CN104865381A - Pig early pregnancy factor colloidal gold test paper card and preparation method thereof - Google Patents
Pig early pregnancy factor colloidal gold test paper card and preparation method thereof Download PDFInfo
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Abstract
The present invention aims to provide a pig early pregnancy factor colloidal gold test paper card for rapid detection of sow early pregnancy factor and clinical diagnosis of sow early pregnancy. The invention is innovative that the cloning technology is employed for artificial synthesis of early pregnancy factor protein with natural early pregnancy factor activity, and a pig early pregnancy factor monoclonal antibody with high sensitivity and good characteristics is obtained on the basis of the protein; and after test paper card assembly condition optimization, the pig early pregnancy factor colloidal gold test paper card is obtained. The test paper card has the advantages of high specificity and sensitivity, and quick and easy detection; the whole testing process does not require specialized equipment, and has low multi-operator requirement; and the test paper card is low in cost and easy to operate, can be widely used in the diagnosis of pig early pregnancy, and lays foundation for understand of pregnant situation of the sows and diagnosis of sow pseudopregnancy.
Description
Technical field
The present invention relates to animal medicine immune detection, diagnostic field, specifically relate to and a kind ofly detect colloidal gold test paper card of pig early pregnancy factor and preparation method thereof.
Background technology
Use diagnosis of early gestation method to improving the very helpful of sow reproductive efficiency and pig industry productivity effect, after insemination of sows, if gestation can be carried out as early as possible make a definite diagnosis, just can reduce the nonpregnant phase of sow or non-breeding number of days, increase the average year farrowing nest number of sow, be conducive to simultaneously to breeding difficulty sow and early treatment process.And the accuracy rate of diagnosis instability had in the diagnosis of early gestation method generally adopted now, the method for operating complex steps had, the use expensive equipment had, have high to operating personnel's skill requirement, in numerous detection method quick detection paper can possess simple to operate, sensitivity is high, specificity good, the plurality of advantages such as cheap and possess application value.
Summary of the invention
Fundamental purpose of the present invention is to provide a boar early pregnancy factor colloidal gold test card, detects sow early pregnancy factor fast, for the clinical diagnosis of sow early pregnancy.Innovative point of the present invention is to utilize gene clone technology, Prof. Du Yucang has the pig early pregnancy factor albumen of natural early pregnancy factor activity, obtain the mouse source pig early pregnancy factor monoclonal antibody that susceptibility is high, characteristic is good on this basis, obtain pig early pregnancy factor colloidal gold test card through test card assembling.
The technical solution adopted in the present invention is:
1, pig early pregnancy factor colloidal gold test card, is characterized in that:
Described pig early pregnancy factor colloidal gold test card is made up of colloidal gold strip and buckle type draw-in groove; Buckle type draw-in groove has the build-in cavities of an accommodating colloidal gold strip, and buckle type draw-in groove is provided with observation area and sample application zone; Colloidal gold strip is primarily of support plate, NC film (nitrocellulose filter), gold mark monoclonal antibody pad, sample pad and absorption pad composition;
Described NC film is arranged on the middle part of support plate, the support plate of NC film side is pasted with successively containing gold mark monoclonal antibody pad and sample pad, the support plate of NC film opposite side is pasted with absorption pad;
There is detection line and a nature controlling line resisted containing sheep anti mouse two containing pig early pregnancy factor polyclonal antibody at the middle part of described NC film;
Colloidal gold strip is arranged in the build-in cavities of draw-in groove, the colour developing district of the corresponding colloidal gold strip in observation area of draw-in groove, the sample pad of the corresponding colloidal gold strip of well;
In described load sample pad, gold mark monoclonal antibody is mouse source pig early pregnancy factor monoclonal antibody; In described NC film, pig early pregnancy factor antibody is rabbit anti-pig early pregnancy factor polyclonal antibody; In described NC film, sheep anti mouse two resists for sheep anti-mouse igg antibody.
