CN113267621B - Stabilizer for ELISA kit coated plate, preparation method of stabilizer, kit coated plate and kit - Google Patents
Stabilizer for ELISA kit coated plate, preparation method of stabilizer, kit coated plate and kit Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54306—Solid-phase reaction mechanisms
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
Abstract
The application relates to the technical field of biological detection, and particularly discloses a stabilizer for an ELISA kit coated plate, a preparation method of the stabilizer, the kit coated plate and a kit. The stabilizer for the ELISA kit coating plate comprises water and the following components in percentage by mass: 5 to 15 percent of glycerol, 0.5 to 1.5 percent of trehalose, 0.005 to 0.015 percent of sodium glutamate and 0.002 to 0.008 percent of thioredoxin; the preparation method comprises the following steps: s1, mixing glycerol, trehalose, sodium glutamate and water uniformly, and adjusting the pH value to 7 to obtain a mixed solution; s2, adding thioredoxin into the mixed solution obtained in the step S1, and uniformly mixing to obtain the thioredoxin. The stabilizer for the ELISA kit coated plate can be used for preparing the ELISA kit coated plate, and the prepared ELISA kit has the advantages of good stability and long storage period.
Description
Technical Field
The application relates to the technical field of biological detection, in particular to a stabilizer for an ELISA kit coated plate, a preparation method of the stabilizer, the kit coated plate and a kit.
Background
Enzyme-linked immunosorbent assay (ELISA) has developed rapidly since the beginning of the 70 s of the 20 th century, and is now widely used in immunology, medicine, biology and other fields. ELISA is a highly sensitive test technique which is based on immunological reactions and combines the specific reactions of antigens and antibodies with the high-efficiency catalytic action of enzyme on substrates.
The ELISA kit can be generally divided into a ready-to-use type and a non-ready-to-use type, and the antigen coated plate of the conventional ready-to-use ELISA kit generally undergoes five steps of coating, washing, blocking, washing and drying in the preparation process, but since the antigen or antibody used for coating is protein, the protein has biological activity, is easily affected by temperature and some organic or inorganic substances, and is inactivated and denatured, the shelf life of the ELISA kit prepared by the steps and the stability of data test are not ideal.
Disclosure of Invention
In order to improve the storage life of an ELISA kit and the stability of data testing and enable the ELISA kit to be stored and used for a long time, the application provides a stabilizer for an ELISA kit coated plate, a preparation method of the stabilizer, the kit coated plate and the kit.
In a first aspect, the present application provides a stabilizer for an ELISA kit coated plate, which adopts the following technical scheme: a stabilizer for an ELISA kit coated plate comprises water and the following components in percentage by mass: 5 to 15 percent of glycerol, 0.5 to 1.5 percent of trehalose, 0.005 to 0.015 percent of sodium glutamate and 0.002 to 0.008 percent of thioredoxin.
By adopting the technical scheme, because the glycerol is adopted and contains a large amount of hydroxyl groups, the glycerol is stable, the glycerol can be adsorbed on the coating plate and forms a layer of protective film on the surface of the antigen to protect the protein structure of the antigen, and in addition, the deposition speed of the antigen and the antibody can be accelerated in the subsequent detection step, so that the combination probability of the enzyme-labeled antibody and the antigen is increased. In addition, trehalose can interact with bound water of protein molecules of the antigen, and shows good protection efficacy. In addition, sodium glutamate can improve the water holding capacity of antigen protein molecules, improve the water holding capacity of the antigen protein molecules, and further maintain the biological activity of the antigen. Thioredoxin has good thermal stability, low molecular weight and good dispersion performance, and can be embedded between a glycerol protective film and antigen protein molecules in a dispersing way, thioredoxin promotes the reduction of other proteins through the interaction of cysteine thiol-disulfide bond, thereby playing a good role in antioxidation, thioredoxin participates in the redox reaction as a hydrogen receptor, greatly reducing the probability of the antigen being damaged by oxidation damage, and the thioredoxin is cooperated with the compounding of trehalose and glycerol to play a good role in protecting an antibody or an antigen, thereby greatly prolonging the storage period of the prepared ELISA kit and improving the stability of test data of the ELISA kit.
Preferably, the stabilizer for the ELISA kit coated plate comprises water and the following components in percentage by mass: 8 to 12 percent of glycerol, 0.7 to 1.3 percent of trehalose, 0.008 to 0.012 percent of sodium glutamate and 0.003 to 0.007 percent of thioredoxin.
By adopting the technical scheme, the ELISA stabilizer compounded according to the proportion has a better stabilizing effect, maintains the immunocompetence of the antigen, reduces the CV values of detection results among ELISA kits with different storage periods, and is more accurate and effective in test results.
Preferably, the thioredoxin is recombinant bacterial thioredoxin.
By adopting the technical scheme, the recombinant bacterial thioredoxin has better clearing effect on active oxygen substances, interacts with harmful substances such as superoxide anions, hydrogen peroxide, free hydroxyl and the like, inhibits the reaction mechanism and activity of the harmful substances, and maintains the redox balance environment of the protein.
Preferably, the mass ratio of the thioredoxin to the glycerol is 1- (1000-3000).
By adopting the technical scheme, on one hand, when the thioredoxin is compounded according to the proportion, the effective embedding sites of the thioredoxin on the protective film formed by the glycerol are uniform and consistent, and a better stabilizing effect on antigen protein molecules can be achieved; on the other hand, the ratio of hydroxyl on the glycerol protective film to cysteine thiol-disulfide bond of thioredoxin is more adaptive, and the hydroxyl can be covalently combined and modified with the corresponding peptide chain segment on the thioredoxin, so that the reactivity of the thioredoxin is improved, the anti-deformability of the thioredoxin is improved, and the anti-oxidation action period of the thioredoxin is prolonged.
Preferably, the component also comprises (0.001% -0.003%) mussel mucin by mass percentage.
