CN109061135A - A kind of method of preparation and use of dog early pregnancy detection reagent item - Google Patents
A kind of method of preparation and use of dog early pregnancy detection reagent item Download PDFInfo
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- CN109061135A CN109061135A CN201810963056.5A CN201810963056A CN109061135A CN 109061135 A CN109061135 A CN 109061135A CN 201810963056 A CN201810963056 A CN 201810963056A CN 109061135 A CN109061135 A CN 109061135A
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- 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
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- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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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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- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract
The present invention provides a kind of method of preparation and use of dog early pregnancy detection reagent item, the processing of sample pad: sample pad is placed in the 0.02mol/L PB buffer containing 1%BSA, 0.5%Tween-20,0.1%NaN3, is placed on abundant drying for standby in 60 DEG C of vacuum ovens after impregnating 3min；The preparation of fluorescent microsphere bonding pad: by the fluorescent nanometer microsphere of the fluorescent nanometer microsphere of RLN label and chicken IgG label, film instrument is drawn using Shanghai gold mark metal spraying and is sprayed on fluorescence bonding pad, 35 DEG C are dried overnight processing；This reagent fills up the domestic blank without dog early pregnancy detection reagent, realize qualitative, quantitative, the quickly detection of dog relaxain, it is 15 days most fast in post-coitum, it can be detected out and whether be pregnant, significantly detecting for 30 days earlier than external observation method, detection in 20 days of Palpation and ultrasonic wave.
Description
Technical field
The present invention is a kind of method of preparation and use of dog early pregnancy detection reagent item, belongs to genetic engineering field.
Background technique
In the prior art, there are many cyesiognosis method of bitch, and method more common at present has external observation method, palpation inspection
Look into method, supersonic sounding etc..Palpation method: when bitch is pregnant 20 days or so, uterus starts to become coarse, touches in stomach wall
It is thicker uterus diameter can obviously to be perceived.The talent that external observation method and Palpation are required to suitable experience can make relatively correct
Diagnosis.Gestation can touch fetus (such as having touched egg size, flexible meat ball) after 25 days.Ultrasonic listening
It can be used for finding the tire dog heartbeat of pregnancy 19-25 days.With the development of tire dog, accuracy rate increases, pregnancy 36-42 days
Accuracy rate is 85%.The too small bitch of figure, due to the bounce of abdominal artery, misdiagnosis rate is slightly higher, so needing a kind of new inspection
Survey method solves the above problems.
Summary of the invention
In view of the deficienciess of the prior art, it is an object of the present invention to provide a kind of preparation of dog early pregnancy detection reagent item and making
With method, to solve the problems mentioned in the above background technology, the present invention is easy to use, and convenient for operation, stability is good, reliability
It is high.
To achieve the goals above, the present invention is to realize by the following technical solutions: a kind of dog early pregnancy detection reagent
The preparation of item, includes the following steps:
S1: the processing of sample pad: sample pad is placed in containing 1%BSA, 0.5%Tween-20,0.1%NaN3
In 0.02mol/L PB buffer, abundant drying for standby in 60 DEG C of vacuum ovens is placed on after impregnating 3min；
S2: the preparation of fluorescent microsphere bonding pad: by the fluorescence nano of the fluorescent nanometer microsphere of RLN label and chicken IgG label
Microballoon is drawn film instrument using Shanghai gold mark metal spraying and is sprayed on fluorescence bonding pad, and 35 DEG C are dried overnight processing；
The preparation of S3:T, C line: using antibody diluent, (PBS buffer solution and volume ratio of pH7.4 is 0.05%
Proclin300 composition) goat IgG and RLN coated antibody R014 are diluted, film instrument, which is drawn, using metal spraying is uniformly sprayed on NC film
Nature controlling line and detection line on, two linear distance 5mm, sprayed after 35 DEG C be dried overnight it is spare；
S4: reagent strip assembling: sample pad, bonding pad, NC film, blotting paper are pasted on PVC bottom plate with successively overlapping, and are used
Cutting machine is cut into the test strips of 4.0mm wide, is placed in the Fresco Bag of desiccant and is sealed.
