CN102539755A - Test strip for detecting influenza A virus antigen in secretion and preparation method thereof - Google Patents
Test strip for detecting influenza A virus antigen in secretion and preparation method thereof Download PDFInfo
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- CN102539755A CN102539755A CN2011104306169A CN201110430616A CN102539755A CN 102539755 A CN102539755 A CN 102539755A CN 2011104306169 A CN2011104306169 A CN 2011104306169A CN 201110430616 A CN201110430616 A CN 201110430616A CN 102539755 A CN102539755 A CN 102539755A
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Abstract
The invention discloses a test strip for detecting influenza A virus antigen in a secretion and a preparation method thereof. The test strip comprises a sample pad, a glass fiber membrane containing a colored latex particle label conjugate, a nitrocellulose membrane and water absorbent paper, which are sequentially stuck in an overlapped manner on a base plate, wherein the nitrocellulose membrane comprises a test region coated with an anti-influenza A virus antibody and a control region coated with a goat anti-rabbit antibody, the colored latex particle label conjugate comprises a micro-signal amplification system and a colored latex-labeled rabbit IgG antibody, and the micro-signal amplification system is a colored latex particle-avidin-biotin-anti-influenza A virus antibody composite. In the double-antibody sandwich ELISA system, the biotin-avidin micro-signal amplification system is added to amplify the signals of the target antibody, so as to improve detection sensitivity and avoid false negative results or missed detection due to weak signals.
Description
Technical field
The invention belongs to field of medical examination, relate in particular to a kind of test strips that detects influenza A virus antigen in the secretion and preparation method thereof.
Background technology
According to statistics, the annual influenza case in the whole world is 600,000,000-1,200,000,000, and wherein the severe influenza is 3,000,000-5,000,000 examples, dead 250,000 people-500,000 people, and the case fatality rate of severe influenza can reach 8%-10%.At present the death toll that causes of the annual influenza of the U.S. surpasses the death toll that traffic accident and AIDS cause, and has become the No.1 formidable enemy of serious harm human health in China and developing country.China is the district occurred frequently of influenza, and influenza popular or local breaks out basically that have every year, has every year people more than 100,000,000 to suffer the puzzlement of influenza, surpasses 500,000 people to the hospitalier of hospital.
Influenza is commonly referred to " influenza ", is the acute respiratory infectious disease that is caused by influenza virus, has very strong infectiousness, mainly through the cough and the propagation of sneezing, and general spring and outburst in winter.Be divided into A type (first type) influenza virus, Type B (B-mode) influenza virus and C type (third type) influenza virus.Influenza A virus has extremely strong variability, and B virus takes second place, and the third type virus is then highly stable.
The method that present stage is detected influenza infection mainly comprises Virus culture method, RT-PCR method and colloidal gold immunity chromatography.Virus culture is the goldstandard that virus detects, but this detection method cycle is very long, generally takes 3～7 days time, and sensitivity is not high; RT-PCR realizes detecting to sample in can 4-8 hour, and sensitivity is very high with specificity, but is not suitable on the airport, the density of population such as port, railway station, hospital are big, flow of the people big, be difficult for letting the public place of the long-time gathering of crowd; Colloidal gold immunity chromatography is owing to the equipment that need not assist, and use in accumulating easily and the basic unit place that is highly suitable for easy and simple to handle, but present stage sensitivity is not high, causes omission easily, still can not distinguish the virus infections hypotype.A kind of new colored latex detection technique that occurs recently, the remolding sensitivity collaurum wants high 10-100 doubly, and can pass through the furnishing various colors, can further be generalized to when realizing disparity items and detect.
Chinese patent CN200910087632.5 discloses a kind of primer special and the kit and the pairing target sequence of this primer that comprise the detection H1N1virus of this primer that belongs to the detection H1N1virus in pathogen gene quick diagnosis field.The pairing target sequence of primer special of the present invention, this kit utilize gene reverse transcription and isothermal amplification technique principle, and A (H 1 N 1) virus nucleic acid is carried out fast detecting, but the testing result naked eyes judge that electrophoresis also capable of using is analyzed.But this method length consuming time, expense is big, is unfavorable for large tracts of land popularization and civil nature.
