CN106940374A - A kind of test card of quick detection vomitoxin - Google Patents
A kind of test card of quick detection vomitoxin Download PDFInfo
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- CN106940374A CN106940374A CN201710319370.5A CN201710319370A CN106940374A CN 106940374 A CN106940374 A CN 106940374A CN 201710319370 A CN201710319370 A CN 201710319370A CN 106940374 A CN106940374 A CN 106940374A
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Abstract
The invention provides a kind of test card of quick detection vomitoxin, including plastic box body and the test strips being packaged in plastic box body；The test strips include bottom support layer and the sample pad being sequentially fixed on the supporting layer, gold mark and antibody binding pad, nitrocellulose filter and absorption pad；Described nitrocellulose filter is provided with the emesis toxin monoclone antibody that colloid gold label is perfused with T lines and C lines, the gold mark and antibody binding pad；Described plastic box body is provided with well and observation window, and the position of the well is corresponding with the sample pad location in the test strips, and the observation window is corresponding with the position of the nitrocellulose filter in test strips.The sensitive height of test card that the present invention is provided, high specificity.
Description
Technical field
The present invention relates to immunochemistry detection technique field, and in particular to a kind of test card of quick detection vomitoxin.
Background technology
Vomitoxin (vomitoxin) also known as deoxynivalenol (deoxynivalenol, DON), are a kind of
Trichothecenes toxin, certain accumulation may be had in vivo by mainly producing .DON by Fusarium graminearum and Fusarlum roseum,
Without special target organ, i.e., a certain specific organ is not damaged, but with very strong cytotoxicity, animal can be caused
Bowel dysfunction, mucous membrane of mouth and dermal lesions, immunity degradation.People and livestock eaten by mistake and produced extensively after the Cereals by endotoxin contamination
General poisonous effect, can cause vomiting, food refusal, the symptom such as weight loss, immune system and hemopoietic system suffer damage, also had
Very strong dermal toxicity and cytotoxicity, can damage hemopoietic system and cause death when serious.Wheat, barley, jade are distributed in DON more
It is the mycotoxin that most serious is polluted after aflatoxin in the cereal seeds such as rice.Because DON harm is serious, cause
The most attention of various countries, has formulated the strict limit standard of DON residual quantities in cereal and feed in succession.
The conventional method of DON residue detections has a microbiology method, fluorescence method, thin-layered chromatography, high performance liquid chromatography, with
And Liquid Chromatography/Mass Spectrometry, the method such as gas chromatography mass spectrometry detection method and Liquid Chromatography-Tandem Mass Spectrometry.These method sensitivity are high, qualitative
Accurately, it may be determined that the molecular weight of compound, molecular formula, or even functional group, it is adapted to quantitatively detect the compound and its metabolin.
But these method high costs are, it is necessary to technical professional, expensive laboratory apparatus and prolonged preliminary preparation.And exempt from
Epidemiology detection method is set up in molecular recognition of the antibody to antigen, and its major advantage is that affinity is high, quick, simple, economical
Practicality, can realize that the small size to biofluid, big flux are detected, be increasingly becoming countries in the world noxious residual chemicals quick
One of method of detection.
The content of the invention
In view of this, the invention provides one kind it is simple to operate, quick and precisely, sensitivity height, low cost, good stability,
The quick detection test paper card of the vomitoxin of batch detection can be carried out.To reach above-mentioned purpose, the present invention uses following technical side
Case：
The invention provides a kind of test card of quick detection vomitoxin, including plastic box body and it is packaged in plastic box body
Test strips；The test strips include bottom support layer and the sample pad being sequentially fixed on the supporting layer, gold mark and antibody knot
Close pad, nitrocellulose filter and absorption pad；Described nitrocellulose filter is provided with T lines and C lines, between the two away from 5mm, the T
Line is located at the emesis toxin Dan Ke that colloid gold label is perfused with the side of sample pad, the gold mark and antibody binding pad
Grand antibody；Described plastic box body is provided with well and observation window, the position of the well and the sample in the test strips
Product pad position is corresponding, and the observation window is corresponding with the position of the nitrocellulose filter in test strips；
The preparation method of the emesis toxin monoclone antibody be using vomitoxin with bovine serum albumin(BSA) conjugate to be immune
Antigen, is immunized Balb/c mouse, prepared by the spleen cell of mouse and myeloma cell SP2/0 into hybridoma, through indirect
ELISA is screened and limiting dilution assay obtains monoclonal antibody hybridoma cell strain, and hybridoma cell strain is injected in into mouse peritoneal
Interior production monoclonal antibody.
