CN110501509A - A kind of anti-feline panleucopenia virus cat source genetic engineering antibody ELISA kit and its detection method - Google Patents
A kind of anti-feline panleucopenia virus cat source genetic engineering antibody ELISA kit and its detection method Download PDFInfo
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- CN110501509A CN110501509A CN201910828147.2A CN201910828147A CN110501509A CN 110501509 A CN110501509 A CN 110501509A CN 201910828147 A CN201910828147 A CN 201910828147A CN 110501509 A CN110501509 A CN 110501509A
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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
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/01—DNA viruses
- G01N2333/015—Parvoviridae, e.g. feline panleukopenia virus, human Parvovirus
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
Abstract
The invention discloses a kind of anti-feline panleucopenia virus cat source genetic engineering antibody ELISA kit and its detection methods, belong to genetic engineering antibody technical field of analysis and detection, which includes the ELISA Plate and enzyme labelled antibody for being coated with antigen；The antigen is FPV-VLPS purifying antigen, and the enzyme labelled antibody is the anti-cat IgG antibody of goat of horseradish peroxidase-labeled.The detection method includes: coated elisa plate, draws standard curve, the anti-cat IgG antibody of goat of horseradish peroxidase-labeled is added, and TMB is shown, terminate liquid terminates, and each hole OD is reacted in measurement450Value, judge according to criterion and etc..The detection of kit provided by the invention and its detection method confrontation feline panleucopenia virus cat source genetic engineering antibody has very high sensibility, specificity and stability, and the research for anti-feline panleucopenia virus cat source genetic engineering antibody level in the animal body before and after detection urgent immunity provides strong support.
Description
Technical field
The present invention relates to genetic engineering antibody technical field of analysis and detection more particularly to a kind of anti-feline panleucopenia virus cat sources
Genetic engineering antibody ELISA kit and its detection method.
Background technique
Feline panleucopenia virus (Feline parvovirus, FPV) also known as Feline Panleukopenia Virus, cat infectiousness intestines
Scorching virus or feline panleukopenia virus.Feline panleucopenia virus is Parvoviridae, parvovirus category member.Feline panleucopenia virus can be in cat and cat family
Proliferation, the tiny disease of cat are replicated in the Various Tissues cell such as animal spleen, the heart, kidney, lung, intestines, testis, bone, adrenal gland and lymph node
Malicious main infection cat and felid (such as tiger, leopard) and mustelid (ermine, ferret), Procyonidae (coati, racoon) animal.
The cat at various ages can be infected, young cat morbidity below in 1 years old is mainly caused.Wherein again with 3 monthly ages to 5 monthly ages without immunization campaign
Young cat it is susceptible, for infection rate up to 70%, case fatality rate is 50%~60%, reaches as high as 80%-90%, is usually expressed as whole nest
Kitten falls ill simultaneously or successively falls ill successively.The probability of feline panleucopenia virus infection increases disease incidence gradually with the susceptible animal age
It reduces, but adult cat is it can also happen that subclinical infection.The immunoprotection that young cat is obtained by colostrum is usually up to 3 months.Sick cat
It is major source of infection with recovery band poison cat.The pregnant mother cat poison that can will be caught an illness in a manner of vertical transmission after being infected with the virus is transmitted to tire
Youngster causes stillborn foetus, the mummification of fetus, monster etc..
It is mainly at present vaccine immunity for the prevention of feline panleucopenia virus, effective vaccine inoculation and thoroughly disinfection are to pre-
Fang Duo cat family and Animal Shelter transmission it is most important.It is most common to have inactivated vaccine and attenuated live vaccine, inactivation
Vaccine safety is high, but generally attenuated vaccine is better than inactivated vaccine effect in terms of controlling disease's spread.In some European states
Family has the horse source antibody preparation of commercialization, can prevent young cat and adult cat and infect from FPV.But if using Ma Yuankang
Body easily leads to fatal allergic reaction.The anti-tiny genetic engineering antibody of cat and the good combination of FPV, at the same with horse source antibody phase
Than reducing heterologous closer to natural cat source antibody, and then make it that can more play effect.