The preparation method of 2, above-mentioned pig early pregnancy factor colloidal gold test card, is characterized in that: comprise the following steps:
(1) NC film is prepared
The preparation of rabbit anti-pig early pregnancy factor polyclonal antibody: adopt prokaryotic expression the pig early pregnancy factor albumen of purifying as immunogene, extract IgG antibody, prepare polyclonal antibody;
The preparation of detection line: adopt three-dimensional spray film instrument, adjustment spouting liquid is 45 ~ 50dots/mL/cm, is buffered liquid dilution rabbit anti-pig early pregnancy factor polyclonal antibody with bag, concentration is 45 ~ 50 μ g/mL, in NC film, appropriate location line, is detection line, dries for subsequent use; Described bag is buffered the CBS damping fluid (carbonate buffer solution) that liquid is 0.05 ~ 0.07M through 0.40 ~ 0.45 μ L membrane filtration, pH9.6;
The preparation of nature controlling line: adopt three-dimensional spray film instrument, adjustment spouting liquid is 45 ~ 48dots/ml/cm, is buffered liquid dilution sheep anti-mouse igg antibody with bag, concentration is 1mg/ml, position line parallel with detection line on NC film, and detection line interval 4 ~ 5mm, be nature controlling line;
NC film assembling preparation: dry detection line and nature controlling line, obtain the NC film containing detection line and nature controlling line; Dry in room temperature 72h with the NC film of closed working fluid by preparation, seal up for safekeeping for subsequent use; Described closed working fluid is the closed working fluid obtained with 0.45 ~ 0.48ul membrane filtration after the CBS damping fluid mixing containing the 2.5%BSA of mass percent, 2.5 ~ 3.0% skimmed milk powers and 0.01M, pH7.0.
(2) coated with gold mark monoclonal antibody pad is prepared
Containing mass percent with 20mM is 1.5%BSA, 0.1%NaN
3with 3% sodium tetraborate dilution buffer (aqueous solution), gold is marked monoclonal antibody and be diluted to final concentration 0.05mg/ml; Cut the glass fibre cotton that specification is 300 × 7mm, gold is marked the unidirectional specking instrument of monoclonal antibody solution, with the speed of 5 μ L/cm (about 20ng/ bar), evenly be sprayed on glass fibre cotton, 56 DEG C of dry 1h, after adding drying agent vacuum seal, room temperature preservation is for subsequent use.
(3) pig early pregnancy factor colloidal gold test card is assembled
The NC film scribbling detection line and nature controlling line is pasted onto the middle part of support plate, the glass fibre membrane containing gold mark monoclonal antibody and sample pad is pasted successively in the side of NC film, absorption pad is pasted at the opposite side of NC film, obtain test paper plate, test paper plate is cut into the test strips of different in width, test strips is placed in draw-in groove and is pig early pregnancy factor colloidal gold test card.
(4) pig early pregnancy factor colloidal gold test card uses and judges
Pig early pregnancy factor colloidal gold test is stuck in diagnosis pig early pregnancy and applies, separation of serum, the serum of gained is dripped in the sample pad of this test card, sample moves along on adsorptive pads, when running into gold mark monoclonal antibody, pig early pregnancy factor is marked mouse-anti pig early pregnancy factor monoclonal antibody be combined with golden, Ag-Ab-Au composite moves to detection line and nature controlling line, the rabbit of pig early pregnancy factor on detection line anti-pig early pregnancy factor polyclonal antibody is combined, form a red detection line, can not in conjunction with gold mark mouse-anti pig early pregnancy factor monoclonal antibody without pig early pregnancy factor in negative serum, so occur without macroscopic detection line, nature controlling line takes on a red color forever, if colourless, represents that test strips lost efficacy.
Further, in step (1), the live width of detection line and nature controlling line is 1mm, and two lines, at a distance of 4mm, are positioned at the centre of film, apart from the back gauge 6 ~ 7mm of NC film.
