CN106018800A - Detecting device for Brucella infection - Google Patents
Detecting device for Brucella infection Download PDFInfo
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- CN106018800A CN106018800A CN201610355671.9A CN201610355671A CN106018800A CN 106018800 A CN106018800 A CN 106018800A CN 201610355671 A CN201610355671 A CN 201610355671A CN 106018800 A CN106018800 A CN 106018800A
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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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
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- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
- G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- G01N2400/50—Lipopolysaccharides; LPS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
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Abstract
The invention discloses a detecting device for Brucella infection. The detecting device comprises a test strip; a Brucella antigen detection area and a Brucella antibody detection area are arranged on a chromatographic membrane of the test strip. According to the detection device, a Brucella antigen and a Brucella antibody in a sample can be detected simultaneously; various outer membrane proteins are applied in a combined manner, so that the occurrence of false positive results can be reduced effectively; a double-antigen sandwich method and a double-antibody sandwich method are adopted, so that the sensitivity and the specificity of detection can be higher than those obtained according to the conventional indirect method, and the rapid detection of brucellosis is realized; the problems of high interference, and high false positive result or false negative result occurrence rate caused by long detection period, poor specificity and low sensitivity in the conventional brucellosis detection process are solved.
Description
Technical field
The present invention relates to field of biological detection, particularly relate to a kind of detection for quickly detecting Infected with Brucella
Device.
Background technology
Brucellosis (Brucellosis is called for short brucellosis) is a kind of serious threat mankind and many animals life and health
Arbo infectious disease.The epidemic disease poultrys such as ill sheep, cattle are the major source of infection of brucellosis, and brucella can be by damaged skin
The approach such as skin mucosa, digestive tract and respiratory tract are propagated.Acute stage case with heating, weak, hyperhidrosis, muscle, arthralgia regulating liver-QI,
Spleen, the enlargement of lymph node mainly show.Chronic phase case shows as joint damage etc. more.Brucellosis is China's Law on the Prevention and Control of Infectious Diseases
The Category B notifiable disease of regulation.According to China CDC statistical report in 2012, people's brucellosis increases newly to people more than 46000, and with annual 10%
Speed rises.The fashion trend of brucellosis occurs substantially to change, i.e. brucellosis epidemic-stricken area is climing to farming-pastoral region, agriculture district and city from pastoral area
Prolong；Form instead of large-scale outbreak of epidemic so that many and scattered point-like is popular；In addition to professional population is invaded, non-professional crowd feels
Dye rate rises the most relatively, becomes one of the most serious public health problem.
At present, the conventional quarantine means of brucellosis are Bu Lu based on bacteriology and Serologic detection technology, bacteriological detection
The goldstandard of Salmonella detection.But, from blood, carry out brucella cultivate more difficulty (brucella poor growth, leakiness
Inspection), the most also take 7 ~ 15 days, and limited by experimental situation and professional operator.Currently, brucellosis serological diagnostic method master
Agglutination test to be included, elisa (ELISA), complement fixation test (CFT) etc..Agglutination test includes Hu Hongping
Plate agglutination test (RBPT), full milk ring test (MRT), tube agglutination test (SAT), card test and buffering flat plates coagulation examination
Test (BBAT) etc., the prescreening method during wherein RBPT Yu MRT becomes international trade and the detection of milch cow brucellosis, people's brucellosis serology
Diagnosis is with agglutination test RBPT primary dcreening operation, and SAT 1:100++ is identified above, and bacteriology's positive is the clinical criteria infected.
Serology test becomes important detection method because of its advantage such as simple and efficient, easy to implement.Due to cloth Shandong
The effective ingredient of antigen used by Salmonella traditional experiment is mainly lipopolysaccharide (LPS), with gram-negative bacteria, especially O:9 type yersinia genus
Salmonella has cross reaction, the accuracy of interference result.It addition, in the period infected in early days or antibody titer is relatively low, especially exist
Infection brucella, in 12 ~ 16 days, is difficult to go out Infected with Brucella by detection antibody test.It addition, brucella is intracellular
Bacterial parasite, in the later stage of Infected with Brucella, cannot detect brucella mark in serum.
Whether brucella epicyte contains O chain (O-chain) according to LPS, be divided into smooth type (smooth, S) and
Rough type (rough, R) two kinds.Owing to smooth type bacterial strain LPS covers the outer membrane protein (outer of phage surface
Membrane proteins, OMPs), make OMPs can not fully expose and make it study and be restricted with value；Slightly
The brucellar OMPs of rough type is exposed because of the covering lacking O chain, such as OMP25, OMP28, OMP31 etc..
