CN113624972A - Dry-type immunofluorescence chromatography influenza A/B virus antigen detection kit - Google Patents
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Abstract
The invention relates to the technical field of medical detection and immunoassay, and relates to a dry immunofluorescence chromatography influenza A/B virus antigen detection kit, which comprises a detection card, a detection chip and a detection buffer solution; the detection card comprises a reagent strip, wherein the reagent strip comprises absorbent paper, a nitrocellulose membrane, a combination pad and a sample pad; the nitrocellulose membrane is sequentially coated with a quality control C line, a detection T1 line and a detection T2 line; the fluorescent antibody formed by the mouse anti-human influenza A/B virus monoclonal antibody marked by the fluorescent substance on the solid phase of the binding pad can simply, conveniently, quickly and accurately detect the influenza A/B virus antigen. The invention adopts the immunofluorescence double antibody sandwich method to detect the influenza virus antigen in the sample, compared with the existing method, the operation is simpler and more convenient, the manpower and material resources are saved, the sensitivity is high, the operation of professional personnel is not needed, the fluorescence immunoassay quantitative analyzer can directly output the detection result, and the method is suitable for all levels of medical units, easy to popularize and wide in applicability.
Description
Technical Field
The invention relates to the technical field of medical detection and immunoassay, in particular to a dry immunofluorescence chromatography influenza A/B virus antigen detection kit.
Background
Influenza viruses are classified into types A, B and C, the influenza A virus has the greatest threat and the influenza B virus has the second highest threat. Influenza a viruses are currently classified into 15H subtypes (H1-H15) and 9N subtypes (N1-N9) depending on the antigenicity of Hemagglutinin (HA) and Neuraminidase (NA) proteins. Influenza a viruses often cause influenza epidemics on a larger scale and even worldwide because nucleic acid sequences encoding HA or NA are susceptible to mutation, resulting in altered epitopes of HA or NA, which defeats the original specific immunity of the human population.
The influenza virus is a virus with extremely high morbidity of upper respiratory tract infection, and the morbidity of people all over the world can reach 80-90%, wherein the susceptible people are the old and the infants. The clinical symptoms of the influenza patients are acute high fever, general aching pain and weakness, and often cause upper respiratory tract infection, and some complications such as bronchitis, bronchopneumonia and the like. The influenza virus has short incubation period, strong infectivity and rapid spread, and is easy to generate variation due to strong pathogenicity of the influenza virus, and is easy to cause outbreak and epidemic if people lack immunity to variation strains, so that the influenza virus causes serious harm to human health and life, and simultaneously forms great threat to social public epidemic prevention systems, so the influenza virus becomes one of main research objects of epidemiology. In recent years, with the marketing of some anti-influenza virus drugs, accurate detection of influenza virus has become an important basis for drug use.
Currently, the influenza virus is generally confirmed by adopting chick embryo or MDCK cell isolation culture and hemagglutination inhibition methods, but the operation is complicated, time-consuming and labor-consuming, and the requirement of rapid diagnosis is difficult to meet. With the development and application of rapid diagnostic techniques, the types of influenza virus detection products currently on the market can be basically divided into three major categories: nucleic acid diagnosis (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and colloidal gold immunochromatography. Among them, the RT-PCR method is the preferred diagnosis method, the accuracy is the highest, but the disadvantages are that the detection cost is high, the detection time is long, the requirements for different reaction conditions and spaces in a laboratory are mutually independent, and the operation is carried out by professional staff through equipment, so that the method has certain limitation and is not suitable for large-scale detection application; the ELISA method has speed and accuracy, and is a classic method in clinical laboratory and disease control system laboratory; the colloidal gold method is fastest, simple and feasible, and is suitable for general screening, but the colloidal gold detection sensitivity is low, so that detection omission is often caused.
Disclosure of Invention
The invention aims to solve the problems in the prior art and provides a dry immunofluorescence chromatography influenza A/B virus antigen detection kit which can simply, conveniently, quickly and accurately detect influenza A/B virus antigens and realize quick typing diagnosis. The invention adopts the immunofluorescence double antibody sandwich method to detect the influenza virus antigen in the sample, compared with the existing method, the operation is simpler and more convenient, the manpower and material resources are saved, the sensitivity is high, the operation of professional personnel is not needed, the fluorescence immunoassay quantitative analyzer can directly output the detection result, and the method is suitable for all levels of medical units, easy to popularize and wide in applicability.
The technical scheme of the invention is as follows:
a dry immunofluorescence chromatography influenza A/B virus antigen detection kit comprises a detection card, a detection chip and a detection buffer solution; the detection card comprises a reagent strip, wherein the reagent strip comprises absorbent paper, a nitrocellulose membrane, a combination pad and a sample pad; the nitrocellulose membrane is sequentially coated with a quality control C line, a detection T1 line and a detection T2 line; the solid phase on the combination pad is provided with fluorescent antibody formed by mouse anti-human influenza A/B virus monoclonal antibody with 0.1-0.5 mg/ml (including 0.1mg/ml, 0.2mg/ml, 0.3mg/ml, 0.4mg/ml and 0.5mg/ml) marked by fluorescent substance; the detection buffer solution is 10mM PBS, 0.5-2 wt% BSA, 0.01-0.05 wt% casein, 0.1-0.5 wt% S9 and 0.5 wt% PC 300.