Beneficial effect:
Colloidal gold test paper card of the present invention utilizes up-to-date epitope screening technology, and Prof. Du Yucang has the pig early pregnancy factor albumen of natural early pregnancy factor activity, obtains the pig early pregnancy factor monoclonal antibody that susceptibility is high, characteristic is good on this basis.Then according to modern times international state-of-the-art colloidal gold immunochromatographimethod technology, optimize test card assembling condition, by NC film in test card sprays the detection line containing rabbit anti-pig early pregnancy factor polyclonal antibody and the nature controlling line containing sheep anti-mouse igg antibody respectively, can clinical detection pig early pregnancy factor, for enterprise of raising pigs provides a kind of test card of clinical diagnosis sow early pregnancy.This test card has high specificity, advantage fast highly sensitive, easy to detect, do not need to use special instrument equipment in whole testing process, multioperation personnel requirement is low, and test card is with low cost, easy to operate, the diagnosis of pig early pregnancy can be widely used in, lay a good foundation for understanding the pregnant feelings of sow and carrying out diagnosis to sow false pregnancy.
Accompanying drawing explanation
Fig. 1 is the structural representation of colloidal gold test paper card of the present invention;
In figure: 1, buckle type draw-in groove 2, support plate, 3, well 4, sample pad 5, golden labeling antibody pad 6, NC film 7, observation area 8, absorption pad
Embodiment
Embodiment 1
Pig early pregnancy factor colloidal gold test card is made up of colloidal gold strip and buckle type draw-in groove; Buckle type draw-in groove has the build-in cavities of an accommodating colloidal gold strip, and buckle type draw-in groove is provided with observation area and sample application zone; Colloidal gold strip is primarily of support plate, NC film, gold mark monoclonal antibody pad, sample pad and absorption pad composition; Described NC film is arranged on the middle part of support plate, the support plate of NC film side is pasted with successively containing gold mark monoclonal antibody pad and sample pad, the support plate of NC film opposite side is pasted with absorption pad; There is detection line and a nature controlling line resisted containing sheep anti mouse two containing pig early pregnancy factor polyclonal antibody at the middle part of described NC film; Colloidal gold strip is arranged in the build-in cavities of draw-in groove, the colour developing district of the corresponding colloidal gold strip in observation area of draw-in groove, the sample pad of the corresponding colloidal gold strip of well; In described load sample pad, gold mark monoclonal antibody is mouse source pig early pregnancy factor monoclonal antibody; In described NC film, pig early pregnancy factor antibody is rabbit anti-pig early pregnancy factor polyclonal antibody; In described NC film, sheep anti mouse two resists for sheep anti-mouse igg antibody.
Embodiment 2
The preparation method of pig early pregnancy factor colloidal gold test card, comprises the following steps:
(1) NC film is prepared
The preparation of rabbit anti-pig early pregnancy factor polyclonal antibody: adopt prokaryotic expression the pig early pregnancy factor albumen of purifying as immunogene, extract IgG antibody, prepare polyclonal antibody;
The preparation of detection line: adopt three-dimensional spray film instrument, adjustment spouting liquid is 45 ~ 50dots/mL/cm, is buffered liquid dilution rabbit anti-pig early pregnancy factor polyclonal antibody with bag, concentration is 45 ~ 50 μ g/mL, in NC film, appropriate location line, is detection line, dries for subsequent use; Described bag is buffered the CBS damping fluid that liquid is 0.05 ~ 0.07M through 0.40 ~ 0.45 μ L membrane filtration, pH9.6;
The preparation of nature controlling line: adopt three-dimensional spray film instrument, adjustment spouting liquid is 45 ~ 48dots/ml/cm, is buffered liquid dilution sheep anti-mouse igg antibody with bag, concentration is 1mg/ml, position line parallel with detection line on NC film, and detection line interval 4 ~ 5mm, be nature controlling line;
NC film assembling preparation: dry detection line and nature controlling line, obtain the NC film containing detection line and nature controlling line; Dry in room temperature 72h with the NC film of closed working fluid by preparation, seal up for safekeeping for subsequent use; Described closed working fluid is the closed working fluid obtained with 0.45 ~ 0.48ul membrane filtration after the CBS damping fluid mixing containing the 2.5%BSA of mass percent, 2.5 ~ 3.0% skimmed milk powers and 0.01M, pH7.0.