Further, the fluorescent nanometer microsphere is used by using partial size 200nm europium ion fluorescent microsphere, antibody label
Be carboxyl on EDC and NHS activation microballoon, the carboxyl after activation makes albumen in Eu-CM coupling in conjunction with the amino on albumen,
It is marked referring to microballoon specification, antibody microballoon is that experiment is marked in 1:10 according to mass ratio, with 0.05mol/l morpholine second
Sulfonate buffer (MES) centrifuge washing europium ion fluorescent microsphere 2 times, according to every milligram of microballoon be separately added into etc. quality EDC and
NHS, concussion reaction 30min, 12000g centrifugation removal supernatant (remove extra EDC and NHS) are buffered using MES at room temperature
Liquid cleaning precipitating 2 times, detection antibody E001 is added according to quantity, room temperature concussion reaction 2h, 12000g centrifugation removal supernatant is used
The washing of pH7.5PBS buffer is precipitated 2 times and is redissolved in PBS buffer solution, and Block buffer is then added (by 0.05mol/l
Tris, 5% bovine serum albumin(BSA) (w/v), 0.05%Triton X-100 (v/v) and 0.09% sodium azide (w/v) composition, envelope
The pH value for closing buffer is 7.5) to be closed, and 4 DEG C of closings overnight, are washed 2 times, 4 DEG C save standby use using PBS.
Further, the detection antibody E001 and coated antibody R014 passes through the dog relaxain and carrier protein that will be purified
KLH crosslinking, immune Balb/C mouse after being mixed in conventional manner with freund adjuvant, 50 micrograms/only, 2 weeks/time are subcutaneous by 5 times
After immune, potency is measured in 1:320000 or more, separating spleen is merged with myeloma cell, with the dog relaxain of purifying
For antigen, indirect elisa method screens relaxain monoclonal antibody, is matched with specificity clone of the A competitive inhibition method to acquisition
Right, screening obtains the detection antibody E001 and coated antibody R014 of pairing antibody.
Further, the dog relaxain is by adding Escherichia coli egg in N-terminal respectively for relaxain B peptide chain and A peptide chain
White signal peptide is designed to secretion peptide chain, in same expression vector in escherichia coli membrane circumferential play secreting, expressing, and in film periphery
The disulfide bond that B peptide chain and A peptide interchain are formed in the oxidation environment in gap, is self-assembled into mature dog relaxain.
A kind of application method of dog early pregnancy detection reagent item, uses antigenic dilution (PBS buffer solution+1%BSA+0.5%
Tween20) dilute serum sample, concussion mix, and take 100 μ l serum suspensions, are added in the well of detection card, after ten minutes
Detect the fluorescence signal of T, C line position respectively by fluorescence detector, the fluorescence signal of T, location of C are converted into digital letter by instrument
Breath, and the ratio of T, location of C fluorescence signal intensity are calculated, with ELISACalc Software on Drawing standard curve.
Beneficial effects of the present invention: a kind of method of preparation and use of dog early pregnancy detection reagent item of the invention, this reagent
Fill up the domestic blank for not having dog early pregnancy detection reagent, realize that the qualitative, quantitative, quick of dog relaxain detects, post-coitum most
Fast 15 days, it can be detected out and whether be pregnant, significantly 30 days earlier than external observation method, detection in 20 days of Palpation and ultrasonic wave
Detection.
Detailed description of the invention
Upon reading the detailed description of non-limiting embodiments with reference to the following drawings, other feature of the invention,
Objects and advantages will become more apparent upon:
Fig. 1 is a kind of stability data statistical chart of dog early pregnancy detection reagent item of the present invention；
Fig. 2 is RLN canonical plotting in a kind of dog early pregnancy detection reagent item of the present invention；
Specific embodiment
To be easy to understand the technical means, the creative features, the aims and the efficiencies achieved by the present invention, below with reference to
Specific embodiment, the present invention is further explained.