Application number (201910535002.3) patent application provides a kind of cat source genetic engineering of anti-feline panleucopenia virus
Antibody is expected to be applied to the treatment of infection feline panleucopenia virus cat.Therefore, anti-feline panleucopenia virus cat source genetic engineering antibody is detected
Horizontal research has important application value to protection cat.Presently commercially available three len antibody kit of Bai Aogao cat does not refer to
It can detecte anti-feline panleucopenia virus genetic engineering antibody, it is anti-to be applicable in feline panleucopenia virus genetic engineering for its detection range
Body, one kind that the application specially designs can resist the detection method and kit of feline panleucopenia virus genetic engineering antibody, it is intended to
Anti- feline panleucopenia virus genetic engineering antibody is horizontal in animal body before and after detection urgent immunity, assesses their immunity function, point
Animal immune effect is analysed, whether judgement is immune succeeds, and achievees the effect that the course of disease controllable, and the diagnosis and prognosis to disease provide side
It helps.
Summary of the invention
The purpose of the present invention is being directed to feline panleucopenia virus genetic engineering antibody, a kind of anti-feline panleucopenia virus cat source base is provided
Because of engineered antibody ELISA kit and a kind of anti-feline panleucopenia virus cat source genetic engineering antibody ELISA detection method, for detection
The research of anti-feline panleucopenia virus cat source genetic engineering antibody level provides strong support in animal body before and after urgent immunity.
To achieve the goals above, technical solution provided by the invention is as follows:
Present invention firstly provides a kind of anti-feline panleucopenia virus cat source genetic engineering antibody ELISA kit, the examinations
Agent box includes the ELISA Plate and enzyme labelled antibody for being coated with antigen, and the antigen is FPV-VLPs (virus-like particle of FPV) purifying
Antigen, the enzyme labelled antibody are the anti-cat IgG antibody of goat of horseradish peroxidase-labeled.
Preferably, in above-mentioned anti-feline panleucopenia virus cat source genetic engineering antibody ELISA kit, the FPV-VLPs
The working concentration of purifying antigen is 2 μ g/mL.
Preferably, in above-mentioned anti-feline panleucopenia virus cat source genetic engineering antibody ELISA kit, the enzyme labelled antibody
Extension rate be 1:6000.
Preferably, in above-mentioned anti-feline panleucopenia virus cat source genetic engineering antibody ELISA kit, the kit is also
Including confining liquid and rinsing liquid, the confining liquid is the PBS buffer solution comprising 2%BSA, and the rinsing liquid is to include 0.05%
The PBS buffer solution of Tween-20.
Preferably, in above-mentioned anti-feline panleucopenia virus cat source genetic engineering antibody ELISA kit, the anti-cat is thin
Small virus cat source genetic engineering antibody includes source of mouse variable region and cat source constant region, the nucleotides sequence of heavy chain variable region
Column are as shown in SEQ ID NO.1, and weight chain constant area gene sequence is as shown in SEQ ID NO.3, the nucleosides of light chain variable region
Acid sequence is as shown in SEQ ID NO.2, and light chain constant region gene sequence is as shown in SEQ ID NO.4.
