An Ebola virus binding protein antibody and its application and Ebola virus detection product

Abstract

The present invention provides Ebola virus binding proteins and their applications as well as Ebola virus detection products. The present invention provides a pair of Ebola virus binding proteins, including protein A and / or protein B, and defines the amino acid sequences of the heavy chain complementary determining region and the light chain complementary determining region of the binding protein. The Ebola virus binding protein has good specificity, high biological activity, strong stability, and high affinity with the Ebola virus, and can be used to prepare products for detecting Ebola virus. The Ebola virus detection products provided by the present invention, including test strips and test kits, have the advantages of simple operation, rapid response, high sensitivity, strong specificity, suitability for rapid on-site detection, and economical and practical, and can be quickly screened during a viral pandemic, and quickly, accurately, and safely detect a large number of suspected cases, asymptomatic infection cases in the incubation period, and asymptomatic virus carriers with higher risks.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an Ebola virus binding protein and an application thereof, and an Ebola virus detection kit. Background Art

[0002] The correct diagnosis of infectious diseases in the early stage is the basis for timely isolation and effective treatment, which can effectively prevent their spread. The inspection of infectious diseases includes the isolation and culture of pathogenic microorganisms, the inspection of pathogenic microorganism antigens, and the nucleic acid inspection of pathogenic microorganisms. Among them, the isolation and culture of pathogenic microorganisms and viruses is an important detection method for infectious diseases. The isolation and culture of pathogenic bacteria or viruses of infectious diseases is of great significance for the prediction of the trend of infectious diseases. The development of molecular biology technology has made nucleic acid detection popular, mainly based on the characteristics that each organism has a specific nucleic acid sequence and DNA sequence. As long as the gene sequence of a specific virus or pathogenic microorganism is detected in the human body, it proves that the person is infected by the pathogenic microorganism or virus. The degree of infection is determined according to the strength of the detected signal. It has the advantages of high sensitivity and good specificity, but it requires professional operators. Pathogenic microorganism antigen detection is an important detection method. It has no special requirements for laboratories and has the advantages of early screening and early diagnosis. It is suitable for large-scale screening in grassroots communities.

[0003] Ebola virus (EBoV) belongs to the Filoviridae family. It is 970 nanometers long, filamentous, and a single-stranded negative-strand RNA virus with 18,959 bases and a molecular weight of 4.17×10 6 . There is an envelope on the outside, the diameter of the virus particle is about 80 nanometers, and the size is 100 nanometers × (300-1500) nanometers. The virus with strong infectivity is generally about 665-805 nanometers long, and there are branched, U-shaped, 6-shaped or ring-shaped ones, and the branched ones are more common. There is an envelope, and there are 8-10 nanometer-long filaments on the surface. The pure virus particle is composed of a spiral ribonucleocapsid complex, containing negative-strand linear RNA molecules and 4 virion structural proteins. The longer and odd-shaped virus particle-related structures can be branched or coiled, up to 10 microns. The Ebola virus is mainly transmitted through the patient's blood, saliva, sweat and secretions.

[0004] It has been determined that the Ebola virus is divided into five subtypes, namely Ebola-Zaire, Ebola-Sudan, Ebola-Reston (EBO-R), Ebola-Ivory Coast (EBO-CI) and Tai Forest virus. Different subtypes have different characteristics. EBO-Z and EBO-S are highly pathogenic and lethal to humans and non-human primates; EBO-R is not pathogenic to humans, but is lethal to non-human primates; EBO-CI is clearly pathogenic to humans, but generally not lethal, and has a high lethality rate to chimpanzees.

[0005] At present, the detection methods of Ebola virus mainly include electron microscopy, molecular biology detection methods, immunology, etc.

[0006] The above methods have the problems of cumbersome detection steps and long detection cycles, which are not conducive to rapid screening during a pandemic, and cannot quickly, accurately and safely detect a large number of suspected cases, asymptomatic infection cases in the incubation period, and asymptomatic virus carriers with higher risks. Solving this problem is of great significance to indirectly solve the insufficient nucleic acid detection capacity, reduce potential infection risks, and prevent the spread of the epidemic. Summary of the invention

[0007] The purpose of the present invention is to provide an Ebola virus binding protein, which can specifically identify Ebola virus; to provide the use of the Ebola virus binding protein in the preparation of Ebola virus detection products; to provide Ebola virus detection products to solve the problems in the prior art.

[0008] In order to achieve the above object, the present invention provides an Ebola virus binding protein, including protein A and / or protein B,

[0009] The protein A comprises a heavy chain complementary determining region whose amino acid sequence is as follows: CDR1-VH whose amino acid sequence is as shown in SEQ ID NO.1, CDR2-VH whose amino acid sequence is as shown in SEQ ID NO.2, and CDR3-VH whose amino acid sequence is as shown in SEQ ID NO.3;

[0010] The protein A comprises a light chain complementary determining region whose amino acid sequence is as follows: CDR1-VL whose amino acid sequence is as shown in SEQ ID NO.4, CDR2-VL whose amino acid sequence is as shown in SEQ ID NO.5, and CDR3-VL whose amino acid sequence is as shown in SEQ ID NO.6;

[0011] The protein B comprises the heavy chain complementary determining regions whose amino acid sequences are as follows: CDR1-VH whose amino acid sequence is shown in SEQ ID NO.7, CDR2-VH whose amino acid sequence is shown in SEQ ID NO.8, and CDR3-VH whose amino acid sequence is shown in SEQ ID NO.9;

[0012] The protein B includes a light chain complementary determining region whose amino acid sequence is as follows: CDR1-VL as shown in SEQ ID NO.10, CDR2-VL as shown in SEQ ID NO.11, and CDR3-VL as shown in SEQ ID NO.12.

