Amantadine hapten, amantadine antigen, preparation method, amantadine monoclonal antibody and amantadine detection kit

Abstract

The present invention provides an amantadine hapten A or B. The amantadine antigen A or B is gradually prepared by the preparation method of the present invention. Then, the anti-amantadine monoclonal antibody 3G7 is obtained. The amantadine ELISA detection kit and the amantadine biochip detection kit prepared by using the above components can detect amantadine in a variety of samples with high sensitivity and high selectivity, the sensitivity is higher than the minimum detection limit of the national standard detection method, and the kit has the characteristics of being fast, simple and inexpensive.

Description

Technical Field

[0001] The present invention relates to an amantadine hapten, an amantadine antigen, a preparation method thereof, and hybridoma cells and monoclonal antibodies for immune screening thereof, as well as an amantadine ELISA detection kit and an amantadine biochip detection kit prepared by using the amantadine antigen and the monoclonal antibody, and the preparation method and application thereof, belonging to the field of biotechnology. Background Art

[0002] Amantadine belongs to the tricyclic amine class and is the earliest antiviral drug used to treat influenza. The United States approved it as a preventive drug in 1966 and confirmed it as a therapeutic drug on the basis of preventive drugs in 1976. In livestock and poultry farming, it is mainly used to treat and prevent avian influenza in chickens and infectious gastroenteritis in pigs. However, long-term use of amantadine can cause toxic side effects such as drowsiness, dizziness, depression, and nausea.

[0003] Traditional methods for detecting adamantane drug residues mainly include high performance liquid chromatography-mass spectrometry, gas chromatography, high performance liquid chromatography, gas chromatography-mass spectrometry, etc. However, these methods cannot meet the requirements of fast and accurate on-site detection because they require complex pretreatment processes, expensive instruments and equipment, and professional operators.

[0004] Immunoassay can make up for the shortcomings of the above detection methods. Immunoassay is an analytical method based on the reaction of antigen-antibody specific binding to detect various substances (such as drugs, hormones, proteins, microorganisms, etc.). It has the characteristics of high sensitivity and high selectivity, and its detection cost is low, fast and accurate, and it is suitable for rapid screening of a large number of samples on site. However, as a banned drug, there are few products with a sensitivity higher than the minimum detection limit (1ppb) of the national standard detection method in the existing literature reports on immunoassay methods and market products. Therefore, it is urgent to establish a faster, simpler, cheaper, more sensitive and specific amantadine screening method. Summary of the invention

[0005] In order to solve the deficiencies of the prior art, the present invention provides an amantadine hapten, which can be used to prepare an amantadine antigen.

[0006] The invention also provides an amantadine antigen, which can be used to prepare a monoclonal antibody that reacts with amantadine in an antigen-antibody manner, and can be used as a coating source for preparing an amantadine detection kit.

[0007] The present invention also provides a monoclonal antibody capable of binding to amantadine with high sensitivity and high specificity.

[0008] The present invention also provides a variable region sequence capable of binding to amantadine with high sensitivity and high specificity.

[0009] The present invention also provides antibodies and antibody fragments capable of binding to amantadine with high sensitivity and high specificity.

[0010] The present invention also provides a hybridoma cell strain capable of secreting a monoclonal antibody that binds to adamantane with high sensitivity and high specificity.

[0011] The invention also provides an amantadine ELISA detection kit capable of detecting amantadine with high sensitivity and high specificity.

[0012] The present invention also provides an amantadine ELISA detection kit for detecting amantadine, the sensitivity of which is higher than the lowest detection limit (1 ppb) of the national standard detection method.

[0013] The invention also provides an amantadine biochip detection kit capable of detecting amantadine with high sensitivity and high specificity.

[0014] The present invention also provides an amantadine biochip detection kit for detecting amantadine with a sensitivity higher than the lowest detection limit (1 ppb) of the national standard detection method. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is the mass spectrum of adamantane hapten A;

[0016] Figure 2 is the mass spectrum of adamantane hapten B;

[0017] Figure 3 Maldi-Tof-Ms for bovine serum albumin BSA;

[0018] Figure 4 Maldi-Tof-Ms for adamantane antigen A;

[0019] Figure 5 Maldi-Tof-Ms for adamantane antigen B;

[0020] Figure 6 The standard curve of amantadine in the amantadine ELISA detection kit prepared in Example 4;

[0021] Figure 7 The figure shows the spotting pattern of each chip well on the chip in the amantadine biochip detection kit. Figure 7 7A, 7B, 7C, 7D, and 7E in the figure respectively show different spotting modes, and a partial enlargement thereof shows a detection chip of a subunit of an adamantane detection chip. Figure 7 7F, 7G, and 7H are kits with one, three, and multiple antibody detection chip subunits, respectively;

[0022] Figure 8It is a built-in standard track for the backend website of the Amantadine Biochip Detection Kit.

[0023] Reference numerals

[0024] 1 is the quality control point 1, 2 is the quality control point 2, 3 is the quality control point 3, 4 is the blank control sampling point, and 5 is the adamantane antigen detection point. DETAILED DESCRIPTION

[0025] definition

[0026] The term "adamantanamine" is a molecular formula C 10 H 17 N, Chinese alias: Adamantane, tricycloalkyneamine, 1-aminoadamantane, 1-adamantaneamine, 1-aminoadamantane, English alias 1-Adamantanamine, 1-Aminoadamantane, Symadine, Symmetrel, Tricyclo[3.3.1.1(3,7)]decane-1-amine, adamantan-1-amine.

[0027] When connecting hapten to carrier protein, the carrier proteins used include but are not limited to OVA, BSA, KLH, BGG, RSA, PSA, LPH, HAS and PLL. Among them, BSA is bovine serum albumin, which is the most stable and soluble albumin with a molecular weight of 67×10 3 Da (containing 59 Lys), with approximately 30-35 main amino groups available for conjugation with linkers, making BSA a very popular carrier protein for weak antigenic compounds. OVA is chicken ovalbumin, with a molecular weight of 45×10 3 Da is generally used as a second carrier protein to verify whether the antibody is specific to the polypeptide rather than the carrier protein (such as BSA). KLH is hemocyanin (Keyhole Limpet Hemocyanin), a free blue respiratory pigment found in the hemolymph of some mollusks and arthropods (spiders and beetles). Hemocyanin contains two copper ions directly connected to the polypeptide chain. Similar to iron-containing hemoglobin, it is easy to bind to oxygen and also easy to dissociate from oxygen. It is the only known copper protein that can reversibly bind to oxygen. It is cyan when oxidized and white when reduced. Its molecular weight is 450,000 to 130,000.

[0028] The term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies constituting the population are identical except for possible spontaneous mutations that may occur in small amounts. Therefore, the modifier "monoclonal" refers to the nature of the antibody as not being a mixture of discrete antibodies. Preferably, the monoclonal antibody includes monovalent or single-chain antibodies, double-chain antibodies, chimeric antibodies, humanized antibodies, derivatives, functional equivalents and homologs of the above antibodies, as well as antibody fragments and any polypeptide containing an antigen binding domain. Antibodies are any specific binding factors covering a binding domain with the desired specificity, and thus, this term covers antibody fragments, derivatives, humanized antibodies, functional equivalents and homologs of antibodies homologous thereto, and also any polypeptide containing an antigen binding domain, whether natural or synthetically produced. Examples of antibodies are immunoglobulin subtypes (such as IgG, IgE, IgM, IgD and IgA) and their subtypes and subclasses; they may also be fragments containing an antigen binding domain such as Fab, scFv, Fv, dAb, Fd; and double-chain antibodies (diabodies). Chimeric molecules or equivalents comprising an antigen binding domain fused to another polypeptide are also included. The cloning and expression of chimeric antibodies are described in EP.A.0120694 and EP.A.0125023. Antibodies can be modified in many ways, and DNA recombinant technology can be used to produce other antibodies or chimeric molecules that retain the specificity of the original antibody. This technology can include introducing DNA encoding the immunoglobulin variable region or complementarity determining region (CDRs) of the antibody into the constant region or constant region plus framework region of different immunoglobulins, see EP.A.184187, GB2188638A or EP.A.239400. Hybridoma cells or other cells that produce antibodies can also be subjected to genetic mutations or other changes, which can change or not change the binding specificity of the produced antibody. The "monoclonal antibody" used in the present invention can also be prepared by the hybridoma method, because the DNA sequence encoding the murinized antibody of the present invention can be obtained by conventional means well known to those skilled in the art, such as artificially synthesizing a nucleotide sequence according to the amino acid sequence disclosed in the present invention or amplifying it by PCR, and thus can also be obtained by recombinant DNA methods, and the sequence can be linked to a suitable expression vector by various methods well known in the art. Finally, the transformed host cells are cultured under conditions suitable for the expression of the antibody of the present invention, and then the monoclonal antibody of the present invention is purified by conventional separation and purification means well known to those skilled in the art. Antibodies contain a geometric body of polypeptide chains connected together by disulfide bridges, and the two polypeptide main chains called light chains and heavy chains constitute all the main structural categories (isotypes) of antibodies. Both heavy chains and light chains can be further divided into some subregions called variable regions and constant regions. The heavy chain includes a single variable region and three different constant regions, while the light chain includes a single variable region (different from the variable region of the heavy chain) and a single constant region (different from the constant region of the heavy chain). The variable regions of the heavy and light chains are responsible for the binding specificity of the antibody.

[0029] The term "heavy chain variable region" refers to a polypeptide having a length of 110 to 125 amino acids, and its amino acid sequence corresponds to the heavy chain amino acid sequence of the monoclonal antibody of the present invention starting from the N-terminal amino acid of the heavy chain. Similarly, the term "light chain variable region" refers to a polypeptide having a length of 95 to 115 amino acids, and its amino acid sequence corresponds to the light chain amino acid sequence of the monoclonal antibody of the present invention starting from the N-terminal amino acid of the light chain. It is obvious to a person of ordinary skill in the art that, based on the amino acid sequences of the heavy chain variable region and light chain variable region of the monoclonal antibody specifically disclosed in the present invention, one or more amino acids can be added, deleted, replaced, etc. by conventional genetic engineering and protein engineering methods to obtain conservative variants, while still being able to maintain specific binding to canine rotavirus. The monoclonal antibodies in the present invention also include active fragments or conservative variants thereof.