The present invention also provides a kind of anti-feline panleucopenia virus cat source genetic engineering antibody ELISA detection methods, including with
Lower step:
S1, using the FPV-VLPs purifying antigen of preparation as envelope antigen, with carbonate buffer solution be diluted to 2 μ g/mL coating
ELISA Plate；
S2, standard items antibody are diluted to 0.25 μ g/mL with PBS, as standard curve initial concentration, 2 doubling dilution 7 later
A gradient draws standard curve；
Standard items preparation method for antibody are as follows: serum is separated from the cat blood of infection FPV, is identified as neutralizing the antibody of FPV,
FPV antibody is purified using purifying Protein A affinity column；First with the combination buffer of 10 times of column volumes
(20mM Na2HPO4 0.15M Nacl pH7.0) balances chromatographic column, then loading, and sample is centrifugation and 0.22 μm of membrane filtration
Cell express supernatant, flow velocity 1mL/min；Elution buffer (0.1M glycine HCI buffer pH2.7) is eventually adding to wash
It is de-；The sample of elution, 2.4mL/ pipe are collected with 5mL EP pipe, each EP pipe domestic demand is previously added neutralization buffer (pH=9.0
1M Tris-HCl) 600 μ L, collect, using 30KD albumen super filter tube be concentrated to get；
After S3, rinsing three times, the anti-cat IgG antibody of goat of 1:6000 times of diluted horseradish peroxidase-labeled is added；
S4, TMB colour developing, terminate liquid terminate, and each hole OD is reacted in measurement450Value, is judged according to criterion, wherein eventually
Only liquid is the sulfuric acid solution of 2M.
Preferably, in above-mentioned anti-feline panleucopenia virus cat source genetic engineering antibody ELISA detection method, step S1 coating
Temperature be 4 DEG C, the coating time be 12h.
Preferably, in above-mentioned anti-feline panleucopenia virus cat source genetic engineering antibody ELISA detection method, step S1 is also wrapped
The step of coated ELISA Plate is closed and rinsed is included, closed process specially includes that the carbonate buffer solution of 2%BSA exists
It closes 2h at 37 DEG C, after closed process, the PBS buffer solution washing comprising 0.05%Tween-20 is added three times.
Preferably, in above-mentioned anti-feline panleucopenia virus cat source genetic engineering antibody ELISA detection method, step S3 is also wrapped
The step of rinsing to ELISA Plate is included, after being incubated for enzyme labelled antibody process, it is slow that the PBS comprising 0.05%Tween-20 is added
Fliud flushing is washed three times.
Preferably, in above-mentioned anti-feline panleucopenia virus cat source genetic engineering antibody ELISA detection method, described in step S4
The determination method of criterion is as follows: with one group of negative control sample OD450Average value add 3 times of standard deviations as positive threshold values,
It is indicated with end point titres, i.e., sample is done serial dilution, highest dilution reactor can occurs and determine criterion, positive
The OD of reactor450> positive threshold value or P/N >=2.
Preferably, in above-mentioned anti-feline panleucopenia virus cat source genetic engineering antibody ELISA detection method, the anti-cat
Parvovirus cat source genetic engineering antibody includes source of mouse variable region and cat source constant region, the nucleotides sequence of heavy chain variable region
Column are as shown in SEQ ID NO.1, and weight chain constant area gene sequence is as shown in SEQ ID NO.3, the nucleosides of light chain variable region
Acid sequence is as shown in SEQ ID NO.2, and light chain constant region gene sequence is as shown in SEQ ID NO.4.
Compared with prior art, technical effect of the invention:
Anti- feline panleucopenia virus cat source provided by the invention genetic engineering antibody ELISA kit and its detection method are right
The detection of feline panleucopenia virus cat source genetic engineering antibody has very high sensibility, specificity and stability, tight to detecting
Anti- feline panleucopenia virus genetic engineering antibody is horizontal in the animal body of anxious immune front and back, assesses their immunity function, analyzing animal
Immune effect, whether judgement is immune succeeds, and provides strong support, achievees the effect that the course of disease controllable, to the diagnosis of disease with
Prognosis provides help, is of great significance to the development for pushing cat source antibody drug.
Detailed description of the invention
In order to illustrate the technical solutions in the embodiments of the present application or in the prior art more clearly, below will be to institute in embodiment
Attached drawing to be used is needed to be briefly described, it should be apparent that, the accompanying drawings in the following description is only one recorded in the present invention
A little embodiments are also possible to obtain other drawings based on these drawings for those of ordinary skill in the art.