[0013] Preferably, the protein A comprises a heavy chain variable region whose amino acid sequence is shown in SEQ ID NO.13 and a light chain variable region whose amino acid sequence is shown in SEQ ID NO.14.

[0014] Preferably, any of the above items is that the protein B comprises a heavy chain variable region whose amino acid sequence is shown in SEQ ID NO.15 and a light chain variable region shown in SEQ ID NO.16.

[0015] Preferably, in any of the above items, the amino acid sequence of the protein A light chain is as shown in SEQ NO ID.17.

[0016] Preferably, in any of the above items, the amino acid sequence of the protein A heavy chain is as shown in SEQ NO ID.18.

[0017] Preferably, in any of the above items, the amino acid sequence of the protein B light chain is as shown in SEQ NO ID.19.

[0018] Preferably, in any of the above items, the amino acid sequence of the protein B heavy chain is as shown in SEQ NO ID.20.

[0019] Preferably, in any of the above items, the amino acid sequence of the light chain constant region of the protein A or protein B is as shown in SEQ NOID.21.

[0020] Preferably, in any of the above items, the amino acid sequence of the heavy chain constant region of the protein A or protein B is as shown in SEQ NOID.22.

[0021] The present invention also provides use of any of the above-mentioned Ebola virus binding proteins in the preparation of Ebola virus detection products.

[0022] The present invention also provides an Ebola virus detection test strip, which comprises a base plate and a sample loading pad, a marking pad, a detection pad and a sample suction pad which are sequentially stacked on the base plate; the marking pad contains any of the above-mentioned Ebola virus binding proteins, and the Ebola virus binding protein is marked with a marker.

[0023] Preferably, in any of the above items, the marker comprises at least one of colloidal gold, colored microspheres, time-resolved fluorescent microspheres or quantum dot microspheres.

[0024] Preferably, any of the above items has a particle size of colloidal gold of 40 to 100 nm; more preferably, it is 40, 50, 60, 80, 100 nm and ranges therebetween.

[0025] Preferably, any of the above items has a particle size of 100-300 nm; more preferably, 100, 200, 300 nm and ranges therebetween.

[0026] Preferably, any of the above items has a particle size of 100-300 nm of the time-resolved fluorescent microspheres; more preferably, 100, 200, 300 nm and ranges therebetween.

[0027] Preferably, any of the above items has a particle size of the quantum dot microspheres of 100-300 nm; more preferably, 100, 200, 300 nm and ranges therebetween.

[0028] Preferably, any of the above items has a mass ratio of Ebola virus binding protein to colloidal gold of (0.04-0.32):1; more preferably, 0.04:1, 0.10:1, 0.15:1, 0.20:1, 0.25:1, 0.30:1, 0.32:1 and ranges therebetween.

[0029] Preferably, in any of the above items, the mass ratio of the Ebola virus binding protein to the colored microspheres is: (0.1-0.4):1; more preferably, it is 0.1:1, 0.2:1, 0.3:1, 0.4:1 and the range therebetween.

[0030] Preferably, in any of the above items, the mass ratio of the Ebola virus binding protein to the time-resolved fluorescent microspheres is: (0.1-0.4):1; more preferably, it is 0.1:1, 0.2:1, 0.3:1, 0.4:1 and the range therebetween.

[0031] Preferably, any of the above items has a mass ratio of the Ebola virus binding protein to the quantum dot microspheres of (0.1-0.4):1; more preferably, 0.1:1, 0.2:1, 0.3:1, 0.4:1 and ranges therebetween.

[0032] Preferably, any of the above items is that a detection line and a quality control line are provided on the detection pad; the detection line is coated with the Ebola virus binding protein; and the quality control line is coated with goat anti-mouse IgG polyclonal antibody.

[0033] Any of the above items is preferably that a detection line and a quality control line are provided on the detection pad; the detection line is coated with the Ebola virus binding protein; the quality control line is coated with sheep anti-chicken IgG; the marking pad contains another Ebola virus binding protein, the Ebola virus binding protein on the marking pad is marked with a marker, and the marker is marked with chicken IgY.

[0034] Preferably, in any of the above items, the diluent for the Ebola virus binding protein and goat anti-mouse IgG polyclonal antibody is a PB buffer containing trehalose.

[0035] Preferably, any of the above items is that the detection line is coated with 1-2 mg / mL of the Ebola virus binding protein; more preferably, it is 1.0, 1.5, 2.0 mg / mL and the range therebetween.

[0036] Preferably, any of the above items is coated with 1-2 mg / mL of goat anti-mouse IgG polyclonal antibody; more preferably, 1.0, 1.5, 2.0 mg / mL and ranges therebetween.

[0037] Preferably, in any of the above items, the diluent for the Ebola virus binding protein and goat anti-mouse IgG polyclonal antibody is a 15-25 mM PB buffer containing trehalose; the PB buffer is further preferably 15, 20, 25 mM and the range therebetween.

[0038] Preferably, any of the above items contains 0.1-0.9 g of trehalose per 100 mL of the diluent, and more preferably 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 g and ranges therebetween.

[0039] Preferably, any of the above items is that the Ebola virus binding protein is of mouse origin.

[0040] Preferably, any of the above items is that the Ebola virus binding protein on the labeling pad is different from the Ebola virus binding protein on the detection pad.

