CN106771208A - Brucella antibody test strip - Google Patents
Brucella antibody test strip Download PDFInfo
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- CN106771208A CN106771208A CN201610987487.6A CN201610987487A CN106771208A CN 106771208 A CN106771208 A CN 106771208A CN 201610987487 A CN201610987487 A CN 201610987487A CN 106771208 A CN106771208 A CN 106771208A
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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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
Abstract
The present invention relates to a kind of Brucella antibody test strip.The present invention has following meaning：(1) China's brucellosis diagnostic method is enriched；(2) for China's brucellosis diagnosis provides a kind of new method simple and efficient, that can various different animals be implemented with field quick detection；(3) this test strips overcomes conventional brucellosis diagnostic method and can not exclude the interference of yersinia enterocolitica O9 and Escherichia coli O 157 antiserum to Brucella antibody, improves the specificity of brucellosis detection.
Description
Technical field
The present invention relates to a kind of Brucella antibody test strip, belong to biological products detection technique field.
Technical background
Brucellosis (Brucellosis, abbreviation cloth disease) is drawn by brucella or brucella (Brucella)
The Amphixenosis being characterized with miscarrying and generating heat for rising, seriously threatens the life and health of people and many animals.This disease is not
But there is serious harm to the breeding and production performance of animal, it is often more important that, after people infection brucella, it tends to be difficult to control
More, so as to cause serious public health problem.Therefore in the country that brucella is popular, it is always public health to eliminate cloth disease
One of most important target in the works.
There is existing history remote in cloth disease, the mankind progressively deepen to the research understanding of the disease, and diagnostic level is or not the world
Disconnected to improve, with the progress of animal cloth disease, the serological detection method of animal cloth disease is updated and improved.Traditional is solidifying
Collection experiment is (such as：The red antigen plate agglutination test of tiger, the experiment of test tube agglutination of antigen, the agglutination test of newborn ring) gradually by sensitiveness it is high,
The new detection techniques such as ELISA method, the fluorescence polarization assay (FPA) of high specificity are replaced.
Agglutination test is a kind of conventional method of brucellosis diagnosis, including serum agglutination is tested, newborn ring precipitation is tried
Test and tested with human immunoglobulins, wherein classical standard tube agglutination test (STAT), plate agglutination test (PAT), with
Upper method is stopped using substantially in developed country, the substitute is buffering brucellergen agglutination test such as tiger red flat
Plate agglutination test (RBPT).The red Avian tubercula plain agglutination test antigen of tiger is through culture, inactivation, centrifugation with the good brucella bacterial strain of antigenicity
It is suspended in lactic acid buffer after being dyeed with brave red after collects thalline and is made, the method sensitivity is high, cheap, operation
Convenient, detection is quick, is suitable to the generaI investigation of animal population brucellosis.It is ox, sheep, traum's disease inspection in international trade
The specified experiment surveyed, the primary dcreening operation of people's brucellosis surveillance is also used in China.Newborn ring precipitation test is still cow cloth Lu Shi
The main method of bacterium monitoring, the method directly detects that screening can be carried out to milk cow colony, and the method is easy to be fast to cow's milk
Speed.
Unanimously think that complement fixation test (CFT) (CFT) is better than its other party in specificity in brucellosis patients inspection
Method, is often used to carry out qualitative inspection to tube agglutination test and rose bengal precipitation test test positive or suspicious case
Survey.Complement fixation test (CFT) is considered as always maximally effective cloth disease diagnostic method, can still be replaced without a kind of diagnostic method so far
Status of the complement fixation test (CFT) in serodiagnosis.But the preparation of the hemolysin required for complement fixation test (CFT) is difficult, experiment
It is cumbersome, it is difficult to clinically to commonly use, and the effect of GPC can be disturbed due to the complement in pig body, cause quick
Perception declines.
ELISA is a kind of diagnostic method suitable with CFT effects, and the method is easy to operate, and sensitivity is high, specificity compared with
It is good, the detection of a large amount of samples can be once completed, both animal population quarantine can be applied to as screening test, it is also used as
Confirmed diagnosis test.ELISA can be applied not only to the detection of serum antibody, apply also for the detection of antibody in milk sample.The cloth of ox
Shandong Salmonella disease ELISA detection method has been one of detection method for specifying in international trade, other kind of ELISA inspection of brucella
Survey method is also the focus of research.Being presently used for the ELISA of brucellosis detection has indirect ELISA (iELISA) and competition
Two kinds of ELISA (cELISA).Patent applicant also successfully develops above two kit, and obtains national novel chiral synthon certificate
(2015 novel chiral synthons demonstrate,prove word 67,2016 novel chiral synthons card word 6), wherein animal brucellosis competitive ELISA antibody test examination
Agent box has obtained the national inventing patent (patent No.：ZL201310213811.5), Brucella abortus indirect ELISA antibody test examination
Agent box has declared national inventing patent (201510477116.9).