(2) coated with gold mark monoclonal antibody pad is prepared
Containing mass percent with 20mM is 1.5%BSA, 0.1%NaN
3with 3% sodium tetraborate dilution buffer, gold is marked monoclonal antibody and be diluted to final concentration 0.05mg/ml; Cut the glass fibre cotton that specification is 300 × 7mm, gold is marked the unidirectional specking instrument of monoclonal antibody solution, with the speed of 5 μ L/cm (about 20ng/ bar), evenly be sprayed on glass fibre cotton, 56 DEG C of dry 1h, after adding drying agent vacuum seal, room temperature preservation is for subsequent use.
(3) pig early pregnancy factor colloidal gold test card is assembled
The NC film scribbling detection line and nature controlling line is pasted onto the middle part of support plate, the glass fibre membrane containing gold mark monoclonal antibody and sample pad is pasted successively in the side of NC film, absorption pad is pasted at the opposite side of NC film, obtain test paper plate, test paper plate is cut into the test strips of different in width, test strips is placed in draw-in groove and is pig early pregnancy factor colloidal gold test card.
(4) pig early pregnancy factor colloidal gold test card uses and judges
Pig early pregnancy factor colloidal gold test is stuck in diagnosis pig early pregnancy and applies, separation of serum, the serum of gained is dripped in the sample pad of this test card, sample moves along on adsorptive pads, when running into gold mark monoclonal antibody, pig early pregnancy factor is marked mouse-anti pig early pregnancy factor monoclonal antibody be combined with golden, Ag-Ab-Au composite moves to detection line and nature controlling line, the rabbit of pig early pregnancy factor on detection line anti-pig early pregnancy factor polyclonal antibody is combined, form a red detection line, can not in conjunction with gold mark mouse-anti pig early pregnancy factor monoclonal antibody without pig early pregnancy factor in negative serum, so occur without macroscopic detection line, nature controlling line takes on a red color forever, if colourless, represents that test strips lost efficacy.
Wherein, in step (1), the live width of detection line and nature controlling line is 1mm, and two lines, at a distance of 4mm, are positioned at the centre of film, apart from the back gauge 6 ~ 7mm of NC film.
Embodiment 3
1 prepares mouse-anti pig early pregnancy factor monoclonal antibody, rabbit anti-pig early pregnancy factor polyclonal antibody
The preparation of 1.1 mouse-anti pig early pregnancy factor gene monoclonal antibodies
According to space conformation and the Antigen Epitope Prediction of (pig early pregnancy factor) gene nucleotide series of pig EPF in GenBank and international current research EPF, Primer Premier 5.0 Software for Design is utilized to go out EPF upstream primer 5'-CGGGATCCATGGCAGGACAAGCTTTTAG-3 ' downstream primer 5'-CCG CTCGAGGGATCCTCTTGGAACCAAGTCGACGTACTTTCCAAGA-3 '.With Animal muscles satellite cell for material, utilize classical TRIzol method to extract total serum IgE, RT-PCR obtains DNA.Utilization designs primer PCR method and obtains the EPF genetic fragment that size is 341bp.Obtaining genetic fragment, to send to the raw work sequencing result display in Shanghai and pig EPF homology be 99.9% not morph.
