CN106771120A - Quick detection bird flu and the method and test strips of human influenza Susceptible population - Google Patents
Quick detection bird flu and the method and test strips of human influenza Susceptible population Download PDFInfo
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- CN106771120A CN106771120A CN201611073359.7A CN201611073359A CN106771120A CN 106771120 A CN106771120 A CN 106771120A CN 201611073359 A CN201611073359 A CN 201611073359A CN 106771120 A CN106771120 A CN 106771120A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
- G01N2400/02—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates involving antibodies to sugar part of glycoproteins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/50—Determining the risk of developing a disease
Abstract
The invention discloses a kind of quick detection bird flu and the method and test strips of human influenza Susceptible population, including sample pad, gold standard pad, prosecution area and the adsorptive pads for surplus liquid in absorption detecting sample set gradually along chromatography direction；The prosecution area includes the detection band and the nature controlling line that set gradually；The gold standard pad includes agglutinin the MAL II or SNA of colloid gold label；The detection band includes stabilization internal reference agglutinin uncombined with agglutinin MAL II or stabilization internal reference agglutinin uncombined with agglutinin SNA.By in sample pad one end insertion saliva sample of test strips or containing in the oral cavity, after 10~15 minutes, test strips are taken out, observe detection band and nature controlling line color status, testing result is obtained, the present invention really realizes detecting bird flu and human influenza virus Susceptible population noninvasive, safe and simplely.
Description
Technical field
The present invention relates to be based on the bird flu of Lateral Flow Strip quick detection and human influenza virus Susceptible population, can also develop into
The bird flu of double agglutinin sandwich method quick detections and human influenza virus Susceptible population based on ELISA Plate detection.
Background technology
Agglutinin is non-immune origin, the class carbohydrate-binding protein without enzymatic activity, can exclusively recognize a certain spy
Specific sugar chain sequence in the monose or glycan of different structure and it is in combination.More than 100 are found from nature so far
Plant the agglutinin of plant source.Aggegation have various applications：Such as malignant tumour disease diagnosis, design antibacterial and antiviral drug,
Design " biological missile " and carry out identification and analysis of sugar chain structure etc..Research neck is learned with chip technology is introduced into sugar group
Domain, agglutinin is more and more noticeable as the important tool of decoding sugar chain structure.Lectin chip is according to agglutinin and sugar chain
Between specificity be mutually distinguishable effect, the agglutinin of various separate sources is fixed on epoxidation, aldehyde radical or through its other party
In the chip base of formula modification, then with mark after the sample incubation reaction to be detected such as glycoprotein, cell and thalline, through a series of follow-up
After treatment, the agglutinin of the sugar chain structure and its identification in sample to be detected can be just obtained.
Host distribution of the influenza virus in nature widely, occurs with complicated gene structure, and its gene
The probability matched somebody with somebody again and be mutated is very high, therefore is in the world periodic epidemic.So far, broken out altogether in human history
Four large-scale influenza pandemic, including the big influenza of Spain (1918), the big influenza in Asia (nineteen fifty-seven), the big influenza in Hong Kong
(nineteen sixty-eight) and new Influenza A H1N1 (at the beginning of 21 century), wherein in spanish influenza is very popular, death toll is up to
2000-4000 ten thousand.Influenza has the incidence of disease and the features such as case fatality rate is high, spread speed fast, to involve scope wide, once break out
Come, it will the mankind are caused with serious disaster.In recent years, H5, H7 and H9 subtype influenza virus and the A type for occurring for 2009
H1N1 influenza viruses all cause larger epidemic situation, some strains energy direct infection mankind of H5N1 and H7N9 types, infection rate high
H9 hypotypes with low pathogenicity equally can also be given people by birds direct contagion, although the virus of these hypotypes does not cause people also
Parainfluenza is very popular, but it is huge that they are potentially hazardous.Influenza virus hemagglutinin (Hemagglutinin, HA) molecule with
The combination of influenza virus host cell surface sialic acid (SA) α 2-3 galactolipins (Gal) or SA α 2-6Gal sugar-chain end acceptors, be
Influenza virus infects the beginning of body.Sugar chain receptor structure on different influenza virus host cell surfaces is different, fowl stream
Influenza Virus HA mainly recognizes and combines the sugar chain acceptor that end is SA α 2-3Gal, and human influenza virus HA is mainly recognized and combined
End is the sugar chain acceptor of SA α 2-6Gal.
New research report points out there are SA α 2-6Gal and SA α 2-3Gal sugar chain acceptors on human respiratory epithelial cell
Distribution, but based on the distribution of SA α 2-6Gal sugar chain acceptors；In addition, SA α 2-6Gal are in high density in people's trachea and bronchus
Distribution, and gradually decreased with the reduction of bronchus classification, it is minimum to alveolar distribution；And SA α 2-3Gal are in trachea-bronchial epithelial cell
And only in a small amount of distribution in bronchiole, its distribution is based on alveolar bronchiole and alveolar.A small amount of SA α 2- on human respiratory
The presence of 3Gal is the most strong evidence of the part H5N1 subtype avian influenza virus direct infection mankind.
