CN109100518A - Bird flu, the test strips of human influenza Susceptible population and test card are quickly detected using competition law - Google Patents
Bird flu, the test strips of human influenza Susceptible population and test card are quickly detected using competition law Download PDFInfo
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- CN109100518A CN109100518A CN201811211047.7A CN201811211047A CN109100518A CN 109100518 A CN109100518 A CN 109100518A CN 201811211047 A CN201811211047 A CN 201811211047A CN 109100518 A CN109100518 A CN 109100518A
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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
- 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
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/11—Orthomyxoviridae, e.g. influenza virus
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4724—Lectins
Abstract
The invention discloses a kind of quickly to detect bird flu, the test strips of human influenza Susceptible population and test card using competition law.The test strips (card) include the I type test strips (card) for detecting bird flu Susceptible population and/or the II type test strips (card) for detecting human influenza Susceptible population, and test strips (card) include sample pad, colloid gold label pad, detection reaction zone and the water absorption pad set gradually along chromatography direction.Test strips (card) detection sensitivity of the present invention is high, and false positive and false negative rate are low, easy to use, is easy observation difference, is suitable for large-scale application in clinical quick diagnosis and base's epidemiological survey etc..
Description
Technical field
The invention belongs to biochemistry detection technical fields, and in particular to one kind is quickly detected based on colloidal gold immunochromatographimethod technology
The test strips (card) of bird flu and human influenza virus Susceptible population.
Background technique
The host's distribution of influenza virus in nature is very extensive, has complicated gene structure, and its gene occurs
The probability of reconfiguration and mutation is very high, therefore is in the world in periodic epidemic.Influenza has disease incidence and case fatality rate height, passes
Broadcast that speed is fast, involves the features such as range is wide, once outburst is got up, it will serious disaster is caused to the mankind.Influenza surface
There are a kind of important structural proteins, exist in the form of H aggressiveness, referred to as hemagglutinin (HA), influenza virus invades the mode of human body
Host is mainly infected in conjunction with host cell surface specificity sugar chain receptor by hemagglutinin (HA) structure on its surface,
The sialic acid molecule that HA is mainly identified can combine the structure to form SA α 2-3Gal by the C atom of its end with α 2-3 and Gal
Or the structure of SA α 2-6Gal is formed, the combination with such host cell surface structure and HA has specificity.Inhomogeneity
The influenza virus of type combines host cell surface sugar-chain end structure type different, wherein human influenza virus's specific recognition
Sialic acid sugar-chain end structure is SA α 2-6Gal；Avian influenza virus mainly identifies SA α 2-3Gal sialic acid sugar-chain end structure.
The sugar-chain end structure of virus surface can be closed due to having in human saliva, how much its content can be used to judge pair
In the neurological susceptibility of influenza virus.
Agglutinin be it is a kind of can be with the non-zymoprotein in conjunction with the sugar chain structure on glycoprotein or glycolipid surface, but it is simultaneously
It is not similar to the immunocompetence of antibody.Golden elder agglutinin (Sambucus Nigra Lectin, SNA) and saddle tree
Agglutinin-II (Maackia Aimrensis Lectin-II, MAL-II) and the region of influenza virus HA specific recognition have
Similitude；Wherein, SNA specific recognition end is the sugar chain receptor structure of SA α 2-6Gal, this and human influenza virus's specificity knot
The site of conjunction has similitude；MAL-II specific recognition end is the sugar chain receptor structure of SA α 2-3Gal, this and avian flu
The site of poison specific binding has similitude." one kind is used for screening bird flu and human influenza virus Susceptible population to Chinese patent
Detection instrument " (publication number: CN103884834A) disclose agglutinin MAL-II and bird flu neurological susceptibility and agglutinin SNA
It with the correlativity of human influenza virus's neurological susceptibility, and points out, in Susceptible population's saliva in conjunction with corresponding agglutinin MAL-II or SNA
The expression of α 2-3 or α 2-6 connection terminal sialic acid sugar chain structure significantly lower, but the patent is only applied to chip examination
Agent box, simplicity, speed and the cost of detection have much room for improvement.
Chinese patent " the quickly method and test strips of detection bird flu and human influenza Susceptible population " (publication number:
CN106771120A the test paper that bird flu and human influenza virus Susceptible population are quickly detected based on colloidal gold chromatographic technology) is disclosed
Item, reaction principle are double agglutinin sandwich methods.But due to agglutinin do not have similar to antibody immunocompetence, agglutinin with
Sialoglycoprotein can not be mentioned in the same breath in the specificity of combination with antigen-antibody binding reaction.For detection sialic acid sugar
This kind of substance with antigenic property of albumen, it is more reasonable using competition law, both embodied the exclusive of agglutinin specific binding
Property, and it is more clear testing principle, the factor for influencing testing result can also become less, to reduce the vacation sun of testing result
Property and false negative.But processing, the agglutinin concentration in gold-labelled pad, the coating object in detection line and its concentration screening of test sample
It is provided with bigger difficulty, needs just to can determine that by research of technique, guarantees accuracy, the reliability of testing result.At present still
Have not seen the report of the colloidal gold strip (card) using the detection bird flu of competition law principle and human influenza Susceptible population.