[0041] Preferably, the Ebola virus binding protein on the marking pad is protein a, and the Ebola virus binding protein on the detection pad is protein b; preferably, the Ebola virus binding protein on the marking pad is protein b, and the Ebola virus binding protein on the detection pad is protein a.

[0042] The present invention also provides an Ebola virus detection kit, comprising any of the above-mentioned Ebola virus detection test strips and a shell, wherein the Ebola virus detection test strip is arranged inside the shell.

[0043] Preferably, any of the above items is that the housing comprises an upper cover and a lower cover which are detachably connected; and the upper cover is provided with an observation window and a sample addition hole.

[0044] Preferably, any of the above items is that the Ebola virus detection kit detects blood samples.

[0045] Any of the above is preferably that the Ebola virus detection kit also includes a sample diluent; the sample diluent is a 90-110 mM Tris buffer, and each 100 mL of 90-110 mM Tris buffer contains 0.9 g NaCl, 0.4 g-0.6 g Tritonx-100, 0.4 g-0.6 g Tween 20, and the pH is 8.5. Further, the Tris buffer is preferably 90, 100, 110 mM and the range therebetween; Tritonx-100 is preferably 0.4, 0.5, 0.6 g and the range therebetween in each 100 mL of 90-110 mM Tris buffer; Tween 20 is preferably 0.4, 0.5, 0.6 g and the range therebetween in each 100 mL of 90-110 mM Tris buffer.

[0046] Compared with the prior art, the present invention has the following beneficial effects:

[0047] The Ebola virus binding protein provided by the present invention has good specificity, high biological activity, strong stability, and high affinity with the Ebola virus, and can be used to prepare products for detecting the Ebola virus.

[0048] The Ebola virus detection kit provided by the present invention has the advantages of simple operation, rapid response, high sensitivity, strong specificity, suitability for rapid on-site detection, and economy and practicality. It can be used for rapid screening during a virus pandemic, and can quickly, accurately and safely detect a large number of people including suspected cases, asymptomatic infection cases in the incubation period, and asymptomatic virus carriers with higher risks, which is of great significance for indirectly solving the insufficient nucleic acid detection capacity, reducing potential infection risks, and preventing the spread of the epidemic. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 It is the upper cover of the Ebola virus detection kit in the preferred embodiment 2 of the present invention;

[0050] Figure 2 It is the lower cover of the Ebola virus detection kit in the preferred embodiment 2 of the present invention;

[0051] Figure 3 This is a structural diagram of the Ebola virus detection test strip in the preferred embodiment 2 of the present invention;

[0052] Figure 4 This is the electrophoresis diagram of the anti-Ebola virus monoclonal antibody in the preferred embodiment 4 of the present invention.

[0053] Figure 5 This is a schematic diagram of the detection results in preferred embodiment 3 of the present invention.

[0054] Icons: 1-Observation window; 2-Sample loading hole; 3-Test card strip loading area; 4-Bottom plate; 5-Test pad; 6-Sample suction pad; 7-Marking pad; 8-Sample loading pad; 9-Quality control line; 10-Test line. DETAILED DESCRIPTION

[0055] The embodiments of the present invention will be described in detail below in conjunction with the embodiments and examples, but it will be appreciated by those skilled in the art that the following embodiments and examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. If specific conditions are not specified, proceed according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0056] It should be noted that the "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy chain or light chain of an antibody. The variable domain of the heavy chain can be referred to as "VH". The variable domain of the light chain can be referred to as "VL". These domains are usually the most variable parts of the antibody and contain the antigen binding site. The light or heavy chain variable region is composed of a framework region interrupted by three hypervariable regions called "complementarity determining regions" or "CDRs". The framework region of an antibody, that is, the combined framework region of the constituent light and heavy chains, plays a role in positioning and aligning the CDRs, which are primarily responsible for binding to the antigen.

[0057] "Framework" or "FR" regions mean the regions of an antibody variable domain excluding those regions defined as CDRs. Each antibody variable domain framework can be further subdivided into contiguous regions (FR1, FR2, FR3, and FR4) separated by CDRs.

[0058] Typically, the variable regions VL / VH of the heavy and light chains can be obtained by arranging and connecting the following numbered CDRs and FRs in the following combinations: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0059] In the present invention, CDR1-VH, CDR2-VH and CDR3-VH refer to the three hypervariable regions of the heavy chain variable region, respectively. Correspondingly, CDR1-VL, CDR2-VL and CDR3-VL refer to the three hypervariable regions of the light chain variable region, respectively.

[0060] In a first aspect, the present invention provides an Ebola virus binding protein selected from protein A and / or protein B;

[0061] The protein A comprises heavy chain complementary determining regions CDR1-VH, CDR2-VH, and CDR3-VH, whose amino acid sequences are sequentially shown in SEQ ID NO.1 to SEQ ID NO.3, and light chain complementary determining regions CDR1-VL, CDR2-VL, and CDR3-VL, whose amino acid sequences are sequentially shown in SEQ ID NO.4 to SEQ ID NO.6;

[0062] The protein B includes heavy chain complementary determining regions CDR1-VH, CDR2-VH, and CDR3-VH whose amino acid sequences are shown in SEQ ID NO.7 to SEQ ID NO.9, and light chain complementary determining regions CDR1-VL, CDR2-VL, and CDR3-VL whose amino acid sequences are shown in SEQ ID NO.10 to SEQ ID NO.12.

[0063] The amino acid sequences shown in SEQ ID NO.1 to SEQ ID NO.12 are shown in Table 1.

[0064] Table 1:

[0065]

[0066] The Ebola virus binding protein provided by the present invention has good specificity, high biological activity, strong stability, and high affinity with the Ebola virus, and can be used to prepare products for detecting the Ebola virus.

