CN103616514A - Rapid diagnosis test strip of cow mastitis candida albicans - Google Patents
Rapid diagnosis test strip of cow mastitis candida albicans Download PDFInfo
- Publication number
- CN103616514A CN103616514A CN201310674116.9A CN201310674116A CN103616514A CN 103616514 A CN103616514 A CN 103616514A CN 201310674116 A CN201310674116 A CN 201310674116A CN 103616514 A CN103616514 A CN 103616514A
- Authority
- CN
- China
- Prior art keywords
- candida albicans
- antibody
- pad
- mastitis
- test strip
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
Abstract
The invention discloses a rapid diagnosis test strip of cow mastitis candida albicans. The test strip comprises an absorbent paper, an analysis membrane, a gold-labelled antibody joint pad, a sample pad and a base plate, wherein the analysis membrane contains a detection line and a quality control line; the detection line is enveloped by a candida abbicans monoclonal antibody; the quality control line is enveloped by goat anti-rabbit IgG; a colloidal gold-labelled anti-rabbit polyclonal antibody of the candida albicans is enveloped by the gold-labelled antibody joint pad. The rapid diagnosis test strip is applied to detection of cow mastitis pathogens, so that the rapid diagnosis test strip is specific, accurate, rapid, simple to operate and the like, and is applicable to basic livestock breeding, husbandry technology popularization, and animal husbandry and animal quarantine inspection. Therefore, the rapid diagnosis test strip has important value on rapid diagnosis of dairy cow mastitis.
Description
Technical field
The invention belongs to mastitis for milk cows detection technique field, relate in particular to a kind of mastitis for milk cows Candida albicans fast diagnose test paper bar.
Background technology
Candida albicans (Candida albicans) claim again candida albicans bacterium, is the most common, topmostly in humans and animals fungal disease to suffer from altogether one of condition pathomycete.This bacterium is invaded mammary gland and breeds in mammary gland, and mastadenitis of cow is taken place frequently, and especially can cause taking place frequently of intractable mammitis, not only restricts the development of milk cow dairy industry, and has a strong impact on the quality of raw material milk, causes very large economic loss to milk cattle cultivating industry; Meanwhile, this is also threatening human health.Although this bacterium diagnosis has the methods such as the separated evaluation of traditional pathogeny microbiology, PCR and TCTA color developing detection, these methods are higher to technical requirement, and time-consuming, uneconomical.The means of take to be easy to sampling, being convenient to detect disease are most important to safeguarding milk cow health in postpartum and cattle farm economic benefit, and set up accurately, diagnostic techniques is to control the basic premise of Candida albicans harm timely.
The advantages such as colloidal gold immunochromatographimethod technology has quick, conveniently because of it, does not need specific installation, and result judgement is directly perceived, are more and more subject to people's attention.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of special, mastitis for milk cows Candida albicans fast diagnose test paper bar accurately and rapidly.
For solving the problems of the technologies described above, the present invention is by the following technical solutions: mastitis for milk cows Candida albicans fast diagnose test paper bar, comprise thieving paper, analyzing film, golden labeling antibody pad, sample pad and base plate, analyzing film is containing the coated detection line (T) of useful anti-candida albicans monoclonal antibody and with the coated nature controlling line of goat anti-rabbit igg (C), the anti-rabbit polyclonal antibody of Candida albicans of the coated colloid gold label of golden labeling antibody pad.
Anti-candida albicans monoclonal antibody is to utilize hybridization integration technology filter out the hybridoma cell strain of Candida albicans monoclonal antibody and obtain; The anti-rabbit polyclonal antibody of Candida albicans is repeatedly immune new zealand white rabbit and obtaining of application Candida albicans particulate antigen.
Analyzing film is nitrocellulose membrane, and golden labeling antibody pad is glass fibre membrane.
The preparation method of above-mentioned mastitis for milk cows Candida albicans fast diagnose test paper bar, comprises the following steps:
(1) prepare Candida albicans antibody
Anti-candida albicans monoclonal antibody is to adopt the Candida albicans of emulsification as antigen, and immune Balb/c mouse repeatedly stage by stage, then carry out Fusion of Cells filters out after positive hybrid cell strain, preparation ascites and obtaining; The anti-rabbit polyclonal antibody of Candida albicans be adopt Candida albicans as antigen repeatedly immune new zealand white rabbit preparation and high-titer anti-candida albicans polyclonal antibody;
(2) prepare analyzing film
Candida albicans monoclonal antibody prepared by step (1) is at the coated detection line that forms of nitrocellulose membrane, and goat anti-rabbit igg is at the coated nature controlling line that forms of nitrocellulose membrane, standby;
(3) prepare golden labeling antibody pad
Adopt trisodium citrate reduction method to prepare colloidal gold solution, and adopt the anti-rabbit polyclonal antibody of mark system mark Candida albicans, by golden labeling antibody immobilization on supporting film;
(4) assembling test strips
The analyzing film of step (2), the golden labeling antibody pad of step (3), thieving paper, sample pad, base plate are assembled into colloidal gold fast detecting test paper strip.