Summary of the invention
The purpose of the present invention is to provide a kind of test paper that bird flu, human influenza Susceptible population are quickly detected using competition law
Item and test card.
In order to achieve the above objectives, the invention adopts the following technical scheme:
A kind of test paper quickly detecting influenza Susceptible population using competition law, the test paper include susceptible for detecting bird flu
The I type test paper of crowd and/or II type test paper for detecting human influenza Susceptible population, the I type test paper and II type test paper wrap
Include the sample pad set gradually along chromatography direction, colloid gold label pad, detection reaction zone and water absorption pad, the detection reaction zone packet
The detection line being arranged successively and nature controlling line are included, the colloid gold label pad of the I type test paper includes the agglutinin for marking colloidal gold
MAL-II (MAL-II-colloid gold label object), the colloid gold label pad of the II type test paper include the agglutinin for marking colloidal gold
SNA (SNA-colloid gold label object), the nature controlling line of the I type test paper includes agglutinin ConA, the nature controlling line of the II type test paper
Including agglutinin STL, the detection line of the I type test paper and II type test paper includes myosin.
Preferably, the colloid gold label pad is the agglutinin MAL-II that will mark colloidal gold or the agglutination for marking colloidal gold
Plain SNA is adsorbed on manufactured on glass fibre membrane or nitrocellulose filter by coating and drying；The detection line be by
Myosin is manufactured on nitrocellulose filter by coating and dry solidification；The nature controlling line is by agglutinin ConA
Or STL by coating and dry solidification where detection line on nitrocellulose filter it is manufactured.
Preferably, the coating of the agglutinin SNA of the agglutinin MAL-II or label colloidal gold of the label colloidal gold is specific
Refer to the agglutinin MAL-II for the label colloidal gold for being 30~60 μ g/mL by concentration or marks the agglutinin SNA spraying of colloidal gold
On glass fibre membrane or nitrocellulose filter；The tire that it is 1~4mg/mL by concentration that the coating of the myosin, which is specifically referred to,
The detection line position of nitrocellulose filter is crossed or be sprayed on to globulin, and the coating of the agglutinin ConA or STL specifically refers to
Agglutinin ConA or STL that concentration is 1~4mg/mL are crossed or are sprayed on the Quality Control line position of nitrocellulose filter.
Preferably, the test paper is fixed in substrate or is arranged in getting stuck with window.
A kind of preparation method of test paper, comprising the following steps:
The preparation of (one) I type test paper
1.1) after the agglutinin MAL-II of the label colloidal gold being coated on glass fibre membrane or nitrocellulose filter
Dry (37~40 DEG C drying 18~24 hours), obtain colloid gold label pad；
1.2) by the myosin and agglutinin ConA coated on nitrocellulose filter, then through drying (20~
30 DEG C) detection line and nature controlling line is prepared；
1.3) contain detection line and matter for what colloid gold label pad that sample pad, step 1.1) obtain, step 1.2) obtained
The nitrocellulose filter and water absorption pad for controlling line are assembled into I type test paper；
The preparation of (two) II type test paper
2.1) by the agglutinin SNA of the label colloidal gold coated on rear dry on glass fibre membrane or nitrocellulose filter
(37~40 DEG C drying 18~24 hours), obtain colloid gold label pad；
2.2) myosin and agglutinin STL are coated on nitrocellulose filter, then through drying (20~30
DEG C) detection line and nature controlling line is prepared；
2.3) contain detection line and matter for what colloid gold label pad that sample pad, step 2.1) obtain, step 2.2) obtained
The nitrocellulose filter and water absorption pad for controlling line are assembled into II type test paper.
Preferably, in the preparation of the colloid gold label pad, by the agglutination for the label colloidal gold that concentration is 30~60 μ g/mL
Plain MAL-II marks the agglutinin SNA of colloidal gold according to 50~200 μ L/cm2Coated on glass fibre membrane or nitrocellulose
On film；It is the myosin and agglutinin ConA of 1~4mg/mL by concentration in the preparation of the detection line and nature controlling line
Or STL is coated on nitrocellulose filter.
