Joint detection test strip for dengue virus and Zika virus antigens and antibodies and preparation method of joint detection test strip

Abstract

The invention provides a joint detection test strip for dengue virus and Zika virus antigens and antibodies and a preparation method of the joint detection test strip. According to the combined detection test strip, the combined detection test strip with the dengue virus NS1 antigen and the dengue virus IgG / IgM antibody adopts a multi-site recombinant antigen labeling or multi-site recombinant antibody coating form, so that the sensitivity of a reagent is improved; the problems of poor specificity and release residue of latex microspheres are solved by optimizing the confining liquid; colored latex microspheres are used as a marker, a detection target is more intuitively distinguished according to line colors, clinical result interpretation is facilitated, the accuracy of dengue virus infection and Zika virus infection is more quickly improved, and the dengue virus NS1, the Zika virus NS1 antigen, the dengue virus IgG / IgM antibody and the Zika virus IgG / IgM antibody can be detected at the same time; and the kit is convenient to carry, simple to operate, convenient for on-site instant detection and capable of performing large-scale preliminary screening.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedical detection, and particularly relates to a combined detection test strip for detecting dengue virus, Zika virus antigen and antibody and a preparation method thereof. BACKGROUND

[0002] Dengue virus belongs to a serotype subgroup of the Flavivirus genus of the Flaviviridae family, and is transmitted by Aedes aegypti and Aedes albopictus, has the characteristics of rapid transmission, high incidence, universal susceptibility of the population, and high mortality of severe types, and is considered to be the most concerned infectious disease in the world; the prevention and control of dengue fever depends on the control of mosquito vectors. At present, there is no specific treatment for dengue fever / severe dengue fever, but early detection and appropriate medical care can greatly reduce the mortality of severe dengue fever.

[0003] Zika virus is a mosquito-borne virus transmitted by infected Aedes mosquitoes (Aedes spp., mainly Aedes aegypti) in tropical and subtropical regions. Most Zika virus diseases show asymptomatic or mild infection, but there is evidence that Zika virus infection can cause neurological complications such as Guillain-Barre syndrome, congenital birth defect syndrome such as microcephaly in children, etc. The occurrence of these diseases may be related to the infection of human neural precursor cells by Zika virus. More seriously, there is a serious cross-reaction between the serum antibodies induced by dengue and Zika viruses, and the persistent infection of different viruses can lead to the occurrence of ADE effect, thereby causing Zika virus infection to trigger the occurrence of severe Zika virus disease in dengue fever epidemic areas; conversely, the epidemic of dengue virus in Zika virus disease epidemic areas can also trigger the occurrence of severe dengue disease, such as dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS).

[0004] Zika virus and dengue virus belong to the same family of Flaviviridae, and the homology of the E proteins of the two viruses is as high as 54%-59%, which is prone to cross; while the homology of the NS1 proteins is relatively low (about 53%-58% for dengue virus / Zika virus), and the concentration of NS1 is high in the early stage of infection. Traditional antibodies are prone to cross-binding with the NS1 proteins of the two viruses, resulting in false positive results. For example, antibodies against Zika virus NS1 may misrecognize dengue virus NS1.

[0005] In addition, after the body is infected, the NS1 antigen appears first, so simultaneous detection of the NS1 antigen and specific IgM and IgG antibodies can effectively diagnose the immune response state of the body to a specific pathogen, and such an antigen-antibody combined detection kit can be used for rapid early diagnosis and screening of patients with dengue / zika virus infection in the early stage, primary infection, and secondary or multiple infections, shortens the window period, and improves the detection rate.

[0006] Serological detection is one of the more reliable indicators of clinical diagnosis recognized at home and abroad. Currently, common dengue / zika antigen and antibody detection methods include ELISA and PCR, and colloidal gold. Enzyme-linked immunoassay (ELISA) and PCR detection technology require special equipment and require professional technicians to perform detection in a professional laboratory. The operation is complicated, the technical difficulty is great, the time is long, the experimental conditions are high, and there are serious biological safety problems, which are not suitable for on-site instant detection, and are suitable as a confirmatory method, but cannot be widely used for primary screening and popularization.

[0007] CN113533732A discloses that the labeled dengue virus antigen monoclonal colloidal gold complex is used in equal amounts, and CN112362870A discloses that four antibodies are coated in a ratio of 2:1:1:1 and at a concentration of 1.0-1.2 mg / mL. The difference is not great because the batch difference makes the ratio unsuitable, and the clinical sample is missed. Both of them need four different subtypes of dengue virus recombinant antigens, and the process is relatively complicated. Moreover, they only detect dengue virus and do not involve joint detection of dengue virus and zika virus.

[0008] Currently, the products on the market are basically single-index detection or dengue virus NS1 antigen and dengue virus IgG / IgM antibody detection combined detection, which can only assist in detecting dengue virus NS1 antigen or dengue virus IgG / IgM antibody, and there is no diagnostic dengue virus and zika virus combined detection chromatography kit. It is easy to miss or misdiagnose. Currently, the colloidal gold immunochromatography or enzyme-linked immunoassay detection has relatively poor sensitivity or complicated operation, cannot simultaneously detect multiple indicators, and has many missed diagnoses and false positives.

[0009] Dengue virus and Zika virus belong to the same family of flaviviruses, and their transmission routes and disease conditions are basically the same. Simultaneous infection of dengue virus and Zika virus can cause more serious phenomena. There is no dengue virus NS1 antigen and Zika antigen combined detection on the market caused by dengue virus. Therefore, there is an urgent need for a kit that can quickly detect dengue virus NS1 antigen, Zika virus NS1 antigen, dengue virus IgG / IgM antibody and Zika virus IgG / IgM antibody in blood, plasma, serum and other samples. Combined antigen and antibody detection at the same time can improve the accuracy of distinguishing dengue virus and Zika infection, and timely distinguish viruses so that patients can receive timely treatment. SUMMARY

[0010] In view of the above-mentioned disadvantages of the prior art, the purpose of the present application is to provide a test strip and kit for simultaneously detecting dengue virus NS1 antigen, Zika virus NS1 antigen and dengue virus IgG / IgM antibody and Zika virus IgG / IgM antibody, which can solve the problems in the prior art.

[0011] To achieve the above-mentioned purposes and other related purposes, the present application is obtained by the following technical solutions.

[0012] In a first aspect of the present application, a combined detection test strip is provided, which comprises: a first test strip; the first test strip is used for combined detection of dengue virus NS1 antigen and Zika virus NS1 antigen;

[0013] The first test strip comprises a first conjugate pad and a first chromatography membrane;

[0014] The first conjugate pad is coated with marker-labeled type I, type II, type III and type IV dengue virus recombinant NS1 antibody and marker-labeled Zika virus NS1 antibody;

[0015] The first chromatography membrane contains a first detection line T1, a second detection line T2 and a first quality control line;

[0016] The first detection line T1 is coated with type I, type II, type III and type IV dengue virus recombinant NS1 antibody, and the type I, type II, type III and type IV dengue virus recombinant NS1 antibody has binding sites for four different serotypes (type I, type II, type III and type IV) of dengue virus, and the second detection line T2 is coated with Zika virus NS1 antibody.

[0017] In some embodiments of the present application, the combined detection test strip further comprises a second test strip and / or a third test strip; the second test strip and / or the third test strip are used for detecting dengue virus IgG / IgM antibody and Zika virus IgG / IgM antibody, respectively;

[0018] The second test strip comprises a second conjugate pad and a second chromatographic membrane;

[0019] The second conjugate pad is coated with marker-labeled type I, type II, type III and type IV dengue virus recombinant E antigens, wherein the type I, type II, type III and type IV dengue virus recombinant E antigens have binding sites for type I, type II, type III and type IV dengue virus IgG / IgM antibodies;

[0020] The second chromatographic membrane contains a second test line T1, a second test line T2 and a second quality control line;

[0021] The second test line T1 is coated with anti-human IgM antibody, and the second test line T2 is coated with anti-human IgG antibody;

[0022] The third test strip comprises a third conjugate pad and a third chromatographic membrane;

[0023] The third conjugate pad is coated with marker-labeled Zika virus E antigen;

[0024] The third chromatographic membrane contains a third test line T1, a third test line T2 and a third quality control line;

[0025] The third test line T1 is coated with anti-human IgM antibody, and the third test line T2 is coated with anti-human IgG antibody;

[0026] In some embodiments of the present application, the first quality control line, the second quality control line and the third quality control line are coated with marker-labeled IgG antibody and Evans blue.

[0027] In some embodiments of the present application, the working concentration of type I, type II, type III and type IV dengue virus recombinant NS1 antibodies of the first test line T1 is 0.5-1.5 mg / mL.

