CN114578047A

CN114578047A - Foot-and-mouth disease virus O-type and A-type antibody joint detection kit and preparation method and application thereof

Info

Publication number: CN114578047A
Application number: CN202011379112.4A
Authority: CN
Inventors: 田克恭; 李玉芳; 张许科
Original assignee: Luoyang Zhongke Biochip Technology Co ltd
Current assignee: Luoyang Zhongke Biochip Technology Co ltd
Priority date: 2020-11-30
Filing date: 2020-11-30
Publication date: 2022-06-03

Abstract

The invention provides a foot-and-mouth disease virus O-type and A-type antibody joint inspection kit, which comprises a foot-and-mouth disease virus O-type and A-type antibody detection chip and an enzyme labeling reagent, wherein sampling points on the foot-and-mouth disease virus O-type and A-type antibody detection chip are respectively coated with a foot-and-mouth disease virus O-type antigen, a foot-and-mouth disease virus A-type antigen, a quality control product and a blank control; the O-type foot-and-mouth disease virus antigen is O-type foot-and-mouth disease virus-like particles, the A-type foot-and-mouth disease virus antigen is A-type foot-and-mouth disease virus-like particles, the quality control product is a goat anti-mouse polyclonal antibody, the blank control is a sample solution, and the enzyme labeling reagent is a solution containing an enzyme-labeled O-type foot-and-mouth disease virus monoclonal antibody 7D2 and an enzyme-labeled A-type foot-and-mouth disease virus monoclonal antibody 3B 4. The antibody joint inspection kit disclosed by the invention not only has high sensitivity, but also overcomes the defect of large difference of detection results among different batches in the prior art, and can be effectively used for various non-immune diagnosis applications such as vaccine quality control and the like.

Description

Foot-and-mouth disease virus O-type and A-type antibody joint detection kit and preparation method and application thereof

Technical Field

The invention relates to a foot-and-mouth disease virus O-type and A-type antibody joint detection kit, a preparation method and application thereof, and belongs to the field of biological medicines.

Background

Foot-and-mouth disease is a febrile, acute, highly contagious infectious disease susceptible to artiodactyl caused by foot-and-mouth disease virus infection. The disease is spread rapidly and has high incidence rate, the outbreak and the epidemic of the disease bring huge economic loss to the global aquaculture industry, the disease is listed as the first class A of virulent infectious diseases by the world animal health Organization (OIE) and the Food and Agriculture Organization (FAO) of the United nations, and China is listed as the first class of infectious diseases. The foot-and-mouth disease virus is divided into 7 serotypes and more than 80 subtypes, and variation is easy to occur, wherein the 7 serotypes comprise A type, O type, Asia I type, C type, SAT1 type, SAT2 type and SAT3 type, and the O type and A type foot-and-mouth disease virus are most popular. According to genetic classification, the type-O foot-and-mouth disease virus mainly epidemics three genetic topotypes (Topo types), which respectively belong to CATHOY type (Chinese type), ME-SA type (middle east-south subtype) and SEA type (southeast subtype). Because of the lack of cross-immune protection between different types of vaccines, different subtypes within the same serotype, i.e., type O, differ in their antigenicity to varying degrees, and the serological cross-reactivity of the antibodies produced varies, which requires that the antibody detection method must be specific and stable.

At present, the prevention and control of the foot-and-mouth disease are mainly based on vaccine immunization, and the monitoring of the antibody level after the vaccine immunization is a necessary measure for controlling the immune effect of the vaccine. The serological detection method for foot-and-mouth disease in the market mainly comprises the following steps: virus Neutralization Test (VNT), liquid phase blocking ELISA method (LPB-ELISA), wherein virus neutralization test is the gold standard for antibody determination, but this method requires the use of live virus and can only be operated in specific high-grade laboratories, is relatively complex, takes about 5 days and presents a biosafety risk; the liquid phase blocking ELISA kit is a commercial kit, is relatively applied, but has the disadvantages of complicated operation steps, about 4 hours of consumed time, unstable detection results among batches, multiple antibodies used by a capture antibody and an enzyme-labeled antibody of the kit, and influence on the detection results due to the fact that the multiple antibodies are often uneven in quality. In addition, the methods are all single-index detection, only one antibody can be detected at one time, and the sample detection amount is large, and time and labor are consumed. Therefore, the kit which can directly prepare antigen plates from antigens in a solid phase and can simultaneously detect FMDV type-O and type-A antibodies at one time is developed, the problem of poor repeatability is solved, the sensitivity and the specificity are improved, and the kit has important significance for diagnosis and prevention of the disease.

Disclosure of Invention

In order to solve the defects of the prior art, the invention provides an O-type and A-type foot-and-mouth disease virus antibody joint inspection kit, wherein the antibody joint inspection kit comprises one or more O-type and A-type foot-and-mouth disease virus antibody detection chips and an enzyme labeling reagent, and sampling points on the O-type and A-type foot-and-mouth disease virus antibody detection chips are respectively coated with an O-type foot-and-mouth disease virus antigen, an A-type foot-and-mouth disease virus antigen, a quality control product and a blank control; the O-type foot-and-mouth disease virus antigen is O-type foot-and-mouth disease virus-like particles, the A-type foot-and-mouth disease virus antigen is A-type foot-and-mouth disease virus-like particles, the quality control product is a goat anti-mouse polyclonal antibody, the blank control is a sample solution, the enzyme labeling reagent is a solution containing an enzyme-labeled O-type foot-and-mouth disease virus monoclonal antibody and an enzyme-labeled A-type foot-and-mouth disease virus monoclonal antibody, the enzyme-labeled O-type foot-and-mouth disease virus monoclonal antibody is an enzyme-labeled O-type foot-and-mouth disease virus monoclonal antibody 7D2 or an enzyme-labeled O-type foot-and-mouth disease virus monoclonal antibody 6G6, and the labeled A-type foot-and-mouth disease virus monoclonal antibody is an enzyme-labeled A-type foot-and-mouth disease virus monoclonal antibody 3B4 or an enzyme-labeled A-type foot-and-mouth disease virus monoclonal antibody 2E 11.

In one embodiment of the invention, in the antibody joint inspection kit, the coating amount of the O-type foot-and-mouth disease virus antigen is 1-128 ng/point; the coating amount of the A-type foot-and-mouth disease virus antigen is 1-128 ng/point; the heavy chain variable region of the O type foot-and-mouth disease virus monoclonal antibody 7D2 is shown as SEQ ID NO.7 or the code of the degenerate sequence thereof, and the light chain variable region is shown as SEQ ID NO.8 or the code of the degenerate sequence thereof; the heavy chain variable region of the O type foot-and-mouth disease virus monoclonal antibody 6G6 is shown as SEQ ID NO.5 or the code of the degenerate sequence thereof, and the light chain variable region is shown as SEQ ID NO.6 or the code of the degenerate sequence thereof; the heavy chain variable region of the A type foot-and-mouth disease virus monoclonal antibody 3B4 is shown as SEQ ID NO.3 or the code of the degenerate sequence thereof, and the light chain variable region is shown as SEQ ID NO.4 or the code of the degenerate sequence thereof; the heavy chain variable region of the A type foot-and-mouth disease virus monoclonal antibody 2E11 is shown as SEQ ID NO.1 or the code of the degenerate sequence thereof, and the light chain variable region is shown as SEQ ID NO.2 or the code of the degenerate sequence thereof; the buffer solution used by the enzyme labeling reagent is a PBS solution containing 20% fetal bovine serum by volume, wherein the final concentrations of the enzyme-labeled O-type foot-and-mouth disease virus monoclonal antibody 7D2 and the enzyme-labeled A-type foot-and-mouth disease virus monoclonal antibody 3B4 are 100ng/ml and 75ng/ml respectively; the enzyme marked by the enzyme labeling reagent is horseradish peroxidase, alkaline phosphatase or beta-D-galactosidase; the quality control sample is applied to quality control sample points 1-3, and the coating amount of the quality control sample point 1 is more than or equal to 1 ng/point and less than 4 ng/point; the coating amount of the quality control product point 2 is 4 ng/point; the coating amount of the quality control product point 3 is more than 4 ng/point and less than or equal to 6 ng/point; the sample solution is prepared by uniformly mixing a 5% glycerol solution, a 5% sorbitol solution, a 0.05% triton solution, a DMSO solution and a PBS (pH6.8) solution according to the volume ratio of 10: 15: 0.1: 50: 100.

The antigen coating amount is further optimized and selected, so that the high sensitivity of detection is ensured.