Through adopting above-mentioned technical scheme, contain a large amount of pyrocatechol group in the mussel mucin, have very high biological affinity and compatibility, by can be in the same place with the comparatively firm combination of antigen on the reaction hole inner wall of ELISA peridium board, constitute the hydrogen bond between the two, constitute stronger physical adsorption, and the phenol group in the mussel mucin can also form stable complex with some harmful inorganic ions, play the effect of shielding, assistance thioredoxin that can be fine protects the antigen.
Preferably, the component also comprises (0.5-1.2%) hydroxytyrosol by mass percentage.
By adopting the technical scheme, the hydroxytyrosol has better water solubility and fat solubility, can be adsorbed and embedded on a protective layer formed by thioredoxin and mussel mucin, can absorb free radicals with higher activity on the one hand, and reduces the probability that the envelope antigen protein molecules are subjected to oxidative attack. On the other hand, the phenolic hydroxyl group of hydroxytyrosol can also be used as a hydrogen acceptor to passivate active oxygen. In addition, hydroxytyrosol can also activate a conserved region in thioredoxin, and the antioxidant activity of the thioredoxin is improved.
Preferably, the weight ratio of the mussel mucin to the hydroxytyrosol is 1 (200- & lt 600- & gt).
Through adopting above-mentioned technical scheme, prepare according to above-mentioned weight ratio, hydroxytyrosol can reduce the inside polymerization power of mussel mucin protective layer, controls the cohesion between the envelope antigen on mussel mucin protective layer and the ELISA kit envelope board to suitable scope, reduces the interference of mussel mucin protective layer to follow-up detection sensitivity.
In a second aspect, the present application provides a method for preparing a stabilizer for an ELISA kit coated plate, comprising the steps of:
s1, mixing glycerol, trehalose, sodium glutamate and water uniformly, and adjusting the pH value to 7 to obtain a mixed solution;
s2, adding thioredoxin into the mixed solution obtained in the step S1, and uniformly mixing to obtain the thioredoxin.
By adopting the technical scheme, the glycerol, the trehalose, the sodium glutamate and the water are uniformly mixed, the solubility of the glycerol, the trehalose and the sodium glutamate is good, a uniform and consistent dispersion system can be quickly formed, a proper biological environment is provided for the subsequently added thioredoxin, and the activity of the thioredoxin is ensured.
In a third aspect, the present application provides an ELISA kit coated plate, using the following technical scheme:
an ELISA kit coated plate is prepared from a porous plate and a raw material comprising the stabilizer for the ELISA coated plate.
By adopting the technical scheme, the protective components in the stabilizer can be fully combined with the antigen coated on the ELISA kit coated plate, easily damaged groups or structures on the antigen are protected, the immunocompetence of the antigen is kept, the antioxidant and anti-allergic capability of the antigen is improved, and meanwhile, the sensitivity of the specificity of the ELISA kit coated plate is ensured.
In a fourth aspect, the present application provides an ELISA kit comprising: sample diluent, stop solution and the ELISA kit coated plate.
By adopting the technical scheme, the shelf life of the ELISA kit is longer, the CV values of the determination structures of the kits in different storage periods are smaller, and the detection result is more stable and accurate.
In summary, the present application has the following beneficial effects:
1. according to the method, thioredoxin, glycerol, trehalose and sodium glutamate are compounded and cooperated with one another, so that the water binding capacity of the antigen is improved, the biological activity of the antigen is maintained, a plurality of layers of protective films are formed between the antigen and the inner wall of a reaction hole of an ELISA coated plate, the capability of the antigen for resisting the damage of the external environment is improved, the preservation time of the ELISA kit can reach more than 24 months, the CV value of sample detection data of the ELISA kit is reduced to about 1.5, and the stability of data test is very good.
2. According to the preparation method of the ELISA kit coated plate, the ELISA kit has better stability by combining the blocking protection effect of the blocking agent and the protection effect of the stabilizing agent.
Drawings
FIG. 1 shows the change tendency of OD value of negative control serum (NC) and OD value of positive control serum (PC) in example 9 of the present application.
FIG. 2 is a graph showing the variation tendency of OD value of serum samples at different dilution times in example 9 of the present application.
FIG. 3 is the trend of the S/P value change of the serum of samples at different dilution times in example 9 of the present application.
Detailed Description
The present application will be described in further detail with reference to examples.
The stabilizer for the ELISA kit coated plate comprises water and the following components in percentage by mass: 5 to 15 percent of glycerol, 0.5 to 1.5 percent of trehalose, 0.005 to 0.015 percent of sodium glutamate and 0.002 to 0.008 percent of thioredoxin.
Further preferably, the stabilizer for the ELISA kit coated plate comprises water and the following components in percentage by mass: 8 to 12 percent of glycerol, 0.7 to 1.3 percent of trehalose, 0.008 to 0.012 percent of sodium glutamate and 0.003 to 0.007 percent of thioredoxin.
The thioredoxin may be a plant thioredoxin, and the plant thioredoxin may be one of a soybean thioredoxin, a wheat thioredoxin, a rice thioredoxin, and a corn thioredoxin.
The thioredoxin can also be animal thioredoxin, and the animal thioredoxin can be one of human thioredoxin, pig thioredoxin, Chinese bee thioredoxin and silkworm thioredoxin.
Preferably, the thioredoxin is a recombinant bacterial thioredoxin. More preferably, the recombinant bacterial thioredoxin is any one of recombinant escherichia coli thioredoxin or recombinant bacillus subtilis thioredoxin. More preferably, the recombinant bacterial thioredoxin is recombinant escherichia coli thioredoxin.
The preparation method of the stabilizer for the ELISA kit coated plate comprises the following steps:
s1, mixing glycerol, trehalose, sodium glutamate and water uniformly, and adjusting the pH value to 7 to obtain a mixed solution;
s2, adding thioredoxin into the mixed solution obtained in the step S1, and uniformly mixing to obtain the thioredoxin.
Preferably, in step S1, the glycerol, the trehalose, the sodium glutamate and the water are uniformly mixed and stirred for 10-20min at a stirring speed of 800-. More preferably, in step S1, the glycerol, trehalose, sodium glutamate, and water are mixed uniformly and stirred for 15min at a stirring speed of 900 rpm.