[0028] In some embodiments of the present application, the working concentration of Zika virus NS1 antibody of the first test line T2 is 0.5-1.5 mg / mL.

[0029] In some embodiments of the present application, the working concentration of anti-human IgM antibody of the second test line T1 is 0.5-1.5 mg / mL.

[0030] In some embodiments of the present application, the working concentration of anti-human IgG antibody of the second test line T2 is 0.5-2.0 mg / mL.

[0031] In some embodiments of the present application, the working concentration of anti-human IgM antibody of the third test line T1 is 0.5-1.5 mg / mL.

[0032] In some embodiments of the present application, the working concentration of the anti-human IgG antibody of the third detection line T2 is 1.0-2.0 mg / mL.

[0033] In some embodiments of the present application, the working concentration of the IgG antibody labeled by the marker of the quality control line is 5-9 mg / mL.

[0034] In some embodiments of the present application, the working concentration of Evans blue of the quality control line is 0.3-1.0 mg / mL.

[0035] In the present application, the IgG antibody and Evans blue can be mixed and coated on the quality control line, or the IgG antibody can be coated first and then the Evans blue is coated. Under the above specific conditions, the coated quality control line will show the blue color of Evans blue; therefore, the color of the marker IgG antibody should be different from blue; preferably red.

[0036] In some embodiments of the present application, the IgG labeled by the marker of the quality control line is a mouse IgG antibody.

[0037] In some embodiments of the present application, the marker is a colored latex microsphere; including: red latex microsphere, blue latex microsphere, green latex microsphere, yellow latex microsphere, brown latex microsphere, etc.

[0038] In some specific embodiments of the present application, the microsphere used on the detection line of the test strip for detecting dengue virus NS1, IgG / IgM is a red microsphere, and the microsphere used on the detection line of the test strip for detecting Zika virus NS1, IgG / IgM is a blue microsphere; but the color of the microsphere is not specially limited, as long as it can achieve the effect of distinguishing.

[0039] In some embodiments of the present application, the particle size of the microsphere is 100-400 nm.

[0040] In some embodiments of the present application, the functional group of the microsphere is carboxyl, sulfate, etc.

[0041] In some embodiments of the present application, the working concentration of the marker-labeled I, II, III and IV type dengue virus recombinant NS1 antibody in the first conjugate pad is 1-5 mg / mL.

[0042] In some embodiments of the present application, the working concentration of the marker-labeled Zika virus NS1 antibody in the first conjugate pad is 1-5 mg / mL.

[0043] In some embodiments of the present application, the working concentration of the marker-labeled I, II, III and IV type dengue virus recombinant E antigen in the second conjugate pad is 1-5 mg / mL.

[0044] In some embodiments of the application, the working concentration of the marker-labeled Zika virus E antigen in the third conjugate pad is 1-5 mg / mL.

[0045] In some embodiments of the application, the method for preparing the latex microsphere-labeled antibody / antigen comprises the following steps: mixing and coupling the latex microspheres with the antibody / antigen to obtain a mixture, and then adding a blocking solution to block to obtain the latex microsphere-labeled antibody / antigen; the blocking solution comprises the following components: 10-15 v / v% rabbit serum, 10-15 wt% PEG6000, and 30-80 mM Tris-HCl.

[0046] In some embodiments of the application, the pH value of the Tris-HCl is 7.5-8.5.

[0047] In some embodiments of the application, the mixing condition is room temperature coupling for 1-4 h.

[0048] In some embodiments of the application, the amount of the blocking solution added is 50-200 μL / mL of the mixture.

[0049] In some embodiments of the application, the blocking time is 0.5-4 h, and the temperature is room temperature, preferably 15-30 °C.

[0050] In some embodiments of the application, before the latex microspheres are mixed with the antibody, the latex microspheres are further subjected to an activation treatment, preferably EDC / NHS activation.

[0051] In some embodiments of the application, the sample pad treatment solution used in the preparation of the sample pad comprises the following components: 15-30 mM Tris-HCl, 0.5-2 wt% EDTA, 0.1-1 wt% PVP-10 (polyvinylpyrrolidone), 0.5-2 wt% Surfynol, 0.5-1.5 wt% BRIJ 35 (polyoxyethylene lauryl ether), 0.01-0.1 v / v% mouse serum, and 0.05-0.2 mg / mL anti-erythrocyte antibody.

[0052] In some embodiments of the application, the conjugate pad treatment solution used in the preparation of the conjugate pad comprises the following components: 5-15 mM HEPES buffer, 0.5-1.5 wt% sodium caseinate, 0.1-1 wt% sodium lauryl polyoxyethylene sulfate, 0.5-2 wt% CHEMAL LA-9, and 0.1-1 wt% Tween-80.

[0053] In some embodiments of the application, the pH value of the HEPES buffer is 7-7.5.

[0054] In some embodiments of the present application, the joint detection test strip further comprises a bottom plate, and the material of the bottom plate comprises but is not limited to PVC.

[0055] In some embodiments of the present application, the first test strip further comprises a first water-absorbing pad, a first sample pad, the first sample pad, the first conjugate pad, the first chromatographic membrane and the first water-absorbing pad are sequentially connected end to end and fixed on the bottom plate.

[0056] In some embodiments of the present application, the second test strip further comprises a second water-absorbing pad, a second sample pad, the second sample pad, the second conjugate pad, the second chromatographic membrane and the second water-absorbing pad are sequentially connected end to end and fixed on the bottom plate.

[0057] In some embodiments of the present application, the third test strip further comprises a third water-absorbing pad, a third sample pad, the third sample pad, the third conjugate pad, the third chromatographic membrane and the third water-absorbing pad are sequentially connected end to end and fixed on the bottom plate.

[0058] In the second aspect of the present application, a preparation method of the above-mentioned quadruple detection test strip is provided, comprising the following steps:

[0059] 1) Preparation of the first chromatographic membrane, the second chromatographic membrane and the third chromatographic membrane:

[0060] After mixing the type I, type II, type III and type IV dengue virus recombinant NS1 antibodies, Zika virus NS1 antibodies, marker-labeled IgG antibodies and Evans blue with the coating diluent respectively, line drawing and drying are performed to obtain the first chromatographic membrane comprising the first detection line T1, the first detection line T2 and the first quality control line;

[0061] After mixing the anti-human IgM antibodies, anti-human IgG antibodies, marker-labeled IgG antibodies and Evans blue with the coating diluent respectively, line drawing, drying and drying are performed to obtain the second chromatographic membrane comprising the second detection line T1, the second detection line T2 and the second quality control line;

[0062] After mixing the anti-human IgM antibodies, anti-human IgG antibodies, marker-labeled IgG antibodies and Evans blue with the coating diluent respectively, line drawing, drying and drying are performed to obtain the third chromatographic membrane comprising the third detection line T1, the third detection line T2 and the third quality control line;

[0063] 2) Preparation of the first conjugate pad, the second conjugate pad and the third conjugate pad:

[0064] After mixing the marker-labeled type I, type II, type III and type IV dengue virus recombinant NS1 antibodies, marker-labeled Zika virus NS1 antibodies with the microsphere storage solution, line drawing and drying are performed to obtain the first conjugate pad;

[0065] The marker-labeled type I, type II, type III and type IV dengue virus recombinant E antigen is mixed with the microsphere preservation solution, then is drawn, dried, and a second binding pad is obtained;

[0066] The marker-labeled Zika virus E antigen is mixed with the microsphere preservation solution, then is drawn, dried, and a third binding pad is obtained.

[0067] In some embodiments of the present application, the spraying amount during drawing is 1-2 μL / cm.

[0068] In some embodiments of the present application, the drying temperature is 33-45℃.

[0069] In some embodiments of the present application, the drying time is 8-12 h.

[0070] In some embodiments of the present application, the microsphere preservation solution comprises the following components: 30-80 mM Tris-HCL buffer, 0.5-2 wt% sodium chloride, 0.01-0.05 wt% Tween-20, 0.5-1 wt% bovine serum albumin, 3-8 wt% trehalose, 0.1-1 wt% PEG6000, and 0.01-0.2 wt% preservative.

[0071] In some embodiments of the present application, the pH of the Tris-HCL buffer is 7.5-8.5.

[0072] In some embodiments of the present application, the preservative comprises Proclin TM 300.

[0073] In some embodiments of the present application, the corresponding antibody and antigen are diluted to a working concentration using a coating diluent, wherein the coating diluent comprises the following components: 10-40 mM HEPES buffer and 3-8 wt% trehalose.