Chinese patent CN200910085475.4 discloses the method that a kind of influenza virus rRT-PCR detects primer and probe and detects influenza virus; Comprise influenza A virus Auele Specific Primer and probe, two groups of new H1N1virus H1 Auele Specific Primers and probe, new influenza A virus Auele Specific Primer and probe etc.; 4 kinds of totally 12 Oligonucleolide primers and probe sequences disclose sample process to be checked, rRT-PCR reaction system and reaction conditions, interpretation of result simultaneously.The deficiency of this method also is owing to expensive, needs the instrument and the operating personnel of specialty need pass through professional training.So also being suitable for, this method do not popularize and civil nature.
Application number is that CN200910040975 has announced a kind of nucleic acid nano-gold biosensor that is used to detect influenza A virus and H1N1virus, is through scribbling the nano gold mark oligonucleotide probe on the spun glass and on nitrocellulose filter, fixing two kinds of oligonucleotide probes and detect object.But detection time, basic need was more than half an hour.
Same; The patent No. is that the patent of CN200810022489.7 has been announced and a kind ofly detected the method for influenza and H5N1 subtype avian influenza virus, patent that application number is CN201110223913.6 by liquid-phase chip and announced that first type/influenza B virus nucleic acid double fluorescent PCR detection kit, application number are that the patent of CN201010171355.9 has been announced and reached new H1N1virus one by people's first, influenza B to manage many detecting methods and kit, application number be 201110149333.7 to have announced double fluorescent quantitative RT-PCR detection kit and application; All there is the long problem of expense height, the professional instrument of needs, sense cycle in these methods, are unfavorable for that influenza test is in family, in basic unit and more extensively promote the use of among the crowd.
As stated, based on colored latex detection technique, add Avidin-biotin signal again and enlarge system, the binding antibody technology of preparing also can realize the influenza A virus detection of antibodies simultaneously overcoming the collaurum under-sensitive.
That this reagent strip has both is easy, characteristics fast and accurately; Being fit to grass-roots unit's primary dcreening operation and family uses; It is high and can not realize the defective of somatotype to remedy present stage chromatography kit sensitivity, can reduce risk that crowd massing infects to greatest extent, lower the harm that disease causes in that epidemic situation is on-the-spot.The design of joint inspection can be saved the medical disposable material that detects each time.These design features meet the national medical system in the policy of the popularization of basic unit and the proposal of environmental protection.
Summary of the invention
The objective of the invention is to solve that the influenza virus testing cost that exists in the prior art is expensive, sense cycle is long, with the low slightly defective of nano colloid gold sensitivity, and the test strips of influenza A virus antigen in a kind of new detection secretion is provided.
For realizing above-mentioned purpose, the present invention has taked following technical scheme:
A kind of test strips that detects influenza A virus antigen in the secretion; It is characterized in that; Said test strips is overlapped in order to stick on the base plate by sample pad, the glass fibre membrane that contains colored latex particulate labels, nitrocellulose filter, thieving paper and constitutes; Said nitrocellulose filter comprises the control zone that is coated with influenza A virus detection of antibodies district and is coated with goat anti-rabbit antibody; Said colored latex particulate labels comprises micro-signal amplification system and colored latex mark rabbit igg antibody, and said micro-signal amplification system is colored latex particle-Avidin-biotin-influenza A virus antibody.The test strips of influenza A virus antigen in the described detection secretion is characterized in that the parallel influenza A virus antibody that encapsulated, and can detect influenza A virus antigen.
Preferably, the consumption of said colored latex particle-Avidin-biotin-influenza A virus antibody complex is 32.5-60 μ g/cm
2The influenza A virus antibody consumption that encapsulates on the said detection zone is 0.144-0.28 μ g/mm; The consumption of said colored latex mark rabbit igg antibody is 20-40 μ g/cm
2Said goat anti-rabbit antibody is a goat anti-rabbit igg antibody, and the consumption of said goat anti-rabbit antibody is 0.18-0.44 μ g/mm.
Preferably, the consumption of said colored latex particle-Avidin-biotin-influenza A virus antibody complex is 55-60 μ g/cm
2, the influenza A virus antibody consumption that encapsulates on the said detection zone is 0.20-0.23 μ g/mm, the consumption of said colored latex mark rabbit igg antibody is 28-32 μ g/cm
2, the consumption of said goat anti-rabbit antibody is 0.30-0.36 μ g/mm.