The structure of 1.2 PET-28 α-EPF/BL21 expression vectors and protein expression
Be that substrate carries out pcr amplification object fragment with PMD-19T-EPF, glue reclaims rear Xho1, BamH1 double digestion, T4 connects, proceed to DH5a, bacterium colony PCR verifies positive bacterium colony, be labeled as PET28 α-EPF/DH5a and extract plasmid PET28 α-EPF, double digestion is verified.Double digestion is verified that correct plasmid checks order.Proceeded to by plasmid PET28 α-EPF and express bacterium BL21, bacterium colony PCR confirms successful construction of expression vector PET-28 α-EPF/BL21.With expression vector PET-28 α-EPF/BL21 for material, IPTG is added by 0.1% amount, in LB nutrient culture media, carry out prokaryotic expression, expression product obtains and expects the consistent product of albumen size 16Kb object band after ultrasound wave process, and destination protein all has great expression in precipitation and supernatant.
The purifying of 1.3 PET-28 α-EPF/BL21 expressing proteins.Adopt NI-NTA to carry out nickel as filler and live purifying, result display purification effect is good, and destination protein purity is higher, and obtaining destination protein concentration through protein concentration detection kit is 450ug/ml.
The preparation of the anti-PET-28 α of 1.4 rabbit-EPF/BL21 protein polyclonal blood serum antibody
After purifying, EPF albumen is after dialysis treatment, collects serum ,-20 DEG C of preservations.
The preparation of 1.5 mouse-anti pig early pregnancy factor monoclonal antibodies
Get mouse spleen lymphocyte and add Macrogol 2000 with the SP2/0 cell of the DMEM medium culture containing 10% calf serum in 10:1 ratio and merge, the supernatant ELISA method merged in hole is detected, determines positive hole.Adopt limiting dilution assay to carry out subclone the hybridoma in the positive hole detected, until the positive rate of hybridoma supernatant reaches 100%, set up hybridoma cell line.Will build be hybridoma expand cultivate, extract and concentrated supernatant in antibody, obtain monoclonal antibody.
2 preparation NC films
The preparation of 2.1 detection line reagent
Taken out from-80 DEG C of refrigerators by anti-for rabbit pig early pregnancy factor polyclonal antibody, equilibrium at room temperature 20min, is mixed with 1.0mg/ml with sterile saline, for subsequent use with 0.22 μm of filtering with microporous membrane.
The preparation of 2.2 nature controlling line reagent
Buy commercial sheep anti-mouse igg two to resist ,-80 DEG C of Refrigerator stores.With 0.22 μm of filtering with microporous membrane before using.
The preparation of 2.3 detection lines and nature controlling line
NC film is put on three-dimensional spray film instrument platform, puts press strip and be fixed; Be that the rabbit anti-pig early pregnancy factor polyclonal antibody of 1.0mg/ml is put in special container by concentration, to be that the sheep anti-mouse igg two of 0.54mg/ml is anti-be put in another container to concentration; Open three-dimensional spray film instrument power supply, start the machine, by anti-for rabbit pig early pregnancy factor polyclonal antibody and the anti-respectively fixed fire of sheep anti-mouse igg two on film, about 50dots/ml/cm, forms two lines, is respectively detection line (T line) and nature controlling line (C line), live width is about 1mm, two lines, at a distance of 4mm, are positioned at the centre of film, and the back gauge apart from film is about 7mm.After natural drying at room temperature 72h, add drying agent sealing, room temperature preservation is for subsequent use.
3 preparation gold mark monoclonal antibody pads
The preparation of 3.1 mouse-anti pig early pregnancy factor monoclonal antibodies
Adopt prokaryotic expression the pig early pregnancy factor albumen of purifying as immunogene, get after spleen cell and SP2/0 oncocyte merge and obtain monoclonal antibody; Be adjusted to 0.54mg/ml with physiological saline, with 0.22 μm of filtering with microporous membrane ,-20 DEG C save backup.
