CN106771121A - A kind of foot and mouth disease virus colloidal gold strip, cause of disease quick detection kit and preparation method thereof - Google Patents
A kind of foot and mouth disease virus colloidal gold strip, cause of disease quick detection kit and preparation method thereof Download PDFInfo
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- CN106771121A CN106771121A CN201611075628.3A CN201611075628A CN106771121A CN 106771121 A CN106771121 A CN 106771121A CN 201611075628 A CN201611075628 A CN 201611075628A CN 106771121 A CN106771121 A CN 106771121A
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
Abstract
A kind of kit the invention provides foot and mouth disease virus colloidal gold strip and comprising the test strips, the reagent strip includes backing, nitrocellulose filter, adsorptive pads, gold standard pad, sample pad, right-hand member is sprayed with rabbit-anti rat immune globulin as quality control band on described nitrocellulose filter, left end is sprayed with foot and mouth disease virus polyclonal antibody as detection band on nitrocellulose filter, and the foot and mouth disease virus monoclonal antibody of colloid gold label is sprayed with gold standard pad.The FMDV group specificity colloidal gold strips that the present invention is developed, optimize detection method colloid gold particle, nitrocellulose specification and adsorptive pads specification etc..The FMDV group specificity colloidal gold strip high specificities, not with the cross reaction of other virus of domestic animal antigens.
Description
Technical field
The invention belongs to biological technical field, and in particular to a kind of foot and mouth disease virus colloidal gold strip, cause of disease are quickly examined
Test agent box and preparation method thereof.
Background technology
Aftosa (Foot-and-mouth disease, FMD), also known as aphthous fever (Aphthous fever), is by mouth hoof
Acute, the hot, height that the artiodactyl that epidemic disease poison (Foot-and-mouth disease virus, FMDV) infection causes suffers from altogether
Degree contagious disease.In addition to the Some Livestocks such as pig, ox, sheep, FMDV can also infect more than 30 kinds of wild animal, infection
Sequela rate is almost up to 100%.There is bubble in the positions such as the mouth of infected animal, tongue, hoof and breast, and ulceration simultaneously forms speck.
People can also infect FMD, and symptom is lighter, and most course of disease is in benign process.FMD epidemic situations are all paid much attention in countries in the world, and the world is moved
Thing health organization (OIE) and the World Food Programme (FAO) are classified as first of A class zoonosis.The outburst of FMD, not only gives
The animal husbandry of popular country causes huge economic loss, and seriously disturbs the society of these countries
Economic order, and cause political crisis and international trade friction.
FMDV belongs to Picornaviridae (Picornaviridae) Hostis (Aphthovirus) member, virus
Genome is single-stranded positive RNA, about 8,500 nucleotides have 7 serotypes and more than a 80 kinds of blood serum subtype, 7 serotypes point
Wei not O-shaped, A types, c-type, the types of SAT I (type of South Africa I), the types of SAT II (south Africa II type), the types of SAT III (type of South Africa III), the types of Asia I
(Asia I type), due to the genetic instability of virus, new virus subtype constantly goes out caused by the continuous change of antigen property
It is existing.FMDV particles are spherical in shape, and regular dodecahedron is symmetrical, a diameter of 20-30nm, are the minimum RNA diseases of known animal without cyst membrane
Poison.Its capsid is by 4 kinds of structural proteins 1A (VP4,85 amino acid), 1B (VP2,218 amino acid), 1C (VP3,220 amino
Acid), 1D (VP1,213 amino acid) each 60 molecular compositions, wherein 1B, 1C, 1D be located at capsid surface (Jackson et
Al., 2003).After FMDV ultracentrifugations, it is seen that particle of different sizes, the first particle is referred to as 146S complete virions, is
The maximum particle that the coat protein being made up of VP1, VP2, VP3 and VP4 of 60 copies is wrapped up, a diameter of 25nm sinks
Drop coefficient 146S, the buoyant density in cesium chloride is 1.43g/cm3, molecular weight is 8.08 × 106u；Second particle is referred to as 75S
Hollow capsid particle, it is a diameter of by VP0 (uncracked VP2 and VP4), VP1 and each 60 molecular compositions of tri- kinds of structural proteins of VP3
21nm, sedimentation coefficient 75S, the buoyant density in cesium chloride are 1.31g/cm3, molecular weight is 4.7 × 106u；The third particle claims
It is 12S capsomere particles, is by the pentamer structure of each 5 molecular composition of VP1, VP2, VP3, a diameter of 7nm, sedimentation
Coefficient 12S, the buoyant density in cesium chloride is 1.5g/cm3, molecular weight is 3.8 × 106u；4th kind of particle is referred to as 5S substances, is
By each 1 molecular composition of VP0, VP1 and VP3.One protein subunit constitutes the icosahedral face of virion, uses
Acid treatment FMDV, can make each virion discharge 20 protein subunits (Liu Ji is saved, 1999).