The characteristics of above-mentioned various cloth disease detection methods respectively have its own, but the common defects for existing are any of the above inspections all
Must be completed in laboratory.Generally require and first separate serum, then carry out laboratory inspection according to the operation sequence of various methods
Test.And immune colloidal gold technique overcomes drawbacks described above, after can directly taking a blood sample, it is added drop-wise in colloidal gold strip well,
Whether scene just can determine that be Infected with Brucella.Although the report of existing correlation collaurum patent of invention, such as Chinese people's solution
(the application numbers such as Fang Jun Military Veterinary Institute, Academy of Military Medical Sciences Wang Xing dragons are waited, Jilin University Yan Guang schemes:
CN200710055740.5, CN200710055741.X, CN200810051726.2) use staphylococcus aureus to recombinate
A albumen (SPA) has the ability of non-specific adsorption IgG molecules due to SPA, therefore can be used for various as gold labeling antibody
Different animals are diagnosed, but different to IgG adsorption capacities in different animals body, therefore to using same test strips pair
Different animals carry out cloth disease detection, and its sensitiveness is simultaneously unstable.The old wound husband of university of Shihezi of Xinjiang waits (application number:
CN201210254837.X) equally using staphylococcal protein A (SPA) as gold labeling antibody, simply vivoexpression
Brucella specific proteins (OMP31 and BP26) substitutes brucella LPS as detection antigen, can not fundamentally solve
To the sensitive question of different animals diagnosis.(the application numbers such as DNA Sci-tech Co., Ltd. Zhang Mingzhou:
CN201310033074.0), a kind of anti-erythrocyte is recycled as nature controlling line envelope antigen using brucella somatic antigen
Monoclonal antibody as competition monoclonal antibody, the collaurum albumen (fluxion strap) being adsorbed by drying, establish sandwich method detection ox cloth
The Test paper card of Shandong Salmonella antigen, due to brucella and some gramnegative bacteriums, particularly Yersinia ruckeri O9 and
Escherichia coli O 157 has stronger somatic antigen similitude, and brucella somatic antigen is used in the patent as collaurum
The adsorption antigen of particle, in theory its testing result can there is certain non-specificity.
Applicant also successfully developed ox cloth in the labeling of monoclonal antibody collaurum using anti-ox IgG Fc ends in 2015
Shandong Salmonella colloidal gold antibody test strip, and successfully developed using the labeling of monoclonal antibody collaurum at anti-sheep IgG Fc ends
Brucella melitensis colloidal gold antibody test strip, and applied for that (application number is respectively national inventing patent
20151047774.8、201510477800.7).Above two test strips are based on the exploitation of indirect method principle and form, and predominantly detect
IgG antibody-likes in serum.For with respect to indirect method, tried using the competition law developed for brucella monoclonal antibody specific
Many animals can not only be carried out cloth disease detection, and the specificity due to monoclonal antibody in itself, and can simultaneously detect serum by paper slip
In IgG and IgM antibody-likes, therefore test strips in terms of Sensitivity and Specificity have more obvious advantage.Inventor relies on
The autonomous monoclonal antibody for preparing, it is preferable to develop a kind of Sensitivity and Specificity, can simultaneously detect cattle and sheep brucellosis antibody,
And cheap domestic Brucella antibody test strip.
Patent of the present invention have it is simple to operate, as a result fast, it is easy to the advantages of judging and preserve, while without any instrument
Equipment, is especially suitable for clinical quick diagnosis.Due to the test strips both can detect ox, also can detect sheep, particularly base-level countryside and
The cloth disease detection of small-scale plant.
The content of the invention
It is an object of the invention to develop it is a kind of it is simple and efficient, various different animals can be implemented field quick detection and
The colloidal gold strip of yersinia enterocolitica O9 and Escherichia coli O 157 interference can be excluded.
Technical scheme
1. a kind of Brucella antibody test strip, it is characterised in that the test strips are by Brucella antibody Test paper
Bar and plastic tips are constituted.Test strip is made up of PVC base plates, sample pad, gold standard pad, nitrocellulose filter and adsorptive pads.
Gold standard pad is S2 plants of lipopolysaccharides (LPS) of smooth type brucella of colloid gold label and the anti-Flag labels in mouse source of colloid gold label
Monoclonal antibody；Detection line is brucella monoclonal antibody 3E4；Nature controlling line is sheep anti-mouse igg antibody.
