CN110346554B

CN110346554B - Enzyme immunochromatography detection kit adopting double-antibody sandwich method, preparation method and application

Info

Publication number: CN110346554B
Application number: CN201810283311.1A
Authority: CN
Inventors: 田克恭; 王莹
Original assignee: Luoyang Zhongke Gene Detection And Diagnosis Center Co ltd; Luoyang Pu Tai Biotechnology Co ltd
Current assignee: Luoyang Zhongke Gene Detection And Diagnosis Center Co ltd; Luoyang Pu Tai Biotechnology Co ltd
Priority date: 2018-04-02
Filing date: 2018-04-02
Publication date: 2023-04-18
Anticipated expiration: 2038-04-02
Also published as: CN110346554A

Abstract

The invention relates to a kit which comprises an enzyme immunochromatography detection test strip, sample treatment liquid, buffer solution and a buffer solution supply unit, wherein the buffer solution comprises phosphate buffer solution, macromolecular protein, tween-20, preservative and any one of carbamide peroxide, carbamide peroxide and hydrogen peroxide; the enzyme immunochromatographic assay test strip comprises a substrate pad, wherein an enzyme substrate which is formed by drying after being diluted by a substrate pad diluent is adsorbed on the substrate pad, and the substrate pad diluent comprises absolute ethyl alcohol, a PBS solution with the pH value of 7.40.1M and 0.5-4%W/V PEG6000 or PEG4000; the enzyme immune chromatography detection test paper strip comprises an enzyme label pad, wherein the enzyme label pad is adsorbed with an enzyme label antibody which is formed by drying after being diluted by an enzyme label antibody diluent, the enzyme label antibody diluent comprises a PBS solution with the pH value of 7.4.1M and any one of 0.5 percent V/V PEG4000, PEG6000 or PEG 8000; the enzyme immunochromatographic assay test strip comprises a detection line and a quality control line, wherein the detection line is fixed with an immobilized antibody diluted by an immobilized antibody diluent, and the immobilized antibody diluent comprises PBS solution with the pH value of 7.4.1M, 0.5% V/V trehalose and 0.1% V/V Tween-20.

Description

Enzyme immunochromatography detection kit adopting double-antibody sandwich method, preparation method and application

Technical Field

The invention relates to a double-antibody sandwich enzyme immunochromatography detection kit and a preparation method and application thereof, belonging to the field of biological detection.

Background

At present, the detection of antigens by using double antibody sandwich is common in the field of biological detection, wherein there are detection methods established by means of enzyme labeling or enzyme-labeled post-chromatography, such as the establishment of double antibody sandwich ELISA method for detecting avian influenza virus antigen reported in Zhang Lihuai and other documents (Zhang Lihuai and other documents), chinese medicine biotechnology, 2009,4 (1): 62-64), ELISA method for detecting avian influenza virus antigen established by monoclonal antibody-polyclonal antibody sandwich principle, and the detection of influenza virus by enzyme chromatography test paper strip established by monoclonal antibody-monoclonal antibody sandwich principle reported in Chinese patent CN104062430A. Among them, detection of an antigen with a monoclonal antibody-monoclonal antibody (bimab) sandwich is most common.

However, such a phenomenon is found in trial and error based on the prior art: after 2 strains of antibodies can be used for double-antibody sandwich detection antigens, the collocation mode research result shows that 1 strain of monoclonal antibody can only be used for marking, and the other 1 strain of monoclonal antibody can only be used as an immobilized antibody on a detection line, if the collocation mode of 2 strains of antibodies is changed, namely the positions of 2 strains of monoclonal antibodies in the product are interchanged, non-specific reaction can be caused or the detection sensitivity is reduced.

In addition, the existing products contain different components in sample treatment solution and buffer solution, so that target antigens are not fully cracked or the reaction is not sufficient in actual detection. Moreover, the sample treatment solutions of different kits are usually effective only for a specific sample from which an object to be detected is obtained, but are not necessarily effective for antigens of epidemic diseases of other animals (see the instruction manual of a commercial kit, and the instruction is marked with 'sample treatment solutions or sample diluents which cannot be used with other kits'), which causes low sensitivity and poor sensitivity when other types of samples are used in actual detection, even causes non-specific reactions, influences the detection speed, and causes illness deterioration or random spread of viruses when serious.

Therefore, the prior art lacks a kit which can exchange a labeled antibody and a fixed antibody of a double-antibody sandwich detection antigen, and also lacks a kit which can fully treat samples from different sources to ensure the detection effect.

Disclosure of Invention

In order to solve the defects of the prior art, the invention provides a double-antibody sandwich enzyme immunochromatography detection kit, which comprises an enzyme immunochromatography detection test strip, a sample treatment solution, a buffer solution and a buffer solution supply unit, and is characterized in that,

the buffer solution comprises phosphate buffer solution, macromolecular protein, tween-20, preservative and any one of carbamide peroxide, carbamide peroxide and hydrogen peroxide; preferably the phosphate buffer is a PBS solution at pH 7.4.1M; preferably the macromolecular protein is one or more of BSA, OVA, fetal calf serum; preferably, the preservative is any one of gentamicin, kanamycin sulfate, sodium azide, merthiolate and salts thereof, and Proclin300; the concentration of the carbamide peroxide, the carbamide peroxide and the hydrogen peroxide is 0.02-0.2 percent by weight; preferably, the concentration of the carbamide peroxide, the carbamide peroxide and the hydrogen peroxide is 0.05-0.1 percent; further preferably comprises pH7.4.0.1M PBS solution, 0.1% -0.5% V/V Tween-20, 0.02% V/V Proclin300, 0.05% V/V urea hydrogen peroxide, and 0.5% -1%W/V of any one of BSA, OVA and fetal calf serum;

the enzyme immunochromatographic assay test strip comprises a substrate pad, wherein an enzyme substrate which is formed by diluting and drying a substrate pad diluent is adsorbed on the substrate pad, and the substrate pad diluent comprises absolute ethyl alcohol, a PBS solution with the pH value of 7.4.0.1M and 0.5-4%W/V PEG6000 or PEG4000; preferably, the substrate pad dilution comprises 2%V/V absolute ethanol, 0.1% V/VPEG4000, 0.1M PBS, pH 7.4;

the enzyme immunochromatographic assay test strip comprises an enzyme label pad, an enzyme-labeled antibody is formed by adsorbing an enzyme-labeled antibody on the enzyme label pad, diluting the enzyme-labeled antibody by an enzyme-labeled antibody diluent and drying the enzyme-labeled antibody, wherein the enzyme-labeled antibody diluent comprises a PBS (phosphate-buffered saline) solution with the pH value of 7.40.1M and any one of 0.5 percent V/V PEG4000, PEG6000 or PEG 8000; preferably, the enzyme-labeled antibody dilution comprises 0.5% V/VPEG4000, 0.1M PBS, pH 7.4;

the enzyme immunochromatographic test strip comprises a detection line and a quality control line, wherein the detection line is fixed with an immobilized antibody diluted by an immobilized antibody diluent, and the immobilized antibody diluent comprises a PBS solution with the pH value of 7.4.0.1M, 0.5% V/V trehalose and 0.1% V/V Tween-20.

The enzyme immunochromatographic assay kit solves the problem that two antibodies in the enzyme immunochromatographic assay of the original double-antibody sandwich method cannot be interchanged through a buffer solution, a substrate pad diluent, an enzyme labeled antibody diluent and a fixed antibody diluent which have specific compositions, further improves the detection sensitivity when the original monoclonal antibody is used for pairing, and has the detection sensitivity equivalent to or better than that before the interchange when the interchanged monoclonal antibody is used for pairing.

As an embodiment of the invention, in the enzyme immunochromatographic assay kit of the present invention, the kit comprises an assay strip, a sample treatment solution, a buffer solution, and a buffer solution supply unit, wherein the assay strip comprises a substrate supply region, a sample supply region, and a detection region in this order from the longitudinal direction; the substrate supply region comprises the substrate pad having adsorbed thereon a dried enzyme substrate, preferably tetramethylbenzidine; the sample supply area comprises the enzyme label pad, an enzyme-labeled antibody is adsorbed on the enzyme label pad, the enzyme is preferably horseradish peroxidase, alkaline phosphatase or beta-galactosidase, and the enzyme substrate can generate color reaction with the enzyme on the enzyme-labeled antibody; the detection area comprises the detection line and the quality control line, and goat anti-mouse polyclonal antibody or goat anti-mouse secondary antibody is immobilized on the quality control line; the buffer solution supply unit is used for supplying the buffer solution to the substrate supply area of the detection test strip and enabling the buffer solution, the sample processing solution diffused along with the buffer solution, the enzyme substrate and the enzyme-labeled antibody to migrate towards one end far away from the substrate supply area along the longitudinal direction of the detection test strip.

In one embodiment of the present invention, the enzyme immunochromatographic detection kit of the present invention comprises an enzyme-labeled antibody diluent, wherein the concentration of the enzyme-labeled antibody dissolved in the enzyme-labeled antibody diluent is 0.5 to 10. Mu.g/ml, the concentration of the immobilized antibody dissolved in the immobilized antibody diluent is 0.5 to 1mg/ml, the concentration of the goat anti-mouse polyclonal antibody or goat anti-mouse secondary antibody dissolved in the immobilized antibody diluent is 1 to 3mg/ml, and the final concentration of the enzyme substrate dissolved in the substrate diluent is 0.5 to 4%W/V.

The concentrations of the enzyme-labeled antibody, the immobilized antibody and the enzyme substrate are in specific ranges, so that the detection sensitivity can be further improved.

As an embodiment of the invention, in the enzyme immunochromatographic assay kit, the distance between the detection line and the quality control line is more than or equal to 5mm.

As an implementation mode of the invention, in the enzyme immunochromatography detection kit, the monoclonal antibody in the enzyme-labeled antibody and the monoclonal antibody in the fixed antibody are any combination of porcine circovirus type 2 monoclonal antibody 3G12 secreted by mouse hybridoma cell 3G12 with CCTCC No: C2014198 and porcine circovirus type 2 monoclonal antibody 2F8 secreted by mouse hybridoma cell 2F8 with CCTCC No: C2014199; or any combination of a porcine epidemic diarrhea virus monoclonal antibody PEDV-McAB2 and a porcine epidemic diarrhea virus monoclonal antibody PEDV-McAB1, wherein the heavy chain variable region of the monoclonal antibody PEDV-McAB2 is shown in SEQ ID No.1, the light chain variable region is shown in SEQ ID No.2, the heavy chain variable region of the monoclonal antibody PEDV-McAB1 is shown in SEQ ID No.3, and the light chain variable region is shown in SEQ ID No. 4; or any combination of a transmissible gastroenteritis virus monoclonal antibody TGEV-3D2 and a transmissible gastroenteritis virus monoclonal antibody TGEV-4B4, wherein the heavy chain variable region of the monoclonal antibody TGEV-3D2 is shown by SEQ ID No.5, the light chain variable region is shown by SEQ ID No.6, the heavy chain variable region of the monoclonal antibody TGEV-4B4 is shown by SEQ ID No.7, and the light chain variable region is shown by SEQ ID No. 8; or any combination of an anti-influenza A virus nucleoprotein monoclonal antibody IV-McAB1 and an anti-influenza A virus nucleoprotein monoclonal antibody IV-McAB2, wherein the heavy chain variable region of the monoclonal antibody IV-McAB1 is shown by SEQ ID No.9, the light chain variable region is shown by SEQ ID No.10, the heavy chain variable region of the monoclonal antibody IV-McAB2 is shown by SEQ ID No.11, and the light chain variable region is shown by SEQ ID No. 12; or the arbitrary combination of the canine parvovirus monoclonal antibody CPV-10B11 secreted by the mouse bone marrow hybridoma cell 10B11 strain with CCTCC No: C201578 and the canine parvovirus monoclonal antibody CPV-10H4 secreted by the mouse bone marrow hybridoma cell 10H4 strain with CCTCC No: C201579; or CCTCC No: monoclonal antibodies CDV-1G5 and CCTCC No of canine distemper virus secreted by C2015201 mouse bone marrow hybridoma cell 1G 5: any combination of canine distemper virus monoclonal antibodies CDV-6E11 secreted by C2015202 mouse bone marrow hybridoma cell 6E11 strain; or any combination of a canine adenovirus monoclonal antibody CAV-5G4 and a canine adenovirus monoclonal antibody CAV-1A1, wherein the heavy chain variable region of the monoclonal antibody CAV-5G4 is shown in SEQ.ID No.13, the light chain variable region is shown in SEQ.ID No.14, the heavy chain variable region of the monoclonal antibody CAV-1A1 is shown in SEQ.ID No.15, and the light chain variable region is shown in SEQ.ID No. 16.