Through experimental research, the invention defines the specific A-type foot-and-mouth disease virus antigen coating amount, the specific O-type foot-and-mouth disease virus antigen coating amount, the specific sheep anti-mouse polyclonal antibody coating amount, the specific enzyme-labeled A-type foot-and-mouth disease virus monoclonal antibody content and the specific enzyme-labeled O-type foot-and-mouth disease virus monoclonal antibody content, and realizes the high-sensitivity detection of serum antibodies in a sample.

Compared with the traditional ELISA kit which is provided with a negative control, a positive control or a standard substance as a quality control substance in the kit, the joint inspection kit provided by the invention is technically improved, the quality control substance is arranged on a reaction carrier, the quality control substance is a commercialized goat anti-mouse polyclonal antibody, and the negative control and the positive control are not required to be arranged in the kit, namely, the complicated preparation steps of immunization or toxicity attack of animals, serum collection and the like, and the cost of manpower and animal test sites are not required to be screened in advance, and the convenience and the accuracy of the operation of the kit are greatly improved. In particular, the trouble that a type of infectious disease virus, foot-and-mouth disease virus, must be handled in a high-grade laboratory is avoided. According to the invention, researches show that the goat anti-mouse polyclonal antibody with a specific content can be used as a quality control product without setting negative control and positive control, and the goat anti-mouse polyclonal antibody has the function of being used as reaction control when no antibody reaction exists in a single detection hole, is used for evaluating the effectiveness of a test and reflects the reaction degree of an enzyme labeling reagent when no blocking exists. The goat anti-mouse polyclonal antibody can be purchased commercially or prepared by a conventional method, and is simple and convenient to operate. According to the invention, the research finds that the sample diluent with specific content and components improves the detection sensitivity and specificity of the kit.

In a preferred embodiment of the invention, in the antibody joint inspection kit, the coating amount of the O-type foot-and-mouth disease virus antigen is 8-32 ng/dot; the coating amount of the A-type foot-and-mouth disease virus antigen is 16-64 ng/point; the coating amount of the goat anti-mouse polyclonal antibody of the quality control sample point 1-3 is respectively 2 ng/point, 4 ng/point and 6 ng/point.

In a more preferred embodiment of the present invention, in the antibody joint inspection kit according to the present invention, the amount of the antigen coating of the foot-and-mouth disease virus type O is 16 ng/spot.

In a more preferred embodiment of the present invention, the amount of the antigen coating of the type-a foot-and-mouth disease virus in the antibody bioassay kit of the present invention is 32 ng/spot.

In a most preferred embodiment of the present invention, in the antibody-based joint inspection kit of the present invention, the amount of the aftosa antigen coated in the aftosa antigen O-type and aftosa antigen a-type is 16 ng/point, and the amount of the aftosa antigen a-type is 32 ng/point.

In a preferred embodiment of the present invention, the enzyme-labeled reagent in the antibody joint inspection kit of the present invention is a solution containing an enzyme-labeled O-type foot-and-mouth disease virus monoclonal antibody 7D2 and an enzyme-labeled a-type foot-and-mouth disease virus monoclonal antibody 3B 4.

In one embodiment of the present invention, in the antibody joint inspection kit of the present invention, the kit further comprises a sample diluent, a substrate solution, and a washing solution; the sample diluent is a PBS solution containing 10% V/V fetal calf serum, 0.05-0.2% V/V Triton-100 and 0.1% V/V Proclin 300; the substrate solution is TMB (3,3',5,5' -tetramethylbenzidine) solution; the washing solution is PBS solution containing 1% V/V Tween 20.

In a preferred embodiment of the present invention, the sample diluent is a PBS solution containing 10% V/V fetal bovine serum, 0.1% V/V Triton-100, and 0.1% V/V Proclin 300.

The Triton-100 content in the sample treatment solution may be 0.05% V/V, 0.06% V/V, 0.07% V/V, 0.08% V/V, 0.09% V/V, 0.1% V/V, 0.11% V/V, 0.12% V/V, 0.13% V/V, 0.14% V/V, 0.15% V/V, 0.16% V/V, 0.17% V/V, 0.18% V/V, 0.19% V/V, 0.2% V/V.

The invention ensures higher sensitivity of detection by further optimizing and selecting components and contents in the substrate solution and the washing solution.

In one embodiment of the present invention, in the antibody joint detection kit of the present invention, the coating amounts of the goat anti-mouse polyclonal antibody of the quality control point (1), the quality control point (2) and the quality control point (3) in the detection chip are respectively 2 ng/point, 4 ng/point, 6 ng/point and blank control point (4) as background detection points, the aftosa a antigen is spotted on the spotting point (5), and the aftosa O antigen is spotted on the spotting point (6); the sampling points of the quality control product point (1), the quality control product point (2), the quality control product point (3) and the blank reference sampling point (4) are respectively positioned at the upper left corner, the lower right corner and the upper right corner of a detection chip, the points (5) and (6) can be respectively positioned at other positions of the detection chip, and the distance between the midpoint of the sampling points (1) to (6) and the point edge is more than or equal to 700 mu m; the distances from the quality control point (1), the quality control point (2), the quality control point (3) and the blank reference point sample point (4) to the upper edge and the lower edge of the detection chip subunit are not less than 8mm, and the distances from the blank reference point sample point to the left edge and the right edge of the detection chip subunit are not less than 5 mm; the antibody joint inspection kit comprises one or more subunits of the foot-and-mouth disease virus O-type and A-type antibody detection chips, and independent detection holes are formed by bottom ends, upper grids and detection chips which are arranged between the bottom ends and the upper grids and clamped by the bottom ends and the upper grids, each detection hole corresponds to one detection chip subunit, and when the antibody joint inspection kit comprises a plurality of the foot-and-mouth disease virus O-type and A-type antibody detection chip subunits, each independent detection chip subunit is integrated into a microarray chip.

The invention also provides a method for preparing the foot-and-mouth disease virus O-type and A-type antibody joint detection kit, wherein the method comprises the following steps: respectively preparing O-type antigen and A-type antigen of foot-and-mouth disease virus, diluting the O-type antigen and A-type antigen of foot-and-mouth disease virus together with goat anti-mouse polyclonal antibody by using a sample solution, and carrying out sample application on a membrane to prepare O-type and A-type antibody detection chips of foot-and-mouth disease virus; step (2) forming independent detection holes by a bottom end, an upper grid and the antibody detection chip in the step (1) arranged between the bottom end and the upper grid, wherein the antibody detection chip is arranged between the bottom end and the upper grid and is clamped by the bottom end and the upper grid, one detection hole corresponds to one detection chip subunit, and when the antibody joint inspection kit comprises a plurality of O-type and A-type antibody detection chip subunits of foot-and-mouth disease viruses, each independent detection chip subunit is integrated into a microarray chip; respectively labeling the O-type foot-and-mouth disease virus monoclonal antibody 7D2 and the A-type foot-and-mouth disease virus monoclonal antibody 3B4 with enzymes, diluting the mixture with an enzyme-labeled diluent according to final concentrations of 100ng/ml and 75ng/ml respectively, and uniformly mixing the diluted mixture to be used as an enzyme-labeled reagent, wherein the enzyme-labeled diluent is a PBS (phosphate buffer solution) containing 20% of fetal calf serum in final volume; preparing a sample diluent, a washing solution and a substrate solution; and (5) assembling the subunit of the foot-and-mouth disease virus type-O or type-A antibody detection chip prepared in the step (2) or a subunit microarray chip thereof, the enzyme labeling reagent prepared in the step (3), the sample diluent prepared in the step (4), the washing solution and the substrate solution into the antibody joint inspection kit.

In one embodiment of the present invention, in the method for preparing the foot-and-mouth disease virus type O and type a antibody joint inspection kit of the present invention, the spotting fluid in the step (1) is a mixture of a 5% glycerol solution, a 5% sorbitol solution, a 0.05% triton solution, a DMSO solution, and a PBS (ph6.8) solution in a volume ratio of 10: 15: 0.1: 50: 100; diluting the goat anti-mouse polyclonal antibody to 100 mug/ml, 200 mug/ml and 300 mug/ml respectively by using the sample solution to be used as quality control sample solution to sample in the spots (1), (2) and (3) respectively; using the sample solution as a blank control to sample a spot (4), wherein the foot-and-mouth disease virus A antigen is sampled at a sample spot (5), and the foot-and-mouth disease virus O antigen is sampled at a sample spot (6); the quality control sample point (1), the quality control sample point (2), the quality control sample point (3) and the blank reference sample point (4) are respectively positioned at the upper left corner, the lower right corner and the upper right corner of the detection chip, the points (5) and (6) can be respectively sampled at other positions of the detection chip, and the distance between the midpoint and the point edge of the sample points (1) to (6) is more than or equal to 700 mu m; the distances from the quality control point (1), the quality control point (2), the quality control point (3) and the blank control point (4) to the upper edge and the lower edge of the detection chip subunit are not less than 8mm, and the distances from the blank control point to the left edge and the right edge of the detection chip subunit are not less than 5 mm; the enzyme-labeled diluent in the step (3) is a PBS solution containing 20% V/V fetal calf serum; the sample diluent in the step (4) is PBS solution containing 10% V/V fetal calf serum, 0.05-0.2% V/V Triton-100 and 0.1% V/V Proclin 300; the washing solution is a PBS solution containing 1% of V/V Tween 20, and the substrate solution is a TMB (3,3',5,5' -tetramethylbenzidine) solution.