[0030] The term "conservative variant" refers to a variant that has substantially retained the characteristics of its parent, such as basic immunological biological characteristics, structural characteristics, regulatory characteristics or biochemical characteristics. Generally, the amino acid sequence of the conservative variant of a polypeptide is different from that of the parent polypeptide, but the difference is limited, so that the sequence of the parent polypeptide is very similar to the conservative variant overall, and is identical in many regions. The difference in the conservative variant and the parent polypeptide amino acid sequence can be, for example, replacement, addition and deletion of one or more amino acid residues and any combination thereof. The replaced or inserted amino acid residues may or may not be encoded by the genetic code. The conservative variant of a polypeptide can occur naturally, or it can be a non-naturally occurring variant. The non-naturally occurring conservative variant of a polypeptide can be produced by mutagenesis techniques or direct synthesis.

[0031] The term "IC 50 " refers to the half-inhibitory concentration (or half-inhibitory rate). It is a very important data in the standard curve of indirect competitive ELISA (abbreviated as ic-ELISA). In ic-ELISA, the OD value of the control well without adding inhibitory substances is B0, and the OD value of the well with added inhibitory substances is B. B / B0 is called the binding rate. The concentration of inhibitory substances corresponding to the binding rate of 50% is called IC 50 .

[0032] The term "ppb" is a concentration unit, 1 ppb = 1 / 1000 ppm = 1 μg / kg = 1 ng / mL = 1 ng / g.

[0033] The term "CV" refers to coefficient of variation, which is the standard deviation of the assay results expressed as a percentage of the mean. DETAILED DESCRIPTION OF THE INVENTION

[0035] The present invention provides an adamantane hapten A or B, which has a chemical structure as shown in structural formula (V) or structure (VII), respectively:

[0036]

[0037] The present invention also provides a method for preparing the adamantane hapten A, wherein the method comprises: step (1) weighing 2g-10g of 3-aminoadamantane-1-carboxylic acid and 1.92g-9.6g of Boc anhydride and adding them into a single-mouth bottle, adding 0.05g-0.26g of 4-dimethylaminopyridine, dissolving them with 30mL of tert-butyl alcohol, and reacting at 70-80°C for 5-8 hours; step (2) performing vacuum rotary evaporation on the liquid after the reaction to remove the solvent tert-butyl alcohol, thereby obtaining a large amount of light yellow oily matter, namely, crude compound 2; step (3) dissolving the crude compound 2 in ethyl acetate, washing twice with 0.1M sulfuric acid solution and pure water, collecting the organic phase, and performing vacuum rotary evaporation to obtain compound 2; step (4) dissolving 0.5g-2g of the compound 2 in 10mL of 80% methanol aqueous solution, adding 0.3g-1.3g EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and 0.25g-1.0g NHS (N-hydroxysuccinimide), react at 20-25°C for 1 hour to obtain a solution of intermediate compound 3; 0.9g-3.6g of 50% m / V aqueous solution of β-alanine is slowly added dropwise to the reaction solution, and the reaction is continued at 20-25°C overnight; step (5) the reaction solution is subjected to reduced pressure rotary evaporation to remove methanol and water, and then 30mL of water and 30mL of ethyl acetate are added again, stirred for 30 minutes, and then separated into layers, the organic phase is collected, and the crude compound 4 is obtained after rotary evaporation, and the crude compound 4 is obtained. Compound 4 is purified by column to obtain 0.5 g of its pure product; step (6) dissolving 0.2-1.0 g of the purified compound 4 in 1 mL of methanol, adding 0.5 mL of concentrated hydrochloric acid, stirring at 20-25° C. for 3-5 hours, adjusting the pH to 6 with 0.5 M sodium hydroxide solution, and then removing the solvent methanol and water by vacuum rotary evaporation to obtain 0.1 g-0.6 g of crude compound 5; and step (7) dissolving the crude compound 5 in methanol again, filtering to remove inorganic salts, and vacuum rotary evaporating the filtrate to obtain a relatively pure compound 5, and further separating and purifying using column silica gel to obtain the adamantane amine hapten A.

[0038] As an embodiment of the present invention, the method for preparing the adamantane hapten A of the present invention comprises: the step (1) weighing 5g of 3-aminoadamantane-1-carboxylic acid and 4.8g of Boc anhydride into a single-mouth bottle, adding 0.13g of 4-dimethylaminopyridine, dissolving with 30mL of tert-butanol, and reacting at 70-80°C for 5-8 hours; the step (2) vacuum-evaporating the liquid after the reaction to remove the solvent tert-butanol to obtain a large amount of light yellow oily substance, which is the crude compound 2; the step (3) dissolving the crude compound 2 in ethyl acetate, washing twice with 0.1M sulfuric acid solution and pure water, collecting the organic phase, and vacuum-evaporating to obtain the compound 2; the step (4) dissolving 1g of the compound 2 in 10mL of 80% methanol aqueous solution, adding 0.65g of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and 0.5g NHS (N-hydroxysuccinimide), react at 20-25°C for 1 hour to obtain a solution of intermediate compound 3; 1.8g of 50% m / V aqueous solution of β-alanine is slowly added dropwise to the reaction solution, and the reaction is continued at 20-25°C overnight; in the step (5), the methanol and water are removed from the reaction solution by vacuum rotary evaporation, and 30mL of water and 30mL of ethyl acetate are added again, and the mixture is stirred for 30 minutes and then separated, and the organic phase is collected and rotary evaporated to obtain a crude compound 4, and the crude compound 4 is purified by column to obtain 0.5g of its pure product ; In the step (6), 0.5 g of the crude compound 4 is dissolved in 1 mL of methanol, 0.5 mL of concentrated hydrochloric acid is added, and the mixture is stirred at 20-25° C. for 3-5 hours, the pH is adjusted to 6 with a 0.5 M sodium hydroxide solution, and then the solvent methanol and water are removed by rotary evaporation under reduced pressure to obtain 0.6 g of crude compound 5; and in the step (7), the crude compound 5 is dissolved in methanol again, and then the inorganic salt is removed by filtration. The filtrate is subjected to rotary evaporation under reduced pressure to obtain a relatively pure compound 5, and column silica gel is used to further separate and purify the compound to obtain the adamantane amine hapten A.

[0039] The present invention also provides a method for preparing the adamantane hapten B, wherein the method comprises: step (1) dissolving 1g-5g of adamantane amine and 0.98g-2.94g of glyoxylic acid in 20mL of dichloromethane, and stirring the mixture for reaction at room temperature for 24 hours; and step (2) subjecting the liquid after the reaction to vacuum rotary evaporation to remove dichloromethane, thereby obtaining a large amount of white solid compound, adding 20mL of 2M hydrochloric acid to dissolve the compound, refluxing the mixture at 100°C for 5-8 hours, and subjecting the mixture to vacuum rotary evaporation to remove water, thereby obtaining 0.5g-3.2g of the hapten B.

[0040] As an embodiment of the present invention, the method for preparing the adamantane amine hapten B of the present invention comprises: the step (1) dissolving 2g of adamantane amine and 1.96g of glyoxylic acid in 20mL of dichloromethane, and stirring the mixture at room temperature for 24 hours; and the step (2) vacuum rotary evaporating the liquid after the reaction to remove the dichloromethane to obtain a large amount of white solid compound, adding 20mL of 2M hydrochloric acid to dissolve the compound, refluxing at 100°C for 5 to 8 hours, and vacuum rotary evaporating the compound to remove water to obtain 1.6g of the hapten B.

[0041] The present invention also provides a method for preparing an adamantane antigen A or B, wherein the preparation method comprises: step (1) dissolving 4 mg to 16 mg of the adamantane hapten A or hapten B in 1 mL of DMF, adding 1.5 mg to 6.0 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 0.9 mg to 3.6 mg of N-hydroxysuccinimide (NHS), and reacting at 25° C. for 1 to 2 hours to form a solution 1; step (2) weighing 10 mg to 30 mg of BSA, OVA, KLH, BGG, RSA, PSA, LPH or HAS carrier protein and dissolving it in 1 mL of 0.01 M NaHCO3 buffer to form solution 2; step (3) slowly adding the solution 1 of the adamantane hapten A or B of step (1) to the solution 2 of step (2), stirring at 4°C for overnight reaction; and step (4) dialyzing the reaction solution of step (3) with 0.01M PBS solution at 4°C for 3 days, replacing the dialysate twice a day; centrifuging the dialysate and taking out the supernatant, which is the adamantane antigen A or B.

[0042] As an embodiment of the present invention, the preparation method of the adamantane antigen A or B of the present invention comprises: step (1) dissolving 4 mg of the adamantane hapten A or hapten B in 1 mL of DMF, adding 2.9 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 1.8 mg of N-hydroxysuccinimide (NHS), and reacting at 25° C. for 1 to 2 hours to form a solution 1; step (2) weighing 13.5 mg of OVA and dissolving it in 1 mL of 0.01 M NaHCO3 buffer to form a solution 2; step (3) slowly dropping the solution 1 of the adamantane hapten A or B in step (1) into the solution 2 in step (2), stirring and reacting at 4° C. overnight; and step (4) dialyzing the reaction solution in step (3) with 0.01 M PBS solution at 4° C. for 3 days, replacing the dialysate twice a day; after centrifuging the dialysate, taking out the supernatant, which is the adamantane antigen A or B.

[0043] The present invention also provides the amantadine antigen A or B prepared by the preparation method of the amantadine antigen A or B.

[0044] The present invention also provides an amantadine monoclonal antibody 3G7, wherein the heavy chain variable region of the monoclonal antibody 3G7 is encoded by SEQ.ID NO 1 or a degenerate sequence thereof, and the light chain variable region of the monoclonal antibody 3G7 is encoded by SEQ.ID NO 2 or a degenerate sequence thereof. The IC of the amantadine monoclonal antibody 3G7 to amantadine is 50 is 0.7~1.2, and the affinity is 1.52×10 9 L / mol, and has good reactivity with adamantane amine.

[0045] The present invention also relates to a variable region sequence of a monoclonal antibody 3G7 that specifically binds to amantadine, wherein the heavy chain variable region of the monoclonal antibody 3G7 is encoded by SEQ.ID No.1 or a degenerate sequence thereof; the light chain variable region of the monoclonal antibody 3G7 is encoded by SEQ.ID No.2 or a degenerate sequence thereof; wherein the amantadine monoclonal antibody 3G7 is obtained by screening after immunization with the amantadine antigen A.

[0046] The present invention also relates to a hybridoma cell strain 3G7, and the hybridoma cell strain 3G7 secretes the monoclonal antibody 3G7.