The present invention also provides the preparation method of above-mentioned test strips, may further comprise the steps:
(1) prepares influenza A virus antibody and colored latex mark rabbit igg antibody by conventional method;
(2) preparation micro-signal amplification system
A, the colored latex mark Avidin of preparation
With Avidin to be marked dialysed overnight in the NaCl of 0.001～0.01mol/L solution in advance, centrifugal, regulate pH value to 7～8 of coloured glue emulsion, the mark Avidin is to form colored latex mark Avidin;
B, prepare biotinylation influenza A virus antibody by conventional method, the mass ratio of said biotin and influenza A virus antibody is 1: 9;
C, completion micro-signal amplification system
The biotinylation influenza A virus antibody that colored latex mark Avidin that step a is obtained and step b obtain mixes; The usage ratio of said colored latex mark Avidin and biotinylation influenza A virus antibody is: add 100 μ l biotinylation influenza A virus antibody in the colored latex mark of the 1ml Avidin solution; Connect and obtain colored latex-Avidin-biotin-first Antibody of Influenza; Washing promptly gets the micro-signal amplification system;
(3) by dilution parameters 20cm
2/ ml～40cm
2/ ml is sprayed onto on the glass fibre membrane drying for standby with the colored latex-Avidin-biotin-influenza A virus antibody and the colored latex-rabbit igg antibody of step (2);
(4) with encapsulating damping fluid dilution influenza A virus antibody, making its concentration is 0.9mg/ml～1.4mg/ml, presses film liquid measure 0.16 μ l/mm～0.2 μ l/mm, and it carefully is sprayed onto on the nitrocellulose filter drying for standby uniformly;
With encapsulating damping fluid dilution goat anti-rabbit antibody, making its concentration is 1.0mg/ml～2.0mg/ml, presses film liquid measure 0.18 μ l/mm～0.22 μ l/mm, and it carefully is sprayed onto on the nitrocellulose filter drying for standby uniformly;
(5) glass fibre membrane, the nitrocellulose filter of step (4), the thieving paper with sample pad, step (3) is pasted on the base plate according to the order of sequence, promptly gets.
Preferably, the consumption of coloured glue breast-Avidin in the said step (3)-biotin-influenza A virus antibody complex is 32.5-60 μ g/cm
2, the consumption of the influenza A virus antibody that encapsulates on the nitrocellulose filter in the said step (4) is 0.144-0.28 μ g/mm, the consumption of said colored latex mark rabbit igg antibody is 20-40 μ g/cm
2, the consumption of said goat anti-rabbit antibody is 0.18-0.44 μ g/mm.
Preferably, the consumption of coloured glue breast-Avidin in the said step (3)-biotin-influenza A virus antibody complex is 55-60 μ g/cm
2, the influenza A virus antibody consumption that encapsulates on the nitrocellulose filter in the said step (4) is 0.20-0.23 μ g/mm, the consumption of said colored latex mark rabbit igg antibody is 28-32 μ g/cm
2, the consumption of said goat anti-rabbit antibody is 0.30-0.36 μ g/mm.
Enlarge not enough problem in order to solve signal; The present invention is based on the solid-phase immunity chromatographic theory, adopt the influenza A virus antigen in colored latex particle labelling technique, micro-signal amplifying technique (Avidin-biotin system and double-antibody sandwich reaction system) the human body secretion.If contain influenza A virus antigen in the sample; Micro-signal cascade system on glass fibre membrane is amplified signal, and influenza A virus antigen is combined with colored latex particle-Avidin-biotin-influenza A virus antibody complex, and is diffused into further chromatography on the nitrocellulose filter; When running into the pairing antibody that the T line (influenza A virus antibody sandwich line) that is coated on detection zone on the nitrocellulose filter locates; Compound then combines with coated antibody again, is trapped in and encapsulates the place, when captive compound reaches some; Then form macroscopic T line; Contain influenza A virus antigen in the interpret sample, if do not occur, the negative or content of interpret sample is lower than the LDL of test strips.Control zone (C line) is as the quality control standard of test strips, and positive and negative sample all can occur when detecting.
Compared with prior art, the present invention has following beneficial effect:
(1) the present invention has adopted new colored latex detection technique to replace traditional collaurum detection technique, and has added biotin-avidin micro-signal cascade amplification system on the basis of existing test strip; Optimized preparation parameter; Obtained influenza A virus antigen in the optimized detection secretion test strips thereby, in the process that detects influenza A virus antigen, enlarged the signal of target antibody; Increase detection sensitivity, avoid occurring false cloudy perhaps omission because of signal is too weak;
(2) test strips of the present invention have handling safety (no radiation pollute), easy (one step of simple operations accomplishes), be fit to single/part detect (put exempt from, enzyme exempt to be not suitable for single/part or small amount of sample detect) with advantages such as (about 15 minutes the result can be arranged) fast, can realize influenza A virus antigen scene and family's self check.