Marked body in step (3) is: colloid gold particle is extremely stable below 20nm, antibody protein is combined optimum pH value with collaurum be 8～9, the antibody protein amount of the suitableeest association colloid gold is 20～40 μ g/mL, the best stabilizer is 10%PEG20000, and the component of best colloidal gold composite damping fluid is that pH value is 8～9 Tris – HCl damping fluid, 1%BSA, 0.5%PEG20000,10% sucrose and 2%Tween – 20.
For easy, quick, the effective mastitis for milk cows Candida albicans of current shortage detection method, inventor has developed mastitis for milk cows Candida albicans fast diagnose test paper bar: the monoclonal antibody of first preparing anti-candida albicans by technology such as immune animal, Fusion of Cells and ELISA detections; Adopt trisodium citrate reduction gold chloride to prepare colloid gold particle, the anti-rabbit polyclonal antibody of mark Candida albicans is as collaurum pad; The fast diagnose test paper bar that detects mammitis fungal pathogen-Candida albicans is made in the nitrocellulose filter, collaurum pad, sample pad and the absorption pad assembling that point are scribbled to anti-candida albicans monoclonal antibody and goat anti-rabbit igg.The present invention is applied to the detection of mastitis for milk cows cause of disease, have spy lead, accurately, the feature such as quick, simple to operate, be suitable for basic unit's livestock-raising, livestock technology is promoted and farming animals quarantine is used, to dairy, development will have far-reaching influence.Therefore, the present invention has very important meaning to quick diagnosis and effective treatment mammitis.
Mastitis for milk cows Candida albicans fast diagnose test paper bar of the present invention has the following advantages:
(1) detect fast, 10-20min goes out result.
(2) specificity is good, and the equal no cross reactions of pathogen such as this test strips and Escherichia coli, streptococcus, staphylococcus are high with the special colour developing of Candida albicans.
(3) highly sensitive, detection sensitivity is 10
2cfumL
-1.
(4) good stability, the test strips stability of preserving after 30 days under 4 ℃ of conditions is still better.
(5) with PCR detection method contrast experiment in, the positive coincidence rate of test strips is 86.67%(13/15), have 2 parts not detect, negative match-rate is 90%(9/10).
Accompanying drawing explanation
Fig. 1 is the assembling assumption diagram of mastitis for milk cows Candida albicans fast diagnose test paper bar of the present invention, in figure: 1 sample pad, 2PVC base plate, 3 p-wires, 4 nitrocellulose filters (NC film), 5 absorbent filters, 6 control lines, 7 collaurum pads.
Embodiment
The preparation of embodiment 1 Candida albicans polyclonal antibody
1 materials and methods
1.1 bacterial strains: reference white candida albicans, clinical identification Candida albicans, staphylococcus aureus type strain, Escherichia coli type strain.
1.2 main agents and instrument: martin's bouillon, Sharpe nutrient agar, the goat anti-rabbit igg of horseradish peroxidase (HRP) mark, Freund's complete adjuvant/incomplete Freund's adjuvant, bag filter, electrophoresis apparatus, electrophoresis tank, ultraviolet gel imaging system, nucleic acid-protein analyser, hypervelocity refrigerated centrifuge.
1.3 animals used as test: new zealand white rabbit (Guangxi University's zoopery base).
2 experimental techniques
The antigen preparation of 2.1 Candida albicanss
The separation of preservation has been identified to Candida albicans rules on Sharpe solid medium, in 37 ℃ of incubators, cultivate 18～48h, the single bacterium colony of picking, in martin's bouillon liquid culture medium, cultivate again after 12～18h, add again 0.4% formalin-inactivated after 48 hours, coated plate carries out bacterial activity detection, after the complete death of bacterium, receives bacterium and puts to 4 ℃ of Refrigerator stores standby.2.2 animal used as test immunity
By the Candida albicans antigen-immunized animal preparing, chose for 2～3 monthly ages, 2 of new zealand white rabbits that body weight is 2～3kg are as immune animal, and one exempts from: 1mL108cfumL
-1abundant ultrasonic emulsification after deactivation Candida albicans mixes with Freund's complete adjuvant equal proportion, subcutaneous branch injection inside four limbs.After 3 weeks, carry out two and exempt from, 0.5 * 10
8cfumL
-1deactivation Candida albicans mixes with incomplete Freund's adjuvant equal proportion, the subcutaneous multi-point injection in back, and the separation of serum of simultaneously taking a blood sample carries out ELISA detection.Every three weeks, carry out respectively three and exempt to exempt from four.Add exempt from four exempt from after one week, blood sampling in a large number, separation of serum, and ELISA detects the specificity of antibody.
2.3 serum antibody purifying
Serum antibody, by positive Xin Liu – ammonium sulfate precipitation method purifying, is measured by foranalysis of nucleic acids instrument, and SDS – PAGE vertical electrophoresis is analyzed.
