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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- 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 present invention relates to emulsion technique immunochromatography field, and in particular to adenovirus detection reagent card, kit and application thereof.
Background technology
All immune diagnostic reagents, using immune response as basic principle, i.e., either qualitative or quantitative reagent, is all
Occur to specifically bind and produce antigen-antibody complex under certain condition with antigen and antibody, with tracer-labelling antigen
Or antibody, carry out the final detection and analysis realized to reaction product.
In the prior art, immune diagnostic reagent card, sets a p-wire, is determined according to the color of p-wire in sample
Whether pathogen antigen is contained.Judged by the color of a p-wire, be easily subject to the shadow of environmental light brightness and color
Ring, and also benefit from the influence of family subjective judgement, produce large error so that testing result, particularly quantitatively testing result is accurate
Exactness is relatively low, and obstacle is brought for the diagnosis of disease.
The content of the invention
In view of the foregoing deficiencies of prior art, it is an object of the invention to provide a kind of adenovirus detection reagent card,
Kit and application thereof, to reduce detection error, improves the accuracy of testing result.
In a first aspect, the present invention provides a kind of adenovirus detection reagent card, including sample application pad, the glass being arranged in order
Cellulose membrane, nitrocellulose filter and water suction gasket；Wherein, the anti-gland of latex particle-the first is contained on the glass fibre element film
Antiviral antibody and latex particle-test antigen；The first p-wire, the second p-wire are disposed with the nitrocellulose filter
And control line；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.
In one embodiment of the present of invention, the test antigen is rabbit igg；The antibody of the test antigen is goat-anti rabbit
IgG。
In one embodiment of the present of invention, first Anti-adenovirus antibody and/or second Anti-adenovirus antibody
For monoclonal antibody.
In one embodiment of the present of invention, the latex particle in the Anti-adenovirus antibody of latex particle-the first and
One adenovirus antibody is connected by chemical coupling, and/or, the latex particle and test in the latex particle-test antigen are anti-
Original is connected by chemical coupling.
Second aspect, the present invention provides a kind of preparation method of adenovirus detection reagent card as described in relation to the first aspect,
Include the following steps：1) the-the first Anti-adenovirus antibody of specking latex particle and latex particle-test resist on glass fibre element film
Former mixed solution；2) using the first pretreatment fluid to will be corresponding to the T1 lines of specking nitrocellulose filter to carry out first pre-
Processing, and in first pretreated the second Anti-adenovirus antibody of nitrocellulose filter specking solution；3) using the second pretreatment
Liquid carries out the second pretreatment to nitrocellulose filter that will be corresponding to the T2 lines of specking, and fine in the second pretreated nitric acid
The plain film specking Adenovirus Antigen solution of dimension；4) on the nitrocellulose filter of predeterminated position specking test antigen antibody-solutions.
In one embodiment of the present of invention, first pretreatment fluid is to contain 1.5-2g/L arginine, 1.2-1.5g/
L Potassium Hydrogen Phthalates, 0.3-0.5g/L sodium hydroxides, the aqueous solution of 2-2.5g/L citric acids.In the implementation of the present invention
In example, second pretreatment fluid is to contain 2-2.5g/L asparagines, 0.5-0.7g/L disodium hydrogen phosphates, 3-4g/L sandlwoods
Sugar, the aqueous solution of 3-3.5g/L glycine.
The third aspect, is stuck in the present invention provides the adenovirus detection reagent described in a kind of first aspect and prepares adenovirus inspection
Purposes in test agent box.
In one embodiment of the present of invention, the purposes is specially in sample using the adenovirus detection kit
Adenovirus Antigen be detected.
In one embodiment of the present of invention, the purposes specifically includes：The value of first p-wire and the second p-wire
Ratio between ratio between value, and/or the value of the first p-wire and the value of control line, for obtaining adenovirus content；Its
In, colour brightness that the value of first p-wire is presented by the latex particle that the first p-wire is enriched with obtains, described second
The colour brightness that the value of p-wire is presented by the latex particle that the second p-wire is enriched with obtains, the value of the control line is by controlling
The colour brightness that the latex particle of line enrichment is presented obtains.
In one embodiment of the present of invention, the color is red.
Fourth aspect, the present invention provides a kind of adenovirus detection kit, including the adenovirus inspection described in first aspect
Test agent card.