[0047] The present invention also provides an amantadine ELISA detection kit, wherein the amantadine ELISA detection kit comprises a support medium coated with the amantadine antigen A, an enzyme label working solution, an amantadine antibody working solution, and an amantadine standard solution; wherein the coating concentration of the amantadine antigen A is 0.10 μg / mL to 0.25 μg / mL; the enzyme label working solution is a solution of HRP-sheep anti-mouse IgG, preferably diluted at 1:5000 to 1:8000 V / V; the amantadine antibody working solution is a solution of the amantadine monoclonal antibody 3G7, and the content of the amantadine monoclonal antibody 3G7 is 0.04 μg / mL; the amantadine standard solution is prepared by mixing the amantadine standard with 0.01 M The PBS diluent is gradiently diluted to concentrations of 0, 0.2 ng / mL, 0.6 ng / mL, 1.8 ng / mL, 5.4 ng / mL, and 16.2 ng / mL; wherein the supporting medium is a microtiter plate, and the adamantane antigen A is coated with 100 μl / well; the enzyme label working solution also contains: 8 g sodium chloride, 2.9 g disodium hydrogen phosphate, 0.24 g potassium dihydrogen phosphate, 0.2 g potassium chloride, 200 mL of newborn calf serum, and 1 mL of proclin300 in 1 L of water; the adamantane antibody working solution also contains: 8 g sodium chloride, 2.9 g disodium hydrogen phosphate, 0.24 g potassium dihydrogen phosphate, 0.2 g potassium chloride, 10 g bovine serum albumin, and 1 mL of proclin300 in 1 L of water; the kit also includes a washing solution, a TMB color developing solution, a stop solution, an extractant, a purifying agent, and a sample diluent; the washing solution is 0.01 M containing 0.1% V / V Tween-20 PBS solution; the TMB color developing solution includes solution A and solution B, wherein solution A is a citric acid-sodium dihydrogen phosphate buffer solution with a pH value of 5, which contains 1.5g citric acid and 6.6g disodium hydrogen phosphate and 1.0g urea peroxide in 100mL water, and solution B is a citric acid-sodium dihydrogen phosphate buffer solution with 0.8% M / V 3,3,5,5-tetramethylbenzidine; the stop solution is a 2mol / L sulfuric acid solution; the extractant is acetonitrile; the purifying agent is n-hexane; and the sample diluent is a 0.01M PBS solution.

[0048] The present invention also provides an amantadine biochip detection kit, wherein the biochip detection kit comprises one or more amantadine chips, an antibody working solution, and an enzyme label working solution, wherein the amantadine chip is coated with the amantadine antigen A, a quality control product, and a blank control point; wherein the quality control product is mouse IgG, and the blank control point is a detection background value; the blank control point is a spotting point where 5% glycerol solution, 5% sorbitol solution, 0.05% Triton solution, DMSO, and PBS (pH 6.8) solution are uniformly mixed in a volume ratio of 10:15:0.1:50:100; the antibody working solution is a sample point containing the amantadine monoclonal antibody The invention relates to a solution of amantadine monoclonal antibody 3G7, wherein the concentration of the amantadine monoclonal antibody 3G7 is 0.06 μg / mL; the enzyme label working solution is a solution containing HRP-sheep anti-mouse IgG, and the HRP-sheep anti-mouse IgG is diluted at 1:200-1:2000 V / V; the coating amount of the amantadine antigen A is 0.6-1.0 ng / point; the coating amount of the mouse IgG is 1-4 ng / point; there are three quality control points in total, the coating amount of quality control point 1 (1) is greater than or equal to 1 ng / point and less than 2 ng / point; the coating amount of quality control point 2 (2) is 2 ng / point; the coating amount of quality control point 3 (3) is greater than 2 ng / point and less than or equal to 4 ng / point.

[0049] Compared with traditional ELISA kits, which are equipped with standard products as quality control products in the kit, the standard products have higher requirements, are difficult to prepare, and are expensive. The biochip detection kit of the present invention is improved by technology, and a quality control product is provided on the reaction carrier. The quality control product is commercialized mouse IgG, which greatly improves the convenience and accuracy of the kit operation. The present invention has found through research that mouse IgG within a certain content range can be used as a quality control product, without the need to set up negative controls and positive controls. The role of mouse IgG is to serve as a reaction control when there is no antibody reaction in a single detection well, which is used to evaluate the effectiveness of the test and reflect the reaction degree of the enzyme-labeled reagent when there is no blocking. Mouse IgG can be purchased commercially or prepared by conventional methods, and is easy to operate.

[0050] As an embodiment of the present invention, the coating amount of the adamantane antigen on the adamantane chip in the biochip detection kit is 0.6 ng / spot to 1.0 ng / spot, and the spotting volume is 20 nl / spot.

[0051] The coating amount of the adamantane antigen can be 0.60 ng / spot, 0.61 ng / spot, 0.62 ng / spot, 0.63 ng / spot, 0.64 ng / spot, 0.65 ng / spot, 0.66 ng / spot, 0.67 ng / spot, 0.68 ng / spot, 0.69 ng / spot, 0.70 ng / spot, 0.71 ng / spot, 0.72 ng / spot, 0.73 ng / spot, 0.74 ng / spot, 0.75 ng / spot, 0.76 ng / spot, 0.77 ng / spot, 0.78 ng / spot, 0.79 ng / spot, g / point, 0.80ng / point, 0.81ng / point, 0.82ng / point, 0.83ng / point, 0.84ng / point, 0.85ng / point, 0.86ng / point, 0.87ng / point, 0.88ng / point, 0.89ng / point, 0.90ng / point, 0.91ng / point, 0.92ng / point, 0.93ng / point, 0.94ng / point, 0.95ng / point, 0.96ng / point, 0.97ng / point, 0.98ng / point, 0.99ng / point, and 1.0ng / point.

[0052] The mouse IgG coating amount can be 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0 ng / spot.

[0053] As an embodiment of the present invention, the biochip detection kit also includes an extractant, a purifying agent, a TMB substrate solution, a washing solution, and a sample diluent; the washing solution is a 0.01M PBS solution containing 0.1% V / V Tween-20; the TMB color developing solution includes A solution and B solution, wherein A solution is a citric acid-sodium dihydrogen phosphate buffer solution with a pH value of 5, which contains 1.5g citric acid and 6.6g disodium hydrogen phosphate, 1.0g urea peroxide in 100mL water, and B solution is a citric acid-sodium dihydrogen phosphate buffer solution with 0.8% M / V 3,3,5,5-tetramethylbenzidine; the stop solution is a 2mol / L sulfuric acid solution; the extractant is acetonitrile; the purifying agent is n-hexane; and the sample diluent is a 0.01M PBS solution.

[0054] As an embodiment of the present invention, the coating amount of the quality control point 1 (1) is 1 ng / point; the coating amount of the quality control point 2 (2) is 2 ng / point; the coating amount of the quality control point 3 (3) is 4 ng / point; the spotting solution is spotted on the blank control spotting point (4) as a blank control, and the adamantane antigen A is spotted on the adamantane antigen detection point (5); the quality control point 1 (1), the quality control point 2 (2), the quality control point 3 (3), and the blank control spotting point (4) are respectively fixed at the upper left corner, lower left corner, lower right corner, and upper right corner of the detection chip, and the adamantane antigen detection point (5) is spotted at other positions of the detection chip, with a distance of ≥700 μm from the edge of any other point, and the quality control point 1 (1), the quality control point 2 (2), the quality control point 3 (3), and the blank control spotting point (4) are respectively fixed at the upper left corner, lower left corner, lower right corner, and upper right corner of the detection chip, and the adamantane antigen detection point (5) is spotted at other positions of the detection chip, with a distance of ≥700 μm from the edge of any other point, and the quality control point 1 (1), the quality control point 2 (2), the quality control point 3 (3), and the blank control spotting point (4) are respectively fixed at the upper left corner, lower left corner, lower right corner, and upper right corner of the detection chip. The shortest distance between the midpoint and the edge of the sample spot (4) and the adamantane antigen detection spot (5) is also ≥700 μm; the distance from the quality control spot 1 (1), the quality control spot 2 (2), the quality control spot 3 (3), and the blank control sample spot (4) to the upper edge and lower edge of the detection chip subunit is ≥8 mm, and the distance to the left edge and right edge of the detection chip subunit is ≥5 mm; the adamantane biochip kit comprises one or more adamantane biochip subunits, which are composed of a bottom, an upper grid, and an adamantane biochip clamped therebetween by the bottom and the upper grid to form an independent detection hole, wherein one detection hole is one adamantane biochip subunit, and when the antibody joint detection kit comprises multiple adamantane biochip subunits, each of the independent detection chip subunits is integrated into a microarray chip.

[0055] Beneficial effects of the present invention:

[0056] The ELISA kit and biochip kit containing the amantadine antigen and amantadine monoclonal antibody of the present invention overcome the problem of low detection limit in the prior art, avoid missed detection of weak positive samples and the occurrence of false negative phenomena, have the advantages of being fast, simple and accurate, and are more conducive to clinical promotion and application.

[0057] The ELISA kit and biochip kit of the present invention can detect multiple targets such as chicken, eggs, pig / beef / mutton, etc., and have high detection accuracy. In particular, the biochip kit can complete the detection within 30 minutes, and there is no need to re-establish the standard curve for complex calculations. The test results can be directly read through the built-in standard curve in the plate.

[0058] The amantadine hapten and antigen disclosed in the invention can be used as raw materials for enzyme-linked immunosorbent assay to obtain high-affinity antibodies, and the sensitivity of the ELISA kit and biochip kit prepared therefrom can reach 0.2 ng / mL.

[0059] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0060] The chemical reagents used in the examples of the present invention were all analytically pure and purchased from Sinopharm Group.

[0061] PBS is a phosphate buffered saline, and its preparation method is as follows: weigh 8.0g NaCl, 0.2g KCl, 1.44g Na2HPO4, and 0.24g KH2PO4 and dissolve them in 800mL distilled water, adjust the solution to 7.4 with HCl, and finally add distilled water to make up to 1L to obtain 0.01M PBS buffer.

[0062] The experimental methods described in the present invention are all conventional methods unless otherwise specified; the biological materials described can be obtained from commercial channels unless otherwise specified.