3. experimental result
Obtained the polyclonal antibody of higher anti-candida albicans, indirect elisa method detects to tire and can reach 1:140 more than ten thousand, and after purifying, its content is 10～30mg/mL, has good susceptibility; Each 6 strains of staphylococcus, streptococcus, Escherichia coli and clinically do not isolate each 6 strains of other bacterial strain of Candida albicans, totally 24 strain bacterium all detect no cross reaction phenomenon.
The preparation of embodiment 2 Candida albicans monoclonal antibodies
1 materials and methods
1.1 experiment materials: Sp2/0 murine myeloma cell, Candida albicans, staphylococcus aureus type strain, Escherichia coli type strain, streptococcus; Tissue Culture Dish, 50mL conical centrifuge tube, 96 hole ELISA Plate, bag filter etc.
1.2 animals used as test: Balb/c mouse.
2. experimental technique
2.1 experimental animal immunity
Select 6 of the Balb/c female mices in 6～7 week age to carry out immunity.Initial immunity, 0.25mL2 * 10
7cfumL
-1deactivation Candida albicans mixes with Freund's complete adjuvant equal proportion, and ultrasonic emulsification, at subcutaneous abdomen multi-point injection.After 3 weeks, the 2nd immunity, 0.25mL2 * 10
7cfumL
-1deactivation Candida albicans mixes with incomplete Freund's adjuvant equal proportion, the subcutaneous multi-point injection in back.Every three weeks, carry out respectively the 3rd time and the 4th immunity.Within after last immunity one week, collect blood, with ELISA, detect specificity and the SDS – PAEG electrophoresis of antibody.
2.2 cell hydridizations are merged
(1) at mixing operation first 24 hours, by two 70cm
2myeloma cell in Tissue Culture Dish departs from and goes down to posterity once with trypsase ethylenediamine tetraacetic acid mixed liquor.
(2) next day, take out the nutrient solution in myeloma cell's double dish, add respectively 5mL GENMED cleaning liquid (Reagent A), clean the cell surface in growth, sop up 5mL cleaning fluid, add respectively 3mL trypsase ethylenediamine tetraacetic acid mixed liquor, be paved with whole cultivation plane and put 37 ℃ of incubators 3 minutes.
(3) with hand, shake double dish, make cell detachment, then add respectively 10mL GENMED cleaning liquid (Reagent A), (myeloma cell 2～5 * 10 to get respectively two kinds of cell suspensions
7, more got mouse spleen cell 1 * 10 ready
8) two parent cells merge to a 50mL taper centrifuge tube, centrifugal 10 minutes, abandon supernatant.Add 10mL GENMED cleaning liquid (Reagent A), after mixing, centrifugal 1000rmin
-110 minutes, remove supernatant, with pointing gently attack centrifuge tube outer wall until cell granulations group is loose.
(4) add 1.5mL GENMED to merge liquid (Reagent B), fully mix, be placed in room temperature lower 1 minute.Add 10mL GENMED cleaning liquid (Reagent A), after mixing gently, centrifugal 1000rmin
-110 minutes, remove supernatant.Add complete cell culture fluid 8mL, fully mix.
(5) move into and have in feeder cells 96 well culture plates, 2～4, every hole adds the suspension of 200 μ L Fusion of Cells, puts 37 ℃ of incubators into.
After (6) 72 hours, start cell screening, until obtain, merge heterozygous cell clone (10～14 days).
The expansion of 2.3 positive hybridoma cells is cultivated
Through blowing and beating positive hybridoma suspension, move on to expansion cultivation in 24 orifice plates that contain feeder cells, add fresh HT nutrient culture media (24 holes add 0.5mL, and foramen primum adds 0.1mL) together with foramen primum, and at 5%CO
2cultivate 24～28 hours, with ELISA, detect, the stronger cell line of secretory antibody stability is done to emphasis and cultivate and preserve, carry out number and names also frozen in time.
2.4 limiting dilution assay monoclonals
In 96 orifice plates, carry out limiting dilution assay monoclonal cell and cultivate, by previously prepared feeder cells the previous day, then carry out Cell-cloned.5,10,50 cells in diluting every milliliter containing 20% hyclone cell culture fluid for the hybridoma of cloning to be done, add respectively in 96 orifice plates of oneself ready feeder cells every hole 100 μ L.During to subclone for the second time or for the third time, only make a dilutability, be adjusted to every milliliter of 5～10 cells, 96 orifice plates are placed on to 37 ℃, 5%CO
2incubator in cultivate.Timing is observed, and treats that cell grows to hole area approximately one half, inhales half nutrient solution and detects, and the cell in positive hole continues monoclonal, and expands cultivation, frozen standby.
The preparation of 2.5 ascites
(1) ascites induction: injection hybridoma one or two weeks ago, by every mouse peritoneal injection IFA0.2mL/ for inducing ascites only, divide cage and strengthen feeding and management.
(2) expand and cultivate hybridoma cell strain: the supernatant antibody hybridoma cell expansion that strong positive detected is cultured to exponential phase, and serves as induction ascites.