Description of drawings
Fig. 1 is the structural representation of the test strips of influenza A virus antigen in the detection secretion of the present invention;
Fig. 2 utilizes test strips of the present invention to detect the positive findings synoptic diagram of influenza A virus antigen;
Fig. 3 utilizes test strips of the present invention to detect the negative findings synoptic diagram of influenza A virus antigen;
Reference numeral: 1. sample pad; 2. glass fibre membrane; 3. nitrocellulose filter; 4. thieving paper; 5. influenza A virus detection zone; 6. Quality Control district; 7. base plate.
Embodiment
Specify the present invention below in conjunction with accompanying drawing and specific embodiment.
Embodiment 1 the present invention detects the test strips of influenza A virus antigen in the secretion
As shown in Figure 1, be a kind of test strips that detects influenza A virus antigen in the secretion of the present invention.The glass fibre membrane that contains colored latex particulate labels 2 that comprises sample pad 1, closely links to each other with said sample pad 1 one ends, the nitrocellulose filter 3 that closely links to each other with said glass fibre membrane 2 other ends and the thieving paper 4 that closely links to each other with the other end of said cellulose membrane 3; Said sample pad 1, glass fibre membrane 2, nitrocellulose filter 3 and thieving paper 4 all are arranged on the base plate 7; Said nitrocellulose filter 3 comprises the control zone 6 that is coated with influenza A virus detection of antibodies district 5 and is coated with goat anti-rabbit antibody; Said colored latex particulate labels comprises micro-signal amplification system and colored latex mark rabbit igg antibody, and said micro-signal amplification system is colored latex particle-Avidin-biotin-influenza A virus antibody.
Reagent strip of the present invention, the said colored latex particle-Avidin-biotin-consumption of influenza A virus antibody complex on glass fibre membrane 2 is 58 μ g/cm
2The consumption of colored latex mark rabbit igg antibody on glass fibre membrane 2 is 30 μ g/cm
2The original concentration of influenza A virus antibody is 3.00mg/ml, and the concentration that encapsulates the detection zone 5 of nitrocellulose membrane 3 is 1.2mg/ml, and encapsulating consumption is 0.216 μ g/mm; Consumption when goat anti-rabbit igg antibody is coated on the control zone is 0.33 μ g/mm.
The preparation method of embodiment 2 embodiment 1 test strips
Adopt colored latex particle mark in the present invention.Colored latex microsphere is through the carboxyl of microsphere surface or amino, under the effect of crosslinking chemical such as EDC, glutaraldehyde or coupling agent, with the amino or the carboxyl generation condensation reaction of albumen, forms stable cross-linking agent.Utilize the antigenicity generation antigen-antibody reaction of albumen, the color of utilizing microballoon to have plays spike, reaches the purpose of reaction.
A kind of preparation process that detects the test strips of influenza A virus antigen in the secretion is following:
1. prepare influenza A virus antibody
Utilize conventional method with the Flu-A antigen of reorganization or the Flu-A antigen immune mouse of deactivation; After the mouse of Flu-A antigen immune and myeloma cell are merged; Differentiate with the ELISA method; The hybridoma that discriminating is come out is cultivated screening positive clone, separation and purification influenza A virus antibody in ascites.ELISA identifies the activity of influenza A virus antibody and tires that purifying is subsequent use.
2, preparation micro-signal amplification system
A, the colored latex mark Avidin of preparation: with Avidin to be marked (Avidin is provided by U.S. Thermo company) 4 ℃ of dialysed overnight in the NaCl of 0.005mol/L pH7.0 solution in advance; To remove unnecessary salt ion; 4 ℃ of 100 centrifugal 1h of 000g removes polymkeric substance then; Regulate pH value to 7～8 of coloured glue emulsion (Merck KGaA (MERCK)) with the PBS of 0.1mol/L, the mark Avidin is to form colored latex mark Avidin;
B, preparation biotinylation influenza A virus antibody: the first connection with biotin (biotin is provided by U.S. Thermo company) with the 6-aminohexose makes long-armed biotin; It is combined with Flu-A antigen; The mass ratio of said biotin and influenza A virus antibody is 1: 9; Then under the effect of carbodiimide with itself and N-hydroxy-succinamide contract with; (N-hydroxy-succinimido-6-biotinyl amido hexanoae BCNHS), is biotinylation influenza A virus antibody to generate long-armed biotin N-maloyl imines ester.Remove free biotin through dialysis.