Referring to Fig. 1, the present invention provides a kind of technical solution: a kind of preparation of dog early pregnancy detection reagent item, including it is as follows
Step:
S1: the processing of sample pad: sample pad is placed in containing 1%BSA, 0.5%Tween-20,0.1%NaN3
In 0.02mol/L PB buffer, abundant drying for standby in 60 DEG C of vacuum ovens is placed on after impregnating 3min；
S2: the preparation of fluorescent microsphere bonding pad: by the fluorescence nano of the fluorescent nanometer microsphere of RLN label and chicken IgG label
Microballoon is drawn film instrument using Shanghai gold mark metal spraying and is sprayed on fluorescence bonding pad, and 35 DEG C are dried overnight processing；
The preparation of S3:T, C line: using antibody diluent, (PBS buffer solution and volume ratio of pH7.4 is 0.05%
Proclin300 composition) goat IgG and RLN coated antibody R014 are diluted, film instrument, which is drawn, using metal spraying is uniformly sprayed on NC film
Nature controlling line and detection line on, two linear distance 5mm, sprayed after 35 DEG C be dried overnight it is spare；
S4: reagent strip assembling: sample pad, bonding pad, NC film, blotting paper are pasted on PVC bottom plate with successively overlapping, and are used
Cutting machine is cut into the test strips of 4.0mm wide, is placed in the Fresco Bag of desiccant and is sealed.
Fluorescent nanometer microsphere uses EDC and NHS by using partial size 200nm europium ion fluorescent microsphere, antibody label
The carboxyl on microballoon is activated, the carboxyl after activation makes albumen in Eu-CM coupling in conjunction with the amino on albumen, referring to microballoon explanation
Book is marked, and antibody microballoon is that experiment is marked in 1:10 according to mass ratio, with 0.05mol/l morpholino b acid buffer
(MES) centrifuge washing europium ion fluorescent microsphere 2 times are separately added into etc. the EDC and NHS of quality according to every milligram of microballoon, at room temperature
Concussion reaction 30min, 12000g centrifugation removal supernatant (remove extra EDC and NHS) uses MES buffer solution for cleaning precipitating 2
It is secondary, detection antibody E001 is added according to quantity, room temperature concussion reaction 2h, 12000g centrifugation removal supernatant is washed with pH7.5PBS buffer
It washs precipitating 2 times and redissolves in PBS buffer solution, it is (pure by 0.05mol/l Tris, 5% ox blood that Block buffer is then added
Albumen (w/v), 0.05%Triton X-100 (v/v) and 0.09% sodium azide (w/v) composition, the pH value of Block buffer are
7.5) it is closed, 4 DEG C of closings overnight, are washed 2 times, 4 DEG C save standby use using PBS.
Detection antibody E001 and coated antibody R014 is by the way that the dog relaxain of purifying to be crosslinked with carrier protein KLH, with normal
Rule method immune Balb/C mouse after being mixed with freund adjuvant, 50 micrograms/only, 2 weeks/time, after 5 subcutaneous inoculations, measurement
Potency is merged in 1:320000 or more, separating spleen with myeloma cell, using the dog relaxain of purifying as antigen, indirectly
ELISA method screens relaxain monoclonal antibody, is matched with specificity clone of the A competitive inhibition method to acquisition, screening is matched
To the detection antibody E001 and coated antibody R014 of antibody.
Dog relaxain is designed to by the way that relaxain B peptide chain and A peptide chain are added e. coli protein signal peptide in N-terminal respectively
Peptide chain is secreted, in same expression vector in escherichia coli membrane circumferential play secreting, expressing, and in the oxidation environment of film circumferential play
The middle disulfide bond for forming B peptide chain and A peptide interchain, is self-assembled into mature dog relaxain.