A method of quickly detecting sialic acid sugar chain using competition law, comprising the following steps:
1) the detection sample of acquisition is pre-processed, to prevent the protein degradation in detection sample, obtains test sample；
2) it will test sample or the test sample after dilution be loaded to I type being prepared and/or II type test paper；
3) there is SA α 2-3Gal structure using the end in the myosin and detection sample in I type and/or II type test paper
Sialic acid sugar chain or end have SA α 2-6Gal structure sialic acid sugar chain competitive binding correspond to the label colloid on test paper
The agglutinin MAL-II or SNA of gold, so that testing result is formed, it is as follows to the judgement of testing result:
If detection line and nature controlling line develop the color, for I type test paper, then determine that detecting end described in sample has SA α
The sialic acid sugar chain of 2-3Gal structure is negative or content is lower, for II type test paper, then determines to detect end described in sample
Sialic acid sugar chain with SA α 2-6Gal structure is negative or content is lower；
If only nature controlling line develops the color, for I type test paper, then determine that detecting end described in sample has SA α 2-3Gal structure
Sialic acid sugar chain be positive or content is higher, for II type test paper, then determine that detecting end described in sample has SA α 2-
The sialic acid sugar chain of 6Gal structure is positive or content is higher；
If nature controlling line does not develop the color, determine that corresponding test paper is invalid.
Preferably, the detection sample is selected from saliva or nasal secretion；The dilution of test sample is using deionized water, note
Jetting, physiological saline (0.9% sodium chloride solution), phosphate buffer or borate buffer solution will pass through pretreated detection
Sample (test sample) dilutes 1~5 times.
Preferably, the pretreatment is the following steps are included: be added albumen into collected detection sample at 0~4 DEG C
It is centrifuged after enzyme inhibitor, protease inhibitors is added again into centrifugation gained supernatant.
Above-mentioned I type and/or II type test paper are preparing the reagent of quickly detection influenza Susceptible population (for example, test strips, test paper
Card etc.) in application (wherein, I type test paper can be used for detecting bird flu Susceptible population, and II type test paper can be used for detecting human influenza easy
Touching group).
The beneficial effects of the present invention are embodied in:
The present invention constitutes layer with the agglutinin MAL-II or SNA of colloid gold label respectively using myosin as detection line
Test paper is analysed, it is fast to realize the sialic acid sugar chain structure (for example, there is SA α 2-3Gal or the end SA α 2-6Gal) based on competition law
Speed detection, test paper detection sensitivity, repetitive rate are high, and high specificity, stability is good, false sun rate and False-Negative Rate can be controlled in 1% with
Under, higher than the level of the existing detection reagent based on sandwich method, testing result is easy observation difference, and it is easy to use, it is suitable for flowing
Clinical quick diagnosis and the base, rural area epidemiological survey large-scale application for feeling neurological susceptibility, can be used for sialic acid sugar chain
The qualitative and half-quantitative detection of structure.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of test strips；
In figure: 1 is PVC board, and 2 be detection line, and 3 be nature controlling line, and 4 be water absorption pad, and 5 be NC film, and 6 be gold-labelled pad, and 7 be sample
Pad.
Specific embodiment
Invention is further described in detail with reference to the accompanying drawings and examples.
(1) the I type test strips preparation of bird flu Susceptible population is detected
(1) prepared by colloidal gold: by gold chloride (HAuCl4) it is made into 1% chlorauric acid solution, take 1mL chlorauric acid solution to be added
In 99mL deionized water, it is heated to that the minor official acid sodium solution of Chinese holly of 2mL 1% is added after boiling immediately, stirs evenly rapidly, continue to add
Heat 10 minutes, is cooled to room temperature, and supplements deionized water to 100mL, obtains colloidal gold solution.
(2) MAL-II-colloid gold label object preparation: the colloidal gold solution for taking 10mL to prepare, with the carbon of 0.1mol/L
Sour potassium solution adjusts pH value to 4.9~5.3 (for example, 5.0), and 120 μ g of agglutinin MAL-II is added, and mixes, room temperature reaction 10
Minute, 4 DEG C of 2000rpm are centrifuged 20 minutes, discard precipitating.Supernatant is centrifuged 60 minutes in 4 DEG C of 12000rpm, is discarded supernatant, and is sunk
It forms sediment in the PBS (including 0.2%BSA) for the 0.01mol/L pH8.0 for being dissolved in 1mL, obtains MAL-II-colloid gold label object solution.
(3) preparation of gold-labelled pad: MAL-II-colloid gold label object solution is taken (to include with the PBS of 0.01mol/L pH8.0
1~2 times (for example, 1 times of dilution, then MAL-II concentration is about 60 μ g/mL) 0.2%BSA) is diluted, by 100 μ L/cm2Uniform spray
Be applied on glass fibre membrane, 30% humidity hereinafter, 40 DEG C drying 24 hours, obtain gold-labelled pad.Gold-labelled pad is sealed with Fresco Bag, interior
Desiccant is filled, is stored at room temperature.
