent pad are covered with protective covering films.
The pre-hepatitis B S2 antigen-bovine serum albumin conjugate is a conjugate formed by connecting amino on BSA and sulfydryl on pre-hepatitis B S2 antigen by using a synthesized pre-hepatitis B S2 antigen polypeptide and bovine serum albumin and a bifunctional coupling agent 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimide ester.
Wherein the amino acid sequence of the pre-hepatitis B S2 antigen is shown as SEQ ID No. 1: CMQWN STAFH QALQD PRVRG LYFPAGG are provided.
The preparation method of the test strip comprises the following steps:
(1) coating of nitrocellulose membrane: diluting pre-hepatitis B S2 antigen-BSA conjugate to 0.01-2mg/mL by using PBS, diluting mouse IgG to 0.01-2mg/mL, respectively spraying the pre-hepatitis B S2 antigen-BSA conjugate and the mouse IgG onto a blank nitrocellulose membrane at a distance of 0.3-1.0cm by using a membrane spraying and scribing device, slightly drying in the air, and drying at 30-60 ℃ for 0.5-2 h; preferably, the pre-hepatitis B S2 antigen-BSA conjugate is diluted to 0.01-1mg/mL, and the mouse IgG is diluted to 0.1-2 mg/mL;
(2) assembling the test strip: fixing a sticky bottom plate on a plane, sequentially adhering a lap joint sample pad, a combination pad, a coated nitrocellulose membrane and a water absorption pad on the sticky bottom plate, overlapping each component by about 1-2mm to ensure smooth surging of liquid, covering a protective transparent coating film on the surface of a test paper board after sticking, and cutting the test paper board into test paper strips with the width of about 3-6mm by using a slitter.
The pre-hepatitis B S2 antigen-BSA conjugate is prepared by the following method:
1) and (3) activation: dissolving BSA in PBS (phosphate buffer solution) with the pH value of 7.0-7.4, adding a bifunctional coupling agent 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimide ester (SMCC for short) to react for 0.5-2h at room temperature in a dark place to activate the amino group on the BSA, wherein the mass ratio of the BSA to the SMCC is 1: 0.1 to 1;
2) desalting: adding activated BSA solution into a PBS balanced desalting column to remove unreacted SMCC;
3) coupling: adding a pre-hepatitis B S2 antigen, placing the mixture on a vertical mixer for overnight coupling reaction at 4 ℃ in a dark place, wherein the mass ratio of BSA to the pre-hepatitis B S2 antigen is 1: 0.1-1.
One preferred scheme is:
1) and (3) activation: dissolving 1mg BSA in PBS (pH 7.0-7.4), adding 0.1-1mg bifunctional coupling agent 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimide ester (SMCC for short) to react for 0.5-2h at room temperature in a dark place to activate the amino group on the BSA;
2) desalting: adding activated BSA solution into a PBS balanced desalting column to remove unreacted SMCC;
3) coupling: 0.1-1mg of pre-hepatitis B S2 antigen is added and the mixture is put on a vertical mixer to carry out coupling reaction overnight at 4 ℃ in the dark.
A method for rapidly detecting hepatitis B virus pre-S2 antigen, comprising the following steps:
(1) pre-incubation: fully and uniformly mixing primary antibody and immunomagnetic beads with a sample to be detected, and standing for 1-30 minutes; the primary antibody is a pre-hepatitis B S2 specific monoclonal antibody; the immunomagnetic beads refer to superparamagnetic nano coupled with goat-anti-mouse secondary antibody4Particles;
(2) fully and uniformly mixing the pre-incubated solution and the chromatographic solution for 5-60 seconds, and dropwise adding the mixture into the middle part of a sample pad of the magnetic immunochromatographic test strip so that the mixture flows forwards;
(3) and (5) judging the result qualitatively or quantitatively.