Further, the fluorescent substance labeling process comprises the following steps:
(1) preparing 25-50 mM MES activation buffer solution by double distilled water, and adjusting the pH to 5.8-6.2, preferably 6.0; adjusting the pH of the prepared MES activation buffer solution to 6.3-6.8 to prepare a coupling buffer solution, preferably to 6.5; MES activation buffer concentrations included 25mM, 30mM, 35mM, 40mM, 45mM, and 50 mM;
(2) uniformly mixing fluorescent microspheres and MES (methyl amino acid) activation buffer solution in a volume ratio of 1:10, and ultrasonically ensuring that the fluorescent microspheres are in a monodisperse state to prepare a microsphere suspension; the fluorescent microspheres are preferably 0.22 mu M fluorescent microspheres;
(3) weighing a proper amount of EDC and a certain volume of MES activation buffer solution, fully and uniformly mixing, and preparing EDC solution with the concentration of 10-20 mg/mL; the concentration is preferably 10mg/mL, 15mg/mL, 20 mg/mL;
(4) weighing a proper amount of Sulfo-NHS and a certain volume of MES (MES) activation buffer solution, fully and uniformly mixing, and preparing a Sulfo-NHS solution with the concentration of 10-20 mg/mL; the concentration is preferably 10mg/mL, 15mg/mL, 20 mg/mL;
(5) adding 100-500 uL of EDC solution and 100-500 uL of Sulfo-NHS solution into the microsphere suspension liquid in the step (2) in sequence, performing ultrasonic and mixed incubation for 20-60 min, and activating microspheres to obtain an activated microsphere solution; specific volumes added include 100uL, 200uL, 300uL, 400uL, 500 uL; the mixing incubation time can be 30min, 40min or 50 min;
(6) centrifuging the microsphere solution activated in the step (5), removing supernatant, adding coupling buffer solution to wash microspheres, and fully and uniformly mixing; centrifuging the uniformly mixed solution again, removing a supernatant, then re-suspending the microspheres by using a coupling buffer solution, and performing ultrasonic treatment to ensure that the microspheres are in a monodisperse state to obtain a microsphere suspension;
(7) respectively transferring a certain volume of microsphere suspension into 1-4 mg/mL (preferably 2mg/mL) of mouse anti-human influenza A virus monoclonal antibody solution prepared by a coupling buffer solution and 1-4 mg/mL (preferably 2mg/mL) of mouse anti-human influenza B virus monoclonal antibody solution prepared by the coupling buffer solution, performing ultrasonic and mixed incubation for 2-6 h, and performing antibody coupling;
the concentration of the mouse anti-human influenza A virus monoclonal antibody solution in the prepared microsphere suspension is 0.1-0.5 mg/mL, preferably 0.4 mg/mL; the concentration of the mouse anti-human influenza B virus monoclonal antibody solution in the prepared microsphere suspension is 0.1-0.5 mg/mL, preferably 0.2 mg/mL;
(8) adding glycine with the final concentration of 100mM into the solution obtained in the step (7) to terminate the reaction, and performing mixed incubation for 20-60 min, preferably for 30 min; then centrifuging the mixed solution, removing the supernatant, adding 25mM Tris blocking buffer solution containing 0.5-5 wt% of casein for heavy suspension, and fully and uniformly mixing; centrifuging the resuspended solution again, removing the supernatant, adding 25mM Tris + 0.5-5 wt% casein blocking buffer solution for resuspending, carrying out ultrasonic treatment, and carrying out mixed incubation for 1.5-2.5 h for blocking reaction; here, the blocking buffer mix incubation time is preferably 2 h;
(9) preparing microsphere preservative solution comprising 25mM Tris +0.5 wt% BSA +0.2 wt% casein +1 wt% trehalose +0.01 wt% NaN3(ii) a And (4) centrifuging the solution obtained in the step (8), removing supernatant, resuspending the microsphere preservation solution, ultrasonically ensuring that the microspheres are in a monodisperse state, and preserving at 4 ℃.
Furthermore, the ultrasound adopts probing ultrasound, and the mixed incubation mode is to adopt a rotating disc type mixer for mixed incubation; the centrifugal rotating speed is 12000-15000 rpm, the centrifugal time is 12-20 min, and the centrifugal temperature is 4 ℃. The centrifugation rotating speed can be selected from any one of 12000rpm, 13000rpm, 14000rpm or 15000rpm, and the centrifugation time comprises 12min, 15min, 18min and 20 min.
Further, the quality control C line is coated with a goat anti-mouse antibody with the concentration of 0.1-3 mg/ml, the goat anti-mouse antibody is diluted by a sample diluent, and the sample diluent is sprayed on the quality control C line after the goat anti-mouse antibody is completely dried so as to increase the quality control stability; the detection T1 coil is coated with a mouse anti-human influenza A virus monoclonal antibody with the concentration of 1.5-3 mg/ml, and the coated antibody is diluted by a sample diluent to increase the stability of the coated antibody; the detection T2 coil is coated with a mouse anti-human influenza B virus monoclonal antibody with the concentration of 1.5-3 mg/ml, and the coated antibody is diluted by sample diluent to increase the stability of the coated antibody; the sample diluent is a PBS buffer solution containing 1-5 wt% of sucrose.
Furthermore, the absorbent paper and the sample pad are respectively arranged at two ends of the nitrocellulose membrane, the combination pad is positioned between the sample pad and the nitrocellulose membrane, and the components are overlapped by 1-2 mm in a cross mode.
Furthermore, the fluorescent antibody is diluted by a solid phase solution and then fixed at the middle position of the combination pad, and the solid phase solution comprises 25mM Tris + 1-10 wt% of sucrose + 1-10 wt% of trehalose.
Further, the fluorescent antibody is diluted to 5-20 times by solid phase liquid, then sprayed to the central position of the bonding pad according to the spraying amount of 2-10 ul/cm, and dried in an oven for 2 hours, so that the fluorescent antibody can be fixed on the bonding pad.
Further, the sample pad needs to be treated by a treatment solution, the treatment solution is 10mM PBS buffer solution, the treatment operation is to place the sample pad in the 10mM PBS buffer solution, soak for 30-35 min, dry in an oven for 4h, and store for later use.
Further, the combined pad needs to be treated by a treatment solution, wherein a buffer system of the treatment solution is 10mM PBS buffer solution, and the buffer system comprises 1-3 wt% of surfactant S9, 0.1-2 wt% of casein, 0.1-2 wt% of PVP and 1-5 wt% of sucrose; and the treatment operation comprises the steps of soaking the bonding pad in the treatment solution for 30-35 min, drying for 4h in an oven, and storing for later use.