As an embodiment of the invention, the enzyme-labeled antibody is PCV2-3G12, and the immobilized antibody is PCV2-2F8; or the enzyme-labeled antibody is PCV2-2F8, and the immobilized antibody is PCV2-3G12.

As an embodiment of the invention, the enzyme-labeled antibody is PEDV-McAB2, and the immobilized antibody is PEDV-McAB1; or the enzyme-labeled antibody is PEDV-McAB1, and the immobilized antibody is PEDV-McAB2.

As an embodiment of the invention, the enzyme-labeled antibody is TGEV-3D2, and the immobilized antibody is TGEV-4B4; or the enzyme-labeled antibody is TGEV-4B4, and the immobilized antibody is TGEV-3D2.

As an embodiment of the invention, the enzyme-labeled antibody is IV-McAB1, and the immobilized antibody is IV-McAB2; or the enzyme-labeled antibody is IV-McAB2, and the immobilized antibody is IV-McAB1.

As an embodiment of the invention, the enzyme-labeled antibody is CPV-10B11, and the immobilized antibody is CPV-10H4; or the enzyme-labeled antibody is CPV-10H4, and the immobilized antibody is CPV-10B11.

As an embodiment of the invention, the enzyme-labeled antibody is CDV-1G5, and the immobilized antibody is CDV-6E11; or the enzyme-labeled antibody is CDV-6E11, and the fixed antibody is CDV-1G5.

As an embodiment of the invention, the enzyme-labeled antibody is CAV-5G4, and the immobilized antibody is CAV-1A1; or the enzyme-labeled antibody is CAV-1A1, and the fixed antibody is CAV-5G4.

In one embodiment of the present invention, in the enzyme immunochromatographic assay kit of the present invention, the sample treatment solution contains a PBS solution with a ph of 7.4.1M, CHAPS, a saponin, and a preservative; preferably, the sample treatment fluid comprises a PBS solution at ph 7.4.4.1M, CHAPS, saponin, and Proclin300; more preferably, said sample treatment solution comprises pH7.4.0.1M PBS solution, 0.5% -2.0%; most preferably, the sample treatment fluid comprises pH7.4.0.1M PBS solution, 0.5% W/V CHAPS, 1%W/V saponin, 0.02% V/V Proclin300.

The invention realizes the detection of a plurality of detection target samples by using the sample treatment fluid with specific composition, and overcomes the defect that one enzyme immunochromatography detection kit in the prior art can only detect one or more detection target samples.

In one embodiment of the present invention, the enzyme immunochromatographic assay kit according to the present invention comprises a solid sample dissolved in the sample treatment solution in an amount of 0.08 to 0.5g/500 to 1500. Mu.l, and a liquid sample dissolved in the sample treatment solution in an amount of 500 to 1500. Mu.l/500 to 1500. Mu.l; the detection dosage of the sample dissolved by the sample treatment solution is 50-150 mul.

In one embodiment of the present invention, in the enzyme immunochromatographic assay kit according to the present invention, the sample is selected from the group consisting of tissue, serum, anal secretions, feces, oronasal secretions, ocular nasal secretions, and virus cultures.

The enzyme immunochromatographic assay kit can be used for detecting various samples.

Still another object of the present invention is to provide a method for preparing the above kit, comprising:

preparing a buffer solution;

diluting a substrate by using a substrate pad diluent, adsorbing the substrate to an adsorbate to obtain a substrate pad, drying the substrate pad, and placing the substrate pad in a detection test strip;

the monoclonal antibody marked in the step (3) is an enzyme-labeled antibody, and is diluted by an enzyme-labeled antibody diluent, and is adsorbed to an adsorbate to obtain an enzyme-labeled pad, and the enzyme-labeled pad is dried and then placed in a detection test strip;

diluting the immobilized antibody by using a solidified antibody diluent, fixing the diluted immobilized antibody in a detection test strip as a detection line, fixing the goat anti-mouse polyclonal antibody or the goat anti-mouse monoclonal antibody on a quality control line, and drying;

preparing a sample treatment solution; and

and (6) assembling the enzyme immunochromatographic detection test strip, the sample treatment solution, the buffer solution and a buffer solution supply unit into a kit.

The kit prepared by the invention solves the technical problem of position interchange of 2 strains of antibodies in the existing product prepared by using a double-antibody sandwich principle, effectively reduces the risk of occurrence of nonspecific reaction, and improves the detection sensitivity.

It is a further object of the present invention to provide a method for detecting a virus in a sample using the above kit for non-diagnostic purposes, the detection method comprising:

step (1) pretreating a sample to be detected by using the sample treatment solution;

step (2) adding the sample pretreated in step (1) into the test strip;

step (3) supplying the buffer solution to the test strip through the buffer solution supply unit, and standing for reaction for 30 minutes; and

observing the test strip after the reaction in the step (4), and judging whether the test strip is invalid or not no matter whether the test strip exists in the test line or not if the quality control line does not have a strip; if the quality control line has a strip, the detection line has a strip which is positive, and the detection line has no strip which is negative.

It is a further object of the present invention to provide a sample treatment solution comprising a PBS solution of ph 7.4.4.1M, CHAPS, saponin, and preservative; preferably, the sample treatment fluid comprises a PBS solution at ph 7.4.4.1M, CHAPS, saponin, and Proclin300; more preferably, said sample treatment solution comprises pH7.4.0.1M PBS solution, 0.5% -2.0%; most preferably, the sample treatment solution comprises pH7.4.0.1M PBS solution, 0.5%/W/V CHAPS, 1%W/V saponin, 0.02%/V Proclin300.

The sample treatment solution can be matched with different commercialized kits for use. When the sample treatment solution is used together with a commercial kit, the detection sensitivity of the sample treatment solution is better than that of the original commercial kit; and can detect a plurality of samples, and overcomes the defect that one enzyme immunochromatography detection kit in the prior art can only detect one or more detection target samples.

In one embodiment of the present invention, the sample to be tested in the sample treatment solution of the present invention is selected from the group consisting of tissue, serum, anal secretions, feces, oronasal secretions, ocular nasal secretions, and viral cultures.

Detailed Description

Hereinafter, some exemplary embodiments of the present invention will be described in detail so that the present invention may be more easily understood by those skilled in the art.

For simplicity and clarity, some embodiments that have been listed in the "summary of the invention" section of the present application are not repeated in the "detailed description" section.

In the present invention, the "enzyme chromatography detection test strip", "enzyme immunochromatographic detection test strip", or "detection test strip" may be used interchangeably.

The "enzyme-labeled antibody" and the "labeled antibody" in the present invention are used interchangeably, and are monoclonal antibodies that are predominantly enzyme-labeled. The term "enzyme" includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, calf intestinal alkaline phosphatase. Wherein, the substrate used by the horseradish peroxidase HRP is o-phenylenediamine (OPD), tetramethyl benzidine (TMB), aminosalicylic acid, o-biphenylmethylamine or 2,2' -diamino-2 (3-ethyl-thiazoline sulfonic acid-6) ammonium salt, preferably tetramethyl benzidine (TMB); the substrate used by the alkaline phosphatase is p-nitrophenyl phosphate (p-NPP) or naphthol-AS-Mx phosphate + diazonium salt; the substrate used by the glucose oxidase is ABTS + HRP + glucose or glucose + methylthiophenol oral liquid + thiazole blue; the substrate used by beta-galactosidase is 4-methylumbelliferyl beta-D galactoside (4 MUG) or nitrophenol galactoside (ONPG).

The fixed antibody is also called a capture antibody, is fixed on a detection line (T line for short) of a detection test strip and is used for capturing a compound formed by an antigen and a labeled antibody which are chromatographed.

The term "phosphate buffer" refers to a solution containing phosphoric acid or a salt thereof and adjusted to a desired pH, and is one of the most widely used buffers in biochemical studies. Typically, phosphate buffers are prepared from phosphoric acid or phosphates (including but not limited to sodium and potassium salts). Some phosphates are known in the art, such as sodium and potassium dihydrogen phosphate, disodium and dipotassium hydrogen phosphate, sodium and potassium phosphate. Phosphate salts are known to exist as hydrates of salts. Due to the secondary dissociation of the buffer, the buffered pH ranges widely, for example, from about pH4 to about pH10, preferably from about pH5 to pH9, more preferably from about pH7 to about pH8, and most preferably about pH7.4. Further preferably, the phosphate buffer is a phosphate buffer containing sodium chloride and potassium chloride. In the present invention, "phosphate buffered saline" and PBS can be used interchangeably.

The term "macromolecular proteins" includes, but is not limited to, bovine serum albumin BSA, ovalbumin OVA, fetal bovine serum.

The term "preservative" includes, but is not limited to, gentamicin, kanamycin sulfate, sodium azide, thimerosal and its salts, proclin300.

The "adsorbate" in the present invention is composed of a material that can adsorb a target and release the adsorbed target in a buffer. The adsorbate does not change the chemical nature of the target to be adsorbed. For example, in some embodiments of the invention, the adsorbate may adsorb a substrate pad diluent to form a substrate pad, which is capable of releasing the substrate under the influence of a buffer. Examples of adsorbates include, but are not limited to, glass fiber membranes, nitrocellulose membranes, and polyester membranes.

The term "glass fiber membrane" is abbreviated as glass fiber, and includes glass cellulose membrane, glass fiber filter membrane and glass fiber filter paper.

The term "saponin" is also known as saponin, alkali saponin, saponin or saponin, and is a glycoside whose aglycone is a triterpene or a spirostanol compound, and can be classified in various ways, including monosaccharide chain saponin, disaccharide chain saponin, trisaccharide chain saponin, acidic saponin, neutral saponin, steroid saponin, and triterpene saponin. The saponin comprises steroid saponin and triterpenoid saponin, preferably digitonin. The term "digitonin" is also known as digitonin, digitoxioside, gitoxil-removing saponin, and digitoxioside.

The term "CHAPS" is a detergent or stain remover, also known as 3- [ (3-cholesterylaminopropyl) dimethylamino ] -1-propanesulfonic acid, 3- ((3-cholaminopropyl) dimethylamine) -1-propanesulfonic acid, 3- [3- (cholamidopropyl) dimethylamino ] propanesulfonic acid inner salt, 3- [ (3-cholesterylaminopropyl) dimethylamino ] -1-propanesulfonic acid (CHAPS), and 3- [3- (cholamidopropyl) dimethylamino ] propanesulfonic acid inner salt.

The term "quality control line" is also known as control line.

