CN110907637A - Influenza virus nucleoprotein antigen protective solution, reagent containing same and kit - Google Patents
Influenza virus nucleoprotein antigen protective solution, reagent containing same and kit Download PDFInfo
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Abstract
The invention provides an influenza virus nucleoprotein antigen protective solution, a reagent containing the same and a kit, and relates to the technical field of diagnostic reagents. The influenza virus nucleoprotein antigen protection solution comprises (a) 20-50 mM Tris; (b)0.5 to 2.5mM aminopyridine; (c) 0.5-10 w/v% bovine serum albumin and/or 0.5-10 w/v% fetal bovine serum; and, optionally, an adjuvant. The influenza virus nucleoprotein antigen protective solution can enable influenza virus nucleoprotein serving as an antigen to have higher reactivity and signal-to-noise ratio, and can enable the influenza virus nucleoprotein antigen to keep better thermal acceleration stability and real-time stability by taking the influenza virus nucleoprotein antigen protective solution as a diluent of the influenza virus nucleoprotein antigen and a coating diluent for coating the influenza virus nucleoprotein antigen on a carrier.
Description
Technical Field
The invention relates to the technical field of diagnostic reagents, in particular to an influenza virus nucleoprotein antigen protective solution, a reagent containing the same and a kit.
Background
Influenza, abbreviated as influenza, is an acute respiratory infectious disease caused by influenza virus. Influenza virus belongs to the family Orthomyxoviridae (Orthomyxoviridae), and influenza virus belongs to the genus, single-stranded negative-sense RNA virus. Influenza viruses can be divided into three serotypes, namely A (A), B (B) and C (C), according to the difference of antigenicity of a nucleocapsid protein and a matrix membrane protein, wherein the influenza A has rapid variation and extremely strong pathogenicity to human beings, poultry and other mammals, and can cause a pandemic worldwide. Influenza b viruses are relatively slow in variation and can only cause small epidemics on a local scale. Influenza c viruses have the slowest mutation and weak pathogenic virulence, and can only infect pregnant women and children with low resistance. Influenza b and c are generally found only in humans. The three types A, B and C not only reflect the chronological order of virus separation, but also reflect the degree of harm to human.
Influenza a viruses are further classified into 16H subtypes (H1-H16) and 9N subtypes (N1-N9) according to antigenic differences between virus surface antigen glycoproteins Hemagglutinin (HA) and Neuraminidase (NA). Influenza virions consist of approximately 1% RNA, 70% protein, 20% lipid, and 5-8% carbohydrate. Lipids are located in the viral membrane, mostly phospholipids, with small amounts of cholesterol and glycolipids. HA, NA, inner membrane matrix protein (M1) and Nucleoprotein (NP) are the main polypeptide molecules causing the antigenicity of the virus, and the specificity of the virus is determined by the high variability of the genes, and different subtypes of virus particles are formed.
The influenza Nucleoprotein (NP) is a non-glycosylated protein encoded by segment 5 and has a molecular weight of about 56kD of 498 amino acids. The influenza virus NP protein is very conservative, the amino acid similarity of homotype influenza virus NP is more than 90%, the gene variation rate is very low, and the influenza virus NP protein has high specificity, so the influenza virus NP protein is an ideal target protein in the serological detection of influenza virus infection.
At present, the reagent kit for detecting influenza by using an immunological method is mainly divided into two types, one type is an influenza antibody detection reagent kit for detecting whether an immune system generates an antibody aiming at influenza virus after the influenza infection, and the other type is an influenza antigen detection reagent kit for detecting whether influenza virus antigens exist in nasal cavities, oral cavities and serum samples after the influenza infection; the influenza NP antigen in the influenza antibody rapid detection reagent can be used as a coating or a marker for the detection reagent. The stability of the influenza NP antigen is therefore particularly important for the stability of the influenza detection reagent. Particularly, when the influenza NP antigen is applied to an in vitro diagnostic reagent for detecting the influenza antigen or antibody, the problem of poor stability of the influenza NP antigen after the influenza NP antigen is solidified under the condition of a liquid buffer solution or on carriers such as a nitrocellulose membrane, a cellulose acetate membrane and the like exists. Therefore, a product capable of improving the stability of influenza NP antigen is needed in the market today.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first purpose of the present invention is to provide an influenza virus nucleoprotein antigen protective solution which can improve the stability of an influenza virus nucleoprotein antigen.