[0074] In some embodiments of the present application, the method further comprises sequentially adhering a first sample pad, a first binding pad, a first chromatography pad, and a first water absorption pad to a bottom plate to prepare a first test strip; sequentially adhering a second sample pad, a second binding pad, a second chromatography pad, and a second water absorption pad to the bottom plate to prepare a second test strip; sequentially adhering a third sample pad, a third binding pad, a third chromatography pad, and a third water absorption pad to the bottom plate to prepare a third test strip; and assembling to form a quadruple detection test strip.

[0075] In a third aspect of the present application, a dengue virus and Zika virus combined detection kit is provided, wherein the combined detection kit comprises the combined detection test strip as described above.

[0076] In some embodiments of the present application, the kit further comprises a sample diluent, which comprises the following components: 5-30 mM HEPES buffer, 0.8-1.0 wt% EDTA, 0.5-1.0 wt% polyvinylpyrrolidone (PVP10), 0.05-0.10 wt% Tween 20 and 0.01-0.02 wt% Proclin TM 300.

[0077] In some embodiments of the present application, the pH of the HEPES buffer is 7-8.

[0078] Advantages:

[0079] (1) Dengue virus and Zika virus have more than 50% homology, resulting in cross phenomena between the two viruses, which makes the combined detection more difficult in terms of technical difficulties, and the specificity of dengue antibodies and Zika antibodies is also higher. High-specificity dengue antibodies and Zika antibodies may also exhibit cross phenomena. In the present application, by screening commercial dengue antibodies and Zika antibodies, the mutual cross phenomena of combined detection are reduced by changing the antibody blocking, binding pad and sample pad, etc. A combined detection test strip based on color latex microspheres is constructed, which can simultaneously detect dengue virus NS1, Zika virus NS1 antigen, dengue virus IgG / IgM antibody and Zika virus IgG / IgM antibody. The combined detection test strip can simultaneously detect dengue virus NS1, Zika virus NS1 antigen, dengue virus IgG / IgM antibody and Zika virus IgG / IgM antibody, more effectively distinguishes dengue virus and Zika virus infection, and combines dengue virus IgG / IgM antibody detection and Zika virus IgG / IgM antibody detection to more quickly improve the accuracy of dengue virus infection and Zika virus infection. Moreover, it is convenient to carry and easy to operate, and can be used for on-site instant detection and large-scale preliminary screening.

[0080] (2) In the combined detection test strip for dengue virus NS1 antigen and dengue virus IgG / IgM antibody, the I+II+III+IV type multi-site recombinant antigen is labeled or the I+II+III+IV type antibody is coated. The dengue recombinant antigen latex labeling solution is prepared by labeling I+II+III+IV type recombinant antigen, and the I+II+III+IV type recombinant monoclonal antibody is coated. Compared with single-site antigen and single-site monoclonal antibody coating technology, the sensitivity of the reagent is maximized, the process flow is reduced, the risk of false positive and false negative brought by the preparation process of multiple antibodies is reduced, and the detection rate is increased.

[0081] (3) The application optimizes the detection of dengue virus IgG / IgM antibodies and Zika virus IgG / IgM antibodies, uses different color microspheres to couple dengue virus antigens and Zika virus antigens, adopts indirect color microsphere chromatography technology, and more intuitively distinguishes the dengue virus IgG / IgM antibody detection and Zika virus IgG / IgM antibody index according to the line color, thereby facilitating clinical result interpretation.

[0082] (4) The particle size of the latex microsphere is larger than that of the colloidal gold, the latex microsphere is easier to observe the result as a marker for the immunochromatography qualitative product, improves the sensitivity of the product, and the latex microsphere has different colors, which can better distinguish different detection targets in the joint detection. However, the latex microsphere is easy to non-specifically adsorb biological molecules such as proteins through hydrophobic interaction, which leads to the phenomenon that the latex microsphere has poor specificity when applied to the immunochromatography product, and hinders the application of high-sensitivity latex microspheres in the immunochromatography product. In view of the problem of poor specificity of the latex microsphere, after coupling the antigen / antibody of the latex microsphere, the application provides a new microsphere combined blocking method, that is, the application first finds that the blocking effect of the combination of rabbit serum and PEG6000 is better than that of commonly used BSA, PEG20000 and sodium casein, which can block the excess binding sites of the color microsphere, reduce the non-specific adsorption of the microsphere itself, and the non-specific binding sites of the coupled antigen / antibody.

[0083] Meanwhile, the application also provides a binding pad treatment liquid formula capable of quickly releasing the latex microsphere, improves the release efficiency of the latex microsphere, avoids 2 times of release in the running plate, overcomes the release residue problem of the color latex microsphere, makes the microsphere release clean on the binding pad, reduces the false positive and false negative phenomenon due to slow chromatography, and the result shows that the sensitivity of the latex microsphere product is about 10 times higher than that of the colloidal gold product, the product uses color latex microspheres as coupling markers, improves the sensitivity of the product, and has a higher positive detection rate than the ordinary colloidal gold product.

[0084] (5)The test strip of the present application adopts "IgG antibody-latex microspheres-Evans blue" as a quality control system sprayed on the nitrocellulose membrane, and the nitrocellulose membrane is a blue line. When the sample solution passes through the quality control line, the blue quality control line (Evans blue) will be chromatographed to the upper layer along with the liquid, and then the IgG antibody-latex microspheres will show a red quality control line, indicating that the sample chromatography is normal, and the quality control line is normal. As a quality control system, "IgG antibody-latex microspheres-Evans blue" minimizes the interference of the quality control system on the detection system, and significantly improves the specificity of the reagent. No secondary antibody commonly used in the art is introduced as a quality control line system, because the secondary antibody will bind to the specific antibody, making the quality control system easily interfered by the sample. The quality control system in the present application avoids introducing other microsphere-coupled antibodies to react with mammalian immunoglobulins (such as rheumatoid factor, autoantibody) to avoid false positives or false negatives, and avoids abnormal detection of plasma, serum, whole blood samples in the quality control line.

[0085] (6)The present application also provides a method for simultaneously detecting dengue virus NS1, Zika virus NS1 antigen and dengue virus IgG / IgM antibody, and Zika virus IgG / IgM antibody, which is simple in steps, strong in operability, and can quickly and conveniently realize the combined detection of dengue virus IgM / IgG antibody, NS1 antigen and Zika virus IgM / IgG antibody, NS1 antigen. It can be used for rapid early diagnosis and screening of patients with primary infection and secondary or multiple infections in the early stage of heat infection, shortening the window period. The method uses a small amount of sample, is strong in operability, short in time consumption, and can obtain a detection result in 10 minutes. DETAILED DESCRIPTION

[0086] The following specific examples illustrate the embodiments of the present application, and those skilled in the art can easily understand other advantages and effects of the present application from the disclosure.

[0087] Before further describing the specific embodiments of the present application, it should be understood that the scope of protection of the present application is not limited to the following specific embodiments; it should also be understood that the terms used in the embodiments of the present application are for describing the specific embodiments, but not for limiting the scope of protection of the present application. The test methods in the following examples are not specified, and are usually carried out according to the conventional conditions or the conditions recommended by the manufacturers.

[0088] When the embodiments give a numerical range, it should be understood that, unless otherwise specified by the present application, both the endpoints of each numerical range and any number within the range can be selected. Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices and materials similar or equivalent to those described in the embodiments of the present application can also be used to implement the present application according to the mastery of the prior art by those skilled in the art and the description of the present application.

[0089] Blue latex microspheres: solid content 5%, particle size 300 nm, American cow biological, product number: DB02; red latex microspheres: solid content 5%, particle size 300 nm, American cow biological, product number: DR02; recombinant dengue virus NS1 antibody, site: I+II+III+IV type NS1, Abeam, product number: AB252707; recombinant mouse anti-dengue virus NS1 antibody, site: I+II+III+IV type NS1, heavy chain biological, product number: HA806-4MB; Zika virus NS1 antibody: site: NS1, Mayjump biological, product number: MC340101; Zika virus NS1 antibody: site: NS1, heavy chain biological, product number: HA816-2M; anti-human IgM monoclonal antibody: Wuhan Aokebotai, product number: A0041; anti-human IgG monoclonal antibody: Wuhan Aokebotai, product number: A0032; I+II+III+IV type recombinant dengue virus E antigen: Mayjump biological, product number: MR320102; Zika virus E antigen: Mayjump biological, product number: ME340202.

