CN106771120B - The quickly method and test strips of detection bird flu and human influenza Susceptible population - Google Patents
The quickly method and test strips of detection bird flu and human influenza Susceptible population Download PDFInfo
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- CN106771120B CN106771120B CN201611073359.7A CN201611073359A CN106771120B CN 106771120 B CN106771120 B CN 106771120B CN 201611073359 A CN201611073359 A CN 201611073359A CN 106771120 B CN106771120 B CN 106771120B
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Abstract
Include sample pad, gold-labelled pad, prosecution area and the water absorption pad for surplus liquid in absorption detecting sample set gradually along chromatography direction the invention discloses a kind of method and test strips of quick detection bird flu and human influenza Susceptible population；The prosecution area includes the detection band and nature controlling line set gradually；The gold-labelled pad includes the agglutinin MAL-II or SNA of colloid gold label；The detection band include with the uncombined stable internal reference agglutinins of agglutinin MAL-II or with the uncombined stable internal reference agglutinins of agglutinin SNA.Sample pad one end of test strips is inserted into saliva sample or containing in the oral cavity, after 10~15 minutes, test strips are taken out, observation detection band and nature controlling line color status, testing result is obtained, the present invention really realizes detects bird flu and human influenza virus Susceptible population noninvasive, safe and simplely.
Description
Technical field
The present invention relates to bird flu and human influenza virus Susceptible population is quickly detected based on Lateral Flow Strip, can also develop into
Double agglutinin sandwich methods based on ELISA Plate detection quickly detect bird flu and human influenza virus Susceptible population.
Background technology
Agglutinin is non-immune origin, a kind of carbohydrate-binding protein without enzymatic activity, can exclusively identify a certain spy
Specific sugar chain sequence in the monosaccharide or glycan of different structure and it is in combination.More than 100 are found from nature so far
The agglutinin of kind plant source.Agglutination is known as various applications：The diagnosis of such as malignant tumour disease, design antibacterial and antiviral drug,
Identification and the analysis etc. for designing " biological missile " and carrying out sugar chain structure.Research neck is learned with chip technology is introduced sugar group
Domain, agglutinin are 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 react with sample incubations to be detected such as glycoprotein, cell and thalline after label, through it is a series of subsequently
After processing, 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 is very extensive, has complicated gene structure, and its gene occurs
It re-matches and the probability of mutation is very high, therefore be in periodic epidemic in the world.So far, it is 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 characteristics that incidence and case fatality rate are high, spread speed is fast, it is wide to involve range, once it breaks out
Come, it will serious disaster is caused to the mankind.In recent years, H5, H7 and H9 subtype influenza virus and the A type occurred in 2009
H1N1 influenza viruses all cause larger epidemic situation, some strains energy direct infection mankind of H5N1 and H7N9 types, high infection rate
H9 hypotypes with low pathogenicity equally also can be by birds direct contagion to people, although the virus of these hypotypes causes people not yet
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 receptors is
Influenza virus infects the beginning of body.Sugar chain receptor structure on different influenza virus host cell surfaces is different, fowl stream
It is the sugar chain receptor of SA α 2-3Gal that Influenza Virus HA, which mainly identifies and combine end, and human influenza virus HA is mainly identified and combined
End is the sugar chain receptor of SA α 2-6Gal.
New research report points out there are SA α 2-6Gal and SA α 2-3Gal sugar chain receptors on human respiratory epithelial cell
Distribution, but based on the distribution of SA α 2-6Gal sugar chain receptors；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, until alveolar distribution is minimum；And SA α 2-3Gal are in trachea-bronchial epithelial cell
And it is distributed based on alveolar bronchiole and alveolar only in a small amount of distribution in bronchiole.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, including nucleic acid, protein, fat
Class, minerals and other small-molecule substances.In addition, there is also the substances inhibited with eliminating bacteria and virus in saliva.Saliva contains
Variation in amount and composition is all likely to result in saliva either caused by oral cavity partial lesion or body integral status
Functional defect.The study found that in addition to containing the basic composition protein of some salivas (in different sexes and all ages and classes in saliva
Section is all almost without differentially expressed protein or glycoprotein) outside, human body health and physiology shape may be reacted by also containing some
Often there is otherness in the crowd of all ages and classes, gender, pathology and physiological status in some protein of condition, these protein
Expression.