Preferably, the pH adjustment in step S1 is performed by using HCl solution and NaOH solution.
Preferably, the step of adding thioredoxin to the mixed solution in the step of S1 in the step of S2 is to mix them uniformly by shaking on a microplate shaker for 5-15 min. Further preferably, in step S2, the step of adding thioredoxin to the mixed solution in step S1 and mixing the mixture uniformly is performed by shaking and mixing the mixture on a microplate shaker for 10 min.
The ELISA kit coated plate is prepared from a porous plate and a raw material comprising the stabilizer for the ELISA coated plate.
The application provides a preparation method of an ELISA kit coated plate, which comprises the following steps:
1) coating: diluting the antigen or the antibody by using a buffer solution to obtain a coating solution, adding the coating solution into a reaction hole of a porous plate, and culturing;
2) washing: sucking out the coating solution cultured in the reaction holes of the porous plate in the step 1), and washing the reaction holes by using a washing solution;
3) and (3) sealing: adding confining liquid into the reaction holes of the porous plate washed in the step 2) for cultivation;
4) washing: sucking out the sealing liquid sealed in the reaction holes of the porous plate in the step 3), and washing the reaction holes by using a washing liquid;
5) and (3) stabilizing: adding the stabilizer into each reaction hole of the washed porous plate in the step 4), and culturing for 0.8-1.2h at the temperature of 25 ℃;
6) and (3) drying: sucking out the stabilizing agent in the reaction holes of the porous plate in the step 5), and drying the porous plate to obtain the composite stabilizer.
Further preferably, the application provides a preparation method of an ELISA kit coated plate, which is prepared by the following steps:
1) coating: diluting the antigen or the antibody by using a buffer solution to obtain a coating solution, adding the coating solution into a reaction hole of a porous plate, and culturing for 12-18h at 4 ℃;
2) washing: sucking out the coating liquid in the reaction holes of the porous plate in the step 1), and washing the reaction holes for 3-5 times by using a washing liquid;
3) and (3) sealing: adding the confining liquid into the reaction holes of the porous plate in the step 2), and culturing for 0.5-1.5h at the temperature of 25 ℃;
4) washing: sucking out the confining liquid in the reaction holes of the porous plate in the step 3), and washing the reaction holes for 3-5 times by using a washing liquid;
5) and (3) stabilizing: adding the stabilizer into each reaction hole of the multi-hole plate in the step 4), and culturing for 0.8-1.2h at the temperature of 25 ℃;
6) and (3) drying: sucking out the stabilizing agent in the reaction holes of the porous plate in the step 5), then placing the porous plate in a constant-temperature incubator upside down, controlling the temperature to be 37 ℃, and drying for 0.5-1.5h to obtain the composite stabilizer.
Preferably, the amount of the coating solution added in step 1) is 50 to 150. mu.L/well. Further preferably, the amount of the coating solution added in step 1) is 100. mu.L/well.
Preferably, the buffer in step 1) is any one of carbonate buffer, phosphate buffer and Tris-HCl buffer. Further preferably, the buffer in step 1) is a carbonate buffer. More preferably, the carbonate buffer has a pH of 9.6 and the carbonate buffer has a concentration of 0.05M.
Preferably, the confining liquid consists of the following raw materials in percentage by mass: 0.1 to 0.3 percent of BSA, and the balance of water.
Preferably, the addition amount of the blocking solution in the step 4) is 150-. Further preferably, the addition amount of the blocking solution in step 4) is 200. mu.L/well.
Preferably, the concentration of antigen or antibody in the coating solution is (5. mu.g-20. mu.g)/ml.
Preferably, the antigen in the coating solution in this example and comparative example is a Porcine Reproductive and Respiratory Syndrome (PRRS) recombinant N protein antigen, and the manufacturer is beijing jinobantai biotechnology limited. In other embodiments, the antigen or antibody in the coating solution may be selected according to the type of disease to be detected.
Preferably, the amount of stabilizer added in step 5) is 50 to 150. mu.L/well. Further preferably, the amount of the stabilizer added in step 5) is 100. mu.L/well.
Preferably, the washing of the reaction well 3 to 5 times with the washing solution in step 2), step 4) is carried out by washing the reaction well 3 times with the washing solution in an amount of 300. mu.L/well each time for 2 to 4 min. Further preferably, each washing time in step 2), step 4) is 3 min.
Preferably, the washing liquid is prepared from the following raw materials: sodium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate dodecahydrate, potassium chloride and tween 20. Further preferably, the content of each raw material in each 1L of washing solution is as follows: 65-85g/L of sodium chloride, 21.5-23.6g/L of potassium dihydrogen phosphate, 30-35.5g/L of disodium hydrogen phosphate dodecahydrate, 1.6-2.2g/L of potassium chloride and 0.25-0.38% of Tween-20 in final concentration.
Further preferably, the content of each raw material in each 1L of washing solution is as follows: 75g/L of sodium chloride, 22.575g/L of monopotassium phosphate, 33.8g/L of disodium hydrogen phosphate dodecahydrate, 1.9g/L of potassium chloride and 0.3% of Tween-20 in final concentration.
Preferably, the ELISA kit of the present application comprises the following components: sample diluent, stop solution and the ELISA kit coated plate.
Further preferably, the ELISA kits of the examples and comparative examples of the present application comprise the following components: the ELISA kit coating plate; 1 bottle of sample diluent, 60ml or 180 ml; 1 bottle of 10 times of washing solution is filled with 125ml or 240 ml; 1 bottle of TMB substrate liquid, and 30ml or 70ml of TMB substrate liquid is filled; 1 bottle of stop solution, 20ml or 40 ml; and (5) sealing a plate film.