(3) injection hybridoma: the hybridoma in collection exponential phase is in 1000～1500rmin
-1centrifugal 5～10min, abandons supernatant, with about 0.5～1mL RPMI1640 serum-free medium re-suspended cell, and every mouse i.p6～32 * 10
5individual hybridoma.
(4) collect ascites: after approximately 7～10 days, observe Balb/c mouse web portion, have obvious belly struttuer, with No. 12 syringe needle puncture abdominal cavity downsides, collect ascites and measure it and tire, deposit in 80 ℃ of – standby.
2.6 titer of ascites and specific detection
Ascites sample detects analysis with ELISA and SDS – PAEG.
2.7 Candida albicans monoclonal antibody ascites cross reaction experiments are with the detection of Candida albicans polyclonal antibody.
The just pungent sulphur-ammonium sulfate precipitation of 2.8 Candida albicans monoclonal antibody (ascites) purifying
3. experimental result
This experiment, by technology such as immune Balb/c mouse, hybridoma integration technology, positive colony cell screening, limiting dilution assay and SDS – PAGE, ELISA detections, has successfully been prepared the monoclonal antibody of anti-candida albicans.Obtained altogether the cell line of 4 strain energy stably excreting anti-candida albicans monoclonal antibodies.With indirect elisa method, detect, more than tiring and can reaching 1:102400.Cross reaction experiment, with staphylococcus, streptococcus, Escherichia coli and do not isolate clinically other bacterial strain sample of Candida albicans and replace antigen, detects cross reaction phenomenon.The mensuration of monoclonal antibody protein concentration after purifying, twice monoclonal antibody protein of purifying crossed protein nucleic acid analysis-e/or determining, and its content reaches respectively 31.32mg/mL, 27.65mg/mL.
The preparation of embodiment 3 glue gold bodies
1. materials and methods
1.1 main agents: HAuCl
4, rabbit anti-candida albicans polyclonal antibody, trisodium citrate, bovine serum albumin(BSA) (BSA), skimmed milk power, PEG20000, K
2cO
3.
1.2 key instruments: high speed freezing centrifuge, electronic platform scale, gas bath constant temperature oscillator, DK – SD type electric heating constant temperature tank, ultraviolet-visible light splitting spectrophotometer.
The collaurum preparation of 1.3 trisodium citrate reduction method
Trisodium citrate reduction method is taked in the preparation of collaurum, with the beaker of 500mL, adds the HAuCl of 2.4mL1%
4in 240mL distilled water (final concentration is 0.01%), be sub-packed in respectively in 6 80mL beakers each 40mL.Electromagnetic oven heating is to boiling, add 1% trisodium citrate, respectively for 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1mL, 1.2mL(are labeled as respectively: 1,2,3,4,5, No. 6), continue heat and stir, the colloidal gold solution change color that detects by an unaided eye, waits that solution is black, purple by xanthochromia, heats 5～10min after claret again.Be cooled to room temperature, each sample complements to 40mL with ultrapure water, continue heating 5min, cooling, supply ultrapure water, in triplicate.
1.4 collaurum ultraviolet light scannings are identified
With ultraviolet-visible light spectrophotometer, 6 kinds of different collaurums are scanned under 400～700nm wavelength, obtain collaurum absorption spectrum, and determine the light absorption value of absorption maximum spike.Colloid gold particle diameter and distribution characteristics according to spectrum, prepare different-diameter colloid gold particle, collaurum is scanned, obtain collaurum maximum absorption band wavelength and particle diameter and draw Y=0.427lX+514.56(r=0.874 in 10～90nm particle size range, Ρ <0.01) (X is grain diameter to straight-line regression pass equation, Y is maximum absorption band wavelength), by Y=0.427lX+514.56 equation, can calculate each colloid gold particle diameter.Again 6 kinds of different colloidal gold solutions are statically placed in to 4 ℃ of a period of times, in the different time periods, check change color and the solids precipitation situation of colloidal gold solution, select a kind of more stable colloidal gold labeled monoclonal antibody and use.
1.5 optimum antibody protein binding capacities
By MeyShi stabilization test side: get 20mL colloidal gold solution 0.lmoL/L K
2cO
3regulate pH value, with 14 1.5mL EP pipes, get respectively 1mL colloidal gold solution, with the rabbit anti-candida albicans polyclonal antibody of variable concentrations, carry out mark again, every kind of concentration make 7 samples, after stirring at room 30min, add 0.1mL10%NaCI solution, standing one or two hour of room temperature, the color of observing colloid gold one antibody-solutions, enter again the OD value of ultraviolet spectrometry degree sweep measuring 520nm/580nm, select the antibody amount of the anti-the best of preparation gold mark.