FMD diagnostic techniques is the important step of the anti-systems of FMD, as developing rapidly for biotechnology is continuous with instrument and equipment
Update, the detection technique of FMD has also obtained unprecedented development.Complement fixation test (CFT) (complement fixation
Test, CFT) be earliest standardized detection method, be successfully used to since FMD Viral typings identify from nineteen forty-three, always by
WRL and each FMD researchs or sizing center applications are so far.The method can identify antigen, can also identify antibody, and can carry out quantitative survey
It is fixed.General constant CFT is that serum to be checked is shaped, and micro CFT is used for hypotype and strain antigenicity analysis, but it is present in the world
Through without this concept of hypotype, because standard strain disunity used in test causes the confusion of Subtypes, and
The immune relationship that virus subtype can not explain in precise term between strain.CFT is cumbersome, is unsuitable for the inspection of extensive sample
Survey；It is not sufficiently stable plus CFT result of the tests, error is larger, therefore, OIE formal recommendation EUSAs
(enzyme-linked immunosorbent assay, ELISA) replaces traditional complement fixation test (CFT).The Anon of OIE
(2000) think that virus neutralization tests (virus neutralization test, VNT) is detection FMD antibody " gold marks
It is accurate ", it is one of detection method that international trade is specified.VNT can both identify antigen, again can antagonist quantitative determination, it is special with type
The opposite sex, is FMD detection methods that are most classical and knowing best, frequently as the reference of other detection methods.International quarantine clause
Regulation this method judges to pass in and out, and whether to infect or carries FMD viral for animal.The shortcoming of VNT is live virus to be used, to operator
Member and environmental requirement are higher, can only be carried out in special laboratory.Above-mentioned traditional serological diagnostic method all cannot distinguish between
The animal population of FMD wild virus infections and animal population (the differentiate infected from of vaccine immunity
vaccinated animals,DIVA).Because FMDV has 7 serotypes and tens kinds of blood serum subtypes, without intersecting between type and type
Immanoprotection action, this adds increased the difficulty of FMD diagnosis, the particularly countries and regions of FMD vaccine immunities will make a definite diagnosis FMD's
Infection is more difficult.
The content of the invention
In view of this, tried it is an object of the invention to provide quick diagnosis aftosa group specificity colloid gold chromatographic test paper strip
Agent box, in being detected for fast and convenient aftosa, applicant of the present invention utilizes the aftosa monoclonal antibody of independent development
Develop can with quick diagnosis aftosa group specificity colloid gold chromatographic test paper strip kit, the popularization and application of achievement of the present invention,
The need for Site Detection being met, reaching significantly reduces testing cost, greatly shortens round of visits, improves port customs clearance speed
With the purpose of Disease monitor efficiency, for the monitoring of China's aftosa, early warning and control provide strong technical support.
I.e. the first object of the present invention is to provide a kind of foot and mouth disease virus colloidal gold strip, including backing, nitric acid are fine
The plain film of dimension, adsorptive pads, gold standard pad, sample pad, the backer board are located at bottommost, and nitrocellulose filter is arranged in liner plate top
Centre, nitrocellulose filter upper right end posts adsorptive pads, and nitrocellulose filter upper left end is provided with gold standard pad, and gold standard pad top is right
End is provided with sample pad, wherein right-hand member is sprayed with rabbit-anti rat immune globulin as quality control band, nitric acid on described nitrocellulose filter
Left end is sprayed with foot and mouth disease virus polyclonal antibody as detection band on cellulose membrane, and the mouth hoof of colloid gold label is sprayed with gold standard pad
Epidemic disease viral monoclonal antibodies.
Preferably, in foot and mouth disease virus colloidal gold strip of the present invention, have between the gold standard pad and sample pad
Overlapping, the adsorptive pads and gold standard pad have overlapping with the joining place of nitrocellulose filter；It is highly preferred that the mark pad and sample pad
Between it is overlapping, be 0.3cm with the adsorptive pads and gold standard pad and the overlapping for joining place of nitrocellulose filter.
Preferably, in foot and mouth disease virus colloidal gold strip of the present invention, the aftosa list of the colloid gold label
Clonal antibody is the O-shaped group specificity monoclonal antibody of aftosa.
Preferably, in foot and mouth disease virus colloidal gold strip of the present invention, the foot and mouth disease virus of the detection band resists
Body is foot and mouth disease virus polyclonal antibody.