[0067] In a preferred embodiment of the present invention, the protein A comprises a heavy chain variable region and a light chain variable region whose amino acid sequences are sequentially shown in SEQ ID NO.13 to SEQ ID NO.14;

[0068] The protein B comprises a heavy chain variable region and a light chain variable region whose amino acid sequences are shown in SEQ ID NO.15 to SEQ ID NO.16;

[0069] Preferably, the Ebola virus binding protein is of murine origin.

[0070] The amino acid sequences shown in SEQ ID NO.13 to SEQ ID NO.14 and SEQ ID NO.15 to SEQ ID NO.16 are shown in Table 2.

[0071] Table 2:

[0072]

[0073] Preferably, the amino acid sequences of the light chain, heavy chain and constant region of protein A (Ebola virus monoclonal antibody 1), and the amino acid sequences of the light chain, heavy chain and constant region of protein B (Ebola virus monoclonal antibody 2) are shown in Table 3.

[0074] Table 3:

[0075]

[0076]

[0077] In a second aspect, the present invention provides the use of the Ebola virus binding protein in the preparation of an Ebola virus detection product.

[0078] In a third aspect, the present invention provides an Ebola virus detection test strip, the detection test strip comprising a base plate and a sample loading pad, a marking pad, a detection pad and a sample suction pad which are sequentially stacked on the base plate;

[0079] The marking pad contains the Ebola virus binding protein, and the Ebola virus binding protein is marked with a marker, and the marker can be detected to identify the location or concentration of the marker.

[0080] The Ebola virus detection test strip provided by the present invention has the advantages of simple operation, rapid response, high sensitivity, strong specificity, suitability for rapid on-site detection, and economy and practicality. It can be used for rapid screening during a virus pandemic, and can quickly, accurately and safely detect a large number of people including suspected cases, asymptomatic infection cases in the incubation period, and asymptomatic virus carriers with higher risks. It is of great significance to indirectly solve the insufficient Norovirus nucleic acid detection capacity, reduce potential infection risks, and prevent the spread of the epidemic.

[0081] In a preferred embodiment of the present invention, the marker includes but is not limited to at least one of colloidal gold, colored microspheres, time-resolved fluorescent microspheres or quantum dot microspheres;

[0082] Preferably, the particle size of the colloidal gold includes, but is not limited to, 40 nm, 60 nm, 80 nm or 100 nm;

[0083] Preferably, the particle size of the colored microspheres, time-resolved fluorescent microspheres and quantum dot microspheres includes, but is not limited to, 100 nm, 150 nm, 200 nm, 250 nm or 300 nm;

[0084] Preferably, the mass ratio of Ebola virus binding protein coupled to colloidal gold is: (0.04-0.32):1; the mass ratio of Ebola virus binding protein to colored microspheres, time-resolved fluorescent microspheres or quantum dot microspheres is: (0.1-0.4):1.

[0085] By adjusting the particle size and dosage of the marker, the sensitivity of the test strip can be made higher.

[0086] In a preferred embodiment of the present invention, a detection line (T line) and a quality control line (C line) are provided on the detection pad;

[0087] The test line is coated with 1-2 mg / mL of the Ebola virus binding protein;

[0088] The quality control line was coated with 1-2 mg / mL of goat anti-mouse IgG polyclonal antibody;

[0089] Preferably, the diluent for the Ebola virus binding protein and goat anti-mouse IgG polyclonal antibody is a 15-25 mM PB buffer containing trehalose, and each 100 mL of the diluent contains 0.1-0.9 g of trehalose.

[0090] In a preferred embodiment of the present invention, the Ebola virus binding protein on the labeling pad is different from the Ebola virus binding protein on the detection pad.

[0091] For example, the protein on the labeling pad is Ebola virus binding protein A, wherein A is Ebola virus monoclonal antibody 1, and the protein on the detection pad is Ebola virus binding protein B, wherein B is Ebola virus monoclonal antibody 2.

[0092] The test strip provided by the present invention is based on the principle of antigen-antibody reaction, and after labeling the Ebola virus monoclonal antibody with colloidal gold, colored microspheres, time-resolved fluorescent microspheres, and quantum dots, the marker is solidified on a glass cellulose membrane. Another monoclonal antibody of the Ebola virus is coated on the detection pad (such as an NC membrane), and based on the principle of antigen-antibody reaction, it can be detected by the naked eye or a matching instrument within the detection time. If the Ebola virus is present in the sample, a double antibody sandwich structure is formed, forming a band visible to the naked eye or a light intensity signal in the instrument. If there is no Ebola virus in the sample, no band appears on the NC membrane or there is no light intensity signal in the instrument. Negative or positive judgment is made based on the presence or absence of the signal, or the viral load is predicted based on the intensity of the light intensity signal.

[0093] By using the test strip provided by the present invention for testing, the test results can be obtained within 15-20 minutes of the entire process, which is fast and efficient, and helps medical personnel to obtain the test results in a timely manner, make comprehensive judgments and take timely measures based on the results, avoid panic, and reduce the spread of the epidemic.

[0094] In a fourth aspect, the present invention provides an Ebola virus detection kit, comprising the Ebola virus detection test strip and a shell, wherein the Ebola virus detection test strip is arranged inside the shell.

[0095] The Ebola virus detection kit provided by the present invention contains an Ebola virus detection test strip, and thus has all the beneficial effects of the Ebola virus detection test strip.

[0096] In a preferred embodiment of the present invention, the housing comprises an upper cover and a lower cover that are detachably connected;

[0097] The upper cover is provided with an observation window and a sample adding hole.