The protein component having in blood exists in saliva, and saliva can reflect part protein level in blood
Variation.By compared with the GO (gene ontology) of mankind's holoprotein group annotations, finding saliva and blood to saliva and blood plasma
It is more to starch Proteomics extracellular matrix components, and intracellular fluid component is less, and saliva and plasma proteins group is prompted to have
The feature of secretory protein group；Analysis result prompt in terms of biological process and molecular function, sialoprotein matter group and blood
Starching protein group has similitude.But, the proteomic comparison of saliva and blood plasma the results show that some protein only in saliva
Proteomics occur, and without appearing in plasma proteins group, show that the two can not be equal.
Saliva resists first of natural cover for defense of respiratory virus infection as human body, and glycoprotein therein plays important
Effect, such as α -2 macroglobulin in saliva, mucoprotein 5B and saliva glycoprotein 340 etc. can pass through (α 2-3/ α 2-6) thereon even
Terminal sialic acid sugar chain structure is connect to be combined with influenza virus HA to neutralize and inhibit the infection of influenza virus.
It is analyzed using the saliva of three age bracket healthy volunteers of high coverage rate lectin chip pair, it was found that one
A bit with age and the relevant saliva glycoprotein sugar chain of gender, wherein：(1) the glycoprotein candy chain structure in children male's saliva is compared with female
Property more horn of plenty；(2) saliva glycoprotein sugar chain differential expression is the most notable between adult male and women；(3) elderly men with
Saliva glycoprotein 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 are expressed and are increased 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 inverted parabolic curve type with age in male's saliva
Expression.
Pass through sialoprotein matter to different age group different sexes healthy volunteer and the comparative studies hair of sugar chain thereon
Existing, with advancing age, protein content in Healthy People saliva is without substantially changeing, and sugar chain structure thereon and quantity
Significant change has occurred, as in sialoprotein matter sialic acid sugar chain express showed increased, and its protein expression have no it is aobvious
It writes and changes.Find simultaneously healthy elderly saliva by provide more α 2-3 and α 2-6 connection terminal sialic acid sugar chain structures and
Influenza virus hemagglutinin combines, the mutual knowledge for neutralizing and inhibiting influenza virus to human upper airway specific cell surface receptors
Not.To speculate that this molecular mechanism can have the fact that stronger ability for resisting influenza with partial interpretation healthy elderly.
However, the people being infected when Influenza Outbreak is mostly the elderly instead, at present to its basic reason there is not yet specific
It explains.According to the clinical observation of various people's influenza virus neurological susceptibility count, 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, usually explanation are the immune systems that chronic disease compromises the elderly, reduce the Immunoresistance of the elderly, remove
Outside the factor of this common-sense, if also have the influence of other physiology or pathological factor so that this part the elderly's easy infection stream
What Influenza Virus, other Susceptible population make and explain again？Therefore, in order to effective guiding clinical diagnosis and the raising public to certainly
The cognition of body risk, there is an urgent need for a kind of easy detection (screening) methods, can be timely without large-scale instrument and tool
Obtain the assessment information of individual bird flu and the susceptible speciality of human influenza virus.
A kind of Chinese patent " detection instrument for screening bird flu and 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
It with the correlativity of the easy neurological susceptibility of human influenza virus, and points out, is tied with corresponding agglutinin MAL-II or SNA in Susceptible population's saliva
α 2-3 or α the 2-6 connection terminal sialic acid sugar chain structure expressions of conjunction are significantly lowered, but the patent is only applied to chip examination
Agent box, simplicity, speed and the cost of detection have much room for improvement.
Test paper detection technique, using gradually increasing, has the characteristics that quick, low price, but mesh in medical diagnosis on disease and detection
It is preceding there is not yet for detect bird flu and human influenza Susceptible population test strips and detection method report.Test strips are usually wrapped
Include sample pad, gold-labelled pad, the cellulose acetate film for being printed with detection band and control line and water suction end.For saliva, no
The existing similitude of sugar chain structure between individual contained by it also has specificity together, and sample is increased from the reliability perspectives of detection
Pad, detection band and control line design and prepare difficulty.