1.6 antibody proteins are combined the selection of optimum pH value with collaurum
By MeyShi stabilization, test: get 8 1.5mL EP pipes, respectively add lmL collaurum, use respectively 0.lmoL/LK
2cO
3after adjusting pH to be 5,6,7,8,9,10,11,12, each pH makees 7 samples, it is the rabbit anti-candida albicans polyclonal antibody 100 μ L of 30 μ g/m1 that every pipe adds concentration, after stirring 30min, each pipe adds respectively 0.1mL10%NaCl liquid, standing l～2h under room temperature, observe its change color, then with ultraviolet spectrophotometer sweep measuring, respectively manage the OD value of 520nm/580nm, select the anti-pH value of best preparation gold mark.
The selection of 1.7 antibody-colloidal gold composite stabilizing agent
In order to prevent that golden labeling antibody from coagulation occurring in long-time, the golden labeling antibody compound that mark is good is selected respectively to BSA, PEG20000 used as stabilizers, and the effect of relatively using between them, select golden labeling antibody to occur to precipitate minimum as stabilizing agent.Its method: with 3 1.5mL EP pipes, add respectively 1mL to prepare golden labeling antibody solution, add respectively stabilizing agent 10%BSA100 μ L; 10%PEG20000100 μ L; 10%BSA50 μ L+10%PEG2000050 μ L places a period of time, and it is red that color still keeps, and what coagulation or coagulation were not less is the best stabilizer.
The selection of 1.8 colloidal gold composite damping fluids
Select different damping fluids to dilute the antibody colloidal gold compound of mark, select and be conducive to the damping fluid that keeps colloidal gold composite in stable condition, three kinds of colloidal gold antibody damping fluids of this experiment apolegamy system.Relatively stability and the colloid gold particle coagulation situation of these three kinds of damping fluids, test by orthogonal design, selects best Jiao Ti Jin – antibody complex damping fluid.
2. experimental result
The colloid gold particle that filters out diameter and be 20nm is more stable, is combined optimum with antibody protein with collaurum
ph value should be 8～9, and the suitableeest binding antibody egg amount is 20 μ g/mL.The best stabilizer 10%PEG20000 has been chosen in this experiment.What according to damping fluid in this experiment, gold mark is resisted has the greatest impact, affecting minimum factor is Tween – 20 concentration, selects Tris – HCl damping fluid, 1%BSA, 0.5%PEG20000,10% sucrose, 2%Tween – 20 that optimum optimization colloidal gold composite buffer composition were pH value is 8～9.
1. materials and methods
1.1 bacterial strains: Candida albicans standard bacterium, staphylococcus aureus type strain, Escherichia coli type strain, streptococcus.
1.2 main agents and consumptive material: the consumptive materials such as nitrocellulose filter (NC film), glass fibre element film, PVC plate.
2. experimental technique
2.1 dot immuno gold filtration assay tests
With dot immuno gold filtration assay test, determine monoclonal antibody and goat anti-rabbit igg package amount, by anti-candida albicans monoclonal antibody, do detection line (T) and goat anti-rabbit igg as nature controlling line (C), use PBS(0.0lmol/L pH7.4) damping fluid dilutes antibody by 1:0～1:128 multiple proportions number, respectively get 2 μ L point samples on 0.45 miillpore filter, after infiltration filter membrane, form spot.Treat that antibody is dry cmpletely.Filter membrane is soaked in 5% skimmed milk power, after room temperature sealing l～2h.With PBST, wash film 2～3 times, each 3～5min.Then washed filter membrane is blotted to water with absorbent filter, then place a period of time and make after film bone dry, drip Candida albicans suspension (1 * 105cfumL-1) and golden labeling antibody bond, standing several minutes of room temperature.Till punctation to be occurred.Then at room temperature with PBST cleansing solution vibration washing 4 times, each 3～5min.
2.2 analyzing film pre-service
NC film (analyzing film) first soaks after half an hour with 0.4% glycerine, is placed in 37 ℃ of drying bakers and dehydrates 2 hours with ankyrin.
The optimization of 2.3 analyzing film confining liquids
With 0.0l mo1/L PBS buffer system, prepare 2 kinds of different confining liquids with different concentration (1%, 2%, 3%, 4%, 5% skimmed milk power, 1%, 2%, 3%, 4%, 5%BSA) respectively to analyzing film sealing l h, drip again colloidal gold solution, according to background colour developing situation on NC film, select confining liquid.
2.4 analyzing film drying modes are selected
With anti-candida albicans monoclonal antibody and goat anti-rabbit igg, make respectively T line and C line is coated on NC film as analyzing film, NC film after sealing and carrying out washing treatment, in the constant situation of other conditions, by it put respectively 37 ℃, room temperature and 4 ℃ dry, observe colour developing and change.
2.3 the preparation of Jiao Ti Jin – antibody complex pad
Glass fibre membrane is cut into the equirotal fritter of specification (0.7 * 10.0cm) as pad, is then soaked in 30min in collaurum one antibody-solutions.Natural drying at room temperature, observes golden labeling antibody state on pad.