The sample application volume of the A type foot-and-mouth disease virus antigen and the O type foot-and-mouth disease virus antigen is 20 nl/point.

In a preferred embodiment of the present invention, in the method for preparing the foot-and-mouth disease virus type O and type a antibody joint inspection kit, the sample diluent is a PBS solution containing 10% V/V fetal bovine serum, 0.1% V/V Triton-100, and 0.1% V/V Proclin 300.

The invention also provides application of the antibody joint inspection kit in preparation of non-immune diagnosis.

As an embodiment of the invention, the antibody joint inspection kit provided by the invention is applied to the preparation of non-immune diagnosis, and the non-immune diagnosis is applied to epidemiological analysis, qualitative foot and mouth disease virus O type and A type antibody detection of isolated serum, international live pig, cattle and sheep trade, import and export inspection and quarantine, and foot and mouth disease virus O type and A type vaccine quality control.

The invention also provides an application method of the joint inspection kit, which comprises the following steps:

pre-diluting a sample in the step (1), and diluting a serum sample by 10 times with a sample diluent;

numbering, namely numbering the chip holes according to the sequence of the samples;

soaking in the step (3), adding 300 mul/hole of washing liquid, and after soaking for 3 minutes, discarding the liquid and patting dry;

adding sample in the step (4), adding 50-100 mul/hole of the diluted sample, and placing the sample in a constant temperature oscillator at 37 ℃ and incubating for 30 minutes at 500 r/min;

adding an enzyme labeling reagent, adding 50-100 mul/hole of the enzyme labeling reagent, and placing the mixture in a constant temperature oscillator at 37 ℃ and incubating for 30 minutes at 500 r/min; discarding the liquid, adding 300 mu l/hole of washing liquid, soaking for 10-20 seconds, discarding the liquid, repeatedly washing for 5 times, and finally drying by beating for the last time;

developing color, adding 100 mu l/hole of substrate solution, and incubating for 15 minutes at 37 ℃;

and (7) measuring, namely vertically discarding the liquid, throwing off the liquid, removing the upper cover of the chip, reversely buckling the chip on the dust-free paper, slightly pressing the chip, measuring the result by using a micropore disk chip imager within 10 minutes, and deriving the blocking rate (1-sample gray value/quality control point gray value).

And (3) testing validity judgment: the gray value of the quality control point is more than or equal to 10000, the gray value of the blank control point is less than or equal to 3000, otherwise, the test is invalid, and the judgment is automatically completed through an internal data processing and analyzing system. The criteria for result determination (which is also done by the internal one-touch intelligent data processing and analyzing system, and does not require the technician to perform data calculation and statistical analysis) are as follows:

calculation of blocking rate: the blocking rate is (1-sample gray value/quality control point gray value) × 100%

And (3) positive judgment: when the blocking rate is more than or equal to 50 percent, the FMDV O and A antibodies are positive;

and (4) negative judgment: FMDV O and A antibodies are negative when the blocking rate is less than 50 percent.

The invention has the advantages that:

different from the traditional ELISA kit, the kit provided by the invention is technically improved, the quality control product is arranged on the reaction carrier, and the quality control product is a commercial goat anti-mouse polyclonal antibody, and no negative control or negative control is required to be additionally arranged in the kit, namely, complex preparation steps such as animal immunization or toxicity attack, serum collection and the like, and labor and animal test site cost are not required to be screened in advance, and the convenience and accuracy of the kit operation are greatly improved.

The joint inspection kit prepared by the invention has the advantages of high integration of detection items, rapid detection, simple and convenient operation, one-key intelligent data processing, elimination of complex data processing and mass data analysis after detection by a conventional method, easier control of the health condition of the swinery and convenience for intensive management of the breeding industry.

The joint inspection kit prepared by the invention has accurate and reliable result and good repeatability, only needs about 1.5 hours for detection, can be used for detecting series serum collected after vaccine immunization and evaluating vaccine immune protection effect, can be used for simultaneously detecting O-type and A-type antibodies in serum of pigs, cows and sheep clinically, and is used for epidemiological investigation.

Drawings

FIG. 1 is a schematic diagram showing spotting patterns of each chip well on a type-O and type-A aftosa virus antibody detection chip, wherein A, B, C, D, E, F, G, H, I, J in FIG. 1 shows different spotting patterns respectively, and 5 and 6 in the same figure can be interchanged in specific positions on the detection chip;

FIG. 2 is a partial enlarged view of three kits having different numbers of subunits of a chip for detecting antibodies against foot-and-mouth disease viruses O and A, wherein O, P, Q in FIG. 2 is a kit having one, three or more subunits of a chip for detecting antibodies.

Reference numerals

1 is a quality control point 1, 2 is a quality control point 2, 3 is a quality control point 3, 4 is a blank control point, 5 is an A-type foot-and-mouth disease virus antigen detection point, and 6 is an O-type foot-and-mouth disease virus antigen detection point.

Detailed Description

Definition of

The term "goat anti-mouse polyclonal antibody" is used interchangeably with goat anti-mouse secondary antibody, goat anti-mouse IgG antibody.

The term "enzyme" includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, beta-D-galactosidase. Wherein, the substrates used by the horseradish peroxidase are o-phenylenediamine (OPD) and Tetramethylbenzidine (TMB), preferably Tetramethylbenzidine (TMB); the substrate used for alkaline phosphatase is p-nitrophenylphosphate (p-NPP); the substrate used for beta-D-galactosidase was 4-methylumbelliferyl-beta-D-galactoside (4 MUG).

The term "virus-like particle", virus-like particles (VLPs), is a viroid capable of self-packaging into a viral coat structure upon expression in vitro and/or in vivo, they are pseudoviruses with a similar coat structure of a virus but without the capacity for viral replication, free of viral nucleic acids, which usually consist of viral capsid proteins or variants or fragments thereof. VLPs are highly immunogenic and immunoreactive because they are structurally very similar to native viral particles. Meanwhile, VLPs do not contain viral genetic materials, have no infection capacity and have high biological safety.

The term "goat anti-mouse polyclonal antibody" refers to a goat anti-mouse polyclonal antibody, also known as a goat anti-mouse secondary antibody or a goat anti-mouse IgG.

The term "heavy chain variable region" refers to a polypeptide of 110 to 125 amino acids in length whose amino acid sequence corresponds to the amino acid sequence of the heavy chain of a monoclonal antibody of the invention starting from the N-terminal amino acid of the heavy chain. Similarly, the term "light chain variable region" refers to a polypeptide of 95 to 115 amino acids in length whose amino acid sequence corresponds to the light chain amino acid sequence of the monoclonal antibody of the invention starting from the N-terminal amino acid of the light chain. It is obvious to those skilled in the art that, based on the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody specifically disclosed in the present invention, modifications such as addition, deletion, substitution and the like of one or more amino acids can be performed by conventional genetic engineering and protein engineering methods to obtain conservative variants, while still maintaining specific binding with feline panleukopenia virus. The monoclonal antibodies of the invention also include active fragments or conservative variants thereof.

The term "microarray chip" refers to a micro-device which is arranged on a substrate in an array capable of processing and analyzing biological or chemical information in a sample in parallel, wherein the dots are arranged in an array with a diameter of 500 mu pi iota and the centers of two adjacent dots are spaced apart from each other by a minimum distance so as not to generate signal cross (see GB/T27990-.

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

The phosphate buffer used in the examples of the present invention was PBS at pH 7.4, 1L volume formulation of which was: NaCl 8.0g, KCl 0.2g, Na₂HPO₄·12H₂O 2.9g、KH₂PO₄0.24g, but this embodiment is not intended to limit the present invention in any way. Goat anti-mouse polyclonal antibodies are commercially available or can be prepared by conventional methods.