[0090] Example 1: Dengue virus NS1 antigen and Zika virus NS1 antigen combined test strip

[0091] The dengue virus NS1 antigen and Zika virus NS1 antigen combined test strip includes a bottom plate, which is a PVC plate, and a sample pad, a first conjugate pad (latex pad), a first chromatography membrane (nitrocellulose membrane) and an absorbent paper are sequentially overlapped and pasted on the bottom plate. The one end of the sample pad layer presses the one end of the first latex pad 1.0-1.5 mm, the other end of the first latex pad layer presses the one end of the first nitrocellulose membrane 1.0-1.5 mm, and the one end of the absorbent layer presses the other end of the first nitrocellulose membrane 1.0-1.5 mm. The first nitrocellulose membrane prepared is pasted at the middle position of the bottom plate, and the absorbent paper is pasted above the position where the first nitrocellulose membrane is fixed on the bottom plate. The first latex pad and the sample pad prepared are pasted below the position where the first nitrocellulose membrane is fixed on the bottom plate. The product is assembled according to the above steps, and cut into a test strip with a width of 3.0 mm.

[0092] The specific preparation process is as follows:

[0093] 1. Glass fiber cellulose membrane was soaked with binding pad treatment solution or sample pad treatment solution, respectively, and the excess binding pad treatment solution or sample pad treatment solution was scraped off with a glass rod, and dried at 37°C for standby;

[0094] The sample pad treatment solution is 20 mM Tris-HCL buffer with a pH value of 8.0±0.1, containing 1wt% EDTA, 0.5wt% PVP-10, 1wt% Surfynol 485, 0.8wt% BRIJ 35, 0.05v / v% mouse serum,

[0095] 0.05mg / mL anti-erythrocyte antibody;

[0096] The binding pad treatment solution is 10 mM HEPES buffer with a pH value of 7.2±0.1, containing 1wt% sodium caseinate, 0.5wt% sodium laureth sulfate, 1wt% CHEMAL LA-9, 0.5wt% Tween-80;

[0097] 2. Color carboxyl latex microspheres (MeiNiu Bio, color microspheres with a particle size of 300 nm) were prepared and coupled with dengue virus / zika virus antibodies, and the coupling method was as follows:

[0098] 2.1. Red latex microspheres coupled with dengue virus NS1 antibody

[0099] (1) 100 μL of red latex microspheres (solid content 5%, particle size 300 nm, Meiniu Bio, product number: DR02) were subjected to solution replacement treatment, and 900 μL of washing buffer was added. In this embodiment, the washing buffer is 10 mM NaH2PO4, 0.01wt% polysorbate 80, with a pH value of 6.3±0.1, and washing was performed 3 times.

[0100] (2) The mass ratio of EDC to Sulfo-NHS was 1:2, specifically: 50 mg / mL 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) activation buffer reagent was slowly added to the microsphere solution obtained in step (1) at a ratio of 100 μL / mL, and then an equal volume of 100 mg / mL N-hydroxysulfosuccinimide (sulfo-NHS) activation buffer reagent was slowly added and mixed, and activated for 30 min. Centrifugation at 10000-15000 rpm for 10-20 min, and the supernatant was discarded to obtain the precipitate.

[0101] (3) Resuspend and disperse with 1 mL of 50 mM 2-(N-morpholino) ethanesulfonic acid (MES) solution with a pH value of 5.3±0.1 to obtain a well-dispersed activated carboxyl microsphere solution.

[0102] (4) According to the mass ratio of microspheres to antibody of 20:1, the dengue virus recombinant NS1 antibody (site: I+II+III+IV type NS1, Abeam, item number: AB252707) to be labeled was added to the activated red latex microsphere solution, and the coupling reaction was carried out at room temperature for 2 h;

[0103] (5) After the coupling reaction was completed, 100 μL / mL of microsphere blocking solution was then added to the reaction solution, wherein the microsphere blocking solution was 50 mM Tris-HCL buffer with a pH of 8.0±0.1, 10 v / v% rabbit serum and 10 wt% PEG6000; blocking was carried out at room temperature for 60 min, centrifugation was carried out, and the precipitate was collected;

[0104] (6) The precipitate was added to 0.5 mL of microsphere storage solution (diluent): 50 mM Tris-HCL buffer with a pH of 8.0±0.1, a sodium chloride concentration of 1 wt%, a Tween-20 mass concentration of 0.02 wt%, a bovine serum albumin mass concentration of 1 wt%, a trehalose mass concentration of 5 wt%, 0.5 wt% PEG6000, Proclin TM 300 mass concentration of 0.05 wt%; ultrasonic, resuspension and dispersion were carried out, and the carboxyl latex microsphere and dengue virus NS1 antibody coupling complex was obtained, with a concentration of 10 mg / mL.

[0105] 2.2. Zika virus NS1 antibody blue latex microsphere coupling

[0106] The coupling method of 2.1 in Example 1 was the same, the microspheres involved were blue latex microspheres (solid content 5%, particle size 300 nm, Meicow Biological, item number: DB02), and the antibody was mouse Zika virus NS1 antibody (site: NS1, Mayi Biological, item number: MC340101); the blue carboxyl latex microsphere and Zika virus NS1 antibody coupling complex was obtained, with a concentration of 10 mg / mL.

[0107] 2.3. Mouse IgG antibody red latex microsphere coupling

[0108] The coupling method of 2.1 in Example 1 was the same, wherein the microspheres used were red latex microspheres (solid content 5%, particle size 300 nm, Meicow Biological, item number: DR02), and the antibody was mouse IgG antibody; the carboxyl latex microsphere and mouse IgG antibody coupling complex was obtained, with a concentration of 10 mg / mL.

[0109] 3. The immunochromatography test paper was prepared according to the following method:

[0110] Preparation of the first nitrocellulose membrane (sheet): mouse dengue virus recombinant NS1 antibody (site: I+II+III+IV type NS1, heavy chain biology, item number: HA806-4MB) was diluted to 0.8 mg / mL with coating diluent (20 mM HEPES buffer, pH 7.2±0.1, 5 wt% trehalose), and Zika virus NS1 antibody (Zika virus NS1 antibody: site: NS1, heavy chain biology, item number: HA816-2M) was diluted to 1.0 mg / mL with coating diluent, and sprayed onto the nitrocellulose membrane as the test line (T1) and the test line (T2), respectively. The mouse IgG antibody-red latex microsphere conjugate solution and the Evans blue solution were mixed, then diluted to 7 mg / mL and 0.6 mg / mL with coating diluent, and sprayed onto the nitrocellulose membrane as the quality control line (C) (the quality control line is blue) using a gold sprayer at 1.5 μL / cm. Then the coated NC membrane was dried in a constant temperature blast drying oven at 37°C for 8-12 h.

[0111] 4. Preparation of the first latex pad: dengue virus recombinant NS1 antibody (site: I+II+III+IV type NS1) monoclonal antibody-latex conjugate solution was used at a concentration of 3 mg / mL, and Zika virus NS1 antibody-latex conjugate solution was used at a concentration of 2 mg / mL. After mixing, the latex pad was sprayed onto the polyester film using a gold sprayer at 1.5 μL / cm. Then the sprayed polyester film was dried in a constant temperature blast drying oven at 37°C for 8-12 h.

[0112] 5. Assembly of the detection card: the water absorption pad, the coated membrane, the latex pad, and the sample pad were sequentially adhered to the PVC base plate, cut into test strips with a width of 3.0 mm, and assembled into the card shell to complete the production of a latex detection card for combined detection of dengue virus NS1 antigen and Zika virus NS1 antigen.

[0113] Example 2:

[0114] The dengue virus IgG / IgM antibody detection test strip includes a base plate, which is a PVC plate. The sample pad layer, the second binding pad (latex pad), the second chromatography membrane (nitrocellulose membrane), and the water absorption paper are sequentially adhered to the base plate. The sample pad is pressed against the first end of the second latex pad by 1.0-1.5 mm, the second latex pad is pressed against the first end of the second nitrocellulose membrane by 1.0-1.5 mm, and the water absorption paper is pressed against the second end of the second nitrocellulose membrane by 1.0-1.5 mm. The second nitrocellulose membrane is adhered to the middle position of the base plate, and the water absorption paper is adhered above the position where the second nitrocellulose membrane is fixed on the base plate. The latex pad and the sample pad are adhered below the position where the second nitrocellulose membrane is fixed on the base plate. The product is assembled according to the above steps and cut into test strips with a width of 3.0 mm.

[0115] The specific preparation process is as follows:

[0116] 1. The glass fiber cellulose membrane was immersed in the conjugate pad treatment solution or the sample pad treatment solution, respectively, and the excess conjugate pad treatment solution or sample pad treatment solution was scraped off with a glass rod. The membrane was dried at 37°C for standby. The specific formula was the same as that in step 1 of Example 1.

