CN109444410A - A kind of veterinary rabies poison neutralizing antibody chemiluminescence detection kit - Google Patents
A kind of veterinary rabies poison neutralizing antibody chemiluminescence detection kit Download PDFInfo
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- CN109444410A CN109444410A CN201811254348.8A CN201811254348A CN109444410A CN 109444410 A CN109444410 A CN 109444410A CN 201811254348 A CN201811254348 A CN 201811254348A CN 109444410 A CN109444410 A CN 109444410A
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
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Abstract
The invention discloses a kind of veterinary rabies poison neutralizing antibody chemiluminescence detection kit, which includes the capillary for being coated with rabies virus antigen, enzyme conjugates, luminous substrate and cleaning solution.Detection method is high-efficient, sensitivity is high, high specificity, high degree of automation, it is easy to operate quickly and detection range is wide, pollution-free, the holding time is long, can effectively improve the deficiency of product on the market at present.
Description
Technical field
The invention belongs to technical field of immunoassay more particularly to a kind of veterinary rabies poison neutralizing antibody chemiluminescence to examine
Test agent box.
Background technique
Rabies are the zoonosis that a kind of infectiousness is strong, harmfulness is big, case fatality rate is high as caused by rabies viruses, mesh
Before there is no and can cure rabic drug.China is rabic high-incidence country, is aimed at prevention for rabic measure.
The animals such as dog, cat are the major storage hosts of rabies viruses and propagate host, therefore, to dog, the cat etc. in rubies epidemiology area
Animal carries out compulsory immunization inoculation antibody titer monitoring after immune and combines, and is the important link of prevention and control measure.In animal body
The height of rabies viruses neutralizing antibody is the important indicator for measuring immune effect of vaccine.
Currently, the method for rabies virus antibodies detection that WHO and OIE recommends has rapid fluorescence stove to inhibit test
(Rapid fluorescent focus inhibition test, RFFIT), fluorescence antibody virus neutralization tests
(Fluorescence antibody virus neutralization test, FAVNT).In recent years, it successively reports both at home and abroad
A variety of rabies virus antibodies detection techniques, such as flow-cytometry method, indirect ELISA technology, competitive ELISA technology, sandwich
Elisa technique, colloidal gold chromatographic diagnosis test paper technology etc..
China Patent Publication No. is that the patent application of CN101936998B discloses a kind of dog anti-rabies virus IgG antibody
ELISA detection kit, China Patent Publication No. are that the patent of CN1547027A discloses a kind of indirect Enzyme immunoassay examination
Test detection rabies poison IgG kit and preparation method.The above patent is ELISA detection method, and with duration, operation is automatic
Change degree is low.
China Patent Publication No. is that the patent application of CN1326101A discloses a kind of colloidal gold immunochromatographimethod rabies disease
Malicious antibody test reagent strip and preparation method, China Patent Publication No. disclose a kind of mad for the patent application of CN104360061A
The immune-gold labeled Test paper of dog disease virus IgG antibody and preparation method.The above patent is colloidal gold detection method, is qualitative
Detection, sensitivity and specificity are lower.
Though RFFIT and FAVNT two methods energy Accurate Determining rabies viruses neutralizing antibody is horizontal, it is both needed to using living cells
It is measured with live virus, is both needed to expensive experiment equipment, and detection time is long, therefore be not suitable for large-scale serum inspection
It surveys.The methods of elisa technique, colloidal gold cannot take into account that high-efficient, sensitivity is high, high specificity and the degree of automation height etc. are excellent
Point, and applied sample amount is larger, needs serum sample amount big.
Summary of the invention
To solve above-mentioned the deficiencies in the prior art, a kind of veterinary rabies poison neutralizing antibody chemiluminescence detection side is provided
Method.This detection method is high-efficient, sensitivity is high, high specificity, high degree of automation, it is easy to operate quickly and detection range it is wide,
It is pollution-free, the holding time is long, can effectively improve the deficiency of product on the market at present.The present invention is applied to detection animal blood serum or blood
The concentration of rabies viruses neutralizing antibody in slurry.
In order to reach above-mentioned technical purpose, the technical scheme adopted by the invention is that:
A kind of veterinary rabies poison neutralizing antibody chemiluminescence detection kit, it is characterised in that: the kit includes coating
Capillary, enzyme conjugates, luminous substrate and the cleaning solution of rabies virus antigen.
The rabies virus antigen is the rabies viruses G egg that active group itself is obtained with active group or by modification
White recombinant antigen, inactivation rabies totivirus or Virus-like particles for pseudorabies virus for same.
The active group is amino, carboxyl, sulfydryl or hydroxyl.
The capillary is high-precision capillary tube, and outer diameter is 1.18 ± 0.02mm, and internal diameter is 0.7 ± 0.005m, length
For 30 ± 1mm；Material is high 3.3 glass of borosilicate.
On the capillary with amino, carboxyl, hydroxyl, sulfydryl, aldehyde radical, epoxy group, cyanic acid ester group, isocyanate group,
One in cyano, isocyano group, maleic amide amino, N-hydroxy-succinamide ester base, phenolic hydroxyl group or phenolic hydroxyl group deriveding group
Kind.
The capillary for being coated with rabies virus antigen is made by the following method: this method includes modification capillary step
Suddenly, rabies virus G protein recombinant antigen, inactivation rabies totivirus or Virus-like particles for pseudorabies virus for same step, coating step are modified
Rapid and closing step；
The modification capillary step specifically:
Capillary is modified using dressing agent, so that having amino, carboxyl, hydroxyl, sulfydryl, aldehyde radical, epoxy group, cyanic acid on capillary
Ester group, isocyanate group, cyano, isocyano group, maleic amide amino, N-hydroxy-succinamide ester base, phenolic hydroxyl group or phenol hydroxyl
One of base deriveding group；
The modification rabies virus G protein recombinant antigen, inactivation rabies totivirus or Virus-like particles for pseudorabies virus for same step tool
Body are as follows:
Using dressing agent modification rabies virus G protein recombinant antigen, rabies totivirus or Virus-like particles for pseudorabies virus for same are inactivated,
So that rabies virus G protein recombinant antigen, inactivation rabies totivirus or Virus-like particles for pseudorabies virus for same connect amino, carboxyl,
Sulfydryl or hydroxyl；
The coating step specifically:
Activation: there are three types of the modes of activation: the first: the capillary after being modified with coupling agent activation, second: living with coupling agent
Rabies virus G protein recombinant antigen, inactivation rabies totivirus or Virus-like particles for pseudorabies virus for same after changing modification, the third,
Coupling agent is added in coating buffer；
Prepare coating buffer: when activation uses first way, preparation coating buffer specifically: by the rabies virus G protein after modification
Recombinant antigen, inactivation rabies totivirus or Virus-like particles for pseudorabies virus for same dilute most 5~30 μ g/mL, and coating buffer I is made；
When activation uses the second way, preparation coating buffer specifically: the rabies virus G protein recombinant antigen after activation, inactivation is mad
Dog disease totivirus or Virus-like particles for pseudorabies virus for same dilute most 5~30 μ g/mL, and coating buffer II is made；When activation uses third
Kind mode, prepares coating buffer specifically: coupling agent and buffer are mixed and made into activation buffer, it then will with activation buffer
Rabies virus G protein recombinant antigen after modification, inactivation rabies totivirus or Virus-like particles for pseudorabies virus for same dilution most 5~
30 μ g/mL, are made coating buffer III；
Coupling: when activation uses first way, specific coupling are as follows: the capillary after activation is immersed in coating buffer I, it is even
After the completion of connection, capillary is washed and dried, obtains the capillary for being coated with rabies virus antigen；When activation uses second of side
Formula, it is specific to be coupled are as follows: the capillary after modification is immersed in coating buffer II, after the completion of coupling, washes and dries capillary,
Obtain the capillary for being coated with rabies virus antigen；When activation uses the third mode, specific coupling are as follows: by the hair after modification
Tubule is immersed in coating buffer III, after the completion of coupling, is washed and dried capillary, is obtained the capillary for being coated with rabies virus antigen
Pipe；
The closing step specifically:
With 1~5%(mass percent by volume) BSA or 1~5%(mass percent by volume) skimmed milk power be dissolved in 0.01~
(0.01~0.1M Tris-HCl preparation method are as follows: weigh 1.57~15.76 g Tris-HCl and be dissolved in the Tris-HCl of 0.1M
1 L injection water；), confining liquid is made；Capillary after coating is immersed in confining liquid, closure temperature is 37 DEG C, when closing
Between be 2~4 h, after the completion of closing, wash and dry capillary, the pH of the confining liquid is 7.2~7.8；
It is cleaned when cleaning capillary in coupling step and closing step with cleaning agent, cleaning agent is the M of 0.02 M~0.2 PBS, pH
It is 7.2~7.8, wherein the percent by volume containing 0.01~0.1%() Tween 20；
The capillary for being coated with rabies virus antigen after closing is vacuum-packed under conditions of temperature is 2~8 DEG C and saves.