The second object of the present invention is to provide a reagent comprising the influenza virus nucleoprotein antigen-protecting solution.
The third object of the present invention is to provide a protective solution for an influenza virus nucleoprotein antigen or a kit comprising the above-mentioned reagent.
In order to solve the technical problems, the invention adopts the following technical scheme:
according to one aspect of the invention, the invention provides an influenza virus nucleoprotein antigen protection solution, which comprises the following components:
(a)20～50mM Tris；
(b)0.5 to 2.5mM aminopyridine;
(c) 0.5-10 w/v% bovine serum albumin and/or 0.5-10 w/v% fetal bovine serum;
and optionally, adjuvants.
Preferably, the pH of the influenza virus nucleoprotein antigen protection solution is 8.0-8.5.
Preferably, the concentration of the Tris is 20-30 mM, and preferably 20 mM.
Preferably, the concentration of the bovine serum albumin is 0.5-5 w/v%, preferably 0.5-1 w/v%, and more preferably 0.5 w/v%.
Preferably, the concentration of the fetal calf serum is 1-5 w/v%.
Preferably, the adjuvant comprises at least one of a salt, a surfactant and a preservative;
preferably, the concentration of the surfactant is 0.05-0.2 w/v%, preferably 0.05-0.1 w/v%, and more preferably 0.1 w/v%;
preferably, the surfactant comprises Tween 20;
preferably, the concentration of the salt is 100-200 mM, preferably 120-160 mM, more preferably 150 mM;
preferably, the salt comprises NaCl;
preferably, the preservative comprises ProClin series preservatives;
preferably, the preservative comprises ProClin 300;
preferably, the concentration of the ProClin300 is 0.01-0.1 w/v%, preferably 0.05-0.1 w/v%, and more preferably 0.05 w/v%.
Preferably, the adjuvant comprises at least one of a salt, a surfactant, a preservative, a lyophilization stabilizer, and a drying stabilizer.
Preferably, the influenza virus nucleoprotein antigen protection solution comprises the following components: 100-200 mM NaCl, 0.1-0.2 w/v% Tween20, 0.05-0.1 w/v% ProClin300, 20-50 mM Tris, 0.5-1 w/v% bovine serum albumin, 0.5-2.5 mM aminopyridine; the pH value of the influenza virus nucleoprotein antigen protection solution is 8.0-8.5.
According to another aspect of the present invention, there is also provided an agent comprising the influenza virus nucleoprotein antigen protective solution described above;
preferably, the agent further comprises an influenza virus nucleoprotein.
According to another aspect of the present invention, there is also provided a kit comprising the above-mentioned influenza virus nucleoprotein antigen-protecting solution, or the above-mentioned reagent;
preferably, the kit comprises an influenza antigen detection kit;
preferably, the kit comprises an influenza antibody detection kit.
Compared with the prior art, the invention has the following beneficial effects:
the influenza virus nucleoprotein antigen protection solution provided by the invention mainly comprises a buffer substance, a protein protective agent and an antioxidant, wherein Tris is used as the buffer substance, bovine serum albumin and/or fetal bovine serum is used as the protein protective agent, aminopyridine is used as the antioxidant, and the influenza virus nucleoprotein used as the antigen has higher reactivity and signal-to-noise ratio by reasonably proportioning the dosage of each component.
The influenza virus nucleoprotein antigen protective solution is used as an antigen diluent and a coating diluent for coating an antigen on a carrier, so that the influenza virus nucleoprotein antigen can keep better thermal acceleration stability and real-time stability: the influenza virus nucleoprotein antigen can maintain good reactivity when being stored for twelve months at the temperature of 2-8 ℃; and the reactivity of the NP antigen after 8 days of treatment at 37 ℃ is similar to that of the NP antigen stored at 2-8 ℃. The influenza virus nucleoprotein antigen protection solution provided by the invention improves the stability of influenza antigens after solidification under the condition of liquid buffer solution or on carriers such as nitrocellulose membranes, cellulose acetate membranes and the like, so that the influenza antigens can be better applied to the detection of target antibodies/antigens.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
According to one aspect of the invention, the invention provides an influenza virus nucleoprotein antigen protection solution which mainly comprises a buffer substance, a protein protective agent and an antioxidant.