The invention provides an enzyme immunochromatography detection kit, which comprises an enzyme immunochromatography detection test strip, a sample treatment solution, a buffer solution and a buffer solution supply unit and is characterized in that,

the buffer solution comprises phosphate buffer solution, macromolecular protein, tween-20, preservative and any one of carbamide peroxide, carbamide peroxide and hydrogen peroxide;

the enzyme immunochromatographic assay test strip comprises a substrate pad, wherein an enzyme substrate which is formed by diluting and drying a substrate pad diluent is adsorbed on the substrate pad, and the substrate pad diluent comprises absolute ethyl alcohol, a PBS solution with the pH value of 7.4.0.1M and 0.5-4%W/V PEG6000 or PEG4000;

the enzyme immunochromatographic assay test strip comprises an enzyme label pad, an enzyme-labeled antibody is formed by adsorbing an enzyme-labeled antibody on the enzyme label pad, diluting the enzyme-labeled antibody by an enzyme-labeled antibody diluent and drying the enzyme-labeled antibody, wherein the enzyme-labeled antibody diluent comprises a PBS (phosphate-buffered saline) solution with the pH value of 7.40.1M and any one of 0.5 percent V/V PEG4000, PEG6000 or PEG 8000;

the enzyme immunochromatographic test strip comprises a detection line on which immobilized antibodies diluted with an immobilized antibody diluent comprising a pH 7.4.1M PBS solution, 0.5-V/V trehalose, 0.1-V Tween-20 are immobilized, and a quality control line.

As one embodiment of the kit of the present invention, the kit comprises a test strip, a sample treatment solution, a buffer solution, and a buffer solution supply unit, wherein,

the detection test strip sequentially comprises a substrate supply area, a sample supply area and a detection area from the longitudinal direction; the substrate supply zone comprises the substrate pad having a dried enzyme substrate adsorbed thereon; the sample supply area comprises the enzyme label pad, an enzyme-labeled antibody is adsorbed on the enzyme label pad, and the enzyme substrate can generate a color reaction with enzyme on the enzyme-labeled antibody; the detection area comprises the detection line and the quality control line, and goat anti-mouse polyclonal antibody or goat anti-mouse secondary antibody is immobilized on the quality control line;

the buffer solution supply unit is used for supplying the buffer solution to the substrate supply area of the detection test strip and enabling the buffer solution, the sample processing solution diffused along with the buffer solution, the enzyme substrate and the enzyme-labeled antibody to migrate to one end far away from the substrate supply area along the longitudinal direction of the detection test strip.

Such buffer supply units capable of performing the above-described functions are known in the art. For example, the buffer solution supply unit may be of a structure as described in chinese patent CN104062430a, the buffer solution is stored in a substrate buffer tank, the buffer solution is supplied to the substrate supply area of the test strip by immersing a developing solution pad connected to the substrate pad of the test strip into the substrate buffer tank, and the buffer solution is transferred to the detection area in the longitudinal direction of the test strip by using the capillary force of a water absorbent pad provided behind the detection area of the test strip. Alternatively, the buffer solution supplied to the substrate supply region may be moved to the detection region by the gravity of the buffer solution itself by placing the test strip on a supporting slope where the substrate supply region is higher and the detection region is lower.

The present invention will be described in detail in example 2 by taking an example of a horseradish peroxidase HRP-labeled antibody and Tetramethylbenzidine (TMB) as a substrate used for horseradish peroxidase HRP. Furthermore, the hydrogen acceptor required by the horseradish peroxidase HRP is hydrogen peroxide H ₂ O ₂ Solid or liquid as main component, including but not limited to carbamide peroxide, hydrogen peroxide, etc.

The swine monoclonal antibody mainly comprises the following components:

(1) The porcine circovirus type 2 monoclonal antibody 3G12 (PCV 2-3G12 for short) is obtained by secreting mouse hybridoma cells 3G12 (CCTCC No: C2014198), and the porcine circovirus type 2 monoclonal antibody 2F8 (PCV 2-2F8 for short) is obtained by secreting mouse hybridoma cells 2F8 (CCTCC No: C2014199). 2 mouse hybridoma cells are all preserved in China center for type culture Collection, the preservation address is Wuhan-Wuhan university in China, and the preservation date is 2014, 11 months and 3 days. See chinese patent CN105717293A.

(2) The porcine epidemic diarrhea virus monoclonal antibody PEDV-McAB2 and the porcine epidemic diarrhea virus monoclonal antibody PEDV-McAB1 are disclosed, wherein the heavy chain variable region of the monoclonal antibody PEDV-McAB2 is shown in SEQ ID No.1, the light chain variable region is shown in SEQ ID No.2, the heavy chain variable region of the monoclonal antibody PEDV-McAB1 is shown in SEQ ID No.3, the light chain variable region is shown in SEQ ID No.4, and the porcine epidemic diarrhea virus monoclonal antibody PEDV-McAB2 and the light chain variable region thereof are shown in Chinese patent CN105461805A.

(3) The swine transmissible gastroenteritis virus monoclonal antibody TGEV-3D2 has the amino acid sequence of the heavy chain variable region of SEQ ID No.5 and the amino acid sequence of the light chain variable region of SEQ ID No.6; the swine transmissible gastroenteritis virus monoclonal antibody TGEV-4B4 has the amino acid sequence of the heavy chain variable region of SEQ ID No.7 and the amino acid sequence of the light chain variable region of SEQ ID No.8.

The avian monoclonal antibody mainly comprises the following components:

(4) The anti-influenza A virus nucleoprotein monoclonal antibody IV-McAB1 and the anti-influenza A virus nucleoprotein monoclonal antibody IV-McAB2 are disclosed, wherein the heavy chain variable region of the monoclonal antibody IV-McAB1 is shown by SEQ ID No.9, the light chain variable region is shown by SEQ ID No.10, the heavy chain variable region of the monoclonal antibody IV-McAB2 is shown by SEQ ID No.11, the light chain variable region is shown by SEQ ID No.12, and the reference is made to Chinese patent CN104062430A.

The canine monoclonal antibody mainly comprises:

(5) The canine parvovirus monoclonal antibody 10B11 (CPV-10B 11 for short) is obtained by secreting a mouse bone marrow hybridoma cell 10B11 strain (with the preservation number of CCTCC No: C201578), and the canine parvovirus monoclonal antibody 10H4 (CPV-10H 4 for short) is obtained by secreting a mouse bone marrow hybridoma cell 10H4 strain (with the preservation number of CCTCC No: C201579). 2 mouse hybridoma cells are preserved in China center for type culture Collection, wherein the preservation address is Wuhan university in Wuhan, and the preservation time is 2015, 05 and 20 days. See chinese patent CN104928258a.

(6) The monoclonal antibody 1G5 (CDV-1G 5 for short) of the canine distemper virus is obtained by secreting mouse bone marrow hybridoma cells 1G5 (the preservation number is CCTCC No: C2015201), and the monoclonal antibody 6E11 (CDV-6E 11 for short) of the canine distemper virus is obtained by secreting mouse bone marrow hybridoma cells 6E11 strain (the preservation number is CCTCC No: C2015202). 2 hybridoma cells are preserved in China center for type culture Collection, wherein the preservation address is Wuhan-Wuhan university in China, and the preservation time is 2015, 11 months and 25 days. See chinese patent CN105695420a.

(7) The heavy chain variable region amino acid sequence of the canine adenovirus monoclonal antibody CAV-5G4 is the amino acid sequence shown in SEQ.ID No.13, and the light chain variable region amino acid sequence is the amino acid sequence shown in SEQ.ID No. 14; and a canine adenovirus monoclonal antibody CAV-1A1, wherein the heavy chain variable region amino acid sequence is the amino acid sequence shown in SEQ.ID No.15, and the light chain variable region amino acid sequence is the amino acid sequence shown in SEQ.ID No. 16.

The invention also provides a buffer solution for the enzyme immunochromatography detection kit, which comprises a phosphate buffer solution, macromolecular protein, tween-20 and a preservative.

The invention also provides a sample treatment solution for the enzyme immunochromatography detection kit, which comprises a PBS solution with the pH value of 7.4.4M, CHAPS, saponin and a preservative.

The sample treatment solution prepared by the invention can be used for dissolving virus solution, excrement, serum, microorganism swabs (including eye-nose swabs, nasopharynx swabs and cloaca swabs) and ground tissue samples, and can be used for accurately detecting the treated samples by using an enzyme-free chromatography detection test strip constructed by a double-monoclonal antibody sandwich principle.

The invention also provides a substrate pad diluent for preparing the enzyme immunochromatography detection kit, which comprises absolute ethyl alcohol, PEG4000 and PBS solution with pH7.4.0.1M, or comprises absolute ethyl alcohol, PEG6000 and PBS solution with pH7.4.0.1M.

The invention also provides an enzyme-labeled antibody diluent for preparing the enzyme immunochromatographic assay kit, which comprises PEG4000, pH7.4.1M PBS solution, or PEG6000, pH7.4.1M PBS solution, or PEG8000, pH7.4.1M PBS solution.

The invention also provides a solidified antibody diluent for preparing the enzyme immunochromatography detection kit, which comprises PBS (phosphate buffer solution) with the pH value of 7.4.0.1M, trehalose and Tween-20.

The invention also provides a preparation method of the kit, which comprises the following steps:

preparing a buffer solution;

the monoclonal antibody marked in the step (3) is an enzyme-labeled antibody, and is diluted by enzyme-labeled antibody diluent, and is adsorbed to an adsorbate to obtain an enzyme-labeled pad, and the enzyme-labeled pad is dried and then placed in a detection test strip;

diluting the fixed antibody by using a solidified antibody diluent, fixing the diluted fixed antibody in a test strip as a detection line, fixing the goat anti-mouse polyclonal antibody or the goat anti-mouse monoclonal antibody in a quality control line, and drying;

preparing a sample treatment solution; and

As an embodiment of the method for preparing the kit of the present invention, the method for preparing comprises:

preparing a buffer solution;

diluting a substrate by using a substrate pad diluent, adsorbing the substrate to an adsorbate to obtain a substrate pad, drying the substrate pad, and placing the substrate pad in a substrate supply area of the detection test strip;

the monoclonal antibody marked in the step (3) is an enzyme-labeled antibody, and is diluted by an enzyme-labeled antibody diluent, and is adsorbed to an adsorbate to obtain an enzyme-labeled pad, and the enzyme-labeled pad is dried and then is placed in a sample supply area of a detection test strip;

diluting the immobilized antibody by using an immobilized antibody diluent, diluting the goat anti-mouse polyclonal antibody or the goat anti-mouse monoclonal antibody by using a phosphate buffer solution or a solidified antibody diluent, fixing the diluted goat anti-mouse polyclonal antibody or the goat anti-mouse monoclonal antibody in a detection area of a detection test strip as a detection line and a quality control line respectively, and drying;

step (5), preparing a sample treatment solution; and

the kit is assembled in the step (6) such that the test strip includes a substrate supply region, a sample supply region, and a detection region in this order from the longitudinal direction, and such that the buffer supply unit can supply the buffer to the substrate supply region of the test strip, and also such that the buffer migrates toward the end away from the substrate supply region along the longitudinal direction of the test strip.

preparing a buffer solution;

diluting a substrate to 0.5% -4%W/V by using a substrate pad diluent, adsorbing the substrate to an adsorbate to obtain a substrate pad, drying the substrate pad, and placing the substrate pad in a substrate supply area of the detection test strip;

the monoclonal antibody marked in the step (3) is an enzyme-labeled antibody, and is diluted to 0.5-10 mu g/ml by using an enzyme-labeled antibody diluent, and is adsorbed on an adsorbate to obtain an enzyme-labeled pad, and the enzyme-labeled pad is dried and then is placed in a sample supply area of a detection test strip;

diluting the fixed antibody to 0.5-1 mg/ml by using a fixed antibody diluent, diluting the goat anti-mouse polyclonal antibody or the goat anti-mouse monoclonal antibody to 1-3 mg/ml by using a phosphate buffer solution or a fixed antibody diluent, fixing the diluted antibodies as a detection line and a quality control line in a detection area of a detection test strip respectively, and drying;

step (5), preparing a sample treatment solution; and

As an embodiment of the preparation method of the kit, in the preparation method, the distance between the detection line and the quality control line is more than or equal to 5mm.