Further, the preparation method of the emesis toxin monoclone antibody, comprises the following steps：
First, animal immune
1-1, experimental animal：The healthy BALB/c mouse of 6-8 week old；
1-2, just exempt from：Antigen physiological saline 1:1 dilution, abdominal cavity 1 point injection fully emulsified with isometric Freund's complete adjuvant,
Dorsal sc 4-6 points are injected；
1-3, two exempt from：2 weeks after initial immunity, with physiological saline 1:1 dilution antigen, it is abundant with isometric incomplete Freund's adjuvant
Emulsification, immunization route is same just to exempt from；
1-4, antibody test：2 weeks after secondary immunity, docking takes blood, separates serum, uses ELISA method detection；
1-5, super exempt from：First 3 days of fusion, takes antigenic solution 100ug abdominal cavities to inject；
2nd, basis prepares
2-1, utensil sterilizing；
2-2, preparation nutrient solution；
2-3, myeloma cell's recovery；
3rd, cell fusion
3-1, myeloma cell collect：Murine myeloma cell is purged with 10ml suction pipes from Tissue Culture Flask, is loaded on
In 50ml centrifuge tubes；
It is prepared by 3-2, splenocyte：Extracing eyeball of mouse draws neck to put to death, and mouse is soaked in 75% alcoholic solution several minutes, with only
Blood pincers tear skin of abdomen, cut off peritonaeum with sterile scissors, use aseptic nipper separating spleen；Spleen is placed in 200 purposes sterile
On nylon wire, several piece is cut into scissors and is ground, rinsed with 1640 culture mediums, until residual tissue is to white, by spleen cell
Centrifuge tube is collected in, and supplements 1640 culture mediums to 40ml；
3-3, cell washing：Above-mentioned myeloma cell and spleen cell are centrifuged 10 minutes in 1000-1200 revs/min, outwelled
Supernatant, myeloma cell and spleen cell are suspended and concentrated in a 50ml centrifuge tube with 1640 culture mediums, supplement
1640 culture mediums are to 40ml, and 1000 revs/min centrifuge 10 minutes, outwell supernatant；
3-4, cell fusion：The centrifuge tube that will be equipped with myeloma cell and spleen cell is placed in 37 DEG C of water-baths, takes 50%PEG
1ml, in being slowly added dropwise in 1 minute in centrifuge tube, side edged is mixed；Add after PEG, continue to mix 30 seconds；Then 1 point is stood
Clock；The culture mediums of 1ml 1640 were slowly added dropwise in 1 minute, side edged is mixed, 3ml 1640 is added dropwise in subsequent 1 minute and cultivates
Base；1640 culture mediums are finally supplemented to 40ml, 1000 revs/min centrifuge 10 minutes, outwell supernatant；
3-5, point plate：Above-mentioned fused cell is washed down with the complete medium containing HAT additives, 120ml selectivity is suspended in
In culture medium, divide and plant in 96 culture plates, per hole 0.2ml, be placed in 5%CO2, saturated humidity, cultivate in 37 DEG C of incubators；
3-6, change liquid：Liquid method is changed using half amount, liquid was changed every 2 days 1 time；The old culture medium of half is suctioned out, new culture medium is supplemented；
Liquid is changed for the first time using HAT complete mediums, uses HT complete mediums instead later；
4th, filtering hybridoma detection and subclone
4-1, detection opportunity：10 days after cell fusion, hybridoma is detected when being paved with the 1/10-1/5 of bottom hole；
4-2, detection method：ELISA method；
4-3, colonized culture：Hybridoma is purged from culture plate, counted with cell counting count board, 96 cells are taken
It is suspended in 20ml complete mediums, divides and plant in 96 well culture plates, be placed in CO2Cultivated in incubator；
4-4, change liquid：The 4th day after clone, observation of cell upgrowth situation under inverted microscope, half amount changed 1640 culture mediums, after
Continuous culture；
4-5, detection：ELISA method detection cells and supernatant is used, the cell for having suppression carries out clone's culture, until positive rate reaches
To 100%；
5th, culture is expanded
Hybridoma after subclone is transferred to Tissue Culture Flask from culture plate, and culture is changed in good time according to cell growth status