The preparation of 3.2 gold medal mark mouse-anti pig early pregnancy factor monoclonal antibodies
3.2.1 pre-treatment is marked: will treat that mark mouse-anti pig early pregnancy factor monoclonal antibody solution loads in bag filter, dislysate is done with physiological saline, in 4 DEG C of numerical control chromatography freezers, magnetic stirrer dialysis 48 ~ 72h, changes 1 dislysate at interval of 6h, takes out the mouse-anti pig early pregnancy factor monoclonal antibody solution after dialysis, the centrifugal 20min of 10000r/min, removing impurity, physiological saline adjustment concentration is to 1.0mg/ml, and-20 DEG C frozen for subsequent use.Collaurum 0.1mol/L K
2cO
3solution regulates the pH to 7.5 of colloidal gold solution, and room temperature preservation is for subsequent use.
3.2.3 the determination of optimum antibody (mouse-anti pig early pregnancy factor monoclonal antibody) labelled amount
Get ELISA Plate 10 hole, every hole 30 μ l DDW shop fixtures, it is that the mouse-anti pig early pregnancy factor monoclonal antibody 10 μ l of 1mg/ml adds 20 μ l DDW that the first hole adds concentration, and carry out multiple proportions afterwards to the 9th hole, the 10th hole is blank, every hole adds the colloidal gold solution of 125 μ l pH 7.5, room temperature reaction 5 ~ 10min, add 100g/L NaCl solution 125 μ l, room temperature reaction 10min, observe colour developing situation, control wells and mouse-anti pig early pregnancy factor monoclonal antibody quantity not sufficient present by the coagulation phenomenon of red stain indigo plant with each hole of stable aurosol, and the mouse-anti pig early pregnancy factor monoclonal anti scale of construction meets or exceeds the quantitative each pipe of minimum steady and still keeps red constant, the hole of the minimum mouse-anti pig early pregnancy factor monoclonal anti scale of construction selecting color not change is best standard gold amount, in this hole, the amount of mouse-anti pig early pregnancy factor monoclonal antibody is multiplied by the amount that 8 are the mouse-anti pig early pregnancy factor monoclonal antibody needed for 1mL collaurum.
3.2.4 colloid gold label mouse-anti pig early pregnancy factor monoclonal antibody
Appropriate mouse-anti pig early pregnancy factor monoclonal antibody solution is added lentamente in the colloidal gold solution of 100mL pH 7.5, after mixing, RT incubation 40min; Slowly adding 10%BSA to its final concentration is 1% (W/V), and after mixing, RT hatches 15min; 12000r/min, 4 DEG C, centrifugal 25min, abandons supernatant; Precipitate resuspended with the BSA of 2%, 12000r/min, 4 DEG C of centrifugal 25min, abandon supernatant; Precipitation TB damping fluid adjusts golden labeling antibody to 0.2mg/mL, and fully after piping and druming, 4 DEG C save backup.
3.2.5 coated with gold labeling antibody pad is prepared
Containing 1.5%BSA and 0.1%NaN3 sodium tetraborate dilution buffer, golden labeling antibody is diluted to final concentration 0.05mg/ml with 20mM; Cut the glass fibre cotton that specification is 300 × 7mm, by the unidirectional specking instrument of golden labeling antibody solution, with the speed of 5ul/cm (about 20ng/ bar), evenly be sprayed on glass fibre cotton, 56 DEG C of dry 1h, after adding drying agent vacuum seal, RT saves backup.
4 preparation pig early pregnancy factor colloidal gold test cards
NC film containing detection line and nature controlling line is pasted onto the middle part of support plate, the glass fibre membrane containing golden labeling antibody and sample pad is pasted successively in the side of NC film, absorption pad is pasted at the opposite side of NC film, obtain test paper plate the semi-manufacture test paper of assembling is put into cutting trough carry out cutting into the wide test strips of 4.08mm, every root test paper is respectively charged in plastics test card, shell is closed by case pressing machine, be prepared into finished product detection test card, test card loads in the aluminium foil bag of lucifuge, capper seals, stamp the production time and batch, room temperature preservation is for subsequent use.
