CN104316703B - A kind of Mycoplasma bovis test strip and its preparation method - Google Patents
A kind of Mycoplasma bovis test strip and its preparation method Download PDFInfo
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- CN104316703B CN104316703B CN201410615939.9A CN201410615939A CN104316703B CN 104316703 B CN104316703 B CN 104316703B CN 201410615939 A CN201410615939 A CN 201410615939A CN 104316703 B CN104316703 B CN 104316703B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56933—Mycoplasma
Abstract
The open a kind of Mycoplasma bovis antibody test test strip of the present invention and its preparation method. The test strip of the present invention by base plate, be arranged on base plate that linearly aligned absorption of sample pad, the colloidal gold pad being coated with antigen, nitrocellulose membrane and absorbent pad form successively, nitrocellulose filter is coated with nature controlling line and detection line, wherein detection line bag quilt is goat anti-ox two anti-igg, and nature controlling line is many anti-igg of rabbit source P48 fusion rotein. The test strip of the present invention can detect Mycoplasma bovis antibody, comprises serum antibody and whey antibody, and it is low to have cost, fast simple, convenient, it is not necessary to specific installation and instrument, detection sensitivity height, high specificity, the advantages such as applied range.
Description
Technical field
The present invention relates to a kind of biological detection reagent preparation method and reagent, the present invention relates to a kind of Mycoplasma bovis antibody test test strip preparation method and prepared test strip thereof exactly.
Background technology
Mycoplasma bovis is a kind of pathogenic pathogen seriously, cattle respiratory disease, mastadenitis of cow, sacroiliitis, keratoconjunctivitis, urogenital tract infection and miscarriage and the multiple disease such as infertile can be caused, and once infect the secondary infection that can cause other cause of disease, such as suppurative Pyrogenes, pasteurella multocida, Mannheimia haemolytica, lethargic sleep influenzae, bovine parainfluenza virus 3 type, simplexvirus 1 type, Bovine Respiratory Syncytial virus, bovine viral diarrhea virus and bovine coronavirus etc. Mycoplasma bovis sickness rate is 50%-100%, and case fatality rate, up to 10%-50%, causes huge financial loss to China's cattle-raising. This disease China actual popularity and epidemiology information is still comparatively deficient accurately, cause the reason of this kind of phenomenon, mainly China lacks the correlation technique of effective Mycoplasma bovis detection at present.
The technology of the detection Mycoplasma bovis existed at present has: 1. Antigen isolation and identification, but Mycoplasma bovis separation is often subject to the impact of clinical application microbiotic, separation difficulty, and separation substratum nutritional requirement height, isolate qualification difficulty is big, sensitivity is low, the method that can not infect as Rapid&Early diagnosis Mycoplasma bovis, also it is difficult to as conventional sense method penetration and promotion (coming from CaswellJL, ArchambaultM.Mycoplasmabovispneumoniaincattle), 2.PCR detection method (comes from ThomasA, DizierI, LindenA, etal.ConservationoftheuvrCgenesequenceinMycoplasmabovisa nditsuseinroutinePCRdiagnosis), this detection method needs samples carries out more complicated process (grinding, centrifugal, extract DNA etc.), though detection method is sensitive, but easily than being easier to, false positive occurs because polluting and deal with improperly, affect the accuracy of result of determination, in addition certain laboratory condition and plant and instrument is also needed, and relatively high cost, be not suitable for clinical or basic unit's use. 3. indirect ELISA applies topmost Serology test at present, successively useful whole bacterial protein bag quilt and expressing protein (come from GrandDL as the method for envelope antigen in the world, CalavasD, BrankM, etal.SerologicalprevalenceofMycoplasmabovisinfectioninsu cklingbeefcattleinFrance), the method specificity deficiency of whole bacterial protein is now seldom applied, expressing protein antigen ELISA method is existing commercially produced product at present, but its which antigen protein used is not announced. in addition, ELISA method is only adapted at laboratory and detects, operation steps relative complex, it is necessary to professional operates, and needs certain laboratory condition and plant and instrument to carry out determination and analysis result. 4. utilizing the indirect hemagglutination detection method that P48 recombinant expression protein bag is set up by sheep red blood cell (SRBC), operation is relatively simple, it also requires 2 ~ 3 hours just can result of determination, susceptibility is weak, it is necessary to specific constant incubator.
Summary of the invention
The present invention provides one can overcome prior art deficiency, can simple and direct, quick, sensitive detection Mycoplasma bovis serum antibody, whey antibody test strip preparation method, the test strip prepared in this way with this.