2.4 sample pad preparations
Glass fibre element film (sample pad) is cut into the band of 2 * 10cm specification size; Glass fibre element film immerses in pretreatment fluid (0.0lmol/L pH8.2 boric acid, 0.5%BSA, 0.1%Tween – 20) hatches after 1～2h in 4 ℃ or 37 ℃, and glass fibre element film is taken out and is placed in 37 ℃, baking oven and fully dries, and saves backup.
The antibody of 2.5 analyzing films is coated
NC film is cut into specification (2.5 * 10.0cm) of the same size band; Film is 1cm place from bottom to top, and the monoclonal antibody 1:2 that goes up anti-candida albicans with liquid-transfering gun point is as detection line (T); From detecting the upper sheep anti mouse 1:16 of band 0.7cm place's point as nature controlling line; Then by NC film in baking oven 37 ℃ fully dry, in 5% skimmed milk power, 37 ℃ of sealing l～2h or 4 ℃ spend the night, and take out kept dry standby.
2.6 adsorptive pads preparations
With filter paper, be used as adsorptive pads, it is cut into the band of specification (3.0 * 10cm) in the same size, be placed in dry environment and save backup.
2.7 test strips assemblings
By processed good various solid phase materials, adhere to successively on PVC base plate, form a complete test strips, idiographic flow is as follows:
(1) toughness base plate is cut into 0.75 * 14cm specification band of the same size.
(2) analyzing film is sticked to position, 4～10cm place on base plate, detection line is front, and nature controlling line is rear.
(3) gold-marking binding pad is affixed on to position, 2cm place in adhesive base, upper end is pressed on the upper end of analyzing film, overlapping 0.5cm.
(4) sample pad film is sticked on base plate, lower end is pressed on the upper end of gold-marking binding pad, overlapping 1cm.
(5) adsorptive pads film sticks on base plate, and upper end is pressed on the lower end of analyzing film, overlapping 1cm..
(6) band that assembling is shaped be stored in dry environment, detect standby.
2.8 ELISA test strip positive findingses are judged
With 2 * 10
5cfumL
-1reference white candida albicans suspension is as positive control, the negative contrast of 0.01mol/L PBS damping fluid.The Candida albicans sample suspension point of clinical identification is added in the sample pad of test strips, treats to observe testing result after sample chromatography.In detection, answer in district, T line and C line all show red stripes, represent object bacterial strain to be detected, are judged to the positive; If only have T line place to show red stripes, object bacterial strain do not detected, be judged to feminine gender; If the not aobvious red stripes of T line, test strips lost efficacy.
2.9 test strips sensitivity detect
By cultured Candida albicans culture, with after 0.4% formalin-inactivated, be diluted to 2 * 10
7, 2 * 10
6, 2 * 10
5, 2 * 10
4, 2 * 10
3, 2 * 10
2, 2 * 10
1cfumL
-1the bacterium liquid suspension of variable concentrations.Respectively get 300 μ L and click and enter above-mentioned test strips, each concentration repeats 5 times, treats to observe testing result after sample chromatography.
The special property of 2.10 test strips detects
With other bacterial strains such as staphylococcus, streptococcus, Escherichia coli, adjust each bacteria suspension concentration to 2 * l0
5cfumL
-1after, with liquid-transfering gun, to draw 300 μ L and click and enter in the sample pad of test strips, each sample repeats 3 times, treats to observe after sample chromatography.
3. experimental result
The equal no cross reactions of pathogen such as the Candida albicans colloidal gold immune chromatography rapid detecting test paper strip of more than preparing and Escherichia coli, streptococcus, staphylococcus, high with the special colour developing of Candida albicans, detection sensitivity is 10
2cfumL
-1, developing time is less than 10～20min.The test strips stability of preserving after 30 days under 4 ℃ of conditions is still better.In PCR detection method contrast experiment, the positive coincidence rate of test strips is 86.67%(13/15), there are 2 parts not detect, negative match-rate is 90%(9/10).
Claims (5)
1. a mastitis for milk cows Candida albicans fast diagnose test paper bar, comprise thieving paper, analyzing film, golden labeling antibody pad, sample pad and base plate, it is characterized in that: described analyzing film is containing the coated detection line of useful anti-candida albicans monoclonal antibody and with the coated nature controlling line of goat anti-rabbit igg, the anti-rabbit polyclonal antibody of Candida albicans of the coated colloid gold label of described golden labeling antibody pad.
2. mastitis for milk cows Candida albicans fast diagnose test paper bar according to claim 1, is characterized in that: described anti-candida albicans monoclonal antibody is to utilize hybridization integration technology filter out the hybridoma cell strain of Candida albicans monoclonal antibody and obtain; The anti-rabbit polyclonal antibody of described Candida albicans is repeatedly immune new zealand white rabbit and obtaining of application Candida albicans particulate antigen.
3. mastitis for milk cows Candida albicans fast diagnose test paper bar according to claim 2, is characterized in that: described analyzing film is nitrocellulose membrane, and golden labeling antibody pad is glass fibre membrane.