2. as claimed in claim 1 a kind of Brucella antibody test strip, it is characterised in that ELISA test strip line makes
With brucella monoclonal antibody specific 3E4, the brucella monoclonal antibody 3E4 cell lines of preparation are in 2013 05
The moon delivers Chinese microorganism strain preservation in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica
Administration committee's common micro-organisms center preservation, deposit number is CGMCCNO.7706；The monoclonal antibody being capable of specificity knowledge
Other brucella LPS, effectively exclusion yersinia enterocolitica O9 and Escherichia coli O 157 are detected to brucellosis
Interference.
3. as claimed in claim 1 a kind of Brucella antibody test strip, it is characterised in that in test strips gold standard pad
It is S2 plants of lipopolysaccharides (LPS) of smooth type brucella of colloid gold particle mark, this LPS pairs for detect Brucella antibody
Common brucella pathogenic autoantibody is respectively provided with good specificity and sensitiveness.
4. the application of the Brucella antibody test strip described in claim 1, it is characterised in that use the test strips
Field quick detection can be carried out to pig, ox, Brucella melitensis.
The specific embodiment of the invention
1. the production strain that selection S2 plants of kind brucella of pig (CVCC70502) is prepared as test strips LPS.
S2 is the most widely used vaccine strain of China, and the vaccine strain immunogenicity is good, is three kinds of China's production at present
In cloth disease vaccine strain (S2 plants, A19 plants and M5 plants), the vaccine strain that virulence is most weak, security is best.In addition, China's cloth disease test tube
Agglutination test country is with reference to product, rose bengal precipitation test country with reference to product, cloth disease full milk ring-type agglutination test antigen country ginseng
According to product, and similar cloth disease diagnostic antigen, using S2 plants as production strain.Analyzed based on antigenicity and bio-safety,
This test strips production antigen strain is S2 plants of pig kind brucella (CVCC70502), identified by China Veterinery Drug Inspection Office,
Preserve and supply.
2. brucella monoclonal antibody 3E4 is selected as the competition antibody of test strips.
Monoclonal antibody largely determines the specific and sensitivity of this kit to antigen reactive specificity
Property.The monoclonal antibody 3E4 specificity identification results used this kit show, monoclonal antibody (cells and supernatant or the preparation
Ascites) with pig kind brucella S1330 antigens, B. abortus A99, brucella melitensis 16M antigens there is aggegation
, there is no agglutinating reaction in reaction, and there is the small intestine of stronger serological cross reaction with brucella with salmonella dublin
There is no aggegation in colitis Yersinia ruckeri O9 and Escherichia coli O 157 antigen, this is also that final can the discriminating of this test strips energy is examined
Disconnected brucella and yersinia enterocolitica O9 and the basic reason of Escherichia coli O 157.
3. the Flag monoclonal antibodies of colloid gold label are used as the instruction of test strips Immune proliferation system effectiveness.
Test strips prepared by this research are the detection methods set up based on competition law, and its principle is to embed to resist in detection line
Brucella LPS monoclonal antibodies, gold standard pad is then sprayed with LPS colloidal gold solutions.After sample is added, because the siphon of blotting paper is made
With the LPS colloidal gold solutions in gold standard pad spread to detection line direction, if measuring samples are free of Brucella antibody, LPS and inspection
The monoclonal antibody 2C7 embedded at survey line reacts, so as to aubergine band occur on detection zone (T), is judged to feminine gender.If treating
Contain Brucella antibody in sample product, antibody will react with LPS colloidal gold solutions, close LPS antigen sites, cause
LPS cannot react with coated monoclonal antibody 2C7 in detection line, so that aubergine band is occurred without on detection zone (T),
So as to be judged to the positive.In order to the validity for confirming Immune proliferation system (is excluded due to being examined caused by Immune proliferation thrashing
Survey line is without band), need the antigen-antibody system of selection other set independence to verify based on the test strips of competition law preparation.For
This, this research is selected the antigen-antibody of Immune proliferation checking system.
By the various commercialization monoclonal antibodies of Integrated comparative, it is found by the applicant that the Flag monoclonal antibodies effect of Sigma companies production
Valency is high, and background is low, and price is low.Further, since it is packaged as 5ml, one can meet the need that this research prepares a collection of test strips
Ask, cause each test strips quality to differ due to there is otherness using different batches and Different Package antibody so as to avoid
Cause.Based on the reason for same, we have selected the sheep anti-mouse igg antibody of Sigma companies production as nature controlling line coated antibody, with
Mouse source Flag monoclonal antibodies constitute corresponding Immune proliferation checking system.