The test strip that the present invention a kind of detects Mycoplasma bovis antibody by base plate, be arranged on base plate that linearly aligned absorption of sample pad, the colloidal gold pad being coated with antigen, nitrocellulose membrane and absorbent pad form successively, nitrocellulose filter is coated with nature controlling line and detection line, the antigen of the colloidal gold pad bag quilt in test strip of the present invention is Mycoplasma bovis P48 fusion rotein, detection line bag quilt be goat anti-ox two anti-igg, nature controlling line is many anti-igg of rabbit source P48 fusion rotein.
The preparation method of the test strip of the detection Mycoplasma bovis antibody of the present invention is: prepared colloidal gold pad with the P48 recombinant protein bag of purified rear dialysis by Radioactive colloidal gold; Nitrocellulose filter wrap by nature controlling line and detection line, wherein: the antigen used of detection line is affinity purification goat anti-ox two anti-igg, nature controlling line antigen is the many anti-igg of P48 albumen, on base plate successively respectively by absorption of sample pad, colloidal gold pad, be coated with detection line and the nitrocellulose filter of nature controlling line and absorbent pad assemble in order.
In the preparation method of the test strip of the detection Mycoplasma bovis antibody of the present invention: Radioactive colloidal gold bag is 6ug/ml by the concentration of P48 albumen, the time of antigen coated 1% gold sol is 30min, process in antigen combination adds 20%BSA solution (20gBSA joins in 100ml deionized water) 50ul/ml and closes, off-period is 30 minutes, utilize re-suspension liquid by resuspended for colloid gold particle preparation colloidal gold pad after centrifugal, then drying treatment, re-suspension liquid formula is: 0.02M sodium tetraborate, 0.5%BSA, 3% sucrose, 0.5% casein, 0.5% polysorbas20, gold mark pad is contributed to better to dissociate by re-suspension liquid, BSA in re-suspension liquid can close the appearance that false positive is avoided in non-specific site, casein and sugar composition can protect the antigen of gold grain bag quilt. wrapping by the anti-ox two anti-igg antigen concentration of affinity purification goat used of nitrocellulose filter detection line is 100ug/ml, and package amount is 1ul/cm, and bag is 200ug/ml by nitrocellulose filter nature controlling line P48 albumen many anti-igg antigen concentration, and package amount is 1ul/cm.
The preparation method of P48 albumen many anti-igg antigen that the present invention uses is: adds 1mg/mlP48 proteantigen 3mL with Freund's complete adjuvant 3mL and prepares Freund's complete adjuvant antigen, and abundant mixing and emulsifying; Add 1mg/mlP48 albumen 3mL with Freund's incomplete adjuvant 3mL and prepare Freund's incomplete adjuvant antigen, and abundant mixing and emulsifying; Selecting healthy male big white rabbit subcutaneous at rabbit two back leg foot, the subcutaneous multiple spot inoculation Freund's complete adjuvant antigen in lymphoglandula place and back, inoculation total amount is 1.5mL; Still inoculate by the subcutaneous multiple spot in back with Freund's complete adjuvant antigen 1 .5ml after 14 days, carry out second time immunity; After 14 days, with Freund's incomplete adjuvant antigen, at every rabbit sole two point, the subcutaneous multiple spot inoculation in back, inoculation total amount is 3mL, is its third time immunity; By ear edge vein exploitating blood separation of serum after 14 days, with IHA test examination blood, immunize rabbit serum I HA tires and reaches 1:512, formal blood sampling, the centrifugal 5min separation of serum of 3000rpm, obtains P48 albumen hyper-immune serum, is purified by the serum being separated to by saturated ammonium sulphate method and obtains the many anti-igg of P48 albumen.
The present invention has the following advantages:
(1) wrapping by the antigen P48 albumen of Radioactive colloidal gold in the present invention is Mycoplasma bovis membranin, has very strong specificity, can truly detect Mycoplasma bovis antibody, comprise serum antibody and whey antibody;
(2) the P48 albumen in the present invention is prokaryotic expression soluble proteins, purity height, active high, detection antibody sensitivity height, and high specificity, has wide range of applications;
(3) cost is low, fast simple, convenient, without the need to specific installation and instrument, whole process completes in 15min, more cost-saving than indirect hemagglutination test and ELISA test, test result presents macroscopic color reaction, it is not necessary to specific apparatus, without the need to specific operator, can conveniently for Mycoplasma bovis epidemiology survey and promote the use of in basic unit;
(4) marker is stablized, and mark sample stores more than 2 year year at 4 DEG C, no signal decay phenomenon;
(5) Radioactive colloidal gold is originally as redness, it is not necessary to adds and sends out a color reagent, eliminates the step of enzyme target carinogenicity substrate and stop buffer in ELISA detection method, to human non-toxic's evil.
