CN107907681A - Adenovirus detection reagent card, kit and application thereof - Google Patents
Adenovirus detection reagent card, kit and application thereof Download PDFInfo
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- CN107907681A CN107907681A CN201711058958.6A CN201711058958A CN107907681A CN 107907681 A CN107907681 A CN 107907681A CN 201711058958 A CN201711058958 A CN 201711058958A CN 107907681 A CN107907681 A CN 107907681A
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Classifications
-
- 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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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Abstract
The present invention relates to emulsion technique immunochromatography field, and in particular to a kind of adenovirus detection reagent card, including sample application pad, glass fibre element film, nitrocellulose filter and the water suction gasket being arranged in order；Wherein, the first Anti-adenovirus antibody of latex particle and latex particle test antigen are contained on the glass fibre element film；The first p-wire, the second p-wire and control line are disposed with the nitrocellulose filter；First p-wire has been coated with the second Anti-adenovirus antibody；Second p-wire has been coated with Adenovirus Antigen；The control line has been coated with the antibody of the test antigen.
Description
Technical Field
The invention relates to the field of latex immunochromatography, in particular to an adenovirus detection reagent card, a kit and application thereof.
Background
All immunodiagnostic reagents, either qualitative or quantitative, are based on the principle of immunoreaction, i.e., antigen and antibody are specifically combined under certain conditions to produce antigen-antibody complex, and the antigen or antibody is labeled with tracer to finally realize the detection and analysis of reaction products.
In the prior art, an immunodiagnostic reagent card is provided with a test line, and whether a sample contains pathogen antigens or not is determined according to the color of the test line. The judgment is carried out through the color of one test line, the influence of the brightness and the color of the environment is easy to be caused, and the influence of the subjective judgment of a user is also caused, so that a larger error is generated, the accuracy of a detection result, particularly a quantitative detection result is lower, and the obstacle is brought to the diagnosis of diseases.
Disclosure of Invention
In view of the above-mentioned shortcomings of the prior art, the present invention aims to provide an adenovirus detection reagent card, a kit and a use thereof, so as to reduce detection errors and improve the accuracy of detection results.
In a first aspect, the invention provides an adenovirus detection reagent card, which comprises a sample adding gasket, a glass cellulose membrane, a nitrocellulose membrane and a water absorption gasket which are arranged in sequence; wherein the glass cellulose membrane contains latex particles-a first anti-adenovirus antibody and latex particles-a test antigen; the nitrocellulose membrane is sequentially provided with a first test line, a second test line and a control line; the first test line is coated with a second anti-adenovirus antibody; the second test line is coated with adenovirus antigen; the control line is coated with an antibody to the test antigen.
In one embodiment of the invention, the test antigen is rabbit IgG; the antibody of the test antigen is goat anti-rabbit IgG.
In one embodiment of the invention, the first anti-adenoviral antibody and/or the second anti-adenoviral antibody is a monoclonal antibody.
In one embodiment of the present invention, the latex particle in the latex particle-first anti-adenovirus antibody and the first adenovirus antibody are linked by chemical coupling, and/or the latex particle in the latex particle-test antigen and the test antigen are linked by chemical coupling.
In a second aspect, the present invention provides a method for preparing an adenovirus detection reagent card according to the first aspect, comprising the following steps: 1) spraying a mixed solution of latex particles, a first anti-adenovirus antibody and latex particles and a test antigen on a glass cellulose membrane; 2) carrying out first pretreatment on a nitrocellulose membrane corresponding to a T1 line to be spotted by using a first pretreatment solution, and spotting a second anti-adenovirus antibody solution on the nitrocellulose membrane subjected to the first pretreatment; 3) carrying out second pretreatment on a nitrocellulose membrane corresponding to a T2 line to be spotted by using a second pretreatment solution, and spotting an adenovirus antigen solution on the nitrocellulose membrane subjected to the second pretreatment; 4) and spraying an antibody solution of the test antigen on the nitrocellulose membrane at a preset position.
In one embodiment of the present invention, the first pretreatment solution is an aqueous solution containing 1.5-2g/L arginine, 1.2-1.5g/L potassium hydrogen phthalate, 0.3-0.5g/L sodium hydroxide, and 2-2.5g/L citric acid. In one embodiment of the invention, the second pretreatment solution is an aqueous solution containing 2-2.5g/L of asparagine, 0.5-0.7g/L of disodium hydrogen phosphate, 3-4g/L of rhamnose and 3-3.5g/L of glycine.