Invention content
The purpose of the present invention is to provide a kind of methods and test paper of quick detection bird flu and human influenza Susceptible population
Item detects bird flu and human influenza virus Susceptible population noninvasive, safe and simplely to really realize.
In order to achieve the above objectives, present invention employs following technical schemes：
The quickly test strips of detection influenza Susceptible population, include for detect the A types test strips of bird flu Susceptible population and/
Or the H-type test strips for detecting human influenza Susceptible population, the A types test strips and H-type test strips include along chromatography direction according to
Sample pad, nanoparticle label pad, prosecution area and the water absorption pad of secondary setting；The prosecution area includes the detection band set gradually
And nature controlling line；The nanoparticle label pad of the A types test strips includes the solidifying of colloidal gold, magnetic fluid or nano-fluorescent grain label
Collect element MAL-II, the nanoparticle label pad of H-type test strips includes the agglutination of colloidal gold, magnetic fluid or nano-fluorescent grain label
Plain SNA；The detection band of the A types test strips includes and the uncombined stable internal reference agglutinins of agglutinin MAL-II, H-type test strips
Detection band include and the uncombined stable internal reference agglutinins of agglutinin SNA.
The detection of the A types test strips has at least one in agglutinin WFA, STL, RCA120, PHA-E, DBA with solidification
Kind, nature controlling line solidification has agglutinin ConA.
The detection band solidification of the H-type test strips has at least one of agglutinin GNA, ECA, HHL, PHA-E, DBA, matter
Control line solidification has agglutinin STL.
The nanoparticle label pad is selected from gold-labelled pad, which is that will mark colloid by solution dipping and drying
The agglutinin MAL-II or SNA of gold are in conjunction on the glass fibers and manufactured；Detection band and nature controlling line be by scribing line,
Corresponding agglutinin is fixed on manufactured on nitrocellulose filter by dry and closing.
The label concentration of agglutinin MAL-II or SNA are 10~12 μ g/mL, detect stroke of agglutinin in band and nature controlling line
A concentration of 1~4mg/mL of line.
Solution dipping refer to using the colloid gold label for being dispersed with a concentration of 4~10 times of enrichments agglutinin it is molten
Liquid impregnates glass fibre；The multiple of the enrichment determines in the following 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 colloidal gold marks compared with low enrichment
Remember that agglutinin solution, the solution I are pH7.5~8.5 0.01mol/L of NaCl containing 0.15mol/L and 0.2%BSA
The pH of HEPES solution, the colloidal gold solution is 7.5~8.5；The scribing line refer to by corresponding one or more agglutinins according to
1~4mg/mL of total concentration is dissolved in the PBS of pH6.5~8.0 0.01mol/L, obtains agglutinin solution, using the solution in nitre
Coating line profile bar band on acid cellulose film.
Preferably, glass fibers are impregnated using the solution of the agglutinin for the colloid gold label for being dispersed with a concentration of 5 times of enrichments
Dimension；A concentration of 2mg/mL of the agglutinin solution used in scribing line.
The preparation method of the test strips of above-mentioned quick detection influenza Susceptible population, includes the following steps：
(1) prepared by colloidal gold
Colloidal gold solution is prepared using aqueous solution of chloraurate；
(2) colloid gold label
Agglutinin MAL-II or SNA, mixing, agglutinin are added after the pH value of colloidal gold solution is adjusted to 7.5~8.5
A concentration of 10~12 μ g/mL of MAL-II or SNA；After reaction, low-speed centrifugal goes to precipitate, and to supernatant high speed centrifugation, then abandons
Supernatant, precipitation are scattered in the 0.01mol/L of the NaCl containing 0.15mol/L and 0.2%BSA of 1mL and the HEPES of pH7.5~8.5
In solution, the colloid gold label agglutinin solution of as 10 times enrichments, by glass fibre 5~10 times of enrichments colloidal gold
It is taken out after being impregnated 5~8 hours in label agglutinin solution, is freeze-dried, obtains gold-labelled pad；
(3) agglutinin is coated with
Will detection be diluted to respectively with the PBS of pH6.5~8.0 0.01mol/L with agglutinin and nature controlling line agglutinin it is dense
Degree is 1~4mg/mL；It crosses on nitrocellulose filter respectively, nitrocellulose filter is closed, cleaned and is done after dry
It is dry, obtain the nitrocellulose filter with detection band and nature controlling line；The detection band agglutinin is WFA, STL, RCA120, PHA-
E, at least one of DBA or at least one of GNA, ECA, HHL, PHA-E, DBA, the nature controlling line agglutinin correspond to
ConA or STL；
(4) assembling of test strips
After step 3, gold-labelled pad, nitrocellulose filter and sample pad, water absorption pad assembling are constituted into test strips.