(4) coating of detection line and nature controlling line: myosin, which is diluted to concentration with the PBS (pH7.2) of 0.01mol/L, is
2mg/mL, crosses (or using the application pattern being sprayed on NC film as substitution scribing line) on NC film, and it is small to spontaneously dry 4 in room temperature
When, then be put in 30 DEG C of aeration cabinets aeration-drying 1 hour, it is solidificated in myosin on NC film, as detection line.From
At 6 millimeters of detection line, use the agglutinin ConA of 2mg/mL draw a line again (or be sprayed on NC film as substitution scribing line coating side
Formula), it spontaneously dries 4 hours in room temperature, then be put in 30 DEG C of aeration cabinets aeration-drying 1 hour, solidifies agglutinin ConA,
As nature controlling line.The coating of detection line and nature controlling line can carry out simultaneously with dry solidification.
(5) assembling of test strips: test strips by sample pad 7, gold-labelled pad 6, solidify the nitric acid for having detection line 2 and nature controlling line 3
Cellulose membrane (NC film 5), water absorption pad 4 and substrate (for example, PVC board 1) composition；Wherein, water adsorption glass fibre can be used in water absorption pad 4
It ties up, surplus liquid in absorption detecting sample；Sample pad 7 can use glass fibre membrane, contact sample to be tested, specific structure ginseng
See Fig. 1.
In PVC board 1, each test paper parts of test strips is sequentially arranged along chromatography direction, wherein in downstream is to inhale
Water cushion 4, upstream end are sample pad 7, and middle section has the NC film 5 of detection line 2 and nature controlling line 3 as detection reaction zone to solidify, in NC
The gold-labelled pad 6 for being adsorbed with the agglutinin MAL-II of colloid gold label is placed between film 5 and sample pad 7.
Test paper is attached in PVC board 1, cuts into the belt strip of 4mm wide；Wherein, first NC film 5 is attached in PVC board 1,
Then water absorption pad 4 and gold-labelled pad 6 are attached in PVC board 1, and partly overlapped respectively with 5 both ends of NC film, finally by sample pad
7 are attached in PVC board 1, and partly overlap with gold-labelled pad 6.In addition, if test paper can be formed in test paper is assembled in getting stuck
Card.It gets stuck and is made of upper plate and lower plate, upper plate has detection window and well, and 7 face well of sample pad detects reaction zone face
Detect window.For cured detection line 2 closer to gold-labelled pad 6, test strips (card) surface has alphabetical " T " and " C " as mark on NC film 5
The symbol of detection line and nature controlling line facilitates differentiation；Test strips (card) save under the conditions of hermetically drying, spare.
(2) the II type test strips preparation of human influenza Susceptible population is detected
(1) prepared by colloidal gold: by gold chloride (HAuCl4) it is made into 1% chlorauric acid solution, take 1mL chlorauric acid solution to be added
In 99mL deionized water, it is heated to that the minor official acid sodium solution of Chinese holly of 2mL 1% is added after boiling immediately, stirs evenly rapidly, continue to add
Heat 10 minutes, is cooled to room temperature, and supplements deionized water to 100mL, obtains colloidal gold solution.
(2) SNA-colloid gold label object preparation: the colloidal gold solution for taking 10mL to prepare, with the carbonic acid of 0.1mol/L
Potassium solution adjusts pH value to 5.8~6.2 (for example, 6.0), and 120 μ g of agglutinin SNA is added, and mixes, and reacts at room temperature 10 minutes,
4 DEG C of 2000rpm are centrifuged 20 minutes, discard precipitating.Supernatant is centrifuged 60 minutes in 4 DEG C of 12000rpm, is discarded supernatant, precipitating is dissolved in
In the PBS (including 0.2%BSA) of the 0.01mol/L pH8.0 of 1mL, SNA-colloid gold label object solution is obtained.
(3) preparation of gold-labelled pad: SNA-colloid gold label object solution is taken (to include with the PBS of 0.01mol/L pH8.0
1~2 times (for example, 1 times of dilution, then SNA concentration is about 60 μ g/mL) 0.2%BSA) is diluted, by 100 μ L/cm2It uniformly sprays
In on glass fibre membrane, 30% humidity hereinafter, 40 DEG C drying 24 hours, obtain gold-labelled pad.Gold-labelled pad is sealed with Fresco Bag, built-in
Desiccant is stored at room temperature.
(4) coating of detection line and nature controlling line: myosin, which is diluted to concentration with the PBS (pH7.2) of 0.01mol/L, is
2mg/mL crosses (or being sprayed on the application pattern on NC film as substitution scribing line) on NC film, spontaneously dries 4 hours in room temperature,
It is put in 30 DEG C of aeration cabinets again aeration-drying 1 hour, is solidificated in myosin on NC film, as detection line.From inspection
At 6 millimeters of survey line, use the agglutinin STL of 2mg/mL draw a line again (or be sprayed on NC film as substitution scribing line coating side
Formula), it spontaneously dries 4 hours in room temperature, then be put in 30 DEG C of aeration cabinets aeration-drying 1 hour, solidifies agglutinin STL,
As nature controlling line.The spraying of detection line and nature controlling line can carry out simultaneously with dry solidification.