Human saliva is secreted by the parotid gland, glandula submandibularis, sublingual gland and some other glandula, comprising nucleic acid, protein, fat
Class, mineral matter and other small-molecule substances.In addition, also exist in saliva suppressing and eliminating bacteria and viral material.Saliva contains
Change in amount and composition, is either caused by oral cavity partial lesion or body integral status, is all likely to result in saliva
Functional defect.Research find, in saliva except containing the basic composition protein of some salivas (in different sexes and all ages and classes
Section is all almost without differentially expressed protein or glycoprotein) outward, human body health and physiology shape may also be reacted containing some
Often there is otherness in the crowd of all ages and classes, sex, pathology and physiological situation in some protein of condition, these protein
Expression.
The protein component having in blood is existed in saliva, and saliva can reflect part protein level in blood
Change.Compared by being annotated with the GO (gene ontology) of mankind's holoprotein group to saliva and blood plasma, find saliva and blood
Slurry Proteomics extracellular matrix components are more, and intracellular fluid component is less, point out saliva and plasma proteins group to have
The feature of secretory protein group；Analysis result prompting in terms of biological process and molecular function, sialoprotein matter group and blood
Slurry protein group has similitude.But, the proteomic comparison result of saliva and blood plasma shows that some protein are only in saliva
Proteomics occur, without appearing in plasma proteins group in, show that both can not be equal to.
Saliva resists first natural cover for defense of respiratory virus infection as human body, and glycoprotein therein plays important
Effect, α -2 macroglobulin such as in saliva, mucoprotein 5B and the grade of SGP 340 can be by (α 2-3/ α 2-6) thereon even
Connect the infection that terminal sialic acid sugar chain structure is combined to neutralize with influenza virus HA and suppresses influenza virus.
The saliva of three age bracket healthy volunteers is analyzed using high coverage rate lectin chip, it was found that one
SGP sugar chain related to age and sex a bit, wherein：(1) the glycoprotein candy chain structure in children male's saliva is compared with female
Property more horn of plenty；(2) SGP sugar chain differential expression is the most notable between adult male and women；(3) elderly men with
SGP sugar chain differential expression is not notable between women.And find Sia α 2-3Gal β 1-4Glc (NAc) and α -1 in saliva,
3 connection Man structures express increase with advancing age；Fuc α 1-2Gal β 1-4Glc (NAc) structure is with female age
Increase expression to reduce, but Fuc α 1-2Gal β 1-4Glc (NAc) structure is in age inverted parabolic curve type in male's saliva
Expression.
Sent out by the sialoprotein matter to different age group different sexes healthy volunteer and the thereon comparative studies of sugar chain
Existing, with advancing age, the protein content in Healthy People saliva is without substantially change, and sugar chain structure thereon and quantity
There occurs significant change, sialic acid sugar chain such as in sialoprotein matter expression showed increased, and its protein expression have no it is aobvious
Write and change.Find simultaneously healthy elderly saliva by provide more α 2-3 and α 2-6 connect terminal sialic acid sugar chain structure with
Influenza virus hemagglutinin is combined, and neutralizes and inhibit mutual knowledge of the influenza virus to human upper airway specific cell surface receptors
Not.So as to speculate the fact that this molecular mechanism can have the ability of stronger resistance influenza with partial interpretation healthy elderly.
However, the people being infected during Influenza Outbreak is generally on the contrary the elderly, at present to its basic reason there is not yet clearly
Explain.According to various people's influenza virus neurological susceptibility clinical observation statistics, diabetes B, hypertension and heart disease, cancer,
Chrome lung, arthritis and pregnant woman etc. are easier to influenza virus infection (not yet distinguishing bird flu or human influenza)；For this kind of feelings
The elderly of condition, generally explanation are the immune systems that chronic disease compromises the elderly, reduce the Immunoresistance of the elderly, are removed
Outside the factor of this common-sense, if the influence of also other physiology or pathological factor so that the elderly's easy infection stream of this part
What Influenza Virus, other Susceptible population make and explain againTherefore, in order to effective guiding clinical diagnosis and the raising public to certainly
The cognition of body risk, needs a kind of easy detection (examination) method badly, can be with timely without large-scale instrument and instrument
Obtain the assessment information of individual bird flu and the susceptible speciality of human influenza virus.
Chinese patent " a kind of for examination bird flu and the detection instrument of human influenza virus Susceptible population " (publication number：
CN103884834A, publication date：2014.06.25 agglutinin MAL-II and bird flu neurological susceptibility and agglutinin SNA) are disclosed
With the dependency relation of the easy neurological susceptibility of human influenza virus, and point out, tied with corresponding agglutinin MAL-II or SNA in Susceptible population's saliva
α 2-3 or α 2-6 the connection terminal sialic acid sugar chain structure expressions of conjunction are significantly lowered, but the patent is only applied to chip examination
Agent box, the simplicity of detection, speed and cost have much room for improvement.