The invention has the beneficial effects that:
(1) the invention provides a rapid detection kit capable of simultaneously detecting influenza A/B virus antigens, which completes clinical typing of influenza viruses through one-time sample adding detection, is suitable for large-scale epidemiological investigation and handling of emergencies, can effectively save cost, is convenient and rapid, judges results in 15 minutes, does not need professionals, achieves real POCT detection, and is generally suitable for outpatient and emergency detection.
(2) The invention adopts a novel fluorescent labeling process, adopts fluorescent microspheres as tracers, modifies carboxyl with proper density on the surfaces of the fluorescent microspheres for covalent coupling with protein or antibody, and improves the labeling stability of the fluorescent probe; the fluorescent microspheres are kept at higher activation efficiency by improving the using amount of the activation buffer solution, and the microspheres and the antibodies are efficiently coupled by improving the pH value of the coupling buffer solution, so that ultrahigh sensitivity is realized, the sensitivity is 10-100 times that of the traditional POCT, the detection limit is obviously reduced, the positivity can be detected in 3-5 days after the patients suffer from the diseases, and the detection window period is shortened.
(3) The invention provides a rapid and accurate dry immunofluorescence chromatography influenza A/B virus antigen detection kit, which is characterized in that more than ten antibody raw materials are screened, the obtained raw materials applied to the kit can pass through national standards, and influenza A/B virus strains have no cross reaction.
(4) The result is read by means of a fluorescence immunoassay quantitative analyzer, so that the problem that a part of weak positive samples are not well interpreted by a colloidal gold immunochromatography method is solved; through the collection to clinical data, upload high in the clouds server, can conveniently acquire the running state of instrument, the demand of test at the background, through the processing to big data, supplementary clinician reads the test result.
Drawings
FIG. 1 is a schematic structural diagram of a detection kit provided by the present invention;
FIG. 2 is a top view of FIG. 1;
FIG. 3 is a top view of the reagent strip of FIG. 1;
in the above figures, 1, a lower cover; 2. an upper cover; 3. a sample pad; 4. a bonding pad; 5. a nitrocellulose membrane; 6. absorbent paper; 7. t2 detection line; 8. t1 detection line; 9. a quality control line C; 10. a sample application hole; 11. and detecting a window.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
For a further understanding of the invention, reference will now be made to the following description taken in conjunction with the accompanying drawings and examples.
Example 1
As shown in FIGS. 1 to 3, the present invention relates to a dry immunofluorescence chromatography influenza A/B virus antigen detection kit, comprising a detection card, a detection chip and a detection buffer solution; the detection card includes lower cover 1, upper cover 2 and reagent strip, and lower cover 1 and the 2 mutual buckle of upper cover are connected, form between lower cover 1 and the upper cover 2 and hold the chamber, and the reagent strip is located and holds the intracavity, and upper cover 2 is provided with application of sample hole 10 and detection window 11.
The reagent strip comprises absorbent paper 6, a nitrocellulose membrane 5, a combination pad 4 and a sample pad 3, wherein the absorbent paper 6 is positioned at one end of the nitrocellulose membrane 5, the sample pad 3 is positioned at the other end of the nitrocellulose membrane 5, the combination pad 4 is positioned between the sample pad 3 and the nitrocellulose membrane 5, and all components are overlapped by 1mm in a cross mode. The cellulose nitrate membrane 5 is sequentially coated with a quality control C line 9, a T1 detection line 8 and a T2 detection line 7, a solid phase on the combination pad 4 is provided with a fluorescent antibody, the sample adding hole 10 corresponds to the sample pad 3, and the position of the detection window 11 corresponds to the positions of the T2 detection line 7, the T1 detection line 8 and the quality control C line 9.
Collecting nasopharyngeal swab samples of a person to be detected according to a standard, putting the collected swab into a detection buffer solution, rotating and squeezing the swab for at least 10 times, squeezing and discarding the swab, and tightly plugging a dripper to obtain the sample to be detected. After 2-3 drops (about 100ul) of a sample to be detected is dripped into a reagent strip sample adding hole 10, if influenza A or B virus exists in the sample to be detected, the sample to be detected migrates to a binding pad 4 under the action of chromatography to form an antigen-antibody complex with a fluorescent antibody, continuously moves upwards along a membrane under the action of chromatography, is captured by a mouse anti-human influenza A virus monoclonal antibody to form a complex when migrating to a T2 detection line 7, is captured by the mouse anti-human influenza A virus monoclonal antibody to form a complex when migrating to a T1 detection line 8, the fluorescent antibody signal of the complex is in direct proportion to the concentration of the influenza A or B virus antigen, and whether the sample has the influenza A/B virus antigen can be calculated after being analyzed by a fluorescence analyzer.
Example 2
A dry immunofluorescence chromatography influenza A/B virus antigen detection kit comprises a detection card, a detection chip and a detection buffer solution; the detection buffer is 10mM PBS +2 wt% BSA +0.02 wt% casein +0.3 wt% S9+0.5 wt% PC 300; the detection card comprises a reagent strip, wherein the reagent strip comprises absorbent paper, a nitrocellulose membrane, a combination pad and a sample pad;
the nitrocellulose membrane is sequentially coated with a quality control C line, a detection T1 line and a detection T2 line;
the quality control C line is coated with a goat anti-mouse antibody with the concentration of 0.1mg/ml, the goat anti-mouse antibody is diluted by adopting PBS (phosphate buffer solution) of 3 wt% of cane sugar, and the PBS buffer solution of 3 wt% of cane sugar is sprayed on the quality control C line after the goat anti-mouse antibody is completely dried so as to increase the quality control stability; the detection T1 line is coated with a mouse anti-human influenza A virus monoclonal antibody with the concentration of 1.5mg/ml, and the coated antibody is diluted by PBS buffer solution of 2 wt% of sucrose to increase the stability of the coated antibody; the T2 detection line is coated with a mouse anti-human influenza B virus monoclonal antibody with the concentration of 1.5mg/ml, the coated antibody is diluted by 4 wt% of sucrose in PBS buffer solution to increase the stability of the coated antibody, and the coated antibody is dried in an oven for 30min for later use.