4. the preparation method of mastitis for milk cows Candida albicans fast diagnose test paper bar according to claim 1, is characterized in that comprising the following steps:
(1) prepare Candida albicans antibody
Anti-candida albicans monoclonal antibody is to adopt the Candida albicans of emulsification as antigen, and immune Balb/c mouse repeatedly stage by stage, then carry out Fusion of Cells filters out after positive hybrid cell strain, preparation ascites and obtaining; The anti-rabbit polyclonal antibody of Candida albicans is to adopt Candida albicans as repeatedly immune new zealand white rabbit preparation and obtaining of antigen;
(2) prepare analyzing film
Candida albicans monoclonal antibody prepared by step (1) is at the coated detection line that forms of nitrocellulose membrane, and goat anti-rabbit igg is at the coated nature controlling line that forms of nitrocellulose membrane, standby;
(3) prepare golden labeling antibody pad
Adopt trisodium citrate reduction method to prepare colloidal gold solution, and adopt the anti-rabbit polyclonal antibody of mark system mark Candida albicans, by golden labeling antibody immobilization on supporting film;
(4) assembling test strips
The analyzing film of step (2), the golden labeling antibody pad of step (3), thieving paper, sample pad, base plate are assembled into colloidal gold fast detecting test paper strip.
5. the preparation method of mastitis for milk cows Candida albicans fast diagnose test paper bar according to claim 4, it is characterized in that the marked body described in step (3) is: colloid gold particle is below 20nm, the pH value that antibody protein is combined with collaurum is 8～9, the antibody protein amount of association colloid gold is 20～40 μ g/mL, stabilizing agent is 10%PEG20000, and the component of colloidal gold composite damping fluid is that pH value is 8～9 Tris – HCl damping fluid, 1%BSA, 0.5%PEG20000,10% sucrose and 2%Tween – 20.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310674116.9A CN103616514A (en) | 2013-12-11 | 2013-12-11 | Rapid diagnosis test strip of cow mastitis candida albicans |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310674116.9A CN103616514A (en) | 2013-12-11 | 2013-12-11 | Rapid diagnosis test strip of cow mastitis candida albicans |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103616514A true CN103616514A (en) | 2014-03-05 |
Family
ID=50167220
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310674116.9A Pending CN103616514A (en) | 2013-12-11 | 2013-12-11 | Rapid diagnosis test strip of cow mastitis candida albicans |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103616514A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104991060A (en) * | 2015-06-01 | 2015-10-21 | 上海凯创生物技术有限公司 | Canidia albicans antigen colloidal gold detection kit |
| CN106290882A (en) * | 2015-06-01 | 2017-01-04 | 上海凯创生物技术有限公司 | A kind of Candida albicans antigen near-infrared fluorescent detection kit and application thereof |
| CN106461651A (en) * | 2014-06-04 | 2017-02-22 | 田中贵金属工业株式会社 | Method for eliminating prozone phenomenon in immunoassay reagent |
| CN110007081A (en) * | 2019-04-22 | 2019-07-12 | 贵州省畜牧兽医研究所 | Sheep Eaton agent pneumonia immuno-gold labeling quick diagnosis method for preparing test paper |
| CN110244049A (en) * | 2019-07-31 | 2019-09-17 | 河南美凯生物科技有限公司 | Pathogenic Candida detection and identification method |
| WO2021057406A1 (en) * | 2019-09-26 | 2021-04-01 | 河北省科学院生物研究所 | Reagent kit for detecting mastitis in dairy cows and usage method therefor |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060068500A1 (en) * | 2004-09-28 | 2006-03-30 | Kimberly-Clark Worldwide, Inc. | Detecting yeast infections using a lateral flow assay |
| US20080311595A1 (en) * | 2007-06-18 | 2008-12-18 | Inger Mattsby-Baltzer | Rapid fungal detection assay and product |
| CN102269763A (en) * | 2011-05-09 | 2011-12-07 | 沈鹤柏 | Quantitative testing test paper for bladder cancer |
| CN102650642A (en) * | 2012-05-08 | 2012-08-29 | 上海师范大学 | Antibody compound for detecting human prepalin retinol binding protein 4, as well as immunochromatography test card and kit |
| CN103204929A (en) * | 2012-01-16 | 2013-07-17 | 天津贻诺琦生物工程有限公司 | Preparation method for candida albicans polyclonal antibody |
-
2013
- 2013-12-11 CN CN201310674116.9A patent/CN103616514A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060068500A1 (en) * | 2004-09-28 | 2006-03-30 | Kimberly-Clark Worldwide, Inc. | Detecting yeast infections using a lateral flow assay |
| US20080311595A1 (en) * | 2007-06-18 | 2008-12-18 | Inger Mattsby-Baltzer | Rapid fungal detection assay and product |
| CN102269763A (en) * | 2011-05-09 | 2011-12-07 | 沈鹤柏 | Quantitative testing test paper for bladder cancer |