The enzyme conjugates is the product after rabies virus G protein recombinant antigen and alkaline phosphatase coupling.
The enzyme conjugates is made by the following method: this method include modification rabies virus G protein recombinant antigen step,
Activation step, coupling step, purification step and dilution step；
The modification rabies virus G protein recombinant antigen step specifically: dressing agent rabies virus G protein recombinant antigen is utilized,
So that rabies virus G protein recombinant antigen connects amino, carboxyl, sulfydryl or hydroxyl；
The activation step specifically: utilize coupling agent activated alkaline phosphatase or rabies virus G protein recombinant antigen；
The coupling agent activated alkaline phosphatase or rabies virus G protein recombinant antigen refer to coupling agent activated alkaline phosphoric acid
The amino of enzyme or rabies virus G protein recombinant antigen, carboxyl, sulfydryl or hydroxyl.
The coupling step specifically: by the rabies virus G protein recombinant antigen after modification and the alkaline phosphatase after activation
Enzyme mixing, coupling form enzyme conjugates；Or mix the rabies virus G protein recombinant antigen after activation with alkaline phosphatase,
Coupling forms enzyme conjugates；
The purification step specifically: the enzyme conjugates after coupling is crossed into gel column, is then purified；
The dilution step specifically: with the M of 0.02 M~0.2, Tris-HCl that pH is 7.2~7.8 is by enzyme knot after purification
It closes object and is diluted to the enzyme conjugates that concentration is 0.5~5 μ g/mL.
It further include anti-corrosion step, anti-corrosion step specifically: BSA or casein are added in enzyme conjugates after dilution,
Final enzyme conjugates is made in Tween 20 and preservative, dispenses, and sealing saves under conditions of temperature is 2~8 DEG C；Most
In whole enzyme conjugates, the percent by volume that the quality percent by volume of BSA or casein is 0.1~5%, Tween 20 is
0.01~0.1%, the percent by volume of preservative is 0.01~0.05%.
The coupling agent is preferably the derivative of carbodiimide, glutaraldehyde, SMCC or SMCC.
The luminous substrate are as follows: APS-5.After the substrate is mixed with alkaline phosphatase (ALP), alkaline phosphatase phosphate radical water
Solution, substrate reaction of decomposing immediately releases photon, within the scope of specific alkaline phosphatase concentration, the photon numbers of release
It is directly proportional to the concentration of solution alkaline phosphatase, therefore APS-5 can be used for the quantitative detection of alkaline phosphatase.The spirit of this substrate
Sensitivity is high, shines to sustainable stability and high efficiency.The luminous substrate packing, sealing are kept in dark place at 2~8 DEG C.
The cleaning solution is formulated by the following method: by Tween 20, Qula is logical, NaCl and preservative, is dissolved in
In the Tris-HCl of the M of 0.02 M~0.2, cleaning solution is made, the percent by volume of Tween 20 is 0.01 in the cleaning solution
~0.1%, the logical volume basis of Qula is 0.01~0.1%: the quality percent by volume of NaCl is 0.85~0.9%, preservative
Percent by volume be 0.01~0.05%, the pH of the cleaning solution is 7.2~7.8.
Used dressing agent is preferably SATA, DTT, TCEP, 2-MEA or 2-IT.
Use the specific steps of present invention measurement veterinary rabies poison neutralizing antibody are as follows: the kit for saving 2~8 DEG C is extensive
It answers to room temperature, takes out coated capillary, contact serum or blood plasma, make capillary full of blood by the siphonage of itself
Clear or blood plasma is put into Full-automatic chemiluminescence immunoassay analysis meter detection luminous value, built-in calibration curve is recycled to calculate animal
The concentration of rabies viruses neutralizing antibody in serum or blood plasma.
The present invention has the following beneficial effects with respect to the prior art:
This kit, using automatic chemiluminescence immunoassay platform, realizes full-automatic chemical using capillary as reaction carriers
Shine detection, and detection process does not need manual operation.The capillary of this kit is in the form of chemical bond and rabies virus G protein
Recombinant antigen or inactivation rabies totivirus or Virus-like particles for pseudorabies virus for same coupling, are not dependent on physisorption coating,
Which raises the stability of antigen coat, and the coupling mode of this chemical bond does not destroy the bioactivity of antigen.Due to hair
Tubule has siphonage, therefore is loaded operation more simply, fast, does not need with auxiliary tools such as pipettors.This kit
Enzyme conjugates is stablized, and 2~8 DEG C can long-term preservation.The luminous substrate plateau of this kit, is long and stablizes, and addition can be effectively reduced
The time difference bring error of generation to be detected such as after luminous substrate.Incubation that this kit detection process is related to, cleaning plus
Enzyme, cleaning plus luminous substrate and etc., it is automatically performed by instrument, therefore Repeatability accesses guarantee.
This kit is valid up to 18 months, has been more than most of colloidal-gold detecting-card, ELISA kit on the market
Attainable shelf life, stability are more preferable.It has been coated with capillary and enzyme conjugates performance is stablized, validity period endoperidium is anti-
Former, enzyme conjugates degradation rate is respectively less than 10%.This kit high sensitivity, high specificity.The range of linearity be 0.03125~
8IU is compared with import reagent box, and correlation is 0.95 or more.CV is respectively less than 10% between criticizing in kit batch.This kit and dog
The animal epidemics antibody no cross reaction such as pest, tiny.
Detailed description of the invention
Fig. 1 is 2-IT(Traut ' s Reagent) modification schematic diagram；
Fig. 2 is SMCC activation coupling schematic diagram；
Fig. 3 is carbodiimide coupling schematic diagram；
Fig. 4 is that glutaraldehyde is coupled schematic diagram.
Specific embodiment
This kit includes the capillary for being coated with rabies virus antigen, enzyme conjugates, luminous substrate and cleaning solution.This examination
The preparation method of agent box each component is as follows.
Capillary is coated with rabies virus antigen
Capillary is carrier to the present invention with high precision, is made on capillary using dressing agent with amino, carboxyl, hydroxyl, sulfydryl, aldehyde
Base, epoxy group, cyanic acid ester group, isocyanate group, cyano, isocyano group, maleic amide amino, N-hydroxy-succinamide ester base,
One of phenolic hydroxyl group and its deriveding group recycle coupling agent and to coated albumen coupling.
Rabies virus G protein recombinant antigen or inactivation rabies totivirus or Virus-like particles for pseudorabies virus for same are lived by itself
Property the group or active group, including amino, carboxyl, sulfydryl, hydroxyl etc. that are obtained by modification, using on coupling agent and capillary
Active group coupling, obtain the capillary of envelope antigen.The coupling agent of application includes but is not limited to carbodiimide, and penta 2
Aldehyde, SMCC.Dressing agent includes but is not limited to SATA, DTT, TCEP, 2-MEA, 2-IT etc..