[0063] Example 1 Preparation and Identification of Amantadine Hapten

[0064] 1.1 Preparation of hapten A

[0065] The synthetic route of adamantane hapten A is as follows:

[0066]

[0067] The preparation method of hapten A is as follows:

[0068] (1) Weigh 5 g of 3-aminoadamantane-1-carboxylic acid (the structural formula is Formula I in the synthetic route) and 4.8 g of Boc anhydride into a single-necked bottle, add 0.13 g of 4-dimethylaminopyridine, dissolve it in 30 mL of tert-butanol, and react at 70-80° C. for 5-8 hours;

[0069] (2) The liquid after the reaction was evaporated under reduced pressure to remove the solvent tert-butyl alcohol, and a large amount of light yellow oil was obtained, which was the crude compound 2 (the structural formula was Formula II in the synthetic route);

[0070] (3) The crude compound 2 was dissolved in ethyl acetate, washed twice with 0.1 M sulfuric acid solution and pure water, and the organic phase was collected and evaporated under reduced pressure to obtain a relatively pure compound 2;

[0071] (4) Dissolve 1 g of relatively pure compound 2 in 10 mL of 80% methanol aqueous solution, add 0.65 g of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and 0.5 g of NHS (N-hydroxysuccinimide), and react at 20-25° C. for 1 hour to obtain intermediate compound 3 (structural formula is Formula III in the synthetic route); slowly add 1.8 g of β-alanine aqueous solution (50%) dropwise to the reaction solution, and continue to react at 20-25° C. overnight;

[0072] (5) The reaction solution was evaporated under reduced pressure to remove methanol and water, and 30 mL of water and 30 mL of ethyl acetate were added again. After stirring for 30 minutes, the layers were separated and the organic phase was collected and evaporated to obtain a crude compound 4 (the structural formula is Formula IV in the synthetic route). The crude compound 4 was purified by column to obtain 0.5 g of its pure product;

[0073] (6) 0.5 g of crude compound 4 was dissolved in 1 mL of methanol, 0.5 mL of concentrated hydrochloric acid was added, and the mixture was stirred at 20-25° C. for 3-5 hours. The pH was adjusted to 6 with 0.5 M sodium hydroxide solution, and then the solvent methanol and water were removed by rotary evaporation under reduced pressure to obtain 0.6 g of crude compound 5 (the structural formula is Formula V in the synthetic route);

[0074] (7) The crude compound 5 was dissolved in methanol again, and then the inorganic salt was removed by filtration. The filtrate was evaporated under reduced pressure to obtain a relatively pure compound 5. The compound 5 was further separated and purified using a silica gel column to obtain a pure adamantane amine hapten A (the structural formula is Formula V in the synthetic route).

[0075] 1.2 Preparation of hapten B

[0076] The synthetic route of adamantane hapten B is as follows:

[0077]

[0078] The steps of preparing hapten B are as follows:

[0079] 1) Dissolve 2 g of adamantane amine (structural formula is Formula VI in the synthetic route) and 1.96 g of glyoxylic acid in 20 mL of dichloromethane and stir at room temperature for 24 hours;

[0080] 2) The liquid after the reaction was evaporated under reduced pressure to remove dichloromethane, and a large amount of white solid compound was obtained. After adding 20 mL of 2M hydrochloric acid to dissolve, it was refluxed at 100° C. for 5 to 8 hours and then evaporated under reduced pressure to remove water, thereby obtaining 1.6 g of pure hapten B (the structural formula is Formula VII in the synthetic route).

[0081] 1.3 Identification of haptens A and B

[0082] The molecular weight of hapten A is 266.3. Figure 1It can be seen that hapten A showed a molecular weight [MH + ] = 265.27, which is consistent with the theoretical molecular weight of 266.3, and thus determined to be hapten A;

[0083] The molecular weight of hapten B is 209.29. Figure 2 It can be seen that hapten B has a molecular weight [MH + ]=208.4, which is consistent with the theoretical molecular weight of 209.29, and thus it is determined to be hapten B.

[0084] By the above method, the linker arms are connected to different positions of the adamantane amine, thereby preparing adamantane amine hapten A and hapten B with different structures, providing raw materials for the subsequent comparison of the immune effects of antigens prepared with different haptens.

[0085] Example 2 Preparation and Identification of Amantadine Antigen

[0086] 2.1 Preparation and identification of immunogens

[0087] 2.2.1 Preparation of immunogens

[0088] 4 mg of the adamantane hapten A or hapten B prepared in Example 1 was weighed and dissolved in 1 mL of DMF, 2.9 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 1.8 mg of N-hydroxysuccinimide (NHS) were added, and the mixture was reacted at 25° C. for 1 to 2 hours to form a solution 1; 20 mg of BSA was weighed and dissolved in 1 mL of 0.01 M NaHCO3 buffer to form a solution 2; the solution 1 was slowly added dropwise to the solution 2, and the mixture was stirred at 4° C. overnight; the reaction solution was dialyzed with 0.01 M PBS solution at 4° C. for 3 days, and the dialyzate was replaced twice a day; after the dialyzate was centrifuged, the supernatant was taken out, which was the adamantane immunogen, and the supernatant was sterile filtered and stored at -20° C.

[0089] 2.2.2 Identification of immunogens

[0090] The protein concentration was detected using a protein quantification kit such as the BCA kit (Biyuntian Biotechnology Co., Ltd.). The protein concentration of immunogen A was 8.5 mg / mL, and the protein concentration of immunogen B was 8.9 mg / mL;

[0091] The carrier protein BSA, the prepared immunogen A, and the immunogen B were identified by Maldi-Tof-Ms (see Figure 3 , 4, 5), the relative molecular masses of immunogens A and B were increased compared with the mass of carrier protein BSA, indicating that the antigen coupling was successful. The coupling ratio of hapten A to carrier protein was 10:1-14:1, and the coupling ratio of hapten B to carrier protein was 11:1-14:1.

[0092] 2.2 Preparation and identification of coating source

[0093] 2.2.1 Preparation of coating

[0094] Weigh 4 mg of adamantane hapten A or hapten B and dissolve it in 1 mL of DMF, add 2.9 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 1.8 mg of N-hydroxysuccinimide (NHS), and react at 25° C. for 1 to 2 hours to form solution 1; weigh 13.5 mg of OVA (ovalbumin) and dissolve it in 1 mL of 0.01 M NaHCO3 buffer to form solution 2; slowly drop solution 1 into solution 2, and stir overnight at 4° C.; dialyze the reaction solution with 0.01 M PBS solution at 4° C. for 3 days, and replace the dialysate twice a day; after centrifuging the dialysate, take out the supernatant, which is the coating source of adamantane, and store it at -20° C. after sterile filtration.

[0095] 2.2.2 Identification of Encapsulation

[0096] The protein concentration was detected using a protein quantification kit such as the BCA kit (Biyuntian Biotechnology Co., Ltd.). The protein concentration of coating original A was 5.3 mg / mL, and the protein concentration of coating original B was 4.8 mg / mL.

[0097] Example 3 Preparation and identification of amantadine monoclonal antibodies

[0098] 3.1 Animal immunization

[0099] The experiment selected healthy female Balb / C mice aged 6 to 8 weeks, with 6 mice in each group. The immunization procedure was as follows: the immunogen was diluted with 1×PBS for the first immunization, mixed with an equal volume of Freund's complete adjuvant and emulsified, and injected subcutaneously at multiple points on the back of the mouse's neck, and the mice were immunized at a dose of 50 μg / mouse; after the first immunization, the booster immunization was performed at an interval of 2 to 3 weeks, and the immunogen was diluted with 1×PBS and emulsified with an equal volume of Freund's incomplete adjuvant, and injected subcutaneously at multiple points on the back of the mouse's neck, and the mice were immunized at a dose of 50 μg / mouse. One week after the third immunization, blood was collected, and the titer and inhibition changes of the serum were monitored by indirect ELISA and indirect competitive ELISA, respectively. For details, see Example 3.2. One week after the fifth immunization, the immunogen diluted with physiological saline was used to impact immunize the mouse abdomen at a dose of 50 μg / mouse, and cell fusion was performed 3 days later.

[0100] 3.2 Evaluation of immune serum titer and inhibitory activity

[0101] 50 μl of blood was collected from the cheek of each mouse, and the serum was separated and collected, diluted 1000 times and then diluted 3 times, and then evaluated by indirect ELISA and indirect competitive ELISA, i.e., icELISA.

[0102] Indirect ELISA:

[0103] Step 1) Add PBS at 50 μl / well, and then add 50 μl of the serum sample diluted in multiples;

[0104] Step 2) Incubate at 37°C for 60 minutes;

[0105] Step 3) Washing: discard the liquid in the wells, add 250 μl 1× washing solution (add 1 mL Tween-20 to 1 L 0.01 M PBS solution, mix well to make the washing solution), repeat washing 4 to 5 times, and pat dry;

[0106] Step 4) Add 100 μl / well of enzyme labeling working solution (use enzyme labeling diluent to dilute HRP-goat anti-mouse IgG at 1:5000-1:8000 V / V. The preparation method of enzyme labeling diluent is as follows: take 8g of sodium chloride, 2.9g of disodium hydrogen phosphate, 0.24g of potassium dihydrogen phosphate, 0.2g of potassium chloride, 600mL of purified water, 200mL of newborn calf serum, and 1mL of proclin300, and make up to 1L with pure water after complete dissolution, and mix well to obtain enzyme labeling antibody diluent);

[0107] Step 5) Incubate at 37°C for 30 minutes;

[0108] Step 6) Washing: discard the liquid in the wells, add 250 μl of 1× washing solution to each well, repeat washing 4 to 5 times, and pat dry;

[0109] Step 7) color development: add 100 μl TMB color development solution (mix solution A and solution B at 1:1 V / V) and react at room temperature for 15 minutes;

[0110] Step 8) Stop, add 50 μl stop solution to each well and vortex gently to mix;

[0111] Step 9) Read the OD value and read the absorbance value B0 of each well at a wavelength of 450 nm or 450 / 630 nm using a microplate reader.

[0112] B0 ≥ 0.15 was used as the criterion for determining serum titer.