The experimental methods are conventional methods unless specified otherwise; the biomaterial is commercially available unless otherwise specified.

Example 1 preparation of raw materials for bivalent test kit for O-type and A-type foot-and-mouth disease Virus

1.1 antigens

Type a foot and mouth disease virus antigen: the preparation and identification method of the A type foot-and-mouth disease virus-like particle is described in Chinese patent CN 105566449A.

Type O foot-and-mouth disease virus antigen: the preparation and identification method of the O-type foot-and-mouth disease virus-like particle is described in Chinese patent CN 105566449A.

1.2 antibodies

Type a foot-and-mouth disease virus monoclonal antibody: 2E11 (the nucleotide sequence of the heavy chain variable region is SEQ ID No.1, the nucleotide sequence of the heavy chain variable region is SEQ ID No.2), 3B4 (the nucleotide sequence of the heavy chain variable region is SEQ ID No.3, and the nucleotide sequence of the heavy chain variable region is SEQ ID No. 4).

Type O foot-and-mouth disease virus monoclonal antibody: 6G6 (the nucleotide sequence of the heavy chain variable region is SEQ ID No.5, the nucleotide sequence of the heavy chain variable region is SEQ ID No.6), 7D2 (the nucleotide sequence of the heavy chain variable region is SEQ ID No.7, and the nucleotide sequence of the heavy chain variable region is SEQ ID No. 8).

Quality control: goat anti-mouse polyclonal antibody.

1.3 preparation and identification of enzyme-labeled antibody

1.3.1 preparation

The monoclonal antibodies 2E11 and 3B4 of the type A foot-and-mouth disease virus and the monoclonal antibodies 6G6 and 7D2 of the type O foot-and-mouth disease virus are respectively labeled with Horse Radish Peroxidase (HRP) by a modified sodium periodate method.

Weighing 20mg of horseradish peroxidase (HRP) and dissolving in 1ml of ultrapure water, adding 1ml of freshly prepared NaIO₄Solution (30mg NaIO)₄Dissolving in 1ml of ultrapure water), mixing, and keeping away from light at 4 ℃ for 30 minutes; adding 40 mul of ethylene glycol into the solution, and keeping away from light at 4 ℃ for 30 minutes; the purified monoclonal antibody 1mg was added to 100. mu.l of the above mixture, mixed well, added to a dialysis bag, mixed well, and dialyzed against CB buffer for 6 hours. The whole operation needs to be carried out in a dark place; the dialyzed mixture was transferred to a 1.5ml EP tube and 10. mu.l of freshly prepared NaBH was added₄Solution (20mg NaBH)₄Dissolved in 1ml of ultrapure water), acted for 2 hours at room temperature, and uniformly mixed once every 30 minutes; adding saturated ammonium sulfate with the same volume, mixing uniformly, and acting for 15 minutes at 4 ℃. Centrifuge at 12000 rpm for 10 minutes and discard the supernatant. The pellet was suspended in a volume of PBS mixed with glycerol equal to the volume of purified antibody (V: V ═ 1: 1).

1.3.2 identification

Appearance: at room temperature, the liquid is reddish brown liquid, and no floccule precipitate is seen.

And (3) quality evaluation: and detecting the absorbance value A of the enzyme-labeled antibody at 403nm and 280nm by using an ultraviolet spectrophotometer. The corresponding enzyme parameters were calculated according to the formula:

enzyme amount (mg/ml) ═ A_403nmX 0.4 x dilution factor.

IgG amount (mg/ml) ═ A_280nm-A_403nm0.3). times.0.62 times the dilution factor.

Gram-molecule ratio (E/P) ═ enzyme amount × 4/IgG amount.

Mark rate is A_403nm/A_280nm。

Through the detection and calculation of light absorption values, the specific results are shown in table 1:

TABLE 1 quality evaluation of different enzyme-labeled antibodies

Example 2 preparation of Dual-detection kit for foot-and-mouth disease Virus type O and A antibodies

2.1 preparation of spotting fluid

Preparation of 5% glycerol solution: precisely weighing 5.00g of glycerol, placing the glycerol in a 100ml volumetric flask, adding a small amount of purified water, slightly rotating the glycerol to fully dissolve the glycerol so as to avoid generating excessive bubbles, adding the purified water to scale marks, and turning and shaking the glycerol up and down for 10 times for later use;

preparation of 5% sorbitol solution: precisely weighing 5.00g of sorbitol, putting the sorbitol into a 250ml beaker, adding a proper amount of purified water, stirring to completely dissolve the sorbitol, completely transferring the sorbitol into a 100ml volumetric flask, adding the purified water to set a scale mark, and turning and shaking up and down for 10 times for later use;

preparation of 0.05% triton solution: measuring 50 μ l of triton with a pipette, placing in a 100ml volumetric flask, adding appropriate amount of purified water to dissolve completely, adding purified water to set scale mark, and shaking for 10 times;

DMSO solution: directly adopting a DMSO reagent;

PBS (pH6.8) solution preparation: preparing 0.2mol/L disodium hydrogen phosphate solution and 0.3mol/L sodium dihydrogen phosphate solution, and mixing the two solutions according to a volume ratio of 49:51 to obtain a phosphate buffer solution with the pH value of 6.8;

the solutions are uniformly mixed according to the volume ratio of 10: 15: 0.1: 50: 100 to be used as a sampling solution.

2.2 preparation of Combined assay kit

The foot-and-mouth disease virus O-type and A-type antibody two-joint detection chip comprises: using the spotting fluid prepared in example 2.1 to dilute the type-O foot-and-mouth disease virus antigen prepared in example 1 to a suitable concentration as a spotting point FMDV O spotting fluid; using the sample solution prepared in example 2.1 to dilute the type a foot-and-mouth disease virus antigen prepared in example 1 to a proper concentration, as a detection point FMDV a sample solution; respectively diluting the goat anti-mouse polyclonal antibody to 100 mug/ml, 200 mug/ml and 300 mug/ml by using a sample application liquid as quality control point sample application liquids, respectively applying samples to points 1, 2 and 3 in the figure 1, setting 3 concentrations, and conveniently selecting corresponding quality control points according to debugging results to perform data statistical analysis; the spotting solution prepared in example 2.1 was spotted on spot 4 as a blank control spot. Starting a sample applicator, setting a program and sample application parameters, applying sample application of a sample application solution of a detection point FMDV A to a point 5, applying sample application solution of a detection point FMDV O to a point 6, applying sample application solution of a quality control point to points 1, 2 and 3 in the figure 1, and applying sample application solution of a blank control point to a point 4 in the figure 1 according to the volume sample application of 20 nL/point. And taking the spotted membrane out, placing the spotted membrane in the middle of a chip bottom plate, adding a cover plate to compress the membrane, clamping the two side strips, assembling the two side strips into a foot-and-mouth disease virus O-type and A-type antibody duplex detection chip, packaging the chip and storing the chip at 2-8 ℃. Except that the points 1, 2, 3 and 4 are respectively fixed at the upper left corner, the lower right corner and the upper right corner of the detection chip, the points 5 and 6 can be respectively spotted at any other positions of the detection chip, the distance between the points and the edges of other points is only required to be more than or equal to 700 mu m, and the shortest distance between any point and the point is also more than or equal to 700 mu m. The distances from the quality control point 1, the quality control point 2, the quality control point 3 and the blank reference point sample point 4 to the upper edge and the lower edge of the detection chip subunit are not less than 8mm, and the distances from the blank reference point sample point 4 to the left edge and the right edge of the detection chip subunit are not less than 5 mm.

The antibody joint inspection kit comprises one or more foot and mouth disease virus O-type and A-type antibody joint inspection detection chip subunits, each detection chip subunit comprises a bottom end, an upper grid and independent detection holes formed by detection chips clamped by the bottom end and the upper grid and arranged between the bottom end and the upper grid, each detection hole corresponds to one detection chip subunit, and when the antibody joint inspection kit comprises a plurality of foot and mouth disease virus O-type and A-type antibody joint inspection detection chip subunits, each independent detection chip subunit is integrated into a microarray chip. FIG. 2 shows a kit with different numbers of O-type and A-type antibodies of foot-and-mouth disease virus for dual detection of chip subunits, O, P, Q in FIG. 2 is a kit with one, three or more antibody detection chip subunits respectively, and its partial enlarged view shows the sampling points on the detection chip in one O-type and A-type antibodies of foot-and-mouth disease virus for dual detection of chip subunits.