Preparation method another object of the present invention is to provide above-mentioned foot and mouth disease virus colloidal gold strip, including it is following
Step：
1) collaurum is prepared：Collaurum is prepared using trisodium citrate reduction method；
2) the foot and mouth disease virus monoclonal antibody of colloid gold label is prepared：Regulating step 1) collaurum pH value, by mouth hoof
Epidemic disease poison monoclonal antibody solution adds colloidal gold solution, and with the free collaurum of bovine serum albumin(BSA) saturation, centrifugation obtains final product colloid
The foot and mouth disease virus monoclonal antibody of gold mark；
3) assembling foot and mouth disease virus colloidal gold strip：The nitrocellulose filter containing detection band and quality control band is prepared, and
Prepare colloidal gold pad, respectively by backer board, sample pad, colloidal gold pad, including detection band, quality control band nitrocellulose filter and
Adsorptive pads into strips, that is, are made different shaped foot and mouth disease virus diagnosis test paper by assembled in sequence from the bottom to top, shearing.
Preferably, in the preparation method of foot and mouth disease virus colloidal gold strip of the present invention, the step 2) prepare
The method of the foot and mouth disease virus monoclonal antibody of colloid gold label is：Use 0.2mol/L K2CO3Or 0.1mol/L HC1 regulation steps
PH8.4~8.7 of the rapid colloidal gold solution for 1) obtaining；
Under magnetic stirring, will be during foot and mouth disease virus monoclonal antibody solution add 50mL colloidal gold solutions, should be by when adding monoclonal antibody
It is added dropwise to, the monoclonal antibody protein about 5min of lmg is added.Minimum stable quantity (20 μ g/ml) basis Jia 10% again~20%
The actual amount of monoclonal antibody as to be marked；
Under magnetic stirring apparatus, the glue for adding the bovine serum albumin(BSA) (BSA) of final concentration of 1% (w/v) to dissociate with saturation
Body gold, continues to stir 30min；
First by gold labeling antibody 1500r/min low-speed centrifugal 1h, precipitation is discarded；Will be centrifuged obtain supernatant again with
15000r/min is centrifuged 1h, supernatant discarded for the second time；The precipitation that second centrifugation is obtained is with the 0.02mol/L TBS of original volume
After pH8.2 (including 1%BSA, 0.05% Sodium azide) dissolvings, repeated centrifugation 2-3 times, precipitation is dissolved in the 1/10TBS of original volume,
Obtain the foot and mouth disease virus monoclonal antibody of colloid gold label.
Preferably, in the preparation method of foot and mouth disease virus colloidal gold strip of the present invention, in the step 3) in
The method of the nitrocellulose filter (2) containing detection band (7) and quality control band (6) is：With concentration for (1.5-2.5mg/mL) is purified
FMDV more resist and rabbit-anti rat immune globulin (0.8-1.2mg/mL) respectively with draw film instrument draw on nitrocellulose membrane.Make respectively
It is detection line and control line, point sample amount is 0.75 μ L/cm, and 37 DEG C dry 2h, and 4 DEG C of sealing preserves are standby.
Preferably, in the preparation method of foot and mouth disease virus colloidal gold strip of the present invention, in the step 3) in
Prepare colloidal gold pad method be using step 2) obtain colloid gold label foot and mouth disease virus monoclonal antibody, with draw film spray
Jin Yi is sprayed on glass fibre element film, and spray sample amount is 1.5-2.0 μ L/cm, takes out and is drained in 4 DEG C of vacuum, adds drier or change
Color silica gel, sealing preserve is in 4 DEG C of refrigerators.
Preferably, in the preparation method of foot and mouth disease virus colloidal gold strip of the present invention, described collaurum is molten
The preparation of liquid：2g BSA are taken, is dissolved in 45mL deionized waters；1g PVP are taken, 20mL deionized waters are dissolved in；Take 1g PEG molten
Solution is in 20mL deionized waters；2g skimmed milk powers are taken, 45mL deionized waters are dissolved in；The casein solutions of 8mL 5%, after dissolving,
12500 ± 200r/min is centrifuged 30min, takes supernatant standby.0.8L deionized waters are taken, 6.4g citric acids, 6.06g is added
Trizma base, 2.8g NaOH, are sufficiently mixed, and adjust pH to 8.1 ± 0.05；0.2g NaN3,3g Tween-20 are added,
25g sucrose, 6.25g trehaloses.All of aforesaid liquid is added in last solution and is sufficiently mixed.Ion is boiled off with three
Water filling-in is to 1L.2-8 DEG C saves backup.
A further object of the present invention is to provide foot and mouth disease virus colloidal gold strip cause of disease quick detection kit, described
Kit includes above-mentioned foot and mouth disease virus colloidal gold strip.