Base simultaneously expands bottle；
6th, cell cryopreservation
6-1, change liquid；
6-2, cell are collected；
6-3, frozen stock solution are prepared；
6-4, cell cryopreservation；
7th, prepared by ascites
7-1, mouse prepare：Inject before cell at least 3 days, sterilized liquid paraffin oil is injected to the BALB/c mouse abdominal cavity of health
0.2ml；
7-2, cell are collected：The good cell of upgrowth situation is purged from blake bottle, and 1000 revs/min centrifuge 10 minutes；
7-3, cell washing：Supernatant is outwelled, original volume PBS is added, ibid centrifuges again once；Supernatant is outwelled, is adjusted with PBS
Cell concentration is to 107/ml；
7-4, injection：Every mouse peritoneal injection 0.5ml cell suspension；
7-5, ascites are collected：Inject after cell about 7-10 days, mouse web portion protuberance is punctured with syringe and extracts ascites, 3000 turns/
Minute centrifugation 10 minutes, draws supernatant ascites, is wanted according to used in amounts, is sub-packed in plastic jar, -70 DEG C of preservations；The ascites of collection
Purified with salting out method or affinity chromatography and produce vomitoxin monoclonal antibody.
A kind of preparation method of the test card of quick detection vomitoxin, comprises the following steps：
1）The preparation of sample pad：The sucrose for being 1% with the BSA, mass concentration for being 2% containing mass concentration by glass fibre cotton, matter
Measure Tween-20, Triton-100 that concentration is 0.1% and the NaN that mass concentration is 0.1%3PBS processing after, dry
It is standby, as sample pad；
2）It is prepared by gold mark and antibody binding pad：Glass fibre cotton is cut into the wide slices of 4mm, it is 2% to be put into containing mass concentration
The NaN that sucrose, 0.5%PEG and the mass concentration that BSA, mass concentration are 5% are 0.05M3PBS in 20min, 37 °C
Constant temperature drying, then by the processed good glass fibre cotton of gold labeling antibody spray, vacuum freeze-drying 4h is that gold mark resists and body knot
Close pad；
3）The preparation of absorption pad：The pure white filter paper made by raw material of plant long fibre is cut into wide thin of 4mm with cutting machine
Bar, blend compounds film carries out one side closed, drying for standby, as absorption pad；
4）The preparation of test card：First by the sample pad of preparation, gold mark and antibody binding pad, nitrocellulose filter and absorption pad
It is sequentially attached on supporting layer, by left-to-right overlapping connection, laminates width 2mm, wide 4mm is then made of cutting machine, it is appropriate long
The test strips of degree, are packaged in the plastics with well and observation window get stuck, that is, test card are made.
Further, the T lines are the detection line of vomitoxin-carrier protein couplet thing solution printing；The C lines are sheep
The nature controlling line of anti-mouse IgG solution printing.
Further, the supporting layer is the PVC board being made up of Corvic and stabilizer and auxiliary material.
Further, the absorption pad is the white filter paper being made up of plant long fibre.
Further, the sample pad and gold mark and antibody binding pad are made by glass fibre cotton.
Further, the sample pad and gold mark and width is laminated for 2mm with antibody binding pad.
Further, the width that laminates of the absorption pad and nitrocellulose filter is 2mm.
Further, covered with rubber protective layer above the sample pad, gold mark and antibody binding pad and absorption pad.
Further, nitrocellulose filter two ends boundary has mark line.
Beneficial effects of the present invention：
1st, the sensitive height of test paper of the invention, high specificity.Vomitoxin remains quick detection test paper card with the high parent of colloid gold label
It is prepared from the monoclonal antibody of power, gold grain is mutually tied with antibody molecule by charges of different polarity Van der Waals force in gold labeling antibody
Close, collaurum influences very little to the specificity and affinity of monoclonal antibody.