Enzyme labeling reagent: and (3) uniformly mixing the PBS solution and 200ml of fetal calf serum, and adding PBS to a constant volume of 1L to be used as an enzyme-labeled diluent. The enzyme-labeled A type foot-and-mouth disease virus monoclonal antibodies 2E11 and 3B4 and the O type foot-and-mouth disease virus monoclonal antibodies 6G6 and 7D2 prepared in example 1 are diluted by the enzyme-labeled A type foot-and-mouth disease virus monoclonal antibody with the final volume of 0.0025% V/V and 0.005% V/V respectively, and then mixed uniformly (the concentrations of the A type enzyme-labeled single antibody and the O type enzyme-labeled single antibody are 75ng/ml and 100ng/ml respectively), filtered by 0.22 mu m, and then aseptically packaged.

Sample diluent: PBS solution containing 10% V/V fetal calf serum, 0.05% -0.2% V/V Triton-100 and 0.1% V/V Proclin 300.

Washing liquid: PBS solution containing 1% V/V Tween 20 was filtered at 0.22 μm and aseptically dispensed. The test was performed by 20-fold dilution with purified water.

Substrate solution: TMB (3,3',5,5' -tetramethylbenzidine) solution, commercial product, sterile packaging.

2.3 detection method establishment

The operation steps are as follows:

(1) the sample is pre-diluted and the serum sample is diluted 10-fold with the sample diluent (e.g., 10. mu.l sample + 90. mu.l sample diluent).

(2) And numbering, namely numbering the chip holes according to the sequence of the samples.

(3) Soaking, adding 300 μ l/hole of washing solution, soaking for 3 min, discarding solution, and patting dry.

(4) The sample is added, 50-100 mul/well of the diluted sample is added, and the mixture is placed on a micropore plate constant temperature oscillator at 37 ℃ and incubated for 30 minutes at 500 rpm.

(5) Adding enzyme standard reagent, adding 50-100 μ l/well of enzyme standard reagent, placing in a microporous plate constant temperature oscillator at 37 deg.C, and incubating at 500 rpm for 30 min; and (4) discarding the liquid, adding 300 mu l/hole of washing liquid, and soaking for 10-20 seconds to discard the liquid. Washing repeatedly for 5 times, and drying for the last time.

(6) Color development, adding substrate solution 100 u l/hole, placed at 37 degrees C temperature 15 minutes incubation.

(7) And measuring, namely vertically discarding liquid, throwing off, removing the upper cover of the chip, reversely buckling the chip on the dust-free paper, slightly pressing the chip, measuring the result by using a micropore disk chip imager within 10 minutes, and deriving the blocking rate (1-sample S value/quality control point N value).

For the purpose of illustration, the kit is described by way of example as a 75. mu.l/well sample.

2.4 determination of antigen coating amount of O-type and A-type foot-and-mouth disease virus antibody duplex detection chip

Serum samples were as follows:

FMDV O antibody positive and FMDV a antibody negative porcine serum (abbreviated P1): the test result of the liquid blocking ELISA detection kit for foot-and-mouth disease virus O-type antibody by animal research of orchid is positive, and the antibody titer is 1: 360; and the liquid-phase blocking ELISA detection kit for foot-and-mouth disease virus A type antibody after being ground by the orchid has negative determination result, and the antibody titer is 1: 16.

FMDV a antibody positive and FMDV O antibody negative porcine serum (abbreviated P2): the liquid phase blocking ELISA detection kit for the A-type antibody of the aftosa virus ground by the orchid has a positive detection result, and the antibody titer is 1: 256; and the test result of the liquid-phase blocking ELISA detection kit for foot-and-mouth disease virus O-type antibody ground by orchid animals is negative, and the antibody titer is 1: 8.

FMDV O antibody negative and FMDV a antibody negative porcine serum (abbreviated N): the liquid-phase blocking ELISA detection kit for the O-type antibody of the orchid-researched foot-and-mouth disease virus and the liquid-phase blocking ELISA detection kit for the A-type antibody of the orchid-researched foot-and-mouth disease virus have negative determination results, and the antibody titer is 1: 8.

The serum is used for selecting the O-type and A-type antibodies of the foot-and-mouth disease virus to detect the antigen coating amount of the chip by two-joint detection, and the principle of the method is an enzyme-linked immunosorbent assay solid-phase competition method, so that the optimal antigen solid-phase condition is selected by calculating the N/P value and selecting the corresponding antigen coating condition when the N/P value is maximum, and the details are as follows:

2.4.1 selection of the amount of antigen coating of foot-and-mouth disease Virus type O

The type O foot and mouth disease virus antigen of example 1 was diluted to an appropriate concentration with a spotting solution as shown in example 2.1, and the spotting volume was 20nl, so that the final coating amounts of the type O foot and mouth disease virus antigen spotted on the membrane were as shown in table 2, respectively, and were not changed. The assay was carried out using P1, N serum according to the method of example 2.3, with the results (see Table 2): the detection is negative under the condition that the coating amount of the A-type foot-and-mouth disease virus antigen is not changed (not shown in table 2); the numerical value of N/P1 is small when the coating amount of the O-type foot-and-mouth disease virus antigen is low, the numerical value is increased along with the increase of the coating concentration, the N/P value is more than 2.9 when the coating amount is 8-32 ng/point, then the N/P1 value of the coating concentration is increased to start to be reduced, the size of the N/P1 value corresponds to the negative and positive distinguishing capability of the method, and the detection condition is more suitable when the data is larger. Therefore, the value of N/P1 is 2.33-3.32 when the coating amount of the O type foot-and-mouth disease virus antigen is 1-128 ng/point, but the value of N/P1 is slightly better (2.97-3.32) when the coating amount is 8-32 ng/point, and the value of N/P1 is optimal (3.32) when the coating amount is 16 ng/point.

TABLE 2 optimization results of O-type foot-and-mouth disease virus antigen coating amount

2.4.2 selection of antigen coating amounts of foot-and-mouth disease Virus type A

The type A foot-and-mouth disease virus antigen of example 1 was diluted to an appropriate concentration with a spotting solution as shown in example 2.1, and the spotting volume was 20nl, so that the final coating amounts of the type A foot-and-mouth disease virus antigen spotted on the membrane were as shown in Table 3, respectively, and were not changed. The assay was carried out using P2, N serum according to the method of example 2.3, with the results (see Table 3): the detection is negative under the condition that the coating amount of the O-type foot-and-mouth disease virus antigen is not changed (not shown in Table 3); the value of N/P2 is small when the antigen coating amount of the A-type foot-and-mouth disease virus is low, the value is increased along with the increase of the coating concentration, the value of N/P2 is more than 3.1 when the coating amount is 16-64 ng/point, then the value of the coating concentration N/P2 is increased to start to be reduced, the size of the value of N/P2 corresponds to the negative and positive distinguishing capability of the method, and the larger the data is, the more appropriate the detection condition is. Therefore, the detection can be satisfied when the coating amount of the type A foot-and-mouth disease virus antigen is 1-128 ng/point, the N/P2 value is 2.08-3.45, but the N/P2 value is slightly better (3.14-3.45) when the coating amount is 16-64 ng/point, and the N/P2 value is optimal (3.45) when the coating amount is 32 ng/point.

TABLE 3 optimization results of antigen coating amount of type A foot-and-mouth disease virus

2.4.3 verification of antigen coating amount of O-type and A-type foot-and-mouth disease viruses

In order to verify whether cross reaction exists when the O-type and A-type foot-and-mouth disease virus antigens are detected to correspond to the antibodies, samples with clear backgrounds and detected by a commercialized kit are selected to further verify the envelope quantity of the O-type and A-type foot-and-mouth disease virus antigens. According to the test result, the O-type foot-and-mouth disease virus antigen is spotted according to 16 ng/spot and the A-type foot-and-mouth disease virus antigen is spotted according to 32 ng/spot to prepare the O-type and A-type antibody dual-joint detection chip for the foot-and-mouth disease virus. And respectively detecting 12 parts of immune serum of the foot-and-mouth disease virus O-type subunit vaccine, 3 parts of immune serum of the foot-and-mouth disease virus A-type subunit vaccine, and 6 parts of specific test serum (comprising 1 part of PCV2 positive serum, 1 part of CSFV positive serum, 1 part of PPV positive serum, 1 part of PRRSV positive serum, 1 part of healthy pig serum and 1 part of PRV positive serum), wherein the corresponding coating amount is the optimal coating amount when the result accords with the standard of 12/12, namely the coincidence rate is 100%. Results (see table 4): the results of 12 samples detected by the prepared foot-and-mouth disease virus O-type and A-type antibody duplex detection chip are consistent with those of a commercial kit, and the coincidence rate is 100 percent.