ELISA test strip method of the invention is：
As shown in Fig. 2 when the antigen in sample reacts with gold mark FMDV monoclonal antibodies first, forming compound.Work as sample
During swimming upward by capillarity along test strips, if contain foot-and-mouth disease virus antigen in test sample, antigen is first
" the gold medal mark FMDV monoclonal antibodies of foot and mouth disease virus one " compound is combined to form with gold mark monoclonal antibody, the compound continues to flow, and fixed
The many anti-bindings of FMDV in detection line form " resisting the gold medal mark FMDV monoclonal antibodies of a foot and mouth disease virus one FMDV " compound, gold grain more
It is enriched with detection line and is presented red；The gold mark FMDV monoclonal antibodies being not bound with continue flow forward, and are fixed on negative control
Rabbit-anti Balb/c mouse immuning ball proteins on line form " the anti-FMDV monoclonal antibodies of the gold medal mark of rabbit-anti Balb/c mouse immuning ball proteins one "
Compound, gold grain is enriched with negative control line and red (control line) is presented, as shown in Figure 2.Appear in detection line simultaneously
It is positive reaction with control line, only red stripes occurs for negative reaction in control line.There are not red stripes in control line,
Show that operating process is incorrect or test strips fail.
From the foregoing, it will be observed that foot and mouth disease virus colloidal gold strip of the invention, foot and mouth disease virus colloidal gold strip cause of disease are fast
Fast detection kit and preparation method thereof, at least has the advantage that：
1) present invention have developed FMDV group specificity colloidal gold strips, optimize detection method colloid gold particle, nitric acid
Cellulose specification and adsorptive pads specification etc..
2) the FMDV group specificity colloidal gold strip high specificities that the present invention is developed, it is not anti-with other virus of domestic animals
Former cross reaction.
Brief description of the drawings
Colloidal gold strip structural representation in Fig. 1 one embodiment of the present of invention；
Fig. 2 foot and mouth disease virus colloidal gold strip reaction principle schematic diagrames of the invention；
The electron microscope of colloid gold particle in Fig. 3 one embodiment of the present of invention；
FMDV group specificities test strips specific test figure in Fig. 4 one embodiment of the present of invention.
Specific embodiment
Further technical scheme is illustrated below by way of specific embodiment, it should be understood that be only below this hair
Bright exemplary illustration, is not intended to limit the invention scope of the claims.
3 plants of O-shaped FMDV cell toxicants are purchased from Lanzhou veterinary institute, O-shaped aftosa immunizing antigen and detection in the present embodiment
Antigen and O, A, C, Asia1 type FMDV, swine vesicular disease virus, the inactivation of viruses antigen of CSFV are studied purchased from Lanzhou animal doctor
Institute.
Serum：O, A, C, AsiaI serotype FMD positive serums, negative serum, swine fever positive serum, SVD are positive
Serum is purchased from Lanzhou veterinary institute, and cavy FMD infection positive serums and negative serum are purchased by Ministry of Agriculture's Disease Diagnosis of Veterinary center
, the pig serum to be checked of FMD difference immune times is bought by Guangdong Wen Shi group companies (picking up from multiple pig farms), the anti-globefish of goat
The mouse IgG-HRP and anti-pig IgG-HRP of goat is purchased from Sigma companies, and rabbit-anti Balb/c mouse immuning ball proteins are produced for Sigma companies
Product.
3 plants of aftosa group specificity monoclonal antibodies, the specificity of the resisting O-type foot and mouth disease virus with potency higher is single
Clonal antibody cell line 1D4,2E11,4C12, are bought by Lanzhou veterinary institute.
The preparation of the foot and mouth disease virus colloidal gold strip of embodiment 1
1st, the preparation of collaurum：Collaurum is prepared using trisodium citrate reduction method
0.01% gold chloride l00mL is placed in conical flask and is heated in micro-wave oven, until boiling, about 3min.
Accurate to draw 1.0mL, 1% trisodium citrate of 1.5mL, 2.0mL, 2.5mL, 3mL, 4mL is rapidly added cone respectively
In shape bottle, rock it is uniform after, be immediately placed in micro-wave oven and continue to boil 15min, flaxen gold chloride water now can be observed
Solution grays quickly after sodium citrate addition, continuous and change into black, is then gradually stable into red, and overall process about 2~
3min.Until after golden yellow gold chloride becomes red, stopping reaction, the amount and gold grain diameter of trisodium citrate are added
Relation, and the fundamental characteristics of gold grain see the table below 1.
The usage amount and the relation of gold grain diameter of trisodium citrate in the preparation of the collaurum of table 1
* 100mL 0.01%HauC14 aequums are reduced
The collaurum suspension of preparation is recovered to add 0.02% NaN to original volume after being cooled to room temperature with ultra-pure water3, warp
Primary sterilization filter is filtered, and 4 DEG C of Refrigerator stores are standby in being fitted into clean Brown Glass Brown glass bottles and jars only.It is the standby colloid gold particle of system of identification
Quality, the diameter and the uniformity of electron microscopic observation collaurum can be used, select the colloid gold particle of suitable diameter.