As one embodiment of the method for preparing the kit of the present invention, in the method for preparing the kit, the buffer solution comprises a phosphate buffer solution, a macromolecular protein, tween-20 and a preservative, preferably the phosphate buffer solution is a PBS solution with ph 7.4.0.1M, preferably the macromolecular protein is one or more of BSA, OVA and fetal calf serum, preferably the preservative is any one of gentamicin, kanamycin sulfate, sodium azide, thimerosal and salts thereof, proclin300, more preferably any one of carbamide peroxide, hydrogen peroxide, and hydrogen peroxide, further preferably any one of PBS solution with ph 7.4.0.1M, PBS solution with ph 0.5-1%W/V BSA, PBS V/V Tween-20 with ph 0.1-0.5, V/V Proclin300 and carbamide peroxide, most preferably 0.1M, 3262 zxft PBS 62/V, vben/V, vtwe/V300, vpwe/300, vpro-0.02, vpro-0.4, ph 7.4; the sample treatment solution comprises a pH7.40.1M PBS solution, CHAPS, saponin and preservative, preferably comprises a pH 7.4.1M PBS solution, CHAPS, saponin and Proclin300, more preferably comprises a pH 7.4.0.1M PBS solution, 0.5% -2.0% W/V CHAPS, 0.5% -2.0% W/V saponin and 0.02% V/V Proclin300, most preferably comprises a pH 7.4.0.1M PBS solution, 0.5% W/V CHAPS, 1%W/V saponin and 0.02% V/V Proclin300; the substrate pad diluent comprises absolute ethanol, PEG4000 and PBS solution with pH 7.4.1M, or comprises absolute ethanol, PEG6000 and PBS solution with pH 7.4.1M, preferably comprises 2%V/V absolute ethanol, 0.1% V/V PEG4000 and PBS solution with pH 7.4.1M, or comprises 2%V/V absolute ethanol, 0.1% V/V PEG6000 and PBS solution with pH 7.4.1M; the enzyme-labeled antibody diluent comprises PEG4000, pH7.4.0.1M PBS solution, or PEG6000, pH7.4.0.1M PBS solution, or PEG8000, pH7.4.0.1M PBS solution, preferably 0.5% V/V PEG4000, pH7.4.0.1M PBS solution, or 0.5% V/V PEG6000, pH7.4.0.1M PBS solution, or 0.5% V/V PEG8000, pH7.4.1M PBS solution; and the solidified antibody diluent comprises pH7.4.0.1M PBS solution, trehalose and Tween-20, preferably comprises pH7.4.0.1M PBS solution, 0.5%.

As an embodiment of the method for preparing the kit of the present invention, in the step (1), the buffer solution is a PBS solution with pH of 7.4.0.1M, 0.5% -1%W/V BSA, 0.1% -0.5% V/V Tween-20, 0.02% V/V Proclin300;

the substrate pad dilution in step (2) is 2%V/V absolute ethanol, 0.1% V/V PEG4000, pH7.4.1M PBS solution, or 2%V/V absolute ethanol, 0.1% V/V PEG6000, pH7.4.0.1M PBS solution;

the enzyme-labeled antibody dilution in the step (3) is 0.5% by volume of V/V PEG4000, pH7.40.1M PBS solution, or 0.5% by volume of V/V PEG6000, pH 7.4.0.1M PBS solution, or 0.5% by volume of V/V PEG8000, pH 7.4.0.1M PBS solution;

the immobilized antibody dilution in step (4) is a PBS solution with pH 7.4.1M, 0.5% V/V trehalose, 0.1% V/V Tween-20;

the sample treatment solution in the step (5) is a PBS solution having a pH of 7.4.1M, and the content of 0.5% by volume of W/V CHAPS, 1%W/V saponin, 0.02% by volume of V/V Proclin300.

As an embodiment of the detection method of the present invention, the detection method includes:

step (1) pretreating a sample to be detected by using the sample treatment solution, wherein the dissolving amount of a solid sample dissolved in the sample treatment solution is 0.08-0.5 g/500-1500 mu l, and the dissolving amount of a liquid sample dissolved in the sample treatment solution is 500-1500 mu l/500-1500 mu l;

step (2) adding 50-150 μ l of the sample pretreated by the step (1) into a sample supply area of the test strip;

observing the detection area of the detection test strip after the reaction in the step (4), and judging whether the detection line has a strip or not to be invalid in the current detection if the quality control line has no strip; if the quality control line has a strip, the detection line has a strip which is positive, and the detection line has no strip which is negative.

The non-diagnosis purpose animal epidemic antigen detection comprises epidemiological analysis, qualitative and quantitative detection of isolated tissues and epitope identification research.

However, this does not exclude the possibility of the kit of the invention being used for other purposes in virus detection activities.

The detoxification way of the sample to be detected is mainly to metabolize through the organism detoxification way after target antigen infection, for example, porcine circovirus is mainly to detoxify through tissues and blood, porcine epidemic diarrhea virus and porcine transmissible gastroenteritis virus are mainly to detoxify through anus swab and excrement, influenza A is mainly to detoxify through pharynx and nose swab, canine parvovirus is mainly to detoxify through anus swab and excrement, canine distemper virus is mainly to detoxify through eye and nose swab, anus swab and excrement, and canine adenovirus is mainly to detoxify through eye and nose swab, anus swab, excrement, blood and the like.

As an embodiment of the detection method of the present invention, the sample to be detected includes, but is not limited to, epidemic antigens produced by pig, fowl and dog. Namely, the application objects of the kit of the invention include, but are not limited to, epidemic antigens produced by pigs, poultry and dogs.

As an embodiment of the detection method of the present invention, the sample to be detected includes porcine circovirus type 2, porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, influenza A virus, canine parvovirus, canine distemper virus and canine adenovirus.

When the buffer solution and the sample treatment solution are used in combination, the positions of the fixed and marked 2 monoclonal antibodies used by the existing product prepared by the double-antibody (especially double-monoclonal-antibody) sandwich principle can be exchanged, the product prepared by the 2 monoclonal antibodies with different collocation modes is completed, non-specific reaction is not caused after the sample is detected, and the sensitivity is properly improved.

The invention is further described below in conjunction with specific embodiments, the advantages and features of which will become apparent as the description proceeds. These embodiments are merely exemplary, and are not intended to limit the scope of the invention in any way, since the invention is not intended to be limited to the specific embodiments described herein. The embodiments described below are intended to cover features of the various embodiments as well as the method steps and sequences for constructing and operating the embodiments. 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 formulation of the PBS buffer used in the examples of the present invention is exemplified below: 8g of sodium chloride, 0.2g of potassium chloride, 1.44g of disodium hydrogen phosphate and 0.24g of monopotassium phosphate, adjusting the pH value to 7.4, and fixing the volume to 1L, including but not limited to the formula; all chemical reagents are analytically pure and purchased from the national pharmaceutical group.

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

EXAMPLE 1 analysis of the monoclonal antibodies used in the existing products

Based on the double-antibody sandwich principle, the technical team and other research and development teams develop a series of products, which are briefly as follows:

when detecting the activity of the porcine circovirus type 2 monoclonal antibody 2F8 (PCV 2-2F8 for short) and 3G12 (PCV 2-3G12 for short) paired antibodies in Chinese patent CN105717293A, the enzyme immunochromatography detection test strip prepared only when the coating antibody is PCV2-2F8 and the labeled antibody PCV2-3G12 can be used, and if the positions of the 2 strains of antibodies are interchanged, the coating antibody is PCV2-3G12 and the labeled antibody PCV2-2F8, the nonspecific reaction exists with a great probability.

In Chinese patent CN105461805A, monoclonal antibodies PEDV-McAB1 and PEDV-McAB2 of porcine epidemic diarrhea virus are paired and studied, and only a colloidal gold detection test strip or an enzyme immunochromatography detection test strip prepared when the immobilized antibody is PEDV-McAB1 and the labeled antibody is PEDV-McAB2 can be applied, and if 2 antibodies are interchanged to prepare a product for detection, the result is false positive with a high probability.

In Chinese patent CN104062430A, monoclonal antibodies 1 (IV-McAB 1 for short) and 2 (IV-McAB 2 for short) of influenza A virus are paired and studied, and it is found that only an enzyme chromatographic detection test strip prepared when the immobilized antibody is IV-McAB2 and the labeled antibody is IV-McAB1 can be used, and if 2 is interchanged to prepare a product for detection, the result shows that a non-specific reaction occurs with a high probability.

In the Chinese patent CN104928258A, the canine parvovirus monoclonal antibodies 10B11 and 10H4 can only be used in an enzyme chromatography detection test strip prepared when the immobilized antibody is 10B11 and the labeled antibody is 10H4 during pairing research, and if 2 are exchanged in position to prepare a product for detection, false negative occurs at a high probability in the result.

In Chinese patent CN105695420A, the monoclonal antibodies 6E11 and 1G5 of canine distemper virus can be used only when the immobilized antibody is 6E11 and the labeled antibody is 1G5, and the enzyme immunochromatographic assay test strip or the colloidal gold assay test strip can be used, if 2 are interchanged to prepare a product for detection, the false negative can be generated with a high probability of the result.

In the Chinese patent CN101149377A, the product prepared only when the fixed antibody is 9D47A1 and the labeled antibody is 7E11A1 in 2 strains of the aspergillus for detection can generate the strongest signal and avoid non-specificity.

Based on this, the present inventors have conducted a series of studies to solve the technical problems that the 2 antibodies cannot exchange positions during the pairing, or the sensitivity is reduced, non-specific reactions are generated, and false positive and false negative are caused after the positions are exchanged.

Considering that the detection targets required by the test strip in clinical application mainly comprise an eye-nose swab, an anus swab, excrement, blood, treated tissues and the like, wherein the excrement contains more impurities, the canine parvovirus enzyme immunochromatographic detection test strip for dogs for detecting excrement is explained in detail as an example.

Example 2 preparation and evaluation of Canine parvoVirus enzyme Immunochromatographic assay kit

In order to better evaluate the canine parvovirus enzyme immunochromatography detection kit, the invention firstly uses a fixed antibody of 10H4 and a labeled antibody of 10B11 to prepare a test strip (corresponding to the part of example 2.1), and uses canine parvovirus CVCC AV298 strain (purchased from China veterinary microbiological culture collection center), canine parvovirus epidemic strain CPV 2a type S2 strain and S0425 strain, canine parvovirus epidemic strain CPV 2B type S0304 strain (see Preparation and application of two monoclonal antibodies against canine parvovirus infection vaccine and field strains, journal of Vaccines and immunity, 2017,3 (1): 001-004), and 20 positive feces and 20 negative feces (respectively positive and negative by canine parvovirus PCR identification results) as sample trays to comprehensively analyze the test strip.

2.1 preparation of buffer

The contents of the buffer components are shown in Table 1, wherein the remaining amount is made up of the components (i.e., PBS or citric acid-0.2M disodium hydrogen phosphate) unless specified. The samples were taken for appearance testing, sterility testing (according to the appendix of the current Chinese veterinary pharmacopoeia) and shelf life (preservation at 2-30 ℃) studies, the results of which are shown in Table 2.

TABLE 1 summary of the components contained in the different buffers

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The results of sequentially preparing buffers A1 to A16, a substrate pad (prepared in example 2.2.A, substrate pad diluent B3), an enzyme-labeled pad (prepared in example 2.2.B, enzyme-labeled antibody diluent C3), a detection zone (prepared in example 2.2.C, immobilized antibody diluent D4) according to example 2.4 to give kits A1 to a16, and a sample plate sample according to example 2.5 diluted with the sample treatment solution F4 prepared in example 2.3 for detection (see Table 2) show: the buffer solution has clear appearance and is qualified in sterile inspection, the retention period is 15 months except for 12 months of A1, and when the buffer solution is A6, the prepared test strip has no false positive result in the detection and has higher sensitivity in detecting CPV strains, particularly epidemic strains.

TABLE 2 summary of different buffer solution test results and test results after preparation of the kit

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Note: the positive excrement and negative excrement results represent the total number of test strip detection results/PCR detection positive or negative samples.