TABLE 4 evaluation results of the detection chip for the two-step detection of foot-and-mouth disease virus type O and A antibodies with the optimal antigen coating amount and comparison with commercial kit

2.5 selection of enzyme-labeled type O foot-and-mouth disease Virus monoclonal antibodies

The O-type foot-and-mouth disease virus monoclonal antibodies 6G6 and 7D2 of the enzyme-labeled O-type foot-and-mouth disease virus of example 1 were diluted to 100ng/ml (1: 20000) using the two-step detection chip for detecting O-type and A-type antibodies of foot-and-mouth disease virus prepared in 2.4.3, respectively. The assay was carried out using P1, N serum as in example 2.3, and the results are shown in Table 5: compared with the N/P1 result detected by 7D2, the 2 enzyme-labeled O-type foot-and-mouth disease virus monoclonal antibody solutions are superior to 6G6, which shows that the monoclonal antibody 7D2 has more advantages in recognizing the epitope of the O-type foot-and-mouth disease virus than the epitope recognized by 6G 6. Subsequent experiments will use enzyme-labeled type O foot-and-mouth disease virus monoclonal antibody 7D2 for the production of the kit.

TABLE 5 results of enzyme-labeled type O foot-and-mouth disease Virus monoclonal antibody selection

Labeled monoclonal antibody	6G6	7D2
			N	36512	38129
P1	10245	6891
			N/P1	3.56	5.53

2.6 selection of enzyme-labeled type A foot-and-mouth disease Virus monoclonal antibodies

The monoclonal antibodies 2E11 and 3B4 of the foot-and-mouth disease virus A of the enzyme-labeled type A of example 1 were diluted to 75ng/ml (1: 40000) respectively using the type-O and type-A antibody double-detection chip prepared in 2.4.3. The assay was carried out using P2, N serum according to the method of example 2.3, with the results (see Table 6): compared with a 3B4 detection result, the N/P2 result of 2 enzyme-labeled A-type foot-and-mouth disease virus monoclonal antibody solutions is superior to that of 2E11, and the monoclonal antibody 3B4 is more advantageous in recognizing the epitope of the A-type foot-and-mouth disease virus than the epitope recognized by 2E 11. Subsequent experiments will use enzyme to mark the monoclonal antibody 3B4 of foot-and-mouth disease virus type A for the production of the kit.

TABLE 6 results of enzyme-labeled type A foot-and-mouth disease Virus monoclonal antibody selection

Labeled monoclonal antibody	2E11	3B4
			N	32516	33813
P2	10813	7917
			N/P2	3.01	4.27

2.7 selection of sample dilutions

A sample diluent is prepared by the foot-and-mouth disease virus O-type and A-type antibody duplex detection chip and an enzyme labeling reagent together with the table 7, N, P1 and P2 samples are detected, and the result is shown in the table 7: after the samples are treated by the 7 sample treatment solutions, the detection values of N/P1 and N/P2 are the maximum and the effect is the best compared with the detection values of the samples treated by the sample diluent 4.

TABLE 7 test results for different sample dilutions

In order to evaluate the Triton-100 content in the sample dilution, the other components were kept unchanged, and the Triton-100 content was adjusted to 0.025%, 0.05%, 0.1%, 0.2%, 0.4%, 0.8% V/V to prepare a sample dilution and tested, as a result: when the Triton-100 contained in the sample treatment solution is 0.025 percent, the sensitivity of detecting FMDV O and A antibodies is reduced by 20-60 times; when the Triton-100 contained in the sample processing liquid is 0.4 percent and 0.8 percent, the specificity of detecting FMDV O and A antibodies is poor; the detection sensitivity and specificity of the prepared sample diluent were comparable to those of the sample diluent 4 when the sample treatment solution contained 0.05% and 0.2% of Triton-100, and the Triton-100 content in the sample diluent was set to 0.05% to 0.2% V/V.

For subsequent evaluation, a foot-and-mouth disease virus type-O and type-A antibody duplex detection chip (FMDV O coating amount is 16 ng/point, FMDV A coating amount is 32 ng/point), an enzyme-labeled reagent (mixed solution diluted by enzyme-labeled type-O foot-and-mouth disease virus monoclonal antibody 7D2 and enzyme-labeled type-A foot-and-mouth disease virus monoclonal antibody 3B4 with final volumes of 1: 20000 and 1: 40000 respectively), a sample diluent 4 (PBS solution containing 10% V/V fetal bovine serum, 0.1% V/V Triton-100 and 0.1% V/V Proclin 300) are prepared together with a substrate solution and a washing solution to carry out subsequent detection.

Example 3 use of the Combined test kit

3.1 sensitivity

The southeast Asia O-type foot-and-mouth disease virus antibody positive serum (the antibody titer is calibrated to be 1: 5760 by an OIE recommendation method) is subjected to 2-fold gradient dilution, the O-type and A-type foot-and-mouth disease virus antibody duplex test kit prepared by the invention is used for determination, the sensitivity of the test kit to the southeast Asia O-type positive serum is determined, and the result is shown in Table 8.

TABLE 8 sensitivity test for gradient diluted southeast Asia type O positive sera

Dilution factor	Blocking Rate (%)	Determination of results
			1∶8	98.1	+
1∶16	96.2	+
			1∶32	91.3	+
1∶64	85.1	+
			1∶128	76.8	+
1∶256	61.2	+
			1∶512	42.5	－

The result shows that the kit detects the southeast Asia O-type positive serum diluted in a gradient way, and the detection titer reaches 1: 256.

The Chinese type O foot-and-mouth disease virus antibody positive serum (the antibody titer is calibrated to be 1: 5760 by the OIE recommendation method) is subjected to 2-fold gradient dilution, the O type and A type antibodies dual detection kit prepared by the invention is used for determination, the sensitivity of the kit to the Chinese type O positive serum is determined, and the result is shown in Table 9.

TABLE 9 sensitivity test for Chinese type O positive serum diluted in gradient

The result shows that the kit detects the southeast Asia O-type positive serum with the gradient dilution, and the detection titer reaches 1: 256.

The A type foot-and-mouth disease virus antibody positive serum (the antibody titer is calibrated by an OIE recommendation method is 1: 2880) is subjected to 2-fold gradient dilution, the O type and A type antibodies dual-detection kit for foot-and-mouth disease virus prepared by the invention is used for determination, the sensitivity of the kit to the positive serum is determined, and the result is shown in Table 10.

TABLE 10 sensitivity test for type A Positive serum in gradient dilution

Dilution factor	Blocking Rate (%)	Determination of results
			1∶8	96.1	+
1∶16	94.3	+
			1∶32	85.6	+
1∶64	72.9	+
			1∶128	53.7	+
1∶256	36.8	－

The result shows that the kit detects the A type positive serum diluted in a gradient way, and the detection titer reaches 1: 128.

The results show that the bivalent detection kit for the foot-and-mouth disease virus type O and type A antibodies has high sensitivity, can be used for carrying out high-sensitivity detection on the foot-and-mouth disease virus type O and type A antibodies, and can also be used for carrying out high-sensitivity detection on the foot-and-mouth disease virus type O antibodies of different subtypes (southeast subtype and Chinese type).

3.2 specificity

The foot-and-mouth disease virus O-type and A-type antibody duplex detection kit prepared by the invention is used for detecting 3 parts of FMDV O-type antibody positive serum (A-type negative), 3 parts of FMDV A-type antibody positive serum (O-type negative) and 3 parts of foot-and-mouth disease O, A-type antibody negative serum, 1 part of Classical Swine Fever Virus (CSFV) antibody positive serum, 1 part of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) antibody positive serum, 1 part of porcine Securitacha virus (SVV) antibody positive serum and 1 part of escherichia coli (E.coli) antibody positive serum, and evaluating the specificity of the FMDV-O-type and A-type antibody duplex detection kit. The results are shown in Table 11.

TABLE 11 results of the evaluation of specificity

The result shows that 3 parts of FMDV O antibody positive serum O-type antibodies detected by the kit are all positive and A-type antibodies are all negative; detecting that all the A type antibodies and all the O type antibodies of 3 FMDV A antibody positive sera are positive; detecting O, A antibodies in3 FMDV negative sera to be negative; the kit is shown to detect O-type and A-type antibodies without cross reaction and has better specificity. The kit detects that the other swine disease virus antibody positive serum O, A antibodies are all negative, which shows that the kit has no cross reaction with other virus antibodies and has better specificity.