The preferred manufacturer of the sample diluent in the ELISA kits of the embodiments and comparative examples of the present application is Cygnus corporation, the supplier is huaya stem cell technology ltd, chongqing, and the model is I028, and the I028 sample diluent comprises the following components by mass percent:
distilled water > 97%;
Tris Hydrochloride<1.0％；
sodium chloride < 1.0%;
bovine serum albumin < 1.0%;
The sample diluent in the ELISA kits of the examples and comparative examples can also be selected from commercially available sample diluents of other ELISA kits.
The stop solution in the ELISA kits of the examples and comparative examples is preferably a 2mol/L sulfuric acid solution.
The TMB substrate liquid was manufactured by Kementec, Italy, having a product number of 4380.
The information on the main raw materials of the examples and comparative examples of the present application is shown in table 1.
TABLE 1 information on main raw materials of examples and comparative examples of the present application
Examples
Example 1
The stabilizer for the ELISA kit coated plate of the embodiment is prepared from the following raw materials in parts by weight: 50g of glycerol, 5g of trehalose, 0.05g of sodium glutamate, 0.02g of thioredoxin and 944.93g of water.
Wherein the thioredoxin is corn thioredoxin.
The preparation method of the stabilizer for the ELISA kit coated plate comprises the following steps:
s1, stirring glycerol, trehalose, sodium glutamate and water at the stirring speed of 900rpm for 15min to obtain a mixed solution, and then adjusting the pH value to 7 by using an HCl solution and an NaOH solution;
and S2, adding thioredoxin into the mixed solution in the step S1, and then oscillating and mixing the mixture on a microplate oscillator for 10min to mix the mixture evenly to obtain the thioredoxin-containing micro-porous membrane.
The confining liquid of the embodiment is prepared from the following raw materials by weight: BSA 1g, water 999 g.
The preparation method of the sealing liquid of the embodiment comprises the following steps: and uniformly mixing BSA and water to obtain the BSA-water emulsion.
The preparation method of the ELISA kit coated plate of the embodiment comprises the following steps:
1) coating: diluting a Porcine Reproductive and Respiratory Syndrome (PRRS) recombinant N protein antigen to a protein concentration of 5 mu g/ml by using a carbonate buffer solution with a concentration of 0.05M to prepare a coating solution, adding the coating solution into a reaction hole of a porous plate, and culturing for 12h at 4 ℃, wherein a producer of the Porcine Reproductive and Respiratory Syndrome (PRRS) recombinant N protein is Beijing Jinobentai Biotech limited;
2) washing: sucking out the coating solution in the reaction holes of the porous plate in the step 1), washing the reaction holes for 3 times by using a plate washing machine, wherein the washing time is 3min each time, and the adding amount of the washing solution is 300 mu L/hole;
3) and (3) sealing: adding the confining liquid into the reaction holes of the porous plate in the step 2), and culturing for 0.5h at the temperature of 25 ℃;
4) washing: sucking out the confining liquid in the reaction holes of the porous plate in the step 3), washing the reaction holes for 3 times by using a plate washing machine, wherein the washing time is 3min each time, and the adding amount of the washing liquid is 300 mu L/hole;
5) and (3) stabilizing: adding 100 mu L of stabilizer into each reaction hole of the multi-hole plate in the step 4), and incubating for 0.8h at the temperature of 25 ℃;
6) and (3) drying: sucking out the stabilizing agent in the reaction holes of the porous plate in the step 5), turning the porous plate to enable the plate to face downwards, placing the plate in a constant-temperature incubator at the set temperature of 37 ℃, drying for 0.5 hour to obtain the coating plate, and taking out the coating plate and sealing the coating plate by using a sealing plate film;
7) packaging: and (3) placing the ELISA coated plate sealed in the step 6) into an aluminum foil bag, placing 1 drying agent in each bag, and sealing.
The ELISA kit of the embodiment of the application comprises the following components:
1) the ELISA kit of this example coated plates;
2) the sample diluent is filled in a bottle of 1 ml of 60ml, wherein the model of the sample diluent is I028, the manufacturer is Cygnus company, the supplier is Chongqing Huaya stem cell technology Co., Ltd, and the I028 sample diluent comprises the following components in percentage by mass: 97 percent of distilled water, 1.0 percent of Tris Hydrochloride, 0.95 percent of sodium chloride, 1 percent of bovine serum albumin,
300Preservative (Preservative) 0.05%;
3) washing liquid 1 bottle, 125ml dress, wherein, the content of each raw materials in per 1L washing liquid is: 75g/L of sodium chloride, 22.575g/L of monopotassium phosphate, 33.8g/L of disodium hydrogen phosphate dodecahydrate, 1.9g/L of potassium chloride and 0.3% of Tween-20 in final concentration;
4) the stop solution is 1 bottle and 20ml, and the stop solution is 2mol/L sulfuric acid solution;
5) 1 bottle of TMB substrate liquid, 30ml of TMB substrate liquid is filled, the manufacturer is Italy kementec company, and the product number is 4380;
6) 1 bottle of positive control serum, 3ml are filled, and the positive control serum is pig serum with specific PRRSV antibody;
7) 1 bottle of negative control serum, 3ml of negative control serum is contained, and the negative control serum is pig serum without specific PRRSV antibody;
8) two or five sealing plate membranes;
9) the PRRSV HRP labeled antibody 1 bottle is packed by 30ml, and the manufacturer is Nanjing Lai Richcel biotechnology limited company with the product number of C00557H.
The amounts (g) of the raw materials added to the stabilizers for ELISA kit-coated plates in examples 2 to 5 are shown in Table 2, and the rest is the same as in example 1.
TABLE 2 addition amounts (g) of respective materials in stabilizers for ELISA kit-coated plates in examples 1 to 5
The preparation method of the stabilizers for ELISA kit-coated plates of examples 2 to 5 was the same as that of example 1.
The blocking solutions of examples 2 to 5 were the same as in example 1.
The blocking solutions of examples 2-5 were prepared in the same manner as in example 1.
The ELISA kit-coated plates of examples 2-5 were prepared in the same manner as in example 1.
The composition of the ELISA kits of examples 2-5 was the same as in example 1.