Preferably, the pre-incubation step is: diluting primary antibody (specific monoclonal antibody of pre-hepatitis B S2 antigen) to 10-1000ng/mL with PBS buffer solution with pH of 7.0-7.4, mixing immunomagnetic beads and sample to be detected, adding primary antibody solution, mixing well for 5-60S, standing for 1-30 min, and completing pre-incubation.
Preferably, the particle size of the superparamagnetic nanoparticle is 100-300 nm; preferably, the superparamagnetic nano-particle is superparamagnetic nano-Fe3O4And (3) granules.
Preferably, the chromatographic solution in step (2) is Borate (BS) buffer containing 5% -20% BSA and 1% -5% tween-20 at a pH of 8.5-9.0. More preferably, the BSA content is 20% and the Tween-20 content is 5%.
In the step (2), the test strip can complete color development within 1-60 min. Preferably 15-40 min.
The immunomagnetic beads in the step (1) are magnetic beads with surfaces marked with antibodies, namely superparamagnetic nano particles coupled with goat-anti-mouse secondary antibodies, and the magnetic beads can be superparamagnetic nano Fe3O4Particles; using carbodiimide and N-hydroxysuccinimide to prepare superparamagnetic nano-scale particles; the particles are covalently connected with a goat-anti-mouse secondary antibody to prepare the conjugate, and the specific preparation method comprises the following steps:
A. and (3) activation: placing superparamagnetic nano-particles (magnetic beads) in a centrifuge tube, washing with 10mM 2- (N-morpholine) ethanesulfonic acid buffer solution (MEST) with pH of 4.5-5.0 containing 0.05% Tween-20, adding NHS and EDC (mass ratio of 1-2: 1), immediately mixing uniformly, ultrasonically dispersing for 30s, vertically mixing uniformly at room temperature for 30min,
B. coupling: fully washing the activated magnetic beads by using 5mM borate Buffer Solution (BST) with pH 8.5-9.0 containing 0.05% Tween-20, transferring the activated magnetic beads to a new centrifuge tube, adding the goat-anti-mouse secondary antibody for coupling, and vertically and uniformly mixing at room temperature, wherein the mass ratio of the magnetic beads to the goat-anti-mouse secondary antibody is 1: 0.01-0.2;
C. and (4) sealing and storing: after washing the beads, BST buffer containing 1% BSA (5 mM borate buffer pH 8.5-9.0 containing 0.05% Tween-20) was added, and the mixture was placed in a refrigerator at 4 ℃ overnight to complete blocking, and then stored at 2-8 ℃ for later use.
Preferably, the step of preparing the immunomagnetic beads comprises:
A. and (3) activation: placing 1mg of magnetic beads in a centrifuge tube, washing the magnetic beads by using 10mM 2- (N-morpholine) ethanesulfonic acid buffer solution (MEST) with pH4.5-5.0 and containing 0.05% Tween-20, and adding a solvent with the mass ratio of 2: 1, immediately mixing the NHS and the EDC, performing ultrasonic treatment for 30s, and vertically mixing the mixture for 30min at room temperature, wherein the mass ratio of the EDC to the magnetic beads is 25: 1.
B. coupling: the activated beads were washed thoroughly with 5mM Borate (BST) buffer pH 8.5-9.0 containing 0.05% Tween-20, transferred to a fresh centrifuge tube, coupled with 10-200. mu.g goat anti-mouse secondary antibody, and mixed vertically for 3h at room temperature.
C. And (4) sealing and storing: and (3) washing the magnetic beads, adding BST buffer solution containing 1% BSA, putting the magnetic beads in a refrigerator at 4 ℃ overnight, sealing the magnetic beads, and storing the magnetic beads at 2-8 ℃ for later use.
Preferably, the step (3) comprises two modes of naked eye qualitative observation and instrumental quantitative determination.