Compared with prior art, the present invention has the advantages that：Using two p-wires ratio or p-wire with
The ratio of control line determines the amount of adenovirus, can effectively reduce error, improves testing result, particularly quantitative detection
As a result accuracy；And avoid interfering with each other between control system and test system；And control system can use
The antigen-antibody reaction thing of not disturbed test system, avoids interfering with each other between control system and test system, improves inspection
The accuracy of survey and precision.
Brief description of the drawings
Fig. 1 is the structure diagram of adenovirus detection reagent card provided in an embodiment of the present invention.
Embodiment
Before the specific embodiment of the invention is further described, it should be appreciated that protection scope of the present invention is not limited to down
State specific specific embodiment；It is also understood that the term used in the embodiment of the present invention is specific specific in order to describe
Embodiment, the protection domain being not intended to be limiting of the invention；In description of the invention and claims, unless in text
In addition explicitly point out, singulative "one", " one " and " this " include plural form.
When embodiment provides number range, it should be appreciated that except non-invention is otherwise noted, two ends of each number range
Any one numerical value can be selected between point and two endpoints.Unless otherwise defined, in the present invention all technologies for using and
Scientific terminology is identical with the normally understood meaning of those skilled in the art of the present technique.Except used in embodiment specific method, equipment,
Outside material, according to grasp of the those skilled in the art to the prior art and the record of the present invention, it can also use and this
Any method, equipment and the material of the similar or equivalent prior art of method, equipment described in inventive embodiments, material come real
The existing present invention.
Unless otherwise stated, disclosed in this invention experimental method, detection method, preparation method using this technology lead
Molecular biology, biochemistry, chromatin Structure and the analysis of domain routine, analytical chemistry, cell culture, recombinant DNA technology and
The routine techniques of association area.These technologies existing perfect explanation in the prior art, for details, reference can be made to Sambrook etc.
MOLECULAR CLONING：A LABORATORY MANUAL, Second edition, Cold Spring Harbor
Laboratory Press, 1989and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY, John Wiley＆Sons, New York, 1987and periodic updates；the
Series METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN
STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS IN
ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic
Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin
Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..
An embodiment of the present invention provides a kind of adenovirus detection reagent card, its structure are as shown in Figure 1.On the reagent card,
Sample application pad, glass fibre element film, nitrocellulose filter, water suction gasket are arranged in order.
Contain the Anti-adenovirus antibody of latex particle-the first and latex particle-test antigen on the glass fibre element film.Institute
Stating latex particle has particular color, can be colored latex particles.The Anti-adenovirus antibody of latex particle-the first and latex
Particle-test antigen can be chromatographed to the nitrocellulose filter.T1 lines, T2 lines and C lines are disposed with nitrocellulose filter.
T1 lines and T2 lines are p-wire, C lines line in order to control.T1 lines have been coated with the second Anti-adenovirus antibody；T2 lines have been coated with adenovirus and have resisted
It is former；C lines have been coated with the antibody of the test antigen.
The Anti-adenovirus antibody of latex particle-the first refers to the first Anti-adenovirus antibody of latex particle mark.
The latex particle-test antigen refers to the test antigen of latex particle mark.
The test antigen refers to the antigen beyond Adenovirus Antigen, such as IgG.
The test antigen of latex particle mark on sample application pad and the antibody of the coated test antigen of C lines, which are used to verify, to be examined
Whether test agent card fails.
Adenovirus detection reagent card provided in an embodiment of the present invention employs latex immunochromatography technique and double antibody folder
Heart method principle.During detection, if with the presence of adenovirus in sample, the first Anti-adenovirus antibody that can be marked first with latex particle
Combine to form latex immune complex, when the latex immune complex is chromatographed to T1 lines, be coated in advance on T1 lines
Two Anti-adenovirus antibodies capture, and latex immune complex is enriched with T1 lines, and adenovirus concentration is higher in sample, and latex is immunized multiple
Enrichment of the compound on T1 lines is more, and T1 line colors are deeper；If adenovirus is less than the anti-adenopathy of latex particle-the first in sample
Malicious antibody, then the first Anti-adenovirus antibody of remaining latex particle mark can chromatograph to T2 lines, adenovirus concentration is got in sample
Low, the latex particle of the enrichment on T2 lines is more, and T2 line colors are deeper；The detection interval or linear zone of reagent card are widened
Between.