Example 6
The stabilizer for the ELISA kit coated plate of the embodiment is prepared from the following raw materials in parts by weight: 100g of glycerol, 10g of trehalose, 0.1g of sodium glutamate, 0.05g of thioredoxin and 889.85g of water.
Wherein the thioredoxin is soybean thioredoxin.
The preparation method of the stabilizer for the ELISA kit coated plate of the present example was the same as that in example 1.
The confining liquid of this example was the same as in example 1.
The blocking solution of this example was prepared in the same manner as in example 1.
The ELISA kit coated plate of this example was prepared in the same manner as in example 1.
The composition of the ELISA kit of this example was the same as that of example 1.
Example 7
This embodiment is different from embodiment 6 in that: thioredoxin was porcine thioredoxin, the rest being the same as in example 6.
The preparation method of the stabilizer for the ELISA kit coated plate of the present example was the same as that in example 6.
The confining liquid of this example was the same as in example 6.
The blocking solution of this example was prepared in the same manner as in example 6.
The ELISA kit coated plate of this example was prepared in the same manner as in example 6.
The composition of the ELISA kit of this example was the same as that of example 6.
Example 8
This embodiment is different from embodiment 6 in that: thioredoxin was silkworm thioredoxin, and the rest was the same as in example 6.
The preparation method of the stabilizer for the ELISA kit coated plate of the present example was the same as that in example 6.
The confining liquid of this example was the same as in example 6.
The blocking solution of this example was prepared in the same manner as in example 6.
The ELISA kit coated plate of this example was prepared in the same manner as in example 6.
The composition of the ELISA kit of this example was the same as that of example 6.
Example 9
This embodiment is different from embodiment 6 in that: thioredoxin is recombinant bacillus subtilis thioredoxin, the rest is the same as in example 6.
The preparation method of the stabilizer for the ELISA kit coated plate of the present example was the same as that in example 6.
The confining liquid of this example was the same as in example 6.
The blocking solution of this example was prepared in the same manner as in example 6.
The ELISA kit coated plate of this example was prepared in the same manner as in example 6.
The composition of the ELISA kit of this example was the same as that of example 6.
Example 10
This embodiment is different from embodiment 6 in that: thioredoxin is recombinant e.coli thioredoxin, the rest is the same as in example 6.
The preparation method of the stabilizer for the ELISA kit coated plate of the present example was the same as that in example 6.
The confining liquid of this example was the same as in example 6.
The blocking solution of this example was prepared in the same manner as in example 6.
The ELISA kit coated plate of this example was prepared in the same manner as in example 6.
The composition of the ELISA kit of this example was the same as that of example 6.
Example 11
The present embodiment is different from embodiment 10 in that: the raw material of the stabilizer for ELISA kit coated plate also included 0.01g of mussel mucin, the rest being the same as in example 10.
The sealing liquid of this example was the same as in example 10.
The preparation method of the stabilizer for the ELISA kit coated plate of the present example is the same as that of example 10.
The ELISA kit coated plate of this example was prepared in the same manner as in example 10.
The composition of the ELISA kit of this example was the same as that of example 10.
The amounts (g) of the raw materials added to the stabilizers for ELISA kit-coated plates in examples 11 to 13 are shown in Table 3, and the rest is the same as in example 10.
TABLE 3 addition amount (g) of each raw material in the stabilizers for ELISA kit coated plates in examples 11 to 13
The preparation method of the stabilizers for ELISA kit-coated plates of examples 12 to 13 was the same as that of example 10.
The blocking solutions of examples 12 to 13 were the same as in example 10.
The blocking solutions of examples 12-13 were prepared in the same manner as in example 10.
The ELISA kit-coated plates of examples 12-13 were prepared in the same manner as in example 10.
The composition of the ELISA kits of examples 12-13 was the same as in example 10.
Example 14
The present embodiment is different from embodiment 12 in that: the raw material of the stabilizer for ELISA kit coated plate also included 5g of hydroxytyrosol, and the rest was the same as in example 12.
The sealing liquid of this example was the same as in example 12.
The preparation method of the stabilizer for the ELISA kit coated plate of the present example is the same as that of example 12.
The ELISA kit coated plate of this example was prepared in the same manner as in example 12.
The composition of the ELISA kit of this example was the same as that of example 12.
The amounts (g) of the raw materials added to the stabilizers for ELISA kit-coated plates in examples 14 to 16 are shown in Table 4, and the rest is the same as in example 12.
TABLE 4 addition amount (g) of each raw material in the stabilizers for ELISA kit coated plates in examples 14 to 16
The preparation method of the stabilizers for the ELISA kit coated plates of examples 15 to 16 was the same as that of example 12.
The blocking solutions of examples 15 to 16 were the same as in example 12.
The blocking solutions of examples 15-16 were prepared in the same manner as in example 12.
The ELISA kit coated plates of examples 15-16 were prepared in the same manner as in example 12.
The composition of the ELISA kits of examples 15-16 was the same as in example 12.
Comparative example
Comparative example 1
The stabilizer for the ELISA kit coated plate of the comparative example is prepared from the following raw materials in parts by weight: 100g of glycerol, 10g of trehalose, 0.1g of sodium glutamate and 889.9g of water.
The preparation method of the stabilizer for the ELISA kit coated plate of the comparative example comprises the following steps: mixing glycerol, trehalose, sodium glutamate and water at 900rpm for 15min to obtain a mixture, and adjusting pH to 7 with HCl solution and NaOH solution.
The sealing liquid of the comparative example is prepared from the following raw materials by weight: 2g of BSA, and 998g of water.
The preparation method of the sealing liquid of the comparative example comprises the following steps: and uniformly mixing BSA and water to obtain the BSA-water emulsion.