A kind of application method of dog early pregnancy detection reagent item, uses antigenic dilution (PBS buffer solution+1%BSA+0.5%
Tween20) dilute serum sample, concussion mix, and take 100 μ l serum suspensions, are added in the well of detection card, after ten minutes
Detect the fluorescence signal of T, C line position respectively by fluorescence detector, the fluorescence signal of T, location of C are converted into digital letter by instrument
Breath, and the ratio of T, location of C fluorescence signal intensity are calculated, with ELISACalc Software on Drawing standard curve.
1. the foundation of standard curve
By the relaxain recombinant antigen of purchase, with Sample dilution accurate dilutions at 0,0.5,1,5,10,50ng/mL 6
Concentration.Fluorescence immune chromatography, each concentration Parallel testing 6 times, reaction time 10min are carried out with the antigen of above-mentioned concentration.With
RLN recombinant antigen standard concentration is abscissa, and T/C value is ordinate, draws standard curve, as shown in Figure 2.Quadruplex parameters
For y=(7.00464-0.01787)/[1+ (x/50.69831) -0.98241]+0.01787, R2=0.99991. chromatography examination
The lowest detection of agent is limited to 0.08ng/ml, detection sensitivity 0.15ng/ml.
2. the rate of recovery is tested
The RLN recombinant antigen of high, medium and low concentration is added into 10 parts of nogestational dog serums, measures the serum rate of recovery.?
Change between 100.14%-110.71% to RLN in the rate of recovery in serum.
The experiment of 2 rate of recovery of table
3. Precision Experiment
It is measured using high, medium and low three standard concentration of the detection kit of the present invention to accurate quantification, it is each to be arranged
10 each multiple holes.As shown in table 2, the variation within batch coefficient of this kit and interassay coefficient of variation are smaller, meet kit regulation
It is required that.
4. stability experiment
This research determines the stability of this immunochromatography reagent, the detection reagent that will be prepared using thermostabilization Acceleration study
It is placed 30 days after sealing in 37 DEG C of electric drying oven with forced convections, the RLN weight of every 5 days detections 10ng/ml, 50ng/ml and 200ng/ml
Group antigen standard, investigates the acceleration for stabilization implementations of this method.The result is shown in Figure 1,37 DEG C accelerate to be equivalent within 30 days 4 DEG C and place to surpass
Spend 2 years.Therefore this immunochromatography reagent can keep relative stability in 4 DEG C of preservations.
Embodiment 1: using antigenic dilution dilute serum sample (15 days and 20 days serum samples it is undiluted, 25 days serum
2 times of Sample Dilution, 30 days and 60 days serum samples dilute 5 times), concussion mixes.Take 100 μ l samples that the well of detection card is added
In, it detects the fluorescence signal of T, C line position respectively by fluorescence detector after ten minutes, it is strong to calculate T, location of C fluorescence signal
The ratio of degree.Meanwhile using the standard items in kit, standard curve is drawn, the RLX of each sample is calculated according to standard curve
Concentration, wherein 15 days pregnant dog serum relaxain concentration of gestation is 0.22 ± 0.05ng/ml, 20 days pregnant dog serum relaxain concentration
For 0.35 ± 0.07ng/ml, 25 days pregnant dog serum relaxain concentration is 4.85 ± 1.23ng/ml, 30 days pregnant dog serum relaxains
Concentration is 100.42 ± 22.8ng/ml, and 60 days pregnant dog serum relaxain concentration is 536.46 ± 104.85ng/ml, present invention examination
Agent item can be used for the detection of dog early pregnancy and dog childbirth monitoring.
The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention, for this field skill
For art personnel, it is clear that invention is not limited to the details of the above exemplary embodiments, and without departing substantially from spirit of the invention or
In the case where essential characteristic, the present invention can be realized in other specific forms.Therefore, in all respects, should all incite somebody to action
Embodiment regards exemplary as, and is non-limiting, the scope of the present invention by appended claims rather than on state
Bright restriction, it is intended that including all changes that fall within the meaning and scope of the equivalent elements of the claims in the present invention
It is interior.Any reference signs in the claims should not be construed as limiting the involved claims.