Detection paper technology in medical diagnosis on disease and detection using gradually increasing, its have quickly, low price the characteristics of, but mesh
It is preceding there is not yet test strips and the report of detection method for detecting bird flu and human influenza Susceptible population.Test strips are generally wrapped
Include sample pad, gold standard pad, be printed with detection band and the cellulose acetate film of control line and water suction end.For saliva, no
The existing similitude of sugar chain structure between individuality contained by it also has specificity together, and sample is increased from the reliability perspectives of detection
The design and preparation difficulty of pad, detection band and control line.
The content of the invention
It is an object of the invention to provide a kind of quick detection bird flu and the method and test paper of human influenza Susceptible population
Bar, so as to really realize detecting bird flu and human influenza virus Susceptible population noninvasive, safe and simplely.
To reach above-mentioned purpose, present invention employs following technical scheme：
The test strips of quick detection influenza Susceptible population, including for detect bird flu Susceptible population A types test strips and/
Or for detecting the H type test strips of human influenza Susceptible population, the A types test strips and H types test strips include along chromatography direction according to
The sample pad of secondary setting, nanoparticle label pad, prosecution area and adsorptive pads；The prosecution area includes the detection band for setting gradually
And nature controlling line；The nanoparticle label pad of the A types test strips includes the solidifying of collaurum, magnetic fluid or nano-fluorescent grain mark
Collecting the nanoparticle label pad of element MAL-II, H type test strips includes the aggegation of collaurum, magnetic fluid or nano-fluorescent grain mark
Plain SNA；The detection band of the A types test strips includes stabilization internal reference agglutinin uncombined with agglutinin MAL-II, H type test strips
Detection band include stabilization internal reference agglutinin uncombined with agglutinin SNA.
The detection band of the A types test strips is solidified with least in agglutinin WFA, STL, RCA120, PHA-E, DBA
Kind, nature controlling line is solidified with agglutinin ConA.
The detection band of the H types test strips is solidified with least one in agglutinin GNA, ECA, HHL, PHA-E, DBA, matter
Control line is solidified with agglutinin STL.
The nanoparticle label pad is selected from gold standard pad, and the gold standard pad is will to mark colloid by solution impregnation and drying
The agglutinin MAL-II or SNA of gold are combined and are made on the glass fibers；The detection band and nature controlling line be by line,
Dry and be fixed on corresponding agglutinin on nitrocellulose filter and be made by closing.
The label concentration of agglutinin MAL-II or SNA is 10~12 μ g/mL, and agglutinin draws in detection band and nature controlling line
Line concentration is 1~4mg/mL.
The solution impregnation refers to use that to be dispersed with concentration molten for 4~10 times of agglutinins of the colloid gold label of enrichment
Immersion bulb glass fiber；The multiple of the enrichment determines in such a way：The agglutinin MAL-II or SNA of 120 μ g are pressed
According to 10~12 μ g/mL label concentration be marked in colloidal gold solution after be scattered in 1mL solution Is, obtain 10 times concentration
The colloid gold label agglutinin solution of degree, after further being diluted using solution I, then obtains other collaurum marks compared with low enrichment
Note agglutinin solution, the solution I is NaCl containing 0.15mol/L and 0.01mol/L pH7.5~8.5 of 0.2%BSA
HEPES solution, the pH of the colloidal gold solution is 7.5~8.5；It is described line refer to by corresponding one or more agglutinin according to
1~4mg/mL of total concentration is dissolved in the PBS of 0.01mol/L pH6.5~8.0, obtains agglutinin solution, using the solution in nitre
Line style band is applied on acid cellulose film.
Preferably, use and be dispersed with concentration for 5 times of solution of the agglutinin of the colloid gold label of enrichment soak glass fibers
Dimension；The concentration of the agglutinin solution used in line is 2mg/mL.
The preparation method of the test strips of above-mentioned quick detection influenza Susceptible population, comprises the following steps：
(1) prepared by collaurum
Colloidal gold solution is prepared using aqueous solution of chloraurate；
(2) colloid gold label
The pH value of colloidal gold solution is adjusted to addition agglutinin MAL-II or SNA after 7.5~8.5, is mixed, agglutinin
The concentration of MAL-II or SNA is 10~12 μ g/mL；After reaction, low-speed centrifugal goes precipitation, to supernatant high speed centrifugation, then abandons
Supernatant, precipitation is scattered in the 0.01mol/L and the HEPES of pH7.5~8.5 of the NaCl containing 0.15mol/L and 0.2%BSA of 1mL
In solution, the as 10 times colloid gold label agglutinin solution of enrichment, by glass fibre in 5~10 times of collaurums of enrichment
Immersion is taken out after 5~8 hours in mark agglutinin solution, and freeze-drying obtains gold standard pad；
(3) agglutinin coating
Detection band agglutinin and nature controlling line agglutinin are diluted to the PBS of 0.01mol/L pH6.5~8.0 respectively dense
It is 1~4mg/mL to spend；Rule on nitrocellulose filter respectively, nitrocellulose filter is closed, cleaned and is done after drying
It is dry, obtain the nitrocellulose filter with detection band and nature controlling line；The detection band agglutinin is WFA, STL, RCA120, PHA-
At least one at least one or GNA, ECA, HHL, PHA-E, DBA in E, DBA, the nature controlling line agglutinin is corresponded to
ConA or STL；
(4) assembling of test strips
By after step 3, gold standard pad, nitrocellulose filter and sample pad, adsorptive pads assembling being constituted into test strips.