The preparation method of the ELISA kit coated plate of the comparative example comprises the following steps:
1) coating: diluting a Porcine Reproductive and Respiratory Syndrome (PRRS) recombinant N protein antigen to a protein concentration of 15 mu g/ml by using a carbonate buffer solution with a concentration of 0.05M to prepare a coating solution, adding the coating solution into a reaction hole of a multi-hole plate, and culturing for 16h at 4 ℃;
2) washing: sucking out the coating solution in the reaction holes of the porous plate in the step 1), washing the reaction holes for 3 times by using a plate washing machine, wherein the washing time is 3min each time, and the adding amount of the washing solution is 300 mu L/hole;
3) and (3) sealing: adding the confining liquid into the reaction holes of the porous plate in the step 2), and culturing for 1h at the temperature of 25 ℃;
4) washing: sucking out the confining liquid in the reaction holes of the porous plate in the step 3), washing the reaction holes for 3 times by using a plate washing machine, wherein the washing time is 3min each time, and the adding amount of the washing liquid is 300 mu L/hole;
5) and (3) stabilizing: adding 100 mu L of stabilizer into each reaction hole of the multi-hole plate in the step 4), and incubating for 1h at 25 ℃;
6) and (3) drying: sucking out the stabilizing agent in the reaction holes of the porous plate in the step 5), turning the porous plate to enable the plate to face downwards, placing the plate in a constant-temperature incubator at the set temperature of 37 ℃, drying for 1 hour to obtain the coating plate, and taking out the coating plate and sealing the coating plate by using a sealing plate film;
7) packaging: and (3) placing the ELISA coated plate sealed in the step 6) into an aluminum foil bag, placing 1 drying agent in each bag, and sealing.
The ELISA kit of the comparative example comprises the following components:
1) the ELISA kit of this comparative example coated plates;
2) the sample diluent is filled in a bottle of 1 ml of 60ml, wherein the model of the sample diluent is I028, the manufacturer is Cygnus company, the supplier is Chongqing Huaya stem cell technology Co., Ltd, and the I028 sample diluent comprises the following components in percentage by mass: 97 percent of distilled water, 1.0 percent of Tris Hydrochloride, 0.95 percent of sodium chloride, 1 percent of bovine serum albumin,
300Preservative (Preservative) 0.05%;
3) washing liquid 1 bottle, 125ml dress, wherein, the content of each raw materials in per 1L washing liquid is: 75g/L of sodium chloride, 22.575g/L of monopotassium phosphate, 33.8g/L of disodium hydrogen phosphate dodecahydrate, 1.9g/L of potassium chloride and 0.3% of Tween-20 in final concentration;
4) the stop solution is 1 bottle and 20ml, and the stop solution is 2mol/L sulfuric acid solution;
5) 1 bottle of TMB substrate liquid, 30ml of TMB substrate liquid is filled, the manufacturer is Italy kementec company, and the product number is 4380;
6) 1 bottle of positive control serum, 3ml are filled, and the positive control serum is pig serum with specific PRRSV antibody;
7) 1 bottle of negative control serum, 3ml of negative control serum is contained, and the negative control serum is pig serum without specific PRRSV antibody;
8) two or five sealing plate membranes;
9) the PRRSV HRP labeled antibody 1 bottle is packed by 30ml, and the manufacturer is Nanjing Lai Richcel biotechnology limited company with the product number of C00557H.
Comparative example 2
The stabilizer for the ELISA kit coated plate of the comparative example is prepared from the following raw materials in parts by weight: 150g of sucrose, 0.1g of sorbitol, 0.01g of sodium glutamate, 0.052g of monopotassium phosphate, 0.1g of bovine serum albumin and 849.738g of water.
The preparation method of the stabilizer for the ELISA kit coated plate of the comparative example comprises the following steps: stirring sucrose, sorbitol, sodium glutamate, potassium dihydrogen phosphate, bovine serum albumin and water at 900rpm for 15min to obtain a mixed solution, and adjusting pH to 7 with HCl solution and NaOH solution.
The sealing liquid of the comparative example is prepared from the following raw materials by weight: BSA2g, water 998 g.
The preparation method of the sealing liquid of the comparative example comprises the following steps: and uniformly mixing BSA and water to obtain the BSA-water emulsion.
The preparation method of the stabilizer for the kit coated plate of the comparative example comprises the following steps:
1) coating: diluting a Porcine Reproductive and Respiratory Syndrome (PRRS) recombinant N protein antigen to a protein concentration of 15 mu g/ml by using a carbonate buffer solution with a concentration of 0.05M to prepare a coating solution, adding the coating solution into a reaction hole of an ELISA coating plate, and culturing for 16h at 4 ℃;
2) washing: sucking out the coating solution in the reaction holes of the porous plate in the step 1), washing the reaction holes for 3 times by using a plate washing machine, wherein the washing time is 3min each time, and the adding amount of the washing solution is 300 mu L/hole;
3) and (3) sealing: adding the confining liquid into the reaction holes of the porous plate in the step 2), and culturing for 1h at the temperature of 25 ℃;
4) washing: sucking out the confining liquid in the reaction holes of the porous plate in the step 3), washing the reaction holes for 3 times by using a plate washing machine, wherein the washing time is 3min each time, and the adding amount of the washing liquid is 300 mu L/hole;
5) and (3) stabilizing: adding 100 mu L of stabilizer into each reaction hole of the multi-hole plate in the step 4), and incubating for 1h at 25 ℃;
6) and (3) drying: sucking out the stabilizing agent in the reaction holes of the porous plate in the step 5), turning the porous plate to enable the plate to face downwards, placing the plate in a constant-temperature incubator at the set temperature of 37 ℃, drying for 1 hour to obtain the coating plate, and taking out the coating plate and sealing the coating plate by using a sealing plate film;
7) packaging: and (3) placing the ELISA coated plate sealed in the step 6) into an aluminum foil bag, placing 1 drying agent in each bag, and sealing.