2nd, it is simple, convenient quick.Using during quick detection test paper card without other any reagents, as long as will processing after
Sample solution with dropper instill test card well in 3-4 drops, 3-5min is observable result.
3rd, testing result image, intuitively.Test card is with red trace line " | " or " ‖ " positive and negative mark for detection line
Note, i.e., the nature controlling line on nitrocellulose filter（C lines）When the red that develops the color is " | " trace, vomitoxin in detection liquid is represented
Residual content >=50ng/ml；If nature controlling line on nitrocellulose filter（C lines）With detection line (T limits) while two red of appearance are
During " ‖ " trace, vomitoxin residual content ＜ 50ng/ml in sample solution are represented.As a result it is visual in image, it is simple accurate, do not allow
Easily judge by accident.
4th, the use of glued membrane can extend testing result observing time, test card good stability.This test card sample absorbs
Pad fully absorbs detection solution, is allowed to fully react with the upper gold labeling antibody of coupling pad, can effectively reduce error rate；Can be with
Prevent that introduced contaminants from disturbing, influence gold labeling antibody and the combination for detecting antigen.
5th, low cost, small investment.Use test card of the present invention, it is not necessary to another with complicated instrument and equipment and expensive examination
Agent, Site Detection is settled at one go, with low cost, instant effect.
6th, it is easy to a wide range of popularization and application.This test card is simple to operate, is adapted to different classes of librarian use, such as laboratory
Detection, customs quarantine control, health supervision, breeding scale and individual production etc., with wide market prospects and larger economic effect
Benefit and social benefit.
Brief description of the drawings
Fig. 1：Test strips structure schematic diagram of the present invention；
Fig. 2：Test strips side view of the present invention；
Fig. 3：Test strips top view of the present invention；
Fig. 4：Testing result is negative result schematic diagram；
Fig. 5：Testing result is positive result schematic diagram；
Fig. 6：The invalid result schematic diagram of testing result.
Brief description of the drawings：1st, plastic box body, 2, test strips, 3, supporting layer, 4, sample pad, 5, gold mark and antibody binding pad, 6,
Nitrocellulose filter, 7, absorption pad, 8, well, 9, observation window, 10, C lines, 11, T lines, 12, rubber protective layer, 13, mark
Line.
Embodiment
The invention will be further described with reference to the accompanying drawings and examples.
As shown in figures 1 to 6, a kind of test card of quick detection vomitoxin, including plastic box body 1 and it is packaged in plastic casing
Test strips 2 in body 1；Test strips 2 include bottom support layer 3 and the sample pad 4 being sequentially fixed on supporting layer 3, gold mark and resisted
Body pad 5, nitrocellulose filter 6 and absorption pad 7；Described nitrocellulose filter 6 is provided with T lines 11 and C lines 10, between the two
Away from 5mm, T lines 11 are located at is perfused with colloid gold label on the side of sample pad 4, described gold mark and antibody binding pad 5
Emesis toxin monoclone antibody；Described plastic box body 1 is provided with well 8 and observation window 9, the position of the well 8
It is corresponding with the position of sample pad 4 in test strips 2, the position of the observation window 9 and the nitrocellulose filter 6 in test strips 2
Put corresponding；The preparation method of the emesis toxin monoclone antibody be using vomitoxin and bovine serum albumin(BSA) conjugate as
Immunizing antigen, is immunized Balb/c mouse, prepared by the spleen cell of mouse and myeloma cell SP2/0 into hybridoma, between
Connect ELISA screenings and limiting dilution assay obtains monoclonal antibody hybridoma cell strain, hybridoma cell strain is injected in mouse abdomen
Production monoclonal antibody in chamber.