A kind of method of quick detection influenza Susceptible population, comprises the following steps：
By in the sample pad of above-mentioned A types test strips and/or H type test strips insertion saliva sample or containing in the oral cavity, saliva
Test strips are taken out after appearing in prosecution area, observe detection band and nature controlling line color status, obtain for bird flu and/or
The testing result of human influenza neurological susceptibility.
For A type test strips, testing result is as follows：
(1) detection band and nature controlling line manifest color change, then show there is the ability of relatively strong resistance avian influenza virus；
(2) only nature controlling line manifests color change, then show susceptible avian influenza virus；
(3) manifest without any color change；Then show that test strips fail；
For H type test strips, testing result is as follows：
(1) detection band and nature controlling line manifest color change, then show there is the ability of relatively strong resistance human influenza virus；
(2) only nature controlling line manifests color change, then show susceptible human influenza virus；
(3) manifest without any color change；Then show that test strips fail.
The invention has the advantages that：
The present invention is used as detection object by taking saliva sample, with scientific and feasibility, and has very easy detection
Advantage.Compared to chip technology, the present invention is easier to use, noninvasive, safe and simple, low cost and be easy to preserve, detect saliva
Individual fowl stream is obtained by the qualitative conclusions that the abundance of α 2-3 and α 2-6 connections terminal sialic acid sugar chain structure draws in liquid in time
Sense and the assessment information of the susceptible speciality of human influenza virus.
Brief description of the drawings
Fig. 1 is the test strips structure schematic diagram for the bird flu of saliva sample quick detection and human influenza Susceptible population, Fig. 1
In：1 is PVC base plates, and 2 is detection band, and 3 is nature controlling line, and 4 is adsorptive pads, and 5 is NC films, and 6 is gold standard pad, and 7 is instruction line, and 8 is sample
Product pad.
Fig. 2 schemes for MAL-II double fastener heart multi-angular analysis Venn.
Fig. 3 schemes for SNA double fastener heart multi-angular analysis Venn.
Specific embodiment
The present invention is described in further details with reference to the accompanying drawings and examples.
(1) test strips signature analysis
The present invention is detected to saliva sample by the chip containing 37 kinds of agglutinins, filtered out steady in people's saliva first
The glycoprotein sugar-type of fixed expression and its agglutinin of identification, as stable internal reference (or house keeper) agglutinin of candidate (in all surveys
In the saliva sample of examination, fluorescence signal value does not have in significant change, or different saliva samples, and the ratio of fluorescence signal value is equal to
Or close to 1), being then capable of identify that in the stable internal reference agglutinin of candidate respectively containing α 2, the agglutinin of 3-SA samples with
MAL-II carries out transactional analysis, and will be capable of identify that containing α 2 in the stable internal reference agglutinin of candidate respectively, 6-SA samples
Agglutinin carries out transactional analysis with SNA, filters out stabilization internal reference agglutinin uncombined with MAL-II or SNA, finally will
These stable internal reference agglutinins preparation for test strips or are fixed on ELISA Plate as detection band, carry out respectively bird flu and
The detection of human influenza virus Susceptible population.
(1) full saliva sample collection：
Healthy People：10~70 one full year of life 240；Patient with breast cancer：200；Patients with lung cancer：200；The male's hepatitis B made a definite diagnosis
Patient 100, liver cirrhosis patient and each 100 of liver cancer patient；Each 100 of diabetes B masculinity and femininity patient；Stomach cancer male
Patient 100.Two hours after meal, between about 9 points to 10 points, physiological saline was gargled three times and gathers the full salivas of naturally secret rapidly afterwards
Liquid.Saliva gathers at least 1mL and is immediately placed on ice, adds protease inhibitors (every milliliter of saliva adds 10 μ L) to prevent albumen
Degraded.Individual example in Fig. 2,3 mentioned by " saliva example grade out stabilization internal reference " refer to it is above-mentioned except hepatopathy (hepatitis B, cirrhosis and
Liver cancer) example.