[0098] The shapes of the observation window and the sample loading hole are not specifically limited in the present invention. The observation window can be, for example, square and located above the test line and the quality control line of the test strip for observing the test results. The sample loading hole can be, for example, a circular hole with a diameter of 0.5-1 cm and located above the sample loading pad.

[0099] In a preferred embodiment of the present invention, the detection sample of the Ebola virus detection kit is a blood sample; the sample includes at least one of serum, plasma or whole blood.

[0100] Preferably, the Ebola virus detection kit further comprises a sample diluent, which is used to process the sample, and then the Ebola virus detection kit of the present invention is used for detection.

[0101] The sample diluent was 90~110 mM Tris buffer. Each 100 mL of 90~110 mM Tris buffer contained 0.9 g NaCl, 0.4 g~0.6 g Tritonx-100, and 0.4 g~0.6 g Tween 20, with a pH of 8.5.

[0102] The sample dilution and sample addition method are as follows:

[0103] Preparation of dilution:

[0104] Take 100 mL as an example to prepare the dilution solution of the blood sample of the Ebola virus antigen detection kit:

[0105] (1) Use a balance to weigh 1.21 g Tris and 0.9 g NaCl and dissolve them in water;

[0106] (2) Use an EP tube to weigh 0.5 g Tritonx-100 and 0.5 g Tween 20 respectively, and repeatedly wash the EP tube with the solution in step 1 to make them completely dissolved in water;

[0107] (3) Adjust the pH to 8.5 with 4 M NaOH or HCl, then add water to 100 mL and use a 100 mL volumetric flask to make up to volume;

[0108] Test kit sample loading:

[0109] (1) When testing samples,

[0110] Plasma and serum samples: Use a disposable plastic dropper to draw 1 drop of liquid sample (about 35-45 μL) and place it in Figure 1 Then add 1 drop of sample diluent (about 40-50 μL) to the sample well;

[0111] Whole blood sample: Use a disposable plastic dropper to draw 1 drop of liquid sample (about 35-45 μL) and place it in Figure 1 Then add 1 drop of sample diluent (about 40-50 μL) to the sample well;

[0112] (2) After 15 minutes, the results were analyzed according to different methodologies.

[0113] The present invention is further described below by means of specific examples and comparative examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0114] It should be noted that, unless otherwise specified in the following examples, the diluent for the Ebola virus binding protein and goat anti-mouse IgG polyclonal antibody is a 20 mM PB buffer containing trehalose, and each 100 mL of the diluent contains 0.5 g of trehalose;

[0115] The sample diluent was 90~110 mM Tris buffer. Each 100 mL of 90~110 mM Tris buffer contained 0.9 g NaCl, 0.4 g~0.6 g Tritonx-100, and 0.4 g~0.6 g Tween 20, with a pH of 8.5.

[0116] The method for preparing the monoclonal antibody described in the present invention is the content of the prior art, and is only briefly described here. The specific operation method can be carried out according to the operation method recorded in the prior art:

[0117] (1) Immunogen and Freund's adjuvant were mixed in equal volumes to an appropriate volume and emulsified completely. Mice were immunized by intraperitoneal injection. Each mouse was injected with 50 μg of immunogen in a volume of 100 μL per mouse, once a week.

[0118] (2) After immunization for 4 times, the titer of the antibody in the serum was tested by the indirect ELISA method, and mice with an OD value greater than 1.0 detected by 16,000-fold serum dilution were screened;

[0119] (3) Using spleen cells from the screened mice to fuse with myeloma cells, the fused cells were plated and cultured using the limiting dilution method;

[0120] (4) Screening monoclonal cell wells and culturing and amplifying them, and taking the monoclonal cell well with the highest OD value detected in the cell culture supernatant as the target hybridoma cell;

[0121] (5) The target hybridoma cells are amplified and injected into the mouse peritoneal cavity. The ascites is collected and the monoclonal antibodies in the ascites are purified using Protein A, which is the target monoclonal antibody.

[0122] Based on the Ebola virus data published by the NCBI database, the Ebola virus VP40 protein (GenBank: ARG44039.1) was analyzed, and the protein recombinant antigen (aa27-aa295) commercially synthesized by Sangon Biotechnology (Shanghai) Co., Ltd. was used as the immunogen for preparing monoclonal antibodies.

[0123] Example 1

[0124] The Ebola virus binding proteins include: Ebola virus monoclonal antibody 1 and Ebola virus monoclonal antibody 2. The variable region sequences of the above binding proteins are shown in Table 2.

[0125] A 12% SDS-PAGE gel was prepared according to the conventional method, and 5 μg of the above antibodies were loaded and electrophoresed using a protein molecular weight standard as a reference. The results showed that the two anti-Ebola virus monoclonal antibodies showed two characteristic bands of about 25 KD and 50 KD, which were the light chain and heavy chain of IgG respectively ( Figure 4 ). After scanning and analysis, the antibody content of the bands was above 90%.

[0126] Figure 4 In the above, 1: Anti-1; 2: Anti-2; wherein, Anti-1 is anti-Ebola virus monoclonal antibody 1; Anti-2 is anti-Ebola virus monoclonal antibody 2.

[0127] The antibodies used in the following examples are the same as those in Example 1.