Colloidal gold solution outward appearance is in claret, bright in colour, sparkling and crystal-clear bright, has light belt in face of daylight is visible；Ultraviolet spectrometry
Photometer measures the ODmax of colloidal gold solution at 525nm；Electric Microscopic observation, it is seen that colloid gold particle is in the same size, distribution is equal
It is even, it is specifically shown in Fig. 3.It can be seen that a diameter of 41nm of the collaurum of preparation.
2nd, the preparation of the foot and mouth disease virus monoclonal antibody of colloid gold label：
The purifying of 2.1 foot and mouth disease virus monoclonal antibodies
The FMDV group specificities monoclonal antibody (3 plants of type group specificity monoclonal antibodies of aftosa 0 of cell line secretion) that will be purified
It is placed in bag filter and is dialysed in distilled water, dialyzate several times is changed in centre, dialyses three days.Take out 50 μ L dialysed it is anti-
Body, adds 1mL 0.5M BaCl2Check whether that dialysis is thorough.The ascites that 3-4mL dialysed is taken, Nai Shi reagents 1-2 is added
Drop, to without color change.Before mark supernatant is taken with 4 DEG C of centrifugation 60min of 5000r/min standby to remove polymer.Use core
Acid albumin instrument determines concentration, according to formula：Mg/mL=(1.450D260-0.740D280) × extension rate, is calculated after purification
AC.
Purified FMDV monoclonal antibodies are diluted, with absorbance of the protein nucleic acid analyzer at 280nm and 260nm, according to
Formula:((1.450D260-0.740D280) × extension rate, calculate monoclonal antibody protein content is mg/mL=：C1=1.00mg/
ML (the first pipe), C2=1.55mg/mL (the second pipe).
The determination of 2.2 collaurums and antibody usage ratio to be marked
During colloid gold label albumen, first have to determine optimum protein labelled amount.It is general to use fixed colloidal gold solution volume
(1mL), the method for changing enzyme protein dosage (volume) is carried out.After nucleic acid-protein instrument determines AC, by antibody stepwise dilution
(by 10 μ g/mL~40 μ g/mL, separately set control tube) afterwards, respectively take 0.lmL (protein solution of 1/10 volume) and be sequentially added into one
In centrifuge tube of the series equipped with lmL collaurums, concussion is mixed, and room temperature places 30min, and 0.1mL is separately added into above-mentioned each pipe
10% (w/v) sodium chloride solution.Carried out according to the order in table 2, after sodium chloride is added, mix standing more than 2h observation knots
Really.Not plus antibody and add the amount of antibody to be not enough to each pipe of stable colloid gold, coagulation phenomenon from red to blue can be presented, and add
Enter amount of antibody and meet or exceed each pipe of minimum stable quantity still to keep red constant.
The colloid gold label FMDV monoclonal antibody quantitative tests of table 2
Note：1st pipe is the control tube for not adding monoclonal antibody, and the 9th pipe is the control tube for not adding NaCl, corresponding to add
The tri-distilled water of 0.1mL.
By the result that table 2 is determined：Colloidal gold solution is heavy poly- quickly after l pipes add the sodium chloride of 0.1mL 10%, from the 2nd pipe
With the 3rd pipe because amount of antibody is not enough to stable colloid gold, colloidal gold solution gradually sinks and gathers, and the color of test tube turns blue or thin out, from
The color that 4th pipe starts collaurum is basically identical with the color of control tube, so this experiment determines colloid gold label FMDV monoclonal antibodies
Minimum flow be 20 μ g.
The optimal pH of 2.3 colloidal gold labeled monoclonal antibodies
The mark of collaurum and albumen, is completed under certain acid-base condition, because the difference of its acid-base value, can be led
The dissociation of albumen and collaurum is caused, so optimal pH must be selected to mark.According to documents and materials, conventional some protein mark
Clock, the pH value used by collaurum is as shown in table 3 below.
Corresponding relation between the collaurum pH of table 3 and protein to be marked
Use 0.2mol/L K2CO3Or 0.1mol/L HC1 regulating steps 1) obtain colloidal gold solution pH it is a little higher than most suitable
0.2~the 0.5pH of PI (pH8.2), i.e. pH8.4~8.7.
Under magnetic stirring, will be during foot and mouth disease virus monoclonal antibody solution add 50mL colloidal gold solutions, should be by when adding monoclonal antibody
It is added dropwise to, the monoclonal antibody protein about 5min of lmg is added.Minimum stable quantity (20 μ g/ml) basis Jia 10% again~20%
The actual amount of monoclonal antibody as to be marked；
Under magnetic stirring apparatus, the glue for adding the bovine serum albumin(BSA) (BSA) of final concentration of 1% (w/v) to dissociate with saturation
Body gold, continues to stir 30min.