4. pair test strips production technology carries out multifactor optimization.
In Brucella antibody test strip preparation process, inventor each reaction condition grope with it is excellent
Change.To colloid gold particle size, colloid gold label optimal pH, colloidal gold solution optimum mark amount, detection line antibody and Quality Control
Line antibody coating concentration is contrasted experiment, final to determine the addition in toward 100ml distilled water by evaluation test result
After the chlorauric acid solutions of 1ml 1% and the sodium citrate aqueous solutions of 1.5ml 1%, colloid gold particle size be 40nm or so (40 ±
4nm), meet research to require；Gold mark optimum pH is 7.5；Colloidal gold solution optimum mark amount is every milliliter of colloidal gold solution
The μ L of middle addition LPS solution (1mg/mL) 14, every milliliter of collaurum adds the μ g of Flag monoclonal antibodies 10；Detection line coated antibody
(brucella monoclonal antibody 3E4, competitive ELISA potency is 1：20000) 1 is made:200 dilutions, nature controlling line coated antibody (goat-anti
Mouse IgG antibody), concentration is 0.5mg/ml.
5. using certain scale, background, clearly negative and positive sample is commented the specificity and sensitiveness of test strips
Valency.
(1) more than the 400 part field samples for collecting seminar between in recent years, through brave red agglutination test primary dcreening operation after, then use test tube
Agglutination test and complement fixation test (CFT) detection, determine 143 parts (wherein 73 parts of cow's serum, 70 parts of sheep blood serum) three kinds of methods equal altogether
It is detected as the positive serum of cloth disease.This 143 parts of serum are detected that positive rate is 100% using 3 batches of test strips.Thus
Determine that this test strips is 100% to the sensitiveness of field sample.
(2) more than the 400 part field samples for collecting seminar between in recent years, through brave red agglutination test, tube agglutination test and
Complement fixation test (CFT) detects, 134 parts (wherein 71 parts of cow's serum, 63 parts of sheep blood serums) is determined altogether and is detected as cloth with three kinds of methods
Sick negative serum.This 134 parts of serum are detected that negative rate is 99.25% with using 3 batches of test strips.Thereby determine that
This test strips is 99.25% to the specificity of field sample.
Brief description of the drawings
Shown in Fig. 1 LPS antigentic specificity inspection result figure figures：2 specific PCR bands, size is respectively 178bp
And 285bp.
Fig. 2 colloid gold particles electromicroscopic photograph (100 000 ×)
The assembling schematic diagram 1 of Fig. 3 immunity colloidal gold test paper strips is sample pad, and 2 is gold standard pad, and 3 is nitrocellulose filter, 4
It is blotting paper, 5 is detection line, and 6 is nature controlling line, and 7 is PVC board.
Microbial resources information of the present invention
Microorganism involved in the present invention is：S2 plants of pig kind brucella (BrucellaSuis) (CVCC70502), comes from
Chinese veterinary microorganism culture presevation administrative center, is pig kind brucella 1 type of biology, is brucellosis attenuated live vaccines
Strain.M28 plants of brucella melitensis (BrucellaMelitensis) (CVCC70003), protect from Chinese veterinary microorganism strain
Administrative center is hidden, is brucella melitensis 1 type of biology, be the strong poison Reference Strains of brucella disease live-vaccine effect evaluation inspection.
Two bacterial strains are protected by China of China Veterinery Drug Inspection Office veterinary microorganism strain of ZhongGuanCun south Street, Haidian District, BeiJing City 8
Administrative center's identification, keeping and supply are hidden (see in China Veterinery Drug Inspection Office, Chinese veterinary microorganism culture presevation management
The heart is write, Chinese animal doctor's strain catalogue (second edition), Scientia Agricultura Sinica technology publishing house, p35, p32 in 2002).Cloth Lu Shi
Bacterium monoclonal antibody 3E4 cell lines delivered the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences in 05 month 2013
China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation in institute of microbiology, deposit number is CGMCC
No.7706 (see Chinese patent ZL 201310213811.5).
Positive effect of the invention
The present invention relates to a kind of Brucella antibody test strip.The present invention has following meaning：(1) China is enriched
Brucellosis diagnostic method；(2) for China's cloth disease diagnosis is provided, one kind is simple and efficient, can implement existing to various different animals
The new method of field quick detection；(3) this test strips overcomes conventional cloth disease diagnostic method and can not exclude enterocolitis yersinia genus
The interference of Salmonella O9 and Escherichia coli O 157 antiserum to Brucella antibody, improves the specificity of cloth disease detection.