In a third aspect, the invention provides a use of the adenovirus detection reagent card of the first aspect in preparing an adenovirus detection kit.
In one embodiment of the invention, the use is specifically to detect adenovirus antigen in a sample by using the adenovirus detection kit.
In one embodiment of the present invention, the use specifically includes: the ratio between the value of the first test line and the value of the second test line, and/or the ratio between the value of the first test line and the value of the control line, is used for obtaining the adenovirus content; wherein the value of the first test line is obtained from the color brightness exhibited by the latex particles enriched in the first test line, the value of the second test line is obtained from the color brightness exhibited by the latex particles enriched in the second test line, and the value of the control line is obtained from the color brightness exhibited by the latex particles enriched in the control line.
In one embodiment of the present invention, the color is red.
In a fourth aspect, the present invention provides an adenovirus detection kit, comprising the adenovirus detection reagent card of the first aspect.
Compared with the prior art, the invention has the following beneficial effects: the ratio of the two test lines or the ratio of the test line to the control line is adopted to determine the amount of the adenovirus, so that the error can be effectively reduced, and the accuracy of a detection result, particularly a quantitative detection result, is improved; and avoids the mutual interference between the control system and the test system; and the control system can adopt an antigen-antibody reactant which does not interfere with the test system, so that the mutual interference between the control system and the test system is avoided, and the accuracy and precision of detection are improved.
Drawings
FIG. 1 is a schematic structural diagram of an adenovirus detection reagent card according to an embodiment of the present invention.
Detailed Description
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.
Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts. These techniques are well described in the literature, and may be found in particular in the study of the MOLECULAR CLONING, Sambrook et al: a LABORATORY MANUAL, Second edition, Cold Spring harbor LABORATORY Press, 1989and Third edition, 2001; ausubel et al, Current PROTOCOLS Inmolecular BIOLOGY, John Wiley & Sons, New York, 1987and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; wolffe, CHROMATINSTRUCUTURE AND FUNCTION, Thirdedition, Academic Press, San Diego, 1998; (iii) Methods Inenzymolygy, Vol.304, Chromatin (P.M. Wassarman and A.P.Wolffe, eds.), academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol.119, chromatography protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999, etc.
The embodiment of the invention provides an adenovirus detection reagent card, and the structure of the adenovirus detection reagent card is shown in figure 1. On the reagent card, a sample adding gasket, a glass cellulose membrane, a nitrocellulose membrane and a water absorption gasket are arranged in sequence.
The glass cellulose membrane contains latex particles-a first anti-adenovirus antibody and latex particles-a test antigen. The latex particles have a specific color and may be colored latex particles. The latex particle-first anti-adenovirus antibody and latex particle-test antigen may be chromatographed onto the nitrocellulose membrane. The nitrocellulose membrane is provided with a T1 line, a T2 line and a C line in sequence. The T1 line and the T2 line are test lines, and the C line is a control line. T1 line coated with a second anti-adenovirus antibody; t2 line is coated with adenovirus antigen; the C-line is coated with antibodies to the test antigen.
The latex particle-first anti-adenovirus antibody refers to a latex particle-labeled first anti-adenovirus antibody.
The latex particle-test antigen refers to a latex particle-labeled test antigen.
The test antigen refers to an antigen other than an adenovirus antigen, such as IgG.
The latex particle marked test antigen on the sample adding pad and the antibody of the test antigen coated by the C line are used for verifying whether the detection reagent card fails.
The adenovirus detection reagent card provided by the embodiment of the invention adopts the latex immunochromatography technology and the principle of a double-antibody sandwich method. When in detection, if adenovirus exists in a sample, the adenovirus is firstly combined with a first anti-adenovirus antibody marked by latex particles to form a latex immune complex, when the latex immune complex is chromatographed to a T1 line, the latex immune complex is captured by a second anti-adenovirus antibody pre-coated on a T1 line, the latex immune complex is enriched at a T1 line, the higher the concentration of the adenovirus in the sample is, the more the latex immune complex is enriched on the T1 line, and the darker the color of the T1 line is; if the sample contains less adenovirus than the latex particle-primary anti-adenovirus antibody, the remaining latex particle-labeled primary anti-adenovirus antibody will chromatographe to the T2 line, the lower the concentration of adenovirus in the sample, the more latex particles will be enriched on the T2 line, and the darker the T2 line will be; the detection area or linear interval of the reagent card is widened.