(5) assembling of test strips: test strips by sample pad 7, gold-labelled pad 6, solidify the nitric acid for having detection line 2 and nature controlling line 3
Cellulose membrane (NC film 5), water absorption pad 4 and substrate (for example, PVC board 1) composition；Wherein, water adsorption glass fibre can be used in water absorption pad 4
It ties up, surplus liquid in absorption detecting sample；Sample pad 7 can use glass fibre membrane, contact sample to be tested, specific structure ginseng
See Fig. 1.
In PVC board 1, each test paper parts of test strips is sequentially arranged along chromatography direction, wherein in downstream is to inhale
Water cushion 4, upstream end are sample pad 7, and middle section has the NC film 5 of detection line 2 and nature controlling line 3 as detection reaction zone to solidify, in NC
The gold-labelled pad 6 for being adsorbed with the agglutinin SNA of colloid gold label is placed between film 5 and sample pad 7.
Test paper is attached in PVC board 1, cuts into the belt strip of 4mm wide；Wherein, first NC film 5 is attached in PVC board 1,
Then water absorption pad 4 and gold-labelled pad 6 are attached in PVC board 1, and partly overlapped respectively with 5 both ends of NC film, finally by sample pad
7 are attached in PVC board 1, and partly overlap with gold-labelled pad 6.In addition, if test paper can be formed in test paper is assembled in getting stuck
Card.It gets stuck and is made of upper plate and lower plate, upper plate has detection window and well, and 7 face well of sample pad detects reaction zone face
Detect window.For cured detection line 2 closer to gold-labelled pad 6, test strips (card) surface has alphabetical " T " and " C " as mark on NC film 5
The symbol of detection line and nature controlling line facilitates differentiation；Test strips (card) save under the conditions of hermetically drying, spare.
(3) testing principle
Detect I type test strips (card) of bird flu Susceptible population: colloid gold label pad (i.e. gold-labelled pad) is adsorbed with MAL-
II-colloid gold label object；Detection line is coated with myosin；Nature controlling line is coated with can be solidifying in conjunction with agglutinin MAL-II
Collect element ConA.Before any sample to be tested is provided, detection line and nature controlling line not display colors.When sample pad is added in sample
Afterwards, the SA α 2-3Gal sugar chain in sample chromatographs swimming to detection reaction zone, due to sample together with MAL-II-colloid gold label object
The SA α 2-3Gal sugar chain competitiveness and MAL-II-colloid gold label object knot in SA α 2-3Gal sugar chain and myosin in product
It closes, when the SA α 2-3Gal sugar chain concentration in sample is more than a certain amount of, MAL-II-colloid gold label object cannot be with detection line
On myosin combine, at this time detect line position do not occur aubergine lines；When do not have in sample SA α 2-3Gal sugar chain or
In sample SA α 2-3Gal sugar chain lower than it is a certain amount of when, MAL-II-colloid gold label object just with the myosin knot in detection line
It closes, to show an aubergine lines in detection line position；Regardless of whether containing SA α 2-3Gal sugar chain, matter in sample
Aubergine lines can all occur in control line position, to show that detection is effective.
Detect II type test strips (card) of human influenza Susceptible population: colloid gold label pad (i.e. gold-labelled pad) is adsorbed with SNA-
Colloid gold label object；Detection line is coated with myosin；Nature controlling line is coated with can be with the agglutinin STL in conjunction with agglutinin SNA.
Before any sample is provided, detection line and nature controlling line not display colors.SA α 2- after sample pad is added in sample, in sample
6Gal sugar chain chromatographs swimming to detection reaction zone, due to the SA α 2-6Gal sugar chain in sample together with SNA-colloid gold label object
With the SA α 2-6Gal sugar chain competitiveness in myosin in conjunction with SNA-colloid gold label object, as the SA α 2-6Gal in sample
When sugar chain concentration is more than a certain amount of, SNA-colloid gold label object cannot detect at this time in conjunction with the myosin in detection line
Line position does not occur aubergine lines；When there is no SA α 2-6Gal sugar chain concentration in SA α 2-6Gal sugar chain or sample low in sample
When a certain amount of, SNA-colloid gold label object is just in conjunction with the myosin in detection line, to show in detection line position
One aubergine lines；Regardless of whether containing SA α 2-6Gal sugar chain in sample, purplish red colo(u)r streak can all occur in Quality Control line position
Item, to show that detection is effective.
Above-mentioned myosin refers to fetuin, and it includes 12 cysteine residues, main constituents are as follows: polypeptide
74%, hexose 8.3%, aminohexose 5.5% and sialic acid 8.7%.The wherein sialic acid content of SA α 2-3Gal sugar chain connection
Sialic acid content for the connection of 4.8%, SA α 2-6Gal sugar chain is 2.8%.Since it contains quantitative sugar chain, set
In the test strips (card) of meter, it can be used for making detection line.