The solid phase on the binding pad is provided with a mouse anti-human influenza A/B virus monoclonal antibody marked by fluorescent substance.
The fluorescent substance labeling process comprises the following steps:
(1) placing 0.533g of MES into a volumetric flask, adding double distilled water to dissolve the MES and fixing the volume to 100mL to prepare 25mM MES, and adjusting the pH to 6.0 by using NaOH solution to prepare MES activation buffer solution; transferring 10mM MES activation buffer solution, and adjusting the pH value to 6.5 by using NaOH solution to prepare coupling buffer solution;
(2) uniformly mixing 0.22 mu M fluorescent microspheres and MES activation buffer solution in a volume ratio of 1:10, ensuring that the microspheres are in a monodisperse state by using probing type ultrasound, and preparing 1mL microsphere suspension;
(3) 5mg EDC and 500uL MES activation buffer were added to a clean centrifuge tube, and mixed by inversion to prepare a 10mg/mL EDC solution (note: the activation reagent was ready to use);
(4) add 5mg of Sulfo-NHS and 500uL of MES activation buffer to a clean centrifuge tube and mix by inversion to prepare a 10mg/mL solution of Sulfo-NHS: (Note: the activating reagent is ready to use as it is);
(5) the microsphere activation step is that 100-500 uL of EDC solution and 100-500 uL of Sulfo-NHS solution are added into the microsphere suspension liquid in the step (2) in sequence, and the mixture is incubated for 30min by probing ultrasonic and mixing with a rotating disc type mixer;
in the above step, the activation ratio was adjusted by changing the addition amounts of EDC solution and Sulfo-NHS solution, both of which were 10mg/mL, EDC: NHS (v/v) is (1-5): (1-5), when 0.5mL of microsphere suspension is marked, adding 100uL of EDC solution and 500uL of Sulfo-NHS solution (activation ratio is 1:5) in the process of marking the influenza A virus monoclonal antibody, adding 100uL of EDC solution and 300uL of Sulfo-NHS solution (activation ratio is 1:3) in the process of marking the influenza B virus monoclonal antibody, detecting by using national references S1-S5 with the lowest detection limit of influenza A/B virus antigen, wherein the detection results are shown in tables 1 and 2, the solution is uniform and does not generate aggregation phenomenon in the microsphere activation process, the activation efficiency is highest, the sensitivity is high, and the microspheres do not generate aggregation after being placed at 2-8 ℃ for 7 days.
TABLE 1 detection results of national reference substances for the lowest detection limit of influenza A virus antigens
TABLE 2 national reference test results for lowest detection limit of influenza B virus antigen
(6) Centrifuging the microsphere suspension activated in the step (5), removing supernatant, adding coupling buffer solution to resuspend the microspheres, and fully mixing uniformly; centrifuging the uniformly mixed solution, removing a supernatant, re-suspending the microspheres by using a coupling buffer solution, and ensuring that the microspheres are in a monodisperse state by using probing type ultrasound to obtain a microsphere suspension;
(7) adding 500 mu L of the microsphere suspension obtained in the step (6) into 2mg/mL of mouse anti-human influenza A virus monoclonal antibody solution prepared by 100 mu L of coupling buffer solution, adding 500 mu L of the microsphere suspension into 2mg/mL of mouse anti-human influenza B virus monoclonal antibody solution prepared by 50 mu L of coupling buffer solution, probing into ultrasonic waves, mixing and incubating for 2h by using a rotating disc type mixer, and carrying out antibody coupling;
in the step, the final concentration of the antibody in the microsphere suspension is selected from 0.1mg/mL, 0.2mg/mL, 0.3mg/mL, 0.4mg/mL and 0.5mg/mL, and when the mouse anti-human influenza A virus monoclonal antibody with the concentration of 0.4mg/mL and the mouse anti-human influenza B virus monoclonal antibody with the concentration of 0.2mg/mL are selected through tests, the microspheres do not have aggregation phenomenon in the antibody coupling process and have the highest labeling efficiency.
(8) Adding glycine into the solution obtained in the step (7) to terminate the reaction, wherein the final concentration of the glycine is 100mM, and mixing for 30min by using a rotating disc type mixer; then centrifuging the mixed solution, removing the supernatant, adding 25mM Tris blocking buffer solution containing 1 wt% of casein, and resuspending the microspheres; centrifuging the resuspended solution, removing the supernatant, adding 25mM Tris +1 wt% casein blocking buffer solution for resuspension, probing in ultrasonic wave to ensure that the microspheres are in a monodisperse state, and mixing for 2h by using a rotating disc type mixer for blocking reaction;
(9) preparation included 25mM Tris +0.5 wt% BSA +0.2 wt% casein +1 wt% trehalose +0.01 wt% NaN3The microsphere preserving fluid of (1); and (4) centrifuging the solution obtained in the step (8), removing the supernatant, adding the prepared microsphere preservation solution, ensuring that the microspheres are in a monodisperse state by using probing type ultrasound, and preserving at 4 ℃.
The centrifugation speed in each step was 12000rpm, the centrifugation time was 15min, and the centrifugation temperature was 4 ℃.
The fluorescent antibody solid phase process comprises the following steps:
diluting the marked fluorescent microsphere antibody by using solid phase liquid, wherein the influenza A virus fluorescent antibody is diluted by 10 times, the influenza B virus fluorescent antibody is diluted by 5 times, the solid phase liquid 25mM Tris +3 wt% sucrose +3 wt% trehalose is sprayed to the central position of the combination pad according to the spraying amount of 5ul/cm, and the combination pad is dried in an oven for 2 hours after the spraying is finished.