The detection result of the adenovirus detection reagent card provided by the embodiment of the invention is characterized by the ratio of T1/T2 or the ratio of T1/C. The T1 value, T2 value and C value are expressed in terms of the color brightness of latex particles enriched on the T1 line, the color brightness of latex particles enriched on the T2 line and the color brightness of latex particles enriched on the C line, respectively. The color brightness can be read by an instrument matched with the reagent card; the user may also take a picture of the reagent card and the color intensity of each line on the picture is read by the computer.
The following situations can occur when the adenovirus detection reagent card provided by the embodiment of the invention is used for detecting adenovirus.
(1) When adenovirus is present in the sample from the beginning, the immune complex formed by the adenovirus and the first adenovirus antigen antibody is more and more, and the free first adenovirus antigen antibody is less and less, so that the latex particles captured by the T1 line are more and more, and the latex particles captured by the T2 line are less and less. The T1 value is gradually increased from none, the T2 value is gradually increased from strong to weak, and the C value is unchanged, then: the T1/T2 is gradually increased from a minimum value, and the T1/C is gradually increased from a minimum value.
(2) When the adenovirus in the sample is in the equivalent band, the immune complex formed by the adenovirus and the first adenovirus antigen antibody is the most, the free first adenovirus antigen antibody is the least, the T1 value is the largest, the T2 value is the smallest, and the C value is unchanged. Then: T1/T2 is big from big to big, and T1/C is gradually strengthened.
Therefore, the content of adenovirus in the sample is obtained according to the ratio of T1/T2 or the ratio of T1/C. In practical applications, a calibration curve of the T1/T2 or T1/C ratio can be fitted with a standard, and then the content of adenovirus in the sample can be calculated according to the standard curve.
While the adenovirus detection reagent card in the prior art only coats one test line, the specific situation in the above case is as follows.
(1) When adenovirus is present in the sample from the absence: t1 becomes stronger from none to gradually and quickly approaches a constant;
(2) when the adenovirus in the sample is within the equivalence band, then the T1 constant.
The amount of the adenovirus is determined by only one test line, so that the error is large, and the accuracy is low particularly for quantitative detection of the adenovirus.
The adenovirus detection reagent card provided by the embodiment of the invention adopts the ratio of the two test lines or the ratio of the test line and the control line to determine the amount of adenovirus, can effectively reduce errors, and can be suitable for quantitative determination of adenovirus.
The test antigen is in particular an antigen different from adenovirus. If the reagent card is specifically used for detecting the human adenovirus, the test antigen is a non-human antigen, so that the human sample cannot be interfered. The test antigen and test antigen antibody do not cross-interfere with the antigen-antibody system of the test line system. The system methodology error is controlled, and the detection accuracy and precision are improved.
In one example, the test antigen is rabbit IgG; the antibody of the test antigen is goat anti-rabbit IgG.
In one example, the first anti-adenoviral antibody and/or the second anti-adenoviral antibody is a monoclonal antibody. Compared with polyclonal antibody, the monoclonal antibody expressed by purified genetic engineering has better specificity and targeting property and more stable quality. Further, the first anti-adenovirus antibody and the second anti-adenovirus antibody are different monoclonal antibodies, for example, the first anti-adenovirus antibody can be AN adenovirus monoclonal antibody of ARC (product No. AN1003M), and the second anti-adenovirus antibody can be AN adenovirus early E1A protein antibody of Novus biologicals (product No. YB-6136R).
In one example, the latex particles are red in color.
The latex particles may be polystyrene latex particles. The particle size of the polystyrene latex particles can be 290-300 nm.
In the embodiment of the present invention, the latex particles are specifically antibodies capable of being chemically coupled with the first anti-adenovirus antibody and the test antigen, so that the latex particles are more firmly combined with the first anti-adenovirus antibody and the test antigen.