A kind of method of quick detection influenza Susceptible population, includes the following steps：
The sample pad of above-mentioned A types test strips and/or H-type test strips is inserted into saliva sample or is contained in the oral cavity, saliva
Test strips are taken out after appearing in prosecution area, observation 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 show color change, then show with the ability for relatively resisting avian influenza virus by force；
(2) only nature controlling line shows color change, then shows susceptible avian influenza virus；
(3) show 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 show color change, then show with the ability for relatively resisting human influenza virus by force；
(2) only nature controlling line shows color change, then shows susceptible human influenza virus；
(3) show without any color change；Then show that test strips fail.
The invention has the advantages that：
The present invention has scientific and feasibility, and have very easy detection by taking saliva sample to be used as detection object
Advantage.Compared to chip technology, the present invention is easier to use, noninvasive, safe and simple, at low cost and be easy to preserve, and detects saliva
α 2-3 connect the qualitative conclusions that the abundance of terminal sialic acid sugar chain structure obtains with α 2-6 in liquid can obtain individual fowl stream in time
The assessment information of sense and the susceptible speciality of human influenza virus.
Description of the drawings
Fig. 1 is the test strips structure schematic diagram that bird flu and human influenza Susceptible population are quickly detected for saliva sample, Fig. 1
In：1 is PVC bottom plates, and 2 be detection band, and 3 be nature controlling line, and 4 be water absorption pad, and 5 be NC films, and 6 be gold-labelled pad, and 7 be index line, and 8 be sample
Product pad.
Fig. 2 is MAL-II double fastener heart multi-angular analysis Venn figures.
Fig. 3 is SNA double fastener heart multi-angular analysis Venn figures.
Specific implementation mode
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 first detected saliva sample by the chip containing 37 kinds of agglutinins, filters out steady in people's saliva
Surely the agglutinin of the glycoprotein sugar-type and its identification expressed, as candidate stabilization internal reference (or house keeper) agglutinin (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), can be identified in stabilization internal reference agglutinin that then respectively will be candidate containing α 2, the agglutinin of 3-SA samples with
MAL-II carries out transactional analysis, and will can be identified containing α 2 in candidate stabilization internal reference agglutinin respectively, 6-SA samples
Agglutinin and SNA carry out transactional analysis, filter out with the uncombined stable internal reference agglutinin of MAL-II or SNA, finally will
These stable internal reference agglutinins as detection bands, for test strips preparation or be fixed on ELISA Plate, carry out respectively bird flu and
The detection of human influenza virus Susceptible population.
(1) full saliva sample acquisition：
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 male and female patient；Gastric cancer male
Patient 100.Between two hours after meal, about 9 points to 10 points, the full saliva of physiological saline rapid acquisition naturally secret after gargling three times
Liquid.Saliva acquires at least 1mL and is immediately placed on ice, and protease inhibitors (10 μ L are added in every milliliter of saliva), which is added, prevents albumen
Degradation.A example in Fig. 2,3 mentioned by " saliva example grade out stable internal reference " refer to it is above-mentioned except hepatopathy (hepatitis B, hepatic sclerosis and
Liver cancer) example.
(2) chip analysis of saliva sample and agglutinin is handled
Collected saliva sample is centrifuged into 1h under the conditions of 4 DEG C, 12000rpm, collect supernatant and uses 0.45 μm of syringe needle
Formula filter is filtered；Protease inhibitors is added into filtered saliva sample supernatant and (suppression of 1 μ L protease is added per 10mL
Preparation), protein quantification is carried out using BCA kits, calculates the protein content in saliva sample, and saliva sample is stored in-
80 DEG C of refrigerators are spare.Empirically room conventional method to saliva sample carry out fluorescent marker, then with lectin chip to label have
The saliva sample of fluorescence is detected and data analysis.