The testing result of adenovirus detection reagent card provided in an embodiment of the present invention is by the ratio of T1/T2 or the ratio of T1/C
To characterize.T1 values, T2 values, C values are specific respectively with the latex being enriched with the colour brightness for the latex particle being enriched with T1 lines, T2 lines
The colour brightness of the latex particle being enriched with the colour brightness of particle, C lines represents.Colour brightness can be with supporting with reagent card
Instrument read；User can also shoot the photo of reagent card, and then the color by each line on computer reading photo is bright
Degree.
The adenovirus detection reagent card provided using inventive embodiments carries out the detection of adenovirus, in fact it could happen that following several
Situation.
(1) when in sample adenovirus from scratch when, adenovirus and the first Adenovirus Antigen antibody are formed immune compound
Thing is more and more, and the first free Adenovirus Antigen antibody is fewer and fewer, and therefore, the latex particle of T1 lines capture is more and more,
And the latex particle of T2 lines capture is fewer and fewer.T1 values to gradual from without becoming strong, and T2 is from extremely strong to decrease, and C is constant, then：T1/T2
By minimum to becoming larger, T1/C is by minimum to becoming larger.
(2) when adenovirus is in equivalence zone in sample, adenovirus and the first Adenovirus Antigen antibody are formed immune multiple
Compound is most, and the first free Adenovirus Antigen antibody is minimum, and T1 values are maximum at this time, and T2 values are minimum, and C values are constant.Then：T1/T2
Become larger greatly by big, T1/C gradually strengthens.
Therefore, the content of adenovirus in sample is obtained according to the ratio of the ratio of T1/T2 or T1/C.In practical application
In, the calibration curve of standard items fitting T1/T2 or T1/C ratios can be utilized, adenopathy in sample is then calculated according to standard curve
The content of poison.
And adenovirus detection reagent card of the prior art has only been coated with a p-wire, specific feelings in the scenario above
Condition is as follows.
(1) when in sample adenovirus from scratch when：T1 tends to constant quickly from without becoming strong to gradual；
(2) when adenovirus is in equivalence zone in sample, then T1 constants.
The amount that adenovirus is determined by means of a p-wire is only relied on, error is larger, accurate especially for the quantitative detection of adenovirus
Exactness is relatively low.
And ratio or p-wire and control of the adenovirus detection reagent card provided in an embodiment of the present invention using two p-wires
The ratio of line processed determines the amount of adenovirus, can effectively reduce error, can be adapted for the quantitative determination of adenovirus.
It is specially the antigen different from adenovirus to test antigen.If the reagent card is specifically used for detection people source adenovirus,
Test antigen and select non-human antigen, humanized's sample will not be interfered.Test antigen and test antigen-antibody and test
The non-cross interference of Ag-Ab system of linear system system.So that system methodology error is controlled, the accurate of detection is improved
Degree and precision.
In one example, the test antigen is rabbit igg；The antibody of the test antigen is goat anti-rabbit igg.
In one example, first Anti-adenovirus antibody and/or second Anti-adenovirus antibody resist for monoclonal
Body.The monoclonal antibody of the gene engineering expression of selection purifying is more preferable compared to polyclonal antibody specificity and targeting, and quality is more
Stablize.Further, first Anti-adenovirus antibody and second Anti-adenovirus antibody are different monoclonal antibodies, example
As first Anti-adenovirus antibody can use the adenovirus monoclonal antibody (article No. AN1003M) of ARC, the second anti-adenopathy
Malicious antibody can use the adenovirus early stage E1A protein antibodies (article No. YB-6136R) of NovusBiologicals.
In one example, the color of the latex particle is red.
The latex particle can be polystyrene latex particulate.The particle diameter of the polystyrene latex particulate can be
290~300nm.
In embodiments of the present invention, latex particle specially can be with the first Anti-adenovirus antibody and test antigen chemistry
The antibody of coupling, so that latex particle and the first Anti-adenovirus antibody and test antigen binding are more firm.