Above-described embodiment is effective embodiment of the present invention, but embodiment of the present invention is not limited to the restriction of above-mentioned embodiment, and according to the production actual conditions of every batch, adjust concrete implementation condition, entirety does not have king-sized change.Other inventions do not deviate from substituting done by this invention cardinal rule and method of operating, combination, optimization etc. all within the scope of available protecting of the present invention.
Claims (3)
1. pig early pregnancy factor colloidal gold test card, is characterized in that:
Described pig early pregnancy factor colloidal gold test card is made up of colloidal gold strip and buckle type draw-in groove; Buckle type draw-in groove has the build-in cavities of an accommodating colloidal gold strip, and buckle type draw-in groove is provided with observation area and sample application zone; Colloidal gold strip is primarily of support plate, NC film, gold mark monoclonal antibody pad, sample pad and absorption pad composition;
Described NC film is arranged on the middle part of support plate, the support plate of NC film side is pasted with successively containing gold mark monoclonal antibody pad and sample pad, the support plate of NC film opposite side is pasted with absorption pad;
There is detection line and a nature controlling line resisted containing sheep anti mouse two containing pig early pregnancy factor polyclonal antibody at the middle part of described NC film;
Colloidal gold strip is arranged in the build-in cavities of draw-in groove, the colour developing district of the corresponding colloidal gold strip in observation area of draw-in groove, the sample pad of the corresponding colloidal gold strip of well;
In described load sample pad, gold mark monoclonal antibody is mouse source pig early pregnancy factor monoclonal antibody;
In described NC film, pig early pregnancy factor antibody is rabbit anti-pig early pregnancy factor polyclonal antibody; In described NC film, sheep anti mouse two resists for sheep anti-mouse igg antibody.
2. the preparation method of pig early pregnancy factor colloidal gold test card as claimed in claim 1, is characterized in that: comprise the following steps:
(1) NC film is prepared
The preparation of rabbit anti-pig early pregnancy factor polyclonal antibody: adopt prokaryotic expression the pig early pregnancy factor albumen of purifying as immunogene, extract IgG antibody, preparation rabbit anti-pig early pregnancy factor polyclonal antibody;
The preparation of detection line: adopt three-dimensional spray film instrument, adjustment spouting liquid is 45 ~ 50dots/mL/cm, is buffered liquid dilution rabbit anti-pig early pregnancy factor polyclonal antibody with bag, concentration is 45 ~ 50 μ g/mL, in NC film, appropriate location line, is detection line, dries for subsequent use; Described bag is buffered the CBS damping fluid that liquid is 0.05 ~ 0.07M through 0.40 ~ 0.45 μ L membrane filtration, pH9.6;
The preparation of nature controlling line: adopt three-dimensional spray film instrument, adjustment spouting liquid is 45 ~ 48dots/ml/cm, is buffered liquid dilution sheep anti-mouse igg antibody with bag, concentration is 1mg/ml, position line parallel with detection line on NC film, and detection line interval 4 ~ 5mm, be nature controlling line; Described bag is buffered the CBS damping fluid that liquid is 0.03M, pH9.4 through 0.40 ~ 0.45 μ l membrane filtration;
NC film assembling preparation: dry detection line and nature controlling line, obtain the NC film containing detection line and nature controlling line; Dry in room temperature 72h with the NC film of closed working fluid by preparation, seal up for safekeeping for subsequent use; Described closed working fluid is the closed working fluid obtained with 0.45 ~ 0.48ul membrane filtration after the CBS damping fluid mixing containing the 2.5%BSA of mass percent, 2.5 ~ 3.0% skimmed milk powers and 0.01M, pH7.0.