The present invention will be further described with reference to the examples below, and described embodiment is only present invention a part
Embodiment is not whole embodiment.Based on the embodiments of the present invention, those skilled in the art are not making
Other embodiments used obtained, belong to protection scope of the present invention under the premise of creative work.
Embodiment 1
It has been coated with the preparation of the capillary of rabies virus antigen:
Rabies virus G protein recombinant antigen or inactivation rabies totivirus or Virus-like particles for pseudorabies virus for same are modified, sulfydryl is connected:
Rabies virus antigen concentration to be coated with is 2mg/mL, and 2-IT usage amount is 2 times of rabies virus antigen mole to be coated with,
The two is mixed in 0.01M PBS (pH 8), reacts at room temperature 0.5h.It crosses desalting column and removes unreacted 2-IT, and replace buffering
Liquid is 0.01M PBS (pH 6.5).
Capillary is connected into amino by dressing agent, activates the amino on modification postcapillary using SMCC:
SMCC is dissolved in dimethyl sulfoxide or n,N-Dimethylformamide or other organic solvents first, is re-dissolved in 0.01M PBS (pH
For the capillary modified and submergence 7), is added, SMCC concentration is 0.1mg/mL.React at room temperature 15min.With 0.02 M Tris-
HCl (pH 7.2) is terminated, dry.
Capillary after SMCC activation and the rabies virus G protein recombinant antigen that has been modified with sulfydryl or to inactivate rabies complete
Virus or Virus-like particles for pseudorabies virus for same coupling.Rabies virus G protein recombinant antigen or the mad dog of inactivation of sulfydryl are connected after modification
Sick totivirus or Virus-like particles for pseudorabies virus for same are configured to the egg to be coated with that concentration is 5 μ g/mL with 0.02M PBS (pH 7.2)
White solution is added SMCC activation capillary and submerges.React at room temperature 0.5h.It is cleaned with cleaning agent, is dry.The capillary of envelope antigen
Effective 1% (quality percent by volume) BSA is dissolved in 37 DEG C of the confining liquid closings of 0.01M Tris-HCl (pH 7.2), closing
Time is 2 h.It is cleaned after closing with cleaning agent, is dry, 2 DEG C of preservations are vacuum-packed.Used cleaning agent is 0.02 M PBS
(pH 7.2), percent by volume containing 0.01%() Tween 20.
The preparation of enzyme conjugates:
Enzyme conjugates is to be diluted to certain density rabies virus G protein recombinant antigen conjugated alkaline phosphoric acid enzyme product.
Rabies virus G protein recombinant antigen is modified, sulfydryl is connected at histone amino.Rabies virus G protein recombinant antigen
Concentration be 2mg/mL, 2-IT (Traut ' s Reagent) usage amount is 2 times of rabies virus G protein recombinant antigen mole,
The two is mixed in 0.01M PBS (pH 8), reacts at room temperature 0.5h.It crosses desalting column and removes unreacted 2-IT, and replace buffer
For 0.01M PBS (pH 6.5).
Utilize the amino on SMCC or derivatives thereof activated alkaline phosphatase.The concentration of alkaline phosphatase is 3mg/mL,
SMCC usage amount is 5 times of alkaline phosphatase mole, and the two is mixed in 0.01M PBS (pH 7), reacts at room temperature 0.5 h.It is de-
Salt plug removes unreacted SMCC.
Alkaline phosphatase after the rabies virus G protein recombinant antigen of sulfhydrylation is activated with SMCC mixes, and coupling forms enzyme
Conjugate.When alkaline phosphatase after rabies virus G protein recombinant antigen and the SMCC activation of sulfhydrylation is coupled, mole 1:
1, coupling temperature is 2 DEG C, and coupling time is 12 h.Gel column is crossed, coupled product is purified, and replacing buffer is 0.02 M
Tris-HCl (pH 7.2) collects rabies virus G protein recombinant antigen conjugated alkaline phosphoric acid enzyme product, and being diluted to concentration is 0.5
0.1% (quality percent by volume) BSA or casein, 0.01% (percent by volume) Tween is added in the enzyme conjugates of μ g/mL
20, and add 0.01%(percent by volume) preservative, it dispenses, sealing, 2~8 DEG C of preservations.
The preparation of luminous substrate:
The luminous substrate of this kit is 4- chlorobenzene sulfydryl 10- methyl-acridan methylene phosphate disodium salt
(APS-5).After the substrate is mixed with alkaline phosphatase (ALP), alkaline phosphatase phosphate radical hydrolysis, substrate is decomposed instead immediately
Photon should be released, within the scope of specific alkaline phosphatase concentration, photon numbers and the solution alkaline phosphatase of release
Concentration is directly proportional, it is possible to the quantitative detection for alkaline phosphatase.This substrate high sensitivity is sent out to sustainable stability and high efficiency
Light.Packing, sealing, 2~8 DEG C are kept in dark place.
The preparation of cleaning solution:
Cleaning solution is prepared: 0.01% (percent by volume) Tween 20,0.01%(percent by volume) Qula is logical, 0.85%(mass
Percent by volume) NaCl, 0.01%(percent by volume) preservative, it is dissolved in 0.02 M Tris-HCl (pH 7.2).Packing, it is close
Envelope, 2~8 DEG C of preservations.
It is semi-finished product after the packing of above-mentioned steps products obtained therefrom.It is assembled into kit finished product after sampling observation is qualified, finished product passes through
It can dispatch from the factory after sampling observation is qualified.
Operating procedure:
The kit that 2 DEG C save is restored to room temperature, coated capillary is taken out, serum or blood plasma is contacted, passes through itself
Siphonage make capillary full of serum or blood plasma, be put into Full-automatic chemiluminescence immunoassay analysis meter detection luminous value, then benefit
The concentration of rabies viruses neutralizing antibody in animal blood serum or blood plasma is calculated with built-in calibration curve.
Embodiment 2
It is only that with the difference of embodiment 1 and utilizes rabies virus G protein recombinant antigen or inactivation rabies totivirus or rabies
Amino after being activated on malicious virus-like particle with SMCC is coupled with the sulfydryl of capillary modification, obtains the capillary of envelope antigen
Pipe.
Embodiment 3
The present embodiment is substantially the same manner as Example 1, unlike: by carbodiimide by rabies virus G protein recombinant antigen or
The carboxyl for inactivating rabies totivirus or Virus-like particles for pseudorabies virus for same and the amino coupled on ammonification capillary, have been coated with
The capillary of antigen.Auxiliary reagent is n-hydroxysuccinimide, improves the stability of intermediate reaction product.
It is complete using carbodiimide and n-hydroxysuccinimide activation rabies virus G protein recombinant antigen or inactivation rabies
Carboxyl in virus or Virus-like particles for pseudorabies virus for same:
Rabies virus G protein recombinant antigen to be activated or inactivation rabies totivirus or Virus-like particles for pseudorabies virus for same concentration are 1
Mg/mL, carbodiimide concentration are 1 mM, and n-hydroxysuccinimide concentration is 2 mM, and buffer is 0.01~0.1M MES (pH
For 5).React at room temperature 15 min.It crosses desalting column and removes unreacted carbodiimide and n-hydroxysuccinimide, and replace buffering
Liquid is 0.01M PBS (pH 7.0).Rabies virus G protein recombinant antigen or inactivation rabies totivirus or mad dog after activation
It is 5 μ g/mL to coating protein solution that virus-virus like particles, which are configured to concentration with 0.01M PBS (pH 7.0), and addition has been repaired
It is decorated with the capillary of amino and submergence.React at room temperature 15 min.It is terminated with 0.02 M Tris-HCl (pH 7.2), it is dry.Packet
The confining liquid 37 of 0.01M Tris-HCl (pH 7.2) is dissolved in by the capillary of antigen effective 1% (quality percent by volume) BSA
DEG C closing, off-period be 2 h.It is cleaned and dried after closing with cleaning agent, 2~8 DEG C of preservations is vacuum-packed.Cleaning agent is 0.02 M
PBS (pH 7.2) contains 0.01% (percent by volume) Tween 20.