The qualitative judgment result method comprises the following steps: and comparing the color depths of the sample and the negative control detection line to judge a negative result and a positive result, wherein the negative result is judged if the color of the sample detection line is close to (or deeper than) that of the negative control, and the positive result is judged if the color of the detection line is lighter than that of the negative control or is not developed. The result of the visual qualitative observation in the step 3) is that the color depth of the sample and the negative control detection line is compared by the naked eye to judge the negative and positive results, if the color of the sample detection line is close to (or deeper than) the negative control, the negative result is obtained, and if the color of the detection line is lighter than the negative control or does not develop the color, the positive result is obtained; if only T line and C line are not colored, the result is invalid.
The quantitative determination method in the step (3) is quantitative determination by using an instrument, measuring the content of the magnetic beads combined on the T line and the C line by using a magnetic signal analyzer, calculating the ratio (T/C) of the T line and the C line, and determining the ratio (B/B) of the T/C between the sample and the negative control0) And establishing a standard curve, and calculating the content of the pre-hepatitis B S2 antigen in the sample.
A kit for rapidly detecting pre-S2 antigen of hepatitis B virus comprises the magnetic immunochromatographic test strip and immunomagnetic beads, wherein the immunomagnetic beads refer to superparamagnetic nano Fe coupled with goat-anti-mouse secondary antibody3O4And (3) granules.
The detection principle of the test strip and the method is as follows:
because the molecular weight of the pre-hepatitis B S2 antigen is small, a competition method is adopted for detection. The method comprises the steps of pre-incubating a sample together with a secondary antibody labeled immunomagnetic bead and an anti-solution, adding an incubation solution to a test strip for detection, and capturing an immunomagnetic bead compound by substances coated on T, C lines when liquid flows through a nitrocellulose membrane. If the sample does not contain the pre-hepatitis B virus S2 antigen, the primary antibody is combined with the immunomagnetic beads to form a primary-secondary antibody magnetic bead complex which can be captured by the pre-hepatitis B virus S2 antigen-BSA fixed on the T line for color development; if the sample contains the pre-hepatitis B S2 antigen, the primary antibody is combined with the pre-hepatitis B S2 antigen and immunomagnetic beads to form a pre-hepatitis B S2 antigen-primary antibody-secondary antibody magnetic bead compound, the primary antibody captured by the T line is reduced, the color development of the T line is reduced or even disappears, and therefore, the content of the pre-hepatitis B S2 antigen in the sample can be qualitatively judged by naked eyes. Furthermore, because the quantity of the magnetic beads captured on the T line and the C line has a certain quantity relation with the content of the pre-hepatitis B S2 antigen in the sample, if a series of pre-hepatitis B S2 antigen samples diluted in a gradient manner are prepared in advance to be tested by a test strip, and a standard curve is established according to the intensity of the magnetic signal on the T, C line, the quantitative analysis of the pre-hepatitis B S2 antigen in the sample can be realized.
According to the invention, the reaction of the target antigen S2 antigen in the sample and the specific antibody is transferred to an external solution system for carrying out through pre-incubation treatment, so that the steric hindrance effect of the immobilized antibody is avoided, the activity of the antibody can be preserved to the maximum extent, and the combination between the antigen and the antibody is more sufficient; the usage amount of the monoclonal antibody is also reduced, and the usage amount of the monoclonal antibody is greatly saved. Cheap secondary antibodies such as goat anti-mouse (rabbit) and the like are used as markers to be coupled with the antibodies, secondary antibody labeled immunomagnetic beads are added into an external solution system to replace the specific monoclonal antibodies modified by the traditional test strip markers, so that the use amount of the specific monoclonal antibodies is reduced, the production cost is greatly reduced, and the detection sensitivity can be improved by utilizing the signal amplification effect of the secondary antibodies. The invention adopts superparamagnetic nanoparticles to replace colloidal gold applied in the traditional test strip as a marker, and utilizes the characteristics of stability and accurate quantification of a magnetic signal, so that the rapid and sensitive quantitative detection is realized while the macroscopic rapid qualitative detection of the pre-S2 antigen of the hepatitis B virus is realized.