The combined pad needs to be treated by a treatment solution, and the buffer system selected by the treatment solution is 10mM PBS, including surfactant S9, casein, PVP and sucrose; the concentration of surfactant S9 was 1 wt%; the concentration of casein was 0.2 wt%; the concentration of PVP is 0.2 wt%; the concentration of sucrose was 3 wt%; the treatment method of the bonding pad comprises the steps of putting the glass fiber into the pretreatment liquid, soaking for 30min, drying for 4h in an oven, and storing for later use.
The sample pad needs to be treated by the treatment solution, the treatment method is to put the glass fiber into 10mM PBS pretreatment solution, soak for 30min, dry in an oven for 4h, and store for later use.
The nitrocellulose membrane, the combination pad, the sample pad and the absorbent paper are sequentially stuck on a PVC plate to be assembled into a test strip, and then the test strip and the clamping plate block shell are assembled into a reagent detection card.
The using method of the kit comprises the following specific operation steps:
(1) collecting nasopharyngeal swab samples of a person to be detected in a standardized manner, putting the collected swabs into a detection buffer solution, rotating and squeezing the swabs for at least 10 times, squeezing and discarding the swabs, and tightly plugging a dripper to obtain a sample to be detected;
(2) inserting a detection chip, and reading chip information matched with the product by an instrument;
(3) and (3) dropwise adding 2-3 drops (about 100ul) of the sample to be detected into a reagent strip sample adding hole 10, horizontally placing at room temperature for 15min, and then inserting into a fluorescence immunoassay quantitative analyzer, wherein the analyzer can automatically judge whether a positive result (the result is more than or equal to 1), a negative result (less than 1) and a detection card are effective or not.
Example 3
A dry immunofluorescence chromatography influenza A/B virus antigen detection kit comprises a detection card, a detection chip and a detection buffer solution; the detection card comprises a reagent strip, wherein the reagent strip comprises absorbent paper, a nitrocellulose membrane, a combination pad and a sample pad; the nitrocellulose membrane is sequentially coated with a quality control C line, a detection T1 line and a detection T2 line; the solid phase on the combination pad is provided with a fluorescent antibody formed by a mouse anti-human influenza A virus/B virus monoclonal antibody with 0.1-0.5 mg/ml marked by a fluorescent substance; the assay buffer was 10mM PBS +0.5 wt% BSA +0.05 wt% casein +0.5 wt% S9+0.5 wt% PC 300.
Further, the fluorescent substance labeling process comprises the following steps:
(1) putting 1.066g of MES into a volumetric flask, adding double distilled water to dissolve the MES and fixing the volume to 100mL, preparing 50mM MES, and adjusting the pH to 5.8; adjusting the pH value of the prepared MES activation buffer solution to 6.8 to prepare a coupling buffer solution;
(2) uniformly mixing fluorescent microspheres and MES (MES) activation buffer solution in a volume ratio of 1:10, and ultrasonically ensuring that the fluorescent microspheres are in a monodisperse state to prepare 1mL of microsphere suspension; the fluorescent microspheres are preferably 0.22 mu M fluorescent microspheres;
(3) weighing a proper amount of EDC and a certain volume of MES activation buffer solution to prepare 15mg/mL EDC solution;
(4) weighing a proper amount of Sulfo-NHS and a certain volume of MES activated buffer solution to prepare a 15mg/mL Sulfo-NHS solution;
(5) adding 100uL of EDC solution and 500uL of Sulfo-NHS solution into the microsphere suspension liquid in the step (2) in sequence, performing ultrasonic treatment and mixed incubation for 60min, and activating microspheres to obtain an activated microsphere solution;
(6) centrifuging the microsphere solution activated in the step (5), removing supernatant, adding coupling buffer solution to wash microspheres, and fully and uniformly mixing; centrifuging the uniformly mixed solution again, removing a supernatant, then re-suspending the microspheres by using a coupling buffer solution, and performing ultrasonic treatment to ensure that the microspheres are in a monodisperse state to obtain a microsphere suspension;
(7) respectively transferring 500 mu L of microsphere suspension, adding the microsphere suspension into 2mg/mL of mouse anti-human influenza A virus monoclonal antibody solution prepared by coupling buffer solution and 2mg/mL of mouse anti-human influenza B virus monoclonal antibody solution prepared by coupling buffer solution, performing ultrasonic treatment and mixed incubation for 3h, and performing antibody coupling;
the concentration of the mouse anti-human influenza A virus monoclonal antibody solution in the prepared microsphere suspension is 0.1-0.5 mg/mL, preferably 0.4 mg/mL; the concentration of the mouse anti-human influenza B virus monoclonal antibody solution in the prepared microsphere suspension is 0.1-0.5 mg/mL, preferably 0.2 mg/mL;
(8) adding glycine with the final concentration of 100mM into the solution obtained in the step (7) to terminate the reaction, and mixing and incubating for 40 min; then centrifuging the mixed solution, removing the supernatant, adding 25mM Tris blocking buffer solution containing 5 wt% of casein for resuspension, centrifuging, removing the supernatant, adding the blocking buffer solution for resuspension, performing ultrasonic treatment, and performing mixed incubation for 2h to perform blocking reaction;
(9) preparing microsphere preservative solution comprising 25mM Tris +0.5 wt% BSA +0.2 wt% casein +1 wt% trehalose +0.01 wt% NaN3(ii) a And (4) centrifuging the solution obtained in the step (8), removing supernatant, resuspending the microsphere preservation solution, ultrasonically ensuring that the microspheres are in a monodisperse state, and preserving at 4 ℃.
The centrifugal speed is 15000rpm, the centrifugal time is 12min, and the centrifugal temperature is 4 ℃.