Wherein, T lines 11 are the detection line of vomitoxin-carrier protein couplet thing solution printing；C lines 10 are sheep anti-mouse igg
The nature controlling line of solution printing；Supporting layer 3 is the PVC board being made up of Corvic and stabilizer and auxiliary material；Absorption pad 7 be by
The white filter paper that plant long fibre is made；Sample pad 4 and gold mark and antibody binding pad 5 are made by glass fibre cotton；
The width that laminates of sample pad 4 and gold mark and antibody binding pad 5 is 2mm.The width that laminates of absorption pad 7 and nitrocellulose filter 6 is
2mm；Sample pad 4, gold mark and antibody binding pad 5 and the top of absorption pad 7 are covered with rubber protective layer 12；6 liang of nitrocellulose filter
End boundary has mark line 13.
The preparation method of the emesis toxin monoclone antibody, comprises the following steps：
First, animal immune
1-1, experimental animal：The healthy BALB/c mouse of 6-8 week old；
1-2, just exempt from：Antigen physiological saline 1:1 dilution, abdominal cavity 1 point injection fully emulsified with isometric Freund's complete adjuvant,
Dorsal sc 4-6 points are injected；
1-3, two exempt from：2 weeks after initial immunity, with physiological saline 1:1 dilution antigen, it is abundant with isometric incomplete Freund's adjuvant
Emulsification, immunization route is same just to exempt from；
1-4, antibody test：2 weeks after secondary immunity, docking takes blood, separates serum, uses ELISA method detection；
1-5, super exempt from：First 3 days of fusion, takes antigenic solution 100ug abdominal cavities to inject；
2nd, basis prepares
2-1, utensil sterilizing；
2-2, preparation nutrient solution；
2-3, myeloma cell's recovery；
3rd, cell fusion
3-1, myeloma cell collect：Murine myeloma cell is purged with 10ml suction pipes from Tissue Culture Flask, is loaded on
In 50ml centrifuge tubes；
It is prepared by 3-2, splenocyte：Extracing eyeball of mouse draws neck to put to death, and mouse is soaked in 75% alcoholic solution several minutes, with only
Blood pincers tear skin of abdomen, cut off peritonaeum with sterile scissors, use aseptic nipper separating spleen；Spleen is placed in 200 purposes sterile
On nylon wire, several piece is cut into scissors and is ground, rinsed with 1640 culture mediums, until residual tissue is to white, by spleen cell
Centrifuge tube is collected in, and supplements 1640 culture mediums to 40ml；
3-3, cell washing：Above-mentioned myeloma cell and spleen cell are centrifuged 10 minutes in 1000-1200 revs/min, outwelled
Supernatant, myeloma cell and spleen cell are suspended and concentrated in a 50ml centrifuge tube with 1640 culture mediums, supplement
1640 culture mediums are to 40ml, and 1000 revs/min centrifuge 10 minutes, outwell supernatant；
3-4, cell fusion：The centrifuge tube that will be equipped with myeloma cell and spleen cell is placed in 37 DEG C of water-baths, takes 50%PEG
1ml, in being slowly added dropwise in 1 minute in centrifuge tube, side edged is mixed；Add after PEG, continue to mix 30 seconds；Then 1 point is stood
Clock；The culture mediums of 1ml 1640 were slowly added dropwise in 1 minute, side edged is mixed, 3ml 1640 is added dropwise in subsequent 1 minute and cultivates
Base；1640 culture mediums are finally supplemented to 40ml, 1000 revs/min centrifuge 10 minutes, outwell supernatant；
3-5, point plate：Above-mentioned fused cell is washed down with the complete medium containing HAT additives, 120ml selectivity is suspended in
In culture medium, divide and plant in 96 culture plates, per hole 0.2ml, be placed in 5%CO2, saturated humidity, cultivate in 37 DEG C of incubators；
3-6, change liquid：Liquid method is changed using half amount, liquid was changed every 2 days 1 time；Suction out the old culture medium of half（About 0.1ml）, supplement is newly
Culture medium（About 0.1ml）；Liquid is changed for the first time using HAT complete mediums, uses HT complete mediums instead later；
4th, filtering hybridoma detection and subclone
4-1, detection opportunity：10 days after cell fusion, hybridoma is detected when being paved with the 1/10-1/5 of bottom hole；
4-2, detection method：ELISA method；
4-3, colonized culture：Hybridoma is purged from culture plate, counted with cell counting count board, 96 cells are taken
It is suspended in 20ml complete mediums, divides and plant in 96 well culture plates, be placed in CO2Cultivated in incubator；
4-4, change liquid：The 4th day after clone, observation of cell upgrowth situation under inverted microscope, half amount changed 1640 culture mediums, after
Continuous culture；
4-5, detection：ELISA method detection cells and supernatant is used, the cell for having suppression carries out clone's culture, until positive rate reaches
To 100%；