C, accomplish the micro-signal amplification system: the biotinylation influenza A virus antibody that colored latex mark Avidin that step a is obtained and step b obtain directly mixes; The usage ratio of said colored latex mark Avidin and biotinylation influenza A virus antibody is: add 100 μ l biotinylation influenza A virus antibody in the colored latex mark of the 1ml Avidin solution; Connect and obtain colored latex-Avidin-biotin-influenza A virus antibody complex; After washing, centrifugal treating, promptly get the micro-signal amplification system.
3, colored latex mark rabbit igg antibody
In pH6.5～7.0 scopes, rabbit igg and colored latex granule protein (minimum amount is 8.0 μ g/ml) mark forms colored latex mark rabbit igg antibody (albumen probe).
4, colored latex-Avidin-biotin-influenza A virus antibody and colored latex mark rabbit igg antibody are sprayed onto on the glass fibre membrane 2, dilution parameters (dilution parameters is the technological parameter of every how many areas of ml spray solution) is 25cm
2/ ml～35cm
2/ ml, the said colored latex particle-Avidin-biotin-consumption of influenza A virus antibody complex on glass fibre membrane 2 is 58 μ g/cm
2The consumption of colored latex mark rabbit igg antibody on glass fibre membrane 2 is 30 μ g/cm
2, and 20～40 ℃ of temperature, humidity 10%-30% is dry more than 12 hours, subsequent use with glass fibre membrane 2.
5, (principal ingredient is 0.01M pH7.2PBS, 5% sucrose, 1% methyl alcohol, 5% glycocoll, 0.05%NaN with encapsulating damping fluid
3) dilution influenza A virus antibody, making its concentration is 1.0mg/ml, presses film liquid measure 0.18 μ l/mm; With its careful respectively being sprayed onto uniformly on the nitrocellulose filter 3; The original concentration of influenza A virus antibody is 3.00mg/ml, and the concentration that encapsulates the detection zone 5 of nitrocellulose membrane 3 is 1.2mg/ml, and encapsulating consumption is 0.216 μ g/mm; Wherein nitrocellulose filter 3 apertures are 5.0 μ m～10.0 μ m; Place 20～40 ℃, oven dry was handled more than 12 hours under humidity 10%～30% condition, and is subsequent use; With encapsulating damping fluid dilution goat anti-rabbit igg antibody, making its concentration is 1.5mg/ml, presses film liquid measure 0.20 μ l/mm; With its careful being sprayed onto uniformly on the nitrocellulose filter 3, place 20～40 ℃, oven dry is handled more than 12 hours under humidity 10%～30% condition; Envelope, subsequent use;
Simultaneously, with encapsulating damping fluid dilution goat anti-rabbit antibody, making its concentration is 1.5mg/ml, presses film liquid measure 0.33 μ g/mm, and it carefully is sprayed onto on the nitrocellulose filter drying for standby uniformly;
6, sample pad 1, glass fibre membrane 2, nitrocellulose filter 3 and thieving paper 4 are pasted on the base plate 7 in regular turn, promptly get test strips of the present invention.
The test strips of influenza A virus antigen can directly be used through packing in the detection secretion of the present invention, perhaps is installed in the kit, cooperates little dropper, serves as reagent card and uses.
Embodiment 3 utilizes the test strips among the embodiment 1 to detect the method for influenza A virus antigen in the secretion
Present embodiment utilizes the test strips of embodiment 1 that the influenza A virus antigen in the juice sample is detected.