The latex particles may be carboxylated latex particles. For example: carboxylated polystyrene latex particles. Specifically, carboxylated polystyrene latex particles can be coupled to a primary anti-adenovirus antibody and a test antigen using the carbodiimide method. The detection reagent card provided by the embodiment of the invention determines the amount of the adenovirus according to the ratio of the two test lines or the ratio of the test line to the control line, effectively reduces errors, and can be suitable for quantitative determination of the adenovirus; and the control system can adopt an antigen-antibody reactant which does not interfere with the test system, so that the mutual interference between the control system and the test system is avoided, and the accuracy and precision of detection are improved. In addition, compared with a colloidal gold reagent, the adenovirus detection reagent card provided by the embodiment of the invention has the advantages of good sensitivity, strong specificity and the like; compared with the PCR method, the method has the advantages of convenience, rapidness and economy.
The embodiment of the invention also provides a preparation method of the adenovirus detection reagent card, which comprises the following steps: 1) spraying a mixed solution of latex particles, a first anti-adenovirus antibody and latex particles and a test antigen on a glass cellulose membrane; 2) carrying out first pretreatment on a nitrocellulose membrane corresponding to a T1 line to be spotted by using a first pretreatment solution, and spotting a second anti-adenovirus antibody solution on the nitrocellulose membrane subjected to the first pretreatment; 3) carrying out second pretreatment on a nitrocellulose membrane corresponding to a T2 line to be spotted by using a second pretreatment solution, and spotting an adenovirus antigen solution on the nitrocellulose membrane subjected to the second pretreatment; 4) and spraying an antibody solution of the test antigen on the nitrocellulose membrane at a preset position.
In one example, the first pretreatment solution is an aqueous solution containing 1.5-2g/L arginine, 1.2-1.5g/L potassium hydrogen phthalate, 0.3-0.5g/L sodium hydroxide, 2-2.5g/L citric acid. In one example, the second pretreatment solution is an aqueous solution containing 2-2.5g/L asparagine, 0.5-0.7g/L disodium hydrogen phosphate, 3-4g/L rhamnose, 3-3.5g/L glycine.
During preparation, the nitrocellulose membrane corresponding to the T1 line is pretreated by using the first pretreatment solution, and the nitrocellulose membrane corresponding to the T2 line is pretreated by using the second pretreatment solution, so that the detection sensitivity and accuracy of the adenovirus detection reagent card can be obviously improved.
The embodiment of the invention also provides the application of the adenovirus detection reagent card in preparing the adenovirus detection reagent card.
In one example, the use is in particular to detect adenovirus antigens in a sample using the adenovirus detection kit.
In one example, the use specifically includes: the ratio between the value of the first test line and the value of the second test line, and/or the ratio between the value of the first test line and the value of the control line, is used for obtaining the adenovirus content; wherein the value of the first test line is obtained from the color depth exhibited by the latex particles enriched in the first test line, the value of the second test line is obtained from the color depth exhibited by the latex particles enriched in the second test line, and the value of the control line is obtained from the color depth exhibited by the latex particles enriched in the control line.
In one example, the color is red.
The embodiment of the invention also provides an adenovirus detection kit, which comprises the adenovirus detection reagent card.
In the following specific examples and comparative examples, the detection effect of the adenovirus detection reagent card provided by the present invention is illustrated by using a labeling recovery method.
Example 1
11. The preparation method of the adenovirus detection reagent card comprises the following steps:
111. preparation of a mixed solution of latex particle-first anti-adenovirus antibody and latex particle-test antigen:
adding the required volume of the adenovirus monoclonal antibody and the carboxylated red polystyrene latex particles into 0.05M PBS (phosphate buffer solution) with the preset volume, performing ultrasonic treatment, centrifuging and other operation steps to obtain a latex particle-first anti-adenovirus antibody solution, diluting the latex particle-first anti-adenovirus antibody solution, and measuring the light absorption value of the suspension at 500nm by using a spectrophotometer to obtain the latex particle-first anti-adenovirus antibody solution. Adding the required volume of rabbit IgG and carboxylated red polystyrene latex particles into 0.05M PBS (phosphate buffer solution) with a preset volume, performing ultrasonic treatment, centrifuging and other operation steps to obtain a latex particle-test antigen solution, diluting the latex particle-test antigen solution, and measuring the light absorption value of the suspension at 500nm by using a spectrophotometer to obtain the latex particle-test antigen solution. Mixing the prepared latex particle-first anti-adenovirus antibody solution and the latex particle-test antigen solution, and storing at 2-8 ℃.