5th, culture is expanded
Hybridoma after subclone is transferred to Tissue Culture Flask from culture plate, and culture is changed in good time according to cell growth status
Base simultaneously expands bottle；
6th, cell cryopreservation
6-1, change liquid；
6-2, cell are collected；
6-3, frozen stock solution are prepared；
6-4, cell cryopreservation；
7th, prepared by ascites
7-1, mouse prepare：Inject before cell at least 3 days, sterilized liquid paraffin oil is injected to the BALB/c mouse abdominal cavity of health
0.2ml；
7-2, cell are collected：The good cell of upgrowth situation is purged from blake bottle, and 1000 revs/min centrifuge 10 minutes；
7-3, cell washing：Supernatant is outwelled, original volume PBS is added, ibid centrifuges again once；Supernatant is outwelled, is adjusted with PBS
Cell concentration is to 107/ml；
7-4, injection：Every mouse peritoneal injection 0.5ml cell suspension；
7-5, ascites are collected：Inject after cell about 7-10 days, mouse web portion protuberance is punctured with syringe and extracts ascites, 3000 turns/
Minute centrifugation 10 minutes, draws supernatant ascites, is wanted according to used in amounts, is sub-packed in plastic jar, -70 DEG C of preservations；The ascites of collection
Purified with salting out method or affinity chromatography and produce vomitoxin monoclonal antibody.
The application method of the test card of quick detection vomitoxin of the present invention, comprises the following steps：
1st, sample：
Feed sample：1g grain is weighed, particle diameter is milled to for 0.5-1.5mm；Add into 10ml centrifuge tubes, add 1.5-
2.5ml working solutions, acutely shake 2min, stand 2min, take supernatant to be used to detect.
Grain sample：0.2g feeds are weighed into centrifuge tube, 10g is added（Or 10ml）Working solution, acutely shakes 2min, stands
5min, takes supernatant to be used to detect.
Urine sample：2min is stood, takes supernatant to be used to detect.
2nd, sample is added dropwise：
After test card is kept flat, result is observed after sample solution is instilled into 2-3 drops in test card well, 3-5min with dropper.
3rd, Analysis of test results：
It is negative：If nature controlling line on nitrocellulose filter（C lines）And detection line（T lines）When there are two red " ‖ " traces simultaneously, table
Show that vomitoxin residual content is less than correspondence test limit in sample solution, is judged to feminine gender.
It is positive：If nature controlling line on nitrocellulose filter（C lines）One red " | " trace of display, and detection line（T lines）Do not show
Color, represents to be detected vomitoxin residual content in sample solution and is more than or equal to correspondence test limit, be judged to the positive.
It is invalid：If nature controlling line on nitrocellulose filter（C lines）Do not develop the color red stripes, then no matter detection line（T lines）Whether
There is red trace, it is invalid that the test card is judged to.
Finally illustrate, the above embodiments are merely illustrative of the technical solutions of the present invention and it is unrestricted, this area is common
Other modifications or equivalent that technical staff is made to technical scheme, without departing from technical solution of the present invention
Spirit and scope, all should cover among scope of the presently claimed invention.
Claims (7)
1. a kind of test card of quick detection vomitoxin, including plastic box body（1）Be packaged in plastic box body（1）Interior test paper
Bar（2）；Characterized in that, the test strips（2）Including bottom support layer（3）Be sequentially fixed at the supporting layer（3）On
Sample pad（4）, gold mark and antibody binding pad（5）, nitrocellulose filter（6）And absorption pad（7）；Described nitrocellulose filter
（6）It is provided with T lines（11）With C lines（10）, between the two away from 5mm, the T lines（11）Positioned at close to sample pad（4）Side, it is described
Gold mark and antibody binding pad（5）On be perfused with the emesis toxin monoclone antibody of colloid gold label；Described plastic box body（1）
It is provided with well（8）And observation window（9）, the well（8）Position and the test strips（2）On sample pad（4）Position
Put corresponding, the observation window（9）With test strips（2）On the nitrocellulose filter（6）Position it is corresponding；It is described anti-to vomit
The preparation method for telling toxin monoclone antibody is, using vomitoxin and bovine serum albumin(BSA) conjugate as immunizing antigen, to be immunized
Balb/c mouse, hybridoma is prepared by the spleen cell of mouse and myeloma cell SP2/0, through indirect ELISA screening and
Limiting dilution assay obtains monoclonal antibody hybridoma cell strain, and hybridoma cell strain is injected in mouse peritoneal and produces monoclonal
Antibody.