May further comprise the steps:
(1) gathers sample: use the polyester sponge swab of aseptic PP (polypropylene) bar to gather sample.Nasal secretion acquisition method: when collecting nasal secretion; Swab is inserted in the nasal cavity the many places of secretion, rotates gently and to the inner swab that promotes of nasal cavity, until concha (from the place of being obstructed of the about 2.0cm in nostril～2.5cm); Paste nasal wall rotation swab three times, take out swab; Throat secretion is gathered: swab is inserted from the oral cavity the throat fully, is the center with the rubescent position of throat wall, maxilla almond, and appropriateness is wiping bilateral pharyngeal tonsils and pharynx rear wall firmly, should avoid touching tongue, takes out swab;
(2) behind the collection saliva sample; Vertical 400 μ l (about 10) the sample extraction liquid that adds in the sample extraction pipe in solution in the insertion of the swab after the sampling sample extraction pipe, rotates about 10 times near inboard wall of test tube; Make sample be dissolved in the solution as far as possible; Cotton swab head along extraction tube inwall extruding swab is stayed in the pipe liquid as far as possible, takes out and discard swab.The sample extraction liquid that adopts viral sample solution or this kit to provide after the collection of specimens is as early as possible handled.As can not handle immediately, sample should place immediately in drying, sterilization and the plastic tube of strict seal and store, can preserve 8 hours under 2 ℃～8 ℃, but-70 ℃ of long preservation;
(3) tear along the aluminium foil bag cutting part, take out strip and keep flat, draw 5 μ l samples and vertically drip in the sample application zone shown in the strip arrow glue; Because capillarity, sample will move with nitrocellulose filter 3 to the plain film 2 of spun glass along test strips, treat sample fully through plain film 2 of spun glass and nitrocellulose filter 3, and the result begins to show;
Observe display result (result displayed is invalid after 30 minutes) after (4) 15 minutes; Interpretation is also write down testing result (Fig. 2 and Fig. 3); If a macroscopic dark line (T line) appears in the detection zone of nitrocellulose filter 35; Show promptly and contain a large amount of influenza A virus antigens in the sample that the human body that i.e. explanation receives the proofer is passive armor type influenza infection (influenza A virus is positive, please simultaneously with reference to figure 2); If a macroscopic dark line does not appear in the detection zone 5 of nitrocellulose filter 3, promptly show and do not contain a large amount of influenza antigens in the sample, explain to receive the proofer not by virus infections (feminine gender is asked simultaneously with reference to figure 3); When the detection zone 5 of sample through nitrocellulose filter 3 moves to control zone 6, no matter in the sample whether influenza A virus antigen is arranged, all can there be a dark line (C line) control zone 6; If control zone 6 no colo(u)r streaks occur, explain that test strips is expired or operate wrong;
(5) behind the EOT, test strips, sample extraction pipe and oral cavity ST after using are handled by the biologic medical discarded object.
The result
Annotate: in order to represent that conveniently A represents influenza A virus antigen in following table
Estimate reagent and reference reagent relatively (immunochromatographic method): detect sample and be included in Guangdong Prov. Disease Prevention-control Center 481 examples, Inspection and Quarantine Technic Center, Guangdong Entry-Exit Inspection and Qu's 300 examples, Guangzhou women and children's medical centre (Guangzhou Children's Hospital) 300 examples, Guangzhou Disease Prevention-Control Center's 300 examples, No.302 Hospital, P.L.A.'s 345 examples, Guangzhou City No.8 People's Hospital's 202 examples, add up to 1928 examples.
Annotate: "+/+" the reagent result is estimated in expression and reference reagent comes to the same thing, and is all positive; "+/-" result is positive for expression evaluation reagent, and the result is negative for reference reagent; "-/+" result is negative for expression evaluation reagent, and the result is positive for reference reagent; "-/-" estimate the reagent testing result and reference reagent comes to the same thing, all negative.Below two product chromatography contrasts identical.
Estimate reagent and reference reagent and PCR method relatively: Inspection and Quarantine Technic Center, Guangdong Entry-Exit Inspection and Qu's 300 examples, Guangzhou women and children's medical centre (Guangzhou Children's Hospital) 300 example and Guangdong Prov. Disease Prevention-control Center 166 routine clinical samples add up to 766 examples.
Annotate: "+/+" (reference) reagent result is estimated in expression and the PCR method comes to the same thing, and is all positive; "+/-" result is positive for expression evaluation (reference) reagent, and the result is negative for the PCR method; "-/+" result is negative for expression evaluation (reference) reagent, and the result is positive for the PCR method; "-/-" estimate (reference) reagent testing result and the PCR method comes to the same thing, all negative.Following PCR method is more identical.