[0117] 2. Color carboxyl latex microspheres (MeiNiu Bio, color microsphere particle size 300 nm) were prepared and coupled with dengue virus antigens. The coupling method was as follows:

[0118] 2.1. Red latex microspheres coupled with I+II+III+IV type dengue virus recombinant E antigens

[0119] The coupling method was the same as that in 2.1 of Example 1, and a red carboxyl latex microsphere and I+II+III+IV type dengue virus recombinant E antigen (Maijie Bio, item number: MR320102) coupled complex was obtained.

[0120] 2.2. Red latex microspheres coupled with mouse IgG antibodies

[0121] The coupling method was the same as that in 2.1 of Example 1, and a red carboxyl latex microsphere and mouse IgG antibody coupled complex was obtained.

[0122] 3. The immunochromatography test paper was prepared as follows:

[0123] Preparation of the second nitrocellulose membrane (sheet) coated membrane: The second detection line T1 was coated with anti-human IgM antibody (Wuhan Aoke Botai, item number: A0041), which was diluted to 0.5 mg / mL with coating buffer and then mixed for coating; the second detection line T2 was coated with anti-human IgG antibody (Wuhan Aoke Botai, item number: A0032), which was diluted to 1.0 mg / mL with coating buffer and then mixed for coating.

[0124] The mouse IgG antibody-microsphere conjugate solution and the Evans blue solution were diluted to 8 mg / mL and 0.6 mg / mL with coating diluent, and sprayed onto the nitrocellulose membrane as the quality control line (C) (at this time the quality control line was blue), and then the coated NC membrane was dried in a constant temperature blast drying oven at 37°C for 8-12 h.

[0125] 4. Preparation of the second latex pad: The I+II+III+IV type dengue virus recombinant E antigen red latex microsphere conjugate was diluted to 2 mg / mL with microsphere storage solution (diluent), and sprayed onto the conjugate pad as the latex pad at 1.2 μL / cm using a gold spraying machine, and then the sprayed polyester film was dried in a constant temperature blast drying oven at 37°C for 8-12 h.

[0126] 5. Detection card assembly: The water-absorbing paper, the second nitrocellulose membrane (sheet), the coated membrane, the latex pad, and the sample pad are sequentially adhered to the PVC base plate, and the test strip is cut into a width of 3.0 mm, and then the card shell is assembled, thereby completing the preparation of a detection card for detecting dengue virus IgG / IgM antibody.

[0127] Example 3

[0128] The Zika virus IgG / IgM antibody detection test strip includes a base plate, which is a PVC plate, and the sample pad layer, the third combination pad (latex pad), the third chromatography membrane (nitrocellulose membrane), and the water-absorbing paper are sequentially adhered to the base plate. One end of the sample pad is pressed against one end of the third latex pad by 1.0-1.5 mm, the other end of the third latex pad is pressed against one end of the third nitrocellulose membrane by 1.0-1.5 mm, and one end of the water-absorbing paper is pressed against the other end of the third nitrocellulose membrane by 1.0-1.5 mm. The prepared third nitrocellulose membrane is adhered to the middle position of the base plate, and the water-absorbing paper is adhered above the position where the third nitrocellulose membrane is fixed on the base plate. The prepared latex pad and sample pad are adhered below the position where the third nitrocellulose membrane is fixed on the base plate. The product is assembled according to the above steps, and the test strip is cut into a width of 3.0 mm.

[0129] The specific preparation process is as follows:

[0130] 1. The glass cellulose membrane is soaked and treated with the combination pad treatment solution or the sample pad treatment solution, and the specific steps are the same as those in step 1 of Example 1.

[0131] 2. Color carboxyl latex microspheres (Mei Niu Biology, color microsphere particle size: 300 nm) coupled with Zika virus antigens are prepared, and the coupling method is as follows:

[0132] 2.1. Blue latex microsphere coupling with Zika virus E antigen

[0133] The coupling method in 2.1 of Example 1 is used to obtain a blue latex microsphere and Zika virus E antigen (Mai Yue Biology, product number: ME340202) coupling complex.

[0134] 2.2. Red latex microsphere coupling with mouse IgG antibody

[0135] The coupling method in 2.1 is used to obtain a red latex microsphere coupling product of mouse IgG antibody.

[0136] 3. The immunochromatography test paper is prepared according to the following method:

[0137] Preparation of the third nitrocellulose membrane (sheet) coated membrane: the third detection line T1 is coated with anti-human IgM antibody (Wuhan Aokebotai, item number: A0041), wherein the anti-human IgM antibody is mixed and coated after being diluted to 1.0 mg / mL with coating buffer; the third detection line T2 is coated with anti-human IgG antibody (Wuhan Aokebotai, item number: A0032), wherein the anti-human IgG antibody is mixed and coated after being diluted to 1.5 mg / mL with coating buffer.

[0138] The mouse IgG antibody-microsphere conjugate solution and the Evans blue solution are diluted to 8 mg / mL and 0.6 mg / mL with coating diluent and sprayed on the nitrocellulose membrane as the quality control line (C) (at this time the quality control line is blue), and then the coated NC membrane is dried in a constant-temperature blast drying oven at 37°C for 8-12 h.

[0139] 4. Preparation of the third latex pad: the Zika virus E antigen blue latex microsphere conjugate solution is diluted to 2.5 mg / mL with microsphere storage solution (diluent), and sprayed on the binding pad as a latex pad at 1.5 μL / cm using a gold sprayer, and then the sprayed polyester film is dried in a constant-temperature blast drying oven at 37°C for 8-12 h.

[0140] 5. Assembly of the detection card: the water-absorbing paper, the third nitrocellulose membrane (sheet) coated membrane, the latex pad, and the sample pad are sequentially adhered to the PVC bottom plate, cut into a test strip with a width of 3.0 mm, and assembled into a card shell to complete the preparation of a blue latex test detection card for detecting Zika virus IgG / IgM antibodies.

[0141] Example 4:

[0142] A dengue virus NS1 antigen, Zika virus NS1 antigen, dengue virus IgG / IgM antibody, and Zika virus IgG / IgM antibody combined detection kit has a card sleeve, and the card sleeve is provided with the dengue virus NS1 antigen and Zika virus NS1 antigen combined detection test strip of Example 1, the dengue virus IgG / IgM antibody test strip of Example 2, and the Zika virus IgG / IgM antibody test strip of Example 3. Further, the card sleeve is provided with a first sample addition hole (S1) corresponding to the dengue virus NS1 antigen and Zika virus NS1 antigen combined detection test strip, a second sample addition hole (S2) corresponding to the dengue virus IgG / IgM antibody test strip, and a third sample addition hole (S3) corresponding to the Zika virus IgG / IgM antibody test strip.

[0143] The kit also includes a 5 μL dropper, a 25 μL dropper, a sample diluent, and an instruction manual, the sample diluent: 10 mM HEPES buffer, pH 7.4±0.1, 1.0 wt% EDTA, 0.5 wt% polyvinylpyrrolidone (PVP10), 0.10 wt% Tween 20, 0.02 wt% Proclin TM 300;

[0144] Example 5:

[0145] The detection method of the Dengue virus NS1 antigen, Zika virus NS1 antigen, Dengue virus IgG / IgM antibody, and Zika virus IgG / IgM antibody combined detection kit is as follows:

[0146] Before use, take the corresponding reagents and samples to be tested out of the storage condition and balance to room temperature (15-30°C).

[0147] Before opening, place the bag at room temperature, take the test box out of the sealed bag, and use it within one hour. Place the test box on a clean, flat surface.

[0148] 1. Dengue virus NS1 antigen and Zika virus NS1 antigen combined detection test strip detection:

[0149] Serum or plasma sample:

[0150] Use a 25 μL dropper: hold the dropper vertically, drop 3 drops of serum or plasma sample (about 75 μL) into the sample well (S) of the test box, and start the timer; the test result should be read within 10 min, and the result read after 20 min is invalid.

[0151] Whole blood sample:

[0152] Use a 25 μL dropper: hold the dropper vertically, transfer 3 drops of whole blood sample (about 75 μL) to the sample well of the test box, then add 1 drop of buffer (about 30 μL), and start the timer. The test result should be read within 10 min, and the result read after 20 min is invalid.

[0153] 2. Dengue virus IgG / IgM antibody test strip and Zika virus IgG / IgM antibody test strip detection:

[0154] Serum or plasma sample:

[0155] Use a 5 μL dropper: hold the dropper vertically, drop 1 drop of serum or plasma sample (about 5 μL) into the sample well (S) of the test box, then add 2 drops of buffer (about 60 μL), and start the timer; the test result should be read within 10 min, and the result read after 20 min is invalid.

[0156] Whole blood sample:

[0157] Using 25 μL dropper: hold the dropper vertically and transfer 3 drops of whole blood specimen (about 5 μL) to the specimen well of the test box, then add 2 drops of buffer (about 60 μL), and start the timer; the test result should be read at 10 min, the result read after 20 min is invalid.