The latex particle can be the latex particle of carboxylated.Such as：The polystyrene latex particulate of carboxylated.Specifically
Carbodiimide method can be used by the polystyrene latex particulate of carboxylated and the first Anti-adenovirus antibody and test antigen
Coupling.Detection reagent card provided in an embodiment of the present invention by the ratio or the ratio of p-wire and control line of two p-wires come
Determine the amount of adenovirus, significantly reduce error, can be adapted for the quantitative determination of adenovirus；And control system can adopt
With the antigen-antibody reaction thing of not disturbed test system, interfering with each other between control system and test system is avoided, is improved
The accuracy of detection and precision.Also, adenovirus detection reagent card provided in an embodiment of the present invention is compared with colloid gold reagent,
Have the advantages that sensitivity is good, high specificity；Compared with PCR methods, have the advantages that convenient, fast, economical.
The embodiment of the present invention additionally provides a kind of preparation method of adenovirus detection reagent card described above, including as follows
Step：1) on glass fibre element film the-the first Anti-adenovirus antibody of specking latex particle and latex particle-test antigen mixing
Solution；2) the first pretreatment is carried out to nitrocellulose filter that will be corresponding to the T1 lines of specking using the first pretreatment fluid, and
In first pretreated the second Anti-adenovirus antibody of nitrocellulose filter specking solution；3) using the second pretreatment fluid to will
Nitrocellulose filter corresponding to the T2 lines of specking carries out the second pretreatment, and in the second pretreated nitrocellulose filter spray
Point Adenovirus Antigen solution；4) on the nitrocellulose filter of predeterminated position specking test antigen antibody-solutions.
In one example, first pretreatment fluid is to contain 1.5-2g/L arginine, 1.2-1.5g/L O-phthalics
Potassium hydrogen phthalate, 0.3-0.5g/L sodium hydroxides, the aqueous solution of 2-2.5g/L citric acids.In one example, second pretreatment
Liquid be containing 2-2.5g/L asparagines, 0.5-0.7g/L disodium hydrogen phosphates, 3-4g/L rhamnoses, 3-3.5g/L glycine water
Solution.
In the preparation, the nitrocellulose filter corresponding to T1 lines is pre-processed using the first pretreatment fluid, and used
Second pretreatment fluid pre-processes the nitrocellulose filter corresponding to T2 lines, can significantly improve adenovirus detection reagent card
Detection sensitivity and accuracy.
The embodiment of the present invention additionally provides a kind of adenovirus detection reagent described above and is stuck in preparation adenovirus detection examination
Purposes in agent card.
In one example, the purposes is specially and the adenovirus in sample is resisted using the adenovirus detection kit
Original is detected.
In one example, the purposes specifically includes：Ratio between the value of first p-wire and the value of the second p-wire
Value, and/or the ratio between the value of the first p-wire and the value of control line, for obtaining adenovirus content；Wherein, described first
The color depth that the value of p-wire is presented by the latex particle that the first p-wire is enriched with obtains, the value of second p-wire by
What the color depth acquisition and the value of the control line that the latex particle of the second p-wire enrichment is presented were enriched with by control line
The color depth that latex particle is presented obtains.
In one example, the color is red.
The embodiment of the present invention additionally provides a kind of adenovirus detection kit, including adenovirus detection reagent described above
Card.
In specific examples below and comparative example, adenovirus provided by the invention is detected using standard samples recovery and is tried
The detection result of agent card is illustrated.
Embodiment 1
11st, the preparation of adenovirus detection reagent card, specific method include the following steps：
111st, the preparation of the mixed solution of the Anti-adenovirus antibody of latex particle-the first and latex particle-test antigen：
The red of the desired amount of above-mentioned adenovirus monoclonal antibody and carboxylated is added in the PBS of the 0.05M of preset vol
The volume of color polystyrene latex particulate, by the operating procedures such as ultrasound, centrifugation, the anti-adenovirus of latex particle-the first of acquisition
Antibody-solutions, after dilution, with light absorption value of the spectrophotometer measurement suspension at 500nm, obtained latex particle-the
One Anti-adenovirus antibody solution.The red that the desired amount of rabbit igg and carboxylated are added in the PBS of the 0.05M of preset vol is gathered
The volume of polystyrene latex particle, by operating procedures such as ultrasound, centrifugations, latex particle-test antigenic solution of acquisition, passes through
After dilution, with light absorption value of the spectrophotometer measurement suspension at 500nm, obtained latex particle-test antigenic solution.Will
The Anti-adenovirus antibody of latex particle-the first solution and latex particle of preparation-test antigenic solution mixing, 2-8 DEG C of preservation.