[0113] Indirect competitive ELISA (ic-ELISA):

[0114] Step 1) Add 30 ppb amantadine standard solution at 50 μl / well, and then add 50 μl of the serum sample diluted in multiples;

[0115] Step 2) Incubate at 37°C for 60 minutes;

[0116] Step 3) Washing: discard the liquid in the wells, add 250 μl 1× washing solution (add 1 mL Tween-20 to 1 L 0.01 M PBS solution, mix well to make the washing solution), repeat washing 4 to 5 times, and pat dry;

[0117] Step 4) Add 100 μl / well of enzyme labeling working solution (use enzyme labeling diluent to dilute HRP-goat anti-mouse IgG at 1:5000-1:8000 V / V. The preparation method of enzyme labeling diluent is as follows: take 8g of sodium chloride, 2.9g of disodium hydrogen phosphate, 0.24g of potassium dihydrogen phosphate, 0.2g of potassium chloride, 600mL of purified water, 200mL of newborn calf serum, and 1mL of proclin300, and make up to 1L with pure water after complete dissolution, and mix well to obtain enzyme labeling antibody diluent);

[0118] Step 5) Incubate at 37°C for 30 minutes;

[0119] Step 6) Washing: discard the liquid in the wells, add 250 μl of 1× washing solution to each well, repeat washing 4 to 5 times, and pat dry;

[0120] Step 7) color development: add 100 μl TMB color development solution (mix solution A and solution B at 1:1 V / V) and react at room temperature for 15 minutes;

[0121] Step 8) Stop, add 50 μl stop solution to each well and vortex gently to mix;

[0122] Step 9) Read the OD value and read the absorbance value B of each well at a wavelength of 450nm or 450 / 630nm using a microplate reader.

[0123] The determination of serum titer and inhibition detection are beneficial for real-time monitoring of the actual immune status of mice. The higher the titer, the better the immune effect of the immunogen; the stronger the inhibition, the better the reaction between the small molecule compound (adamantane) and the antibody, thus making it more likely to prepare highly sensitive antibodies.

[0124] Some test results of the mouse sera after the fourth booster immunization (see Table 1): the indirect ELISA method detected that the highest serum titer of mice immunized with immunogen A was 1:27000, and the highest serum titer of mice immunized with immunogen B was 1:27000; the indirect competitive ELISA method (ic-ELISA) detected that the inhibition rate of immunogen A was much higher than that of immunogen B (inhibition rate = 1-ic-ELISA / ELISA).

[0125] Table 1 Mouse serum titer and inhibitory evaluation

[0126]

[0127] In summary, the potency of immunogen A and immunogen B is basically the same, but the blocking effect of amantadine on mouse serum and antigen of immunogen A is more obvious, indicating that immunogen A prepared by amantadine hapten A is more suitable for immunizing animals. Subsequent experiments were carried out using coated antigen A prepared by hapten A in Example 2.2 and immunogen A prepared by Example 2.1.

[0128] 3.3 Screening of monoclonal antibodies

[0129] Mice with high titer and best inhibitory ability were selected, and spleen cells from mice were mixed with myeloma cells in logarithmic growth phase at a ratio of 1:5 to 1:10, and then cell fusion was performed within 1 minute using fusion agent (PEG1500) preheated at 37°C. Hybridoma cells were screened using ic-ELISA method, and after multiple screenings and subcloning, three hybridoma cell lines with good evaluation results, 1H4, 3G7, and 5B1, were obtained. Ascites was prepared separately, and three pure antibodies were obtained after purification by protein G chromatography column. The three monoclonal antibodies were identified by ic-ELISA.

[0130] 3.4 Preparation and identification of monoclonal antibodies

[0131] 3.4.1 Purity and protein content determination

[0132] The purified monoclonal antibodies 1H4, 3G7 and 5B1 were identified by SDS-PAGE gel electrophoresis. The results showed that the purity of the three monoclonal antibodies was no less than 85%.

[0133] The BCA protein quantification kit was used to quantify the content of the purified monoclonal antibodies. The results showed that the protein contents of monoclonal antibodies 1H4, 3G7, and 5B1 were 2.5 mg / mL, 1.8 mg / mL, and 2.2 mg / mL, respectively.

[0134] 3.4.2IC 50 Detection

[0135] The IC values ​​of monoclonal antibodies 1H4, 3G7, and 5B1 were detected by the indirect competitive ELISA method described in Example 3.2. 50The results (see Table 2) showed that the IC50 of the monoclonal antibody secreted by the hybridoma cell line 3G7 was the lowest, indicating that the small molecule compound had the highest inhibition rate on the antigen / antibody reaction and had the best detection sensitivity when used as the raw material of the kit. Therefore, the antibody secreted by the 3G7 cell line was used as the raw material for preparing the kit, and the half-inhibitory concentration of the monoclonal antibody secreted by the 3G7 cell line to AMA was determined to be 1.2 ng / mL, and the sensitivity was 0.2 ng / mL.

[0136] Table 2 Half inhibitory concentration (IC 50 ) determination

[0137]

[0138] 3.4.3 Specificity identification

[0139] The indirect ELISA method described in Example 3.2 was used to identify the specificity of monoclonal antibody 3G7 and amantadine drugs. The results (see Table 3) showed that the cross-reaction rate of monoclonal antibody 3G7 with amantadine was 100%, and the cross-reaction rate with rimantadine was ≤7%. There was no cross-reaction with other similar drugs such as acetaminophen, ribavirin, ofloxacin / chloramphenicol, tetracycline, quinolone metabolites and other antibiotics, indicating good specificity.

[0140] Table 3 Specificity determination of amantadine monoclonal antibody

[0141]

[0142] In summary, the monoclonal antibody secreted by hybridoma cell line 3G7 is not only more sensitive than the monoclonal antibodies of the other two cell lines, but also has good specificity, and is preferably used as the raw material of the kit.

[0143] 3.4.4 Identification of monoclonal antibody types and subtypes

[0144] The type and subtype of monoclonal antibody 3G7 were identified using a monoclonal antibody subtype identification kit. The results showed that the subtype of monoclonal antibody 3G7 was IgG1, and the light chain was κ chain.

[0145] 3.4.5 Affinity identification of monoclonal antibodies

[0146] The affinity of monoclonal antibodies was determined using the ic-ELISA method. The steps are as follows:

[0147] Step 1) The antigen plate was coated with 0.1 μg / mL of the coating source A prepared in Example 2.2, and then blocked with 1% OVA at 37° C. for 2 h and washed with PBS.

[0148] Step 2) In a microplate, a concentration gradient of 0.1 nmol / L to 1 μmol / L antigen PBS solution was established by limiting dilution method, and antibody solution (concentration ≤ 0.5 μmol / L) was added to make the total volume 100 μl.

[0149] Step 3) After incubation at room temperature for 30 minutes, add 90 μl of the reaction mixture to the microwells coated with the antigens. Add 10 μl of PBS to the microwells and incubate for 8 minutes, but the time should not exceed 10 minutes (the time should not be too long, as it will destroy the reaction equilibrium system in the mixture and the final experimental data will be inaccurate). Wash the antigen plate thoroughly.

[0150] Step 4) Add enzyme-labeled secondary antibody, incubate for 1 h, and wash with PBS.

[0151] Step 5) Add chromogenic substrate to develop color. After 30 minutes of color development, add stop solution to stop the reaction and read the OD value.

[0152] The measured OD values ​​were recorded and fitted curves were drawn in combination with the gradient dilution antigen concentrations. Finally, the point with half of the strongest signal was found. The inverse of the corresponding antigen concentration was the affinity constant Ka. The samples were measured three times in parallel, and the results were 1.52×10 9 L / mol, 1.46×10 9 L / mol, 1.58×10 9 L / mol, the results were averaged, and the affinity was 1.52×10 9 L / mol.

[0153] 3.4.6 Monoclonal Antibody Variable Region Sequencing

[0154] According to the sequence characteristics of mouse monoclonal antibodies, the heavy chain variable region primer sequences were designed:

[0155] P1: 5'-ACTAGTTGACGTGGTCTCTAGGGTCACTTTAGTTTCCT-3'P2: 5'-CGGAAGCTTCCAGCGRCCARKCCATATACIGRTGG-3'

[0156] Design light chain variable region primer sequences:

[0157] P3: 5'-GCCATCTCATGRAGWCATTKWCYCAAGTCTTT-3'

[0158] P4: 5'-CGGACGCTTACTGCCTGGTAAGAAGATGGA-3'

[0159] Hybridoma cell 3G7 was collected, RNA was extracted and reverse transcribed as a template, the variable region sequence was amplified using the above primers, the amplified product was purified and identified by agarose gel electrophoresis, and then sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for sequencing. Results: The gene sequence of the heavy chain variable region of monoclonal antibody 3G7 is shown in SEQ ID No.1, and the gene sequence of the light chain variable region is shown in SEQ ID No.2.

[0160] Example 4 Preparation of Amantadine ELISA Detection Kit and Establishment of Detection Method

[0161] 4.1 Preparation of Amantadine ELISA Detection Kit

[0162] Washing solution: Add 1 mL of Tween-20 to 1 L of 0.01 M PBS solution and mix well to obtain the washing solution.

[0163] Coating plate: dilute the coating source A prepared in Example 2.2 to 0.10-0.25 μg / mL with 0.05 M CBS buffer, add 100 μL per well, and coat at 4°C overnight or at 37°C for 1-3 hours; wash with 0.01 M PBST washing solution, add 250 μL per well, wash 4 times, add 1% OVA solution to block at 37°C for 2 hours, wash 4 times with 250 μL / well 0.01 M PBST washing solution, pat dry, dry at 25°C for 6-8 hours, and then vacuum pack in aluminum foil bag.

[0164] Enzyme labeling working solution: dilute HRP-goat anti-mouse IgG with enzyme labeling diluent at 1:5000-1:8000 V / V. The preparation method of enzyme labeling diluent is as follows: take 8g sodium chloride, 2.9g disodium hydrogen phosphate, 0.24g potassium dihydrogen phosphate, 0.2g potassium chloride, 600mL purified water, 200mL newborn calf serum, 1mL proclin300, and make up to 1L with pure water after complete dissolution. Mix well to obtain enzyme labeling antibody diluent.

[0165] Amantadine antibody working solution: dilute the purified amantadine monoclonal antibody 3G7 in Example 3 to 0.04 μg / mL with antibody diluent. The antibody diluent is prepared as follows: 8 g of sodium chloride, 2.9 g of disodium hydrogen phosphate, 0.24 g of potassium dihydrogen phosphate, 0.2 g of potassium chloride, 600 mL of purified water, 10 g of bovine serum albumin, and 1 mL of proclin300. After completely dissolving, dilute to 1 L with pure water, and mix to obtain the antibody diluent.

[0166] Amantadine standard solution: dilute the amantadine standard with 0.01 M PBS diluent in a gradient manner to 0, 0.2 ng / mL, 0.6 ng / mL, 1.8 ng / mL, 5.4 ng / mL, and 16.2 ng / mL.