Embodiment
Following examples are not limited the invention to further illustrate the present invention.
Embodiment 1
--- the preparation of test strips
1. the preparation of test strips LPS antigens
Choose S2 plants of brucella vaccine and produce seed as antigen, after S2 is cultivated through TSA, the LPS of extraction is purified
After quantitative, the position of the nitrocellulose filter correspondence detection line for being fixed on colloidal gold strip.
(1) LPS antigens are set up to prepare with the quality standard of brucella S2 pigs.
1) colonial morphology colony edge is neat, mellow and full, reveals drop-wise, skew ray irradiation, backlight observation micro-strip blue-opalescent.
2) dyeing form is coccobacillus, single to be dispersed in, and gemma and pod membrane are not formed.Size is between 0.3~0.6 μm.Leather
Blue Albert'stain Albert is feminine gender, and Ke's Albert'stain Albert is red.
3) purely by existing《Chinese veterinary pharmacopoeia》Annex is carried out, should be pure.
4) specificity
1. serological specificity is made antigen with culture, aggegation should occurs with smooth type brucella positive serum, with
Rough type serum occurs without aggegation.
2. following 4 primers of PCR specificity synthesis, are made into primer mixing storing liquid, and each primer concentration is 25 μM.
Feri：5 '-GCGCCGCGAA GAACTTATC A A-3 ' 21 (sequence 1)
Reri:5 '-CGCCATGTTA GCGGCGGTGA -3 ' 20 (sequence 2)
Fsuis：5 '-GCGCGGTTTT CTGAAGGTTC AGG-3 ' 23 (sequence 3)
RIS711:5 '-TGCCGATCAC TTAAGGGCCT TCAT-3 ' 24 (sequence 4)
Template：S2 cultivates the S2 genomic DNAs that bacterium colony or kit are extracted.
PCR reaction systems：In the reaction system of 50 μ L, containing 5 μ L 10 × Buffer, 8 μ L2.5mMdNTPs, mix primer
Storing liquid 2 μ L, Taq enzyme 2U, the μ L (or picking colony is a little) of template DNA 1.
PCR response procedures：After 95 DEG C of 5min, 28 circulations are carried out by 94 DEG C of 1min, 60 DEG C of 1.5min, 72 DEG C of 1.5min,
Last 72 DEG C of 10min.
As a result：Amplified production carries out electroresis appraisal with 1.5% Ago-Gel, 2 specific PCR bands should occurs, greatly
It is small to be respectively 178bp and 285bp (see accompanying drawing 1).
2. the size of label L PS colloid gold particles is determined
(1) preparation of colloid gold particle takes 250ml round-bottomed flasks, measures 100ml distilled water and adds the gold chlorides of 1ml 1% molten
Liquid, is heated with stirring to and boils.Add the sodium citrates of different volumes 1% such as 0.5ml, 1.0ml, 1.5ml, 2.0ml, 2.5ml water-soluble
In liquid to above-mentioned aqueous solution of chloraurate, stir and evenly mix, and keep the 10min that seethes with excitement.Stop heating after 10min, after solution is cooled down,
Add distilled water and be settled to 100ml, this is colloidal gold solution.The colloidal gold solution that will be prepared is placed in 2~8 DEG C of preservations.With branch
The nickel screen for holding film dips gold mark solution, is directly observed under transmission electron microscope after natural drying, calculates 100 collaurum protein bodies
Average diameter, while colloidal gold solution is made into 1 with 1 × PBS (contain 1%BSA):OD is determined after 20 dilutions520nmValue.
(2) the selection colloid gold particle size of colloid gold particle size is inversely proportional (table 1) with the addition of sodium citrate.Plus
It is claret, particle to enter the collaurum color that the sodium citrates of 1.5ml 1% are prepared in the chlorauric acid solution of 100ml 0.01%
Size meets the requirements than more uniform (electron microscopic observation colloid gold particle, as shown in Figure 2).Compiled according to the Kangxu ruler of great talent and bold vision《Immune colloid
Technology for gold clinical practice》Requirement and this laboratory prepare practical experience show, confirm the collaurum used in this research
Average diameter of particles is about 40nm (40 ± 4nm), colloidal gold solution OD520nmBetween 0.2~0.3.