The quality control C line is coated with a goat anti-mouse antibody with the concentration of 1mg/ml, the goat anti-mouse antibody is diluted by adopting a PBS (phosphate buffer solution) of 2 wt% of cane sugar, and the PBS buffer solution of 4 wt% of cane sugar is sprayed on the quality control C line after the goat anti-mouse antibody is completely dried so as to increase the quality control stability; the detection T1 line is coated with a mouse anti-human influenza A virus monoclonal antibody with the concentration of 2mg/ml, and the coated antibody is diluted by a PBS buffer solution of 5 wt% of sucrose to increase the stability of the coated antibody; the T2 detection line is coated with a mouse anti-human influenza B virus monoclonal antibody with the concentration of 2.5mg/ml, the coated antibody is diluted by PBS buffer solution of 2 wt% of cane sugar to increase the stability of the coated antibody, and the coated antibody is dried in an oven for 30min for later use.
The fluorescent antibody is diluted by solid phase liquid and then fixed at the middle position of the binding pad, and the solid phase liquid comprises 25mM Tris +5 wt% sucrose +5 wt% trehalose.
And (3) diluting the fluorescent antibody to 10 times by using solid phase liquid, spraying the diluted fluorescent antibody to the central position of the binding pad according to the spraying amount of 2ul/cm, and drying in an oven for 2 hours to fix the fluorescent antibody on the binding pad.
The sample pad needs to be treated by a treatment solution, the treatment solution is 10mM PBS buffer solution, the treatment operation is to place the sample pad in the 10mM PBS buffer solution, soak for 30min, dry the sample pad in an oven for 4h, and store the sample pad for later use.
The combined pad needs to be treated by a treatment solution, wherein the buffer system of the treatment solution is 10mM PBS buffer solution, and the treatment solution comprises 3 wt% of surfactant S9, 2 wt% of casein, 0.1 wt% of PVP and 5 wt% of cane sugar; the treatment operation comprises soaking the bonding pad in the above treatment solution for 30min, oven drying for 4h, and storing for use.
The nitrocellulose membrane, the combination pad, the sample pad and the absorbent paper are sequentially stuck on a PVC plate to be assembled into a test strip, and then the test strip and the clamping plate block shell are assembled into a reagent detection card.
Example 4
1. Carrying out stability investigation on the assembled kit
The kit is placed at 37 ℃ for destructive test, and the stability detection standard of the kit is periodically examined as follows:
(1) and (3) negative quality control quality detection results:
6 parts of negative quality control materials are used for detection, the detection results are shown in table 3, and the influenza A virus and the influenza B virus are negative.
TABLE 3 detection results of negative quality control products of kit after accelerated destruction at 37 deg.C
(2) Positive quality control quality detection result:
detecting by using 6 parts of positive quality control substances, wherein the detection results are shown in table 4, wherein the quality control substances 1-3 are positive for influenza B virus and negative for influenza A virus; the quality control products 4-6 are positive for influenza A virus and negative for influenza B virus.
TABLE 4 test results of positive quality control products of the kit after accelerated destruction at 37 deg.C
The test shows that the kit can be stable for at least 60 days at 37 ℃, and according to the stability experiment principle, the Arrhenius formula: d (Ink)/dT (Ea/RT 2 Ea), is preserved for 24 months at normal temperature, is equivalent to 60 days of destruction at 37 ℃, can meet the clinical requirements of hospital clinics and health quarantine departments, and can also be used for disease diagnosis research of colleges and universities and scientific research institutions.
Example 5
1. The performance evaluation is carried out by taking the influenza A/B virus antigen detection kit prepared by the invention in an industry-universal mode, and the results are as follows:
(1) positive reference detection result
Detecting by using national reference products with positive influenza A/B virus antigens, and strictly operating according to a specification, wherein the influenza A viruses are all positive and the influenza B viruses are all negative from P1 to P4; the influenza A viruses from P5 to P10 are all positive, and the influenza B viruses are all negative; the positive match rate (+/+) was 10/10. The results are shown in Table 5.
TABLE 5 detection results of positive reference compliance rates
(2) Negative reference detection result
The national reference substance with negative influenza A/B virus antigen is used for detection, the operation is strictly carried out according to the specification, the influenza A viruses N1-N6 and the influenza B viruses are negative, and the negative coincidence rate (-/-) is 6/6. The results are shown in Table 6.
TABLE 6 negative reference coincidence rate test results
| Negative reference substance | N1 | N2 | N3 | N4 | N5 | N6 |
| FluA | 0.3- | 0.4- | 0.2- | 0.3- | 0.3- | 0.1- |
| FluB | 0.3- | 0.2- | 0.3- | 0.1- | 0.4- | 0.2- |
(3) National reference substance detection result of lowest detection limit of influenza A/B virus antigen
The detection is carried out by national reference products S1-S5 with the lowest detection limit of influenza A/B virus antigens and strictly operated according to the instructions, and the detection results are shown in Table 7.
S1: the titer is not higher than 1.22X 104TCID50The detection result is positive influenza A virus and negative influenza B virus;
s2: the titer is not higher than 3.25 multiplied by 104TCID50The detection result is positive influenza A virus and negative influenza B virus;
s3: the titer is not higher than 5.25X 105TCID50The detection result is positive for the influenza B virus and negative for the influenza A virus;
s4: the titer is not higher than 1.00 multiplied by 104TCID50The detection result is positive for influenza B virus and negative for influenza A virus;
s5: the titer is not higher than 1.25X 103TCID50The detection result is positive for influenza A virus and negative for influenza B virus.