Embodiment 4
The present embodiment is substantially the same manner as Example 3, is only that with the difference of embodiment 3 and utilizes rabies virus G protein recombinant antigen
Or the carboxyl of amino and the capillary modification of inactivation rabies totivirus or Virus-like particles for pseudorabies virus for same is coupled, and has been coated with
The capillary of antigen.
Embodiment 5
The present embodiment is substantially the same manner as Example 1, unlike: carbodiimide concentration 0.2mM, buffer are 0.01M MES
(pH 5), is configured to activation buffer:
Rabies virus G protein recombinant antigen or inactivation rabies totivirus or Virus-like particles for pseudorabies virus for same are matched with activation buffer
It is 5 μ g/mL to coating protein solution that concentration, which is made, and ammonification capillary is added, reacts at room temperature 0.5 h.With 0.02 M Tris-
HCl (pH 7.2) is terminated, dry.The capillary of envelope antigen effective 1% (quality percent by volume) skimmed milk power is dissolved in 0.01M
37 DEG C of the confining liquid closings of Tris-HCl (pH 7.2), off-period are 2 h.It is cleaned and dried after closing with cleaning agent, vacuum
2~8 DEG C of preservations of packaging.Cleaning agent is 0.02 M PBS (pH 7.2), contains 0.01% (percent by volume) Tween 20.
Embodiment 6
The present embodiment is substantially the same manner as Example 1, unlike: glutaraldehyde is coupled rabies virus G protein recombinant antigen or inactivation
Rabies totivirus or the amino of the amino of Virus-like particles for pseudorabies virus for same and capillary modification:
With 0.01M PBS, (pH is 7 activation buffers for being configured to that concentration is 1% to glutaraldehyde, and the capillary for being modified with amino is added
It manages and submerges, react at room temperature 2h.It is cleaned and dried with 0.01MPBS (pH 7).Rabies virus G protein recombinant antigen or inactivation is mad
Dog disease totivirus or Virus-like particles for pseudorabies virus for same are configured to the egg to be coated with that concentration is 5 μ g/mL with 0.01M PBS (pH 7)
White solution is added glutaraldehyde treated ammonification capillary and submerges, reacts at room temperature 2h.With 0.02 M Tris-HCl, (pH is
7.2) termination is cleaned with cleaning agent, dry.It is coated with capillary effective 1% (quality percent by volume) defatted milk of rabies virus antigen
Powder is dissolved in 37 DEG C of the confining liquid closings of 0.01M Tris-HCl (pH 7.2), off-period 2h.It is clear with cleaning agent after closing
Drying is washed, 2~8 DEG C of preservations are vacuum-packed.Cleaning agent is 0.02 M PBS (pH 7.2), contains 0.01% (percent by volume)
Tween 20。
Embodiment 7
The present embodiment is substantially the same manner as Example 1, is only that enzyme conjugates is to utilize rabies viruses G egg with the difference of embodiment 1
Amino after white recombinant antigen SMCC activation, is coupled with the sulfydryl obtained after alkaline phosphatase enzyme modification, obtains coupled product.
Embodiment 8
The present embodiment is substantially the same manner as Example 1, unlike: enzyme conjugates is by carbodiimide by rabies virus G protein
The coupled product that the carboxyl of recombinant antigen and the amino coupled of alkaline phosphatase obtain, auxiliary reagent are that N- hydroxysuccinimidyl acyl is sub-
Amine improves the stability of intermediate reaction product:
Utilize the carboxyl on carbodiimide and n-hydroxysuccinimide activation rabies virus G protein recombinant antigen.Rabies viruses G
Protein reconstitution antigen concentration is 1mg/mL, and carbodiimide concentration is 1 mM, and n-hydroxysuccinimide concentration is 2 mM, buffer
For 0.01M MES (pH 5).React at room temperature 15min.It crosses desalting column and removes unreacted carbodiimide and N- hydroxysuccinimidyl acyl Asia
Amine, and replacing buffer is 0.01M PBS (pH 7.0).The alkaline phosphatase of mole 1:1 is added, mixes, room temperature reaction 2
h.The azanol that final concentration of 10 mM is added terminates.Gel column is crossed, coupled product is purified, and replacing buffer is 0.02 M
MTris-HCl (pH 7.2) collects rabies virus G protein recombinant antigen conjugated alkaline phosphoric acid enzyme product, and being diluted to concentration is
0.1%(mass percent by volume is added in the enzyme conjugates of 0.5 μ g/mL) BSA or casein, 0.01% (percent by volume)
Tween 20, and 0.05% (percent by volume) preservative is added, it dispenses, sealing, 2~8 DEG C of preservations.
Embodiment 9
The present embodiment is substantially the same manner as Example 8, is only that enzyme conjugates is to utilize rabies viruses G egg with the difference of embodiment 3
White recombinant antigen amino is coupled, the coupled product of acquisition with the carboxyl of alkaline phosphatase.
Embodiment 10
It has been coated with the preparation of the capillary of rabies virus antigen:
Rabies virus G protein recombinant antigen or inactivation rabies totivirus or Virus-like particles for pseudorabies virus for same are modified, sulfydryl is connected:
Rabies virus antigen concentration to be coated with is 4mg/mL, and 2-IT usage amount is the 20 of rabies virus antigen mole to be coated with
Times, the two is mixed in 0.1M PBS (pH 9), reacts at room temperature 1.5 h.It crosses desalting column and removes unreacted 2-IT, and replace slow
Fliud flushing is 0.1M PBS (pH 7.5).
Capillary is connected into amino by dressing agent, activates the ammonia on modification postcapillary using SMCC or derivatives thereof
Base:
SMCC is dissolved in dimethyl sulfoxide or n,N-Dimethylformamide or other organic solvents first, is re-dissolved in 0.1M PBS (pH
For the capillary modified and submergence 9), is added, SMCC concentration is 2mg/mL.React at room temperature 60 min.With 0.2 M Tris-
HCl (pH 7.8) is terminated, dry.
Capillary after SMCC activation and the rabies virus G protein recombinant antigen that has been modified with sulfydryl or to inactivate rabies complete
Virus or Virus-like particles for pseudorabies virus for same coupling.Rabies virus G protein recombinant antigen or the mad dog of inactivation of sulfydryl are connected after modification
Sick totivirus or Virus-like particles for pseudorabies virus for same with 0.2 M Tris-HCl (pH 7.8) be configured to concentration be 30 μ g/mL to
Coating protein solution is added SMCC activation capillary and submerges.React at room temperature 2 h.It is cleaned with cleaning agent, is dry.Envelope antigen
The effective 5%(mass percent by volume of capillary) BSA is dissolved in 37 DEG C of confining liquid of 0.1M Tris-HCl (pH 7.8) closings,
Off-period is 4 h.It is cleaned after closing with cleaning agent, is dry, 2~8 DEG C of preservations are vacuum-packed.Used cleaning agent is 0.2
M PBS (pH 7.8), percent by volume containing 0.1%() Tween 20.
The preparation of enzyme conjugates:
Enzyme conjugates is to be diluted to certain density rabies virus G protein recombinant antigen conjugated alkaline phosphoric acid enzyme product.
Rabies virus G protein recombinant antigen is modified, sulfydryl is connected at histone amino.Rabies virus G protein recombinant antigen
Concentration be 4mg/mL, 2-IT usage amount is 20 times of rabies virus G protein recombinant antigen mole, and the two is mixed in 0.1M
PBS (pH 9) reacts at room temperature 1.5 h.It crosses desalting column and removes unreacted 2-IT, and replacing buffer is that (pH is 0.1M PBS
7.5)。
Utilize the amino on SMCC or derivatives thereof activated alkaline phosphatase.The concentration of alkaline phosphatase is 5mg/mL,
SMCC usage amount is 20 times of alkaline phosphatase mole, and the two is mixed in 0.1M PBS (pH 8), reacts at room temperature 1 h.Desalination
Column removes unreacted SMCC.