To evaluate whether the content of the major components in buffer A6 affects the test results, a specific range (see Table 1 Nos. A6 to A13) was further adjusted, and the results (see Table 2) are shown: when the buffer solution is A6-A9 (corresponding to the buffer solution with main components of 0.5-1%W/VBSA, 0.1-0.5% V/VTween-20), the prepared test strip has high detection sensitivity (10) ^3.2 ～10 ^3.5 TCID ₅₀ Per ml), the detection result of the clinical sample is consistent with that of the PCR; when the buffer solution is A10-A13, the detection sensitivity of the prepared test strip is reduced (more than 10) ^3.5 TCID ₅₀ Ml), the probability of non-specific reactions occurring in the detection of clinical samples is high.

To evaluate the final concentration of carbamide peroxide in the buffer, the concentration of carbamide peroxide in buffer A6 was adjusted to 0.01%, 0.02%, 0.2%, 0.3% w/V in the order stated to prepare test strips, and as a result: when the test strip prepared with 0.01% w/V of urea hydrogen peroxide was used for the detection of positive feces, there was a problem that a negative result was likely to occur, and when the test strip prepared with 0.3% w/V of urea hydrogen peroxide was used for the detection of negative feces, there was a positive result, i.e., false positive and false negative; on the other hand, the test strips prepared when the concentration of urea hydrogen peroxide was 0.02% or 0.2% W/V were equivalent to the A6 results in detection sensitivity and correct results for both positive and negative feces, so that the final concentration of urea hydrogen peroxide in the buffer was set to 0.02% to 0.2% W/V.

For the evaluation of other macromolecular proteins such as OVA in place of BSA for debugging (see a14 in table 1), the test results (see table 2) were consistent with the test results of buffer-assembled test strips containing BSA, indicating that: other macromolecules can also be used as buffer components and can achieve better detection effect.

In addition, urea hydrogen peroxide and hydrogen peroxide are used for debugging instead of urea hydrogen peroxide (see A15-A16 in Table 1), and the detection result (see Table 2) is consistent with the detection result of the test strip assembled by the buffer solution containing urea hydrogen peroxide, which shows that: the urea hydrogen peroxide and the hydrogen peroxide can also be used as buffer solution components and can achieve better detection effect.

2.2 preparation of test paper

2.2.a preparation of substrate pads for substrate areas

The substrate Tetramethylbenzidine (TMB) was diluted with a substrate pad diluent (see Table 3) so that the TMB concentration was 0.5 to 4%W/V, and the resulting solution was adsorbed on a glass fiber membrane as an adsorbate and dried for use.

TABLE 3 substrate pad Diluent composition

Numbering	Substrate pad diluent composition
		B1	2%V/V Anhydrous ethanol
B2	2%V/V absolute ethanol, 0.1% V/VPEG2000, 0.1M PBS, pH7.4
		B3	2%V/V absolute ethanol, 0.1% V/VPEG4000, 0.1M PBS, pH7.4
B4	2%V/V anhydrous ethanol, 0.1%/VPEG 6000, 0.1M PBS, pH7.4
		B5	2%V/V absolute ethanol, 0.1% V/VPEG8000, 0.1M PBS of pH7.4

Buffer A6 (prepared as in example 2.1), substrate pad (TMB final concentration of 1%), enzyme-labeled pad (prepared as in example 2.2.B, enzyme-labeled antibody dilution C3), detection zone (as per example 2.1. B)

Example 2.2.C preparation, D4 as immobilized antibody diluent), kits b1 to b5 were prepared in the order of example 2.4, and the sample plate samples were diluted with the sample treatment solution F4 prepared in example 2.3 for detection as in example 2.5, and the results (see table 4) showed: when the substrate pad diluent is B3 or B4, the prepared test strip has higher sensitivity to CPV strains, particularly epidemic strains during detection (10) ^3.2 ～10 ^3.5 TCID ₅₀ Per ml), detecting that the clinical sample is consistent with the PCR detection result; when the substrate pad diluents are B1, B2 and B5, the detection sensitivity of the prepared test strip is reduced (more than 10) ^3.5 TCID ₅₀ Ml), detecting the occurrence of non-specific reaction in clinical samples.

TABLE 4 summary of the test results after preparation of the kit from different substrate pad dilutions

Note: the positive excrement and negative excrement results represent the total number of positive or negative samples of the test strip/PCR detection.

To evaluate the final concentration of TMB upon adsorption to the substrate pad, test strips were prepared by diluting TMB to a final concentration of 0.4%, 0.5%, 4%, 5%W/V with substrate pad diluent, and as a result: the test strip prepared when TMB was 0.4% W/V was used for detection of positive feces, and the test strip prepared when TMB was 5%W/V was used for detection of negative feces, and the test strip was positive in the case where the test strip was used for detection of negative feces, that is, there was a problem of false positive and false negative; when TMB is 0.5% and 4%W/V, the detection sensitivity of the prepared test strip is equivalent to the result of B3, and the results of detecting positive excrement and negative excrement are both correct, so that the final concentration of the TMB is set to be 0.5% -4%W/V.

2.2.b preparation of enzyme-labeled pads in sample supply area

2.2.b.1 preparation of enzyme-labeled antibodies

And (3) adopting a modified sodium periodate method to label the horseradish peroxidase HRP. Weighing 20mg of horse radish peroxidase HRP, dissolving in 1ml of ultrapure water, adding 1ml of freshly prepared NaIO ₄ Solution (30 mg NaIO) ₄ Dissolved in1ml of ultrapure water), mixing uniformly, and keeping away from light at 2-8 ℃ for 30 minutes; adding 40 mul of glycol into the solution, and keeping away from light at the temperature of 2-8 ℃ for 30 minutes; the monoclonal antibody purified by the octanoic acid-saturated ammonium sulfate precipitation method was mixed with 100. Mu.l of the above mixture in a ratio of 1mg to 100. Mu.l, and the mixture was added to a dialysis bag, mixed, and dialyzed against a coating buffer (pH 9.6,0.05mol/L carbonate 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 added ₄ Solution (20 mg NaBH) ₄ Dissolved 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 12000r/min for 10 min, discard the supernatant. The pellet was suspended with a volume of PBS mixed with glycerol equal to the volume of purified antibody (V: V = 1: 1).

2.2.b.2 identification of enzyme-labeled antibodies

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

And (3) quality evaluation: detecting the absorbance value A of the enzyme-labeled antibody at 403nm and 280nm by using an ultraviolet spectrophotometer according to the mark rate = A _403nm /A _280nm "calculating the mark rate; the amount of monoclonal antibody after enzyme labeling, i.e., the amount of IgG, was expressed as "amount of IgG (mg/ml) = (A) _280nm -A _403nm X 0.3) x 0.62 x dilution times ". The results are shown in Table 5:

TABLE 5 summary of the quality evaluation results of enzyme-labeled antibodies

Name of labeled antibody	Amount of IgG (mg/ml)	Mark rate
			CPV-10B11	1.0	60％
CPV-10H4	1.5	58％

2.2.b.3 preparation of enzyme-labeled pad

The enzyme-labeled antibody is diluted with different enzyme-labeled antibody diluents (see table 6) until the final concentration of the monoclonal antibody is 0.5-10 mug/ml, adsorbed on a glass fiber membrane, and dried for use.

TABLE 6 dilution Components of enzyme-labeled antibodies

Numbering	Enzyme-labeled antibody diluent component
		C1	0.1M PBS, pH7.4
C2	0.5% of V/VPEG2000, 0.1M PBS of pH7.4
		C3	0.5% of 0.1M PBS V/VPEG4000, pH7.4
C4	0.5% of V/VPEG6000, 0.1M PBS of pH7.4
		C5	0.5% of 0.1M PBS V/VPEG8000, pH7.4

The results of sequentially preparing the buffer A6 (prepared in example 2.1), the substrate pad (prepared in example 2.2.A, substrate pad diluent B3), the enzyme-labeled pad (enzyme-labeled antibody content: 2. Mu.g/ml), the detection zone (prepared in example 2.2.C, immobilized antibody diluent D4), kits c1 to c5 in the order of example 2.4, and diluting the sample plate sample with the sample treatment solution F4 prepared in example 2.3 in example 2.5 for detection (see Table 7) show: when the enzyme-labeled antibody diluent is C3, C4 or C5, the prepared test strip has high detection sensitivity on CPV strains (10) ^3.0 ～10 ^3.5 TCID ₅₀ Per ml), detecting that the clinical sample is consistent with the PCR detection result; when the substrate pad diluent is C1 or C2, the detection sensitivity of the prepared test strip is reduced (more than 10) ^4.0 TCID ₅₀ Ml), detecting the occurrence of non-specific reaction in clinical samples.

TABLE 7 summary of the detection results after preparation of the kit from different enzyme-labeled antibody dilutions

In order to evaluate the use concentration of the content of the enzyme-labeled antibody during the preparation of the enzyme-labeled pad, the enzyme-labeled antibody is diluted into the working concentration of 0.4, 0.5, 10 and 12 mu g/ml by using an enzyme-labeled antibody diluent to prepare a test strip, and the results are as follows: when the working concentration of the enzyme-labeled antibody is 0.4 mug/ml, the sensitivity of the prepared test strip for detecting CPV epidemic strains is reduced by 10-100 times, and when the working concentration of the enzyme-labeled antibody is 12 mug/ml, the positive result, namely the false positive result, appears in the result of detecting the negative feces by using the prepared test strip; when the working concentration of the enzyme-labeled antibody is 0.5 and 10 mug/ml, the detection sensitivity of the prepared test strip is equivalent to the result of C3, and the results of detecting positive excrement and negative excrement are both correct, so that the working concentration of the enzyme-labeled antibody is set to be 0.5-10 mug/ml.

2.2.c preparation of detection zone

The immobilized antibody is diluted to 0.5-1 mg/ml by using an immobilized antibody (see table 8) diluent, the antibody for the quality control line is diluted to 1mg/ml by using a PBS buffer solution with the pH value of 7.4.0.1M or a solidified antibody diluent D4, the two antibodies are respectively adsorbed at one end of a nitrocellulose membrane, the distance between two lines is not less than 5mm, and the antibody is used after being dried.

TABLE 8 set of solidified antibody diluent Components

The results of preparing the buffer A6 (prepared in example 2.1), the substrate pad (prepared in example 2.2.A, substrate pad diluent B3), the enzyme-labeled pad (prepared in example 2.2.B, enzyme-labeled antibody diluent C3), the nitrocellulose membrane (fixed antibody concentration on the fixing line is 0.8 mg/ml), preparing the kits d1 to d6 in the order of example 2.4, and diluting the sample plate sample with the sample treatment solution F4 prepared in example 2.3 for detection in example 2.5 (see Table 9) show: when the detection line is prepared, the test strip prepared when the solidified antibody diluent is D4 has higher sensitivity for detecting CPV strains (10) ^3.0 ～10 ^3.5 TCID ₅₀ Per ml), detecting that the clinical sample is consistent with the PCR detection result; when the solidified antibody diluent is D1, D2, D3, D5 or D6, the sensitivity of the prepared test strip is reduced when a sample is detected (> 10) ^4.0 TCID ₅₀ /ml) and non-specific reactions occurred in the clinical samples.

TABLE 9 summary of the test results after preparation of the kit from different dilutions of the immobilized antibody

To evaluate the immobilized antibody concentration at the time of preparation of the nitrocellulose membrane, the immobilized antibody was diluted with an immobilized antibody diluent to working concentrations of 0.4, 0.5, 1.0, 1.2mg/ml to prepare test strips, and as a result: when the working concentration of the immobilized antibody is 0.4mg/ml, the sensitivity of the prepared test strip for detecting CPV epidemic strains is reduced by 3-100 times, and when the working concentration of the immobilized antibody is 1.2mg/ml, the prepared test strip has a positive result with a high probability when detecting negative feces, namely a false positive result exists; when the concentration of the immobilized antibody is 0.5 and 1.0mg/ml, the detection sensitivity of the prepared test strip is equivalent to the D4 result, and the results of detecting positive excrement and negative excrement are correct, so that the working concentration of the immobilized antibody is set to be 0.5-1.0 mg/ml.