(2) chip analysis for the treatment of saliva sample and agglutinin
The saliva sample that will be collected is centrifuged 1h under the conditions of 4 DEG C, 12000rpm, collects supernatant and uses 0.45 μm of syringe needle
Formula filter is filtered；(1 μ L protease is added to press down per 10mL to protease inhibitors is added in the saliva sample supernatant after filtering
Preparation), carry out protein quantification using BCA kits, calculate the protein content in saliva sample, and saliva sample is stored in-
80 DEG C of refrigerators are standby.Empirically room conventional method carries out fluorescence labeling to saliva sample, then with lectin chip to mark have
The saliva sample of fluorescence is detected and data analysis.
(3) stabilization internal reference agglutinin uncombined with MAL-II
The glycoprotein sugar-type of stabilization expression in people's saliva and its agglutinin of identification are filtered out by step (2), as time
Stable internal reference (or house keeper) agglutinin of choosing, then will be capable of identify that containing α 2,3-SA in the stable internal reference agglutinin of candidate respectively
The agglutinin of sample carries out transactional analysis with MAL-II, then filters out stabilization internal reference agglutinin uncombined with MAL-II
WFA, STL, RCA120, PHA-E, DBA (Fig. 2).
(4) stabilization internal reference agglutinin uncombined with SNA
The glycoprotein sugar-type of stabilization expression in people's saliva and its agglutinin of identification are filtered out by step (2), as time
Stable internal reference (or house keeper) agglutinin of choosing, then will be capable of identify that containing α 2,6-SA in the stable internal reference agglutinin of candidate respectively
The agglutinin of sample carries out transactional analysis with SNA, then filters out stabilization internal reference agglutinin GNA, ECA uncombined with SNA,
HHL, PHA-E, DBA (Fig. 3).
Analysis and experiment conclusion based on more than, the present invention devise it is a kind of for the bird flu of saliva sample quick detection and
The method of human influenza Susceptible population and the test strips of use, test strips are divided to two kinds (A types or H types test strips)：Two kinds of test strips
Include the sample pad, gold standard pad, the nitrocellulose filter that are set gradually along chromatography direction, and for many in absorption detecting sample
The adsorptive pads of extraction raffinate body, adhesion indicates the adhesive tape of instruction line in sample pad；The gold standard pad is to be adsorbed with coagulating for colloid gold label
The glass fibre of collection element MAL-II (A types test strips) or SNA (H types test strips)；Detection is identified with nitrocellulose filter successively
Band and nature controlling line；Wherein, the detection band is solidified with agglutinin WFA, STL, RCA120, PHA-E, at least one (A in DBA
Type test strips) or GNA, ECA, HHL, PHA-E, DBA at least one (H types test strips), nature controlling line C is solidified with agglutinin
ConA (A types test strips) or STL (H types test strips), for combining MAL-II (A types test strips) or SNA (H types test strips).Institute
A types test strips are stated for detecting bird flu Susceptible population, H types test strips are used to detect human influenza Susceptible population.
(2) prepared by test strips
1) prepared by collaurum：Gold chloride (HAuCl4) aqueous solution of chloraurate of mass fraction 1% is made into, take 1mL additions
99mL deionized waters, be heated to seething with excitement adds the minor official acid sodium aqueous solution of Chinese holly of 2mL mass fractions 1% immediately, stirs rapidly, after
Continuous heating 10 minutes, is cooled to room temperature, and supplement deionized water obtains colloidal gold solution to 100mL.
2) optimum mark pH value is determined：The pH value of colloidal gold solution is respectively adjusted to the solution of potassium carbonate of 0.1mol/L
6.0th, 6.5,7.0,7.5,8.0,8.5 and 9.0, respectively take 1mL and add 20 μ g agglutinins (MAL-II or SNA), it is well mixed, room temperature
Reaction 10 minutes, stands 2 hours, observation solution colour change.Then, 12000rpm is centrifuged 10 minutes, removes supernatant, adds
PBS (PBS, pH8.0) dissolution precipitation of 0.01mol/L 1%BSA containing volume fraction (bovine serum albumin(BSA)), with
It is optimal that precipitation is completely dispersed and suspends in the pH value of uniform transparent purplish red solution pipe.Result shows the optimal of colloid gold label
PH is 8.0.
3) optimum mark agglutinin MAL-II or SNA concentration is determined：By the pH regulations of colloidal gold solution to optimum value, 5
It is each in branch test tube to add 1mL colloidal gold solutions, 2.5~40 μ g agglutinins (MAL-II or SNA) are separately added into, react 10 minutes
Afterwards, each pipe adds the μ L of 10% sodium chloride solution 100, stands 2 hours, observes each pipe color change.Then, 12000rpm centrifugations 20
Minute, remove supernatant, PBS (pH8.0) dissolution precipitations of 0.01mol/L containing 1%BSA are added, being completely dispersed suspension with precipitation is in
The error that minimum albumen (MAL-II or SNA) concentration of uniform purplish red solution pipe adds 20% is optimum protein concentration.As a result
Each pipe precipitation is completely dissolved after display, 10 μ g/mL, therefore optimum mark agglutinin concentration is 12 μ g/mL.