[0167] TMB color developing solution: includes solution A and solution B, wherein solution A is a buffer solution of citric acid-sodium dihydrogen phosphate with a pH value of 5 (1.5g citric acid and 6.6g disodium hydrogen phosphate are dissolved in 100mL pure water, and then 1.0g urea peroxide is added), and solution B is a buffer solution of citric acid-sodium dihydrogen phosphate with a concentration of 0.8% M / V 3,3,5,5-tetramethylbenzidine.

[0168] Stop solution: 2 mol / L sulfuric acid solution.

[0169] Extraction solvent: acetonitrile (analytical grade).

[0170] Purification agent: n-hexane (analytical grade).

[0171] Sample diluent: 0.01M PBS solution.

[0172] The above components are assembled into a kit, namely, an amantadine ELISA detection kit, referred to as kit 1.

[0173] 4.2 Establishment of the detection method of the amantadine ELISA detection kit

[0174] The samples were pre-treated before testing. The pre-treatment method was as follows:

[0175] Homogenize 3.0±0.03g of sample (such as chicken, fish, chicken liver, eggs, pork, beef, mutton, etc.) and add it to a 50mL centrifuge tube, add 9ml of extractant, mix immediately to shake the sample, vortex for 5 minutes, and centrifuge at 4000 rpm for 5 minutes; take 4mL of supernatant into a clean centrifuge tube, blow dry with nitrogen at 50℃~60℃, add 2ml of purification agent and vortex for 1 minute to mix; add 660μl of sample diluent, vortex for 1 minute, and centrifuge at 4000 rpm for 5 minutes; discard the supernatant and middle layer impurities, leaving the clarified liquid in the test tube to obtain the processed sample.

[0176] ELISA test kit: The test method of the amantadine ELISA test kit includes:

[0177] Step 1) Return the temperature to room temperature (25°C) for 1 to 2 hours;

[0178] Step 2) Add the treated sample or each standard solution to each well at 50 μl / well, then add 25 μl of enzyme label working solution and 25 μl of antibody working solution;

[0179] Step 3) Incubate at room temperature for 25 minutes;

[0180] Step 4) Washing: discard the liquid in the wells, add 250 μl 1× washing solution to each well, repeat washing 4 to 5 times, and pat dry;

[0181] Step 5) color development: add 100 μl TMB color development solution (mix solution A and solution B at 1:1 V / V) and react at room temperature for 15 minutes;

[0182] Step 6) Stop, add 50 μl of stop solution to each well and vortex gently to mix;

[0183] Step 7) Read the OD value and read the absorbance value B of each well at a wavelength of 450nm or 450 / 630nm using a microplate reader.

[0184] Divide the absorbance value B of each standard concentration by the absorbance value B0 of the 0 standard concentration, multiply by 100%, and obtain the relative absorbance B / B0. Draw a standard curve with the logarithm of the concentration (ng / mL) of the amantadine standard as the abscissa and B / B0 as the ordinate, as shown in the following figure: Figure 6 As shown; then calculate B / B0% according to each sample well, the residual concentration of adamantane in the sample can be calculated from the standard curve, and then multiplied by the dilution factor of the sample 0.66 to obtain the actual residual concentration of adamantane in the sample.

[0185] 4.3ELISA test kit

[0186] Optimization of the coating concentration of the coating source (coating source A prepared in Example 2.2) in the amantadine ELISA detection kit

[0187] The amantadine coating source A was prepared into 6 gradient amounts: 0.01 μg / mL, 0.05 μg / mL, 0.10 μg / mL, 0.15 μg / mL, 0.20 μg / mL, and 0.25 μg / mL. The ELISA plate was prepared according to the coating conditions of Example 4.1. The antibody concentrations were 0.01 μg / mL, 0.02 μg / mL, 0.04 μg / mL, and 0.06 μg / mL. The absorbance values ​​B0 at different antigen / antibody concentrations were detected according to Example 4.2. The OD value ranged from 1.5 to 2.0 as the optimal coating source concentration. The absorbance B was measured to confirm that the preferred coating concentration of the amantadine coating source A was 0.10 μg / mL to 0.20 μg / mL. The specific data are shown in Table 4:

[0188] Table 4 Optimization of coating concentration of amantadine coating original A

[0189]

[0190] Example 5 Preparation of Amantadine Biochip Detection Kit and Establishment of Detection Method

[0191] 5.1 Preparation of Amantadine Biochip Detection Kit

[0192] Preparation of spotting solution:

[0193] Preparation of 5% glycerol solution: accurately weigh 5.00g glycerol and place it in a 100mL volumetric flask, add a small amount of purified water and gently rotate it to dissolve it fully, avoiding excessive bubbles, then add purified water to the scale line, turn it upside down and shake it 10 times, and set aside;

[0194] Preparation of 5% sorbitol solution: accurately weigh 5.00g of sorbitol and place it in a 250mL beaker, add an appropriate amount of purified water and stir to completely dissolve it, then transfer it completely to a 100mL volumetric flask, add purified water to the scale line, shake it upside down 10 times, and set aside;

[0195] Preparation of 0.05% Triton solution: Use a pipette to measure 50 μl of Triton and place it in a 100 mL volumetric flask. Add an appropriate amount of purified water to completely dissolve it. Add purified water to the scale mark, shake it upside down 10 times, and set aside.

[0196] DMSO solution: Use DMSO reagent directly;

[0197] Preparation of PBS (pH 6.8) solution: first prepare 0.2 mol / L disodium hydrogen phosphate solution and 0.3 mol / L sodium dihydrogen phosphate solution, and then mix the two in a volume ratio of 49:51 to obtain a phosphate buffer solution with a pH value of 6.8;

[0198] The above solutions were mixed evenly in a volume ratio of 10:15:0.1:50:100 to prepare the spotting solution.

[0199] Amantadine chip: The amantadine coating original A prepared in Example 2 was diluted to 0.03 mg / mL to 0.04 mg / mL with a spotting solution, which was used as the detection point amantadine spotting solution; mouse IgG was diluted to 50 μg / ml, 100 μg / ml, and 200 μg / ml with a spotting solution, respectively, as the quality control point spotting solution, and the samples were spotted on Figure 7 The quality control sample points 1, 2, and 3 are set with 3 concentrations to facilitate the selection of the corresponding quality control points for data statistical analysis according to the debugging results; the spotting solution is used as the blank control spotting solution and is spotted at the blank control spotting point 4. Turn on the spotting instrument, set the program and spotting parameters, 20nl / point, spot the test point adamantane spotting solution at adamantane antigen test point 5, and the quality control point spotting solution at Figure 7 The quality control samples are spotted at points 1, 2, and 3, and the blank control spotting solution is spotted at Figure 7 The blank control spot 4 is placed in the middle of the chip bottom plate, and the cover plate is added to press it tightly, and the two sides are fixed with side strips to assemble into an amantadine chip, which is packaged and stored at 2-8°C.

[0200] The coating amount of mouse IgG is 1-4 ng / point; the quality control points are three points (corresponding to Figure 7 1, 2, and 3), the coating amount of control point 1 is greater than or equal to 1 ng / point and less than 2 ng / point; the coating amount of control point 2 is 2 ng / point; the coating amount of control point 3 is greater than 2 ng / point and less than or equal to 4 ng / point.

[0201] Among the above points, except for the quality control points 1, 2, 3, and the blank control spot 4, which are respectively fixed at the upper left corner, lower left corner, lower right corner, and upper right corner of the detection chip, the adamantane antigen detection point 5 can be spotted at any other position of the detection chip, as long as the distance from the edge of other points is ≥700μm, and the shortest distance between any points is also ≥700μm. The distance from the quality control point 1, the quality control point 2, the quality control point 3, and the blank control spot 4 to the upper and lower edges of the detection chip subunit is ≥8mm, and the distance from the left and right edges of the detection chip subunit is ≥5mm.

[0202] The adamantane biochip detection kit comprises one or more adamantane chip subunits, which are composed of a bottom end, an upper grid, and a detection chip arranged therebetween and clamped by the bottom end and the upper grid to form an independent detection hole. Each detection hole corresponds to one of the detection chip subunits. When the antibody joint detection kit comprises multiple adamantane chip subunits, each of the independent detection chip subunits is integrated into a microarray chip. Figure 7 Kits with different numbers of amantadine chip subunits are shown. Figure 7 7F, 7G, and 7H are test kits with one, three, and multiple antibody detection chip subunits, respectively, and the partial enlarged image thereof shows the spotting points on the detection chip in an adamantane chip subunit.

[0203] Enzyme labeling working solution: dilute HRP-goat anti-mouse IgG with enzyme labeling diluent at 1:200 to 1:2000 V / V.

[0204] Antibody working solution: dilute the amantadine antibody to 0.06 μg / mL.

[0205] TMB substrate solution: TMB (3,3',5,5'-tetramethylbenzidine) solution, commercial product, sterile packaging.

[0206] Washing solution: Add 1 mL of Tween-20 to 1 L of 0.01 M PBS solution and mix well to obtain the washing solution.

[0207] Extraction solvent: acetonitrile (analytical grade).

[0208] Purification agent: n-hexane (analytical grade).

[0209] Sample diluent: 0.01M PBS solution.

[0210] When preparing the adamantane biochip, 4 quality control points (1 spotting solution quality control point and 3 mouse IgG quality control points) are simultaneously spotted in each well during spotting.

[0211] Assemble the above components together with dust-free paper to obtain the adamantane biochip detection kit, referred to as kit 2.

[0212] 5.2 Establishment of the detection method of the amantadine biochip detection kit

[0213] The samples were pre-treated before detection, and the pre-treatment method was carried out according to Example 4.2.

[0214] The detection method of the amantadine biochip detection kit includes:

[0215] Step (1) equilibration: equilibrate the components of the kit at room temperature for 30 minutes;

[0216] Step (2) Infiltration: add 250 μl of washing solution to each well (about 4 to 5 drops per well), let it soak for 3 minutes, shake off and pat dry the liquid in the well;

[0217] Step (3) adding samples, adding 50 μl of sample or standard solution into the microwells of the chip, adding 25 μl of enzyme-labeled secondary antibody, and adding 25 μl of antibody working solution;

[0218] Step (4) incubation: incubate in a microplate incubator at 30°C and 1000 rpm for 15 minutes;

[0219] Step (5) washing: discard the liquid in the wells, add 250 μl of washing solution to each well (about 4 to 5 drops per well, do not overflow), soak 4 times, soak for about 10 seconds each time, and pat dry on absorbent paper;

[0220] Step (6) color development: add 100 μl of TMB substrate solution, and shake in a microplate constant temperature oscillator at 30° C. and 1000 rpm for 10 minutes;

[0221] Step (7) Read the data, shake off the liquid, open the chip holder, press the chip moderately with dust-free paper, absorb the liquid on the chip membrane surface, and detect with a microplate chip imager. The data must be read within 5 minutes to check the test results.