Fig. 1 is the OD of the antigen of different operating concentration provided by the invention and the standard antibody of various concentration450Value figure；
Fig. 2 is the OD of the enzyme labelled antibody of different extension rates provided by the invention450Value figure；
Fig. 3 is specificity experiments result figure provided by the invention；
Fig. 4 is sensitivity experiments result figure provided by the invention.
Specific embodiment
Present invention firstly provides a kind of anti-feline panleucopenia virus cat source genetic engineering antibody ELISA kit, the examinations
Agent box includes the ELISA Plate and enzyme labelled antibody for being coated with antigen, and the antigen is FPV-VLPs purifying antigen, the enzyme labelled antibody
For the anti-cat IgG antibody of goat of horseradish peroxidase-labeled.The kit further includes confining liquid, rinsing liquid and terminate liquid,
The confining liquid is the PBS buffer solution comprising 2%BSA, and the rinsing liquid is the PBS buffer solution comprising 0.05%Tween-20,
Terminate liquid is the sulfuric acid solution of 2M.
The present invention also provides a kind of anti-feline panleucopenia virus cat source genetic engineering antibody ELISA detection methods, including with
Lower step:
S1, using the FPV-VLPs purifying antigen of preparation as envelope antigen, with carbonate buffer solution dilute coated elisa plate；
S2, standard items antibody dilute 2 doubling dilution, 7 gradients with PBS, draw standard curve；
After S3, rinsing three times, the anti-cat IgG antibody of goat of horseradish peroxidase-labeled is added；
S4, TMB colour developing, terminate liquid terminate, and each hole OD is reacted in measurement450Value, is judged according to criterion, wherein eventually
Only liquid is the sulfuric acid solution of 2M.
Wherein, the coated temperature of step S1 is 4 DEG C, and the coating time is 12h；Step S1 further include to coated ELISA Plate into
The step of row closing and rinsing, closed process specially include that the carbonate buffer solution of 2%BSA closes 2h at 37 DEG C, are closed
After process, the PBS buffer solution washing comprising 0.05%Tween-20 is added three times；Step S3 further includes carrying out to ELISA Plate
After being incubated for enzyme labelled antibody process, the PBS buffer solution washing comprising 0.05%Tween-20 is added three times in the step of rinsing；
The determination method of criterion described in step S4 is as follows: with one group of negative control sample OD450Average value add 3 times of standard deviations to make
It for positive threshold value, is indicated with end point titres, i.e., sample is done serial dilution, highest dilution reactor's determination can occurs and sentence
Calibration is quasi-, the OD of reactor450> positive threshold value or P/N >=2.
Preferably, anti-feline panleucopenia virus cat source to be detected genetic engineering antibody, including source of mouse variable region and cat source
Constant region, the nucleotide sequence of heavy chain variable region is as shown in SEQ ID NO.1, weight chain constant area gene sequence such as SEQ ID
Shown in NO.3, the nucleotide sequence of light chain variable region is as shown in SEQ ID NO.2, light chain constant region gene sequence such as SEQ
Shown in ID NO.4.
In order to make those skilled in the art more fully understand technical solution of the present invention, below in conjunction with attached drawing to this hair
It is bright to be further detailed.Obviously, described embodiments are only a part of the embodiments of the present invention, rather than whole
Embodiment.Based on the embodiments of the present invention, those of ordinary skill in the art are obtained without making creative work
The every other embodiment obtained, shall fall within the protection scope of the present invention.Experimental method in following embodiments, such as without special theory
It is bright, it is conventional method.Test material as used in the following examples is unless otherwise specified from conventional biochemical reagent quotient
What shop was commercially available.