4) colloid gold label：The pH value regulation of the colloidal gold solution that 10mL is prepared adds agglutinin to optimum value
(MAL-II or SNA) 120 μ g mix (i.e. label concentration is 12 μ g/mL), and room temperature reaction 10 minutes, 4 DEG C of 2000rpm are centrifuged 20 points
Clock, goes precipitation (this is to remove the agglomerate polymer for being formed).4 DEG C of 12000rpm of supernatant are centrifuged 20 minutes, abandon supernatant, precipitate
(being the sediment of loose kermesinus) dispersion is suspended in HEPES solution (the hydroxyethyl piperazine second thiosulfonic acid, 0.01mol/L of 1mL
PH7.5, and NaCl containing 0.15mol/L and volume fraction 0.2%BSA) in, the as 10 times colloid gold label aggegations of enrichment
Plain solution, is then diluted to 5 or 4 times of colloid gold label agglutinin solution of enrichment, by glass with above-mentioned HEPES solution
Fiber is soaked in 10 or 5 or 4 times of colloid gold label agglutinin solution of enrichment (5 times of enrichments are optimal), fully immersion (5
~8 hours) take out afterwards, freeze-drying more than 6 hours, as gold standard pad.
Above with respect to content of the agglutinin to colloid gold label in gold standard pad and solidifying in the agglutinin in detection band
The restriction of content, the application employs the usual describing mode in this area, and compared to other limiting modes, this restriction is for this
Art personnel (including user of test strips) are not only clearly, and the composition in the clearly test strips and effect side
Face is more meaningful.
5) stabilization internal reference agglutinin and Quality Control agglutinin coating：Above-mentioned stabilization internal reference uncombined with MAL-II or SNA
Agglutinin (in WFA, STL, RCA120, PHA-E, DBA at least one or GNA, ECA, HHL, PHA-E, DBA at least one
Kind, if using if various according to waiting mass mixing) to be diluted to concentration with the PBS (pH7.2) of 0.01mol/L be that 1~4mg/mL is each
1mL (being optimal with 2mg/mL).Line (makes accordingly to stablize internal reference agglutinin and is solidificated on NC films, as detection on NC films
Band).From stablizing at 6 millimeters of internal reference agglutinin line, it is 4mg/mL's to be diluted to concentration with the PBS (pH7.2) of 0.01mol/L
Agglutinin ConA or STL draw a line (as nature controlling line) again.Film is completely soaked in the PBS of the pH7.2 containing 1%BSA after drying
In, 4 DEG C 1 hour, closed.Then take out NC films to be washed with PBS 3 times, hang in room temperature and dry more than 3 hours, then put
In aeration-drying 1 hour in 30 DEG C of aeration cabinets.
6) assembling (such as Fig. 1) of test strips.
Test strips are made up of sample pad 8, gold standard pad 6, nitrocellulose filter (NC films 5) and adsorptive pads 4, by chromatography direction according to
Secondary arrangement, combines closely between adjacent two parts.Epimere is handgrip part (adsorptive pads：It is absorbent filter, absorption detecting sample
Middle surplus liquid), stage casing is detection zone and quality control region (nitrocellulose filter：Detection band 2 and nature controlling line including sentence read result
3) gold standard pad of the agglutinin MAL-II or SNA that are adsorbed with colloid gold label, is placed between middle hypomere；Hypomere is sample pad
(water adsorption glass fiber contacts detected sample), sample pad is adhered to as the adhesive tape of instruction line 7, and sample-adding is identified with instruction line
Sample area.Whole test strips are attached at the center of PVC base plates 1.Cut into 4mm belt strips wide.Hermetically drying is preserved, standby.
(3) ELISA test strip bird flu and human influenza Susceptible population are used
1) application method of test strips：By in sample area (before indicating line position) insertion saliva sample to be measured of sample pad
Or containing in the oral cavity, when sample is appeared on NC films within 10~15 minutes, test strips are taken out, keep flat on the table, in order to
Observation result, observes the change of detection line shade.
2) result judges：
For the detection (using A types test strips) of bird flu Susceptible population
By in sample pad one end of chromatograph test strip insertion saliva sample or containing in the oral cavity, saliva sample after 10~15 minutes
Originally appear on NC films；Test strips are taken out, detection band and nature controlling line color status are observed, following three kinds of testing results are obtained
One of：
(1) detection band and nature controlling line manifest, in aubergine；Then show the α 2-3 connections end contained in saliva sample
Sialic acid sugar chain structure abundance is higher, the ability with relatively strong resistance avian influenza virus；
(2) only nature controlling line manifests, in aubergine；Then show the α 2-3 connection terminal sialic acid sugar chain structures in saliva sample
Abundance is relatively low, susceptible avian influenza virus；
(3) manifest without aubergine band；Then show that test strips fail, the detection process is invalid.