First by gold labeling antibody 1500r/min low-speed centrifugal 1h, precipitation is discarded；Supernatant is centrifuged with 15000r/min again
1h, supernatant discarded, to remove the protein being not associated with supernatant.
Dissolved precipitating with the 0.02mol/L TBS pH8.2 (including 1%BSA, 0.05% Sodium azide) of original volume, repeated
Centrifugation 2-3 times, precipitation is dissolved in the 1/10TBS of original volume.4 DEG C save backup.
3rd, assembling foot and mouth disease virus colloidal gold strip
1) preparation of gold mark film
After prepared by gold mark monoclonal antibody, it is sprayed on glass fibre element film with film gold spraying instrument is drawn, spray sample amount is respectively 1.0,1.5,
2.0,2.5,3.0 μ L/cm, it is 1.5-2.0 μ L/cm to obtain most preferably spray sample amount, takes out and is drained in 4 DEG C of vacuum, add drier or
Discoloration silica gel, sealing preserve is in 4 DEG C of refrigerators.
2) nitrocellulose filter containing detection band and quality control band is prepared
The FMDV polyclonal antibodies purified with various concentrations (with the purification process of the monoclonal antibody of embodiment 1 distinguish only by purification process
It is by three kinds of monoclonal antibody equal proportions while using) (1.0,1.5,2.0,2.5,3.0mg/mL) and rabbit-anti rat immune globulin
(0.75,1.0,1.5,2.0,2.5mg/mL) respectively as detection line and control line reagent.By two kinds of reagents respectively with a stroke film instrument
Draw on nitrocellulose membrane, most preferably drawn film concentration, the how anti-concentration of FMDV of purifying is (1.5-2.5mg/mL), and rabbit-anti is exempted from by mouse
Epidemic disease globulin concentration is (0.8-1.2mg/mL), and it is 0.75 μ L/cm to draw film amount.37 DEG C dry 2h, and 4 DEG C of sealing preserves are standby.
3) assembling of test strips
As shown in Figure 1, by the smooth center in backing of NC films.It is noted that stretching, extension is smooth, can not there are space or bubble after patch.
The smooth lower section in NC film T lines of glue gold pad, overlaps NC film 0.3cm, and sample pad is smooth in glue gold pad again, overlaps glue gold pad one
Point.Blotting paper is lain against one end of NC film C lines, NC films 0.3cm is overlapped.And in CM4000 cutting machines, it is cut into 3mm wide
Test strips, it is standby in 4 DEG C of kept dries.
FMDV group specificity colloidal gold strip testing result decision methods are：
The FMDV group specificity colloidal gold strip testing results set up are judged to：
It is positive：In peep hole, detection line area (T) and control line area (C) is while there is purplish red colo(u)r streak；
It is negative：In peep hole, only there is a purplish red colo(u)r streak in control line area (C)；
Failure：In peep hole, control line area (C) and detection line area (T) all occurs without colo(u)r streak；Or only detection line area (T)
There is colo(u)r streak.
The foot and mouth disease virus ELISA test strip experimental technique of embodiment 2
1 specific test
With test strips to O, A, Asia1 type FMDV, swine vesicular disease virus (VSV), CSFV inactivation of viruses antigen
(CSPV), distilled water and normal cell culture detected respectively, observes result.
As a result test strips are only reacted with various FMDV, as shown in figure 4, illustrating that the test strips have specificity well.Its
In, test strips are from left to right followed successively by Fig. 4：
1 O-shaped FMDV, 2Asia1 type FMDV, 3VSV, 4 normal cell cultures, 5CSPV, 6BTV, 7WNV, 8 normal pig bloods
Clearly, 9 distilled water.
2 sensitivity tests
Standard antigen is done into doubling dilution, respectively by test strips insert different diluent in, with reverse indirect hemagglutination assay
Compare the amount for determining and detecting virus.Nucleic acid-protein instrument bioassay standard antigen protein content is used again, determines minimum with test strips
Detected level.
5 times of the FMDV antigens (protein content is 0.84mg/mL after measured) to purifying make continuous doubling dilution after diluting, and use
Test strips test its limit of identification.Test result shows that FMDV antigens can still be detected when diluting for 2560 times, it is seen that test strips
Limit of identification to purifying FMDV antigens is 0.31 μ g/mL.It is approximately equivalent to 100-200ECID50's in virus neutralization tests
Viral level, sensitivity is very high, and substantially more simple and easy to do than other methods.
The sensitivity tests of the FMDV group specificity colloidal gold strips of table 4
Note：+++ represent and show most deep, strong positive；+ it is the positive ,-it is feminine gender.
3 replica tests
The random test strips for taking out different batches, 5 test strips of each batch picking are examined respectively with 1 after 3,6 months
Survey, to do repeated between criticizing and test.With 5 parts of works, 5 repetitions of O, Asia1 type FMDV inactivation of viruses antigen, result is observed.