Accompanying drawing explanation
Fig. 1 is the test strip schematic diagram of the present invention, in figure: 1 absorption of sample pad, 2 colloidal gold pad, 3 detection line T, 4 nature controlling line C, 5 absorbent pad, 6 base plates, 7 nitrocellulose membranes.
Fig. 2 is the P48 recombinant protein SDS-PAGE of purifying, wherein: M. protein molecular quality standard; 1 swimming lane is albumen before purifying; 2 swimming lanes are the albumen of first column volume of wash-out after purifying; 3 swimming lanes are the albumen of the 2nd column volume of wash-out after purifying; 4. swimming lane is the albumen of the 3rd column volume of wash-out after purifying; 5 swimming lanes are the albumen of the 4th column volume of wash-out after purifying; 6 swimming lanes are the albumen of the 5th column volume of wash-out after purifying; 7. swimming lane is the albumen of the 6th column volume of wash-out after purifying.
Fig. 3 be the test strip of the present invention to standard positive serum and negative serum detected result figure, wherein: 1 is the blank of sample diluting liquid; 2 is negative serum; 3 is Mycoplasma bovis positive standard serum; (note: owing to serum glues thick, negative serum and positive serum are all diluted 10 times and detects).
Fig. 4 is the detected result figure of test strip of the present invention for the positive standard serum of different gradient dilution, and 1 is the blank of sample diluting liquid; 2 is negative serum; 3 is that Mycoplasma bovis positive standard serum 1:10 dilutes; 4. it is that Mycoplasma bovis positive standard serum 1:100 dilutes; 5 is that Mycoplasma bovis positive standard serum 1:1000 dilutes; 6 is that Mycoplasma bovis positive standard serum 1:10000 dilutes; 7 is that Mycoplasma bovis positive standard serum 1:100000 dilutes; 8 is that Mycoplasma bovis positive standard serum 1:200000 dilutes.
Fig. 5 is test strip specificity testing inspection of the present invention, is 1. Mycoplasma bovis positive standard serum; 2. Mycoplasma mycoides subsp.capri positive standard serum; 3. mycoplasma hyopneumoniae positive standard serum; 4. ox pasteurellosis bacillus positive standard serum; 5. pleuropneumonia positive standard serum; 6. ox genital tract mycoplasma positive standard serum; 7. mycoplasma ovine pneumoniae positive standard serum; 8. goat mycoplasma goat pneumonia subspecies positive standard serum; (serum is 1:10 dilution).
Embodiment
The present invention is below in conjunction with embodiment explanation.
1.P48 the Expression and purification of albumen
1.1 sequent synthesis and construction of recombinant plasmid
Mycoplasma bovis p48 gene (Genebank:NC_014760.1), this stream cipher is optimized by the present invention, it is optimized according to the hobby of codon intestinal bacteria, in this sequence of Mycoplasma bovis 4 are expressed the TGA(UGA of tryptophane simultaneously) it is optimized to TGG(UGG), because this codon is terminator codon in intestinal bacteria, adding restriction enzyme site BamHI and XhoI at its two ends, its gene order is SEQID �� 1 simultaneously;
By optimize after p48 gene order transfer to Invitrogen company to be synthesized in pet32a expression vector plasmid, after synthesis, carry out order-checking qualification, enzyme cuts qualification, by identify the positive recombinant plasmid called after Pet32-a after successfully (+)-p48.
1.2P48 the expression of albumen
By the recombinant plasmid pet32a (+)-p48 be transformed into competent cell BL21(DE3) with goal gene, choose and get 2 single bacterium colonies being transformed into BL21 (DE3) and be inoculated in 5ml respectively containing in the LB liquid nutrient medium of penbritin, it is positioned in 37 DEG C of shaking tables, about 220rpm concussion is cultured to OD value to 0.6, then the bacterium liquid drawing this 10ml is inoculated into the LB liquid nutrient medium of new 1L containing penbritin 100ug/ml, at 16 DEG C, abduction delivering 20h under 0.01mmol/lIPTG concentration, collect thalline, resuspended with the PH7.2PBS of 100ml, and carry out ultrasonication 30min on ice. then the liquid of supersound process is placed in the centrifugal 30min of whizzer 11000rpm, abandons precipitation, collect supernatant 100ml.