TABLE 7 national reference test results for minimum detection limits for influenza A/B virus antigens
| Minimum detection limit | S1 | S2 | S3 | S4 | S5 |
| FluA | 1.6+ | 1.5+ | 0.3- | 0.2- | 1.9+ |
| FluB | 0.3- | 0.3- | 1.4+ | 1.6+ | 0.3- |
(4) Detection result of antigen repetitive national reference substance of influenza A/B virus
Taking the same batch of kit, performing parallel detection 10 times by using repetitive national reference products (CV1 and CV2) of influenza A/B virus antigen, strictly operating according to the instruction, and calculating the average value of 10 detection results
And Standard Deviation (SD), calculating Coefficient of Variation (CV) according to equation (1),
in the formula:
CV-coefficient of variation;
SD-Standard deviation;
And (3) detection results: CV1 is positive for influenza A virus and negative for influenza B virus, wherein CV of the positive detection result of influenza A virus is 7.5%; CV2 is positive for influenza B virus and negative for influenza A virus, wherein CV of positive detection result of influenza B virus is 10.4%, CV of positive result is less than 15.0%, CV of negative result meets requirement, CV of negative result does not do requirement, and detection result is shown in Table 8.
TABLE 8 influenza A/B Virus antigen repetitive national reference test results
| Reference article | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| CV1(FluA) | 6.3+ | 5.9+ | 5.4+ | 6.3+ | 6.5+ | 5.5+ | 5.6+ | 5.9+ | 6.2+ | 5.2+ |
| CV1(FluB) | 0.3- | 0.2- | 0.1- | 0.3- | 0.3- | 0.2- | 0.1- | 0.2- | 0.2- | 0.4- |
| CV2(FluB) | 4.7+ | 5.1+ | 4.4+ | 4.6+ | 4.8+ | 4.2+ | 4.4+ | 4.9+ | 5.3+ | 5.9+ |
| CV2(FluA) | 0.1- | 0.2- | 0.4- | 0.3- | 0.2- | 0.3- | 0.3- | 0.2- | 0.4- | 0.2- |
Test examples
1. Sample testing
The kit and a kit on the market are used for carrying out comparative test, 30 nasopharyngeal swabs of respiratory tract patients and 100 nasopharyngeal swabs of healthy physical examination are collected from clinic, the kit and the kit on the market are used for simultaneous detection and comparison, the detection results of the nasopharyngeal swabs of the respiratory tract patients are shown in the following table 9, the specificity and the detection rate are shown in the following table 10, and the total coincidence rate is shown in the following table 11. Wherein the total coincidence rate of the two kits of the influenza A virus and the influenza B virus is 98.5 percent, and the total coincidence rate is good, so the kit can be used for clinical diagnosis.
TABLE 9 comparison of the detection results of nasopharyngeal swabs of 30 respiratory tract patients in the present kit and the marketed kit
TABLE 10 comparison of specificity and detection Rate of two different nasopharyngeal swab kits
Table 11130 samples Total coincidence rate of two kits
2. Screening by raw material manufacturer
The method is characterized in that the antibodies of different manufacturers are screened by national reference products with positive influenza A/B virus antigens, only representative manufacturer results are listed, wherein the manufacturer A and the manufacturer C have the condition that the national reference products with positive influenza A virus antigens are missed to be detected, the manufacturer D has the condition that influenza A virus strains and influenza B virus strains have cross reaction, only P1-P4 influenza B viruses in the detection results of the manufacturer B are positive, the influenza A viruses are negative, the influenza A viruses P5-P10 influenza A viruses are positive, the influenza B viruses are negative, the detection level meets the requirement, and the influenza A/B virus strains have no cross reaction. The results are shown in Table 12.
TABLE 12 detection results of national reference positive for influenza A/B virus antigens
The invention provides a rapid, specific and dry immunofluorescence chromatography influenza A/B virus antigen detection kit, which solves the problem that a colloidal gold immunochromatography method is not good for distinguishing partial weak positive samples by means of reading of a fluorescence immunoassay quantitative analyzer; by adopting a novel fluorescent labeling process and adopting fluorescent microspheres as tracers, the labeling stability of the fluorescent probe is improved, the ultrahigh sensitivity is realized, the positivity can be detected after the patient is attacked for 3-5 days, and the detection window period is shortened.
Through screening more than ten antibody raw materials, the obtained raw materials applied to the kit can pass through national standards, and influenza A/B virus strains have no cross reaction; meanwhile, the kit can simultaneously detect influenza A/B virus antigens, complete clinical typing of influenza viruses through one-time detection, is suitable for large-scale epidemiological investigation and handling of emergencies, is simple and quick to operate, does not need professionals, achieves real POCT detection, and is generally suitable for outpatient and emergency detection.
Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art will understand that various changes, modifications and substitutions can be made without departing from the spirit and scope of the present invention. Any modification, equivalent replacement, or modification made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (9)
1. A dry immunofluorescence chromatography influenza A/B virus antigen detection kit is characterized by comprising a detection card, a detection chip and a detection buffer solution; the detection card comprises a reagent strip, wherein the reagent strip comprises absorbent paper, a nitrocellulose membrane, a combination pad and a sample pad; the nitrocellulose membrane is sequentially coated with a quality control C line, a detection T1 line and a detection T2 line; the solid phase on the combination pad is provided with a fluorescent antibody formed by a mouse anti-human influenza A virus/B virus monoclonal antibody with 0.1-0.5 mg/ml marked by a fluorescent substance; the detection buffer solution is 10mM PBS, 0.5-2 wt% BSA, 0.01-0.05 wt% casein, 0.1-0.5 wt% S9 and 0.5 wt% PC 300.