(3) with the uncombined stable internal reference agglutinins of MAL-II
It is filtered out by step (2) and stablizes the glycoprotein sugar-type of expression and its agglutinin of identification in people's saliva, as time
Then stabilization internal reference (or house keeper) agglutinin of choosing will can identify in candidate stabilization internal reference agglutinin containing α 2,3-SA respectively
The agglutinin of sample carries out transactional analysis with MAL-II, then filters out and the uncombined stable internal reference agglutinins of MAL-II
WFA, STL, RCA120, PHA-E, DBA (Fig. 2).
(4) with the uncombined stable internal reference agglutinins of SNA
It is filtered out by step (2) and stablizes the glycoprotein sugar-type of expression and its agglutinin of identification in people's saliva, as time
Then stabilization internal reference (or house keeper) agglutinin of choosing will can identify in candidate stabilization internal reference agglutinin containing α 2,6-SA respectively
The agglutinin of sample carries out transactional analysis with SNA, then filter out with SNA uncombined stable internal reference agglutinin GNA, ECA,
HHL, PHA-E, DBA (Fig. 3).
Based on the above analysis and experiment conclusion, the present invention devise it is a kind of for saliva sample quickly detect bird flu with
The method of human influenza Susceptible population and the test strips of use, test strips are divided to two kinds (A types or H-type test strips)：Two kinds of test strips
Include the sample pad, gold-labelled pad, nitrocellulose filter set gradually along chromatography direction, and for more in absorption detecting sample
The water absorption pad of extraction raffinate body, in sample pad adherency indicate the adhesive tape of index line；The gold-labelled pad is to be adsorbed with coagulating for colloid gold label
Collect the glass fibre of element MAL-II (A types test strips) or SNA (H-type test strips)；Detection is identified on nitrocellulose filter successively
Band and nature controlling line；Wherein, the detection band solidification has agglutinin WFA, STL, RCA120, PHA-E, at least one of DBA (A
Type test strips) or GNA, ECA, HHL, PHA-E, at least one of DBA (H-type test strips), nature controlling line C, which cures, agglutinin
ConA (A types test strips) or STL (H-type test strips), for combining MAL-II (A types test strips) or SNA (H-type test strips).Institute
A type test strips are stated for detecting bird flu Susceptible population, H-type test strips are for detecting human influenza Susceptible population.
(2) prepared by test strips
1) prepared by colloidal gold：Gold chloride (HAuCl4) it is made into the aqueous solution of chloraurate of mass fraction 1%, take 1mL to be added
99mL deionized waters are heated to the minor official acid sodium aqueous solution of Chinese holly that 2mL mass fractions 1% are added in boiling immediately, stir evenly 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
6.0,6.5,7.0,7.5,8.0,8.5 and 9.0, it respectively takes 1mL that 20 μ g agglutinins (MAL-II or SNA) are added, is uniformly mixed, room temperature
Reaction 10 minutes stands 2 hours, observation solution colour variation.Then, 12000rpm is centrifuged 10 minutes, removes supernatant, is added
PBS (phosphate buffer solution, pH8.0) the dissolving precipitations of 0.01mol/L 1%BSA containing volume fraction (bovine serum albumin(BSA)), with
It is best that precipitation, which is completely dispersed and suspends in the pH value of uniform transparent purplish red solution pipe,.As a result the best of colloid gold label is shown
PH is 8.0.
3) optimum mark agglutinin MAL-II or SNA concentration is determined：The pH of colloidal gold solution is adjusted to optimum value, 5
1mL colloidal gold solutions are respectively added in branch test tube, are separately added into 2.5~40 μ g agglutinins (MAL-II or SNA), react 10 minutes
Afterwards, 10% sodium chloride solution, 100 μ L are added in each pipe, stand 2 hours, observe each pipe color change.Then, 12000rpm centrifugations 20
Minute, remove supernatant, PBS (pH8.0) dissolving precipitations of the 0.01mol/L containing 1%BSA is added, being completely dispersed suspension with precipitation is in
Minimum albumen (MAL-II or SNA) concentration of uniform purplish red solution pipe adds 20% error for optimum protein concentration.As a result
It shows, each pipe precipitation is completely dissolved after 10 μ g/mL, therefore a concentration of 12 μ g/mL of optimum mark agglutinin.