Tris is used as a buffer substance, Tris is Tris hydroxymethyl aminomethane, and Tris is used as a buffer substance of an influenza virus nucleoprotein antigen protective solution, so that the reactivity of NP protein as an antigen can be effectively improved. The concentration of Tris in the influenza virus nucleoprotein antigen protection solution is 20-50 mM, for example, but not limited to, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM or 50mM, preferably 20-30 mM, more preferably 20 mM. Tris is used as a buffer substance and can be prepared into a Tris buffer solution, and the Tris buffer solution is mixed with other components after the pH value is adjusted to form the influenza virus nucleoprotein antigen protective solution. The pH of the influenza virus nucleoprotein antigen protection solution provided by the invention is preferably 8.0-8.5, and can be, for example but not limited to, 8, 8.1, 8.2, 8.3, 8.4 or 8.5.
Bovine Serum Albumin (BSA) and/or fetal bovine serum are/is used as a protein protective agent, and the BSA and the fetal bovine serum respectively have the functions of preventing protein denaturation and decomposition and stabilizing protein. Optionally, the protein protectant employs BSA; optionally, the protein protectant employs fetal bovine serum; optionally, the protein protectant is used in combination with BSA and fetal bovine serum. When BSA is contained in the influenza virus nucleoprotein antigen-protecting solution, the concentration of BSA may be 0.5 to 10 w/v%, for example, but not limited to, 0.5 w/v%, 1 w/v%, 2 w/v%, 3 w/v%, 4 w/v%, 5 w/v%, 6 w/v%, 7 w/v%, 8 w/v%, 9 w/v%, or 10 w/v%, preferably 0.5 to 1 w/v%, more preferably 0.5 w/v%. When the influenza virus nucleoprotein antigen protective solution contains fetal bovine serum, the concentration of the fetal bovine serum is 0.5-10 w/v%, for example, but not limited to, 0.5 w/v%, 1 w/v%, 2 w/v%, 3 w/v%, 4 w/v%, 5 w/v%, 6 w/v%, 7 w/v%, 8 w/v%, 9 w/v%, or 10 w/v%, preferably 1-5 w/v%.
Aminopyraline is used as an antioxidant, and the concentration of the Aminopyraline in the influenza virus nucleoprotein antigen protection solution is 0.5-2.5 mM, and can be, for example, but not limited to, 0.5mM, 1mM, 1.5mM, 2mM or 2.5 mM.
The influenza virus nucleoprotein antigen protection solution provided by the invention can also comprise conventional auxiliary materials optionally selected in the field, and in some preferred embodiments, the auxiliary materials comprise at least one of salt, surface activity, preservative, freeze-drying stabilizer and drying stabilizer. Optionally one of salt, surfactant, preservative, freeze-drying stabilizer or oven-drying stabilizer; optionally containing salts and surface active agents; optionally a preservative and a kinetic stabilizer; optionally containing a surfactant and a drying stabilizer. In some preferred embodiments, the influenza virus nucleoprotein antigen protection solution comprises a salt, a surfactant and a preservative.
Wherein, alternative examples of salts include but are not limited to NaCl, Na2SO4、KCl、Mg2Cl、NH4Cl、K2SO4、Mg2SO4Or (NH)4)2SO4The amount of the salt is preferably 100 to 200mM, and may be, for example, but not limited to, 100mM, 110mM, 120mM, 130mM, 140mM, 150mM, 160mM, 170mM, 180mM, 190mM or 200mM, and the salt concentration is preferably 120 to 160mM, more preferably 150 mM.
Examples of the surfactant include, but are not limited to, Tween20, Tween80, NP40, etc., and the amount of the surfactant is preferably 0.05 to 0.2 w/v%, and for example, may be, but is not limited to, 0.05 w/v%, 0.1 w/v%, 0.15 w/v%, or 0.2 w/v%, preferably 0.05 to 0.1 w/v%, more preferably 0.1 w/v%.
Examples of preservatives include, but are not limited to, sodium azide, thimerosal, antibiotics, or ProClin-series preservatives, etc., preferably ProClin-series preservatives are used, the active ingredients of which are mainly 2-methyl-4-isothiazolin-3-one (MCI) and 5-chloro-2-methyl-4-isothiazolin-3-one (CMCI). MCI and CMCI can inhibit cell growth and promote apoptosis, can effectively control the growth of microorganisms in-vitro diagnostic reagents, have broad-spectrum antibacterial activity, and can inhibit the growth of microorganisms such as bacteria, fungi, yeasts and the like in a relatively long time. ProClin series preservatives include, but are not limited to ProClin150, ProClin200, ProClin300 or ProClin5000, ProClin300 being preferably used. The concentration of ProClin300 is preferably 0.01-0.1 w/v%, and may be, for example, but not limited to, 0.01 w/v%, 0.02 w/v%, 0.03 w/v%, 0.04 w/v%, 0.05 w/v%, 0.06 w/v%, 0.07 w/v%, 0.08 w/v%, 0.09 w/v%, or 0.1 w/v%, preferably 0.05-0.1 w/v%, and more preferably 0.05 w/v%.