(4) detect bird flu, human influenza Susceptible population test strips (card) false sun rate and False-Negative Rate test experiments
4.1, saliva sample acquisition and pretreatment: between Healthy People 50, after meal two hours, about 9 points to 10 points, physiology salt
After water is gargled three times, the full saliva of naturally secret, every at least 1mL are acquired rapidly, and is immediately placed on ice, and protease suppression is added
Preparation (10 μ L are added in every milliliter of saliva) prevents protein degradation.Collected saliva sample is merged into (50 mixing), 4 DEG C,
It is centrifuged 1h (removing desalivation residue and cast-off cells) under the conditions of 12000rpm, collects supernatant；To on the saliva sample after centrifugation
Protease inhibitors (1 μ L protease inhibitors is added in every 10mL) is added in clear, it is spare (4 DEG C) to can be used as sample to be tested,
Protease Inhibitor Cocktail of the above-mentioned protease inhibitors for example, by using SIGMA company.
4.2, in sample SA α 2-6Gal sugar chain, SA α 2-3Gal sugar chain removal:
1, the preparation of agglutinin magnetic particle compound
The centrifuge tube for taking 50 2mL, is placed on magnetic separation rack, each to draw 3mg epoxidation magnetic particle (epoxidation magnet
It is suspended in aqueous solution and saves, about 14mg/mL) it is placed in above-mentioned centrifuge tube.Magnetic Isolation abandons supernatant after supernatant is limpid.Respectively
Addition 1mL boric acid borax coupling buffer (composition: 5mL 0.05mol/L borax soln, 45mL0.2mol/L boric acid solution, and
0.15mol/L NaCl adjusts pH with hydrochloric acid or sodium hydroxide and is adjusted to 7.4), and oscillation is mixed to be placed on magnetic separation rack and be cleaned
Magnetic grain, Magnetic Isolation 3min abandon supernatant after supernatant is limpid.By 600 μ L SNA/MAL-II solution, (1mg/mL, solvent are that coupling is slow
Fliud flushing) it is added in cleaned epoxidation magnetic particle, mixing is placed on constant temperature oscillator, at 25 DEG C, under the conditions of 180rpm
Oscillating reactions 3h, after coupling reaction, Magnetic Isolation 3min abandons supernatant.By confining liquid (aqueous solution, containing 2% ethanol amine and
0.1% BSA) 1mL is separately added into above-mentioned centrifuge tube, at 25 DEG C, oscillating reactions 1 hour under the conditions of 180rpm.Closing terminates
Afterwards, it is placed on magnetic separation rack, after Magnetic Isolation, discards supernatant, respectively obtain SNA magnetic particle compound and MAL-II is magnetic
Particle complex.
2, the removal of α 2-6 connection terminal sialic acid sugar chain
The saliva sample handled well in 4.1 is taken, is added in the above-mentioned SNA magnetic particle compound prepared, every pipe is added
700 μ L (saliva), the oscillating reactions 3 hours under the conditions of 25 DEG C, 180rpm.To which after reaction, taking-up is placed in magnetic separation rack
After upper separation, supernatant is collected.Obtain the saliva sample of removal SA α 2-6Gal sugar chain.
3, the removal of α 2-3 connection terminal sialic acid sugar chain
The saliva sample of above-mentioned removal SA α 2-6Gal sugar chain is taken, it is compound that the above-mentioned MAL-II magnetic particle prepared is added
In object, the oscillating reactions 3 hours under the conditions of 25 DEG C, 180rpm.It is separated to after reaction, take out to be placed on magnetic separation rack
Afterwards, supernatant is collected.Obtain the saliva sample of removal SA α 2-6Gal sugar chain and SA α 2-3Gal sugar chain.
4.3, false positive is tested
1, I type test strips (card) of bird flu Susceptible population are detected
(1) saliva sample (the removal SA α 2-6Gal sugar chain and SA α 2-3Gal sugar chain) 5mL handled well is taken.
(2) by 100 μ L of 100 μ L of saliva sample and deionized water, I type test strips of detection bird flu Susceptible population are instilled
(card) sample pad, does 50 altogether.Observation in 5 minutes is as a result, determine whether contain SA α 2-3Gal sugar chain in sample.
Judgment basis is as follows:
Negative (nothing): aubergine is presented in detection line and nature controlling line；
Positive (having): only aubergine is presented in nature controlling line；
Invalid: aubergine is not presented in nature controlling line, then no matter whether detection line is presented aubergine, the detection knot of test strips (card)
Fruit is judged in vain；
Determining result are as follows: 1 part of saliva sample is positive, and 49 parts are negative, all effectively, false positive rate 2%, with sample
Product example increases, and can control below 1%.
2, II type test strips (card) of human influenza Susceptible population are detected
(1) saliva sample (the removal SA α 2-6Gal sugar chain and SA α 2-3Gal sugar chain) 5mL handled well is taken.
(2) by 100 μ L of 100 μ L of saliva sample and deionized water, II type test strips of detection human influenza Susceptible population are instilled
(card) sample pad, does 50 altogether.Observation in 5 minutes is as a result, determine whether contain SA α 2-6Gal sugar chain in sample.