Relation in the collaurum preparation process of table 1 between trisodium citrate addition and colloid gold particle size
3. determine that the main technique of colloidal gold strip determines
(1) determination of colloidal gold solution optimum mark amount take respectively S2 plants of lipopolysaccharides of smooth type brucella to be marked and
Commercialization Flag monoclonal antibody (concentration is 1mg/ml) 0 μ L, 4 μ L, 8 μ L, 12 μ L, 16 μ L, 20 μ L are added in 1ml collaurums, effect
100 μ L 10%NaCl are added after 45 minutes, 2~8 DEG C stand 2 hours.There is no color change in claret LPS colloidal gold solutions
Minimized content be 12 μ L, therefore, rule of thumb, Jia 20% on this basis, i.e., the μ containing LPS 14 in every milliliter colloidal gold solution
L(1mg/mL).The minimized content that claret commercialization Flag monoclonal antibody colloidal gold solutions do not occur color change is 8 μ L, i.e., containing business
The μ g of product Flag monoclonal antibodies 8, therefore, Jia 20% on this basis, i.e., the μ g of monoclonal antibody containing Flag 10 in every milliliter colloidal gold solution.
(2) the determination brucella monoclonal antibody 3E4 of detection line antibody and nature controlling line antibody coating concentration is used as nitric acid
Detection line coated antibody on cellulose membrane.By brucella odd contradictive hydroperitoneum, (competitive ELISA potency is 1:20000) with a stroke film bag
By liquid (containing 3% methyl alcohol, 1% sucrose, 0.05% Sodium azide 0.02M phosphate buffers, pH7.4) make 1:100、1:200、
1:400、1:800 dilutions, are sprayed onto with 0.1 μ l/mm with point film instrument (Bio-Dot, USA) and are attached on nitrocellulose filter in PVC board
Detection line (T lines) position.The increase of concentration is coated with antibody, the color of detection line is also gradually deepened, and works as brucella
Odd contradictive hydroperitoneum makees 1 with film coating buffer is drawn:200 dilutions, detection line is bright-colored, obvious, be easy to interpretation, it is thus determined that being detection line
Optimum antibody is coated with concentration.
(3) the nature controlling line coated antibody concentration on nitrocellulose filter determines to use stroke film coating buffer by sheep anti-mouse igg antibody
0.25mg/mL, 0.5mg/mL, 1.0mg/mL, 2.0mg/mL are diluted to, are sprayed onto respectively with 0.1 μ l/mm with point film instrument and is attached on
Nitrocellulose filter nature controlling line (C lines) position in PVC board.Result of the test shows：The increase of concentration, nature controlling line are coated with antibody
Color also gradually deepen, when sheep anti-mouse igg antibody concentration is 0.5mg/ml, nature controlling line is bright-colored, obvious, be easy to sentence
Read, it is thus determined that for nature controlling line optimum antibody is coated with concentration.
4. the assembling of test strips
(1) assembling of immune colloid gold system by nitrocellulose filter (3) as shown in figure 3, first paste PVC board (7)
Relevant position, then by sample pad (1), gold standard pad (2), blotting paper (4) pastes the relevant position of PVC board (7) successively.Make gold
Mark pad (2) and nitrocellulose filter (3) part contact, about 1~2mm；Blotting paper (4) is set partly to be connect with nitrocellulose filter (3)
Touch, about 2~3mm.3mm small bars wide are cut into cutting machine, are placed in upper and lower cover plates.
(2) the assembling aluminium foil bag of test strips is by 1 test strips, and 1 dropper and a drying prescription of being responsible for a task until it is completed pack into cloth Shandong
Salmonella antibody test test strips；Every 10 test strips and 1 part of specification, are packaged into 1 box.
Embodiment 2
--- the source of the main supplementary material in Brucella antibody test strip part and the method for inspection
1.Flag monoclonal antibodies are purchased from Sigma companies, for the Flag monoclonal antibodies of different batches, are using preceding need
It is checked as follows：
(1) proterties colourless clear liquid.
(2) product analysis that supplier provides reports that the product analysis report provided supplier is checked, examines it
Whether the parameters such as protein content, mole ratio, specificity are in prescribed limit.
(3) steriling test is by existing《Chinese veterinary pharmacopoeia》(the Chinese veterinary pharmacopoeia committee, Republic of China Veterinary Pharmacopoeia,
Two 〇, mono- 〇 versions three, Chinese agriculture publishing house, 2011, hereinafter referred to as《Chinese veterinary pharmacopoeia》) annex tests, should be without bacterium
Growth.
(4) survey report is shown in Table 2.
The mouse source Flag monoclonal antibody survey reports of table 2
2. sheep anti-mouse igg antibody is purchased from Sigma companies, for the sheep anti-mouse igg antibody of different batches, right using preceding need
It is checked as follows：
(1) proterties pale yellow powder.