The concentration of each component in the influenza virus nucleoprotein antigen protection solution refers to the concentration of each component in the influenza virus nucleoprotein antigen protection solution when the influenza virus nucleoprotein antigen protection solution is used. It is understood that the influenza virus nucleoprotein antigen protective solution can also be prepared into a concentrated solution, such as a 2 × influenza virus nucleoprotein antigen protective solution, a 5 × influenza virus nucleoprotein antigen protective solution or a 10 × influenza virus nucleoprotein antigen protective solution; and dry powder preparations, etc., which are diluted or dissolved to the use concentration of the influenza virus nucleoprotein antigen protective solution when used, it is understood that these other forms of influenza virus nucleoprotein antigen protective solutions also belong to the influenza virus nucleoprotein antigen protective solutions to be protected by the present invention.
The influenza virus nucleoprotein antigen protection solution composed of the components and the formula amount can enable NP protein as an antigen to have higher reactivity and signal-to-noise ratio. The influenza virus nucleoprotein antigen protective solution is used as a dilution solution of NP antigen and a coating dilution solution for coating the NP antigen on a carrier, so that the NP antigen can keep better thermal acceleration stability and real-time stability: the NP protein can maintain good reactivity when stored for twelve months at the temperature of 2-8 ℃; and after treating at 37 ℃ for 8 days, the reactivity was similar to that of NP protein stored at 2-8 ℃. Therefore, the influenza virus nucleoprotein antigen protection solution provided by the invention can keep good antigen activity of NP protein after long-time storage, and the reactivity of the NP protein can be improved by using the influenza virus nucleoprotein antigen protection solution.
In some preferred embodiments, the influenza virus nucleoprotein antigen protection solution consists of NaCl, Tween20, ProClin300, Tris buffer, BSA and aminopyridine. The preferred formula of the influenza virus nucleoprotein antigen protection solution is as follows: 100-200 mM NaCl, 0.1-0.2 w/v% Tween20, 0.05-0.1 w/v% ProClin300, 20-50 mM Tris, 0.5-1 w/v% bovine serum albumin, 0.5-2.5 mM aminopyridine; the pH value of the influenza virus nucleoprotein antigen protection solution is 8.0-8.5.
According to another aspect of the present invention, there is also provided a reagent comprising the above-mentioned influenza virus nucleoprotein antigen-protecting solution, which can be used as a stock solution for storing NP protein; a diluent for diluting the NP protein; the NP protein is used as a diluent and a working solution when the antigen is used; or a coating diluent in which the NP protein is coated on a carrier as an antigen. In some preferred embodiments, the reagent comprises an influenza virus nucleoprotein antigen protective solution and an NP antigen, and the reagent can be used as a positive quality control product, a calibrator, an internal reference product and the like in an influenza antigen detection product; can also be used as coating or label in influenza antibody detection products.
According to another aspect of the present invention, there is also provided a kit comprising the above-mentioned influenza virus nucleoprotein antigen-protecting solution, or the above-mentioned reagent. In some alternative embodiments, the kit comprises an influenza antibody detection kit, and the influenza virus nucleoprotein antigen protective solution can be used as a protective solution for an NP antigen of a positive quality control product, a calibrator or an internal reference product in the kit, or the reagent containing the NP antigen and the influenza virus nucleoprotein antigen protective solution can be directly used as the positive quality control product, the calibrator or the internal reference product in the kit. In some alternative embodiments, the kit comprises an antibody detection kit comprising an influenza virus nucleoprotein antigen protection solution as described above as a coating diluent for the NP antigen. When used as a coating diluent, the influenza virus nucleoprotein antigen protective solution preferably further comprises a lyophilization stabilizer or an oven drying stabilizer. The influenza virus nucleoprotein antigen protection solution in the kit can enable NP antigen in the kit to have high reactivity and signal to noise ratio, and the kit contains the influenza virus nucleoprotein antigen protection solution, so that the kit can be stored for a long time, and the NP antigen in the kit has good reactivity after being stored for a long time. It will be appreciated that reagents conventional in the art, including but not limited to buffers, eluents, chromogenic reagents and labels, and the like, may also be included in the kit; consumables conventional in the art may also be included, including but not limited to reagent vials, microplate, centrifuge tubes, or pipette tips, among others.