[0128] Example 2

[0129] An Ebola virus detection kit, such as Figure 1~Figure 3 As shown, it includes an Ebola virus detection test strip and a shell, and the Ebola virus detection test strip is arranged inside the shell. The Ebola virus detection test strip includes a bottom plate 4 and a sample loading pad 8, a marking pad 7, a detection pad 5 and a sample suction pad 6 which are sequentially stacked on the bottom plate; a detection line 10 and a quality control line 9 are arranged on the detection pad; the shell includes a detachably connected upper cover and a lower cover; the upper cover is provided with an observation window 1 and a sample loading hole 2, and the lower cover is provided with a detection card strip area 3.

[0130] Example 3

[0131] Example 3 provides a colloidal gold-labeled antibody for preparing an Ebola virus antigen detection kit.

[0132] 1 Main Materials

[0133] 1.1 Antibodies: Mouse monoclonal antibodies are protein A and protein B provided by the present invention, which are labeled and coated respectively; Sheep anti-mouse IgG antibody: a product of Shenzhen Feipeng Biological Co., Ltd., used for coating the nitrocellulose membrane quality control line.

[0134] 1.2 Nitrocellulose membrane: NC membrane is a product of Sartorius.

[0135] 1.3 Other consumables: PVC boards and other consumables are products of Beacon Labs; commonly used reagents are all analytical grade reagents.

[0136] 1.4 Acquisition of recombinant antigens: Based on the Ebola virus data published by the NCBI database, the Ebola virus VP40 protein (GenBank: ARG44039.1) was analyzed and protein recombinant antigens (aa27-aa295) were commercially synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.

[0137] 2 Methods

[0138] 2.1 Preparation of colloidal gold labeling pad:

[0139] The steps for preparing the colloidal gold labeling pad are as follows:

[0140] (1) Take 1 mL of colloidal gold solution with a particle size of 40 nm and adjust the pH to 8.5 with 0.2 M K2CO3;

[0141] (2) Add 25 μg of monoclonal antibody 1, the mass ratio of antibody to colloidal gold particles is 0.1:1, adjust the rotary shaker to a certain speed, rotate and label at room temperature for 1.5 h, then add 20 μL of blocking solution;

[0142] (3) Centrifuge at 12,000 rpm for 15 min and discard the supernatant;

[0143] (4) Add 100 μL of colloidal gold solution;

[0144] (5) The above concentrate was diluted in a ratio of 1:7, sprayed with gold, and placed in a drying oven at 37°C for 2 h for later use;

[0145] 2.2 NC membrane coating:

[0146] Monoclonal antibody 2 was diluted to 1.5 mg / mL and goat anti-mouse IgG antibody was diluted to 2 mg / mL using 0.02 M PB containing 0.5% trehalose. Then, the test line T and the quality control line C were drawn on the nitrocellulose membrane using a film sprayer. After coating, the NC membrane was dried in an oven at 37°C for 24 h for later use.

[0147] 2.3 Assembly of the kit:

[0148] Place the coated nitrocellulose membrane in the middle of the plastic support plate in a drying room and stick it. Overlap the marker colloidal gold pad on one side of the T line of the nitrocellulose membrane (1 / 3 of the colloidal gold pad) and stick it. Overlap the sample pad on the other side of the colloidal gold pad (1 / 5 of the colloidal gold pad). Overlap the sample suction pad on one side of the C line of the nitrocellulose membrane (1 / 10 of the sample suction pad). Then use a cutting machine to cut the pasted plastic plate into test strips of a certain width, and then load them into the test card to form an Ebola virus antigen detection kit.

[0149] 2.4 Detection:

[0150] Step 1, take out the test kit and the sample to be tested and equilibrate to room temperature;

[0151] Step 2. Open the sealed aluminum foil bag, take out the test kit and place it flat on the table;

[0152] Step 3, add sample:

[0153] Plasma and serum samples: Use a disposable plastic dropper to draw 1 drop of liquid sample (about 35-45 μL) and place it in Figure 1 Then add 1 drop of sample diluent (about 40-50 μL) to the sample well;

[0154] Whole blood sample: Use a disposable plastic dropper to draw 1 drop of liquid sample (about 35-45 μL) and place it in Figure 1 Then add 1 drop of sample diluent (about 40-50 μL) to the sample well;

[0155] Step 4: Set the timer to count, and read the result after 15 minutes. Note that if the sample does not undergo lateral chromatography or diafiltration within 1 minute after adding the sample, it may be because the sample is too viscous and needs to be pretreated with saline.

[0156] 3 Results

[0157] Under the action of lateral flow chromatography, when Ebola virus is present in the sample, the test line will show color, and the quality control line will also show color ( Figure 5 a in the figure); when there is no Ebola virus in the sample, the test line does not show color, but the quality control line shows color ( Figure 5 b); After loading, if the quality control line does not show color, the result is considered invalid regardless of whether the test line shows color or not ( Figure 5 c in Figure 5 d) in.

[0158] Example 4

[0159] Example 4 provides a time-resolved fluorescent microsphere-labeled antibody for preparing an Ebola virus antigen detection kit.

[0160] A pair of Ebola virus monoclonal antibodies (protein A and protein B) provided by the present invention is used to prepare an Ebola virus antigen detection kit. The kit comprises a detection card and a test strip. The detection card is divided into an upper cover and a lower bottom. The test strip has a monoclonal antibody 1 labeled with time-resolved fluorescent microspheres embedded in a fluorescent pad, and a detection line is coated with a monoclonal antibody 2. The Ebola virus antigen in a sample is quantitatively detected by a double antibody sandwich method.