112. Diluting the mixed solution obtained in the step 111 by PBS (phosphate buffer solution) with the pH value of 6.8-7.2 and the concentration of 0.01-0.05M until the OD720 is 2.0-4.0, spraying the obtained solution onto a glass cellulose membrane, wherein the spraying amount is 2-5mg/ml, and drying at 35-45 ℃ to obtain the glass cellulose membrane containing the latex particle-first anti-adenovirus antibody and the latex particle-test antigen.
113. C, preparation of line solution: using PBS (0.01-0.05M, pH6.8-7.2) to prepare goat anti-rabbit IgG solution, wherein the final concentration of the solution is 1.0-2.0 mg/ml.
114. Preparation of T1 line solution: and (3) preparing a second anti-adenovirus antibody solution by using PBS (phosphate buffer solution) with the pH value of 0.01-0.05M and the pH value of 6.8-7.2, wherein the second anti-adenovirus antibody adopts the adenovirus early E1A protein antibody, and the final concentration of the solution is 1.0-2.0 mg/ml.
115. Preparation of T2 line solution: the adenovirus antigen solution is prepared by PBS (0.01-0.05M, pH6.8-7.2), and the final concentration of the solution is 1.0-2.0 mg/ml.
116. Pretreating the cellulose nitrate membrane corresponding to the T1 line to be sprayed with a first pretreatment solution, wherein the formula of the first pretreatment solution is as follows: 1.5g/L of arginine, 1.2g/L of potassium hydrogen phthalate, 0.5g/L of sodium hydroxide, 2.0g/L of citric acid, water as a solvent, and the pH value of 6.5, wherein the pretreatment comprises the following specific steps:
1161. uniformly dropwise adding 5 mu l of first pretreatment solution on a T1 line, and airing at room temperature;
1162. step 1161 is repeated twice.
117. Pretreating the cellulose nitrate membrane corresponding to the T2 line to be sprayed with a second pretreatment solution, wherein the formula of the second pretreatment solution is as follows: 2.5g/L of asparagine, 0.5g/L of disodium hydrogen phosphate, 4g/L of rhamnose, 3.5g/L of glycine, water as a solvent, and a pH value of 7.6, wherein the pretreatment comprises the following specific steps:
1171. uniformly dropwise adding 5 mu l of second pretreatment buffer solution to a T2 line, and airing at room temperature;
1172. step 1171 is repeated twice.
118. Dot spraying of a C line, a T1 line and a T2 line on the nitrocellulose membrane by using a C line solution, a T1 line solution and a T2 line solution respectively: washing a micro-protein spot membrane system by using PBS (phosphate buffer solution) with the concentration of 0.01M and the pH value of 7.2, adjusting parameters of a membrane spraying instrument, connecting an inlet pipeline and an outlet pipeline, respectively putting a C pipeline, a T1 pipeline and a T2 pipeline into solutions of a C line, a T1 line and a T2 line, adjusting the spraying speed and the membrane moving speed of the system to enable each 1 cm-length membrane band to be sprayed with 1-3 mul of the solutions of the C line, the solutions of the T1 line and the solutions of the T2 line, arranging the three lines on the nitrocellulose membrane in sequence of the T1 line, the T2 line and the C line from the sample adding end, putting the sprayed membrane into a vacuum pump, vacuumizing and drying to obtain the nitrocellulose membrane sequentially provided with the first test line, the second test line and the control line, and reserving the nitrocellulose membrane.
119. Film pasting: and sequentially attaching the prepared sample adding gasket, the glass cellulose membrane, the nitrocellulose membrane and the water absorption gasket on the rubber plate from top to bottom. And preparing a reagent large plate.
1110. Cutting: the reagent large plate is longitudinally cut into test strips with the width of 3-5mm by a cutter, and each test strip is 1 part.
1111. Assembling: and correspondingly installing each 1 person of test paper strips into each 1 plastic card to obtain the reagent card.