In addition, it should be understood that although this specification is described in terms of embodiments, but not each embodiment is only wrapped
Containing an independent technical solution, this description of the specification is merely for the sake of clarity, and those skilled in the art should
It considers the specification as a whole, the technical solutions in the various embodiments may also be suitably combined, forms those skilled in the art
The other embodiments being understood that.
Claims (5)
1. a kind of preparation of dog early pregnancy detection reagent item, it is characterised in that include the following steps:
S1: sample pad the processing of sample pad: is placed in the 0.02mol/L containing 1%BSA, 0.5%Tween-20,0.1%NaN3
In PB buffer, abundant drying for standby in 60 DEG C of vacuum ovens is placed on after impregnating 3min；
S2: the preparation of fluorescent microsphere bonding pad: the fluorescent nanometer microsphere that the fluorescent nanometer microsphere of RLN label and chicken IgG are marked,
It draws film instrument using Shanghai gold mark metal spraying to be sprayed on fluorescence bonding pad, 35 DEG C are dried overnight processing；
The preparation of S3:T, C line: using antibody diluent, (PBS buffer solution and volume ratio of pH7.4 is 0.05%Proclin300
Composition) goat IgG and RLN coated antibody R014 are diluted, using metal spraying draw film instrument be uniformly sprayed on NC film nature controlling line and
In detection line, two linear distance 5mm, sprayed after 35 DEG C be dried overnight it is spare；
S4: reagent strip assembling: sample pad, bonding pad, NC film, blotting paper are pasted on PVC bottom plate with successively overlapping, with slitting
Machine-cut is placed in the Fresco Bag of desiccant and is sealed at the test strips of 4.0mm wide.
2. a kind of preparation of dog early pregnancy detection reagent item according to claim 1, it is characterised in that: the fluorescence nano is micro-
For ball by using partial size 200nm europium ion fluorescent microsphere, antibody label uses EDC and NHS to activate the carboxyl on microballoon, living
Carboxyl after change makes albumen in Eu-CM coupling in conjunction with the amino on albumen, is marked referring to microballoon specification, antibody microballoon
It is that experiment is marked in 1:10 according to mass ratio, it is glimmering with 0.05mol/l morpholino b acid buffer (MES) centrifuge washing europium ion
Light microballoon 2 times, the EDC and NHS of quality are separately added into etc. according to every milligram of microballoon, at room temperature concussion reaction 30min, 12000g from
The heart removes supernatant (remove extra EDC and NHS), is precipitated 2 times using MES buffer solution for cleaning, detection antibody is added according to quantity
E001, room temperature concussion reaction 2h, 12000g centrifugation removal supernatant, washs precipitating 2 times with pH7.5PBS buffer and redissolves in PBS
In buffer, Block buffer is then added (by 0.05mol/l Tris, 5% bovine serum albumin(BSA) (w/v), 0.05%
Triton X-100 (v/v) and 0.09% sodium azide (w/v) composition, the pH value of Block buffer are 7.5) to be closed, 4 DEG C
Closing overnight, is washed 2 times, 4 DEG C save standby use using PBS.
3. a kind of preparation of dog early pregnancy detection reagent item according to claim 1, it is characterised in that: the detection antibody
E001 and coated antibody R014, which pass through, is crosslinked the dog relaxain purified with carrier protein KLH, in conventional manner with freund adjuvant
After mixing be immunized Balb/C mouse, 50 micrograms/only, 2 weeks/time, after 5 subcutaneous inoculations, measure potency 1:320000 with
On, separating spleen is merged with myeloma cell, and using the dog relaxain of purifying as antigen, indirect elisa method screens relaxain
Monoclonal antibody is matched with specificity clone of the A competitive inhibition method to acquisition, and screening obtains the detection antibody of pairing antibody
E001 and coated antibody R014.