[0222] Kit 2 is a built-in marker song on the backend website (where the marker song is as follows Figure 8 ), there is no need to re-establish the standard curve, and the test results can be read directly by the microplate chip imager.

[0223] 5.3 Optimization of Amantadine Chip Coating Concentration in Kit 2

[0224] The adamantane antigen was prepared into 8 concentrations of 5 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL for spotting, and the spotting volume was 20 nl. Different monoclonal antibody gradient concentrations (0.01 μg / mL, 0.02 μg / mL, 0.04 μg / mL, and 0.08 μg / mL) were added to the chip and the reaction was carried out according to the steps of Example 5.2.

[0225] The optimal concentration of the antigen was determined based on the grayscale value (the grayscale value represents the color depth of the antigen spot after reaction, which can be directly read by the microplate imager without networking through the reading software). The antigen concentration when the grayscale value is 15000-18000 is the preferred antigen concentration of the adamantane kit. The results (see Table 5) show that the adamantane antigen coating concentration is preferably 30-50 μg / mL (i.e. 0.6-1 ng / spot).

[0226] Table 5 Optimization of the coating concentration of amantadine antigen

[0227]

[0228] Example 6 Evaluation of Kit 1 and Kit 2

[0229] 6.1 Sensitivity determination

[0230] According to the detection methods of Kit 1 and Kit 2, the adamantane standard was gradiently diluted to 6 concentrations of 0 ng / mL, 0.2 ng / mL, 0.6 ng / mL, 1.8 ng / mL, 5.4 ng / mL, and 16.2 ng / mL, and a standard curve was established to determine the sensitivity and half-inhibitory concentration of the kit.

[0231] Results: IC of Kit 1 50 The sensitivity is 0.2 ng / mL, which is equivalent to 0.2 ppb. The IC value of kit 2 is 0.9-1.2 ng / mL, and the sensitivity is 0.2 ng / mL, which is equivalent to 0.2 ppb. 50 The sensitivity of kits 1 and 2 is higher than the detection limit of 1.0 ng / mL of the national standard test method.

[0232] At the same time, the recovery rates of 20 samples with the same matrix were measured with spiked adamantane at different concentrations. The actual residual concentration of the samples was calculated from the standard curve, and the detection limit of the kit was confirmed based on the average concentration of the 20 samples and 3 times the standard deviation. The data results showed that the sensitivity of kit 1 and kit 2 were both 0.2 ng / mL, which were higher than the detection limit of 1.0 ng / mL of the national standard detection method (national standard detection method GB31660.5-2019).

[0233] 6.2 Specificity detection

[0234] According to the detection methods of kit 1 and kit 2, the different drug standard solutions were gradiently diluted with PBS, and the standard curves were drawn to calculate the IC values ​​of different drugs. 50 And cross-reaction rate. The smaller the cross-reaction rate with other drugs, the better the specificity of the amantadine test kit for detecting amantadine. The test results are shown in the following table:

[0235] Table 6 Specificity detection of amantadine monoclonal antibodies

[0236]

[0237] 6.3 Accuracy determination

[0238] Different concentrations of amantadine standard were added to negative samples such as chicken, so that the final residual concentration of the sample was 0.5μg / kg, 1.0μg / kg, and 2.0μg / kg, and the pretreatment method of the kit was used to obtain sample solutions with different added concentrations; three kits were drawn from three batches of the two kits, and the coefficient of variation between different batches and between different kits in the same batch was calculated. Each experiment was repeated 3 times. The results are shown in the following table: The recovery rates of different concentrations of kit 1 and kit 2 were all within the range of 100% ± 20%, the intra-batch coefficient of variation was less than 7%, and the inter-batch coefficient of variation was less than 11%, and the accuracy of the kit detection value was good.

[0239] Table 7 Kit 1

[0240]

[0241]

[0242] Table 8 Kit 2

[0243]

[0244] 6.4 Stability determination

[0245] The ELISA kit and the biochip test kit were stored at 2-8°C. After 15 months of continuous monitoring, the IC values ​​of the two kits were 50 , the maximum S / N value and the change of the added concentration of amantadine were all within the normal range. Considering the unexpected situations that may occur during actual transportation and storage, the test kit was subjected to a thermal stability test at 37°C for 9 days and a frozen test at -20°C for 9 days. The experimental results showed that the indicators of the test kit were normal. The above data show that the test kit has good stability and can be stored at 4-8°C for at least one year.

[0246] 6.5 Clinical sample testing

[0247] Three samples of different matrices (chicken, duck, goose, chicken / duck / goose liver, beef, mutton, pork, eggs, etc.) were purchased from Inner Mongolia, Harbin, Shenyang, Hebei, Luoyang, Zhumadian, Chengdu, Guangzhou, Xiamen, Hangzhou, Nanjing, Guangxi and other places and different supermarkets. The samples were tested according to the national standard method GB31660.5-2019. After confirming as negative samples, Kit 1 and Kit 2 were used for spiked recovery rate detection. A large amount of data showed that the spiked recovery rates of Kit 1 and Kit 2 for samples with different matrices were within the normal range (100% ± 20%) and could be used for the detection of multiple targets.

[0248] At the same time, two brands of adamantane detection kits were purchased from the market (operation and determination were performed according to the kit instructions) to confirm the IC values ​​of the kits from different manufacturers. 50 , sensitivity and detection limit, as well as the repeatability of the detection values ​​at different sample addition concentrations (negative samples are chicken and duck meat), the data are as follows;

[0249] Table 9 Sensitivity and IC of different kits 50 Determination

[0250]

[0251]

[0252] Table 10 CV value detection of three wells of different kits

[0253]

[0254] From the above data, it can be seen that Kit 1 and Kit 2 are superior to the other two brands of detection kits in terms of sensitivity and repeatability.

[0255] Example 7 Preparation and Application of Genetically Engineered Antibodies

[0256] The heavy chain variable region and light chain variable region gene sequences of the monoclonal antibody 3G7 in Example 3 were amplified respectively, and the heavy chain variable region gene and the light chain variable region gene were linked by connecting peptides to construct recombinant plasmids 3G7-ScFv, and the ScFv gene was inserted into the pCDNA-3.1 vector to construct the pCDNA-3G7-ScFv eukaryotic expression system, and MDCK cells were transfected for expression.

[0257] The expressed single-chain antibody 3G7 was subjected to ic-ELISA assay according to the ic-ELISA method described in Example 3 (the concentration of adamantane spiked was 1 ppb). The results are shown in Table 11: The inhibition rate of the small molecule compound on the antigen / antibody reaction was normal, indicating that the single-chain antibody 3G7 had good reaction characteristics with the adamantane small molecule and its antigen.

[0258] Table 11 ic-ELISA assay of single-chain antibody 3G7

[0259] Antigen coating concentration ELISA ic-ELISA Inhibition rate 0.1 μg / mL 1.6 0.8 50.0％ 0.2 μg / mL 2.2 1.3 41.0％ 0.3 μg / mL 2.9 2.2 24.2％

[0260] The above results show that SEQ.ID No.1 and SEQ.ID No.2 can be used for the preparation of amantadine engineered antibodies.

[0261] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention. SEQUENCE LISTING <110> Luoyang Zhongke Biochip Technology Co., Ltd. <120> Amantadine hapten, amantadine antigen, preparation method, amantadine monoclonal antibody and amantadine detection kit <160> 2 <170> PatentIn version 3.3 <210> 1 <211> 366 <212> DNA <213> Hybridoma <400> 1 gatgtgcacc cggtggagtc tgggggaggc ttagtgcagc ctggagggtc ccggaaactc 60 tcctgtgcag cctctggatt cactttcagt gcttatggaa tgcactgggt tcgtcaggct 120 ccagagaagg gactggagtg ggtcgcatat ataagtagtg gcagtagtac cgtctactat 180 gcagacacag tgaagggccg attcaccatc tccagagaca atcccaagaa caccctgttc 240 ctgcaaatga ccagtctgag gtctgaggac acggccattt attactgtgc aagaggggat 300 tactacggta gaacctccta tgctttggac tactggggtc aaggaacctc agtcaccgtc 360 tcctca 366 <210> 2 <211> 323 <212> DNA <213> Hybridoma (hybridoma) <400> 2 agtattgtga tgacccagac tcccaaattc ctgcttgtat caccaggaga cagggttacc 60 attacctgca aggccagtca gagtgtgagt aataatgtag cttggtacca acagaagcca 120 gggcagtccc ctaaactgct tatatactct gcatccactc gctacactgg agtccctgat 180 cgtttcactg gcagtggata tgggacggat ttcactttca ccatcagcac tgtgcaggct 240 gaagacctgg cagtttattt ctgtcagcag gtctctaact atccgttcgc gttcggaggg 300 gggaccaagc tggaaataaa acg 323

Claims

1. Amantadine hapten A, which has a chemical structure as shown in structural formula (V):

2. A method for preparing the amantadine hapten A according to claim 1, wherein: The method comprises: Step (1) weighing 2g-10g of 3-aminoadamantane-1-carboxylic acid and 1.92-9.6g of Boc anhydride into a single-mouth bottle, adding 0.05-0.26g of 4-dimethylaminopyridine, dissolving with 30mL of tert-butanol, and reacting at 70-80°C for 5-8 hours; Step (2) The liquid after the reaction is subjected to reduced pressure rotary evaporation to remove the solvent tert-butyl alcohol, thereby obtaining a large amount of light yellow oily substance, which is the crude compound 2; Step (3) dissolving the crude compound 2 in ethyl acetate, washing twice with 0.1 M sulfuric acid solution and pure water, collecting the organic phase, and performing vacuum rotary evaporation to obtain compound 2; Step (4) dissolving 0.5-2 g of the compound 2 in 10 mL of 80% methanol aqueous solution, adding 0.3-1.3 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.25-1.0 g of N-hydroxysuccinimide, and reacting at 20-25° C. for 1 hour to obtain a solution of the intermediate compound 3; slowly adding 0.9-3.6 g of a 50% m / V aqueous solution of β-alanine dropwise to the reaction solution, and continuing to react at 20-25° C. overnight; Step (5) The reaction solution was subjected to reduced pressure rotary evaporation to remove methanol and water, and 30 mL of water and 30 mL of ethyl acetate were added again. After stirring for 30 minutes, the layers were separated, and the organic phase was collected and rotary evaporated to obtain a crude compound 4. The crude compound 4 was purified by column to obtain 0.5 g of its pure product; Step (6) dissolving 0.2-1.0 g of the purified compound 4 in 1 mL of methanol, adding 0.5 mL of concentrated hydrochloric acid, stirring at 20-25° C. for 3-5 hours, adjusting the pH to 6 with 0.5 M sodium hydroxide solution, and then removing the solvent methanol and water by rotary evaporation under reduced pressure to obtain 0.1-0.6 g of crude compound 5; and Step (7) The crude compound 5 is dissolved in methanol again, and then the inorganic salt is removed by filtration. The filtrate is evaporated under reduced pressure to obtain a relatively pure compound 5, and column silica gel is used to further separate and purify the compound to obtain the adamantane amine hapten A.