For the detection (using H types test strips) of human influenza Susceptible population
By in sample pad one end of chromatograph test strip insertion saliva sample or containing in the oral cavity, saliva sample after 10~15 minutes
Originally appear on NC films；Test strips are taken out, detection band and nature controlling line color status are observed, following three kinds of testing results are obtained
One of：
(1) detection band and nature controlling line manifest, in aubergine；Then show the α 2-6 connections end contained in saliva sample
Sialic acid sugar chain structure abundance is higher, the ability with relatively strong resistance human influenza virus；
(2) only nature controlling line manifests, in aubergine；Then show the α 2-6 connection terminal sialic acid sugar chain structures in saliva sample
Abundance is relatively low, susceptible human influenza virus；
(3) manifest without aubergine band；Then show that test strips fail, the detection process is invalid.
(4) Analysis of test results
Through analysis, the present invention by using " in hepatopathy stabilization internal reference " and " saliva example grade out stable internal reference " the two
The screening conditions of " the stable internal reference agglutinin of candidate ", improve the accuracy and reliability of Susceptible population's detection.
Claims (10)
1. test strips of quick detection influenza Susceptible population, it is characterised in that：Including the A types for detecting bird flu Susceptible population
Test strips and/or the H type test strips for detecting human influenza Susceptible population, the A types test strips and H types test strips are included along layer
Sample pad, nanoparticle label pad, prosecution area and adsorptive pads that analysis direction sets gradually；The prosecution area includes setting gradually
Detection band and nature controlling line；The nanoparticle label pad of the A types test strips includes collaurum, magnetic fluid or nano-fluorescent grain
The nanoparticle label pad of agglutinin MAL-II, H the type test strips of mark includes collaurum, magnetic fluid or nano-fluorescent grain mark
The agglutinin SNA of note；The detection band of the A types test strips includes stabilization internal reference agglutinin uncombined with agglutinin MAL-II, H
The detection band of type test strips includes stabilization internal reference agglutinin uncombined with agglutinin SNA.
2. test strips according to claim 1, it is characterised in that：The detection band of the A types test strips is solidified with agglutinin
At least one in WFA, STL, RCA120, PHA-E, DBA, nature controlling line is solidified with agglutinin ConA.
3. test strips according to claim 1, it is characterised in that：The detection band of the H types test strips is solidified with agglutinin
At least one in GNA, ECA, HHL, PHA-E, DBA, nature controlling line is solidified with agglutinin STL.
4. test strips according to claim 1, it is characterised in that：The nanoparticle label pad is selected from gold standard pad, the gold
Mark pad is by solution impregnation and drying will mark agglutinin MAL-II or the SNA combination of collaurum to make on the glass fibers
Into；The detection band and nature controlling line are that corresponding agglutinin is fixed on into cellulose nitrate by line, dry and closing
It is made on plain film.
5. test strips according to claim 4, it is characterised in that：The label concentration of agglutinin MAL-II or SNA be 10~
12 μ g/mL, the line concentration of agglutinin is 1~4mg/mL in detection band and nature controlling line.
6. test strips according to claim 5, it is characterised in that：The solution impregnation refers to use that to be dispersed with concentration be 4
~10 times of solution immersion glass fibres of the agglutinin of the colloid gold label of enrichment；The multiple of the enrichment is according to lower section
Formula determines：It is scattered in 1mL solution Is after the agglutinin MAL-II or SNA of 120 μ g are marked in colloidal gold solution, is obtained
To 10 times of colloid gold label agglutinin solution of enrichment, after further being diluted using solution I, then other are obtained compared with low enrichment
Colloid gold label agglutinin solution, the solution I is NaCl containing 0.15mol/L and the 0.01mol/L pH7.5 of 0.2%BSA
~8.5 HEPES solution, the pH of the colloidal gold solution is 7.5~8.5；The line refers to coagulate corresponding one or more
Collection element is dissolved in the PBS of 0.01mol/L pH6.5~8.0 according to 1~4mg/mL of total concentration, agglutinin solution is obtained, using this
Solution applies line style band on nitrocellulose filter.
7. test strips according to claim 6, it is characterised in that：It is 5 times of collaurums of enrichment to use and be dispersed with concentration
The solution immersion glass fibre of the agglutinin of mark；The concentration of the agglutinin solution used in line is 2mg/mL.
8. a kind of preparation method of the test strips of quick detection influenza Susceptible population, it is characterised in that：Comprise the following steps：
(1) prepared by collaurum
Colloidal gold solution is prepared using aqueous solution of chloraurate；
(2) colloid gold label
The pH value of colloidal gold solution is adjusted to addition agglutinin MAL-II or SNA after 7.5~8.5, is mixed, agglutinin MAL-II
Or the concentration of SNA is 10~12 μ g/mL；After reaction, low-speed centrifugal goes precipitation, to supernatant high speed centrifugation, then abandons supernatant,
Precipitation is scattered in the 0.01mol/L and the HEPES solution of pH7.5~8.5 of the NaCl containing 0.15mol/L and 0.2%BSA of 1mL
In, the as 10 times colloid gold label agglutinin solution of enrichment, by glass fibre in 5~10 times of colloid gold labels of enrichment
Taken out after being soaked 5~8 hours in agglutinin solution, freeze-drying obtains gold standard pad；
(3) agglutinin coating
Detection band agglutinin and nature controlling line agglutinin are diluted into concentration with the PBS of 0.01mol/L pH6.5~8.0 respectively is
1~4mg/mL；Rule on nitrocellulose filter respectively, nitrocellulose filter is closed, cleaned and is dried after drying, obtained
To the nitrocellulose filter with detection band and nature controlling line；The detection band agglutinin is WFA, STL, RCA120, PHA-E, DBA
In at least one or GNA, ECA, HHL, PHA-E, DBA at least one, the nature controlling line agglutinin correspond to ConA or
STL；
(4) assembling of test strips
By after step 3, gold standard pad, nitrocellulose filter and sample pad, adsorptive pads assembling being constituted into test strips.