| CN103204929A (en) * | 2012-01-16 | 2013-07-17 | 天津贻诺琦生物工程有限公司 | Preparation method for candida albicans polyclonal antibody |
| CN102650642A (en) * | 2012-05-08 | 2012-08-29 | 上海师范大学 | Antibody compound for detecting human prepalin retinol binding protein 4, as well as immunochromatography test card and kit |
Non-Patent Citations (1)
| Title |
|---|
| AGNES MAROT-LEBLOND，ET AL.: "Evaluation of a Rapid Immunochromatographic Assay for Identification of Candida albicans and Candida dubliniensis", 《JOURNAL OF CLINICAL MICROBIOLOGY》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106461651A (en) * | 2014-06-04 | 2017-02-22 | 田中贵金属工业株式会社 | Method for eliminating prozone phenomenon in immunoassay reagent |
| US10466237B2 (en) | 2014-06-04 | 2019-11-05 | Tanaka Kikinzoku Kogyo K.K. | Method for excluding prozone phenomenon in immunological measurement reagent |
| CN104991060A (en) * | 2015-06-01 | 2015-10-21 | 上海凯创生物技术有限公司 | Canidia albicans antigen colloidal gold detection kit |
| CN106290882A (en) * | 2015-06-01 | 2017-01-04 | 上海凯创生物技术有限公司 | A kind of Candida albicans antigen near-infrared fluorescent detection kit and application thereof |
| CN110007081A (en) * | 2019-04-22 | 2019-07-12 | 贵州省畜牧兽医研究所 | Sheep Eaton agent pneumonia immuno-gold labeling quick diagnosis method for preparing test paper |
| CN110244049A (en) * | 2019-07-31 | 2019-09-17 | 河南美凯生物科技有限公司 | Pathogenic Candida detection and identification method |
| CN110244049B (en) * | 2019-07-31 | 2022-05-06 | 河南美凯生物科技有限公司 | Pathogenic candida detection and identification method |
| WO2021057406A1 (en) * | 2019-09-26 | 2021-04-01 | 河北省科学院生物研究所 | Reagent kit for detecting mastitis in dairy cows and usage method therefor |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103616514A (en) | Rapid diagnosis test strip of cow mastitis candida albicans | |
| Morrill et al. | Estimate of colostral immunoglobulin G concentration using refractometry without or with caprylic acid fractionation | |
| CN103123354B (en) | Immunodetection assay for mycobacterium tuberculosis complex | |
| CN103509756A (en) | Mycoplasma bovis monoclonal antibody, and preparation method and application thereof | |
| CN103333864B (en) | Monoclonal antibody of toxoplasma gondii resistant MIC3 protein and application monoclonal antibody | |
| CN105044365A (en) | Preparation method of time-resolved fluoroimmunoassay test paper for detection of Enrofloxacin residue | |
| CN110161239A (en) | A kind of double-antibody sandwich colloid gold test paper based on EFTu, staphylococcus detection method and application | |
| CN102183653A (en) | Cryptosporidium parvum immune colloidal gold detection test paper strip and production method thereof | |
| CN103797370B (en) | For detecting the immunoassay of antibiotic | |
| CN105137068A (en) | Florfenicol on-site test paper, and preparation and use methods thereof | |
| CN105334323A (en) | Method and test strip for detecting zilpaterol, and application of test strip | |
| CN102020713A (en) | Immune colloidal gold test strip used for detecting aureomycin residue and preparation method thereof | |
| CN104749368B (en) | Duck Tembusu virus monoclonal antibody and application | |
| CN103777018A (en) | Preparation method of test strip for rapidly diagnosing porcine contagious pleuropneumonia actinobacillus | |
| CN108918875A (en) | Pigeon with newcastle disease monoclonal antibody and the application in preparation diagnosis and detection kit | |
| CN104965083B (en) | A kind of kit of detection H3N2 hypotype canine influenza virus | |
| CN103509757A (en) | Hybridoma cell strain No. 15, and pyrethroid-resistant mass selection monoclonal antibody produced by hybridoma cell strain No. 15 | |
| CN101004419B (en) | Colloid selenium test paper for detecting pulpy kidney, and type of pathogenesis bacteria | |
| CN104911150B (en) | The foundation of monoclonal antibody hybridoma cell strain and its preparation and application of monoclonal antibody of a kind of H3N2 canine influenza virus | |
| CN104267189B (en) | Human alpha-lactalbumin screening test strip and preparation method and application in milk | |
| CN103336121B (en) | Swine influenza virus H3 subtype colloidal gold detection kit as well as preparation method thereof | |
| CN111303282B (en) | Paired anti-MRJP 2 monoclonal antibody and application thereof in qualitative or quantitative detection of MRJP2 | |
| CN102146138B (en) | Monoclonal antibody of chloramphenicol and application thereof | |
| CN102012432B (en) | Colloidal gold immunochromatographic test paper based on recombinant UL51 protein as well as preparation method and application thereof | |
| CN104777302A (en) | Immunochromatographic assay test strip for bacterial fruit blotch of melons |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20140305 |