[0158] 3. Test result interpretation

[0159] 3.1 Dengue virus NS1 antigen and Zika virus NS1 antigen combined test strip test result interpretation:

[0160] The quality control line is not tested as a blue line, after adding the sample, Evans blue will be eluted with the liquid chromatography, and the Evans blue will fade, exposing the lower layer of red latex microsphere marker antibody, and the quality control line will become a red line.

[0161] Negative: only the quality control area corresponding to the quality control line appears a red band, and there is no red band in the corresponding T1 and T2 detection area. Negative result indicates that Dengue virus NS1 antigen / Zika virus NS1 antigen is not detected in the sample.

[0162] Positive:

[0163] Two red bands appear: one in the above T1 detection area, and the other in the above first quality control area. It indicates that the sample contains Dengue virus NS1 antigen.

[0164] One red band appears one blue band appears: one in the above T2 detection area (blue band), and the other in the above first quality control area (red band). It indicates that the sample contains Zika virus NS1 antigen.

[0165] Two red bands appear one blue band appears: one in the above T1 detection area (red band), one in the above T2 detection area (blue band), and the other in the above first quality control area (red band). It indicates that the sample contains both Dengue virus NS1 antigen and Zika virus NS1 antigen.

[0166] Invalid: no red band appears in the quality control area, indicating incorrect operation process or reagent has deteriorated. In this case, the instructions should be read again carefully, and the new reagent should be tested again. If the problem still exists, the use of this batch number product should be stopped immediately, and the local supplier should be contacted

[0167] 3.2 Dengue virus IgG / IgM antibody test strip test result interpretation:

[0168] The quality control line is not tested as a blue line. After adding the sample, the Evans blue will be eluted with the liquid, the Evans blue will fade, and the lower layer of red latex microsphere labeled antibody will be exposed, and the quality control line will become a red line.

[0169] Negative: Only a red band appears in the quality control area corresponding to the quality control line, and no red band appears in the IgG and IgM detection areas. A negative result indicates that no dengue virus IgG / IgM antibodies are detected in the sample.

[0170] Positive:

[0171] Two red bands appear: one in the above IgG detection area and the other in the above first quality control area. It indicates that the sample contains dengue virus IgG antibodies.

[0172] Two red bands appear: one in the above IgM detection area and the other in the above first quality control area. It indicates that the sample contains dengue virus IgM antibodies.

[0173] Three red bands appear: one in the above IgG detection area, one in the above IgM detection area, and the other in the above first quality control area. It indicates that the sample contains dengue virus IgG and IgM antibodies.

[0174] Invalid: No red band appears in the quality control area, indicating incorrect operation or that the reagent has deteriorated. In this case, the instructions should be carefully read again, and the new reagent should be tested again. If the problem still exists, the product of this batch number should be immediately stopped and the local supplier should be contacted.

[0175] 3.3 Zika virus IgG / IgM antibody test strip test result interpretation:

[0176] Negative: Only a red band appears in the quality control area corresponding to the quality control line, and no red band appears in the IgG and IgM detection areas. A negative result indicates that no dengue virus IgG / IgM antibodies are detected in the sample.

[0177] Positive: One red band and one blue band appear: one in the above IgG detection area (blue band) and the other in the above first quality control area (red band). It indicates that the sample contains Zika virus IgG antibodies.

[0178] One red band and one blue band appear: one in the above IgM detection area (blue band) and the other in the above first quality control area (red band). It indicates that the sample contains Zika virus IgM antibodies.

[0179] A red band and two blue bands appear: one in the IgG detection zone (blue band), one in the IgM detection zone (blue band), and the other in the first quality control zone (red band). It indicates that:

[0180] The sample contains both Zika virus IgG and IgM antibodies.

[0181] Invalid: The quality control zone does not appear a red band, indicating improper operation or the reagent has deteriorated. In this case, the instructions should be carefully read again, and the new reagent should be retested. If the problem still exists, the product of this batch number should be immediately stopped and the local supplier should be contacted.

[0182] Example 6:

[0183] Performance verification of the kit

[0184] 1. Sensitivity test results:

[0185] 1.1 Dengue virus NS1 antigen and Zika virus NS1 antigen combined detection test strip

[0186] Using the dengue virus NS1 antigen and Zika virus NS1 antigen combined detection test strip in Example 1, the dengue virus NS1 recombinant antigen, dengue virus NS1 positive plasma and Zika virus NS1 antigen, Zika virus NS1 positive plasma were detected after gradient dilution, each concentration was tested 3 times, the results are shown in Table 1;

[0187] Table 1

[0188]

[0189] “+” indicates that the result is positive; “-” indicates that the result is negative;

[0190] 1.2 Dengue virus IgG / IgM antibody detection test strip

[0191] Using the dengue virus IgG / IgM antibody detection test strip in Example 2, the dengue virus IgM, IgG positive plasma was detected after gradient dilution, each concentration was tested 3 times, the results are shown in Table 2;

[0192] Table 2

[0193]

[0194]

[0195] “+” indicates that the result is positive; “-” indicates that the result is negative;

[0196] 1.3 Zika virus IgG / IgM antibody test reagent strip

[0197] The Zika virus IgG / IgM antibody test reagent strip in Example 3 was used to detect the gradient dilution of Zika virus IgM, IgG positive plasma, each concentration was tested 3 times, the results were shown in Table 3;

[0198] Table 3

[0199]

[0200]

[0201] " +" means the result is positive; "-" means the result is negative

[0202] Conclusion: The detection of dengue virus NS1 recombinant protein antigen was 0.025 ng / mL, the detection of Zika virus NS1 protein antigen was 0.1 ng / mL, the detection of dengue virus NS1 antigen positive plasma was diluted to 1:32, and the detection of Zika virus NS1 antigen positive plasma was diluted to 1:16 using the combined detection test reagent strip of dengue virus NS1 antigen and Zika virus NS1 antigen in Example 1.

[0203] The dengue virus IgG / IgM antibody test reagent strip in Example 2 detected dengue virus IgG antibody positive plasma diluted to 1:128, and detected dengue virus IgM antibody positive plasma diluted to 1:16; the Zika virus IgG / IgM antibody test reagent strip in Example 3 detected Zika virus IgG antibody positive plasma diluted to 1:64, and detected Zika virus IgM antibody positive plasma diluted to 1:16.

[0204] 2. Specificity

[0205] 2.1 Cross reaction

[0206] The quadruple detection test strip of dengue virus NS1 antigen, Zika virus NS1 antigen, dengue virus IgG / IgM antibody and Zika virus IgG / IgM antibody in Example 4 was used to detect RF, hepatitis B, syphilis, AIDS, HAMA, Hp, rubella IgM and toxoplasma IgM positive samples respectively, 10 samples of each cross substance were detected, the results were shown in Table 4;

[0207] Table 4

[0208]

[0209]

[0210] "-(10 / 10)": "-" means negative result; "10 / 10" means that the results of 10 samples are consistent, all negative.

[0211] From the above table, it can be seen that there is no cross-reaction between dengue virus NS1, Zika virus NS1 antigen and dengue virus IgG / IgM antibody detection, Zika virus IgG / IgM antibody combined detection kit and the cross-reacting substances in the above table.

[0212] 2.2 Interference reaction

[0213] The quadruple detection test strips of dengue virus NS1 antigen, Zika virus NS1 antigen and dengue virus IgG / IgM antibody, Zika virus IgG / IgM antibody in Example 4 were used for detection according to the following table of interference substance concentration, each interference substance was detected for 3 times, and the results are shown in Table 5.

[0214] Table 5

[0215]

[0216]

[0217] "-(3 / 3)": "-" represents a negative result; "3 / 3" represents that the results of 3 repeated tests are consistent, and all are negative.

[0218] From the above table, it can be seen that there is no interference phenomenon between dengue virus NS1, Zika virus NS1 antigen and dengue virus IgG / IgM antibody detection, Zika virus IgG / IgM antibody combined detection kit and the interference substances in the above table.

[0219] 3. Different anticoagulants test

[0220] The quadruple detection test strips of dengue virus NS1 antigen, Zika virus NS1 antigen and dengue virus IgG / IgM antibody, Zika virus IgG / IgM antibody in Example 4 were used for detection on samples in different anticoagulants, each anticoagulant was detected for 30 times, and the results are shown in Table 6.

[0221] Table 6

[0222]

[0223] "-(30 / 30)": "-" represents a negative result; "30 / 30" represents that the results of 30 samples are consistent, and all are negative.