112nd, the step 111 gained mixed solution PBS of pH6.8-7.2,0.01-0.05M is diluted, is diluted to OD720
=2.0~4.0, by resulting solution specking to glass fibre element film, specking amount is 2-5mg/ml, and 35-45 DEG C of drying, contains to obtain the final product
There is the glass fibre element film of the Anti-adenovirus antibody of latex particle-the first and latex particle-test antigen.
113rd, the preparation of C lines solution：Goat anti-rabbit igg solution is prepared with the PBS of 0.01-0.05M, pH6.8-7.2, solution
Final concentration of 1.0-2.0mg/ml.
114th, the preparation of T1 lines solution：It is molten that the second Anti-adenovirus antibody is prepared with the PBS of 0.01-0.05M, pH6.8-7.2
Liquid, the second Anti-adenovirus antibody use above-mentioned adenovirus early stage E1A protein antibodies, the final concentration of 1.0-2.0mg/ml of solution.
115th, the preparation of T2 lines solution：Adenovirus Antigen solution, solution are prepared with the PBS of 0.01-0.05M, pH6.8-7.2
Final concentration of 1.0-2.0mg/ml.
116th, nitrocellulose filter that will be corresponding to the T1 lines of specking is pre-processed using the first pretreatment fluid,
The formula of one pretreatment fluid is：Arginine 1.5g/L, Potassium Hydrogen Phthalate 1.2g/L, sodium hydroxide 0.5g/L, citric acid
2.0g/L, solvent are water, pH value=6.5, and pretreatment concretely comprises the following steps：
1161st, toward 5 the first pretreatment fluids of μ l are dropped evenly on T1 lines, room temperature is dried；
1162nd, repeat step 1161 is twice.
117th, nitrocellulose filter that will be corresponding to the T2 lines of specking is pre-processed using the second pretreatment fluid,
The formula of two pretreatment fluids is：Asparagine 2.5g/L, disodium hydrogen phosphate 0.5g/L, rhamnose 4g/L, glycine 3.5g/L, it is molten
Agent is water, pH value=7.6, and pretreatment concretely comprises the following steps：
1171st, 5 the second pre-treatment buffers of μ l are dropped evenly toward T2 lines, room temperature is dried；
1172nd, repeat step 1171 is twice.
118th, respectively using C lines solution, T1 lines solution, T2 line solution specking C lines, T1 lines, T2 on nitrocellulose filter
Line：It is with the PBS buffer of 0.01M, pH7.2 that trace of albumin point membranous system is cleaned, spray each parameter of film instrument is debugged, is connected
Inlet/outlet pipeline, C pipelines, T1 pipelines, T2 pipelines are respectively put into C lines, T1 lines, T2 line solution, the spray of regulating system speed and
Film-passing speed, so as to can respectively spray the C lines solution, T1 lines solution, T2 line solution of 1-3 μ l, cellulose nitrate per the film strips of 1cm length
The distributing order of three lines is on plain film, and T1 lines, T2 lines and C lines are followed successively by since being loaded end, and the film sprayed is put into vacuum
Drying is vacuumized in pump, is disposed with the nitrocellulose filter of the first p-wire, the second p-wire and control line to obtain the final product, it is standby
With.
119th, pad pasting：On offset plate, from top to bottom, the above-mentioned sample application pad prepared, glass fibre are sticked successively
Plain film, nitrocellulose filter and water suction gasket.The big plate of reagent is made.
1110th, cutting：The test strips for being 3-5mm into width by the big plate rip cutting of reagent with cutter, each are 1 person-portion.
1111st, assemble：Every 1 person-portion test strips are corresponded to and are installed in every 1 plastic clip, up to reagent card.
12nd, Adenovirus Antigen is detected using adenovirus detection reagent card provided in this embodiment.
121st, calibration curve is made.