In addition, the test strip can realize the trace magnetic detection from the surface to the inside of the reaction area on the nitrocellulose membrane by a small-sized magnetic signal analyzer after the detection is finished, thereby greatly improving the detection sensitivity, and being a great improvement compared with the traditional test strip which can only carry out surface optical signal quantification. The method also has strong universality, and if a new detection object is developed, the product can be rapidly developed only by using a corresponding specific antibody and coating a corresponding substance on the test strip T line without replacing the immunomagnetic beads.
The method has the characteristics of rapidness, accuracy, high sensitivity and capability of qualitative analysis by naked eyes of the traditional test strip, and simultaneously meets the requirements of low-cost development and real-time quantitative detection, thereby providing a solution for hospitals and clinical detection centers at all levels, particularly basic institutions with poor conditions and sensitive cost.
Therefore, compared with the prior art, the invention has the advantages that:
1. the invention fills the blank of the related test strip for quantitative detection of the pre-hepatitis B S2 antigen on the market, and has positive significance for the development of the field of rapid detection of hepatitis B.
2. The invention adopts the second antibody labeled immunomagnetic beads as the markers, changes the specific monoclonal antibody connected with the markers of the traditional test strip into the cheap and easily obtained second antibody, further improves the detection performance and effectively reduces the production cost. And the second antibody has a signal amplification effect, so that the sensitivity can be further improved. Meanwhile, the single use amount of the specific monoclonal antibody is reduced, so that the cost of the test strip production condition optimization process is reduced, and the test strip product can be rapidly developed at the minimum cost.
3. The invention also introduces a pre-incubation treatment method, and the reaction of the antigen to be detected and the specific antibody is transferred to a solution system for carrying out, so that the activity of the antibody can be furthest preserved, the antigen and the antibody can be more fully combined, and the detection accuracy is improved. Compared with the traditional method for capturing the antigen by the labeled monoclonal antibody, the capture efficiency of the same antigen by combining the indirect secondary antibody labeling and the pre-incubation method can be improved by up to 66.2 percent.
4. The invention also introduces a magnetic signal quantitative analysis instrument into the detection of the pre-S2 hepatitis B virus antigen, can not only carry out rapid qualitative judgment on the pre-S2 hepatitis B virus antigen through visual inspection, but also realize the quantitative analysis of the test result of the test strip by combining the magnetic signal analyzer so as to avoid the inaccuracy of the interpretation of the result of a low-concentration sample, and can calculate the content by establishing a standard curve, thereby realizing the quantitative detection of the pre-S2 hepatitis B virus antigen and greatly improving the detection sensitivity, reliability and convenience of use.
5. The method established by the invention is simple to operate and short in time consumption, the whole detection process can be completed within 40min, and the test strip and the quantitative instrument have the advantages of low cost, small volume, light weight and convenience in carrying, and can be used for quickly detecting the pre-hepatitis B S2 antigen in medical institutions, particularly primary hospitals and institutions.
6. The invention also establishes a coupling method of the polypeptide antigen of the pre-hepatitis B S2 antigen and macromolecular proteins such as BSA and the like, and the method has the advantages of no need of additional instruments and equipment, high and stable coupling efficiency and easy implementation.
7. The hepatitis B pre-S2 antigen is used as a detection target in the detection system of the invention, is positioned on the surfaces of HBV mature virus particles and subviral particles, contains the binding sites of fibronectin, transferrin and polymeric human serum albumin (pHSA), has a trans-activation function, and is closely related to the invasion, release and replication of HBV. The research proves that the antigen can be used for judging the chronic degree of hepatitis B and the virus replication condition, compared with the conventional detection of hepatitis B surface antigen and hepatitis B pre-S1 antigen, the hepatitis B pre-S2 antigen is beneficial to judging the HBV infection stage, identifying occult hepatitis B carriers and evaluating treatment response and prognosis, and has important significance in early diagnosis, medium-term immune escape and late-stage course evaluation of hepatitis B.