1 antigen coat concentration of embodiment and standard curve initial concentration determine
Standard antigen FPV-VLPs albumen the preparation method comprises the following steps: recombinant baculovirus is inoculated with suspension by MOI=0.1 respectively
Sf9 cell, culture harvest after 4 days.The direct 3000rpm of the Sf9 cell for being vaccinated with FPV antigen is centrifuged 30min, abandons supernatant, carefully
Born of the same parents are with isometric 25mM NaHCO3It suspends, acts on 30min on ice, 3000rpm is centrifuged 30min, obtains supernatant, as contains FPV
The harvest liquid of virus-like particle carries out the measurement of hemagglutinative titer, HA 1:216 using pig blood.As the standard of this experiment
Antigen protein.
Standard items preparation method for antibody are as follows: serum is separated from the cat blood of infection FPV, is identified as neutralizing the antibody of FPV,
FPV antibody is purified using purifying Protein A affinity column；First with the combination buffer of 10 times of column volumes
(20mM Na2HPO4 0.15M Nacl pH7.0) balances chromatographic column, then loading, and sample is centrifugation and 0.22 μm of membrane filtration
Cell express supernatant, flow velocity 1mL/min；Elution buffer (0.1M glycine HCI buffer pH2.7) is eventually adding to wash
It is de-；The sample of elution, 2.4mL/ pipe are collected with 5mL EP pipe, each EP pipe domestic demand is previously added neutralization buffer (pH=9.0
1M Tris-HCl) 600 μ L, collect, using 30KD albumen super filter tube be concentrated to get；
Standard antigen FPV-VLPs albumen is pressed into 8 μ g/mL, 4 μ, g/mL, 2 μ g/mL ..., 0.00390625 μ g/mL concentration,
Doubling dilution from left to right, every 100 μ L of hole are placed in 4 DEG C of 12h.Standard items antibody presses 4 μ g/mL, 2 μ g/mL, 1 μ g/mL, 0.5 μ
0.0625 μ g/mL of g/mL ..., doubling dilution from top to bottom, using PBS as negative control.By single-factor variable method, same mark
The positive antibody standard items of different dilutions are added in quasi- antigen diluent degree, and 100 μ L are added in every hole, form square matrix.According to above-mentioned
ELISA detection method operating procedure is measured, positive group OD450Value >=1.0, negative control OD450It is determined when value < 0.1-0.2
Most suitable 2 μ g/mL of antigen coat concentration and 0.25 μ g/mL of standard curve initial concentration, as shown in Figure 1.
The determination of the anti-cat IgG antibody best effort concentration of the goat of 2 horseradish peroxidase-labeled of embodiment
According to above-mentioned ELISA detection method operating procedure and determine that most suitable 2 μ g/mL of antigen coat concentration and standard curve rise
The HRP anti-cat IgG antibody of goat marked is only diluted to 1:3000,1: 4000,1: 5000,1 by 0.25 μ g/mL of beginning concentration:
6000, it 1: 7000,1: 8000 is tested, remaining operation is constant.Retest 3 times, according to the OD of yin and yang attribute serum450Value is surveyed
Determine OD450When value is close to 1, the best effort concentration of the anti-cat IgG antibody of goat of HRP label is determined.I.e. when the goat of HRP label
When anti-cat IgG antibody is diluted by 1:6000 times, OD450Value is determined as best effort concentration, as shown in Figure 2 close to 1.
The preparation of 3 anti-feline panleucopenia virus cat source genetic engineering antibody ELISA kit of embodiment
According to the optimum optimizing condition that embodiment before determines, antigen FPV-VLPs albumen is pressed with 2 μ g/mL working concentrations
ELISA Plate, 4 DEG C of placement 12h are added according to 100 holes μ l/；After the PBS cleaning solution washing three times of 0.05%Tween-20, it is added 2%
Close 2h in 100 hole μ L/ BSA；It pats dry, is placed in hermetic bag.According to dosage by enzyme label antibody reagent, positive control sample and yin
Property control sample, rinsing liquid, developing solution, terminate liquid are sub-packed in reagent bottle.By ELISA Plate and reagent, sealing plate film is assembled into reagent
Box, 2-8 DEG C of preservation, is tested spare.