3.3 repeatability

Using 3 chip boards of different batches, performing an in-batch repeatability test on one board, performing an inter-batch repeatability test on 3 boards of different batches, measuring a gray value and a blocking rate, and calculating a coefficient of variation. The coefficient of variation is less than 15%, which indicates that the repeatability and stability of the kit are good. The results show that the detection results of 3 chip boards in different batches are consistent, the variation coefficients are all less than 10%, and the repeatability of the kit is good.

Coefficient of Variation (CV) calculation formula:

CV (%) - (standard deviation/average) × 100%

3.3 clinical applications

191 parts of serum with O-type foot-and-mouth disease in southeast Asia and A-type foot-and-mouth disease in negative (calibrated by an OIE recommendation method) are subjected to contrast detection by using three batches of the O-type and A-type foot-and-mouth disease virus dual-detection kit prepared by the invention and three batches of the commercialized liquid phase blocking kit. The results are shown in Table 12.

TABLE 12 kit for detecting, comparing and testing southeast Asia O-type foot-and-mouth disease

The result shows that the kit can detect the O-type foot-and-mouth disease antibody positivity of southeast Asia in 100% in three batches, and the commercial liquid phase blocking kit has different detection results of 96.3% at the lowest and 97.9% at the highest, so that the sensitivity is lower and the batch-to-batch difference is realized.

172 parts of serum with Chinese type O foot-and-mouth disease as positive and A type foot-and-mouth disease as negative (calibrated by an OIE recommendation method), three batches of the foot-and-mouth disease virus O-type and A-type antibody duplex detection kit prepared by the invention and three batches of the commercialized liquid phase blocking kit are respectively used for comparative detection. The results are shown in Table 13.

Table 13 kit for Chinese type O foot-and-mouth disease detection and comparison experiment results

The results show that the kit can detect Chinese type O foot-and-mouth disease antibody positivity by 100% in three batches, and the commercial liquid phase blocking kit has different detection results of 94.2% at the lowest and 97.7% at the highest, so that the kit has lower sensitivity and has different batches.

183 parts of serum with A-type foot-and-mouth disease positive and O-type foot-and-mouth disease negative (calibrated by an OIE recommendation method) are subjected to contrast detection by using three batches of the foot-and-mouth disease virus O-type and A-type antibody duplex detection kit prepared by the invention and three batches of the commercialized liquid phase blocking kit. The results are shown in Table 14.

TABLE 14 test results of the kit for detecting and comparing A-type foot-and-mouth disease

The results show that the kit can detect the positive A-type foot-and-mouth disease antibody by 100% in three batches, and the detection results of the liquid phase blocking kit in the three batches are different, wherein the detection results are 93.4% at the lowest and 97.8% at the highest, so that the sensitivity is lower and the batch-to-batch differences are realized.

149 parts of serum (89 parts of southeast Asia O-type and 60 parts of Chinese-type O-type) with positive foot-and-mouth disease types O and A (calibrated by an OIE recommendation method) are subjected to contrast detection by using three batches of the foot-and-mouth disease virus O-type and A-type antibody dual-detection kit prepared by the invention and three batches of the commercialized liquid phase blocking kit. The results are shown in Table 15.

Table 15 kit comparison test results for O-type and A-type positive samples of foot-and-mouth disease

The result shows that the kit for detecting O-type and A-type antibodies in foot-and-mouth disease negative serum in three batches is 100% positive, and has good specificity. The detection results of three batches of the commercial liquid phase blocking kit are all lower than 100%, and the three batches have differences.

151 parts of serum with O type and A type of foot-and-mouth disease virus negative (calibrated by an OIE recommendation method) are subjected to comparative detection by using three batches of the foot-and-mouth disease virus O type and A type antibody duplex detection kit prepared by the invention and three batches of the commercialized liquid phase blocking kit. The results are shown in Table 16.

TABLE 16 test results of the comparison of foot-and-mouth disease type O and type A negative samples with the kit

The result shows that the kit disclosed by the invention detects O-type and A-type antibodies in foot-and-mouth disease negative serum in three batches, and the specificity is good, wherein the O-type and A-type antibodies are all negative 100%. And the detection results of three batches of commercial liquid phase blocking kits are unstable and some are lower than 100%.

In conclusion, the foot-and-mouth disease virus O-type and A-type antibody duplex detection kit has better detection capability for different genetic topology type antibodies, and can also better solve the problems of larger batch difference and poor stability of the existing liquid blocking ELISA kit.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

SEQUENCE LISTING

<110> Luoyang Zhongke biochip technology Co., Ltd

<120> foot-and-mouth disease virus O-type and A-type antibody joint detection kit, preparation method and application thereof

<160> 8

<170> PatentIn version 3.3

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caggtccaac tgcagcaacc tgggtctgag ctggtgaggc ctggagcttc agtgaagctg 60

tcctgcaagg cttctggcta cacattcacc agctactgga tgcactgggt gaagcagagg 120

cctggacaag gccttgagtg gattggaaat atttatcctg atagtggtag ttttaattac 180

gatgagaaat tcaagagcaa ggccacactg actgtagaca catcctccag cacagcctac 240

atgcagctca gcagcctgac atctgaggac tctgcggtct attattgtac aagagtccac 300

tataggcacg ggtttgctta ctggggccaa gggactctgg tcactgtctc tgca 354

<210> 8

<211> 321

<212> DNA

<213> hybridoma cell (hybridoma)

<400> 8

gacattgtga tgacccagtc tcacaaattc atgtccacat cagtaggaga cagggtcagc 60

atcacctgca aggccagtca ggatgtgagt actgctgtag tctggtatca agagaaacca 120

ggacaatctc ctaaactact aatttactcg gcatccttcc ggtactctgg agtccctgat 180

cgcttcactg gcagtggatc tgggacggat ttcactttca ccatcagcag tgttcaggct 240

gaagacctgg cagtttatta ctgtcagcaa cattatacta ctccgctcac gttcggtgct 300

gggaccaagc tggagctgaa a 321

Claims

1. A foot-and-mouth disease virus O-type and A-type antibody joint inspection kit comprises one or more foot-and-mouth disease virus O-type and A-type antibody detection chips and an enzyme labeling reagent, wherein sampling points on the foot-and-mouth disease virus O-type and A-type antibody detection chips are respectively coated with a foot-and-mouth disease virus O-type antigen, a foot-and-mouth disease virus A-type antigen, a quality control product and a blank control; the O-type foot-and-mouth disease virus antigen is O-type foot-and-mouth disease virus-like particles, the A-type foot-and-mouth disease virus antigen is A-type foot-and-mouth disease virus-like particles, the quality control product is a goat anti-mouse polyclonal antibody, the blank control is a sample solution, the enzyme labeling reagent is a solution containing an enzyme-labeled O-type foot-and-mouth disease virus monoclonal antibody and an enzyme-labeled A-type foot-and-mouth disease virus monoclonal antibody, the enzyme-labeled O-type foot-and-mouth disease virus monoclonal antibody is an enzyme-labeled O-type foot-and-mouth disease virus monoclonal antibody 7D2 or an enzyme-labeled O-type foot-and-mouth disease virus monoclonal antibody 6G6, and the labeled A-type foot-and-mouth disease virus monoclonal antibody is an enzyme-labeled A-type foot-and-mouth disease virus monoclonal antibody 3B4 or an enzyme-labeled A-type foot-and-mouth disease virus monoclonal antibody 2E 11.