Alkaline phosphatase after the rabies virus G protein recombinant antigen of sulfhydrylation is activated with SMCC mixes, and coupling forms enzyme
Conjugate.When alkaline phosphatase after rabies virus G protein recombinant antigen and the SMCC activation of sulfhydrylation is coupled, mole 1:
1, coupling temperature is 8 DEG C, and coupling time is 18 h.Gel column is crossed, coupled product is purified, and replacing buffer is 0.2 M
Tris-HCl (pH 7.8) collects rabies virus G protein recombinant antigen conjugated alkaline phosphoric acid enzyme product, and being diluted to concentration is 5 μ
5% (quality percent by volume) BSA or casein, 0.1%(percent by volume is added in the enzyme conjugates of g/mL) Tween 20,
And 0.05% (percent by volume) preservative is added, it dispenses, sealing, 2~8 DEG C of preservations.
The preparation of luminous substrate:
The luminous substrate of this kit is 4- chlorobenzene sulfydryl 10- methyl-acridan methylene phosphate disodium salt
(APS-5).After the substrate is mixed with alkaline phosphatase (ALP), alkaline phosphatase phosphate radical hydrolysis, substrate is decomposed instead immediately
Photon should be released, within the scope of specific alkaline phosphatase concentration, photon numbers and the solution alkaline phosphatase of release
Concentration is directly proportional, it is possible to the quantitative detection for alkaline phosphatase.This substrate high sensitivity is sent out to sustainable stability and high efficiency
Light.Packing, sealing, 2~8 DEG C are kept in dark place.
The preparation of cleaning solution:
Cleaning solution is prepared: 0.1% (percent by volume) Tween 20,0.1%(percent by volume) Qula is logical, 0.9%(mass
Percent by volume) NaCl, 0.05%(percent by volume) preservative, it is dissolved in 0.2 M Tris-HCl (pH 7.8).Packing, it is close
Envelope, 2~8 DEG C of preservations.
Embodiment 11
It has been coated with the preparation of the capillary of rabies virus antigen:
Rabies virus G protein recombinant antigen or inactivation rabies totivirus or Virus-like particles for pseudorabies virus for same are modified, in albumen ammonia
Ji Chu connects sulfydryl:
Rabies virus antigen concentration to be coated with is 3mg/mL, and 2-IT usage amount is the 10 of rabies virus antigen mole to be coated with
Times, the two is mixed in 0.08M PBS (pH 8.5), reacts at room temperature 1 h.It crosses desalting column and removes unreacted 2-IT, and replace slow
Fliud flushing is 0.06M PBS (pH 7).
Capillary is connected into amino by dressing agent, activates the ammonia on modification postcapillary using SMCC or derivatives thereof
Base:
SMCC is dissolved in dimethyl sulfoxide or n,N-Dimethylformamide or other organic solvents first, is re-dissolved in 0.05M PBS (pH
For the capillary modified and submergence 8), is added, SMCC concentration is 1mg/mL.React at room temperature 40 min.With 0.1 M Tris-
HCl (pH 7.5) is terminated, dry.
Capillary after SMCC activation and the rabies virus G protein recombinant antigen that has been modified with sulfydryl or to inactivate rabies complete
Virus or Virus-like particles for pseudorabies virus for same coupling.Rabies virus G protein recombinant antigen or the mad dog of inactivation of sulfydryl are connected after modification
It is 25 μ g/mL's that sick totivirus or Virus-like particles for pseudorabies virus for same, which are configured to concentration with 0.08 M Tris-HCl (pH 7.6),
To coating protein solution, SMCC activation capillary is added and submerges.React at room temperature 1.5 h.It is cleaned with cleaning agent, is dry.Coating
The effective 3%(mass percent by volume of the capillary of antigen) skimmed milk power is dissolved in the confining liquid of 0.04M Tris-HCl (pH 7.4)
37 DEG C of closings, off-period are 3 h.It is cleaned after closing with cleaning agent, is dry, 2~8 DEG C of preservations are vacuum-packed.Used is clear
Lotion is 0.1 M PBS (pH 7.6), percent by volume containing 0.07%() Tween 20.
The preparation of enzyme conjugates:
Enzyme conjugates is to be diluted to certain density rabies virus G protein recombinant antigen conjugated alkaline phosphoric acid enzyme product.
Rabies virus G protein recombinant antigen is modified, sulfydryl is connected at histone amino.Rabies virus G protein recombinant antigen
Concentration be 2.5mg/mL, 2-IT usage amount is 16 times of rabies virus G protein recombinant antigen mole, and the two is mixed in
0.06M PBS (pH 8.8) reacts at room temperature 1.2 h.It crosses desalting column and removes unreacted 2-IT, and replacing buffer is 0.06M
PBS (pH 7).
Utilize the amino on SMCC activated alkaline phosphatase.The concentration of alkaline phosphatase is 3.5mg/mL, SMCC usage amount
It is 15 times of alkaline phosphatase mole, the two is mixed in 0.08M PBS (pH 7.5), reacts at room temperature 0.8 h.Desalting column removes
Remove unreacted SMCC.
Alkaline phosphatase after the rabies virus G protein recombinant antigen of sulfhydrylation is activated with SMCC mixes, and coupling forms enzyme
Conjugate.When alkaline phosphatase after rabies virus G protein recombinant antigen and the SMCC activation of sulfhydrylation is coupled, mole 1:
1, coupling temperature is 7 DEG C, and coupling time is 15 h.Gel column is crossed, coupled product is purified, and replacing buffer is 0.08 M
Tris-HCl (pH 7.6) collects rabies virus G protein recombinant antigen conjugated alkaline phosphoric acid enzyme product, and being diluted to concentration is 4 μ
4%(mass percent by volume is added in the enzyme conjugates of g/mL) BSA or casein, 0.05%(percent by volume) Tween
20, and 0.03% (percent by volume) preservative is added, it dispenses, sealing, 2~8 DEG C of preservations.
The preparation of luminous substrate:
The luminous substrate of this kit is 4- chlorobenzene sulfydryl 10- methyl-acridan methylene phosphate disodium salt
(APS-5).After the substrate is mixed with alkaline phosphatase (ALP), alkaline phosphatase phosphate radical hydrolysis, substrate is decomposed instead immediately
Photon should be released, within the scope of specific alkaline phosphatase concentration, photon numbers and the solution alkaline phosphatase of release
Concentration is directly proportional, it is possible to the quantitative detection for alkaline phosphatase.This substrate high sensitivity is sent out to sustainable stability and high efficiency
Light.Packing, sealing, 2~8 DEG C are kept in dark place.
The preparation of cleaning solution:
Cleaning solution is prepared: 0.08% (percent by volume) Tween 20,0.06%(percent by volume) Qula is logical, 0.88%(mass
Percent by volume) NaCl, 0.04%(percent by volume) preservative, it is dissolved in 0.18 M Tris-HCl (pH 7.5).Packing, it is close
Envelope, 2~8 DEG C of preservations.
Embodiment 12
The present embodiment is substantially the same manner as Example 3, unlike:
Rabies virus G protein recombinant antigen to be activated or inactivation rabies totivirus or Virus-like particles for pseudorabies virus for same concentration are 10
Mg/mL, carbodiimide concentration are 5 mM, and n-hydroxysuccinimide concentration is 10 mM, and buffer is 0.1M MES (pH 7).