4 specificity experiments of embodiment
Adopt kit prepared with embodiment 3 according to above-mentioned ELISA detection method operating procedure and optimal conditions, respectively
Feline panleucopenia virus cat source genetic engineering antibody sample is added (to prepare using the method for application number 201910535002.3, concentration
For 100.58 μ g/mL), feline herpetovirus (Feline herpesvirus, FHV) and feline calicivirus (Feline
Calicivurus, FCV) (through cat FHV vaccine, cat FCV vaccine immunity 8-9 monthly age children cat, blood sampling separates blood to positive control serum
Positive serum obtained clearly), it is detected, as a result such as Fig. 3, it can be seen from the figure that FPV group is the positive, FHV group, FCV group are
It is negative.It follows that ELISA kit provided by the invention and its detection method are to FPV antiviral antibody with good special
Property.
5 sensitivity experiments of embodiment
Kit prepared with embodiment 3 is adopted according to above-mentioned ELISA detection method operating procedure and optimal conditions, only will
The antibody (being prepared using the method for application number 201910535002.3) of preparation is according to 1:400,1:800,1: 1600,1: 3200,1
: 6400,1:12800 dilution proportion, other operations are constant.Figure 4, it is seen that detection method provided by the invention is to sample
Sensibility be greater than 1:1600.Illustrate good using the sensibility of kit provided by the invention and its detection method test sample
It is good.
Embodiment 6 repeats to test
The kit of same batch and different 3 method of batch embodiment preparations is respectively adopted, detects FPV gene work respectively
Engineered antibody sample 1-4 (is prepared) using the method for application number 201910535002.3, and every part of sample is repeated 3 times, and the results are shown in Table 1 institute
Show.
1 repeated experiment result of table
A, B, C indicate duplicate measurements OD in measuring samples batch in table450Value；D, E and F indicates weight between measuring samples batch
Repetition measurement amount OD450Value.As can be seen from the table, the testing result coefficient of variation of kit is distinguished between kit and batch in batch
For 2.8%-3.1% and 4.2%-4.5%, it is therefore seen that the ELISA detection method and kit established have good precision
Degree.
It is above that certain exemplary embodiments of the invention are only described by way of explanation, undoubtedly, for ability
The those of ordinary skill in domain without departing from the spirit and scope of the present invention can be with a variety of different modes to institute
The embodiment of description is modified.Therefore, above-mentioned attached drawing and description are regarded as illustrative in nature, and should not be construed as to the present invention
The limitation of claims.
Sequence table
<110>Xinuo Biological Science & Technology Co., Ltd., Changchun
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cggagtgatg ctcagccatc tgtctttctc ttccaaccat ctctggacga gttacataca 60
ggaagtgcct ctatcgtgtg catattgaat gacttctacc ccaaagaggt caatgtcaag 120
tggaaagtgg atggcgtagt ccaaaacaaa ggcatccagg agagcaccac agagcagaac 180
agcaaggaca gcacctacag cctcagcagc accctgacga tgtccagtac ggagtaccaa 240
agtcatgaaa agttctcctg cgaggtcact cacaagagcc tggcctccac cctcgtcaag 300
agcttcaaca ggagcgagtg tcagagagag 330
Claims (10)
1. a kind of anti-feline panleucopenia virus cat source genetic engineering antibody ELISA kit, which is characterized in that the kit includes
It is coated with the ELISA Plate and enzyme labelled antibody of antigen, the antigen is FPV-VLPs purifying antigen, and the enzyme labelled antibody is horseradish mistake
The anti-cat IgG antibody of goat of oxide enzyme label.