2. The antibody joint inspection kit according to claim 1, wherein the coating amount of the O-type foot-and-mouth disease virus antigen is 1-128 ng/spot; preferably, the coating amount of the O-type foot-and-mouth disease virus antigen is 8-32 ng/point; more preferably, the coating amount of the O type foot-and-mouth disease virus antigen is 16 ng/point;

the coating amount of the A-type foot-and-mouth disease virus antigen is 1-128 ng/point; the coating amount of the A-type foot-and-mouth disease virus antigen is 16-64 ng/point; the coating amount of the A-type foot-and-mouth disease virus antigen is 32 ng/point;

the heavy chain variable region of the O type foot-and-mouth disease virus monoclonal antibody 7D2 is shown as SEQ ID NO.7 or the code of the degenerate sequence thereof, and the light chain variable region is shown as SEQ ID NO.8 or the code of the degenerate sequence thereof;

the heavy chain variable region of the O type foot-and-mouth disease virus monoclonal antibody 6G6 is shown as SEQ ID NO.5 or the code of the degenerate sequence thereof, and the light chain variable region is shown as SEQ ID NO.6 or the code of the degenerate sequence thereof;

the heavy chain variable region of the A-type foot-and-mouth disease virus monoclonal antibody 3B4 is shown as SEQ ID NO.3 or the code of the degenerate sequence thereof, and the light chain variable region is shown as SEQ ID NO.4 or the code of the degenerate sequence thereof;

the heavy chain variable region of the A-type foot-and-mouth disease virus monoclonal antibody 2E11 is shown as SEQ ID NO.1 or the code of the degenerate sequence thereof, and the light chain variable region is shown as SEQ ID NO.2 or the code of the degenerate sequence thereof;

preferably, the enzyme labeling reagent is a solution containing an enzyme-labeled O-type foot-and-mouth disease virus monoclonal antibody 7D2 and an enzyme-labeled A-type foot-and-mouth disease virus monoclonal antibody 3B 4;

the buffer solution used by the enzyme labeling reagent is a PBS solution containing 20% fetal bovine serum by volume, wherein the final concentrations of the enzyme-labeled O-type foot-and-mouth disease virus monoclonal antibody 7D2 and the enzyme-labeled A-type foot-and-mouth disease virus monoclonal antibody 3B4 are 100ng/ml and 75ng/ml respectively; the enzyme marked by the enzyme labeling reagent is horseradish peroxidase, alkaline phosphatase or beta-D-galactosidase;

the quality control sample is applied to quality control sample points 1-3, and the coating amount of the quality control sample point 1 is more than or equal to 1 ng/point and less than 4 ng/point; the coating amount of the quality control product point 2 is 4 ng/point; the coating amount of the quality control product point 3 is more than 4 ng/point and less than or equal to 6 ng/point; preferably, the coating amount of the goat anti-mouse polyclonal antibody of the quality control product point 1-3 is 2 ng/point, 4 ng/point and 6 ng/point respectively;

the sample solution is prepared by uniformly mixing a 5% glycerol solution, a 5% sorbitol solution, a 0.05% triton solution, a DMSO solution and a PBS (pH6.8) solution according to the volume ratio of 10: 15: 0.1: 50: 100;

further preferably, the coating amount of the foot-and-mouth disease virus O-type antigen in the foot-and-mouth disease virus O-type and A-type antibody joint detection kit is 16 ng/point, and the coating amount of the foot-and-mouth disease virus A-type antigen is 32 ng/point.

3. The antibody joint inspection kit of claim 1, wherein the kit further comprises a sample diluent, a substrate solution, a washing solution;

the sample diluent is a PBS solution containing 10 percent of V/V fetal calf serum, 0.05 to 0.2 percent of V/V Triton-100 and 0.1 percent of V/V Proclin 300; preferably, the sample dilution contains 0.1% V/V Triton-100;

the substrate solution is TMB (3,3',5,5' -tetramethylbenzidine) solution;

the washing solution is PBS solution containing 1% V/V Tween 20.

4. The antibody joint inspection kit according to claim 1 to 3, wherein the coating amounts of the goat anti-mouse polyclonal antibody of the quality control point (1), the quality control point (2) and the quality control point (3) in the detection chip are respectively 2 ng/point, 4 ng/point, 6 ng/point and a blank control point (4) as background detection points, the foot-and-mouth disease virus A-type antigen is spotted on the point (5), and the foot-and-mouth disease virus O-type antigen is spotted on the point (6); the sampling points of the quality control product point (1), the quality control product point (2), the quality control product point (3) and the blank reference sampling point (4) are respectively positioned at the upper left corner, the lower right corner and the upper right corner of a detection chip, the points (5) and (6) can be respectively positioned at other positions of the detection chip, and the distance between the midpoint of the sampling points (1) to (6) and the point edge is more than or equal to 700 mu m; the distances from the quality control point (1), the quality control point (2), the quality control point (3) and the blank reference point sample point (4) to the upper edge and the lower edge of the detection chip subunit are not less than 8mm, and the distances from the blank reference point sample point to the left edge and the right edge of the detection chip subunit are not less than 5 mm;

the antibody joint inspection kit comprises one or more subunits of the foot-and-mouth disease virus O-type and A-type antibody detection chips, and independent detection holes are formed by bottom ends, upper grids and detection chips which are arranged between the bottom ends and the upper grids and clamped by the bottom ends and the upper grids, each detection hole corresponds to one detection chip subunit, and when the antibody joint inspection kit comprises a plurality of the foot-and-mouth disease virus O-type and A-type antibody detection chip subunits, each independent detection chip subunit is integrated into a microarray chip.

5. A method for preparing the foot-and-mouth disease virus type O and type A antibody joint detection kit of claim 1 to 4, wherein the method comprises the following steps:

respectively preparing O-type antigen and A-type antigen of foot-and-mouth disease virus, diluting the O-type antigen and A-type antigen of foot-and-mouth disease virus together with goat anti-mouse polyclonal antibody by using a sample solution, and carrying out sample application on a membrane to prepare O-type and A-type antibody detection chips of foot-and-mouth disease virus;

step (2) forming independent detection holes by a bottom end, an upper grid and the antibody detection chip in the step (1) arranged between the bottom end and the upper grid, wherein the antibody detection chip is arranged between the bottom end and the upper grid and is clamped by the bottom end and the upper grid, one detection hole corresponds to one detection chip subunit, and when the antibody joint inspection kit comprises a plurality of O-type and A-type antibody detection chip subunits of foot-and-mouth disease viruses, each independent detection chip subunit is integrated into a microarray chip;

respectively labeling the O-type foot-and-mouth disease virus monoclonal antibody 7D2 and the A-type foot-and-mouth disease virus monoclonal antibody 3B4 with enzymes, diluting the mixture with an enzyme-labeled diluent according to final concentrations of 100ng/ml and 75ng/ml respectively, and uniformly mixing the diluted mixture to be used as an enzyme-labeled reagent, wherein the enzyme-labeled diluent is a PBS (phosphate buffer solution) containing 20% of fetal calf serum in final volume;

preparing a sample diluent, a washing solution and a substrate solution; and

and (5) assembling the subunit of the foot-and-mouth disease virus type-O or type-A antibody detection chip prepared in the step (2) or a subunit microarray chip thereof, the enzyme labeling reagent prepared in the step (3), the sample diluent prepared in the step (4), the washing solution and the substrate solution into the antibody joint inspection kit.

6. The method according to claim 5, wherein the spotting solution in the step (1) is prepared by uniformly mixing a 5% glycerol solution, a 5% sorbitol solution, a 0.05% triton solution, a DMSO solution and a PBS (pH6.8) solution according to a volume ratio of 10: 15: 0.1: 50: 100; diluting the goat anti-mouse polyclonal antibody to 100 mug/ml, 200 mug/ml and 300 mug/ml respectively by using the sample solution to be used as quality control sample solution to sample in the spots (1), (2) and (3) respectively; using the sample solution as a blank control to sample a spot (4), wherein the foot-and-mouth disease virus A antigen is sampled at a sample spot (5), and the foot-and-mouth disease virus O antigen is sampled at a sample spot (6);

the quality control sample point (1), the quality control sample point (2), the quality control sample point (3) and the blank reference sample point (4) are respectively positioned at the upper left corner, the lower right corner and the upper right corner of the detection chip, the points (5) and (6) can be respectively sampled at other positions of the detection chip, and the distance between the midpoint and the point edge of the sample points (1) to (6) is more than or equal to 700 mu m; the distances from the quality control point (1), the quality control point (2), the quality control point (3) and the blank control point (4) to the upper edge and the lower edge of the detection chip subunit are not less than 8mm, and the distances from the blank control point to the left edge and the right edge of the detection chip subunit are not less than 5 mm;

the enzyme-labeled diluent in the step (3) is a PBS solution containing 20% V/V fetal calf serum;

the sample diluent in the step (4) is PBS solution containing 10% V/V fetal calf serum, 0.05-0.2% V/V Triton-100 and 0.1% V/V Proclin 300; preferably, the sample dilution contains 0.1% V/V Triton-100;

the washing solution is a PBS solution containing 1% V/V Tween 20, and the substrate solution is a TMB (3,3',5,5' -tetramethylbenzidine) solution.

7. Use of the antibody joint inspection kit according to claims 1-4 for the preparation of a non-immunological diagnosis; preferably, the applications in the non-immune diagnosis comprise epidemiological analysis, qualitative foot and mouth disease virus type O and A antibody detection on isolated serum, international live pig, cattle and sheep trade, import and export inspection and quarantine, foot and mouth disease virus type O and A vaccine quality control.