[0161] 1 Kit preparation operation process:

[0162] The monoclonal antibody 1 prepared by the method of the present invention is labeled on the surface of time-resolved fluorescent microspheres. The specific embodiments are as follows:

[0163] Time-resolved fluorescent microsphere antibody labeling: 1 mL of 1% carboxyl time-resolved fluorescent microspheres, add 9 mL of MES buffer, then add 25 μL of EDC solution (10 mg / mL) and 25 μL of NHS solution (10 mg / mL), shake at room temperature for 30 min, and collect the precipitate by centrifugation. Add HEPES reconstitution solution, ultrasonically disperse evenly, add 1 mL of 1 mg / mL monoclonal antibody 1, shake at room temperature for 120 min, and collect the precipitate by centrifugation. Then add 1 mL of blocking solution, shake at room temperature for 120 min. Collect the microsphere precipitate by centrifugation and reconstitute with reconstitution solution.

[0164] Preparation of fluorescent pad: The labeled time-resolved fluorescent microspheres were diluted with the microsphere complex solution, and the fluorescent pads were sprayed with a gold spray film instrument at a spraying rate of 3 μL / cm and a spraying interval of 6 mm. After spraying, they were placed in a 37°C low humidity (<30%) oven for 2 h.

[0165] NC membrane coated CT line: T line uses monoclonal antibody 2 with a concentration of 1.5 mg / mL, C line uses goat anti-mouse IgG antibody with a concentration of 1 mg / mL, 1 μL / cm line is drawn, and then placed in 37℃ low humidity (<30%) for drying for 24 hours.

[0166] Sample pad treatment: The sample pad treatment solution is composed of buffer salt, slow-release agent, cosolvent, blocking agent, etc. The specific formula is 20 mM Tris buffer, and each 100 mL Tris buffer contains 1g BSA, 0.5g Tween 20, and 2g sucrose. 2 Use 1mL of sample treatment solution to treat. After evenly treating, place in a 37℃ low humidity (<30%) oven to dry for 2h.

[0167] Test strip assembly: NC film, sample suction pad, fluorescent pad, and sample loading pad are sequentially pasted on the PVC board. The sample suction pad and fluorescent pad are pressed 1~2mm on the NC film, and the sample loading pad is pressed 1~2mm on the fluorescent pad. After assembly, the test strip is cut into a width of 4±0.4mm and installed in a card shell. The card shell and desiccant are placed in an aluminum foil bag and sealed. After labeling and boxing, the finished test card is obtained.

[0168] Test sample preparation: The commercially synthesized protein recombinant antigen produced by Shanghai Bioengineering Co., Ltd. was tested after gradient dilution.

[0169] 2 Kit testing process

[0170] Place the test card on a clean and flat surface, draw 90~100μL of the treated sample and drop it into the sample adding end of the test card, and set up a PBS control group at the same time.

[0171] The standard curve of the test strip was introduced. After 15 minutes, the fluorescent immunoassay analyzer was used to scan the detection area to obtain the fluorescent signal. After the test, the concentration value corresponding to the Ebola virus antigen was displayed. The test results are shown in Tables 4 and 5 below.

[0172] Table 4 Test results of dilution samples

[0173] Table 4-1 Standard curve

[0174]

[0175] Table 4-2 Dilution test results

[0176]

[0177] Table 5 Positive recombinant antigen test results

[0178]

[0179] Example 5

[0180] Example 5 provides a kit for preparing an Ebola virus antigen detection kit using colored microspheres labeled with antibodies.

[0181] A pair of Ebola virus monoclonal antibodies (protein A and protein B) provided by the present invention is used to prepare an Ebola virus antigen detection test strip, wherein the test strip has a monoclonal antibody 1 labeled with colored microspheres embedded on a marking pad, and a detection line is coated with a monoclonal antibody 2, and a double antibody sandwich method is used to qualitatively detect Ebola virus antigens in a sample.

[0182] 1. Preparation of test strips:

[0183] 1.1 Colored microspheres labeled with antibodies

[0184] (1) Adjust the pH of 100 nm colored microspheres to 8.0 with 0.1 M K2CO3;

[0185] (2) Monoclonal antibody 1 labeled microspheres: Take 1 mL of the above pH-adjusted solution, add 30 μg of monoclonal antibody 1, the mass ratio of antibody to colored microspheres is 0.3:1, react at room temperature for 1 hour, then centrifuge and discard the supernatant;

[0186] Chicken IgY antibody-labeled microspheres: add 5 μg of chicken IgY to 1 mL of the pH-adjusted solution, with the mass ratio of antibody to colored microspheres being 0.05:1. After reacting at room temperature for 1 hour, centrifuge and discard the supernatant.

[0187] (3) Add 1 mL of 20% BSA to block for 2 h, then centrifuge and discard the supernatant;

[0188] (4) After re-dissolving the above microspheres with 100 μL of pH 8.0 reconstitution solution, the two microspheres were mixed at a ratio of 5:1, and the mixture was diluted with the reconstitution solution at a ratio of 15%;

[0189] 1.2 Preparation of labeling pad

[0190] The dilution of the labeled colored microsphere antibody complex was sprayed on the marking pad using a gold spray film instrument at a spraying rate of 7.5 μL / cm and a spraying interval of 6 mm. After spraying, it was placed in a 37°C low humidity (<30%) drying room for 2 hours.

[0191] 1.3 Preparation of sample pad

[0192] The sample pad treatment solution is composed of buffer salt, slow-release agent, cosolvent, blocking agent, etc. The specific formula is 20mM Tris, 1% BSA, 0.5% Tween 20, 2% sucrose. 2 Use 1mL of sample treatment solution to treat. After evenly treating, place in a 37℃ low humidity (<30%) oven to dry for 2h.