2. test card according to claim 1, it is characterised in that the T lines（11）For vomitoxin-carrier protein couplet
The detection line of thing solution printing；The C lines（10）The nature controlling line printed for sheep anti-mouse igg solution.
3. test card according to claim 1, it is characterised in that the supporting layer（3）Be by Corvic with it is steady
Determine the PVC board that agent and auxiliary material are made.
4. test card according to claim 1, it is characterised in that the sample pad（4）, gold mark and antibody binding pad（5）
And absorption pad（7）Top is covered with rubber protective layer（12）.
5. test card according to claim 1, it is characterised in that the nitrocellulose filter（6）Two ends boundary has mark
Remember line（13）.
6. test card according to claim 1 or 2, it is characterised in that the preparation of the emesis toxin monoclone antibody
Method, comprises the following steps：
First, animal immune
1-1, experimental animal：The healthy BALB/c mouse of 6-8 week old；
1-2, just exempt from：Antigen physiological saline 1:1 dilution, abdominal cavity 1 point injection fully emulsified with isometric Freund's complete adjuvant,
Dorsal sc 4-6 points are injected；
1-3, two exempt from：2 weeks after initial immunity, with physiological saline 1:1 dilution antigen, it is abundant with isometric incomplete Freund's adjuvant
Emulsification, immunization route is same just to exempt from；
1-4, antibody test：2 weeks after secondary immunity, docking takes blood, separates serum, uses ELISA method detection；
1-5, super exempt from：First 3 days of fusion, takes antigenic solution 100ug abdominal cavities to inject；
2nd, basis prepares
2-1, utensil sterilizing；
2-2, preparation nutrient solution；
2-3, myeloma cell's recovery；
3rd, cell fusion
3-1, myeloma cell collect：Murine myeloma cell is purged with 10ml suction pipes from Tissue Culture Flask, is loaded on
In 50ml centrifuge tubes；
It is prepared by 3-2, splenocyte：Extracing eyeball of mouse draws neck to put to death, and mouse is soaked in 75% alcoholic solution several minutes, with only
Blood pincers tear skin of abdomen, cut off peritonaeum with sterile scissors, use aseptic nipper separating spleen；Spleen is placed in 200 purposes sterile
On nylon wire, several piece is cut into scissors and is ground, rinsed with 1640 culture mediums, until residual tissue is to white, by spleen cell
Centrifuge tube is collected in, and supplements 1640 culture mediums to 40ml；
3-3, cell washing：Above-mentioned myeloma cell and spleen cell are centrifuged 10 minutes in 1000-1200 revs/min, outwelled
Supernatant, myeloma cell and spleen cell are suspended and concentrated in a 50ml centrifuge tube with 1640 culture mediums, supplement
1640 culture mediums are to 40ml, and 1000 revs/min centrifuge 10 minutes, outwell supernatant；
3-4, cell fusion：The centrifuge tube that will be equipped with myeloma cell and spleen cell is placed in 37 DEG C of water-baths, takes 50%PEG
1ml, in being slowly added dropwise in 1 minute in centrifuge tube, side edged is mixed；Add after PEG, continue to mix 30 seconds；Then 1 point is stood
Clock；The culture mediums of 1ml 1640 were slowly added dropwise in 1 minute, side edged is mixed, 3ml 1640 is added dropwise in subsequent 1 minute and cultivates
Base；1640 culture mediums are finally supplemented to 40ml, 1000 revs/min centrifuge 10 minutes, outwell supernatant；
3-5, point plate：Above-mentioned fused cell is washed down with the complete medium containing HAT additives, 120ml selectivity is suspended in
In culture medium, divide and plant in 96 culture plates, per hole 0.2ml, be placed in 5%CO2, saturated humidity, cultivate in 37 DEG C of incubators；
3-6, change liquid：Liquid method is changed using half amount, liquid was changed every 2 days 1 time；The old culture medium of half is suctioned out, new culture medium is supplemented；