The detection of nose swab sample: 202 examples of Guangdong Prov. Disease Prevention-control Center (sanitary inspection center, Guangdong Province) 128 examples, Inspection and Quarantine Technic Center, Guangdong Entry-Exit Inspection and Qu's 300 examples and Guangzhou City No.8 People's Hospital, three families add up to 630 examples.
The detection of throat swab sample: Guangzhou Disease Prevention-Control Center (sanitary inspection center, Guangzhou) 300 examples, 323 examples at Guangdong Prov. Disease Prevention-control Center (sanitary inspection center, Guangdong Province), 300 examples of Guangzhou Children's Hospital and 345 examples of No.302 Hospital, P.L.A., four families add up to 1268 examples.
Annotate: "+/+" represent that evaluation reagent and reference reagent testing result are with positive; "+/-" represent that evaluation reagent is positive, reference reagent is negative; "-/+" represent that evaluation reagent is negative, reference reagent is positive; "-/-" represent that evaluation reagent and reference reagent are with negative
Embodiment 4 present embodiments detect the test strips of influenza A virus antigen in the secretion
The described a kind of test strips that detects influenza A virus antigen in the secretion of present embodiment.The glass fibre membrane that contains colored latex particulate labels 2 that comprises sample pad 1, closely links to each other with said sample pad 1 one ends, the nitrocellulose filter 3 that closely links to each other with said glass fibre membrane 2 other ends and the thieving paper 4 that closely links to each other with the other end of said cellulose membrane 3; Said sample pad 1, glass fibre membrane 2, nitrocellulose filter 3 and thieving paper 4 all are arranged on the base plate 7; Said nitrocellulose filter 3 comprises the control zone 6 that is coated with influenza A virus detection of antibodies district 5 and is coated with goat anti-rabbit antibody; Said colored latex particulate labels comprises micro-signal amplification system and colored latex mark rabbit igg antibody, and said micro-signal amplification system is colored latex particle-Avidin-biotin-influenza A virus antibody.
The reagent strip of present embodiment, the said colored latex particle-Avidin-biotin-consumption of influenza A virus antibody complex on glass fibre membrane 2 is 40 μ g/cm
2The consumption of colored latex mark rabbit igg antibody on glass fibre membrane 2 is 20 μ g/cm
2The original concentration of influenza A virus antibody is 3.00mg/ml, and the concentration that encapsulates the detection zone 5 of nitrocellulose membrane 3 is 1.2mg/ml, and encapsulating consumption is 0.150 μ g/mm; Consumption when goat anti-rabbit igg antibody is coated on the control zone is 0.20 μ g/mm.
The preparation method of the said reagent strip of present embodiment and embodiment 2 are basic identical.
Adopt Inspection and Quarantine Technic Center, Guangdong Entry-Exit Inspection and Qu's 200 examples and Guangdong Prov. Disease Prevention-control Center 200 routine clinical samples; Totally 400 routine clinical definite samples; Compare detection with embodiment 1 with embodiment 4 described diagnosis test papers and Hangzhou Creative Company first type detection kit, the result is following in comparation and assessment.
The sensitivity and the specificity result of embodiment 1,4 and commercially available prod are following:
Can find out that from last table embodiment 1 scheme obviously is being superior to embodiment 4 schemes aspect sensitivity and the specificity, and the sensitivity of comparing with Hangzhou Creative Company like product is higher, more helps detecting of influenza A virus.
More than be to the specifying of possible embodiments of the present invention, but this embodiment is not in order to limiting claim of the present invention, does not allly break away from the equivalence that skill spirit of the present invention does and implement or change, all should be contained in the claim of the present invention.
Claims (9)
1. test strips that detects influenza A virus antigen in the secretion; It is characterized in that; Said test strips is overlapped in order to stick on the base plate by sample pad, the glass fibre membrane that contains colored latex particulate labels, nitrocellulose filter, thieving paper and constitutes; Said nitrocellulose filter comprises the control zone that is coated with influenza A virus detection of antibodies district and is coated with goat anti-rabbit antibody; Said colored latex particulate labels comprises micro-signal amplification system and colored latex mark rabbit igg antibody, and said micro-signal amplification system is colored latex particle-Avidin-biotin-influenza A virus antibody.
2. the test strips of influenza A virus antigen is characterized in that in the detection secretion according to claim 1, and the consumption of said colored latex particle-Avidin-biotin-influenza A virus antibody complex is 32.5-60 μ g/cm
2
3. the test strips of influenza A virus antigen is characterized in that in the detection secretion according to claim 1, and the influenza A virus antibody consumption that said detection zone encapsulates is 0.144-0.28 μ g/mm.