(2) product analysis that supplier provides reports that the product analysis report provided supplier is checked, examines it
Whether the parameters such as protein content, mole ratio, specificity are in prescribed limit.
(3) steriling test is by existing《Chinese veterinary pharmacopoeia》Annex is tested, should be without bacterial growth.
(4) survey report sees attached list 3.
The sheep anti-mouse igg antibody survey report of table 3
Embodiment 3
--- competitive ELISA method determines monoclonal antibody (3E4) titer of ascites and determines
1. the ascites that ascites dilution will be prepared makees 1 with 1 × PBS solution:10000、1:15000、1:20000、1:25000、
1:30000、1:35000 and 1:40000 dilutions.
2.cELISA determine take antigen coated microplate (according to sample how much, removable gradation is used), with the μ of 1 × cleaning solution 300
L/ holes board-washing 1 time, discards cleaning solution.The positive control serum and negative control sera that will have been diluted are added separately to elisa plate
In, 50 μ l/ holes, its positives and negative control sera respectively does 2 repetitions.After sample-adding terminates, the monoclonal that addition has diluted
Antibody, per the μ l of hole 50, vibration mixes 5min.After 37 DEG C are incubated 30min, reaction plate is taken out, discard reaction solution, 300 μ are added per hole
1 × cleaning solutions of l, after washing 3 times, dry.The goat anti-mouse IgG antibodies that HRP is marked make 1 with corresponding dilution:100 is dilute
After releasing, 100 μ l are added per hole, after 37 DEG C are incubated 30min, ibid method is washed 3 times, is dried.Substrate solution A and B are pressed 1:1
(V/V) after mixing, it is added to immediately in ELISA reaction plates, 100 μ l/ holes, after room temperature lucifuge colour developing 15min, added for 50 μ l ends per hole
Only liquid terminating reaction.After reaction terminating, OD is determined with ELIASA in 15 minutes450nmValue.
3. computing formula PI (inhibiting rate)=(negative control OD450nm- positive control OD450nm)/negative control OD450nm×
100%
4. potency judges to make the highest dilution of PI average value >=90% of positive control serum in each dilution factor of ascites
It is the potency of ascites.
Embodiment 4
--- the sensitivity tests of test strips
1. the sensitivity test of the Brucella antibody Quality Control positive serum of pair gradient dilution
After Brucella antibody positive serum is carried out into 2 times of gradient dilutions, make test strips by oneself with 3 batches of laboratories and examined
Survey, determine sensitivity of the test strips to the positive control serum of gradient dilution, the results are shown in Table 4.
Sensitivity technique of the table 4 to the positive control serum of gradient dilution
Note：The criterion of Brucella antibody test strip is：Only there is an aubergine band in quality control region (C),
Occur without aubergine band in detection zone (T), be judged to positive (being designated as " P ")；When quality control region (C) and detection zone (T) occur
During aubergine band, it is judged to negative (being designated as " N ").
It is consistent from 4,3 batches of results of ELISA test strip gradient dilution Quality Control positive serum of table, can detect to work 1:
64 times of Quality Control positive serums of dilution.
2. the sensitivity tests of positive sample known to pair
143 parts of Brucella antibody positive serums are detected using 3 batches of Brucella antibody test strips, as a result
It is shown in Table 2.Every batch of sensitiveness of test strips is calculated, 5 are the results are shown in Table.
The sensitivity Detection of positive sample known to 5 pairs, table
Note：1. the criterion of Brucella antibody test strip is：Only there is a purplish red vitta in quality control region (C)
Band, occurs in detection zone (T) without aubergine band, is judged to positive (being designated as " P ")；When quality control region (C) and detection zone (T) go out
During existing aubergine band, it is judged to negative (being designated as " N ").2. numbering 1~73 is cow's serum sample；74~143 is sheep blood serum sample.
From table 5, for 143 portions of oxen, Brucella melitensis Positive Sera, 3 batches of test strips inspection prepared by laboratory
Test result and be the positive.3 batches of ELISA test strip interpretations of result are shown in Table 6.
The sensitivity analysis of positive serum known to 6 pairs, table
Table 6 shows that 3 batches of Brucella antibody test strips prepared by laboratory are quick to the sick positive sample of 143 parts of cloth
Perception is 100%, shows that test strips sensitiveness is higher, and detection leakage phenomenon is less prone to during as clinical detection.