2. The kit according to claim 1, wherein the fluorescent substance labeling process comprises the steps of:
(1) preparing 25-50 mM MES activation buffer solution by double distilled water and adjusting the pH value to 5.8-6.2; adjusting the pH value of the prepared MES activation buffer solution to 6.3-6.8 to prepare a coupling buffer solution;
(2) uniformly mixing fluorescent microspheres and MES (methyl amino acid) activation buffer solution in a volume ratio of 1:10, and ultrasonically ensuring that the fluorescent microspheres are in a monodisperse state to prepare a microsphere suspension;
(3) weighing a proper amount of EDC and a certain volume of MES activation buffer solution, fully and uniformly mixing, and preparing EDC solution with the concentration of 10-20 mg/mL;
(4) weighing a proper amount of Sulfo-NHS and a certain volume of MES (MES) activation buffer solution, fully and uniformly mixing, and preparing a Sulfo-NHS solution with the concentration of 10-20 mg/mL;
(5) adding 100-500 uL of EDC solution and 100-500 uL of Sulfo-NHS solution into the microsphere suspension liquid in the step (2) in sequence, performing ultrasonic and mixed incubation for 20-60 min, and activating microspheres to obtain an activated microsphere solution;
(6) centrifuging the microsphere solution activated in the step (5), removing supernatant, adding coupling buffer solution to wash microspheres, and fully and uniformly mixing; centrifuging the uniformly mixed solution again, removing a supernatant, then re-suspending the microspheres by using a coupling buffer solution, and performing ultrasonic treatment to ensure that the microspheres are in a monodisperse state to obtain a microsphere suspension;
(7) respectively transferring a certain volume of microsphere suspension into a mouse anti-human influenza A virus monoclonal antibody solution prepared by a coupling buffer solution and a mouse anti-human influenza B virus monoclonal antibody solution prepared by the coupling buffer solution, performing ultrasonic and mixed incubation for 2-6 h, and performing antibody coupling;
the concentration of the mouse anti-human influenza A virus monoclonal antibody solution in the prepared microsphere suspension is 0.1-0.5 mg/mL, preferably 0.4 mg/mL; the concentration of the mouse anti-human influenza B virus monoclonal antibody solution in the prepared microsphere suspension is 0.1-0.5 mg/mL, preferably 0.2 mg/mL;
(8) adding glycine with the final concentration of 100mM into the solution obtained in the step (7) to terminate the reaction, and performing mixed incubation for 20-60 min; then centrifuging the mixed solution, removing the supernatant, adding 25mM Tris blocking buffer solution containing 0.5-5 wt% of casein for heavy suspension, and fully and uniformly mixing; centrifuging the resuspended solution again, removing the supernatant, adding 25mM Tris + 0.5-5 wt% casein blocking buffer solution for resuspending, carrying out ultrasonic treatment, and carrying out mixed incubation for 1.5-2.5 h for blocking reaction;
(9) preparing microsphere preservative solution comprising 25mM Tris +0.5 wt% BSA +0.2 wt% casein +1 wt% trehalose +0.01 wt% NaN3(ii) a And (4) centrifuging the solution obtained in the step (8), removing supernatant, resuspending the microsphere preservation solution, ultrasonically ensuring that the microspheres are in a monodisperse state, and preserving at 4 ℃.
3. The kit according to claim 2, wherein the ultrasound is probe ultrasound, and the mixed incubation mode is mixed incubation by using a rotating disc mixer; the centrifugal rotating speed is 12000-15000 rpm, the centrifugal time is 12-20 min, and the centrifugal temperature is 4 ℃;
adjusting the pH value of MES activation buffer solution to 6.0 and adjusting the pH value to 6.5 in the step (1) to prepare coupling buffer solution; the fluorescent microspheres obtained in the step (2) are 0.22 mu M fluorescent microspheres; the concentration of the mouse anti-human influenza A virus monoclonal antibody solution prepared by the coupling buffer solution in the step (7) is 1-4 mg/mL, and the concentration of the mouse anti-human influenza B virus monoclonal antibody solution prepared by the coupling buffer solution is 1-4 mg/mL; after the glycine termination reaction in the step (8), mixing and incubating for 30 min; resuspending, sonicating, mixing with blocking buffer and incubating for 2 h.
4. The kit according to claim 1, wherein the quality control C line is coated with a goat anti-mouse antibody with a concentration of 0.1-3 mg/ml, the goat anti-mouse antibody is diluted by a sample diluent, and the sample diluent is sprayed on the quality control C line after the goat anti-mouse antibody is completely dried to increase the quality control stability; the detection T1 coil is coated with a mouse anti-human influenza A virus monoclonal antibody with the concentration of 1.5-3 mg/ml, and the coated antibody is diluted by a sample diluent to increase the stability of the coated antibody; the detection T2 coil is coated with a mouse anti-human influenza B virus monoclonal antibody with the concentration of 1.5-3 mg/ml, and the coated antibody is diluted by sample diluent to increase the stability of the coated antibody; the sample diluent is a PBS buffer solution containing 1-5 wt% of sucrose.
5. The kit according to claim 1, wherein the absorbent paper and the sample pad are respectively arranged at two ends of the nitrocellulose membrane, the combination pad is positioned between the sample pad and the nitrocellulose membrane, and the components are overlapped by 1-2 mm in a cross manner.
6. The kit of claim 1, wherein the fluorescent antibody is diluted with a solid solution and immobilized at the middle position of the binding pad, and the solid solution has a composition of 25mM Tris + 1-10 wt% sucrose + 1-10 wt% trehalose.
7. The kit according to claim 6, wherein the fluorescent antibody is diluted to 5-20 times by the solid phase solution, then sprayed to the central position of the bonding pad according to the spraying amount of 2-10 ul/cm, and dried in an oven for 2 hours, so that the fluorescent antibody can be fixed on the bonding pad.
8. The kit according to claim 1, wherein the sample pad is treated with a treatment solution, the treatment solution is 10mM PBS buffer solution, the treatment operation is to place the sample pad in 10mM PBS buffer solution, soak for 30-35 min, dry in an oven for 4h, and store for later use.
9. The kit of claim 1, wherein the conjugate pad is treated with a treatment solution, and the buffer system of the treatment solution is 10mM PBS buffer solution, which comprises 1-3 wt% of surfactant S9, 0.1-2 wt% of casein, 0.1-2 wt% of PVP and 1-5 wt% of sucrose; and the treatment operation comprises the steps of soaking the bonding pad in the treatment solution for 30-35 min, drying for 4h in an oven, and storing for later use.
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