12. Adenovirus antigen was detected using the adenovirus detection reagent card provided in this example.
121. And (5) making a calibration curve.
Adenovirus antigen solutions with concentrations of 0, 50IFU/ml, 200IFU/ml, 1000IFU/ml, 5000IFU/ml and 10000IFU/ml were dropped onto the sample addition pad, 5 replicates were set for each concentration (the detection result was an average of 5 replicates), and after 10 minutes of membrane chromatography, color brightness of T1 line and T2 line was collected using an immunoassay instrument. When the preset adenovirus concentration is 0, the color brightness value of the T1 line is 0, namely the T1 value is 0, and the color brightness value of the T2 line is 100, namely the T2 value is 100; when the concentration of the adenovirus is 10000IFU/ml, the T1 value is 100, and the T2 value is 1; wherein 0 to 100 is a linear transition. The immunoassay instrument calculates color brightness values of the collected T1 line and the T2 line according to preset calculation, and calculates the value of T1/T2. Calibration curves were constructed from the values of the line T1/T2, where the Y-axis is the value of T1/T2 and the X-axis is the true value of the standard.
122. The adenovirus detection reagent card prepared in this example detects a sample to be detected.
Samples to be tested with adenovirus antigen concentrations of 30IFU/ml, 300IFU/ml, 1200IFU/ml and 6000IFU/ml are prepared, and PBS buffer is used as a control. And (3) dripping the sample to be detected on the sample adding pad, setting 5 repetitions for each sample (the detection result is an average value of the 5 repetitions), after 10 minutes of film chromatography, comparing a T1/T2 value obtained during sample detection with a standard curve to obtain a detection value, and comparing the detection value with an actual value to obtain an accuracy influence deviation value. The data obtained for the content of adenovirus antigen detected in samples 1-4 were 29IFU/ml, 316IFU/ml, 1182IFU/ml and 6100IFU/ml, respectively, and no adenovirus antigen was detected in the blank.
The result shows that the accuracy influence deviation value of the adenovirus antigen detection antigen provided by the invention is less than or equal to 10%, and the adenovirus antigen with the concentration as low as 30IFU/ml can be accurately detected.
Example 2
21. And (3) preparing an adenovirus detection reagent card.
The specific preparation method is described with reference to step 111-1111 of example 1, wherein the difference is that the formula of the first pretreatment solution in this example is as follows: 1.75g/L of arginine, 1.5g/L of potassium hydrogen phthalate, 0.3g/L of sodium hydroxide, 2.5g/L of citric acid and water as a solvent, wherein the pH value is 6.0. The second pretreatment liquid formula is as follows: 2.0g/L of asparagine, 0.7g/L of disodium hydrogen phosphate, 3.5g/L of rhamnose, 3.0g/L of glycine and water as a solvent, wherein the pH value is 8.0.
22. Adenovirus antigen was detected using the adenovirus detection reagent card provided in this example.
The detection process refers to steps 121 and 122 in embodiment 1. The detection results are respectively 30IFU/ml, 293IFU/ml, 1220IFU/ml and 6070IFU/ml, and no adenovirus antigen is detected in the blank control.
The result shows that the accuracy influence deviation value of the adenovirus antigen detection antigen provided by the invention is less than or equal to 10%, and the adenovirus antigen with the concentration as low as 30IFU/ml can be accurately detected.
Example 3
31. And (3) preparing an adenovirus detection reagent card.
The specific preparation method is described with reference to step 111-1111 of example 1, wherein the difference is that the formula of the first pretreatment solution in this example is as follows: 2.0g/L of arginine, 1.35g/L of potassium hydrogen phthalate, 0.4g/L of sodium hydroxide, 2.25g/L of citric acid and water as a solvent, wherein the pH value is 6.3. The second pretreatment liquid formula is as follows: 2.25g/L of asparagine, 0.6g/L of disodium hydrogen phosphate, 3g/L of rhamnose, 3.25g/L of glycine and water as a solvent, wherein the pH value is 7.8.
32. Adenovirus antigen was detected using the adenovirus detection reagent card provided in this example.
The detection process refers to steps 121 and 122 in embodiment 1. The detection results are 31IFU/ml, 313IFU/ml, 1180IFU/ml and 6090IFU/ml respectively, and no adenovirus antigen is detected in the blank control.
The result shows that the accuracy influence deviation value of the adenovirus antigen detection antigen provided by the invention is less than or equal to 10%, and the adenovirus antigen with the concentration as low as 30IFU/ml can be accurately detected.