[0193] 1.4 C / T line coating

[0194] The T line uses monoclonal antibody 2 at a concentration of 1.5 mg / mL (the C line uses goat anti-chicken IgY antibody at a concentration of 2 mg / mL), 1 μL / cm is used for drawing lines, and after completion, it is placed in a 37℃ low humidity (<30%) oven to dry for 24 hours.

[0195] 1.5 Test strip assembly

[0196] The PVC board is pasted with the sample suction pad, NC film, marking pad, and sample loading pad in sequence. The sample suction pad and marking pad are pressed 1~2mm by the NC film, and the sample loading pad is pressed 1~2 mm by the marking pad. After assembly, the test strip is cut into a width of 4±0.4mm and installed in the card shell. The card shell and desiccant are placed in an aluminum foil bag. The finished test kit is obtained by labeling and boxing.

[0197] 1.6 Test sample preparation: The commercially synthesized protein recombinant antigen produced by Shanghai Bioengineering Co., Ltd. was serially diluted for testing.

[0198] 2. Detection

[0199] Place the test card on a clean and flat table, draw 90~100μL of the prepared recombinant antigen sample and drop it into the sample end of the test card, and set up a PBS control group. Observe the test card window after 10 minutes. There are 2 positive lines in the window and only one C line is negative. The test results are as follows: The results are shown in Table 6, showing that the antigen compliance rate of the test strips assembled with 1 pair of monoclonal antibodies is 100%.

[0200] Table 6 Results of the additional test for the preparation of Ebola virus antigen detection reagents with colored microsphere-labeled antibodies

[0201]

[0202] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An Ebola virus binding protein, comprising protein A or protein B, characterized in that: The protein A is an Ebola virus monoclonal antibody 1, comprising a heavy chain complementary determining region whose amino acid sequence is as follows: a CDR1-VH whose amino acid sequence is as shown in SEQ ID NO.1, a CDR2-VH whose amino acid sequence is as shown in SEQ ID NO.2, and a CDR3-VH whose amino acid sequence is as shown in SEQ ID NO.3; The protein A comprises a light chain complementary determining region whose amino acid sequence is as follows: CDR1-VL whose amino acid sequence is as shown in SEQ ID NO.4, CDR2-VL whose amino acid sequence is as shown in SEQ ID NO.5, and CDR3-VL whose amino acid sequence is as shown in SEQ ID NO.6; The protein B is Ebola virus monoclonal antibody 2, comprising heavy chain complementary determining regions whose amino acid sequences are as follows: CDR1-VH whose amino acid sequence is shown in SEQ ID NO.7, CDR2-VH whose amino acid sequence is shown in SEQ ID NO.8, and CDR3-VH whose amino acid sequence is shown in SEQ ID NO.9; The protein B comprises a light chain complementary determining region whose amino acid sequence is as follows: CDR1-VL whose amino acid sequence is shown in SEQ ID NO.10, CDR2-VL whose amino acid sequence is shown in SEQ ID NO.11 and CDR3-VL whose amino acid sequence is shown in SEQ ID NO.

12.

2. The Ebola virus binding protein according to claim 1, characterized in that The protein A comprises a heavy chain variable region whose amino acid sequence is shown in SEQ ID NO.13 and a light chain variable region whose amino acid sequence is shown in SEQ ID NO.14; The protein B comprises a heavy chain variable region whose amino acid sequence is shown in SEQ ID NO.15 and a light chain variable region shown in SEQ ID NO.

16.

3. Use of the Ebola virus binding protein according to claim 1 or 2 in the preparation of an Ebola virus detection product.

4. An Ebola virus detection test strip, characterized in that: The test strip comprises a base plate and a sample loading pad, a marking pad, a detection pad and a sample suction pad which are sequentially stacked on the base plate; The labeling pad contains the Ebola virus binding protein according to claim 1 or 2, and the Ebola virus binding protein is labeled with a marker. The detection pad is provided with a detection line and a quality control line; the detection line is coated with the Ebola virus binding protein according to claim 1 or 2; the quality control line is coated with goat anti-mouse IgG polyclonal antibody, The Ebola virus binding protein on the labeling pad is different from the Ebola virus binding protein on the detection pad.

5. The Ebola virus detection test strip according to claim 4, characterized in that: The marker includes at least one of colloidal gold, colored microspheres, time-resolved fluorescent microspheres or quantum dot microspheres.

6. The Ebola virus test strip according to claim 4, characterized in that: The detection line is coated with 1-2 mg / mL of the Ebola virus binding protein according to claim 1 or 2; the quality control line is coated with 1-2 mg / mL of goat anti-mouse IgG polyclonal antibody; the diluent for the Ebola virus binding protein and the goat anti-mouse IgG polyclonal antibody is a 15-25 mM PB buffer containing trehalose, and each 100 mL of the diluent contains 0.1-0.9 g of trehalose.

7. An Ebola virus detection kit, characterized in that: The invention comprises the Ebola virus detection test strip according to any one of claims 4 to 6 and a shell, wherein the Ebola virus detection test strip is arranged inside the shell.

8. The Ebola virus detection kit according to claim 7, characterized in that: The housing comprises an upper cover and a lower cover which are detachably connected; the upper cover is provided with an observation window and a sample addition hole.

9. The Ebola virus detection kit according to claim 7, characterized in that: The Ebola virus detection kit detects blood samples; the Ebola virus detection kit also includes a sample diluent; the sample diluent is a 90-110 mM Tris buffer, and each 100 mL of 90-110 mM Tris buffer contains 0.9 g NaCl, 0.4 g-0.6 g Tritonx-100, 0.4 g-0.6 g Tween 20, and the pH is 8.5.

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