The ELISA kit of the comparative example comprises the following components:
1) the ELISA kit of this comparative example coated plates;
2) the sample diluent is filled in a bottle of 1 ml of 60ml, wherein the model of the sample diluent is I028, the manufacturer is Cygnus company, the supplier is Chongqing Huaya stem cell technology Co., Ltd, and the I028 sample diluent comprises the following components in percentage by mass: 97 percent of distilled water, 1.0 percent of Tris Hydrochloride, 0.95 percent of sodium chloride, 1 percent of bovine serum albumin,
300Preservative (Preservative) 0.05%;
3) washing liquid 1 bottle, 125ml dress, wherein, the content of each raw materials in per 1L washing liquid is: 75g/L of sodium chloride, 22.575g/L of monopotassium phosphate, 33.8g/L of disodium hydrogen phosphate dodecahydrate, 1.9g/L of potassium chloride and 0.3% of Tween-20 in final concentration;
4) the stop solution is 1 bottle and 20ml, and the stop solution is 2mol/L sulfuric acid solution;
5) 1 bottle of TMB substrate liquid, 30ml of TMB substrate liquid is filled, the manufacturer is Italy kementec company, and the product number is 4380;
6) 1 bottle of positive control serum, 3ml are filled, and the positive control serum is pig serum with specific PRRSV antibody;
7) 1 bottle of negative control serum, 3ml of negative control serum is contained, and the negative control serum is pig serum without specific PRRSV antibody;
8) two or five sealing plate membranes;
9) the PRRSV HRP labeled antibody 1 bottle is packed by 30ml, and the manufacturer is Nanjing Lai Richcel biotechnology limited company with the product number of C00557H.
Performance test
Information of the enzyme-linked immunosorbent assay device:
1) the type of the detector is as follows: and (4) an Epoch.
2) Software version: 3.03.14.
3) the sequence number of the detector is as follows: 170719A.
4) Plate type 96WELL PLATE.
5) The detection wavelength is 450 nm.
6) And (5) detecting the speed to be normal.
7) Delay 100 msec.
8) Measurement/data point: 8.
The detection method comprises the following steps:
1. reagent preparation
1) All reagents were returned to room temperature (25. + -. 3 ℃ C.) and used.
2) Preparation of 1 time washing solution: the 10-fold washing solution was diluted 10-fold with ultrapure water, and the diluted washing solution was stored at 2-8 ℃ for at most 1 week.
2. Sample preparation
1) A fresh whole blood sample of the pig is collected, and then the serum sample is obtained by taking supernatant after centrifugation.
2) Serum samples were stored at 2-8 ℃ (up to 5 days).
3. Sample dilution
Samples were diluted 50-fold: 245 μ L of sample diluent was added to each well of the dilution plate, and 5 μ L of serum sample was added to each well and mixed well. (negative control serum and positive control serum were not diluted)
4. Procedure for the preparation of the
1) Taking out all components of the ELISA kit, placing the ELISA kit in a constant temperature box (25 +/-3 ℃) for 60 minutes, and recovering to room temperature;
2) adding 100 mu L of sample diluted by 50 times in a dilution plate in advance into a reaction hole of an ELISA kit coated plate;
3) adding 100 mu L of undiluted negative control serum (NC) and undiluted positive control serum (PC) into four reaction wells of an ELISA kit coated plate, wherein two reaction wells of the negative control serum (NC) and the positive control serum (PC) are respectively marked as NC1 and NC2, and two reaction wells of the positive control serum (PC) are marked as PC1 and PC 2;
4) covering a sealing plate membrane, and incubating for 30 minutes at room temperature (25 +/-3 ℃);
5) washing the plate by using an ELISA plate washing machine or a micropipette, discarding liquid in the hole, adding 300 mu L of 1 times washing liquid into each hole, washing for three times, 3min each time, and slightly patting the ELISA kit coated plate on absorbent paper after the last washing;
6) adding 100 mu L PRRSV HRP labeled antibody into each well;
7) covering a sealing plate membrane, and incubating for 30 minutes at room temperature (25 +/-3 ℃);
8) repeating the step 5);
9) adding 100 mu L of TMB substrate solution into each hole;
10) covering a sealing plate membrane, and incubating for 15 minutes at the temperature of 25 +/-3 ℃;
11) adding 50 mu L of stop solution into each hole to stop the enzymatic reaction;
12) measuring absorbance values using a detector at a wavelength of 450 nm;
13) and judging and calculating a result.
5. Determination
1) Conditions for establishing experiment
The positive control OD (450nm) mean value is more than 0.4;
negative control OD (450nm) mean < 0.4;
if the detection result is not in the defined range, the detection needs to be carried out again.
2) Calculation method
NC OD (450nm) average value ═ NC1+ NC 2)/2;
PC OD (450nm) average value ═ (PC1+ PC 2)/2;
3) sample S/P value calculation formula
4) Criteria for determination
Positive: S/P is more than or equal to 0.4
Negative: S/P is less than or equal to 0.4
The kit of example 9 was stored at 4 ℃ under dry conditions, samples were taken out every three months for testing, and spot-checked to the 24 th month, and the OD value change trends of finished product shelf life tests NC and PC of the ELISA kit of example 9 are shown in fig. 1.
Taking the kit of example 9, storing at 4 ℃ under dry condition, taking out samples every three months for detection, sampling and checking to 24 months, diluting the samples according to different concentrations to prepare 8 standard samples (CAL1-CAL8), wherein the diluted concentration gradient is as follows: (CLA 1: 250 times, CLA 2: 500 times, CLA 3: 1000 times, CLA 4: 2000 times, CLA 5: 4000 times, CLA 6: 16000 times, CLA 7: 64000 times, CLA 8: 128000 times), the OD values of the 8 standards were measured, and the test results of the OD value change trend of the finished product shelf life test of the ELISA kit of example 9 are shown in FIG. 2.
Taking the kit of example 9, storing at 4 ℃ under dry condition, taking out samples every three months for detection, sampling and checking to 24 months, diluting the samples according to different concentrations to prepare 8 standard samples (CAL1-CAL8), wherein the diluted concentration gradient is as follows: (CLA 1: 250 times, CLA 2: 500 times, CLA 3: 1000 times, CLA 4: 2000 times, CLA 5: 4000 times, CLA 6: 16000 times, CLA 7: 64000 times, CLA 8: 128000 times), the OD values of the 8 standards were measured, and the results of the test of the variation tendency of the S/P value in the product shelf life test of the ELISA kit of example 9 are shown in FIG. 3.