9. the method for the quick detection influenza Susceptible population of a kind of test strips as claimed in claim 1, it is characterised in that：Including with
Lower step：
By in the sample pad insertion saliva sample of A types test strips and/or H type test strips or containing in the oral cavity, saliva appears in inspection
Test strips are taken out after in control area, observes detection band and nature controlling line color status, obtain easy for bird flu and/or human influenza
The testing result of perception.
10. method according to claim 9, it is characterised in that：
For A type test strips, testing result is as follows：
(1) detection band and nature controlling line manifest color change, then show there is the ability of relatively strong resistance avian influenza virus；
(2) only nature controlling line manifests color change, then show susceptible avian influenza virus；
(3) manifest without any color change；Then show that test strips fail；
For H type test strips, testing result is as follows：
(1) detection band and nature controlling line manifest color change, then show there is the ability of relatively strong resistance human influenza virus；
(2) only nature controlling line manifests color change, then show susceptible human influenza virus；
(3) manifest without any color change；Then show that test strips fail.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109021094A (en) * | 2018-08-01 | 2018-12-18 | 陕西医药控股医药研究院有限公司 | A kind of method and test strips quickly detecting influenza Susceptible population using sialic acid artificial antigen |
| CN109100518A (en) * | 2018-10-17 | 2018-12-28 | 陕西医药控股医药研究院有限公司 | Bird flu, the test strips of human influenza Susceptible population and test card are quickly detected using competition law |
| CN111264937A (en) * | 2020-03-13 | 2020-06-12 | 北京理工大学 | Gauze mask with prevention and detection function |
| CN111672544A (en) * | 2020-06-08 | 2020-09-18 | 江苏师范大学 | Paper-based biochemical reagent storage method based on water two-phase system |
| CN113176411A (en) * | 2021-03-10 | 2021-07-27 | 北京大学口腔医学院 | Biomarker for detecting novel coronavirus pneumonia by using saliva and application thereof |
| CN113519926A (en) * | 2020-04-20 | 2021-10-22 | 高俊阁 | Intelligent wearable device, health monitoring method and system |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109021094A (en) * | 2018-08-01 | 2018-12-18 | 陕西医药控股医药研究院有限公司 | A kind of method and test strips quickly detecting influenza Susceptible population using sialic acid artificial antigen |
| CN109100518A (en) * | 2018-10-17 | 2018-12-28 | 陕西医药控股医药研究院有限公司 | Bird flu, the test strips of human influenza Susceptible population and test card are quickly detected using competition law |
| CN111264937A (en) * | 2020-03-13 | 2020-06-12 | 北京理工大学 | Gauze mask with prevention and detection function |
| CN113519926A (en) * | 2020-04-20 | 2021-10-22 | 高俊阁 | Intelligent wearable device, health monitoring method and system |
| CN111672544A (en) * | 2020-06-08 | 2020-09-18 | 江苏师范大学 | Paper-based biochemical reagent storage method based on water two-phase system |
| CN113176411A (en) * | 2021-03-10 | 2021-07-27 | 北京大学口腔医学院 | Biomarker for detecting novel coronavirus pneumonia by using saliva and application thereof |
| CN113176411B (en) * | 2021-03-10 | 2023-12-26 | 北京大学口腔医学院 | Biomarker for detecting novel coronavirus infection by saliva and application thereof |
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Address after: 712000 No. 16 West tableland Road, Shaanxi, Xianyang Patentee after: Shaanxi Institute of traditional Chinese medicine (Shaanxi Medical Information Center) Address before: 712000 No. 16 West tableland Road, Shaanxi, Xianyang Patentee before: SHAANXI Research Institute OF TRADITIONAL CHINESE MEDICINE |
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Application publication date: 20170531 Assignee: MEDICINE Research Institute OF SHAANXI PHARMACEUTICAL HOLDING COOPERATION Assignor: Shaanxi Research Institute of Traditional Chinese Medicine Contract record no.: X2022980005847 Denomination of invention: Methods and test strips for rapid detection of avian influenza and human influenza susceptible people Granted publication date: 20181030 License type: Exclusive License Record date: 20220523 |