The dialysis of 1.3 affinity chromatography column purification P48 albumen and purifying protein
P48 recombinant protein is purified by nickel ion affinity chromatograph method. First by 5mLNi-NTAHisBindResin(purchased from Nanjing Jin Sirui biotechnology company) load empty chromatography column, after allowing liquid lean on action of gravity naturally to drip and, the chromatography column balance Buffer of 20 times of column volumes washs, then Sample supernatants is added in chromatography column, then allows it flow out in conjunction with 60min on ice; The chromatography column NTA-10 of 20 times of column volumes washs foreign protein; Then carrying out wash-out with the NTA-60 wash-out Buffer of 6 column volumes respectively, every 5ml collects once, obtains albumen after purifying, and electrophorogram of its detection is shown in accompanying drawing 2.
By the albumen of first volume purifying, the albumen of the 2nd volume purifying, three or four volume purifying protein merges, five or six volume purifying protein merges, and is respectively charged into dialysis tubing, puts into the large beaker that 5L is equipped with dialyzate, dialyzate is 0.1MPB damping fluid, put into 4 DEG C of refrigerator dialysed overnight, within the 2nd day, take out, the P48 albumen dialysed is loaded EP pipe and puts into-40 DEG C of Refrigerator stores (1mL/ pipe).
2. the preparation process of colloidal gold strip
2.1 Radioactive colloidal gold preparation process
Getting 1% aqueous solution of chloraurate 1ml adds in 100ml deionized water, it is heated to boiling at temperature control stirring device mouth, disposable rapidly again add 1% trisodium citrate 1.5ml, continue heated and boiled 15min, then mend with water and namely obtain the colloidal gold solution of homogeneous transparent to 100ml together.
The preparation of 2.2 Radioactive colloidal gold mark pads
After the purifying obtained with aforesaid method, the P48 recombinant protein bag of dialysis by Radioactive colloidal gold and prepares colloidal gold pad, antigen is 7.4 in conjunction with the pH value of Radioactive colloidal gold, Radioactive colloidal gold bag is 6ug/ml by the optimum concn of P48 albumen, the time of antigen coated gold grain is 30min, adding 20%BSA50ul/ml after antigen combines to close, off-period is 30 minutes, utilizes re-suspension liquid that colloid gold particle is resuspended after centrifugal, prepare Radioactive colloidal gold mark pad, 37 DEG C of dryings 3 hours. Its re-suspension liquid formula is: 0.02M sodium tetraborate, 0.5%BSA, 3% sucrose, 0.5% casein, 0.5% polysorbas20.
The bag quilt of nature controlling line and detection line on 2.3 nitrocellulose filters
Test strip nitrocellulose filter model of the present invention is M135, buy in MILLIPORE (U.S.), wrapping by the antigen used of nitrocellulose filter detection line is 100ug/ml affinity purification goat anti-ox two anti-igg, wrapping tested survey line 1ul/cm, anti-ox of affinity purification goat two is anti-buys in ImmunoReagents Reagent Company for this.
Bag is the many anti-igg of P48 albumen by nitrocellulose filter nature controlling line antigen, utilizes its concentration for 200ug/ml, and bag is by nature controlling line 1ul/cm. This IgG obtains through purifying with hyper-immune serum. The preparation method of hyper-immune serum is as follows:
The preparation of Freund's complete adjuvant antigen used and Freund's incomplete adjuvant antigen in hyper-immune serum preparation, wherein: Freund's complete adjuvant antigen: Freund's complete adjuvant 3mL adds 1mg/mlP48 proteantigen 3mL; Freund's incomplete adjuvant antigen: Freund's incomplete adjuvant 3mL adds 1mg/mlP48 albumen 3mL.
Above-mentioned adjuvant antigen is all prepared before immunization, and abundant mixing and emulsifying.
P48 albumen hyper-immune serum preparation process:
First, a. the big white rabbit of healthy Male New Zealand at about 2kg, 12 monthly ages is selected.
B. above-mentioned adequately emulsified Freund's complete adjuvant antigen is got, subcutaneous in every rabbit two back leg foot, the subcutaneous multiple spot inoculation in lymphoglandula place and back, inoculation total amount is 1.5mL; Still inoculate by the subcutaneous multiple spot in back with Freund's complete adjuvant antigen 1 .5ml after 14 days, carry out second time immunity; After 14 days, with above-mentioned Freund's incomplete adjuvant antigen, by every rabbit sole two point, the subcutaneous multiple spot inoculation in back, inoculation total amount is 3mL, is its third time immunity;
C. by ear edge vein exploitating blood separation of serum after 14 days, with IHA test examination blood, immunize rabbit serum I HA tires and reaches 1:512, formally takes a blood sample, and the centrifugal 5min separation of serum of 3000rpm, is P48 albumen hyper-immune serum.