The ELISA kits of examples 1 to 16 and comparative examples 1 to 2 were stored at 4 ℃ under dry conditions, and were sampled every month until the 12 th month, and CV values were measured, and the data of the CV values in the experiments of the ELISA kits of examples 1 to 16 and comparative examples 1 to 2 are shown in Table 5.
TABLE 5 CV values of the experiments of ELISA kits of examples 1 to 16 and comparative examples 1 to 2
Analyzing examples 1-5 and comparative examples 1-2 in combination with table 5, it can be seen that the corn thioredoxin significantly reduced the experimental CV value of the ELISA kit, the CV value was still lower than 6 after 12 months of continuous spot inspection, and the data testing stability was better, whereas the ELISA kits of comparative examples 1 and 2 failed in the fourth month and the fifth month, respectively.
As can be seen from the analysis of example 9 and the combination of fig. 1, in the sampling test data of twenty-four months, the OD values of the negative control serum (NC) and the OD value of the positive control serum (PC) of the ELISA kit of example 9 are relatively average, and the data fluctuation is small, which represents the good data test stability.
Analysis of example 9 and fig. 2 shows that the OD values of the serum samples at different dilution times are more average in the twenty-four month spot check test, the data fluctuation is small, the end point titer exceeds 2000 times, and the sensitivity is better.
Analysis example 9 and combination of fig. 3 show that the S/P value of the serum samples with different dilution times has smaller fluctuation in the twenty-four month spot check test, and it can be clearly seen that the stability of the test data of the ELISA kit is better in a longer spot check period.
Comparing example 9 and examples 6 to 8 with table 5, it can be seen that the influence of soybean thioredoxin, corn thioredoxin, swine thioredoxin and silkworm thioredoxin on the stability of the data test of the ELISA kit is not very different, wherein the stabilization effect of swine thioredoxin is slightly better. In summary, the stabilization effect of animal thioredoxin is better than that of plant thioredoxin.
Comparing examples 9-10, examples 6-8 and combining table 5, it can be seen that the stabilization effect of recombinant bacterial thioredoxin is better than that of soybean thioredoxin, corn thioredoxin, pig thioredoxin and silkworm thioredoxin, wherein the stabilization performance of recombinant escherichia coli thioredoxin is better than that of recombinant bacillus subtilis thioredoxin.
Comparing examples 9-10, examples 11-13 and with Table 5, it can be seen that the addition of mussel mucin further improves the stability of the data test of the ELISA kit, and that the effect is better when the amount of mussel mucin added is 0.02 g.
As can be seen by comparing example 12, examples 14-16 and Table 5, the addition of hydroxytyrosol, which is synergistic with mussel mucin, further improves the stability of data testing of ELISA kits.
As described above, when examples 1 to 16 and comparative examples 1 to 2 are analyzed in combination with table 2 and fig. 1 to 3, it can be seen that the ELISA kit of the present application has advantages of long shelf life and small CV value of test data of the kits of different shelf life and lot.
The present embodiment is only for explaining the present application, and it is not limited to the present application, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present application.
Claims (10)
1. A stabilizer for an ELISA kit coated plate is characterized in that: the paint comprises water and the following components in percentage by mass: 5 to 15 percent of glycerol, 0.5 to 1.5 percent of trehalose, 0.005 to 0.015 percent of sodium glutamate and 0.002 to 0.008 percent of thioredoxin.
2. The stabilizer for ELISA kit coated plate according to claim 1, wherein: the paint comprises water and the following components in percentage by mass: 8 to 12 percent of glycerol, 0.7 to 1.3 percent of trehalose, 0.008 to 0.012 percent of sodium glutamate and 0.003 to 0.007 percent of thioredoxin.
3. The stabilizer for ELISA kit coated plate according to claim 2, wherein: the thioredoxin is recombinant bacterial thioredoxin.
4. The stabilizer for ELISA kit coated plate according to claim 1, wherein: the weight ratio of the thioredoxin to the glycerol is 1 (1000-3000).
5. The stabilizer for ELISA kit coated plate according to claim 1, wherein: the components also comprise 0.001-0.003 mass percent of mussel mucin.
6. The stabilizer for ELISA kit coated plate according to claim 5, wherein: the components also comprise 0.5 to 1.2 mass percent of hydroxytyrosol.
7. The stabilizer for ELISA kit coated plate according to claim 6, wherein: the weight ratio of the mussel mucin to the hydroxytyrosol is 1 (200-600).
8. A method of preparing a stabiliser for an ELISA coated plate as claimed in any one of claims 1 to 2 or 4 wherein: the method comprises the following steps:
s1, mixing glycerol, trehalose, sodium glutamate and water uniformly, and adjusting the pH value to 7 to obtain a mixed solution;
s2, adding thioredoxin into the mixed solution obtained in the step S1, and uniformly mixing to obtain the thioredoxin.
9. An ELISA kit coating plate, which is characterized in that: the method comprises the following steps: 1) coating: diluting the antigen or the antibody by using a buffer solution to obtain a coating solution, adding the coating solution into a reaction hole of a porous plate, and culturing; 2) washing: sucking out the coating solution cultured in the reaction holes of the porous plate in the step 1), and washing the reaction holes by using a washing solution; 3) and (3) sealing: adding confining liquid into the reaction holes of the porous plate washed in the step 2) for cultivation; 4) washing: sucking out the sealing liquid sealed in the reaction holes of the porous plate in the step 3), and washing the reaction holes by using a washing liquid; 5) and (3) stabilizing: adding the stabilizer for the ELISA coated plate according to any one of claims 1 to 7 into each reaction well after washing of the multi-well plate in the step 4), and incubating; 6) and (3) drying: sucking out the stabilizing agent in the reaction holes of the porous plate in the step 5), and drying the porous plate to obtain the composite stabilizer.
10. An ELISA kit, characterized in that: comprises the following components: a sample diluent, a stop solution and an ELISA kit coated plate according to claim 9.
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