Liquid is changed for the first time using HAT complete mediums, uses HT complete mediums instead later；
4th, filtering hybridoma detection and subclone
4-1, detection opportunity：10 days after cell fusion, hybridoma is detected when being paved with the 1/10-1/5 of bottom hole；
4-2, detection method：ELISA method；
4-3, colonized culture：Hybridoma is purged from culture plate, counted with cell counting count board, 96 cells are taken
It is suspended in 20ml complete mediums, divides and plant in 96 well culture plates, be placed in CO2Cultivated in incubator；
4-4, change liquid：The 4th day after clone, observation of cell upgrowth situation under inverted microscope, half amount changed 1640 culture mediums, after
Continuous culture；
4-5, detection：ELISA method detection cells and supernatant is used, the cell for having suppression carries out clone's culture, until positive rate reaches
To 100%；
5th, culture is expanded
Hybridoma after subclone is transferred to Tissue Culture Flask from culture plate, and culture is changed in good time according to cell growth status
Base simultaneously expands bottle；
6th, cell cryopreservation
6-1, change liquid；
6-2, cell are collected；
6-3, frozen stock solution are prepared；
6-4, cell cryopreservation；
7th, prepared by ascites
7-1, mouse prepare：Inject before cell at least 3 days, sterilized liquid paraffin oil is injected to the BALB/c mouse abdominal cavity of health
0.2ml；
7-2, cell are collected：The good cell of upgrowth situation is purged from blake bottle, and 1000 revs/min centrifuge 10 minutes；
7-3, cell washing：Supernatant is outwelled, original volume PBS is added, ibid centrifuges again once；Supernatant is outwelled, is adjusted with PBS
Cell concentration is to 107/ml；
7-4, injection：Every mouse peritoneal injection 0.5ml cell suspension；
7-5, ascites are collected：Inject after cell about 7-10 days, mouse web portion protuberance is punctured with syringe and extracts ascites, 3000 turns/
Minute centrifugation 10 minutes, draws supernatant ascites, is wanted according to used in amounts, is sub-packed in plastic jar, -70 DEG C of preservations；The ascites of collection
Purified with salting out method or affinity chromatography and produce vomitoxin monoclonal antibody.
7. a kind of preparation method of test card according to any one of 1-6 quick detection vomitoxins, comprises the following steps：
1）The preparation of sample pad：The sucrose for being 1% with the BSA, mass concentration for being 2% containing mass concentration by glass fibre cotton, matter
Measure Tween-20, Triton-100 that concentration is 0.1% and the NaN that mass concentration is 0.1%3PBS processing after, dry
It is standby, as sample pad；
2）It is prepared by gold mark and antibody binding pad：Glass fibre cotton is cut into the wide slices of 4mm, it is 2% to be put into containing mass concentration
The NaN that sucrose, 0.5%PEG and the mass concentration that BSA, mass concentration are 5% are 0.05M3PBS in 20min, 37 °C
Constant temperature drying, then by the processed good glass fibre cotton of gold labeling antibody spray, vacuum freeze-drying 4h is gold mark and antibody knot
Close pad；
3）The preparation of absorption pad：The pure white filter paper made by raw material of plant long fibre is cut into wide thin of 4mm with cutting machine
Bar, blend compounds film carries out one side closed, drying for standby, as absorption pad；
4）The preparation of test card：First by the sample pad of preparation, gold mark and antibody binding pad, nitrocellulose filter and absorption pad
It is sequentially attached on supporting layer, by left-to-right overlapping connection, laminates width 2mm, wide 4mm is then made of cutting machine, it is appropriate long
The test strips of degree, are packaged in the plastics with well and observation window get stuck, that is, test card are made.
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Application publication date: 20170711 |