3. The method according to claim 2, wherein: The method comprises: In the step (1), 5 g of 3-aminoadamantane-1-carboxylic acid and 4.8 g of Boc anhydride are weighed and added into a single-mouth bottle, 0.13 g of 4-dimethylaminopyridine is added, dissolved in 30 mL of tert-butanol, and reacted at 70-80° C. for 5-8 hours; In the step (2), the liquid after the reaction is evaporated under reduced pressure to remove the solvent tert-butyl alcohol, thereby obtaining a large amount of light yellow oily substance, which is the crude compound 2; In step (3), the crude compound 2 is dissolved in ethyl acetate, washed twice with a 0.1M sulfuric acid solution and pure water, and then the organic phase is collected and subjected to vacuum rotary evaporation to obtain compound 2; In the step (4), 1 g of the compound 2 is dissolved in 10 mL of 80% methanol aqueous solution, 0.65 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.5 g of N-hydroxysuccinimide are added, and the mixture is reacted at 20 to 25° C. for 1 hour to obtain a solution of the compound 3; 1.8 g of a 50% m / V aqueous solution of β-alanine is slowly added dropwise to the reaction solution, and the reaction is continued at 20 to 25° C. overnight; In the step (5), the methanol and water are removed from the reaction solution by rotary evaporation under reduced pressure, and 30 mL of water and 30 mL of ethyl acetate are added again, and the mixture is stirred for 30 minutes and then separated into layers. The organic phase is collected and rotary evaporated to obtain a crude compound 4, and the crude compound 4 is purified by column to obtain 0.5 g of its pure product; The step (6) is to dissolve 0.5 g of the crude compound 4 in 1 mL of methanol, add 0.5 mL of concentrated hydrochloric acid, stir at 20-25° C. for 3-5 hours, adjust the pH to 6 with 0.5 M sodium hydroxide solution, and then remove the solvent methanol and water by rotary evaporation under reduced pressure to obtain 0.6 g of crude compound 5; and In the step (7), the crude compound 5 is dissolved in methanol again, and then the inorganic salt is removed by filtration. The filtrate is evaporated under reduced pressure to obtain a relatively pure compound 5, and column silica gel is used to further separate and purify the compound to obtain the adamantane amine hapten A.

4. A method for preparing an amantadine antigen A, wherein: The preparation method comprises: Step (1) dissolving 4-16 mg of the adamantane amine hapten A described in claim 1 in 1 mL of DMF, adding 1.5-6.0 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.9-3.6 mg of N-hydroxysuccinimide, and reacting at 25° C. for 1-2 hours to form a solution 1; Step (2) weighing 10-30 mg of BSA, OVA, KLH, BGG, RSA, PSA, LPH or HAS carrier protein and dissolving it in 1 mL of 0.01 M NaHCO3 buffer to form solution 2; Step (3) slowly adding the solution 1 of the adamantane amine hapten A of step (1) dropwise into the solution 2 of step (2), stirring at 4° C. for overnight reaction; and Step (4) The reaction solution of step (3) is dialyzed with 0.01M PBS solution at 4°C for 3 days, and the dialysate is replaced twice a day; after the dialysate is centrifuged, the supernatant is taken out, which is the adamantane antigen A.

5. The method according to claim 4, wherein: The method comprises: Step (1) dissolving 4 mg of the adamantane amine hapten A in 1 mL of DMF, adding 2.9 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1.8 mg of N-hydroxysuccinimide, and reacting at 25° C. for 1 to 2 hours to form a solution 1; Step (2) 13.5 mg of OVA was weighed and dissolved in 1 mL of 0.01 M NaHCO3 buffer to form solution 2; Step (3) slowly adding the solution 1 of the adamantane amine hapten A of step (1) dropwise into the solution 2 of step (2), stirring at 4° C. for overnight reaction; and Step (4) dialyzing the reaction solution of step (3) with 0.01M PBS solution at 4°C for 3 days, replacing the dialysate twice a day; centrifuging the dialysate, and taking out the supernatant, which is the adamantane antigen A.

6. Amantadine antigen A prepared according to the method for preparing amantadine antigen A according to claim 4 or 5.

7. An amantadine ELISA test kit, wherein: The amantadine ELISA detection kit comprises a support medium coated with the amantadine antigen A according to claim 4, an enzyme label working solution, an amantadine antibody working solution, and an adamantane standard solution; Wherein, the coating concentration of the adamantane antigen A is 0.10 μg / mL to 0.25 μg / mL; The enzyme labeling working solution is a solution of HRP-sheep anti-mouse IgG; The adamantane antibody working solution is a solution of adamantane monoclonal antibody 3G7, the content of the adamantane monoclonal antibody 3G7 is 0.04 μg / mL, wherein the heavy chain variable region of the monoclonal antibody 3G7 is encoded by SEQ.ID NO: 1, and the light chain variable region thereof is encoded by SEQ.ID NO: 2; The adamantane standard solution is prepared by gradiently diluting the adamantane standard with 0.01M PBS diluent to concentrations of 0, 0.2 ng / mL, 0.6 ng / mL, 1.8 ng / mL, 5.4 ng / mL, and 16.2 ng / mL; Wherein, the supporting medium is a microtiter plate, and the adamantane antigen A is coated with 100 μl / well; The enzyme label working solution also contains: 8g sodium chloride, 2.9g disodium hydrogen phosphate, 0.24g potassium dihydrogen phosphate, 0.2g potassium chloride, 200mL newborn calf serum, and 1mL proclin300 in 1L water; The adamantane antibody working solution also contains: 8 g sodium chloride, 2.9 g disodium hydrogen phosphate, 0.24 g potassium dihydrogen phosphate, 0.2 g potassium chloride, 10 g bovine serum albumin, and 1 mL proclin300 in 1 L of water; The kit also includes a washing solution, a TMB color developing solution, a stop solution, an extractant, a purification agent, and a sample diluent; The washing solution is a 0.01M PBS solution containing 0.1% V / V Tween-20; The TMB color developing solution includes solution A and solution B, wherein solution A is a buffer solution of citric acid-sodium dihydrogen phosphate with a pH value of 5, which contains 1.5g citric acid, 6.6g disodium hydrogen phosphate, and 1.0g urea peroxide in 100mL water, and solution B is a buffer solution of citric acid-sodium dihydrogen phosphate with 0.8% M / V 3,3,5,5-tetramethylbenzidine; The stop solution is a 2 mol / L sulfuric acid solution; The extractant is acetonitrile; The purifying agent is n-hexane; and The sample diluent is 0.01 M PBS solution.

8. An amantadine biochip detection kit, wherein: The biochip detection kit comprises one or more amantadine chips, antibody working solution, and enzyme label working solution, wherein the amantadine chip is coated with the amantadine antigen A according to claim 4, a quality control product, and a blank control point; Wherein, the quality control product is mouse IgG, and the blank control point is the detection background value; The blank control point is a spot where 5% glycerol solution, 5% sorbitol solution, 0.05% Triton solution, DMSO, and pH 6.8 PBS solution are mixed evenly in a volume ratio of 10:15:0.1:50:100; The antibody working solution is a solution of the amantadine monoclonal antibody 3G7, the concentration of the amantadine monoclonal antibody 3G7 is 0.06 μg / mL, wherein the heavy chain variable region of the monoclonal antibody 3G7 is encoded by SEQ.ID NO: 1, and the light chain variable region thereof is encoded by SEQ.ID NO: 2; The enzyme labeling working solution is a solution containing HRP-goat anti-mouse IgG, and HRP-goat anti-mouse IgG is diluted at 1:200 to 1:2000 V / V; The coating amount of the adamantane antigen A is 0.6-1.0 ng / point; the coating amount of the mouse IgG is 1-4 ng / point; there are three quality control points in total, the coating amount of quality control point 1 (1) is greater than or equal to 1 ng / point and less than 2 ng / point; the coating amount of quality control point 2 (2) is 2 ng / point; the coating amount of quality control point 3 (3) is greater than 2 ng / point and less than or equal to 4 ng / point.

9. The amantadine biochip detection kit according to claim 8, wherein: The coating amount of the quality control point 1 (1) is 1 ng / point; the coating amount of the quality control point 2 (2) is 2 ng / point; the coating amount of the quality control point 3 (3) is 4 ng / point; Spotting the spotting solution as a blank control at the blank control spotting point (4), and spotting the adamantane antigen A at the adamantane antigen detection point (5); The quality control product spot 1 (1), the quality control product spot 2 (2), the quality control product spot 3 (3), and the blank control spot (4) are respectively fixed at the upper left corner, lower left corner, lower right corner, and upper right corner of the detection chip, and the adamantane antigen detection spot (5) is spotted at other positions of the detection chip, and is ≥700μm away from the edge of any other point, and the shortest distance between the midpoint and the edge of the quality control product spot 1 (1), the quality control product spot 2 (2), the quality control product spot 3 (3), the blank control spot (4), and the adamantane antigen detection spot (5) is also ≥700μm; the quality control product spot 1 (1), the quality control product spot 2 (2) , the distance from the quality control sample point 3 (3) and the blank control sample point (4) to the upper edge and lower edge of the detection chip subunit is ≥8mm, and the distance from the left edge and right edge of the detection chip subunit is ≥5mm; the adamantane biochip kit comprises one or more adamantane biochip subunits, which are composed of a bottom, an upper grid, and an adamantane biochip clamped therebetween by the bottom and the upper grid to form an independent detection hole, wherein one detection hole is one adamantane biochip subunit, and when the antibody joint detection kit comprises multiple adamantane biochip subunits, each of the independent detection chip subunits is integrated into a microarray chip.

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