8. The invention uses B/B0The ratio (inhibition rate) was used to establish a quantitative curve, B/B0The reagent is commonly used in quantitative analysis of competitive immunoassay, and can reflect relative change of the determination result of a sample and a negative control. The prior quantitative method of the immunochromatographic test strip is mainly based on a T-line signal value or a T/C ratio, but the T-line signal value or the T/C ratio can be greatly changed in samples from different sources or test strips in different batches due to the matrix effect and the difference between the test strip batches, so that the invention utilizes B/B0And a standard curve is established according to the ratio so as to reduce the influence of matrix and batch difference on the detection result and ensure that the result is more accurate.
Drawings
FIG. 1 is a schematic structural diagram of a magnetic immunochromatographic test strip for detecting a pre-S2 antigen of hepatitis B virus according to the present invention, wherein 1-a sample pad, 2-a binding pad, 3-a nitrocellulose membrane, 4-a water-absorbing pad, 5-a detection line, and 6-a quality control line.
FIG. 2 is a schematic diagram showing the color development of the test strip of the present invention when detecting different pre-hepatitis B S2 antigen content samples.
FIG. 3 is a sensitivity verification real-time image of the test strip of the present invention, which is negative, 9.8, 19.5, 39, 78, 156, 313, 625, 1250, 2500, 5000ng/ml antigen diluent samples in sequence from left to right.
FIG. 4 is a quantitative curve of the sensitivity results of the test strip of the present invention.
FIG. 5 shows the result of the specificity test of the test strip of the present invention.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and examples, in order to explain technical features and aspects of the present invention in detail. The pre-hepatitis B S2 antigen polypeptide used in the examples was artificially synthesized, except that the reagents and materials used were all commercially available products.
EXAMPLE 1 preparation of test strip for hepatitis B Virus pre-S2 antigen
The invention relates to a magnetic bead immunochromatographic test strip for qualitatively/quantitatively detecting a hepatitis B virus pre-S2 antigen, which comprises a sticky bottom plate, a sample pad 1, a combination pad 2, a nitrocellulose membrane 3, a water absorption pad 4 and a protective film covered on the surface, wherein the sample pad 1, the combination pad 2, the nitrocellulose membrane 3, the water absorption pad 4 and the protective film are sequentially adhered and overlapped on the sticky bottom plate as shown in figure 1. Wherein the cellulose nitrate membrane is coated with a detection line (T line) 5 of a hepatitis B pre-S2 antigen-bovine serum albumin (hepatitis B pre-S2 antigen-BSA) conjugate and a quality control line (C line) 6 of mouse IgG.
The preparation method of the test strip specifically comprises the following steps:
1. test strip assembly
The invention provides a method for assembling a test strip. Fixing a PVC (polyvinyl chloride) adhesive bottom plate on a certain plane, sequentially adhering and overlapping a sample pad 1, a combination pad 2, a nitrocellulose membrane 3 and a water absorption pad 4 on the PVC adhesive bottom plate, overlapping each component by about 1-2mm to ensure smooth surging of liquid on the PVC adhesive bottom plate, covering a layer of transparent protective covering film on the surface of a test paper board after the PVC adhesive bottom plate is well adhered, cutting the PVC adhesive bottom plate into test paper strips with the width of about 5mm by using a programmable slitter, and placing the test paper strips and a small amount of drying agent in a sealing bag for drying and light-proof storage. The structure is shown in fig. 1.
(1) Preparation of pre-hepatitis B S2 antigen-BSA
The invention also provides a preparation method of the polypeptide antigen conjugate, which is used for coating the T line.
And (3) activation: 1mg of carrier protein BSA was dissolved in 1mL of PBS (pH 7.4), 0.2mg of bifunctional coupling agent 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester (abbreviated as SMCC) was added, the reaction vessel was wrapped with tinfoil, and the mixture was reacted at room temperature in the dark for 2 hours to activate the amino groups on the surface of the carrier protein.