2.3 preparation of sample treatment solution

The following sample treatment solutions (see table 10) were prepared and sampled for appearance testing, sterility testing (according to the supplement of the current pharmacopoeia of china) and shelf life studies, the results of which are shown in table 11.

TABLE 10 summary of sample treatment fluid components

The results of sequentially preparing the buffer A6 (prepared as in example 2.1), the substrate pad (prepared as in example 2.2.A, substrate pad diluent B3), the enzyme-labeled pad (prepared as in example 2.2.B, enzyme-labeled antibody diluent C3), the detection zone (prepared as in example 2.2.C, immobilized antibody diluent D4), the kits F1 to F6 according to example 2.4, and the sample plate samples diluted with the sample treatment solutions F1 to F6 prepared in example 2.3 according to example 2.5 for detection (see Table 11) show that: when the sample treatment fluid is F4, the sensitivity for detecting the CPV strain is higher (10) ^3.2 ～10 ^3.5 TCID ₅₀ Per ml), detecting that the clinical sample is consistent with the PCR detection result; when the sample treatment solutions were F1, F2, F3, F5, and F6, the sensitivity of the detection sample decreased (> 10) ^4.0 TCID ₅₀ Ml) and non-specific reactions occurred in clinical samples.

TABLE 11 summary of assay results after preparation of kits with different sample treatment solutions

To evaluate the CHAPS content of the sample treatment solution, the other components were kept constant, and the CHAPS content was adjusted to 0.4%, 0.5%, 2.0%, 2.5% by weight W/V to prepare a kit and to perform detection, as a result: when the CHAPS contained in the sample treatment solution is 0.4%, the sensitivity of the prepared kit for detecting CPV is reduced by 10-100 times; when the CHAPS contained in the sample treatment solution is 2.5 percent, the prepared kit detects that positive excrement is negative and negative excrement is positive, and serious non-specificity exists; whereas, when the CHAPS contents of the sample treatment solution were 0.5% and 2.0%, the detection sensitivity of the kit prepared was comparable to the F4 result, and the results were correct for both positive and negative stools, so the CHAPS content of the sample treatment solution was set to 0.5% -2.0% w/V.

To evaluate the saponin content in the sample treatment solution, the other components were kept unchanged, the saponin content was adjusted to 0.4%, 0.5%, 2.0%, 3.0% w/V to prepare a kit and perform detection, and as a result: when the saponin contained in the sample treatment solution is 0.4%, the sensitivity of the prepared kit for detecting CPV is reduced by 10 times, and the feces with negative detection is positive; when the CHAPS contained in the sample treatment solution is 3.0 percent, the prepared kit detects that positive excrement is negative and negative excrement is positive, and serious non-specificity exists; when the saponin content in the sample treatment solution is 0.5% or 2.0%, the detection sensitivity of the prepared kit is equivalent to the F4 result, and the results of detecting positive feces and negative feces are correct, so that the saponin content in the sample treatment solution is set to 0.5% -2.0% w/V.

In addition, in order to evaluate whether or not the main components of the buffer solution A6, the substrate pad diluent B3 or B4, the enzyme-labeled antibody diluent C3 or C4 or C5, and the immobilized antibody diluent D4 after the test have affected the test results of the test strip, the positions thereof were interchanged and evaluated. The results show that: the detection of negative feces after the exchange of the main components of the solutions has non-specific reaction, and the sensitivity of detecting CPV epidemic strains is reduced by 10 to 1000 times. Thus, sensitivity and specificity of the kit in detection can be realized only by using corresponding buffer solution and diluent at the fixed or specific position of the kit. In other words, unlike the positions of the 2 monoclonal antibodies (which essentially represent the reaction sequence), the positions of the buffer and the diluent cannot be interchanged.

2.4 preparation method of kit

Preparing a buffer solution;

diluting the substrate to 0.5% -4%W/V by using a diluent for the substrate pad, adsorbing the substrate to an adsorbate to obtain the substrate pad, drying the substrate pad, and placing the dried substrate pad in a substrate supply area of the detection test strip;

diluting the fixed antibody to 0.5-1 mg/ml by using a fixed antibody diluent, diluting the goat anti-mouse polyclonal antibody or the goat anti-mouse monoclonal antibody to 1-3 mg/ml by using a phosphate buffer solution or a fixed antibody diluent, fixing the diluted antibodies as a detection line and a quality control line in a detection area of a detection test strip respectively, and drying the diluted antibodies, wherein the distance between the two lines is more than or equal to 5 mm;

preparing a sample treatment solution with the volume of 1ml per tube; and

step (6) assembling the kit, connecting the test strip to a buffer solution supply unit, and injecting the buffer solution prepared in the step (1) into the buffer solution supply unit so that the buffer solution supply unit can supply the buffer solution to a substrate supply area of the test strip; the detection test strip comprises a substrate supply area (the left end of the nitrocellulose membrane comprises a substrate pad, a dry enzyme substrate is adsorbed on the substrate pad), a sample supply area (the middle of the nitrocellulose membrane comprises an enzyme label pad, an enzyme-labeled antibody is adsorbed on the enzyme substrate, the enzyme substrate can generate a color reaction with enzyme on the enzyme-labeled antibody, and migrates to the far end away from the buffer solution supply unit on the nitrocellulose membrane), and a detection area (the right end of the nitrocellulose membrane comprises a detection line and a quality control line, the detection line is immobilized with an immobilized antibody, and the quality control line is immobilized with goat-anti-mouse polyclonal antibody or goat-anti mouse secondary antibody), wherein the substrate pad and the enzyme label pad are sequentially adhered to the nitrocellulose membrane from left to right in the whole section; and (4) filling the sample treatment solution filled in the tube prepared in the step (5) into a kit.

2.5 detection method of kit

The detection method of the kit comprises the following steps:

the method comprises the following steps of (1) collecting a sample to be detected according to a detoxification way of animal epidemic disease antigens, and dissolving a solid sample and/or a liquid sample in a sample treatment solution.

Dropwise adding the treated sample into a sample supply area added with the detection test strip;

observing the detection area of the detection test strip after the reaction in the step (4), and judging whether the detection line has a strip or not to be invalid if the quality control line has no strip; if the quality control line has a strip, the detection line has a strip which is positive, and the detection line has no strip which is negative.

The test amount of the CPV epidemic strain S2 and positive feces was adjusted, and the test was performed with a kit (a combination of buffer solution A6, substrate pad diluent B3, enzyme-labeled antibody diluent C3 on an enzyme-labeled pad, immobilized antibody diluent D4, and sample treatment solution F4), and the results were obtained (see table 12): under the condition that the sample detection dosage is 100 mul, when the amount of the solid sample dissolved in 500-1500 mul of sample treatment fluid is 0.05g, the detection result is negative (false negative exists), when the amount of the solid sample dissolved is 0.6g, the sample treatment fluid can not finish the chromatography process, so that the result is invalid, and only when the amount of the solid sample is 0.08-0.5 g, the detection result is valid and accurate; similarly, in the case where the amount of the sample to be tested is 100. Mu.l, the test results are negative (false negative) when the amount of the liquid sample dissolved in 500 to 1500. Mu.l of the sample treatment liquid is 300. Mu.l or 2000. Mu.l, and the results are correct only when the amount of the liquid sample dissolved in 500 to 1500. Mu.l is present. When the detection dosage of the sample passing through the sample treatment solution is 25 mul, the detection result is invalid because the chromatography process is stopped after half, when the detection dosage of the sample is 200 mul, the false negative result is caused because of excessive reaction, and the detection result is valid and accurate only when the detection dosage of the sample is 50-150 mul.

TABLE 12 test results of the amount of the sample to be tested and the amount to be added

According to the above debugging results, the present invention will be described in detail only with respect to the same kind of kits prepared under the conditions (i.e., the combination of the buffer solution A6, the substrate pad diluent B3, the enzyme-labeled antibody diluent C3, the immobilized antibody diluent D4, and the sample treatment solution F4, the amount of the solid sample dissolved in the sample treatment solution is 0.08 to 0.5g/500 to 1500. Mu.l, the amount of the liquid sample dissolved in the sample treatment solution is 500 to 1500. Mu.l/500 to 1500. Mu.l, and the amount of the sample dissolved in the sample treatment solution is 50 to 150. Mu.l) and the evaluation and application thereof.

2.6 Kit evaluation prepared by different collocation modes of 2 monoclonal antibodies

2.6.a comparison of sensitivity of kits prepared in different collocation modes

The 2 monoclonal antibodies in the test strip were transposed (see table 13) and evaluated using a sample tray, while a commercial Han Guoan jie canine parvovirus colloidal gold test strip was used for parallel testing, and the results (see table 13): CPV kit 3 (see Chinese patent CN 104928258A) prepared in 2 monoclonal antibody pairing mode according to prior art has the sample sensitivity of 10 in a detection sample disk ^3.2 ～10 ^3.5 TCID ₅₀ Per ml; the CPV kit 2 with unchanged monoclonal antibody position, which is obtained according to the invention, has the sensitivity of 10 in a detection sample plate ^2.0 ～10 ^2.5 TCID ₅₀ Perml, specific CPV kit 1 and commercially available conventional colloidal gold productsThe sensitivity is 10 to 50 times higher; after the matching mode of the existing 2 monoclonal antibodies is changed, namely the positions of the monoclonal antibodies are exchanged, the detection sensitivity of the CPV kit 1 obtained according to the invention is equivalent to or better than that of the CPV kit 3, the results of detecting positive and negative samples are accurate, and no non-specific reaction occurs.

TABLE 13 summary of test results after preparation of kits with different collocation patterns

2.6.b kit specificity detection prepared by different collocation modes

CPV kits 1, 2 and 3 are respectively used for detecting specific samples including canine distemper virus liquid, canine parainfluenza virus liquid, canine adenovirus type 1 virus liquid, canine adenovirus type 2 virus liquid, canine rabies virus liquid, healthy dog feces, canine distemper virus positive dog feces, canine rotavirus positive dog feces and canine coronavirus cytotoxin samples, and the results are negative, which indicates that the specificity is good.

2.6.c repeatability research on kits prepared in different collocation modes

The CPV kits 1 and 2 are respectively subjected to batch-to-batch and batch-to-batch repeatability researches on samples in sample trays and specific samples, and the results are as follows: the CPV kits 1 and 2 have good repeatability between batches and in batches.

2.6.d shelf life study of kits prepared in different collocation models

Placing the CPV kits 1 and 2 at 2-8 ℃ for 6, 9, 12 and 15 months respectively for sensitivity and specificity detection, and obtaining the following results: the coincidence rate of the detection results is 100%, which indicates that the kits 1 and 2 can be stored at the temperature of 2-8 ℃ for 12 months or even 15 months.

Clinical application of kit prepared by different collocation modes of 2.6.e

200 parts of clinical samples which are identified as positive and 200 parts of clinical samples which are identified as negative by PCR are respectively detected by CPV kits 1 and 2, and the results are shown in a table 14: the CPV kit 1 has a total detection coincidence rate of 94 percent, and the CPV kit 2 has a total detection coincidence rate of 97 percent, and can replace a PCR detection method which is time-consuming and tedious in operation and can be realized only by special technicians and special instruments and equipment to carry out clinical rapid and accurate identification.