[0224] From the above table, it can be seen that there is no phenomenon in the detection of different anticoagulant plasma by dengue virus NS1, Zika virus NS1 antigen and dengue virus IgG / IgM antibody detection, Zika virus IgG / IgM antibody combined detection kit.

[0225] 4. Serum / plasma / whole blood consistency test

[0226] The quadruple detection test strip of dengue virus NS1 antigen, Zika virus NS1 antigen and dengue virus IgG / IgM antibody and Zika virus IgG / IgM antibody in Example 4 was used to detect different sample types (serum, plasma, whole blood), and each sample type was detected for 10 times, and the condition of the quality control line was observed, and the results are shown in Table 7.

[0227] Table 7

[0228]

[0229]

[0230]

[0231] “-(10 / 10)”: “-” represents a negative result; “10 / 10” represents that the results of 10 samples are consistent, and all are negative.

[0232] “+(10 / 10)”: “+” represents a positive result; “10 / 10” represents that the results of 10 samples are consistent, and all are positive.

[0233] Through different types of serum / plasma / whole blood samples, the dengue virus NS1, Zika virus NS1 antigen and dengue virus IgG / IgM antibody detection, Zika virus IgG / IgM antibody combined detection kit has little difference in positive and negative results in three sample types, and the special quality control line used in the kit has no effect on the quality control line of different types of samples.

[0234] Example 7:

[0235] The difference from Example 1 is that the microsphere sealing liquid: 50mM Tris-HCL buffer solution PH is 8.0±0.1, 15v / v% rabbit serum and 15wt% PEG6000 combination; the rest is the same as Example 1.

[0236] Comparative Example 1:

[0237] The difference from Example 1 is that the microsphere sealing liquid: 50mM Tris-HCL buffer solution PH is 8.0±0.1, 10v / v% rabbit serum; the rest is the same as Example 1.

[0238] Comparative Example 2:

[0239] The difference from Example 1 is that the microsphere sealing liquid: 50mM Tris-HCL buffer solution PH is 8.0±0.1, 10wt% PEG 6000; the rest is the same as Example 1.

[0240] Comparative Example 3:

[0241] The difference from Example 1 is that the microsphere blocking solution: 50 mM Tris-HCL buffer with pH 8.0±0.1, 10 w / v% BSA (a conventional protein blocking agent); the rest are the same as Example 1.

[0242] Comparative Example 4:

[0243] The difference from Example 1 is that the microsphere blocking solution: 50 mM Tris-HCL buffer with pH 8.0±0.1, 10 w / v% BSA (a conventional protein blocking agent) and 10 wt% PEG6000 combination; the rest are the same as Example 1.

[0244] Comparative Example 5:

[0245] The difference from Example 1 is that the microsphere blocking solution: 50 mM Tris-HCL buffer with pH 8.0±0.1, 10 v / v% rabbit serum and 10 wt% Tween-20 combination; the rest are the same as Example 1.

[0246] Comparative Example 6:

[0247] The difference from Example 1 is that the microsphere blocking solution: 50 mM Tris-HCL buffer with pH 8.0±0.1, 5 v / v% rabbit serum and 5 wt% PEG6000 combination; the rest are the same as Example 1.

[0248] Comparative Example 7:

[0249] The difference from Example 1 is that the microsphere blocking solution: 50 mM Tris-HCL buffer with pH 8.0±0.1, 10 v / v% mouse serum and 10 wt% PEG6000 combination; the rest are the same as Example 1.

[0250] Comparative Example 8:

[0251] The difference from Example 1 is that the microsphere blocking solution: 50 mM Tris-HCL buffer with pH 8.0±0.1, 10 v / v% rabbit serum and 10 wt% PEG20000 combination; the rest are the same as Example 1.

[0252] Example 8:

[0253] Example 1 and Comparative Examples 1-8 simultaneously detect negative plasma, serum, whole blood, respectively detect dengue virus NS1 recombinant antigen positive and Zika virus NS1 antigen positive, the results are shown in Table 8;

[0254] Table 8

[0255]

[0256]

[0257] "(20 / 20)": "-" means negative result; "20 / 20" means 20 samples were tested, and all of them were negative.

[0258] "(19 / 20)": "-" means negative result; "19 / 20" means 20 samples were tested, and 19 of them were negative, and 1 of them was positive.

[0259] "(5 / 5)": "+" means positive result; "5 / 5" means 5 samples were tested, and all of them were positive.

[0260] "(4 / 5)": "+" means positive result; "4 / 5" means 5 samples were tested, and 4 of them were positive, and 1 of them was negative.

[0261] From the above results, it can be seen that in Comparative Examples 1-6, the conventional blocking agent (BSA) or the single blocking method (10 v / v% rabbit serum or 10 wt% PEG6000) cannot completely block the non-specific sites of dengue virus NS1 antibody and Zika virus NS1 antibody. The blocking effect of BSA+PEG6000 in Comparative Example 4 is also poor. In Example 1 and Example 7, 10 v / v% rabbit serum and 10 wt% PEG6000 combination and 15 v / v% rabbit serum and 15 wt% PEG6000 combination applied in blocking latex microsphere coupled antibody did not find false positive phenomenon, 5 v / v% rabbit serum and 5 wt% PEG6000 combination applied in blocking latex microsphere coupled antibody in Comparative Example 6 was not completely blocked, and there were false positive phenomena in Comparative Examples 7 and 8, wherein the positive detection limit of dengue virus NS1 antigen in Comparative Example 8 was weakened. Considering comprehensively, 10 v / v% rabbit serum and 10 wt% PEG6000 combination applied in blocking latex microsphere coupled antibody is selected, which improves the specificity of latex coupled antigen / antibody and improves the accuracy in clinical samples.

[0262] Comparative Example 9:

[0263] The difference between Example 1 is that the conjugate pad is treated with conjugate pad treatment liquid, which is prepared according to the following formula: 10 mM HEPES buffer, pH 7.2±0.1, containing 1 wt% sodium casein, 0.5 wt% Tween-80; the rest is the same as Example 1, Example 2 and Example 3.

[0264] Comparative Example 10:

[0265] The difference between Example 1 is that the conjugate pad is treated with conjugate pad treatment liquid, which is prepared according to the following formula: 10 mM HEPES buffer, pH 7.2±0.1, containing 1 wt% sodium casein, 0.5 wt% Tween-80; the rest is the same as Example 1, Example 2 and Example 3.

[0266] Comparative Example 11:

[0267] The difference from Example 1 is that the conjugate pad is treated with a conjugate pad treatment solution, which is prepared according to the following formula: 10 mM HEPES buffer, pH 7.2 ± 0.1, containing 1 wt% sodium caseinate, 1 wt% CHEMAL LA-9, 0.5 wt% Tween-80; the rest is the same as Example 1, Example 2 and Example 3.

[0268] Example 9

[0269] Example 1 and Comparative Examples 9-11 simultaneously detect negative plasma, serum, whole blood, respectively detect dengue virus NS1 recombinant antigen (0.05 mg / mL) positive and Zika virus NS1 (0.1 mg / mL) positive, read the results, observe the release of latex microspheres on the latex conjugate pad, the results are shown in Table 9;

[0270] Table 9

[0271]

[0272]

[0273] From the above results, compared with Comparative Examples 9-11, the release of latex microspheres in Example 1 is very clean, which is conducive to the improvement of sensitivity, and the uniformity of the reagent strip is good.

[0274] Comparative Example 12:

[0275] The difference from Example 1, Example 2, Example 3 and Example 4 is that the antigen / antibody to be labeled is labeled with a colloidal gold dispersion solution with a particle size of 40-60 nm and a concentration of 0.02 wt%, and the labeling process is labeled according to the conventional process of colloidal gold. The rest is the same as Example 1, Example 2, Example 3 and Example 4, and a dengue virus NS1 antigen, Zika virus NS1 antigen, dengue virus IgG / IgM antibody, Zika virus IgG / IgM antibody combined detection kit based on colloidal gold method is prepared.

[0276] Example 10

[0277] The effect of the combined detection test strip in Example 4 and Comparative Example 12 is detected according to the steps in Reference Example 7, and the results are shown in Tables 10-12.

[0278] 1.1 Dengue virus NS1 antigen and Zika virus NS1 antigen combined detection test strip

[0279] Table 10

[0280]

[0281]

[0282] 1.2 Dengue virus IgG / IgM antibody detection test strip

[0283] Table 11

[0284]

[0285] 1.3 Zika virus IgG / IgM antibody detection test strip

[0286] Table 12

[0287]

[0288]

[0289] The above results show that the dengue virus NS1 antigen and Zika virus NS1 antigen combined detection test strip (latex method) in Example 4 has a sensitivity about 10 times higher than that of the dengue virus NS1 antigen and Zika virus NS1 antigen combined detection test strip (colloidal gold method) in Comparative Example 12 in detecting dengue virus NS1 antigen and Zika virus NS1 antigen; the dengue virus IgG / IgM antibody detection test strip (latex method) has a sensitivity about 4 times higher than that of the dengue virus IgG / IgM antibody detection test strip (colloidal gold method) in detecting positive IgG / IgM antibody plasma; and the Zika virus IgG / IgM antibody detection test strip (latex method) has a sensitivity about 4 times higher than that of the Zika virus IgG / IgM antibody detection test strip (colloidal gold method) in detecting positive IgG / IgM antibody plasma.