The technical solution and the advantages of the present invention will be further explained with reference to the preferred embodiments.
Wherein the influenza NP antigen is from Wuhan virus research institute, and the purity of the recombinant antigen is more than 90%.
Influenza antibody/antigen positive samples: is provided by a maternal and child health care institute in Zhuhai City.
The influenza antibody/antigen detection reagent was prepared by the beauty bead reagent company itself.
Example 1
BSA, NaCl, Tween20 and ProClin300 were dissolved in the following buffers, respectively: phosphate (pH7.4), citric acid buffer (pH6.0), 2-morpholinoethanesulfonic acid (pH7.4), borate (pH8.0), carbonate (pH9.0), acetate (pH5.0), 4-hydroxyethylpiperazineethanesulfonic acid (pH7.0), Tris buffer (pH7.4) to concentrations of BSA0.5 w/v%, NaCl150mM, Tween 200.1w/v% and ProClin 3000.05w/v%, respectively; the concentration of the buffer substance in each buffer was 20mM, and the pH value in parentheses was the pH of the buffer. The influenza NP antigens with different concentrations are prepared by diluting influenza virus nucleoprotein antigen protective solution prepared by different buffer solutions and are used as samples, and a fluorescent influenza antigen detection reagent (double-antibody sandwich method) is adopted to detect the influenza NP antigens and record a fluorescent signal value. The reactivity was measured with an influenza antigen detection reagent.
The results are shown in Table 1.
TABLE 1
As can be seen from the above, the influenza virus nucleoprotein antigen protection solution prepared by taking Tris as a buffer substance as a basic buffer solution has higher reactivity and signal-to-noise ratio (higher fluorescence signal value compared with a negative sample) for antigen sample samples with different concentrations. Therefore, the influenza virus nucleoprotein antigen protective solution taking Tris as a buffer substance has better effect.
Example 2
BSA, NaCl, Tween20 and ProClin300 were dissolved in Tris buffer solutions of the respective concentrations and the respective pHs shown in Table 2, respectively, until the concentrations of the respective components were BSA0.5 w/v%, NaCl150mM, Tween 200.1 w/v% and ProClin 3000.05w/v%, respectively, and the prepared influenza virus nucleoprotein antigen protective solutions were diluted to prepare influenza NP antigens of different concentrations as samples, which were detected with a fluorescent influenza NP antigen detection reagent (double antibody sandwich method) and the fluorescent signal values were recorded. The results are shown in Table 2.
TABLE 2
As can be seen from the above, the fluorescence signal value of the influenza virus nucleoprotein antigen protection solution is high at pH 8-8.5, and the fluorescence signal value of Tris is high at concentration of 20-50 mM in the influenza virus nucleoprotein antigen protection solution, wherein the effects of 20mM Tris buffer (pH8.0) and 20mM Tris buffer (pH8.5) are the best, and the reactivity and the signal-to-noise ratio (higher fluorescence signal value compared with negative samples) of antigen sample samples with different concentrations are high.
Example 3
An influenza virus nucleoprotein antigen protection solution was prepared containing 20mM Tris (pH8.0), 150mM NaCl, 0.1 w/v% Tween20 and 0.05 w/v% ProClin300 and protein protectants as shown in Table 3, including Bovine Serum Albumin (BSA), casein, fetal bovine serum, horse serum, at concentrations ranging from 0.5 w/v% to 10 w/v%. Influenza NP antigens with different concentrations are prepared by diluting the influenza virus nucleoprotein antigen protective solution and are used as samples to examine the reactivity of the samples by using an influenza antigen detection reagent, and the results are shown in a table 3:
TABLE 3
As can be seen from the above, the effect of using BSA and fetal calf serum as protein protectant is better than that of other protein protectant, wherein 0.5 w/v% of bovine serum albumin, 1 w/v% of fetal bovine serum and 5 w/v% of fetal bovine serum as protein protectant have higher reactivity and signal-to-noise ratio (higher fluorescence signal value relative to negative sample) for antigen sample samples with different concentrations, and at the same time, according to the empirical value, the sample gradient is from 5ng/ml to 50ng/ml, and the fluorescence signal value is more reasonable by 6-7 times, so at least one of 0.5 w/v% of bovine serum albumin, 1 w/v% of fetal bovine serum and 5 w/v% of fetal bovine serum is more preferably used as protein protectant.