4) colloid gold label：The pH value of the 10mL colloidal gold solutions prepared is adjusted to optimum value, agglutinin is added
(MAL-II or SNA) 120 μ g mixings (i.e. label concentration is 12 μ g/mL), react at room temperature 10 minutes, 4 DEG C of 2000rpm centrifuge 20 points
Clock goes precipitation (this is to remove the agglomerate polymer formed).4 DEG C of 12000rpm of supernatant are centrifuged 20 minutes, abandon supernatant, are precipitated
(being the sediment of loose kermesinus) disperses the HEPES solution (hydroxyethyl piperazine second thiosulfonic acid, the 0.01mol/L that are suspended in 1mL
PH7.5, and NaCl containing 0.15mol/L and volume fraction 0.2%BSA) in, the colloid gold label agglutination of as 10 times enrichments
Then plain solution is diluted to the colloid gold label agglutinin solution of 5 or 4 times of enrichments, by glass with above-mentioned HEPES solution
Fiber is soaked in the colloid gold label agglutinin solution of 10 or 5 or 4 times of enrichments (5 times of enrichments are best), fully impregnates (5
~8 hours) it takes out afterwards, be freeze-dried 6 hours or more, as gold-labelled pad.
The agglutinin taken in detection above with respect to content of the agglutinin to colloid gold label in gold-labelled pad and solidification
The restriction of content, the application uses the usual describing mode in this field, this to limit for this compared to other limited ways
Field technology personnel (user for including test strips) are not only clearly, and in the ingredient of the clearly test strips and effect side
Face is more meaningful.
5) stablize internal reference agglutinin and Quality Control agglutinin coating：The above-mentioned and uncombined stable internal references of MAL-II or SNA
Agglutinin (at least one at least one of WFA, STL, RCA120, PHA-E, DBA or GNA, ECA, HHL, PHA-E, DBA
Kind, if using if a variety of according to equal mass mixings) with the PBS (pH7.2) of 0.01mol/L to be diluted to a concentration of 1~4mg/mL each
1mL (being best with 2mg/mL).Scribing line (makes accordingly to stablize internal reference agglutinin and be solidificated on NC films, as detection on NC films
Band).From stablizing at 6 millimeters of internal reference agglutinin scribing line, it is diluted to a concentration of 4mg/mL's with the PBS (pH7.2) of 0.01mol/L
Agglutinin ConA or STL are drawn a line again (as nature controlling line).It is completely soaked in the PBS of the pH7.2 containing 1%BSA after film drying
In, 4 DEG C 1 hour, closed.It then takes out NC films to be washed 3 times with PBS, hangs in room temperature 3 hours dry or more, 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 of sample pad 8, gold-labelled pad 6, nitrocellulose filter (NC films 5) and water absorption pad 4, by chromatography direction according to
Secondary arrangement is combined closely between adjacent two parts.Epimere is handgrip part (water absorption pad：For absorbent filter, absorption detecting sample
Middle surplus liquid), stage casing is detection zone and quality control region (nitrocellulose filter：Detection band 2 including sentence read result and nature controlling line
3) gold-labelled pad of the agglutinin MAL-II or SNA that are adsorbed with colloid gold label, are placed between middle and lower sections；Hypomere is sample pad
(water adsorption glass fiber contacts detected sample), sample pad adheres to the adhesive tape as index line 7, with index line mark sample-adding
Sample area.Entire test strips are attached at 1 center of PVC bottom plates.Cut into the belt strip of 4mm wide.Hermetically drying preserves, spare.
(3) ELISA test strip bird flu and human influenza Susceptible population are used
1) application method of test strips：The sample area of sample pad is inserted into (before instruction line position) in saliva sample to be measured
Or containing in the oral cavity, when sample appears on NC films within 10~15 minutes, test strips are taken out, keep flat on the table, in order to
Observation is as a result, observe the variation of detection line shade.