[0290] In general, the sensitivity of the dengue virus NS1, Zika virus NS1 antigen and dengue virus IgG / IgM antibody detection, Zika virus IgG / IgM antibody combined detection test kit (latex product) of the present application is much higher than that of the colloidal gold method. It is more convenient and faster to distinguish dengue virus and Zika virus, as well as the infection status, so that the patient can receive the corresponding treatment in a timely manner.

[0291] The above examples are only illustrative of the principles and effects of the present application, and are not intended to limit the present application. Any person skilled in the art can modify or change the above examples without departing from the spirit and scope of the present application. Therefore, all equivalent modifications or changes made by those skilled in the art without departing from the spirit and technical thought disclosed by the present application should be covered by the claims of the present application.

Claims

1. A combined test strip; said combined test strip includes a first test strip; The first test strip includes a first conjugation pad and a first chromatographic membrane; The first binding pad is coated with recombinant NS1 antibodies against dengue virus types I, II, III and IV labeled with markers and NS1 antibodies against Zika virus labeled with markers; The first chromatography membrane includes a first detection line T1, a first detection line T2, and a first control line; The first detection line T1 is coated with recombinant NS1 antibodies against dengue virus types I, II, III and IV, and the second detection line T2 is coated with Zika virus NS1 antibodies. The first control line is coated with labeled IgG antibody and Evans blue; The marker is a colored latex microsphere.

2. The combined test strip according to claim 1, characterized in that, The combined test strip further includes a second test strip and / or a third test strip; the second test strip includes a second conjugation pad and a second chromatography membrane; The second binding pad is coated with recombinant E antigens of dengue virus types I, II, III and IV that are labeled with markers; The second chromatography membrane contains a second detection line T1, a second detection line T2, and a second control line; The second detection line T1 is coated with anti-human IgM antibody, and the second detection line T2 is coated with anti-human IgG antibody. The third test strip includes a third conjugation pad and a third chromatography membrane; The third binding pad is coated with Zika virus E antigen labeled with a marker; The third chromatography membrane contains a third detection line T1, a third detection line T2, and a third quality control line; The third detection line T1 is coated with anti-human IgM antibody, and the third detection line T2 is coated with anti-human IgG antibody. The second and third control lines are coated with labeled IgG antibodies and Evans blue.

3. The combined test strip according to claim 2, characterized in that, The preparation method of the marker-labeled antibody or marker-labeled antigen is as follows: the marker is mixed with the antibody or antigen to obtain a mixture, and then a blocking solution is added for blocking to obtain the marker-labeled antibody or marker-labeled antigen. Preferably, the blocking solution comprises the following components: 5-15 v / v% rabbit serum, 5-15 wt% PEG6000 and 30-80 mM Tris-HCl; Preferably, the conjugate pad treatment solution used in conjugate pad preparation includes the following components: 5-15 mM HEPES buffer, 0.5–1.5 wt% sodium caseinate, 0.1–1 wt% sodium lauryl ether sulfate, 0.5–2 wt% CHEMAL LA-9, 0.1–1 wt% Tween-80.

4. The combined test strip according to claim 2, characterized in that, The working concentration of recombinant NS1 antibodies against dengue virus types I, II, III, and IV in the first detection line T1 is 0.5-1.5 mg / mL; and / or, The working concentration of Zika virus NS1 antibody in the first detection line T2 is 0.5-1.5 mg / mL; and / or, The working concentration of the anti-human IgM antibody in the second detection line T1 is 0.5-1.5 mg / mL; and / or, The working concentration of the anti-human IgG antibody in the second detection line T2 is 0.5-2.0 mg / mL; and / or, The working concentration of the anti-human IgM antibody in the third detection line T1 is 0.5-1.5 mg / mL; and / or, The working concentration of the anti-human IgG antibody in the third detection line T2 is 1.0-2.0 mg / mL; and / or, The working concentration of the marker-labeled IgG in the quality control line is 5–9 mg / mL; and / or, The working concentration of Evans blue in the quality control line is 0.3–1.0 mg / mL.

5. The combined test strip according to claim 2, characterized in that, The working concentration of the marker-labeled recombinant NS1 antibody against dengue virus types I, II, III, and IV in the first binding pad is 1–5 mg / mL; and / or, The working concentration of the Zika virus NS1 antibody labeled with the marker in the first conjugate pad is 1–5 mg / mL; and / or, the working concentration of the recombinant E antigen of dengue virus types I, II, III, and IV labeled with the marker in the second conjugate pad is 1–5 mg / mL; and / or, The working concentration of the Zika virus E antigen labeled in the third binding pad is 1–5 mg / mL.

6. The combined test strip according to any one of claims 2 to 5, characterized in that, The combined test strip includes a base plate; Preferably, the first test strip further includes a first absorbent pad and a first sample pad, wherein the first sample pad, the first binding pad, the first chromatography membrane and the first absorbent pad are connected end to end and fixed on the base plate. The second test strip also includes a second absorbent pad and a second sample pad. The second sample pad, the second binding pad, the second chromatography membrane, and the second absorbent pad are connected end to end and fixed on the base plate. The third test strip also includes a third absorbent pad and a third sample pad, wherein the third sample pad, the third binding pad, the third chromatography membrane and the third absorbent pad are connected end to end and fixed on the base plate.

7. The combined test strip according to claim 6, characterized in that, The sample pad preparation solution used in sample pad preparation contains the following components: 15–30 mM Tris-HCl, 0.5–2 wt% EDTA, 0.1–1 wt% PVP-10, 0.5–2 wt% Surfynol, 0.5–1.5 wt% BRIJ 35, 0.01–0.1 v / v% mouse serum, and 0.05–0.2 mg / mL anti-erythrocyte antibody.

8. A method for preparing the combined test strip according to any one of claims 2 to 7, comprising the following steps; Preparation of the first, second, and third chromatography membranes: Recombinant NS1 antibodies against dengue virus of types I, II, III and IV, NS1 antibodies against Zika virus, and labeled IgG antibodies and Evans blue were mixed with coating diluent, streaked, and dried to obtain a first chromatographic membrane containing a first detection line T1, a first detection line T2 and a first control line. Anti-human IgM antibody, anti-human IgG antibody, labeled IgG antibody and Evans blue were mixed with coating diluent, streaked and dried to obtain a second chromatographic membrane containing a second detection line T1, a second detection line T2 and a second control line. Anti-human IgM antibody, anti-human IgG antibody, labeled IgG antibody and Evans blue were mixed with coating diluent, streaked and dried to obtain a third chromatographic membrane containing a third detection line T1, a third detection line T2 and a third control line. Preparation of the first, second, and third binding pads: The recombinant NS1 antibodies of dengue virus types I, II, III and IV labeled with markers, and the NS1 antibody of Zika virus labeled with markers were mixed with microsphere preservation solution, streaked, and dried to obtain the first binding pad. The recombinant E antigens of dengue virus types I, II, III and IV labeled with markers were mixed with microsphere preservation solution, streaked, and dried to obtain the second binding pad; The Zika virus E antigen labeled with the marker is mixed with the microsphere preservation solution, streaked, and dried to obtain the third binding pad; preferably, the spray volume during streaking is 1-1.5 μL / cm; and / or, the drying temperature is 33-45°C; and / or, the drying time is 8-12 h; and / or, the microsphere preservation solution contains the following components: 30-80 mM Tris-HCl, 0.5-2 wt% sodium chloride, 0.01-0.05 wt% Tween-20, 0.5-1 wt% bovine serum albumin, 3-8 wt% trehalose, 0.1-1 wt% PEG6000, and 0.01-0.2 wt% preservative.

9. A dengue virus and Zika virus combined detection kit, wherein the combined detection kit comprises the combined detection test strip as described in any one of claims 1-7.

10. The reagent kit according to claim 9, characterized in that, The kit also includes a sample diluent comprising the following components: 5–30 mM HEPES buffer, 0.8–1.0 wt% EDTA, 0.5–1.0 wt% polyvinylpyrrolidone, 0.05–0.10 wt% Tween 20, and 0.01–0.02 wt% preservative.

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