Desalting: washing a desalting column by using 15mL of PBS for three times, adding 2.5mL of activated solution, supplementing the activated solution by using PBS if the sample solution is less than 2.5mL to remove unreacted SMCC, and collecting eluent to perform the next reaction;
coupling: adding 1mg of pre-hepatitis B S2 antigen polypeptide dissolved in PBS (trace amount of DMSO can be added to aid dissolution if the solubility of the polypeptide is poor), wrapping the reaction container with tinfoil, and placing on a vertical mixer for overnight reaction at 4 deg.C in the dark.
(2) Coating of nitrocellulose membranes
The method for coating T, C wire material on the nitrocellulose membrane is to dilute the pre-hepatitis B S2 antigen-BSA to 0.01-2mg/mL by PBS, dilute the mouse IgG to 0.01-2mg/mL, spray the two on the blank nitrocellulose membrane at the interval of 0.5cm by using a membrane spraying and scribing device, wherein the spraying amount is 1 muL/cm, and respectively form a detection line (T line, pre-hepatitis B S2 antigen-BSA) 5 and a quality control line (C line, mouse IgG) 6. Drying at 30-60 deg.C for 0.5-2 hr, and placing in a sealed bag together with a small amount of desiccant. In this example, the concentration of pre-HBV S2 antigen-BSA was 0.1mg/mL, the concentration of mouse IgG was 1.5mg/mL, the baking temperature was 37 ℃ and the baking time was 2 h.
Example 2 preparation of immunomagnetic beads
The invention also provides a preparation method of the immunomagnetic beads, wherein the immunomagnetic beads refer to magnetic beads with the surfaces marked with antibodies, and the immunomagnetic beads are prepared by the following method:
and (3) activation: 1mg of magnetic beads are sucked and placed in a centrifuge tube, the magnetic beads are washed by using 10mM 2- (N-morpholine) ethanesulfonic acid (MEST) buffer solution with the pH value of 4.5-5.0 and containing 0.05% Tween-20, 10-25mg of NHS and EDC are added in sequence, then the mixture is immediately mixed and is subjected to ultrasonic treatment for 30s, and then the mixture is vertically mixed for 30min at room temperature. In this example, MEST buffer pH was 5.0, NHS was 25mg, and EDC was 12.5 mg. The magnetic beads can be selected from superparamagnetic nano Fe with the particle size of 100-3O4And (3) granules.
Coupling: the activated beads were washed thoroughly with 5mM borate Buffered Saline (BST) pH 8.5-9.0 containing 0.05% Tween-20, transferred to a new centrifuge tube, and 10-200. mu.g of goat anti-mouse secondary antibody was added for coupling, followed by mixing vertically at room temperature for 3 hours. In this example, BST buffer pH was 9.0 and goat anti-mouse secondary antibody was added at 100. mu.g.
And (4) sealing and storing: after washing the magnetic beads, adding BST buffer solution containing 1% BSA, placing the immune magnetic beads in a refrigerator at 4 ℃ overnight, sealing the immune magnetic beads, and storing the immune magnetic beads at 2-8 ℃ for later use.
Example 3 detection of Pre-S2 antigen of hepatitis B Virus
The embodiment provides a method for detecting pre-hepatitis B S2 antigen by using the test strip and immunomagnetic beads, which comprises three steps of pre-incubation, sample detection and result judgment.
(1) Pre-incubation: diluting a specific monoclonal antibody (primary antibody for short) of the pre-S2 antigen of the hepatitis B virus to 100ng/mL by using PBS (phosphate buffer solution) with the pH value of 7.4, mixing 1-8 mu L of immunomagnetic beads with a sample to be detected, adding an equal volume of primary antibody solution, fully mixing for 15S, standing at room temperature for 5min, and finishing primary antibody incubation.