Comparative example 1
41. The preparation method of the reagent card for detecting the adenovirus specifically comprises the following steps:
411. preparation of a mixed solution of latex particle-first anti-adenovirus antibody and latex particle-test antigen:
preparation of a mixed solution of latex particle-first anti-adenovirus antibody and latex particle-test antigen:
adding the required volume of the adenovirus monoclonal antibody and the carboxylated red polystyrene latex particles into 0.05M PBS (phosphate buffer solution) with the preset volume, performing ultrasonic treatment, centrifuging and other operation steps to obtain a latex particle-first anti-adenovirus antibody solution, diluting the latex particle-first anti-adenovirus antibody solution, and measuring the light absorption value of the suspension at 500nm by using a spectrophotometer to obtain the latex particle-first anti-adenovirus antibody solution. Adding the required volume of rabbit IgG and carboxylated red polystyrene latex particles into 0.05M PBS (phosphate buffer solution) with a preset volume, performing ultrasonic treatment, centrifuging and other operation steps to obtain a latex particle-test antigen solution, diluting the latex particle-test antigen solution, and measuring the light absorption value of the suspension at 500nm by using a spectrophotometer to obtain the latex particle-test antigen solution. Mixing the prepared latex particle-first anti-adenovirus antibody solution and the latex particle-test antigen solution, and storing at 2-8 ℃.
412. Diluting the mixed solution obtained in the step 411 by PBS (phosphate buffer solution) with the pH value of 6.8-7.2 and the concentration of 0.01-0.05M until the OD720 is 2.0-4.0, spraying the obtained solution onto a glass cellulose membrane, wherein the spraying amount is 2-5mg/ml, and drying at 35-45 ℃ to obtain the glass cellulose membrane containing the latex particle-first anti-adenovirus antibody and the latex particle-test antigen.
413. C, preparation of line solution: using PBS (0.01-0.05M, pH6.8-7.2) to prepare goat anti-rabbit IgG solution, wherein the final concentration of the solution is 1.0-2.0 mg/ml.
414. Preparing a T line solution: and (3) preparing a second anti-adenovirus antibody solution by using PBS (phosphate buffer solution) with the pH value of 0.01-0.05M and the pH value of 6.8-7.2, wherein the second anti-adenovirus antibody adopts the adenovirus early E1A protein antibody, and the final concentration of the solution is 1.0-2.0 mg/ml.
415. Dot C, T lines on nitrocellulose membrane: the microalbumin membrane system was cleaned with 0.01M phosphate buffer pH 7.2. And (3) adjusting parameters of the micro-protein spot membrane system, connecting an inlet pipeline and an outlet pipeline, and respectively putting the C, T pipelines into C, T-line solution. The spraying speed and the film running speed of the system were adjusted so that 1 to 3. mu.l of C, T line solution could be sprayed per 1cm length of film tape. And (4) putting the sprayed film into a vacuum pump, vacuumizing and drying for later use.
416. Film pasting: and sequentially attaching the prepared sample adding gasket, the glass cellulose membrane, the nitrocellulose membrane and the water absorption gasket on the rubber plate from top to bottom. And preparing a reagent large plate.
417. Cutting: the reagent large plate is longitudinally cut into test strips with the width of 3-5mm by a cutter, and each test strip is 1 part.
418. Assembling: and correspondingly installing each 1 person of test paper strips into each 1 plastic card to obtain the reagent card.
42. The invention relates to an adenovirus detection reagent card and a detection effect of a contrast adenovirus detection reagent card.
421. The adenovirus detection reagent card provided by the embodiment of the invention is used for detecting adenovirus antigens.
4211. And (5) making a calibration curve.
Adenovirus antigen solutions with concentrations of 0, 50IFU/ml, 200IFU/ml, 1000IFU/ml, 5000IFU/ml and 10000IFU/ml were dropped on the sample addition pad, 5 replicates were set for each concentration (the detection result was an average of 5 replicates), and after 10 minutes of membrane chromatography, T-line color brightness was collected using an immunoassay instrument. When the concentration of the adenovirus is preset to be 0, the color brightness value of the T line is 0, namely the T1 value is 0; when the concentration of the adenovirus is 10000IFU/ml, the T value is 100; wherein 0 to 100 is a linear transition. And the immunoassay instrument calculates the acquired T line color brightness value according to the preset calculation and calculates the T value. And establishing a calibration curve according to the value of the T line, wherein the Y axis is the T1 value, and the X axis is the true value of the standard product.