TABLE 14 summary of the results of the different kits for testing clinical samples

Reagent kit	Positive/identification Total Positive	Negative/identification Total negative	Total rate of agreement
				CPV kit 1	176/200	224/200	94％
CPV kit 2	187/200	213/200	97％

EXAMPLE 3 preparation of other enzyme Immunochromatographic assay kits

In order to further evaluate whether each condition optimized in example 2 can meet the application in diagnosis and detection of animal epidemic diseases such as other antigens of pig, poultry, cat and dog, the method specifically debugs and evaluates porcine circovirus type 2, porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, influenza A virus, canine distemper virus, canine adenovirus and the like one by one, and results are as follows: (1) The sensitivity of the kit prepared by the original collocation mode is superior to that of the kit sold on the market or the existing kit (10-50 times higher), and the sensitivity of the kit prepared by the collocation mode after transposition is equivalent to or better than that of the kit prepared by the collocation mode before debugging of the original collocation mode; (2) The specificity is good, and no non-specificity phenomenon occurs; (3) good repeatability; (4) the storage life is all met; (5) The detection application effect is good, and the detection can be quickly, on-site and accurately carried out. The following is a brief explanation of the porcine circovirus type 2 and type A influenza viruses as representatives.

3.1 porcine circovirus type 2 enzyme-free chromatography detection test strip

3.1.A comparison of sensitivity of kits prepared in different collocation modes

The positions of 2 monoclonal antibodies in the test strip are exchanged (see table 15), and different subtype strains of PCV2 and clinical positive serum and negative serum identified by PCR are used for evaluation, and the results (see table 15, positive tissue, negative tissue, positive serum and negative serum are all identified by PCR): PCV2 kit 3 prepared in 2 monoclonal antibody pairing mode according to the prior art (see Chinese patent CN 105717293A) has the sample sensitivity of 10 in a detection sample tray ^4.0 ～10 ^4.2 TCID ₅₀ Per ml; the sensitivity of PCV2 kit 2 obtained according to the invention in the detection of a sample plate is 10 ^3.8 ～10 ^4.0 TCID ₅₀ Per ml, the sensitivity is 10 to 15 times higher than that of the PCV2 kit 1 in the prior art; after the monoclonal antibody pairing mode is changed, the detection sensitivity of the PCV2 kit 1 obtained according to the invention is equivalent to or better than that of the PCV2 kit 3, and the results of detecting positive and negative samples are both accurate without non-specific reaction.

TABLE 15 summary of assay results after preparation of kits with different collocation patterns

3.1.b kit specificity detection prepared by different collocation modes

The PCV2 kits 1 and 2 are respectively used for detecting specific samples, including porcine circovirus type 1 virus liquid, porcine reproductive and respiratory syndrome virus liquid, highly pathogenic porcine reproductive and respiratory syndrome virus liquid, porcine parvovirus liquid, porcine pseudorabies virus liquid, hog cholera virus liquid and porcine epidemic diarrhea virus liquid, and the results are negative, which indicates that the specificity is good.

3.1.c. kit repeatability research prepared by different collocation modes

The PCV2 kits 1 and 2 are respectively subjected to batch-to-batch and batch-to-batch repeatability researches on samples in sample trays and specific samples, and the results are as follows: the PCV2 kits 1 and 2 have good repeatability between batches and in batches.

3.1.D shelf life study of kits prepared in different collocation models

Placing PCV2 kits 1 and 2 at 2-8 ℃ for 6, 9, 12 and 15 months respectively for sensitivity and specificity detection, and obtaining the following results: the coincidence rate of the detection results is 100%, which indicates that the kits 1 and 2 can be stored at the temperature of 2-8 ℃ for 12 months or even 15 months.

3.1.e clinical application of kits prepared in different collocation modes

250 positive clinical samples and 250 negative clinical samples (containing serum and ground tissue samples) which are identified by virus separation and PCR (polymerase chain reaction) are respectively detected by PCV2 kits 1 and 2, and the results are shown in Table 16: the PCV2 kit 1 detection total coincidence rate is 91%, and the PCV2 kit 2 detection total coincidence rate is 94%, which are both superior to the results of the PCV2 kit 3 (original collocation mode, the prior art), and can replace the virus separation identification or PCR detection method which is time-consuming and tedious in operation and can be realized only by special technicians and special instruments and equipment to carry out clinical rapid and accurate identification.

TABLE 16 summary of the results of the different kits for testing clinical samples

Reagent kit	Positive/identification Total Positive	Negative/identification Total negative	Total rate of agreement
				PCV2 kit 1	205/250	295/250	91％
PCV2 kit 2	220/250	280/250	94％
				PCV2 kit 3	200/250	300/250	90％

3.2 influenza A enzyme-free chromatography detection test strip

3.2.A comparison of sensitivity of kits prepared in different collocation modes

The 2 monoclonal antibodies in the test strip were transposed (see table 17), and evaluated using a sample tray consisting of different subtype strains of influenza and microbial swabs (i.e., negative swabs, positive swabs) collected before and after the animal challenge test, and the results (see tables 17 to 18): the sensitivity of the IV kit 3 (see Chinese patent CN 104062430A) prepared according to the prior art by using the original 2 monoclonal antibodies in the matching mode for detecting the sample disc sample is 0.01-0.1 HA or 10 ^4.5 ～10 ^5.5 EID ₅₀ 100 mul; the sensitivity of the IV kit 2 obtained according to the invention in a detection sample plate is 0.005-01 HA or 10 according to the original 2 monoclonal antibody matching mode ^3.5 ～10 ^4.0 EID ₅₀ 100 mul, which is 10 to 50 times higher than the sensitivity before optimization; after the original 2 monoclonal antibodies are matched in a mode of changing, namely, after exchange positions, the detection sensitivity of the IV kit 1 obtained according to the invention is equivalent to that of the IV kit 3Or better, and the results of detecting positive and negative samples are accurate without non-specific reaction.

TABLE 17 preparation of kits with different collocation patterns

TABLE 18 summary of test results of kits prepared with different collocation patterns

3.1.b kit specificity detection prepared by different collocation modes

The IV kits 1 and 2 are used for respectively detecting specific samples, including negative SPF chicken throat and cloaca swab samples, healthy pig nasopharynx swab samples and healthy dog nasopharynx swab samples, and the results are negative, which indicates that the specificity is good.

3.1.c repeatability Studies on kits prepared in different collocation models

The samples in the sample trays and the specific samples of the IV kits 1 and 2 are respectively subjected to batch-to-batch and batch-to-batch repeatability researches, and the results are as follows: the inter-batch and intra-batch repeatability of the IV kits 1 and 2 is good.

3.1.D shelf life study of kits prepared in different collocation modes

The IV kits 1 and 2 are respectively placed at 2-8 ℃ for 6, 9, 12 and 15 months for sensitivity and specificity detection, and the results are as follows: the coincidence rate of the detection results is 100 percent, which indicates that the kits 1 and 2 can be stored for 12 months or even 15 months at the temperature of 2-8 ℃.

3.1.e clinical application of kits prepared in different collocation modes

Clinical samples which are identified as positive 200 parts and negative 200 parts by an SPF chick embryo inoculation virus separation method and an RT-PCR method are respectively detected by IV kits 1, 2 and 3, and the results (shown in a table 19) are as follows: the total detection coincidence rate of the IV kit 1 is 94 percent, the total detection coincidence rate of the IV kit 2 is 96 percent, the total detection coincidence rate is superior to 92 percent of the detection coincidence rate of the IV kit 3 (the original collocation mode and the prior art), and the method can replace the SPF chick embryo inoculation virus separation and identification method or the PCR detection method which is time-consuming and tedious in operation and can be realized by special technicians and special instruments for clinical quick and accurate identification.

TABLE 19 summary of clinical sample results from different kits

Reagent kit	Positive/identification Total Positive	Negative/identification Total negative	Total rate of agreement
				IV kit 1	176/200	224/200	94％
IV kit 2	185/200	215/200	96％
				IV kit 3	168/200	232/200	92％

Example 4 enzyme-labeled antibody-prepared enzyme-free chromatography detection kit and application thereof

4.1 other enzyme-labeled antibodies

Alkaline phosphatase-labeled antibody: labeling the monoclonal antibody by a glutaraldehyde two-step method, dissolving alkaline phosphatase by 2% glutaraldehyde solution until the final concentration is 20mg/ml, standing overnight in a biochemical incubator at 25 ℃, transferring to a dialysis bag, dialyzing overnight in PBS solution at 2-8 ℃, adjusting the hydroformylation concentration to 2mg/ml, mixing uniformly with 1mg monoclonal antibody, transferring to the dialysis bag, dialyzing overnight by coating buffer solution (pH9.6, 0.05mol/L carbonate buffer solution, changing the solution for 3 times), then changing to PBS solution (changing the solution for 3 times), continuously dialyzing overnight, taking out the dialysate, adding glycerol with the same volume, and freezing for later use. The alkaline phosphatase-labeled antibody was identified in the same manner as the HRP enzyme-labeled antibody in reference example 2, and the results are shown in Table 20. Beta-galactosidase-labeled antibody: labeling a monoclonal antibody by a glutaraldehyde method, dissolving 5mg of the monoclonal antibody and 10mg of beta-galactosidase in 2ml of 0.1mol/L potassium phosphate buffer solution (pH 6.8), mixing the mixture at 2-8 ℃ for overnight dialysis, diluting glutaraldehyde to 1% by 0.1mol/L potassium phosphate buffer solution (pH 6.8), adding 100 mu L of diluted glutaraldehyde into the dialysis mixed solution, stirring the solution at room temperature for three hours, adding 2mol/L glycine solution to make the final concentration of the solution be 0.1mol/L, standing the solution at room temperature for 2 hours, dialyzing the mixed solution at 2-8 ℃ for overnight by PBS, centrifuging the mixed solution at 10000 r/min and 4 ℃ for 30 minutes, taking supernatant, adding isovolumetric glycerol, and freezing and storing the supernatant for later use. The beta-galactosidase-labeled antibody was identified in the same manner as the HRP-labeled antibody in reference example 2, and the results are shown in Table 20.

TABLE 20 summary of the quality evaluation results of other enzyme-labeled antibodies

4.2 Assembly of enzyme-linked immunosorbent assay kit prepared by other enzyme-labeled antibodies and application thereof

The alkaline phosphatase-labeled antibody and the β -galactosidase-labeled antibodies H1 to H4 prepared in example 4.1 were used to prepare canine parvovirus enzyme immunoassay detection kits H1 to H4 according to example 2, respectively, and the results of detection using the samples in the sample plate (see Table 21) indicated that: the enzyme-free chromatography detection kit prepared by other enzyme-labeled antibodies can also realize effective detection, and the detection results are equivalent after the positions of two strains of antibodies in the kit are interchanged.

TABLE 21 summary of the results of the detection after preparation of the kit with other enzyme-labeled antibodies

In summary, when the buffer solution, the substrate pad diluent and the test strip prepared from the enzyme-labeled antibody diluent and the immobilized antibody diluent of the kit are combined, the positions of the fixed and marked 2 monoclonal antibodies used in the enzyme-linked immunosorbent assay test strip prepared by the double-monoclonal-antibody sandwich principle can be interchanged, so that the detection kit can be prepared from the 2 monoclonal antibodies with different collocation modes, and the kit can not cause non-specific reaction and can improve the sensitivity after a sample is detected.

SEQUENCE LISTING

<110> Luoyang Pluley Ke Motai Biotechnology Limited

Luoyang Zhongke gene detection and Diagnosis Center Co.,Ltd.