(2) Sample detection: sucking 105 mu L of the pre-incubated solution in the step (1) and fully mixing the pre-incubated solution with 15 mu L of chromatography liquid for 15s, dropwise adding the mixture to the middle part of the sample pad to enable the liquid to flow forwards, and finishing color development in 5-60min by using a test strip. Observations were made after 20 min. The chromatography liquid is BS buffer solution containing 20% BSA and 5% Tween-20 and having pH of 9.0
(3) And (4) judging a result: the method comprises two modes of naked eye qualitative observation and instrumental quantitative determination.
The visual qualitative observation is that the color depth of the sample and the negative control detection line is compared by the visual method to judge the negative and positive results, if the color of the sample detection line is close to (or deeper than) the negative control, the negative result is obtained, and if the color of the detection line is lighter than the negative control or does not develop the color, the positive result is obtained. As shown in fig. 2.
The quantitative determination of the instrument refers to determining the content of magnetic beads combined on a T line and a C line by using a magnetic immunochromatographic analyzer (MICAD), calculating the ratio (T/C) of the T line and the C line, and determining the ratio (B/B) of the T/C between a sample and a negative control0) And establishing a standard curve, and calculating the content of the pre-hepatitis B S2 antigen in the sample.
Example 4 test strip sensitivity test for hepatitis B Virus pre-S2 antigen and establishment of Standard Curve
This example provides a sensitivity assay for the detection of pre-hepatitis B S2 antigen. The test strips prepared in example 1 were subjected to sensitivity test using dilutions of 9.8, 19.5, 39, 78, 156, 313, 625, 1250, 2500, 5000ng/ml of pre-hepatitis b S2 antigen, and using equal amounts of PBS buffer as negative control. The results are shown in FIG. 3, and data analysis was performed using Origin, and a four parameter standard curve was fitted as shown in FIG. 4, with the fitting equation:
wherein y is B/B0The ratio, x, is the concentration of the pre-hepatitis B S2 antigen, R2Is 0.994, which indicates that B/B on the test strip is0The ratio has strong correlation with the concentration of the pre-hepatitis B S2 antigen, and can be applied to the competitive method for detecting the concentration of the pre-hepatitis B S2 antigen.
Example 5 test strip specificity for hepatitis B Virus pre-S2 antigen
This example provides a specific assay for the detection of pre-hepatitis B S2 antigen. Samples were assayed using 10. mu.g/mL of pre-hepatitis B S1 antigen, hepatitis B surface antigen, hepatitis B e antigen, and HIV surface antigen, and specificity tests were performed on the test strips prepared in example 1 using the same concentration of pre-hepatitis B S2 antigen as a positive control. The results of the measurement are shown in FIG. 5, and the pre-hepatitis B S2 antigen sample B/B0The ratio is less than 0.1, which indicates a strong positive result; pre-hepatitis B S1 antigen, hepatitis B surface antigen, hepatitis B e antigen, and HIV surface antigen groups B/B0The ratio is close to 1.0, and the test paper shows that the test paper has no cross reaction with other types of virus antigens and has good specificity on the pre-hepatitis B S2 antigen.
Example 6 hepatitis B Virus Pre-S2 antigen test strip clinical serum sample analysis
This example provides a clinical serum sample assay for detection of pre-hepatitis B S2 antigen. The test paper and the commercial pre-hepatitis B S2 antigen kit have the same consistency rate with the hepatitis B surface antigen method, namely 93.3 percent (23/25), and the test paper positive detection rate is higher than the commercial pre-hepatitis B S2 antigen kit, so that the test paper is an effective method for identifying HBV infection.