Sequence table
<110>China Veterinery Drug Inspection Office
<120>Brucella antibody test strip
<130>
<160> 4
<170> Patentin version 3.5
<210> 1
<211> 21
<212> DNA
<213>Artificial sequence
<223>Description to artificial sequence：Primers F eri
<400> 1
GCGCCGCGAA GAACTTATCA A 21（Sequence 1）
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<223>Description to artificial sequence：Primer Reri
<400> 2
CGCCATGTTA GCGGCGGTGA 20（Sequence 2）
<210> 3
<211> 23
<212> DNA
<213>Artificial sequence
<223>Description to artificial sequence：Primers F suis
<400> 3
GCGCGGTTTT CTGAAGGTTC AGG 23（Sequence 3）
<210> 4
<211> 24
<212> DNA
<213>Artificial sequence
<223>Description to artificial sequence：Primer RIS711
<400> 4
TGCCGATCAC TTAAGGGCCT TCAT 24（Sequence 4）
1
Claims (4)
1. a kind of Brucella antibody test strip, it is characterised in that the test strips by Brucella antibody test strip and
Plastic tips are constituted；Test strip is made up of PVC base plates, sample pad, gold standard pad, nitrocellulose filter and adsorptive pads, wherein
Gold standard pad is the list of the anti-Flag labels in mouse source of S2 plants of lipopolysaccharides of smooth type brucella and colloid gold label of colloid gold label
Clonal antibody, detection line are brucella monoclonal antibody 3E4, nature controlling line is sheep anti-mouse igg antibody.
2. as claimed in claim 1 a kind of Brucella antibody test strip, it is characterised in that ELISA test strip line is used
Brucella monoclonal antibody specific 3E4, the brucella monoclonal antibody 3E4 cell lines of preparation sent in 05 month 2013
Hand over Chinese microorganism strain preservation management in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica
Committee's common micro-organisms center preservation, deposit number is CGMCCNO.7706；The monoclonal antibody being capable of specific recognition cloth
Shandong Salmonella LPS, effectively excludes the interference that yersinia enterocolitica O9 and Escherichia coli O 157 are detected to brucellosis.
3. as claimed in claim 1 a kind of Brucella antibody test strip, it is characterised in that be used in test strips gold standard pad
Detection Brucella antibody is S2 plants of lipopolysaccharides of smooth type brucella of colloid gold particle mark, and the lipopolysaccharides is to common
Brucella pathogenic autoantibody is respectively provided with good specificity and sensitiveness.
4. the application of the Brucella antibody test strip described in claim 1, it is characterised in that can be right using the test strips
Pig, ox, Brucella melitensis carry out field quick detection.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108588247A (en) * | 2018-05-02 | 2018-09-28 | 杭州优思达生物技术有限公司 | A kind of kit and its detection method of brucella nucleic acid |
| CN113045648A (en) * | 2021-04-06 | 2021-06-29 | 江南大学 | Yersinia enterocolitica monoclonal antibody hybridoma cell strain and application thereof |
| CN113092755A (en) * | 2021-05-18 | 2021-07-09 | 中国兽医药品监察所 | Colloidal gold test strip for detecting brucella antigen by sandwich method |
| CN115166238A (en) * | 2022-08-02 | 2022-10-11 | 中国农业科学院北京畜牧兽医研究所 | Integrated antibody detection test strip for primary screening and accurate diagnosis of brucellosis in sheep |
| CN115166239A (en) * | 2022-08-02 | 2022-10-11 | 中国农业科学院北京畜牧兽医研究所 | Integrated antibody detection test strip for primary screening and accurate diagnosis of bovine brucellosis |
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| CN101706499A (en) * | 2009-11-06 | 2010-05-12 | 河南省生物工程技术研究中心 | FLAG fusion tag colloidal gold test strip and preparation method thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108588247A (en) * | 2018-05-02 | 2018-09-28 | 杭州优思达生物技术有限公司 | A kind of kit and its detection method of brucella nucleic acid |
| CN113045648A (en) * | 2021-04-06 | 2021-06-29 | 江南大学 | Yersinia enterocolitica monoclonal antibody hybridoma cell strain and application thereof |
| CN113092755A (en) * | 2021-05-18 | 2021-07-09 | 中国兽医药品监察所 | Colloidal gold test strip for detecting brucella antigen by sandwich method |
| CN115166238A (en) * | 2022-08-02 | 2022-10-11 | 中国农业科学院北京畜牧兽医研究所 | Integrated antibody detection test strip for primary screening and accurate diagnosis of brucellosis in sheep |
| CN115166239A (en) * | 2022-08-02 | 2022-10-11 | 中国农业科学院北京畜牧兽医研究所 | Integrated antibody detection test strip for primary screening and accurate diagnosis of bovine brucellosis |
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