It is respectively 0,50IFU/ml, 200IFU/ml, 1000IFU/ml, 5000IFU/ml, 10000IFU/ml by concentration
Adenovirus Antigen solution is added dropwise on sample application pad, and each concentration sets 5 repetitions (testing result takes the average value of 5 repetitions),
After film layer is analysed 10 minutes, the colour brightness of T1 lines and T2 lines is gathered using immunoassay instrument.Default adenovirus concentration is 0
When, the colour intensity value of T1 lines is 0, i.e. T1 values 0, and the colour intensity value of T2 lines is 100, i.e., T2 values are 100；Adenovirus concentration is
During 10000IFU/ml, T1 values are that 100, T2 values are 1；Wherein, 0 to 100 is linear transitions.Immunoassay instrument is according to pre-designed
The T1 lines and T2 line color brightness values collected, and calculate the value of T1/T2.Calibration curve is established according to the value of T1/T2 lines, its
Middle Y-axis is the value of T1/T2, and X-axis is standard items actual value.
122nd, adenovirus detection reagent card manufactured in the present embodiment is detected sample to be tested.
It is respectively treating for 30IFU/ml, 300IFU/ml, 1200IFU/ml, 6000IFU/ml to prepare Adenovirus Antigen concentration
Sample, using PBS buffer as control.Sample to be tested is added dropwise on sample application pad, each sample sets 5 repetitions (detection knot
Fruit takes the average value of 5 repetitions), after film layer is analysed 10 minutes, the T1/T2 values and the standard curve ratio that will be obtained when detecting sample
Compared with, detected value is obtained, detected value and actual value are compared, acquisition accuracy influence deviation.The inspection that sample 1-4 is obtained
The content data of the Adenovirus Antigen of survey is respectively 29IFU/ml, 316IFU/ml, 1182IFU/ml, 6100IFU/ml, blank pair
Adenovirus Antigen is not detected by according in.
The above results show that the accuracy of Adenovirus Antigen detection antigen provided by the invention influences deviation≤10%,
Can the accurate detectable concentration as low as Adenovirus Antigen of 30IFU/ml.
Embodiment 2
21st, the preparation of adenovirus detection reagent card.
In specific preparation method reference embodiment 1 described in step 111-1111, wherein, difference is, in this implementation
Example in the first pretreatment fluid formula be：Arginine 1.75g/L, Potassium Hydrogen Phthalate 1.5g/L, sodium hydroxide 0.3g/L, lemon
Lemon acid 2.5g/L, solvent are water, pH value=6.0.Second pretreatment fluid formula is：Asparagine 2.0g/L, disodium hydrogen phosphate
0.7g/L, rhamnose 3.5g/L, glycine 3.0g/L, solvent are water, pH value=8.0.
22nd, Adenovirus Antigen is detected using adenovirus detection reagent card provided in this embodiment.
Detection process is with reference to the step 121 and step 122 in embodiment 1.Testing result for be respectively 30IFU/ml,
293IFU/ml, 1220IFU/ml, 6070IFU/ml, Adenovirus Antigen is not detected by blank control.
The above results show that the accuracy of Adenovirus Antigen detection antigen provided by the invention influences deviation≤10%,
Can the accurate detectable concentration as low as Adenovirus Antigen of 30IFU/ml.
Embodiment 3
31st, the preparation of adenovirus detection reagent card.
In specific preparation method reference embodiment 1 described in step 111-1111, wherein, difference is, in this implementation
Example in the first pretreatment fluid formula be：Arginase 12 .0g/L, Potassium Hydrogen Phthalate 1.35g/L, sodium hydroxide 0.4g/L, lemon
Lemon acid 2.25g/L, solvent are water, pH value=6.3.Second pretreatment fluid formula is：Asparagine 2.25g/L, disodium hydrogen phosphate
0.6g/L, rhamnose 3g/L, glycine 3.25g/L, solvent are water, pH value=7.8.
32nd, Adenovirus Antigen is detected using adenovirus detection reagent card provided in this embodiment.
Detection process is with reference to the step 121 and step 122 in embodiment 1.Testing result for be respectively 31IFU/ml,
313IFU/ml, 1180IFU/ml, 6090IFU/ml, Adenovirus Antigen is not detected by blank control.
The above results show that the accuracy of Adenovirus Antigen detection antigen provided by the invention influences deviation≤10%,
Can the accurate detectable concentration as low as Adenovirus Antigen of 30IFU/ml.