Judgment basis is as follows:
Negative (nothing): aubergine is presented in detection line and nature controlling line；
Positive (having): only aubergine is presented in nature controlling line；
Invalid: aubergine is not presented in nature controlling line, then no matter whether detection line is presented aubergine, test strips (card) testing result
It is invalid to be judged to；
Determining result are as follows: 2 parts of saliva sample are positive, and 48 parts are negative, all effectively, false positive rate 4%, with sample
Product example increases, and can control below 1%.
4.4, false negative is tested
1, sample preparation:
The saliva sample 10mL except SA α 2-6Gal sugar chain and SA α 2-3Gal sugar chain is removed, 16.4mg myosin is added,
Dissolution is used as test specimen after mixing.
2, I type test strips (card) of bird flu Susceptible population are detected
(1) the test specimen 5mL prepared is taken.
(2) by 100 μ L of 100 μ L of test specimen and deionized water, I type test strips of detection bird flu Susceptible population are instilled
(card) sample pad, does 50 altogether.Observation in 5 minutes is as a result, determine whether contain SA α 2-3Gal sugar chain in sample.
Judgment basis is as follows:
Negative (nothing): aubergine is presented in detection line and nature controlling line；
Positive (having): only aubergine is presented in nature controlling line；
Invalid: aubergine is not presented in nature controlling line, then no matter whether detection line is presented aubergine, the detection knot of test strips (card)
Fruit is judged in vain；
Determining result are as follows: 1 part of saliva sample is negative, and 49 parts are positive, all effectively, false negative rate 2%, with sample
Product example increases, and can control below 1%.
3, II type test strips (card) of human influenza Susceptible population are detected
(1) the test specimen 5mL prepared is taken.
(2) by 100 μ L of 100 μ L of test specimen and deionized water, II type test strips of detection human influenza Susceptible population are instilled
(card) sample pad, does 50 altogether.Observation in 5 minutes is as a result, determine whether contain SA α 2-6Gal sugar chain in sample.
Judgment basis is as follows:
Negative (nothing): aubergine is presented in detection line and nature controlling line；
Positive (having): only aubergine is presented in nature controlling line；
Invalid: aubergine is not presented in nature controlling line, then no matter whether detection line is presented aubergine, the detection knot of test strips (card)
Fruit is judged in vain；
Determining result are as follows: 1 part of saliva sample is negative, and 49 parts are positive, all effectively, false negative rate 2%, with sample
Product example increases, and can control below 1%.
Claims (10)
1. a kind of test paper for quickly detecting influenza Susceptible population using competition law, it is characterised in that: the test paper includes for detecting
I type test paper of bird flu Susceptible population and/or II type test paper for detecting human influenza Susceptible population, the I type test paper and II
Type test paper includes sample pad (7), colloid gold label pad, detection reaction zone and the water absorption pad (4) set gradually along chromatography direction, institute
Stating detection reaction zone includes detection line (2) and nature controlling line (3), and the colloid gold label pad of the I type test paper includes label colloidal gold
Agglutinin MAL-II, the colloid gold label pad of the II type test paper includes the agglutinin SNA for marking colloidal gold, the I type examination
The nature controlling line (3) of paper includes agglutinin ConA, and the nature controlling line (3) of the II type test paper includes agglutinin STL, I type and II
The detection line (2) of type test paper includes myosin.
2. a kind of test paper for quickly detecting influenza Susceptible population using competition law according to claim 1, it is characterised in that: institute
State colloid gold label pad be will mark colloidal gold agglutinin MAL-II or mark colloidal gold agglutinin SNA by coating and
Dry be adsorbed on glass fibre membrane or nitrocellulose filter and it is manufactured；The detection line (2) is that myosin is passed through to painting
It is coated with and dry solidification is manufactured on nitrocellulose filter；The nature controlling line (3) is that agglutinin ConA or STL are passed through painting
It is coated with and dry solidification is manufactured on nitrocellulose filter.
3. a kind of test paper for quickly detecting influenza Susceptible population using competition law according to claim 2, it is characterised in that: institute
It states the agglutinin MAL-II of label colloidal gold or the coating of the agglutinin SNA of colloidal gold is marked to specifically refer to concentration be 30~60
The agglutinin MAL-II of the label colloidal gold of μ g/mL marks the agglutinin SNA of colloidal gold to be sprayed on glass fibre membrane or nitric acid
On cellulose membrane；The coating of the myosin specifically refers to that the myosin that concentration is 1~4mg/mL is crossed or is sprayed on
The detection line position of nitrocellulose filter, the coating of the agglutinin ConA or STL specifically refer to concentration be 1~4mg/mL's
Agglutinin ConA or STL cross or are sprayed on the Quality Control line position of nitrocellulose filter.