2) result judges：
For the detection (using A types test strips) of bird flu Susceptible population
Sample pad one end of chromatograph test strip is inserted into saliva sample or is contained in the oral cavity, saliva sample after 10~15 minutes
Originally it appears on NC films；Test strips are taken out, observation detection band and nature controlling line color status obtain following three kinds of testing results
One of：
(1) detection band and nature controlling line show, and are in aubergine；Then show the α 2-3 connections end contained in saliva sample
Sialic acid sugar chain structure abundance is higher, has the ability for relatively resisting avian influenza virus by force；
(2) only nature controlling line shows, and is 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) show without aubergine band；Then show that test strips fail, the detection process is invalid.
For the detection (using H-type test strips) of human influenza Susceptible population
Sample pad one end of chromatograph test strip is inserted into saliva sample or is contained in the oral cavity, saliva sample after 10~15 minutes
Originally it appears on NC films；Test strips are taken out, observation detection band and nature controlling line color status obtain following three kinds of testing results
One of：
(1) detection band and nature controlling line show, and are in aubergine；Then show the α 2-6 connections end contained in saliva sample
Sialic acid sugar chain structure abundance is higher, has the ability for relatively resisting human influenza virus by force；
(2) only nature controlling line shows, and is 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) show 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 stablize internal reference " and " saliva example grade out stable internal reference " the two
The screening conditions of " candidate stabilization internal reference agglutinin " improve the accuracy and reliability of Susceptible population's detection.
Claims (10)
1. the quickly test strips of detection influenza Susceptible population, it is characterised in that：Include the A types for detecting bird flu Susceptible population
Test strips and/or H-type test strips for detecting human influenza Susceptible population, the A types test strips and H-type test strips include along layer
Sample pad, nanoparticle label pad, prosecution area and the water absorption pad that analysis direction is set gradually；The prosecution area includes setting gradually
Detection band and nature controlling line；The nanoparticle label pad of the A types test strips includes colloidal gold, magnetic fluid or nano-fluorescent grain
The nanoparticle label pad of the agglutinin MAL-II of label, H-type test strips include colloidal gold, magnetic fluid or nano-fluorescent grain mark
The agglutinin SNA of note；The detection band of the A types test strips includes and the uncombined stable internal reference agglutinins of agglutinin MAL-II, H
The detection band of type test strips includes and the uncombined stable internal reference agglutinins of agglutinin SNA；
The detection band solidification of the A types test strips has at least one of agglutinin WFA, STL, RCA120, PHA-E, DBA；Institute
The detection band solidification for stating H-type test strips has at least one of agglutinin GNA, ECA, HHL, PHA-E, DBA.
2. test strips according to claim 1, it is characterised in that：The nature controlling line solidification of the A types test strips has agglutinin
ConA。
3. test strips according to claim 1, it is characterised in that：The nature controlling line solidification of the H-type test strips has agglutinin
STL。
4. test strips according to claim 1, it is characterised in that：The nanoparticle label pad is selected from gold-labelled pad, the gold
Mark pad is the agglutinin MAL-II or SNA of colloidal gold will to be marked in conjunction with by system on the glass fibers by solution dipping and drying
At；The detection band and nature controlling line are that corresponding agglutinin is fixed on cellulose nitrate by scribing line, drying and closing
It is manufactured 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 detect a concentration of 1~4mg/mL of scribing line of agglutinin in band and nature controlling line.
6. test strips according to claim 5, it is characterised in that：The solution dipping refers to using to be dispersed with a concentration of 4
The solution of the agglutinin of the colloid gold label of~10 times of enrichments impregnates glass fibre；The multiple of the enrichment is according to lower section
Formula determines：It is scattered in 1 mL solution Is, obtains after the agglutinin MAL-II or SNA of 120 μ g are marked in colloidal gold solution
To the colloid gold label agglutinin solution of 10 times of enrichments, after further being diluted using solution I, then other are obtained compared with low enrichment
Colloid gold label agglutinin solution, the solution I be the 0.01mol/L containing 0.15 mol/L NaCl and 0.2% BSA
The pH of the HEPES solution of pH7.5~8.5, the colloidal gold solution is 7.5~8.5；The scribing line refers to will be corresponding a kind of or more
Kind agglutinin is dissolved in the PBS of pH6.5~8.0 0.01mol/L according to 1~4mg/mL of total concentration, obtains agglutinin solution, profit
With the solution on nitrocellulose filter coating line profile bar band.