React at room temperature 60 min.It crosses desalting column and removes unreacted carbodiimide and n-hydroxysuccinimide, and replace buffer and be
0.1M PBS (pH 7.5).Rabies virus G protein recombinant antigen or inactivation rabies totivirus or rabies viral disease after activation
It is 30 μ g/mL to coating protein solution that malicious sample particle, which is configured to concentration with 0.1M PBS (pH 7.5), and addition has been modified with ammonia
The capillary of base and submergence.React at room temperature 60 min.It is terminated with 0.2 M Tris-HCl (pH 7.8), it is dry.Envelope antigen
Capillary effective 5% (quality percent by volume) BSA be dissolved in 37 DEG C of confining liquid of 0.1M Tris-HCl (pH 7.8) closings,
Off-period is 4 h.It is cleaned and dried after closing with cleaning agent, 2~8 DEG C of preservations is vacuum-packed.Cleaning agent is 0.2 M PBS (pH
7.8), to contain 0.1% (percent by volume) Tween 20.
Embodiment 13
The present embodiment is substantially the same manner as Example 3, unlike: rabies virus G protein recombinant antigen to be activated or the mad dog of inactivation
Sick totivirus or Virus-like particles for pseudorabies virus for same concentration are 8 mg/mL, and carbodiimide concentration is 4 mM, n-hydroxysuccinimide
Concentration is 6 mM, and buffer is 0.05M MES (pH 6).React at room temperature 30 min.It crosses desalting column and removes unreacted two Asia of carbon
Amine and n-hydroxysuccinimide, and replacing buffer is 0.06M PBS (pH 7.2).Rabies virus G protein weight after activation
Group antigen or inactivation rabies totivirus or Virus-like particles for pseudorabies virus for same are configured to concentration with 0.06M PBS (pH 7.2) and are
15 μ g/mL to coating protein solution, capillary and the submergence for being modified with amino is added.React at room temperature 45 min.With 0.08
M Tris-HCl (pH 7.6) is terminated, dry.The capillary of envelope antigen effective (quality percent by volume) 3% skimmed milk power is molten
In 37 DEG C of the confining liquid closings of 0.06M Tris-HCl (pH 7.5), off-period is 3 h.It is cleaned after closing with cleaning agent
It is dry, 2~8 DEG C of preservations are vacuum-packed.Cleaning agent is 0.1 M PBS (pH 7.6), contains 0.06% (percent by volume) Tween
20。
Embodiment 14
The present embodiment is substantially the same manner as Example 5, unlike: carbodiimide concentration is 2 mM, and buffer is 0.1M MES (pH
7), to be configured to activation buffer:
Rabies virus G protein recombinant antigen or inactivation rabies totivirus or Virus-like particles for pseudorabies virus for same are matched with activation buffer
It is 30 μ g/mL to coating protein solution that concentration, which is made, and ammonification capillary is added, reacts at room temperature 1 h.With 0.2 M Tris-HCl
(pH 7.8) is terminated, dry.The effective 5%(mass percent by volume of the capillary of envelope antigen) BSA is dissolved in 0.01~0.1M
37 DEG C of the confining liquid closings of Tris-HCl (pH 7.8), off-period are 4 h.It is cleaned and dried after closing with cleaning agent, vacuum
2~8 DEG C of preservations of packaging.Cleaning agent is 0.2 M PBS (pH 7.8), contains 0.1% (percent by volume) Tween 20.
Embodiment 15
The present embodiment is substantially the same manner as Example 5, unlike: carbodiimide concentration 1mM, buffer are 0.8M MES (pH
6), to be configured to activation buffer:
Rabies virus G protein recombinant antigen or inactivation rabies totivirus or Virus-like particles for pseudorabies virus for same are matched with activation buffer
It is 25 μ g/mL to coating protein solution that concentration, which is made, and ammonification capillary is added, reacts at room temperature 0.8h.With 0.1 M Tris-
HCl (pH 7.6) is terminated, dry.The capillary of envelope antigen effective 4% (quality percent by volume) skimmed milk power is dissolved in 0.06M
37 DEG C of the confining liquid closings of Tris-HCl (pH 7.6), off-period are 3 h.It is cleaned and dried after closing with cleaning agent, vacuum
2~8 DEG C of preservations of packaging.Cleaning agent is 0.1 M PBS (pH 7.6), contains 0.08% (percent by volume) Tween 20.
Embodiment 16
The present embodiment is substantially the same manner as Example 6, unlike: it is 5% that glutaraldehyde, which is configured to concentration with 0.1M PBS (pH 8),
Capillary and the submergence for being modified with amino is added in the activation buffer of (percent by volume), reacts at room temperature 4 h.Use 0.1M
PBS (pH 8) is cleaned and dried.By rabies virus G protein recombinant antigen or inactivation rabies totivirus or rabies viruses virus-like
It is 30 μ g/mL to coating protein solution that particle, which is configured to concentration with 0.1M PBS (pH 8), is added glutaraldehyde treated ammonia
Change capillary and submerge, reacts at room temperature 4 h.It is cleaned with 0.2 M Tris-HCl (pH 7.8) termination with cleaning agent, it is dry.Packet
Being dissolved in 0.1M Tris-HCl by the capillary of rabies virus antigen effective 5% (quality percent by volume) skimmed milk power, (pH is
7.8) 37 DEG C of confining liquid closings, off-period are 4 h.It is cleaned and dried after closing with cleaning agent, 2~8 DEG C of guarantors is vacuum-packed
It deposits.Cleaning agent is 0.2 M PBS (pH 7.8), contains 0.1% (percent by volume) Tween 20.
Embodiment 17
The present embodiment is substantially the same manner as Example 6, unlike: glutaraldehyde is configured to concentration with 0.06M PBS (pH 7.5) and is
Capillary and the submergence for being modified with amino is added in 3% activation buffer, reacts at room temperature 3 h.With 0.06M PBS, (pH is
7.5) it is cleaned and dried.Rabies virus G protein recombinant antigen or inactivation rabies totivirus or Virus-like particles for pseudorabies virus for same are used
It is 15 μ g/mL to coating protein solution that 0.08M PBS (pH 7.6), which is configured to concentration, is added glutaraldehyde treated ammonification
Capillary simultaneously submerges, and reacts at room temperature 3.5 h.It is cleaned with 0.12M Tris-HCl (pH 7.5) termination with cleaning agent, it is dry.Packet
By the effective 3%(mass percent by volume of the capillary of rabies virus antigen) BSA is dissolved in the envelope of 0.07M Tris-HCl (pH 7.4)
37 DEG C of liquid closings are closed, off-period is 2.5 h.It is cleaned and dried after closing with cleaning agent, 2~8 DEG C of preservations is vacuum-packed.Cleaning
Agent is 0.12 MPBS (pH 7.6), percent by volume containing 0.05%() Tween 20.
Embodiment 18
The present embodiment is substantially the same manner as Example 8, unlike: rabies virus G protein recombinant antigen concentration is 10 mg/mL, carbon
Diimine concentration is 5 mM, and n-hydroxysuccinimide concentration is 10 mM, and buffer is 0.1M MES (pH 7).Room temperature reaction
60 min.It crosses desalting column and removes unreacted carbodiimide and n-hydroxysuccinimide, and replacing buffer is 0.1M PBS
(pH 7.5).The alkaline phosphatase of mole 1:1 is added, mixes, reacts at room temperature 4 h.The azanol of final concentration of 20 mM is added
It terminates.Gel column is crossed, coupled product is purified, and replacing buffer is 0.2 M Tris-HCl (pH 7.8), collects rabies viruses
G-protein recombinant antigen conjugated alkaline phosphoric acid enzyme product is diluted to the enzyme conjugates that concentration is 5 μ g/mL, 5%(mass volume is added
Percentage) casein, 0.1% (percent by volume) Tween 20, and add 0.05%(percent by volume) and preservative, packing,
Sealing, 2~8 DEG C of preservations.