TABLE 1
Sequence listing
<120> magnetic immunochromatographic test strip and method for rapidly detecting pre-S2 antigen of hepatitis B virus
<130> W-20-1-02079
<150> 2020111977330
<151> 2020-10-31
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 27
<212> PRT
<213> hepatitis B Virus pre-S2 antigen (2 Ambystoma laterale x Ambystoma jeffersonanum)
<400> 1
Cys Met Gln Trp Asn Ser Thr Ala Phe His Gln Ala Leu Gln Asp Pro
1 5 10 15
Arg Val Arg Gly Leu Tyr Phe Pro Ala Gly Gly
20 25
Claims (10)
1. A magnetic immunochromatographic test strip for rapidly detecting a pre-S2 antigen of hepatitis B virus is characterized by comprising a sticky bottom plate, and a sample pad (1), a combination pad (2), a nitrocellulose membrane (3) and a water absorption pad (4) which are sequentially adhered and overlapped on the sticky bottom plate;
the nitrocellulose membrane (3) is coated with a detection line (5) and a quality control line (6); the detection line is hepatitis B virus pre-hepatic S2 antigen-bovine serum albumin conjugate, and the quality control line is mouse IgG.
2. The magnetic immunochromatographic strip for rapid detection of hepatitis B virus pre-S2 antigen according to claim 1, wherein the hepatitis B virus pre-S2 antigen-bovine serum albumin conjugate is a conjugate in which the synthetic hepatitis B pre-S2 antigen polypeptide and bovine serum albumin are linked by the amino group on BSA and the thiol group on hepatitis B virus pre-S2 antigen polypeptide using the bifunctional conjugate 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimide ester.
3. The magnetic immunochromatographic test strip for rapidly detecting the hepatitis B virus pre-S2 antigen of claim 2, wherein the amino acid sequence of the hepatitis B pre-S2 antigen polypeptide is shown in SEQ ID No. 1: SEQ ID No. 1: CMQWN STAFH QALQD PRVRG LYFPA GG are provided.
4. A method for rapidly detecting pre-S2 antigen of hepatitis B virus, which is characterized by comprising the following steps:
(1) pre-incubation: fully and uniformly mixing primary antibody and immunomagnetic beads with a sample to be detected, and standing for 1-30 minutes; the primary antibody is a hepatitis B virus pre-S2 specific monoclonal antibody; the immunomagnetic beads refer to superparamagnetic nanoparticles coupled with goat-anti-mouse secondary antibodies;
(2) fully and uniformly mixing the pre-incubated solution and the chromatographic solution for 5-60 seconds, and dropwise adding the mixture to a sample pad of the magnetic immunochromatographic test strip according to any one of claims 1-3;
(3) and (5) judging the result qualitatively or quantitatively.
5. The detection method according to claim 4, wherein the immunomagnetic beads in the step (1) are prepared by covalently linking superparamagnetic nanoparticles with goat-anti-mouse secondary antibodies using carbodiimide and N-hydroxysuccinimide.
6. The detection method according to claim 4 or 5, wherein the particle size of the superparamagnetic nanoparticle is 100-300 nm.
7. The detection method according to claim 4, wherein the chromatographic solution in the step (2) is a BS buffer solution containing 5% -20% BSA and 1% -5% Tween-20.
8. The detection method according to claim 4, wherein the qualitative judgment result in the step (3) is as follows: and comparing the color depths of the sample and the negative control detection line to judge a negative result and a positive result, wherein if the color of the sample detection line is close to or darker than that of the negative control, the negative result is judged, and if the color of the detection line is lighter than that of the negative control or no color is developed, the positive result is judged.
9. The detection method according to claim 4, wherein the quantitative determination result in the step (3) is quantitative determination by an instrument, and the magnetic signal analyzer is used to determine the content of the magnetic beads bound to the detection line and the quality control line, and calculate the ratio T/C of the two, and the ratio B/B of the T/C between the sample and the negative control is used as the ratio0And establishing a standard curve, and calculating the content of the pre-hepatitis B S2 antigen in the sample.
10. A kit for rapidly detecting pre-S2 antigen of hepatitis B virus, which is characterized by comprising the magnetic immunochromatographic test strip of any one of claims 1 to 3 and immunomagnetic beads, wherein the immunomagnetic beads are superparamagnetic nanoparticles coupled with goat-anti-mouse secondary antibodies.
Applications Claiming Priority (2)
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