(2) coated with gold mark monoclonal antibody pad is prepared
Containing mass percent with 20mM is 1.5%BSA, 0.1%NaN
3with 3% sodium tetraborate dilution buffer, gold is marked monoclonal antibody and be diluted to final concentration 0.05mg/ml; Cut the glass fibre cotton that specification is 300 × 7mm, gold is marked the unidirectional specking instrument of monoclonal antibody solution, with the speed of 5 μ L/cm (about 20ng/ bar), evenly be sprayed on glass fibre cotton, 56 DEG C of dry 1h, after adding drying agent vacuum seal, room temperature preservation is for subsequent use.
(3) pig early pregnancy factor colloidal gold test card is assembled
The NC film scribbling detection line and nature controlling line is pasted onto the middle part of support plate, the glass fibre membrane containing gold mark monoclonal antibody and sample pad is pasted successively in the side of NC film, absorption pad is pasted at the opposite side of NC film, obtain test paper plate, test paper plate is cut into the test strips of different in width, test strips is placed in draw-in groove and is pig early pregnancy factor colloidal gold test card.
(4) pig early pregnancy factor colloidal gold test card uses and judges
Pig early pregnancy factor colloidal gold test is stuck in diagnosis pig early pregnancy and applies, separation of serum, the serum of gained is dripped in the sample pad of this test card, sample moves along on adsorptive pads, when running into gold mark monoclonal antibody, pig early pregnancy factor is marked mouse-anti pig early pregnancy factor monoclonal antibody be combined with golden, Ag-Ab-Au composite moves to detection line and nature controlling line, the rabbit of pig early pregnancy factor on detection line anti-pig early pregnancy factor polyclonal antibody is combined, form a red detection line, can not in conjunction with gold mark mouse-anti pig early pregnancy factor monoclonal antibody without pig early pregnancy factor in negative serum, so occur without macroscopic detection line, nature controlling line takes on a red color forever, if colourless, represents that test strips lost efficacy.
3. the preparation method of pig early pregnancy factor colloidal gold test card as claimed in claim 2, it is characterized in that: in step (1), the live width of detection line and nature controlling line is 1mm, two lines, at a distance of 4mm, are positioned at the centre of film, apart from the back gauge 6 ~ 7mm of NC film.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510193313.8A CN104865381A (en) | 2015-04-22 | 2015-04-22 | Pig early pregnancy factor colloidal gold test paper card and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510193313.8A CN104865381A (en) | 2015-04-22 | 2015-04-22 | Pig early pregnancy factor colloidal gold test paper card and preparation method thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105675890A (en) * | 2015-12-31 | 2016-06-15 | 湖南农业大学 | Apparatus for detecting pregnancy of laboratory mice, and method thereof |
| CN106771153A (en) * | 2016-11-21 | 2017-05-31 | 百奥森（江苏）食品安全科技有限公司 | It is a kind of for feed, the vomitoxin colloidal-gold detecting-card of feedstuff |
| CN110954705A (en) * | 2019-12-30 | 2020-04-03 | 深圳市绿诗源生物技术有限公司 | Detection test paper card for rapidly detecting early pregnancy factor of dairy cow |
| CN113203865A (en) * | 2021-06-03 | 2021-08-03 | 甘肃农业大学 | Preparation method of yak early pregnancy diagnosis reagent |
-
2015
- 2015-04-22 CN CN201510193313.8A patent/CN104865381A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105675890A (en) * | 2015-12-31 | 2016-06-15 | 湖南农业大学 | Apparatus for detecting pregnancy of laboratory mice, and method thereof |
| CN106771153A (en) * | 2016-11-21 | 2017-05-31 | 百奥森（江苏）食品安全科技有限公司 | It is a kind of for feed, the vomitoxin colloidal-gold detecting-card of feedstuff |
| CN110954705A (en) * | 2019-12-30 | 2020-04-03 | 深圳市绿诗源生物技术有限公司 | Detection test paper card for rapidly detecting early pregnancy factor of dairy cow |
| CN113203865A (en) * | 2021-06-03 | 2021-08-03 | 甘肃农业大学 | Preparation method of yak early pregnancy diagnosis reagent |
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