<120> enzyme immunochromatographic assay kit adopting double-antibody sandwich method, preparation method and application

<160> 16

<170> PatentIn version 3.3

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130 135

<210> 12

<211> 131

<212> PRT

<213> hybridoma cell (hybridoma)

<400> 12

Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser Trp Ile Met Ser Lys Gly

1 5 10 15

Gln Ile Trp Leu Ser Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly

20 25 30

Glu Lys Val Thr Met Thr Cys Thr Ala Ser Ser Ser Val Thr Ser Ser

35 40 45

Tyr Leu His Val Tyr Gln Gln Arg Pro Gly Ser Ser Pro Arg Leu Val

50 55 60

Ile Tyr Ser Thr Ser Thr Leu Ala Ser Gly Val Pro Ala Lys Phe Ser

65 70 75 80

Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu

85 90 95

Ala Glu Asp Ala Ala Thr Tyr Tyr Cys His Gln Tyr His Leu Ser Pro

100 105 110

Val Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Lys Ala Asp Ala

115 120 125

Ala Pro Thr

130

<210> 13

<211> 117

<212> PRT

<213> hybridoma cell (hybridoma)

<400> 13

Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Thr Ile Ser Gly Leu Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile

35 40 45

Gly Arg Phe Asp Pro Val Asn Val Asn Ser Lys Tyr Asp Pro Lys Tyr

50 55 60

Gln Gly Lys Ala Thr Ile Thr Ser Asp Thr Ser Ser Asn Thr Ala Tyr

65 70 75 80

Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Asn Ser Ala Met Asp Tyr Trp Gly Gln Gly Ser Ser

100 105 110

Val Thr Val Ser Ser

115

<210> 14

<211> 107

<212> PRT

<213> hybridoma cell (hybridoma)

<400> 14

Asp Val Val Met Thr Pro Thr Pro Lys Phe Leu Leu Val Ser Pro Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asn Asp

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Ala Ser His Arg Tyr Thr Gly Val Pro Val Arg Phe Thr Gly

50 55 60

Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Thr Val Gln Ala

65 70 75 80

Glu Asp Leu Ala Ile Tyr Phe Cys Gln Gln Asp Phe Ala Ser Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 15

<211> 120

<212> PRT

<213> hybridoma cell (hybridoma)

<400> 15

Val Phe Ala Ser Gly Ala His Gly Glu Gly Val Met Ile Arg Thr Thr

1 5 10 15

Lys Phe Gly Lys Arg Ser Pro Gly Val His Leu Val Asp Met Lys Met

20 25 30

Trp Val Asn Trp Val Lys Glu Ala Pro Gly Lys Gly Leu Lys Trp Met

35 40 45

Gly Arg Ile Asn Thr Asn Asn Glu Val Ser Thr Tyr Ala Glu Glu Phe

50 55 60

Lys Gly Arg Phe Ala Phe Ser Leu Glu Ala Ser Ala Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Asn Asp Leu Thr Asn Glu Asp Ser Ala Thr Tyr Phe Cys

85 90 95

Ala Arg Met Asp Ser Ser Gly Tyr Val Trp Phe Thr Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ala

115 120

<210> 16

<211> 107

<212> PRT

<213> hybridoma cell (hybridoma)

<400> 16

Asp Thr Val Met Thr Gln Ser Gln Lys Phe Ile Ser Thr Ser Ile Gly

1 5 10 15

Asp Arg Val Ser Val Thr Cys Thr Ala Ser Gln Asn Val Gly Thr Phe

20 25 30

Val Val Trp Tyr Gln Arg Lys Ser Gly Gln Ser Pro Lys Ala Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Lys Ser

65 70 75 80

Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Asp Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

Claims

1.A double-antibody sandwich enzyme immunochromatography detection kit comprises an enzyme immunochromatography detection test strip, a sample treatment solution, a buffer solution and a buffer solution supply unit, and is characterized in that,

the buffer solution comprises phosphate buffer solution, macromolecular protein, tween-20, proclin300 and any one of carbamide peroxide, carbamide peroxide and hydrogen peroxide;

the phosphate buffer solution is a PBS solution with the pH value of 7.4.1M;

the macromolecular protein is BSA or OVA;

the concentration of the carbamide peroxide, the carbamide peroxide and the hydrogen peroxide is 0.02-0.2 percent by weight;

the enzyme immunochromatographic assay test strip comprises an enzyme label pad, an enzyme-labeled antibody is formed by adsorbing an enzyme-labeled antibody on the enzyme label pad, diluting the enzyme-labeled antibody by an enzyme-labeled antibody diluent and drying the enzyme-labeled antibody, wherein the enzyme-labeled antibody diluent comprises a PBS (phosphate-buffered saline) solution with the pH value of 7.4.1M and any one of 0.5 percent V/V PEG4000, PEG6000 or PEG 8000;

the enzyme immunochromatographic test strip comprises a detection line on which immobilized antibodies diluted with an immobilized antibody diluent comprising a pH 7.4.1M PBS solution, 0.5-V/V trehalose, 0.1-V Tween-20 are immobilized, and a quality control line;

monoclonal antibody in enzyme-labeled antibody and monoclonal antibody in immobilized antibody

Is any combination of porcine circovirus type 2 monoclonal antibody 3G12 secreted by mouse hybridoma cell 3G12 with CCTCC No. C2014198 and porcine circovirus type 2 monoclonal antibody 2F8 secreted by mouse hybridoma cell 2F8 with CCTCC No. C2014199; or

The porcine epidemic diarrhea virus monoclonal antibody PEDV-McAB2 and the porcine epidemic diarrhea virus monoclonal antibody PEDV-McAB1 are combined randomly, the heavy chain variable region of the monoclonal antibody PEDV-McAB2 is shown in SEQ ID No.1, the light chain variable region is shown in SEQ ID No.2, the heavy chain variable region of the monoclonal antibody PEDV-McAB1 is shown in SEQ ID No.3, and the light chain variable region is shown in SEQ ID No. 4; or

The antibody is any combination of a swine transmissible gastroenteritis virus monoclonal antibody TGEV-3D2 and a swine transmissible gastroenteritis virus monoclonal antibody TGEV-4B4, wherein a heavy chain variable region of the monoclonal antibody TGEV-3D2 is shown in SEQ ID No.5, a light chain variable region is shown in SEQ ID No.6, a heavy chain variable region of the monoclonal antibody TGEV-4B4 is shown in SEQ ID No.7, and a light chain variable region is shown in SEQ ID No. 8; or

The monoclonal antibody IV-McAB1 is any combination of an anti-influenza A virus nucleoprotein monoclonal antibody IV-McAB1 and an anti-influenza A virus nucleoprotein monoclonal antibody IV-McAB2, the heavy chain variable region of the monoclonal antibody IV-McAB1 is shown in SEQ ID No.9, the light chain variable region is shown in SEQ ID No.10, the heavy chain variable region of the monoclonal antibody IV-McAB2 is shown in SEQ ID No.11, and the light chain variable region is shown in SEQ ID No. 12; or

Is any combination of a canine parvovirus monoclonal antibody CPV-10B11 secreted by a CCTCC No. C201578 mouse bone marrow hybridoma cell 10B11 strain and a canine parvovirus monoclonal antibody CPV-10H4 secreted by a CCTCC No. C201579 mouse bone marrow hybridoma cell 10H4 strain; or

Is CCTCC No: monoclonal antibodies CDV-1G5 and CCTCC No of canine distemper virus secreted by C2015201 mouse bone marrow hybridoma cell 1G 5: any combination of canine distemper virus monoclonal antibodies CDV-6E11 secreted by C2015202 mouse bone marrow hybridoma cell 6E11 strain; or alternatively

Is any combination of a canine adenovirus monoclonal antibody CAV-5G4 and a canine adenovirus monoclonal antibody CAV-1A1, wherein the heavy chain variable region of the monoclonal antibody CAV-5G4 is shown by SEQ ID No.13, the light chain variable region is shown by SEQ ID No.14, the heavy chain variable region of the monoclonal antibody CAV-1A1 is shown by SEQ ID No.15, and the light chain variable region is shown by SEQ ID No. 16.

2. The enzyme immunochromatography detection kit according to claim 1, wherein the concentration of urea hydrogen peroxide, urea hydrogen peroxide and hydrogen peroxide is 0.05% to 0.1% w/V.

3. The enzyme immunochromatographic assay kit of claim 1, wherein the buffer solution comprises pH7.4.0.1M PBS solution, 0.1% -0.5% V/V Tween-20, 0.02% V/V Proclin300, 0.05% V/V carbamide peroxide, and 0.5% -1%W/V BSA or OVA.

4. The enzyme immunochromatographic assay kit according to claim 1, wherein the substrate pad diluent comprises 2%V/V absolute ethanol, 0.1% V/VPEG4000, 0.1M PBS at ph7.4.

5. The enzyme immunochromatographic assay kit according to claim 1, wherein the enzyme-labeled antibody diluent comprises 0.5% V/VPEG4000, 0.1M PBS, pH7.4.

6. The enzyme immunochromatographic assay kit according to claim 1, comprising an assay strip, a sample treatment solution, a buffer solution and a buffer solution supply unit, wherein,

the detection test strip sequentially comprises a substrate supply area, a sample supply area and a detection area from the longitudinal direction; the substrate supply region comprises the substrate pad having adsorbed thereon a dried enzyme substrate which is tetramethylbenzidine; the sample supply area comprises the enzyme label pad, an enzyme-labeled antibody is adsorbed on the enzyme label pad, and the enzyme substrate can generate a color reaction with enzyme on the enzyme-labeled antibody; the detection area comprises the detection line and the quality control line, and goat anti-mouse polyclonal antibody or goat anti-mouse secondary antibody is immobilized on the quality control line;

the buffer solution supply unit is used for supplying the buffer solution to the substrate supply area of the detection test strip and enabling the buffer solution, the sample processing solution diffused along with the buffer solution, the enzyme substrate and the enzyme-labeled antibody to migrate towards one end far away from the substrate supply area along the longitudinal direction of the detection test strip.

7. The enzyme immunochromatographic assay kit according to claim 6, wherein the concentration of the enzyme-labeled antibody dissolved in the enzyme-labeled antibody diluent is 0.5 to 10 μ g/ml, the concentration of the immobilized antibody dissolved in the immobilized antibody diluent is 0.5 to 1mg/ml, the concentration of the goat-anti-mouse polyclonal or goat-anti-mouse secondary antibody dissolved in the phosphate buffer or the immobilized antibody diluent is 1 to 3mg/ml, and the final concentration of the enzyme substrate dissolved in the substrate pad diluent is 0.5 to 4%W/V.

8. The enzyme immunochromatographic assay kit according to claim 6, wherein the distance between the detection line and the quality control line is not less than 5mm.

9. The enzyme immunochromatographic detection kit according to claim 1, wherein said sample treatment solution comprises a PBS solution of ph 7.4.4.1M, CHAPS, saponin and Proclin300.

10. The enzyme immunochromatographic detection kit of claim 9, wherein the sample treatment solution comprises pH 7.4.0.1M PBS solution, 0.5% -2.0% W/V CHAPS, 0.5% -2.0% W/V saponin and 0.02% V/V Proclin300.

11. The enzyme immunochromatographic assay kit of claim 10, wherein the sample treatment solution comprises pH7.4.0.1M PBS solution, 0.5% W/V CHAPS, 1%W/V saponin, 0.02% V/V Proclin300.

12. The enzyme immunochromatographic detection kit according to claim 9, wherein the amount of dissolution of a solid sample into the sample treatment liquid is 0.08 to 0.5g/500 to 1500 μ l, and the amount of dissolution of a liquid sample into the sample treatment liquid is 500 to 1500 μ l/500 to 1500 μ l; the detection dosage of the sample dissolved by the sample treatment solution is 50-150 mul;

the sample is selected from the group consisting of tissue, serum, anal secretions, pharyngeal secretions, ocular and nasal secretions, and viral cultures.

13. Use of a sample treatment fluid comprising a PBS solution at ph 7.4.1M, CHAPS, saponin and Proclin300 for the preparation of a kit according to any of claims 1-12.

14. The use of claim 13, wherein the sample processing solution comprises a PBS solution at ph 7.4.0.1M, 0.5% to 2.0% w/V CHAPS, 0.5% to 2.0% w/V saponin, and 0.02% V/V Proclin300.

15. The use of claim 14, wherein said sample treatment solution comprises a PBS solution at ph 7.4.0.1M, 0.5% w/V CHAPS, 1%W/V saponin, 0.02% V/V Proclin300.

16. The use according to claim 13, wherein the sample is selected from the group consisting of tissue, serum, anal secretions, pharyngeal secretions, ocular nasal secretions and viral cultures.

17. A method of making a kit according to any one of claims 1 to 12, comprising:

preparing a buffer solution;

step (5), preparing a sample treatment solution; and