Example 4
An influenza virus nucleoprotein antigen protective solution was prepared containing 20mM Tris (pH8.0), 150mM NaCl, 0.1 w/v% Tween20, 0.05 w/v% ProClin300 and antioxidants (including mercaptoethanol, dithiothreitol, aminopyridine and reduced glutathione) as shown in Table 4 in the order of 0.1mM to 5 mM. Influenza NP antigens with different concentrations are prepared by diluting the influenza virus nucleoprotein antigen protective solution and used as samples, and the reactivity of the samples is examined by using an influenza antigen detection reagent, and the results are shown in Table 4.
TABLE 4
As can be seen from the above, the influenza virus nucleoprotein antigen protection solution containing 0.5 mM-2.5 mM aminopyridine has higher reactivity and signal to noise ratio (higher fluorescence signal value compared with negative samples) for different concentrations of antigen sample samples.
Example 5
Configuration method
1. This example uses an influenza virus nucleoprotein antigen protection solution of the following formulation: 20mM Tris, 150mM NaCl, 0.5 w/v% BSA, 0.2 w/v% T ween20, 2.5mM aminopyridine and 0.1 w/v% ProClin300 preservative, the pH of the influenza virus nucleoprotein antigen protection solution being 8.0. The influenza virus nucleoprotein antigen protection solution is filtered by a filter membrane with the diameter of 0.2 mu m for later use.
2. The influenza virus nucleoprotein antigen protective solution is used as a diluent, an antigen mother solution is diluted to be 0.5ng/ml, 5ng/ml, 10ng/ml, 50ng/ml, 100ng/ml and 500ng/ml to be used as an antigen calibrator, and the influenza virus nucleoprotein antigen protective solution is used to be 20ng/ml to be used as an antigen positive quality control.
3. The antigen is subpackaged according to 1.0 ml/tube, sealed and stored. The long-term storage stability of the calibrator and the quality control products stored at the temperature of 2-8 ℃ is checked, and the thermal acceleration stability of the thermal acceleration treatment at the temperature of 37 ℃ is checked. The stability assessment reagent is an influenza antigen detection kit (fluorescence chromatography) prepared from a Zhuhaili bead reagent.
And (3) testing results:
(1) and (3) performing 37 ℃ thermal accelerated stability reactivity examination, taking out 0.5ng/ml, 5ng/ml, 10ng/ml, 50ng/ml, 100ng/ml and 500ng/ml quality control products and 20ng/ml calibrator after placing for 8 days at 37 ℃, detecting with a fluorescent influenza antigen detection reagent together with the quality control products and calibrator stored at 2-8 ℃, and recording a fluorescence signal value, wherein the result is shown in Table 5.
TABLE 5
(2) And (3) performing real-time stability and reactivity investigation at the temperature of 2-8 ℃, taking out the quality control product and the calibrator which are placed at the temperature of 2-8 ℃ in the months 1, 3, 6, 9, 10, 11 and 12 respectively, detecting by using the fluorescent influenza antigen detection reagent, and recording the fluorescence signal value, wherein the result is shown in the table 6.
The experimental results show that the influenza virus nucleoprotein antigen protection solution provided by the invention can be used as an antigen diluent, so that the NP antigen thermal acceleration stability can be kept well, and the real-time stability at the temperature of 2-8 ℃ can be kept well (the fluorescence signal value is attenuated less). And (4) conclusion: the influenza virus nucleoprotein antigen protection solution is used as a diluent of an influenza NP antigen, and can keep better thermal acceleration stability and real-time stability.
Example 6
1. This example uses an influenza virus nucleoprotein antigen protection solution of the following formulation: 20mM Tris, 150mM NaCl, 0.5 w/v% BSA, 0.2 w/v% Tween20, 2.5mM aminopyridine and 0.1 w/v% ProClin300 preservative, the pH of the influenza virus nucleoprotein antigen protective solution is 8.0. The influenza virus nucleoprotein antigen protection solution is filtered by a filter membrane with the diameter of 0.2 mu m for later use.