Comparative example 1
41st, the preparation of adenovirus detection reagent card is contrasted, specific method includes the following steps：
411st, the preparation of the mixed solution of the Anti-adenovirus antibody of latex particle-the first and latex particle-test antigen：
The preparation of the mixed solution of the Anti-adenovirus antibody of latex particle-the first and latex particle-test antigen：
The red of the desired amount of above-mentioned adenovirus monoclonal antibody and carboxylated is added in the PBS of the 0.05M of preset vol
The volume of color polystyrene latex particulate, by the operating procedures such as ultrasound, centrifugation, the anti-adenovirus of latex particle-the first of acquisition
Antibody-solutions, after dilution, with light absorption value of the spectrophotometer measurement suspension at 500nm, obtained latex particle-the
One Anti-adenovirus antibody solution.The red that the desired amount of rabbit igg and carboxylated are added in the PBS of the 0.05M of preset vol is gathered
The volume of polystyrene latex particle, by operating procedures such as ultrasound, centrifugations, latex particle-test antigenic solution of acquisition, passes through
After dilution, with light absorption value of the spectrophotometer measurement suspension at 500nm, obtained latex particle-test antigenic solution.Will
The Anti-adenovirus antibody of latex particle-the first solution and latex particle of preparation-test antigenic solution mixing, 2-8 DEG C of preservation.
412nd, the step 411 gained mixed solution PBS of pH6.8-7.2,0.01-0.05M is diluted, is diluted to OD720
=2.0~4.0, by resulting solution specking to glass fibre element film, specking amount is 2-5mg/ml, and 35-45 DEG C of drying, contains to obtain the final product
There is the glass fibre element film of the Anti-adenovirus antibody of latex particle-the first and latex particle-test antigen.
413rd, the preparation of C lines solution：Goat anti-rabbit igg solution is prepared with the PBS of 0.01-0.05M, pH6.8-7.2, solution
Final concentration of 1.0-2.0mg/ml.
414th, the preparation of T lines solution：It is molten that the second Anti-adenovirus antibody is prepared with the PBS of 0.01-0.05M, pH6.8-7.2
Liquid, the second Anti-adenovirus antibody use above-mentioned adenovirus early stage E1A protein antibodies, the final concentration of 1.0-2.0mg/ml of solution.
415th, specking C, T lines on nitrocellulose filter：With 0.01M, the phosphate buffer of PH7.2 is by trace of albumin point
Membranous system is cleaned.Each parameter of trace of albumin point membranous system has been debugged, inlet/outlet pipeline has been connected, C, T pipeline is respectively put into
C, in T lines solution.The spray speed and film-passing speed of regulating system, so that C, T line that 1-3 μ l can be sprayed per the film strips of 1cm length are molten
Liquid.The film sprayed is put into vacuum pump and vacuumizes drying, it is spare.
416th, pad pasting：On offset plate, from top to bottom, the above-mentioned sample application pad prepared, glass fibre are sticked successively
Plain film, nitrocellulose filter and water suction gasket.The big plate of reagent is made.
417th, cutting：The test strips for being 3-5mm into width by the big plate rip cutting of reagent with cutter, each are 1 person-portion.
418th, assemble：Every 1 person-portion test strips are corresponded to and are installed in every 1 plastic clip, up to reagent card.
42nd, the detection result of adenovirus detection reagent card of the present invention and contrast adenovirus detection reagent card.
421st, Adenovirus Antigen is detected using adenovirus detection reagent card provided in an embodiment of the present invention.
4211st, calibration curve is made.
It is respectively 0,50IFU/ml, 200IFU/ml, 1000IFU/ml, 5000IFU/ml, 10000IFU/ml by concentration
Adenovirus Antigen solution is added dropwise on sample application pad, and each concentration sets 5 repetitions (testing result takes the average value of 5 repetitions),
After film layer is analysed 10 minutes, T line color brightness is gathered using immunoassay instrument.When default adenovirus concentration is 0, the face of T lines
Colour brightness value is 0, i.e. T1 values 0；When adenovirus concentration is 10000IFU/ml, T values are 100；Wherein, 0 to 100 is linear transitions.
The T line color brightness values that immunoassay instrument is collected according to default calculating, and calculate T values.It is bent that calibration is established according to the value of T lines
Line, wherein Y-axis are T1 values, and X-axis is standard items actual value.
4212nd, the testing result of adenovirus detection reagent card of the present invention.
It is respectively treating for 30IFU/ml, 300IFU/ml, 1200IFU/ml, 6000IFU/ml to prepare Adenovirus Antigen concentration
Sample, using PBS buffer as control.Sample to be tested is added dropwise on sample application pad, each sample sets 5 repetitions (detection knot
Fruit takes the average value of 5 repetitions), after film layer is analysed 10 minutes, by the T values obtained when detecting sample compared with standard curve, obtain
Detected value is obtained, detected value and actual value are compared, obtaining accuracy influences deviation.