4. a kind of preparation of dog early pregnancy detection reagent item according to claim 3, it is characterised in that: the dog relaxain is logical
It crosses and relaxain B peptide chain and A peptide chain is designed to secretion peptide chain in N-terminal addition e. coli protein signal peptide respectively, in same table
Up to carrier in escherichia coli membrane circumferential play secreting, expressing, and B peptide chain and A peptide chain are formed in the oxidation environment of film circumferential play
Between disulfide bond, be self-assembled into mature dog relaxain.
5. a kind of application method of dog early pregnancy detection reagent item according to claim 3, it is characterised in that: dilute using antigen
Liquid (PBS buffer solution+1%BSA+0.5%Tween20) dilute serum sample is released, concussion mixes, takes 100 μ l serum suspensions, add
In the well for entering detection card, the fluorescence signal of T, C line position is detected by fluorescence detector respectively after ten minutes, instrument by T,
The fluorescence signal of location of C is converted into digital information, and calculates the ratio of T, location of C fluorescence signal intensity, soft with ELISACalc
Part draws standard curve.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109813893A (en) * | 2019-01-28 | 2019-05-28 | 深圳市亚辉龙生物科技股份有限公司 | CEA detection test strip and preparation method thereof, CEA detection device |
| WO2023041845A1 (en) * | 2021-09-14 | 2023-03-23 | Bellylabs Oy | Assay method for relaxin |
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| WO1991002251A1 (en) * | 1989-08-07 | 1991-02-21 | International Canine Genetics, Inc. | Detection of pregnancy by identification of the c peptide of relaxin in body fluids of animals |
| US5108897A (en) * | 1988-09-30 | 1992-04-28 | Cornell Research Foundation, Inc. | Relaxin testing for early detection of pregnancy in dogs |
| CN104730245A (en) * | 2014-11-28 | 2015-06-24 | 威海纽普生物技术有限公司 | D-dimer detection kit and D-dimer detection method |
| CN106811470A (en) * | 2015-11-30 | 2017-06-09 | 四川农业大学 | A kind of preparation method of dog relaxation precipitinogen recombinant protein and dog relaxation precipitinogen polyclonal antibody |
| CN108318685A (en) * | 2018-05-04 | 2018-07-24 | 广州敏捷生物技术有限公司 | Immunofluorescence for detecting canine coronavirus antigen chromatographs detection card and preparation method |
-
2018
- 2018-08-22 CN CN201810963056.5A patent/CN109061135A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5108897A (en) * | 1988-09-30 | 1992-04-28 | Cornell Research Foundation, Inc. | Relaxin testing for early detection of pregnancy in dogs |
| WO1991002251A1 (en) * | 1989-08-07 | 1991-02-21 | International Canine Genetics, Inc. | Detection of pregnancy by identification of the c peptide of relaxin in body fluids of animals |
| CN104730245A (en) * | 2014-11-28 | 2015-06-24 | 威海纽普生物技术有限公司 | D-dimer detection kit and D-dimer detection method |
| CN106811470A (en) * | 2015-11-30 | 2017-06-09 | 四川农业大学 | A kind of preparation method of dog relaxation precipitinogen recombinant protein and dog relaxation precipitinogen polyclonal antibody |
| CN108318685A (en) * | 2018-05-04 | 2018-07-24 | 广州敏捷生物技术有限公司 | Immunofluorescence for detecting canine coronavirus antigen chromatographs detection card and preparation method |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109813893A (en) * | 2019-01-28 | 2019-05-28 | 深圳市亚辉龙生物科技股份有限公司 | CEA detection test strip and preparation method thereof, CEA detection device |
| WO2023041845A1 (en) * | 2021-09-14 | 2023-03-23 | Bellylabs Oy | Assay method for relaxin |
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