2. And (2) taking the influenza virus nucleoprotein antigen protection solution prepared in the step (1) as a diluent, taking an influenza NP antigen diluted by the diluent as a coating, and preparing the influenza NP antigen by a fluorescence chromatography test strip preparation process. The reagent combining pad is coated with influenza antigen marked by fluorescent microspheres and anti-chicken IgY antibody, the detection object of the nitrocellulose membrane is coated with influenza NP antigen diluted by the diluent, and the quality control area is coated with chicken IgY antibody, thus preparing the detection reagent.
3. The long-term storage stability of the reagent stored at the temperature of 2-8 ℃ is checked, and the thermal acceleration stability of the reagent subjected to thermal acceleration treatment at the temperature of 37 ℃ is checked. The stability assessment sample is provided by a maternal and child health care institute in Zhuhai City.
(1) The results of the reactivity test for the thermal stability at 37 ℃ are shown in Table 7.
TABLE 7
(2) The real-time stability and reactivity at 2-8 ℃ were examined, and the results are shown in Table 8.
TABLE 8
From the experimental results, the influenza virus nucleoprotein antigen protection solution provided by the invention is used for coating detection of an influenza antibody detection reagent, the detection reagent prepared after being solidified on a nitrocellulose membrane carrier has better thermal acceleration stability, and the real-time stability at 2-8 ℃ is better maintained (the fluorescence signal value of the detection reagent is less attenuated).
And (4) conclusion: the influenza virus nucleoprotein antigen protection solution provided by the invention is used as a coating diluent of NP antigen, and can keep better thermal acceleration stability and real-time stability.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. An influenza virus nucleoprotein antigen protective solution is characterized by comprising the following components:
(a)20～50mM Tris；
(b)0.5 to 2.5mM aminopyridine;
(c) 0.5-10 w/v% bovine serum albumin and/or 0.5-10 w/v% fetal bovine serum;
and optionally, adjuvants.
2. The influenza virus nucleoprotein antigen-protecting solution of claim 1, wherein the pH of the influenza virus nucleoprotein antigen-protecting solution is 8.0 to 8.5.
3. The influenza virus nucleoprotein antigen protecting solution of claim 1, wherein the concentration of Tris is 20-30 mM, preferably 20 mM.
4. The influenza virus nucleoprotein antigen protective solution of claim 1, wherein the concentration of bovine serum albumin is 0.5-5 w/v%, preferably 0.5-1 w/v%, and more preferably 0.5 w/v%.
5. The influenza virus nucleoprotein antigen protective solution of claim 1, wherein the concentration of fetal bovine serum is 1-5 w/v%.
6. The influenza virus nucleoprotein antigen protective solution of claim 1, wherein the excipients comprise at least one of a salt, a surfactant, a preservative, a lyophilization stabilizer, and a baking stabilizer;
preferably, the concentration of the surfactant is 0.05-0.2 w/v%, preferably 0.05-0.1 w/v%, and more preferably 0.1 w/v%;
preferably, the surfactant comprises Tween 20;
preferably, the concentration of the salt is 100-200 mM, preferably 120-160 mM, more preferably 150 mM;
preferably, the salt comprises NaCl;
preferably, the preservative comprises ProClin series preservatives;
preferably, the preservative comprises ProClin 300;
preferably, the concentration of the ProClin300 is 0.01-0.1 w/v%, preferably 0.05-0.1 w/v%, and more preferably 0.05 w/v%.
7. The influenza virus nucleoprotein antigen protective solution of claim 1, wherein the excipients comprise a salt, a surfactant and a preservative.
8. The influenza virus nucleoprotein antigen protecting solution of any one of claims 1 to 7, which comprises the following components: 100-200 mM NaCl, 0.1-0.2 w/v% Tween20, 0.05-0.1 w/v% ProClin300, 20-50 mM Tris, 0.5-1 w/v% bovine serum albumin, 0.5-2.5 mM aminopyridine; the pH value of the influenza virus nucleoprotein antigen protection solution is 8.0-8.5.
9. An agent comprising the influenza virus nucleoprotein antigen protective solution of any one of claims 1 to 8;
preferably, the agent further comprises an influenza virus nucleoprotein.
10. A kit comprising an influenza virus nucleoprotein antigen protecting solution of any one of claims 1 to 8, or a reagent of claim 9;
preferably, the kit comprises an influenza antigen detection kit;
preferably, the kit comprises an influenza antibody detection kit.
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