4. a kind of test paper for quickly detecting influenza Susceptible population using competition law according to claim 1, it is characterised in that: institute
Test paper is stated to be fixed in substrate or be arranged in getting stuck with window.
5. a kind of preparation method for the test paper for quickly detecting influenza Susceptible population using competition law as described in claim 1,
It is characterized in that: the following steps are included:
1) the agglutinin MAL-II of the label colloidal gold is obtained coated on drying rear on glass fibre membrane or nitrocellulose filter
To colloid gold label pad；
2) then coated on nitrocellulose filter detection is prepared through drying in the myosin and agglutinin ConA
Line (2) and nature controlling line (3)；
3) by sample pad (7), colloid gold label pad, nitrocellulose filter and the water suction for containing detection line (2) and nature controlling line (3)
Pad (4) is assembled into the I type test paper for detecting bird flu Susceptible population；
Alternatively,
1) the agglutinin SNA of the label colloidal gold is obtained coated on drying rear on glass fibre membrane or nitrocellulose filter
Colloid gold label pad；
2) then coated on nitrocellulose filter detection is prepared through drying in the myosin and agglutinin STL
Line (2) and nature controlling line (3)；
3) by sample pad (7), colloid gold label pad, nitrocellulose filter and the water suction for containing detection line (2) and nature controlling line (3)
Pad (4) is assembled into the II type test paper for detecting human influenza Susceptible population.
6. according to the method described in claim 5, it is characterized by: being the mark of 30~60 μ g/mL by concentration in the step 1)
Remember the agglutinin MAL-II of colloidal gold or marks the agglutinin SNA of colloidal gold according to 50~200 μ L/cm2Coated on glass fibre
On film or nitrocellulose filter；In the step 2), by concentration be 1~4mg/mL myosin and agglutinin ConA or
STL is coated on nitrocellulose filter.
7. a kind of method for quickly detecting sialic acid sugar chain using competition law, it is characterised in that: the following steps are included:
1) the detection sample of acquisition is pre-processed, to prevent the protein degradation in detection sample, obtains test sample；
2) it will test sample or the test sample after dilution be loaded to I type and/or II type test paper, the I type test paper and II
Type test paper includes sample pad (7), colloid gold label pad, detection reaction zone and the water absorption pad (4) set gradually along chromatography direction, institute
Stating detection reaction zone includes detection line (2) and nature controlling line (3), and the colloid gold label pad of the I type test paper includes label colloidal gold
Agglutinin MAL-II, the colloid gold label pad of the II type test paper includes the agglutinin SNA for marking colloidal gold, the I type examination
The nature controlling line (3) of paper includes agglutinin ConA, and the nature controlling line (3) of the II type test paper includes agglutinin STL, I type and II
The detection line (2) of type test paper includes myosin；
3) there is the saliva of SA α 2-3Gal structure using the end in the myosin and detection sample in I type and/or II type test paper
Liquid acid sugar chain or end correspond to the label colloidal gold on test paper with the sialic acid sugar chain competitive binding of SA α 2-6Gal structure
Agglutinin MAL-II or SNA form testing result, as follows to the judgement of testing result:
If detection line (2) and nature controlling line (3) develop the color, for I type test paper, then determine that detecting end described in sample has SA
The sialic acid sugar chain of α 2-3Gal structure is negative or content is lower, for II type test paper, then determines to detect end described in sample
Sialic acid sugar chain with SA α 2-6Gal structure is negative or content is lower；
If only nature controlling line (3) develops the color, for I type test paper, then determine that detecting end described in sample has SA α 2-3Gal structure
Sialic acid sugar chain is positive or content is higher, for II type test paper, then determines that detecting end described in sample has SA α 2-6Gal
The sialic acid sugar chain of structure is positive or content is higher；
If nature controlling line (3) does not develop the color, determine that corresponding test paper is invalid.
8. the method according to claim 7 for quickly detecting sialic acid sugar chain using competition law, it is characterised in that: the inspection
Test sample sheet is selected from saliva or nasal secretion；The dilution of test sample uses deionized water, injection water, physiological saline, phosphate
Buffer or borate buffer solution, extension rate are 1~5 times.
9. the method according to claim 7 for quickly detecting sialic acid sugar chain using competition law, it is characterised in that: described pre-
Processing into the detection sample of acquisition after addition protease inhibitors the following steps are included: be centrifuged, to centrifugation gained supernatant
Protease inhibitors is added in liquid again, obtains test sample.
10. a kind of application of test paper as described in claim 1 in the preparation quickly reagent of detection influenza Susceptible population.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811211047.7A CN109100518A (en) | 2018-10-17 | 2018-10-17 | Bird flu, the test strips of human influenza Susceptible population and test card are quickly detected using competition law |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811211047.7A CN109100518A (en) | 2018-10-17 | 2018-10-17 | Bird flu, the test strips of human influenza Susceptible population and test card are quickly detected using competition law |
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ID=64868905
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