Test paper between the different batches of random picking is carried out to repeat experiment, 5 test strips of each batch picking are right respectively
Detected with portion standard positive sample, as a result found that the testing result of test strips in each batch is completely the same, and it is different
Testing result between batch is also completely the same, so as to the detection repeatability for showing the method is good.
4 shelf-lifves tested
After taking 40 times of standard antigen dilution, with 4 DEG C of test strips of preservation different time (1,2,3,4,5,6,7,8,9 months)
Each 40 (30 examination criteria antigens, 10 detection normal cell nutrient solutions) carry out qualitative detection.
Result finds that preserved in 7 months at 4 DEG C, ELISA test strip result does not have any change, is preserved 8 months used in 4 DEG C
During with the ELISA test strip of 9 months, respectively there are 2 parts for false positive；The test strips difference of 7,8 and 9 months is preserved at ambient temperature
During detection, respectively there are 2 parts, 2 parts and 3 parts for false positive；Different temperatures preserves 10 parts of negative samples of ELISA test strip of different time
Feminine gender is, detailed results are as shown in table 3.Experiment shows that the test strips can preserve 7 months and preserve at room temperature half at 4 DEG C
Year, Sensitivity and Specificity is unaffected.
The test strips shelf-life of table 5 tests
Note：N/m, represents positive quantity/negative sample quantity.
5 clinical samples are detected
50 parts of doubtful samples of clinic are detected using the FMDV group specificities colloidal gold strip developed, and and Lanzhou
The reverse indirect hemagglutination detection kit contrast that veterinary institute is provided, all of testing result is consistent.
The repeatability of the test strips that the embodiment of the present invention is obtained is preferable, and stability is also fine, can just be protected in room temperature
Half a year is deposited, its clinical practicality is increased, it is not necessary to abnormal test conditions, it is as a result objective, visually it is easy to judge.Institute is in this way
It is a up-and-coming technology, is particularly suitable in vast grass-roots unit's promotion and application.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of foot and mouth disease virus colloidal gold strip, including backing, nitrocellulose filter, adsorptive pads, gold standard pad, sample pad,
The backer board is located at bottommost, and nitrocellulose filter is arranged at liner plate center upper portion, and nitrocellulose filter upper right end is posted
Adsorptive pads, nitrocellulose filter upper left end is provided with gold standard pad, and the upper right end of gold standard pad is provided with sample pad, it is characterised in that
Right-hand member is provided with rabbit-anti rat immune globulin as quality control band on described nitrocellulose filter, and left end is provided with nitrocellulose filter
Foot and mouth disease virus polyclonal antibody is sprayed with the foot and mouth disease virus monoclonal antibody of colloid gold label as detection band in gold standard pad.
2. foot and mouth disease virus colloidal gold strip according to claim 1, it is characterised in that the gold standard pad and sample pad
Between have overlapping, the adsorptive pads and gold standard pad have overlapping with the joining place of nitrocellulose filter.
3. foot and mouth disease virus colloidal gold strip according to claim 1, it is characterised in that the mouth of the colloid gold label
Fever aphthous monoclonal antibody is the O-shaped group specificity monoclonal antibody of aftosa.
4. foot and mouth disease virus colloidal gold strip according to claim 1, it is characterised in that the aftosa of the detection band
Antiviral antibody is polyclonal antibody.
5. a kind of preparation method of foot and mouth disease virus colloidal gold strip as described in Claims 1 to 4 any one, its feature
It is to comprise the following steps：
1) collaurum is prepared：Collaurum is prepared using trisodium citrate reduction method；
2) the foot and mouth disease virus monoclonal antibody of colloid gold label is prepared：Regulating step 1) collaurum pH value, by hoof-and-mouth disease
Malicious monoclonal antibody solution adds colloidal gold solution, and with the free collaurum of bovine serum albumin(BSA) saturation, centrifugation obtains final product collaurum mark
The foot and mouth disease virus monoclonal antibody of note；
3) assembling foot and mouth disease virus colloidal gold strip：The nitrocellulose filter containing detection band and quality control band is prepared, and is prepared
Colloidal gold pad, respectively by backer board, sample pad, colloidal gold pad, including detection band, quality control band nitrocellulose filter and water suction
Pad into strips, that is, is made different shaped foot and mouth disease virus diagnosis test paper by assembled in sequence from the bottom to top, shearing.