Testing result is shown, is not detect that adenovirus resists in 30IFU/ml, 300IFU/ml and blank control from concentration
It is former.And actual concentrations be 1200IFU/ml and 6000IFU/ml sample to be tested testing result be respectively 780IFU/ml and
5190IFU/ml。
In conclusion detection kit provided by the present invention has good sensitivity and accuracy, effectively overcome
Various shortcoming of the prior art and have high industrial utilization.
The above-described embodiments merely illustrate the principles and effects of the present invention, not for the limitation present invention.It is any ripe
Know the personage of this technology all can carry out modifications and changes under the spirit and scope without prejudice to the present invention to above-described embodiment.Cause
This, those of ordinary skill in the art is complete without departing from disclosed spirit and institute under technological thought such as
Into all equivalent modifications or change, should by the present invention claim be covered.
Claims (10)
1. a kind of adenovirus detection reagent card, it is characterised in that including the sample application pad, glass fibre element film, nitre being arranged in order
Acid cellulose film and water suction gasket；Wherein,
Contain the Anti-adenovirus antibody of latex particle-the first and latex particle-test antigen 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.
2. adenovirus detection reagent card according to claim 1, it is characterised in that the test antigen is rabbit igg；It is described
The antibody for testing antigen is goat anti-rabbit igg.
3. adenovirus detection reagent card according to claim 1, it is characterised in that first Anti-adenovirus antibody and/
Or second Anti-adenovirus antibody is monoclonal antibody.
4. adenovirus detection reagent card according to claim 1, it is characterised in that the anti-adenopathy of latex particle-the first
Latex particle and the first adenovirus antibody in malicious antibody are connected by chemical coupling, and/or, the latex particle-test resists
Latex particle in original is connected with test antigen by chemical coupling.
5. the preparation method of adenovirus detection reagent card, includes the following steps as described in claim any one of 1-4：
1) on glass fibre element film the-the first Anti-adenovirus antibody of specking latex particle and latex particle-test antigen mixing
Solution；
2) the first pretreatment carried out to nitrocellulose filter that will be corresponding to the T1 lines of specking using the first pretreatment fluid, and
First pretreated the second Anti-adenovirus antibody of nitrocellulose filter specking solution；
3) the second pretreatment carried out to nitrocellulose filter that will be corresponding to the T2 lines of specking using the second pretreatment fluid, and
Second pretreated nitrocellulose filter specking Adenovirus Antigen solution；
4) on the nitrocellulose filter of predeterminated position specking test antigen antibody-solutions.
6. preparation method according to claim 5, it is characterised in that further include any one of following characteristics or multinomial：
First pretreatment fluid is to contain 1.5-2g/L arginine, 1.2-1.5g/L Potassium Hydrogen Phthalates, 0.3-0.5g/L hydrogen-oxygens
Change sodium, the aqueous solution of 2-2.5g/L citric acids；Second pretreatment fluid is to contain 2-2.5g/L asparagines, 0.5-0.7g/L
Disodium hydrogen phosphate, 3-4g/L rhamnoses, the aqueous solution of 3-3.5g/L glycine.
7. as claim 1-4 any one of them adenovirus detection reagents are stuck in the use prepared in adenovirus detection kit
On the way.
8. purposes according to claim 7, it is characterised in that the purposes is specially to use the adenovirus detection kit
Adenovirus Antigen in sample is detected.
9. purposes according to claim 8, it is characterised in that the purposes specifically includes：
Ratio between the value of first p-wire and the value of the second p-wire, and/or the value of the first p-wire and the value of control line
Between ratio, for obtaining adenovirus content；Wherein,
The colour brightness that the value of first p-wire is presented by the latex particle that the first p-wire is enriched with obtains, described second
The colour brightness that the value of p-wire is presented by the latex particle that the second p-wire is enriched with obtains, the value of the control line is by controlling
The colour brightness that the latex particle of line enrichment is presented obtains.
10. a kind of adenovirus detection kit, it is characterised in that detected including claim 1-5 any one of them adenovirus
Reagent card.
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Application publication date: 20180413 |