Embodiment 19
The present embodiment is substantially the same manner as Example 8, unlike: rabies virus G protein recombinant antigen concentration is 8 mg/mL, carbon
Diimine concentration is 4 mM, and n-hydroxysuccinimide concentration is 6 mM, and buffer is 0.05M MES (pH 6).Room temperature reaction
55 min.It crosses desalting column and removes unreacted carbodiimide and n-hydroxysuccinimide, and replacing buffer is 0.07M PBS
(pH 7.3).The alkaline phosphatase of mole 1:1 is added, mixes, reacts at room temperature 3.8h.The azanol of final concentration of 15 mM is added
It terminates.Gel column is crossed, coupled product is purified, and replacing buffer is 0.18 M Tris-HCl (pH 7.6), collects rabies
Malicious G-protein recombinant antigen conjugated alkaline phosphoric acid enzyme product is diluted to the enzyme conjugates that concentration is 3 μ g/mL, 3% (mass body is added
Product percentage) BSA, 0.08% (percent by volume) Tween 20, and 0.03% (percent by volume) preservative is added, packing,
Sealing, 2~8 DEG C of preservations.
English abbreviation is explained:
SMCC:4- (N- maleimidomehyl) hexamethylene -1- carboxylic acid succinimide ester
SATA:N- succinimide-S- acetylthioacetate
DTT: dithiothreitol (DTT)
TCEP: three (2- carboxyethyl) phosphines
2-MEA:2- mercaptoethylmaine hydrochloric acid
2-IT:2- iminothiolane hydrochloride
PBS: phosphate buffer
MES:2- morpholino b acid buffer
Tween: tween
Tris-HCl: trishydroxymethylaminomethane-hydrochloric acid only contains Tris in Tris-HCl buffer, is adjusted with sodium hydroxide
pH
BSA: bovine serum albumin(BSA)
APS-5:(4- chlorobenzene sulfydryl) (10- methyl-acridan methylene) disodic alkaliine.
Claims (9)
1. a kind of veterinary rabies poison neutralizing antibody chemiluminescence detection kit, it is characterised in that: the kit includes coating
The capillary of rabies virus antigen, enzyme conjugates, luminous substrate and cleaning solution.
2. a kind of veterinary rabies poison neutralizing antibody chemiluminescence detection kit according to claim 1, feature exist
In: the rabies virus antigen is itself with active group or by the rabies virus G protein weight of modification acquisition active group
Group antigen, inactivation rabies totivirus or Virus-like particles for pseudorabies virus for same.
3. a kind of veterinary rabies poison neutralizing antibody chemiluminescence detection kit according to claim 1, feature exist
In: on the capillary with amino, carboxyl, hydroxyl, sulfydryl, aldehyde radical, epoxy group, cyanic acid ester group, isocyanate group, cyano,
One of isocyano group, maleic amide amino, N-hydroxy-succinamide ester base, phenolic hydroxyl group or phenolic hydroxyl group deriveding group.
4. a kind of veterinary rabies poison neutralizing antibody chemiluminescence detection kit according to claim 1, feature exist
In: the capillary for being coated with rabies virus antigen is made by the following method: this method includes modification capillary step, is repaired
Rabies virus G protein recombinant antigen, inactivation rabies totivirus or Virus-like particles for pseudorabies virus for same step are adornd, step and envelope are coated with
Close step；
The modification capillary step specifically: using dressing agent modify capillary so that on capillary with amino, carboxyl,
Hydroxyl, sulfydryl, aldehyde radical, epoxy group, cyanic acid ester group, isocyanate group, cyano, isocyano group, maleic amide amino, N- hydroxysuccinimidyl
One of acid imide ester group, phenolic hydroxyl group or phenolic hydroxyl group deriveding group；
The modification rabies virus G protein recombinant antigen, inactivation rabies totivirus or Virus-like particles for pseudorabies virus for same step tool
Body are as follows: utilize dressing agent modification rabies virus G protein recombinant antigen, inactivation rabies totivirus or rabies viruses virus-like
Grain, so that rabies virus G protein recombinant antigen, inactivation rabies totivirus or Virus-like particles for pseudorabies virus for same connect amino, carboxylic
Base, sulfydryl or hydroxyl；
The coating step specifically:
Activation: there are three types of the modes of activation: the first: the capillary after being modified with coupling agent activation, second: living with coupling agent
Rabies virus G protein recombinant antigen, inactivation rabies totivirus or Virus-like particles for pseudorabies virus for same after changing modification, the third,
Coupling agent is added in coating buffer；
Prepare coating buffer: when activation uses first way, preparation coating buffer specifically: by the rabies virus G protein after modification
Recombinant antigen, inactivation rabies totivirus or Virus-like particles for pseudorabies virus for same dilute most 5~30 μ g/mL, and coating buffer I is made；
When activation uses the second way, preparation coating buffer specifically: the rabies virus G protein recombinant antigen after activation, inactivation is mad
Dog disease totivirus or Virus-like particles for pseudorabies virus for same dilute most 5~30 μ g/mL, and coating buffer II is made；When activation uses third
Kind mode, prepares coating buffer specifically: coupling agent and buffer are mixed and made into activation buffer, it then will with activation buffer
Rabies virus G protein recombinant antigen after modification, inactivation rabies totivirus or Virus-like particles for pseudorabies virus for same dilution most 5~
30 μ g/mL, are made coating buffer III；
Coupling: when activation uses first way, specific coupling are as follows: the capillary after activation is immersed in coating buffer I, it is even
After the completion of connection, capillary is washed and dried, obtains the capillary for being coated with rabies virus antigen；When activation uses second of side
Formula, it is specific to be coupled are as follows: the capillary after modification is immersed in coating buffer II, after the completion of coupling, washes and dries capillary,
Obtain the capillary for being coated with rabies virus antigen；When activation uses the third mode, specific coupling are as follows: by the hair after modification
Tubule is immersed in coating buffer III, after the completion of coupling, is washed and dried capillary, is obtained the capillary for being coated with rabies virus antigen
Pipe；
The closing step specifically: be dissolved in Tris-HCl with BSA or skimmed milk power, confining liquid is made；By the hair after coating
Tubule is immersed in confining liquid, after closing 2~4 h, washes and dries capillary.
5. a kind of veterinary rabies poison neutralizing antibody chemiluminescence detection kit according to claim 4, feature exist
In: in the closing step, in the confining liquid, the quality percent by volume of BSA or skimmed milk power is 1~5%, Tris-HCl
Concentration be 0.01~0.1M, the pH of confining liquid is 7.2~7.8, and closure temperature is 37 DEG C.
6. a kind of veterinary rabies poison neutralizing antibody chemiluminescence detection kit according to claim 1, feature exist
In: the enzyme conjugates is made by the following method: this method includes modification rabies virus G protein recombinant antigen step, activation
Step, coupling step, purification step and dilution step；
The modification rabies virus G protein recombinant antigen step specifically: make to modify rabies virus G protein weight using dressing agent
Group antigen, so that rabies virus G protein recombinant antigen connects amino, carboxyl, sulfydryl or hydroxyl；
The activation step specifically: utilize coupling agent activated alkaline phosphatase or rabies virus G protein recombinant antigen；
It is described using coupling agent activated alkaline phosphatase refer to the amino of coupling agent activated alkaline phosphatase, carboxyl, sulfydryl or
Hydroxyl；
The coupling step specifically: mix the rabies virus G protein recombinant antigen after modification with the alkaline phosphatase after activation
It closes, coupling forms enzyme conjugates；Or mix the rabies virus G protein recombinant antigen after activation with alkaline phosphatase, it is coupled
Form enzyme conjugates；
The purification step specifically: the enzyme conjugates after coupling is crossed into gel column, is then purified；
The dilution step specifically: with the M of 0.02 M~0.2, Tris-HCl that pH is 7.2~7.8 is by enzyme knot after purification
It closes object and is diluted to the enzyme conjugates that concentration is 0.5~5 μ g/mL.
7. a kind of veterinary rabies poison neutralizing antibody chemiluminescence detection kit according to claim 6, feature exist
In: it further include anti-corrosion step, anti-corrosion step specifically: BSA or casein, Tween 20 are added in enzyme conjugates after dilution
Final enzyme conjugates is made with preservative, dispenses, sealing saves under conditions of temperature is 2~8 DEG C.