7. test strips according to claim 6, it is characterised in that：Using the colloidal gold for being dispersed with a concentration of 5 times of enrichments
The solution of the agglutinin of label impregnates glass fibre；A concentration of 2mg/mL of the agglutinin solution used in scribing line.
8. a kind of preparation method of the test strips of quick detection influenza Susceptible population, it is characterised in that：The detection influenza is susceptible
The test strips of crowd include A types test strips for detecting bird flu Susceptible population and/or for detecting human influenza Susceptible population
H-type test strips, the preparation method comprises the following steps：
（1）It is prepared by colloidal gold
Colloidal gold solution is prepared using aqueous solution of chloraurate；
（2）Colloid gold label
Agglutinin MAL-II or SNA, mixing, agglutinin MAL-II are added after the pH value of colloidal gold solution is adjusted to 7.5~8.5
Or a concentration of 10~12 μ g/mL of SNA；After reaction, low-speed centrifugal goes to precipitate, and to supernatant high speed centrifugation, then abandons supernatant,
Precipitation is scattered in the 0.01mol/L containing 0.15 mol/L NaCl and 0.2%BSA of 1 mL and the HEPES solution of pH7.5~8.5
In, the colloid gold label agglutinin solution of as 10 times enrichments, by glass fibre 5 ~ 10 times of enrichments colloid gold label
It is taken out after being impregnated 5~8 hours in agglutinin solution, is freeze-dried, obtains gold-labelled pad；Wherein, the gold-labelled pad of the A types test strips
Agglutinin MAL-II including colloid gold label, the gold-labelled pad of the H-type test strips includes the agglutinin SNA of colloid gold label；
（3）Agglutinin is coated with
Will detection be diluted to respectively with the PBS of pH6.5~8.0 0.01mol/L with agglutinin and nature controlling line agglutinin it is a concentration of
1~4mg/mL；It crosses on nitrocellulose filter respectively, nitrocellulose filter is closed, cleaned and is dried after dry, is obtained
To the nitrocellulose filter with detection band and nature controlling line；The detection band agglutinin is WFA, STL, RCA120, PHA-E, DBA
At least one of or at least one of GNA, ECA, HHL, PHA-E, DBA, the nature controlling line agglutinin correspond to ConA or
STL；Wherein, the detection of the A types test strips has at least one in agglutinin WFA, STL, RCA120, PHA-E, DBA with solidification
Kind, the detection band solidification of the H-type test strips has at least one of agglutinin GNA, ECA, HHL, PHA-E, DBA；The A types
The nature controlling line solidification of test strips has agglutinin ConA, the nature controlling line solidification of the H-type test strips to have agglutinin STL；
（4）The assembling of test strips
By step（3）Afterwards, gold-labelled pad, nitrocellulose filter and sample pad, water absorption pad assembling are constituted into test strips.
9. a kind of application of test strips as described in claim 1 in preparing the quickly reagent of detection influenza Susceptible population,
It is characterized in that：The detection includes the following steps：
The sample pad of A types test strips and/or H-type test strips is inserted into saliva sample or containing in the oral cavity, saliva appears in inspection
Test strips are taken out after in control area, observation detection band and nature controlling line color status obtain easy for bird flu and/or human influenza
The testing result of perception.
10. application 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 show color change, then show with the ability for relatively resisting avian influenza virus by force；
（2）Only nature controlling line shows color change, then shows susceptible avian influenza virus；
（3）No any color change shows；Then show that test strips fail；
For H-type test strips, testing result is as follows：
（1）Detection band and nature controlling line show color change, then show with the ability for relatively resisting human influenza virus by force；
（2）Only nature controlling line shows color change, then shows susceptible human influenza virus；
（3）No any color change shows；Then show that test strips fail.
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| 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 |
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| CN111672544B (en) * | 2020-06-08 | 2022-03-01 | 江苏师范大学 | Paper-based biochemical reagent storage method based on water two-phase system |
| 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 |