6. method according to claim 5, it is characterised in that the step 2) prepare the foot and mouth disease virus of colloid gold label
The method of monoclonal antibody is：Use 0.2mol/L K2CO3Or 0.1mol/L HC1 regulating steps 1) colloidal gold solution that obtains
PH is 8.4~8.7；
Under magnetic stirring, by foot and mouth disease virus monoclonal antibody solution addition 50mL colloidal gold solutions, should dropwise add when adding monoclonal antibody
Enter, the monoclonal antibody protein about 5min of 1mg is added.Jia 10% again on the basis of minimum stable quantity (20 μ g/ml)~20% it is
The actual amount of monoclonal antibody to be marked；
Under magnetic stirring apparatus, the collaurum for adding the bovine serum albumin(BSA) (BSA) of final concentration of 1% (w/v) to dissociate with saturation,
Continue to stir 30min；
First by gold labeling antibody 1500r/min low-speed centrifugal 1h, precipitation is discarded；The supernatant for obtaining will be centrifuged again with 15000r/min
Second centrifugation 1h, supernatant discarded；The precipitation that second centrifugation is obtained is (interior with the 0.02mol/L TBS pH8.2 of original volume
Containing 1%BSA, 0.05% Sodium azide) after dissolving, repeated centrifugation 2-3 time is precipitated and is dissolved in the 1/10TBS of original volume, that is, obtain glue
The foot and mouth disease virus monoclonal antibody of body gold mark.
7. method according to claim 5, it is characterised in that in the step 3) in contain detection band (7) and quality control band
(6) method of nitrocellulose filter (2) is：With concentration be (1.5-2.5mg/mL) purify FMDV more resist and rabbit-anti mouse be immunized
Globulin (0.8-1.2mg/mL) is drawn on nitrocellulose membrane with stroke film instrument respectively.Respectively as detection line and control line, point sample
It is 0.75 μ L/cm to measure, and 37 DEG C dry 2h, and 4 DEG C of sealing preserves are standby.
8. method according to claim 5, it is characterised in that in the step 3) in prepare the method for colloidal gold pad to make
With step 2) obtain colloid gold label foot and mouth disease virus monoclonal antibody, with draw a film gold spraying instrument be sprayed on glass fibre element film
On, spray sample amount is 1.5-2.0 μ L/cm, is drained in 4 DEG C of vacuum, adds drier or discoloration silica gel, and sealing preserve is in 4 DEG C of refrigerators
In.
9. method according to claim 5, it is characterised in that the preparation of described colloidal gold solution：2g BSA are taken, is dissolved
In 45mL deionized waters；1g PVP are taken, 20mL deionized waters are dissolved in；Take 1g PEG and be dissolved in 20mL deionized waters；Take 2g
Skimmed milk power, is dissolved in 45mL deionized waters；The casein solutions of 8mL 5%, after dissolving, 12500 ± 200r/min centrifugations
30min, takes supernatant standby；0.8L deionized waters are taken, adds 6.4g citric acids, 6.06g trizma base, 2.8g NaOH to fill
Divide mixing, adjust pH to 8.1 ± 0.05；Add 0.2g NaN3,3g Tween-20,25g sucrose, 6.25g trehaloses；Will be all
Aforesaid liquid be added to last solution in be sufficiently mixed；Ionized water filling-in to 1L is boiled off with three.
10. a kind of foot and mouth disease virus colloidal gold strip cause of disease quick detection kit, it is characterised in that the kit is included
Foot and mouth disease virus colloidal gold strip as described in Claims 1 to 4 any one.
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|---|---|---|---|
| CN201611075628.3A CN106771121A (en) | 2016-11-30 | 2016-11-30 | A kind of foot and mouth disease virus colloidal gold strip, cause of disease quick detection kit and preparation method thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108593919A (en) * | 2018-03-13 | 2018-09-28 | 深圳市第二人民医院 | A kind of colloidal gold immune chromatography test and its preparation method and application |
| CN110297088A (en) * | 2019-07-17 | 2019-10-01 | 中国农业科学院兰州兽医研究所 | Foot and mouth disease virus quantitative testing test paper card and preparation method thereof |
| CN110907638A (en) * | 2020-02-14 | 2020-03-24 | 北京纳百生物科技有限公司 | Antibody dispersant for immune colloidal gold homogeneous phase labeling and application thereof |
-
2016
- 2016-11-30 CN CN201611075628.3A patent/CN106771121A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108593919A (en) * | 2018-03-13 | 2018-09-28 | 深圳市第二人民医院 | A kind of colloidal gold immune chromatography test and its preparation method and application |
| CN110297088A (en) * | 2019-07-17 | 2019-10-01 | 中国农业科学院兰州兽医研究所 | Foot and mouth disease virus quantitative testing test paper card and preparation method thereof |
| CN110907638A (en) * | 2020-02-14 | 2020-03-24 | 北京纳百生物科技有限公司 | Antibody dispersant for immune colloidal gold homogeneous phase labeling and application thereof |
| CN110907638B (en) * | 2020-02-14 | 2020-09-22 | 北京纳百生物科技有限公司 | Antibody dispersant for immune colloidal gold homogeneous phase labeling and application thereof |
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