2. anti-feline panleucopenia virus cat source as described in claim 1 genetic engineering antibody ELISA kit, which is characterized in that
The working concentration of the FPV-VLPs purifying antigen is 2 μ g/mL.
3. anti-feline panleucopenia virus cat source as described in claim 1 genetic engineering antibody ELISA kit, which is characterized in that
The extension rate of the enzyme labelled antibody is 1:6000.
4. anti-feline panleucopenia virus cat source as described in claim 1 genetic engineering antibody ELISA kit, which is characterized in that
The kit further includes confining liquid and rinsing liquid, and the confining liquid is the PBS buffer solution comprising 2%BSA, and the rinsing liquid is
PBS buffer solution comprising 0.05%Tween-20.
5. anti-feline panleucopenia virus cat source as described in claim 1 genetic engineering antibody ELISA kit, which is characterized in that
The anti-feline panleucopenia virus cat source genetic engineering antibody includes source of mouse variable region and cat source constant region, weight chain variable
The nucleotide sequence in area is as shown in SEQ ID NO.1, and weight chain constant area gene sequence is as shown in SEQ ID NO.3, light chain
The nucleotide sequence of variable region is as shown in SEQ ID NO.2, and light chain constant region gene sequence is as shown in SEQ ID NO.4.
6. a kind of anti-feline panleucopenia virus cat source genetic engineering antibody ELISA detection method, which is characterized in that including following step
It is rapid:
S1, using the FPV-VLPs purifying antigen of preparation as envelope antigen, with carbonate buffer solution be diluted to 2 μ g/mL coating enzyme mark
Plate；
S2, standard items antibody are diluted to 0.25 μ g/mL with PBS, as standard curve initial concentration, 2 doubling dilution, 7 ladders later
Degree draws standard curve；
After S3, rinsing three times, the anti-cat IgG antibody of goat of 1:6000 times of diluted horseradish peroxidase-labeled is added；
S4, TMB colour developing, terminate liquid terminate, and each hole OD is reacted in measurement450Value, is judged, wherein terminate liquid according to criterion
For the sulfuric acid solution of 2M.
7. anti-feline panleucopenia virus cat source as claimed in claim 6 genetic engineering antibody ELISA detection method, feature exist
In the coated temperature of step S1 is 4 DEG C, and the coating time is 12h.
8. anti-feline panleucopenia virus cat source as claimed in claim 6 genetic engineering antibody ELISA detection method, feature exist
In step S1 further includes the steps that coated ELISA Plate is closed and rinsed, and closed process includes specially 2%BSA's
Carbonate buffer solution closes 2h at 37 DEG C, after closed process, the PBS buffer solution comprising 0.05%Tween-20 is added and washes
It washs three times.
9. anti-feline panleucopenia virus cat source as claimed in claim 6 genetic engineering antibody ELISA detection method, feature exist
In the determination method of criterion described in step S4 is as follows: with one group of negative control sample OD450Average value add 3 times of standard deviations
It as positive threshold value, is indicated with end point titres, i.e., sample is done serial dilution, highest dilution reactor can occurs and determine
Criterion, the OD of reactor450> positive threshold value or P/N >=2.
10. anti-feline panleucopenia virus cat source as claimed in claim 6 genetic engineering antibody ELISA detection method, feature exist
In the anti-feline panleucopenia virus cat source genetic engineering antibody includes source of mouse variable region and cat source constant region, and heavy chain can
Become the nucleotide sequence in area as shown in SEQ ID NO.1, weight chain constant area gene sequence is light as shown in SEQ ID NO.3
The nucleotide sequence of chain variable region is as shown in SEQ ID NO.2, and light chain constant region gene sequence is as shown in SEQ ID NO.4.
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| CN113956362A (en) * | 2021-09-01 | 2022-01-21 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | Recombinant feline parvovirus VP2 protein antigen and application thereof in antibody diagnosis and vaccine preparation |
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Application publication date: 20191126 |