4212. The invention relates to a detection result of an adenovirus detection reagent card.
Samples to be tested with adenovirus antigen concentrations of 30IFU/ml, 300IFU/ml, 1200IFU/ml and 6000IFU/ml are prepared, and PBS buffer is used as a control. Dropping a sample to be detected on the sample adding gasket, setting 5 repetitions for each sample (the detection result is an average value of the 5 repetitions), after 10 minutes of film chromatography, comparing a T value obtained when the sample is detected with a standard curve to obtain a detection value, and comparing the detection value with an actual value to obtain an accuracy influence deviation value.
The results showed that no adenovirus antigen was detected from the concentrations of 30IFU/ml, 300IFU/ml and the blank. And the detection results of the samples to be detected with the actual concentrations of 1200IFU/ml and 6000IFU/ml are 780IFU/ml and 5190IFU/ml respectively.
In conclusion, the detection kit provided by the invention has good sensitivity and accuracy, effectively overcomes various defects in the prior art, and has high industrial utilization value.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
Claims (10)
1. An adenovirus detection reagent card is characterized by comprising a sample adding gasket, a glass cellulose membrane, a nitrocellulose membrane and a water absorption gasket which are sequentially arranged; wherein,
the glass cellulose membrane contains latex particles, a first anti-adenovirus antibody and latex particles, a test antigen;
the nitrocellulose membrane is sequentially provided with a first test line, a second test line and a control line;
the first test line is coated with a second anti-adenovirus antibody;
the second test line is coated with adenovirus antigen;
the control line is coated with an antibody to the test antigen.
2. The adenovirus detection reagent card of claim 1, wherein the test antigen is rabbit IgG; the antibody of the test antigen is goat anti-rabbit IgG.
3. The adenovirus detection reagent card of claim 1, wherein the first anti-adenovirus antibody and/or the second anti-adenovirus antibody is a monoclonal antibody.
4. The adenovirus detection reagent card of claim 1, wherein the latex particle in the latex particle-first anti-adenovirus antibody and the first adenovirus antibody are linked by chemical coupling, and/or the latex particle in the latex particle-test antigen and the test antigen are linked by chemical coupling.
5. The method for preparing the adenovirus detection reagent card according to any one of claims 1-4, comprising the following steps:
1) spraying a mixed solution of latex particles, a first anti-adenovirus antibody and latex particles and a test antigen on a glass cellulose membrane;
2) carrying out first pretreatment on a nitrocellulose membrane corresponding to a T1 line to be spotted by using a first pretreatment solution, and spotting a second anti-adenovirus antibody solution on the nitrocellulose membrane subjected to the first pretreatment;
3) carrying out second pretreatment on a nitrocellulose membrane corresponding to a T2 line to be spotted by using a second pretreatment solution, and spotting an adenovirus antigen solution on the nitrocellulose membrane subjected to the second pretreatment;
4) and spraying an antibody solution of the test antigen on the nitrocellulose membrane at a preset position.
6. The method of claim 5, further comprising any one or more of the following features: the first pretreatment solution is an aqueous solution containing 1.5-2g/L arginine, 1.2-1.5g/L potassium hydrogen phthalate, 0.3-0.5g/L sodium hydroxide and 2-2.5g/L citric acid; the second pretreatment solution is an aqueous solution containing 2-2.5g/L of asparagine, 0.5-0.7g/L of disodium hydrogen phosphate, 3-4g/L of rhamnose and 3-3.5g/L of glycine.
7. Use of an adenovirus detection reagent card according to any one of claims 1-4 in the preparation of an adenovirus detection kit.
8. The use according to claim 7, wherein the use is in particular to detect adenovirus antigens in a sample using the adenovirus detection kit.
9. Use according to claim 8, characterized in that it comprises in particular:
the ratio between the value of the first test line and the value of the second test line, and/or the ratio between the value of the first test line and the value of the control line, is used for obtaining the adenovirus content; wherein,
the value of the first test line is obtained from the color brightness exhibited by the latex particles enriched in the first test line, the value of the second test line is obtained from the color brightness exhibited by the latex particles enriched in the second test line, and the value of the control line is obtained from the color brightness exhibited by the latex particles enriched in the control line.
10. An adenovirus detection kit comprising the adenovirus detection reagent card according to any one of claims 1 to 5.
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Application publication date: 20180413 |