8. a kind of veterinary rabies poison neutralizing antibody chemiluminescence detection kit according to claim 7, feature exist
In: in final enzyme conjugates, the quality percent by volume of BSA or casein is the volume basis of 0.1~5%, Tween 20
Than being 0.01~0.1%, the percent by volume of preservative is 0.01~0.05%.
9. a kind of veterinary rabies poison neutralizing antibody chemiluminescence detection kit according to claim 1, feature exist
In: the cleaning solution is formulated by the following method: by Tween 20, Qula is logical, NaCl and preservative, is dissolved in 0.02 M
In the Tris-HCl of~0.2 M, cleaning solution is made, in the cleaning solution percent by volume of Tween 20 be 0.01~
0.1%, the logical percent by volume of Qula is 0.01~0.1%: the quality percent by volume of NaCl is 0.85~0.9%, preservative
Percent by volume be 0.01~0.05%, the pH of the cleaning solution is 7.2~7.8.
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Citations (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1989000290A1 (en) * | 1987-07-02 | 1989-01-12 | In Vitro Technologies, Inc. | Capillary device for immunoassay of multiple analytes |
| JPH06148075A (en) * | 1992-11-12 | 1994-05-27 | Olympus Optical Co Ltd | Chemiluminescence measuring instrument |
| US5976896A (en) * | 1994-06-06 | 1999-11-02 | Idexx Laboratories, Inc. | Immunoassays in capillary tubes |
| US20040219163A1 (en) * | 2001-10-03 | 2004-11-04 | Frelinger John G. | Human glandular kallikrein (hk2)-specific monoclonal antibodies that enhance or inhibit the enzymatic activity of hk2 |
| CN101017170A (en) * | 2006-06-21 | 2007-08-15 | 中国人民解放军军事医学科学院军事兽医研究所 | Rabies antibody gold immunochromatography assay testing indicator paper and preparation technique |
| JP2010085126A (en) * | 2008-09-29 | 2010-04-15 | Adtec Kk | Determination instrument for determining neutralizing antibody titer of rabies virus, and method for measuring neutralizing antibody titer of rabies virus |
| CN101936998A (en) * | 2010-08-19 | 2011-01-05 | 武汉中博生物股份有限公司 | Antirabies virus IgG (Immunoglobulin G) antibody ELISA (Enzyme Linked Immunosorbent Assay) detection kit for dogs |
| CN102175867A (en) * | 2010-12-23 | 2011-09-07 | 北京民海生物科技有限公司 | Enzyme-linked immunoassay kit for detecting rabies virus antibody |
| US20110262940A1 (en) * | 2008-12-19 | 2011-10-27 | Hideaki Hisamoto | Capillary for immunoassay, and capillary immunoassay method using same |
| CN102520169A (en) * | 2011-11-29 | 2012-06-27 | 唐山怡安生物工程有限公司 | ELISA (Enzyme-Linked Immuno Sorbent Assay) detection kit of animal rabies neutralizing antibody and application thereof |
| CN103323588A (en) * | 2012-03-19 | 2013-09-25 | 郑州中道生物技术有限公司 | Method for detecting canine rabies virus antibody and detection kit |
| CN203825011U (en) * | 2013-11-08 | 2014-09-10 | 洛阳莱普生信息科技有限公司 | Chemiluminescence kit for swine pseudorabies virus antibody |
| CN107091923A (en) * | 2017-06-02 | 2017-08-25 | 成都普利泰生物科技有限公司 | A kind of capillary chemistry luminescence detection apparatus and its detection method |
| CN107860920A (en) * | 2017-11-01 | 2018-03-30 | 杭州微瑞科技有限公司 | Rabies virus antibodies Quantitative detection card and application method |
| CN107957495A (en) * | 2017-11-17 | 2018-04-24 | 南通伊仕生物技术股份有限公司 | A kind of CK-MB detection kits and its application method |
| CN108700584A (en) * | 2017-01-20 | 2018-10-23 | 深圳市新产业生物医学工程股份有限公司 | Labeled complex and preparation method thereof, kit, application and detecting system |
-
2018
- 2018-10-26 CN CN201811254348.8A patent/CN109444410A/en active Pending
Patent Citations (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1989000290A1 (en) * | 1987-07-02 | 1989-01-12 | In Vitro Technologies, Inc. | Capillary device for immunoassay of multiple analytes |
| JPH06148075A (en) * | 1992-11-12 | 1994-05-27 | Olympus Optical Co Ltd | Chemiluminescence measuring instrument |
| US5976896A (en) * | 1994-06-06 | 1999-11-02 | Idexx Laboratories, Inc. | Immunoassays in capillary tubes |
| US20040219163A1 (en) * | 2001-10-03 | 2004-11-04 | Frelinger John G. | Human glandular kallikrein (hk2)-specific monoclonal antibodies that enhance or inhibit the enzymatic activity of hk2 |
| CN101017170A (en) * | 2006-06-21 | 2007-08-15 | 中国人民解放军军事医学科学院军事兽医研究所 | Rabies antibody gold immunochromatography assay testing indicator paper and preparation technique |
| JP2010085126A (en) * | 2008-09-29 | 2010-04-15 | Adtec Kk | Determination instrument for determining neutralizing antibody titer of rabies virus, and method for measuring neutralizing antibody titer of rabies virus |
| US20110262940A1 (en) * | 2008-12-19 | 2011-10-27 | Hideaki Hisamoto | Capillary for immunoassay, and capillary immunoassay method using same |
| CN101936998A (en) * | 2010-08-19 | 2011-01-05 | 武汉中博生物股份有限公司 | Antirabies virus IgG (Immunoglobulin G) antibody ELISA (Enzyme Linked Immunosorbent Assay) detection kit for dogs |
| CN102175867A (en) * | 2010-12-23 | 2011-09-07 | 北京民海生物科技有限公司 | Enzyme-linked immunoassay kit for detecting rabies virus antibody |
| CN102520169A (en) * | 2011-11-29 | 2012-06-27 | 唐山怡安生物工程有限公司 | ELISA (Enzyme-Linked Immuno Sorbent Assay) detection kit of animal rabies neutralizing antibody and application thereof |
| CN103323588A (en) * | 2012-03-19 | 2013-09-25 | 郑州中道生物技术有限公司 | Method for detecting canine rabies virus antibody and detection kit |
| CN203825011U (en) * | 2013-11-08 | 2014-09-10 | 洛阳莱普生信息科技有限公司 | Chemiluminescence kit for swine pseudorabies virus antibody |
| CN108700584A (en) * | 2017-01-20 | 2018-10-23 | 深圳市新产业生物医学工程股份有限公司 | Labeled complex and preparation method thereof, kit, application and detecting system |
| CN107091923A (en) * | 2017-06-02 | 2017-08-25 | 成都普利泰生物科技有限公司 | A kind of capillary chemistry luminescence detection apparatus and its detection method |
| CN107860920A (en) * | 2017-11-01 | 2018-03-30 | 杭州微瑞科技有限公司 | Rabies virus antibodies Quantitative detection card and application method |
| CN107957495A (en) * | 2017-11-17 | 2018-04-24 | 南通伊仕生物技术股份有限公司 | A kind of CK-MB detection kits and its application method |
Non-Patent Citations (6)
| Title |
|---|
| ALI Z , LIANG W , JIN L , ET AL: "Development of Magnetic Nanoparticles Based Nucleic Acid Extraction Method and Application in Hepatitis C Virus Chemiluminescent Detection", 《SCIENCE OF ADVANCED MATERIALS》 * |
| 冯燕: "SMCC法抗体定向偶联技术的优化与应用研究", 《中国优秀硕士学位论文全文数据库（医药卫生科技辑）》 * |
| 吴俊英 等: "《临床免疫学检验》", 30 March 2014 * |
| 徐开先 等: "《传感器实用技术》", 31 December 2016 * |
| 秦晓松: "《2018临床医学检验技术（师）应试指南》", 30 November 2017 * |
| 韩晓军: "《生物功能化界面》", 30 January 2017 * |
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