D. by saturated ammonium sulphate method the serum being separated to purified and obtain the many anti-igg of P48 albumen.
2.4 test strip assemblings
Operation carries out to comprise the nitrocellulose filter of detection line and nature controlling line respectively from top to bottom on PVC backing plate as follows, colloidal gold pad, sample pad, and absorption pad assembles in order, PVC liner plate is located at bottommost, stage casing, liner plate top is provided with nitrocellulose filter, nitrocellulose filter upper left end posts absorption pad, nitrocellulose filter upper right end is provided with gold mark pad, the upper right end of gold mark pad is provided with sample pad, with spray gun, the many anti-igg of P48 of aforementioned affinity purification goat anti-ox two anti-igg that obtains and purifying are made detection line and nature controlling line on nitrocellulose filter respectively again, in test strip of the present invention, detection line and nature controlling line two distance between centers of tracks are from remaining on 0.8mm. test paper is cut into the wide strip of 4mm again, namely makes the colloidal gold strip schematic diagram of the detection Mycoplasma bovis antibody of the structure the present invention such as accompanying drawing 1.
The test strip principle of the present invention is dual-antigen sandwich method detection antibody, and wherein criterion is under the prerequisite of nature controlling line colour developing, and detection line colour developing is positive; Detection line, without colour developing, is negative. If nature controlling line does not develop the color, then lost efficacy, it is necessary to again measure.
Positive serum and negative serum are detected by colloidal gold strip of the present invention respectively, the results are shown in accompanying drawing 3, and from accompanying drawing 3, positive serum display detection line and nature controlling line two lines are redness; Negative nature controlling line colour developing, detection line does not develop the color; It is nature controlling line colour developing that blank is only sample diluting liquid result, and detection line does not develop the color. Wherein sample diluting liquid is the 0.1MPBS damping fluid containing 0.5% polysorbas20.
The different extent of dilution of positive serum is detected by colloidal gold strip of the present invention, the results are shown in accompanying drawing 4, and from accompanying drawing 4, the equal nature controlling line colour developing of negative serum, sample diluting liquid blank, detection line does not develop the color. Positive serum 1:10,1:100,1:1000,1:10000,1:100000 are nature controlling line and detection line all develops the color, and present the positive; And positive serum 1:200000 only nature controlling line colour developing, present feminine gender, therefore in this invention, test strip sensitivity can reach, and the Mycoplasma bovis indirect hemagglutination detection method set up before only can detect 1:4096, so this invention substantially increases detection sensitivity.
Test strip specificity testing inspection of the present invention the results are shown in accompanying drawing 5, wherein have detected Mycoplasma bovis positive standard serum, Mycoplasma mycoides subsp.capri positive standard serum, mycoplasma hyopneumoniae positive standard serum, ox pasteurellosis bacillus positive standard serum, pleuropneumonia positive standard serum, ox genital tract mycoplasma positive standard serum, mycoplasma ovine pneumoniae positive standard serum and goat mycoplasma goat pneumonia subspecies positive standard serum, result only presents the positive by Mycoplasma bovis positive serum, all the other similar mycoplasmas and the pathogenic autoantibody of ox similar conditions can be caused all to present feminine gender, prove that this test strip specificity is good, reliable results.
The detection paper of import ELISA kit and the present invention is utilized to contrast, detect the negative sample (containing whey, serum) totally 83 parts of clear and definite background, positive (containing whey, serum) totally 189 parts, wherein import ELISA kit detection negative serum 90 parts, positive serum 182 parts, and test strip detected result negative serum 86 parts in this invention, positive serum 186 parts. With the detection contrast of import ELISA kit, negative match-rate is 95.6%, positive coincidence rate is 97.8%, and the cost of import ELISA kit detection sample is up to 5 yuan/part, and manipulation require 3 hours just can go out detected result, and test strip cost 0.5 yuan/part in the present invention, in 15min, only just can present result clear, that understand, greatly reduce cost, it is to increase efficiency.