4. the test strips of influenza A virus antigen is characterized in that in the detection secretion according to claim 1, and the consumption of said colored latex mark rabbit igg antibody is 20-40 μ g/cm
2
5. the test strips of influenza A virus antigen is characterized in that in the detection secretion according to claim 1, and said goat anti-rabbit antibody is a goat anti-rabbit igg antibody, and the consumption of said goat anti-rabbit antibody is 0.18-0.44 μ g/mm.
6. the test strips of influenza A virus antigen is characterized in that in the detection secretion according to claim 1, and the consumption of said colored latex particle-Avidin-biotin-influenza A virus antibody complex is 55-60 μ g/cm
2, the influenza A virus antibody consumption that said detection zone encapsulates is 0.20-0.23 μ g/mm, the consumption of said colored latex mark rabbit igg antibody is 28-32 μ g/cm
2, the consumption of said goat anti-rabbit antibody is 0.30-0.36 μ g/mm.
7. a method for preparing the test strips of influenza A virus antigen in each described detection secretion of claim 1-6 is characterized in that, may further comprise the steps:
(1) prepares influenza A virus antibody and colored latex mark rabbit igg antibody by conventional method;
(2) preparation micro-signal amplification system
A, the colored latex mark Avidin of preparation
With Avidin to be marked dialysed overnight in the NaCl of 0.001～0.01mol/L solution in advance, centrifugal, regulate pH value to 7～8 of coloured glue emulsion, the mark Avidin is to form colored latex mark Avidin;
B, prepare biotinylation influenza A virus antibody by conventional method, the mass ratio of said biotin and influenza A virus antibody is 1: 9;
C, completion micro-signal amplification system
The biotinylation influenza A virus antibody that colored latex mark Avidin that step a is obtained and step b obtain mixes; The usage ratio of said colored latex mark Avidin and biotinylation influenza A virus antibody is: add 100 μ l biotinylation influenza A virus antibody in the colored latex mark of the 1ml Avidin solution; Connect and obtain colored latex-Avidin-biotin-influenza A virus antibody; Washing promptly gets the micro-signal amplification system;
(3) by dilution parameters 20cm
2/ ml～40cm
2/ ml is sprayed onto on the glass fibre membrane drying for standby with the colored latex-Avidin-biotin-influenza A virus antibody and the colored latex-rabbit igg antibody of step (2);
(4) with encapsulating damping fluid dilution influenza A virus antibody, making its concentration is 0.9mg/ml～1.4mg/ml, presses film liquid measure 0.16 μ l/mm～0.2 μ l/mm, and it carefully is sprayed onto on the nitrocellulose filter drying for standby uniformly;
With encapsulating damping fluid dilution goat anti-rabbit antibody, making its concentration is 1.0mg/ml～2.0mg/ml, presses film liquid measure 0.18 μ l/mm～0.22 μ l/mm, and it carefully is sprayed onto on the nitrocellulose filter drying for standby uniformly;
(5) glass fibre membrane, the nitrocellulose filter of step (4), the thieving paper with sample pad, step (3) is pasted on the base plate according to the order of sequence, promptly gets.
8. preparation according to claim 7 detects the method for the test strips of influenza A virus antigen in the secretion, it is characterized in that, the consumption of coloured glue breast-Avidin in the said step (3)-biotin-influenza A virus antibody complex is 32.5-60 μ g/cm
2, the consumption of the influenza A virus antibody that encapsulates on the nitrocellulose filter in the said step (4) is 0.144-0.28 μ g/mm, the consumption of said colored latex mark rabbit igg antibody is 20-40 μ g/cm
2, the consumption of said goat anti-rabbit antibody is 0.18-0.44 μ g/mm.
9. preparation according to claim 7 detects the method for the test strips of influenza A virus antigen in the secretion, it is characterized in that, the consumption of coloured glue breast-Avidin in the said step (3)-biotin-influenza A virus antibody complex is 55-60 μ g/cm
2, the influenza A virus antibody consumption that encapsulates on the nitrocellulose filter in the said step (4) is 0.20-0.23 μ g/mm, the consumption of said colored latex mark rabbit igg antibody is 28-32 μ g/cm
2, the consumption of said goat anti-rabbit antibody is 0.30-0.36 μ g/mm.
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