<110>Chinese Academy of Agricultural Sciences Lanzhou veterinary institute
<120>a kind of Mycoplasma bovis antibody test test strip and its preparation method
<160>1
<210>1
<211>1419
<212>DNA
<213>through optimize after P48 gene order
<400>
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Claims (3)
1. one kind is detected the preparation method of the test strip of Mycoplasma bovis antibody, it is characterised in that: prepared colloidal gold pad with the P48 recombinant protein bag of purified rear dialysis by Radioactive colloidal gold, nitrocellulose filter wrap by nature controlling line and detection line, wherein: the antibody used of detection line is affinity purification goat anti-ox two anti-igg, nature controlling line antibody is the many anti-igg of P48 albumen, successively respectively by absorption of sample pad on base plate, colloidal gold pad, the nitrocellulose filter and the absorbent pad that are coated with detection line and nature controlling line assemble in order, Radioactive colloidal gold bag is 6 �� g/ml by the concentration of P48 recombinant protein, bag is 30min by the time, after antigen combines, add 20%BSA solution 50 �� l/ml close, off-period is 30 minutes, re-suspension liquid is utilized to prepare colloidal gold pad by resuspended for Radioactive colloidal gold after centrifugal, then drying treatment, re-suspension liquid formula is: 0.02M sodium tetraborate, 0.5%BSA, 3% sucrose, 0.5% casein, 0.5% polysorbas20, wrapping by the anti-ox two anti-igg concentration of affinity purification goat used of nitrocellulose filter detection line is 100 �� g/ml, package amount is 1 �� l/cm, bag is 200 �� g/ml by P48 albumen many anti-igg concentration that nitrocellulose filter nature controlling line is used, package amount is 1 �� l/cm.
2. the preparation method of a kind of test strip detecting Mycoplasma bovis antibody described in claim 1, it is characterised in that add 1mg/mlP48 proteantigen 3ml with Freund's complete adjuvant 3ml and prepare Freund's complete adjuvant antigen, and abundant mixing and emulsifying; Add 1mg/mlP48 albumen 3ml with Freund's incomplete adjuvant 3ml and prepare Freund's incomplete adjuvant antigen, and abundant mixing and emulsifying; Selecting healthy male big white rabbit subcutaneous at rabbit two back leg foot, the subcutaneous multiple spot inoculation Freund's complete adjuvant antigen in lymphoglandula place and back, inoculation total amount is 1.5ml; Still inoculate by the subcutaneous multiple spot in back with Freund's complete adjuvant antigen 1 .5ml after 14 days, carry out second time immunity; After 14 days, with Freund's incomplete adjuvant antigen, at every rabbit sole two point, the subcutaneous multiple spot inoculation in back, inoculation total amount is 3ml, is its third time immunity; By ear edge vein exploitating blood separation of serum after 14 days, with IHA test examination blood, immunize rabbit serum I HA tires and reaches 1:512, formal blood sampling, the centrifugal 5min separation of serum of 3000rpm, obtains P48 albumen hyper-immune serum, is purified by the serum being separated to by saturated ammonium sulphate method and obtains the many anti-igg of P48 albumen.
3. the test strip of the detection Mycoplasma bovis antibody that prepared by method described in claim 1 or 2.
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| CN105695417B (en) * | 2016-01-04 | 2018-11-06 | 甘肃农业大学 | One plant of hybridoma cell strain for secreting Mycoplasma bovis monoclonal antibody and its application |
| CN109541199B (en) * | 2018-11-26 | 2022-02-11 | 重庆理工大学 | Immune colloidal gold test paper for rapidly detecting mycoplasma bovis and preparation method thereof |
| CN109459570A (en) * | 2018-12-26 | 2019-03-12 | 重庆市职业病防治院（重庆市第六人民医院） | The colloid gold immune test paper preparation method of lead ion in a kind of quick detection blood of human body |
| CN111323605A (en) * | 2020-02-27 | 2020-06-23 | 四川新健康成生物股份有限公司 | Method for improving chromatographic linear range and β -HCG fluorescence chromatography detection test strip |
| EP4320438A1 (en) * | 2021-04-09 | 2024-02-14 | Pictor Limited | Multiplex immunoassay for the detection of mycoplasma bovis infection |
| CN113884674A (en) * | 2021-09-14 | 2022-01-04 | 石河子大学 | Mycoplasma bovis colloidal gold immunoassay test strip, preparation method and application thereof |
| CN113754744B (en) * | 2021-09-22 | 2023-06-02 | 宁夏农林科学院动物科学研究所(宁夏草畜工程技术研究中心) | Mycoplasma bovis protein SBP-2 and application thereof |
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