Sum it up, there is presently no a kind of detection method reliably in brucellosis context of detection.
Summary of the invention
The technical problem to be solved there is provided the detection device of a kind of Infected with Brucella, and it detects simultaneously
Brucellergen and antibody in sample, the use in conjunction of multiple outer membrane protein, it is effectively reduced the appearance of false positive results, double
Antigen sandwich and the more traditional indirect method of double antibody sandwich method can improve susceptiveness and the specificity of detection, for brucella
Sick quick detection；Solve detection cycle length, poor specificity, low the asking of susceptiveness present in the detection of current brucellosis
Inscribe and occur that false positive or false-negative problem by force, easily occurs in interference.
The technical problem to be solved is achieved by the following technical programs:
The detection device of a kind of Infected with Brucella, including test strips；The chromatographic film of test strips is provided with brucellergen inspection
Survey district, Brucella antibody detection zone；Detection of antigen district is coated with capture antibody, the detection antibody of nanoparticle label and detection
The coated antibody sandwich detection brucellergen in district；Antibody test district is coated with capture antigen, the detection of nanoparticle label
Antigen detects Brucella antibody with the capture antigen sandwich in antibody test district.
The detection device of Infected with Brucella as above, described brucellergen detection zone is used for specific detection
Brucella lipopolysaccharide in sample；Described Brucella antibody detection zone is for detecting the brucella outer membrane protein in sample
Antibody.
In the detection device of Infected with Brucella as above, described detection antibody and capture antibody at least one
For the monoclonal antibody specific binding with brucella lipopolysaccharide O chain.
The detection device of Infected with Brucella as above, described detection antigen and capture antigen are brucella adventitia
Albumen；Described brucella outer membrane protein be OMP10 albumen, OMP19 albumen, OMP25 albumen, OMP28 albumen, OMP31 albumen,
One or more in OMP16 albumen, or the protein of two or more above-mentioned protein fusion expressions, or above-mentioned egg
The polypeptide fragment of white matter and the conjugate of carrier protein, or the protein that the different polypeptide fragments of above-mentioned protein are in series
Molecule.
The detection device of Infected with Brucella as above, described capture antigen is that brucella is derived from OMP28 albumen
The covalent coupling thing of polypeptide fragment and carrier protein.
The detection device of Infected with Brucella as above, described chromatographic film is followed successively by absorption pad direction by sample pad
It is coated the Detection of antigen district of brucella lipopolysaccharide, is coated the antibody test district of OMP28 albumen.
The detection device of Infected with Brucella as above, described chromatographic film is followed successively by absorption pad direction by sample pad
The antibody test district being coated OMP16 albumen, the Detection of antigen district being coated brucella lipopolysaccharide, it is coated the antibody of OMP25 albumen
Detection zone, it is coated the antibody test district of OMP28 albumen.
The detection device of Infected with Brucella as above, described chromatographic film is followed successively by absorption pad direction by sample pad
The antibody test district being coated OMP31 albumen, the Detection of antigen district being coated brucella lipopolysaccharide, it is coated the antibody of OMP19 albumen
Detection zone, it is coated the antibody test district of OMP28 albumen.
The detection device of Infected with Brucella as above, described chromatographic film is followed successively by absorption pad direction by sample pad
The antibody test district being coated OMP25 albumen, the Detection of antigen district being coated brucella lipopolysaccharide, it is coated the antibody of OMP28 albumen
Detection zone.
The detection device of Infected with Brucella as above, described chromatographic film is followed successively by absorption pad direction by sample pad
The Detection of antigen district being coated brucella lipopolysaccharide, the antibody test district being coated OMP10 albumen, it is coated the antibody of OMP31 albumen
Detection zone, it is coated the antibody test district of OMP28 albumen.
The detection device of Infected with Brucella as above, described chromatographic film is followed successively by absorption pad direction by sample pad
It is coated the Detection of antigen district of brucella lipopolysaccharide monoclonal antibody, is coated the antibody test district of fusion protein；Described fusion egg
The white protein molecule being in series for the polypeptide fragment of OMP28-OMP31 fusion protein or OMP10, OMP28, OMP31.
There is advantages that existing brucella immunological detection method generally also exists false positive rate
High problem.Bacteriological detection (bacterium cultivation) is though being the goldstandard of brucella detection, but time-consuming long, missing inspection (false negative) problem
Prominent.Additionally, there is brucella and mark thereof in initial infection, serum, but antibody does not produces, and after infection
Phase, brucella parasitizes intracellular, does not has thalline and mark thereof in serum.The antibody combined detection of brucellergen
Complementary action can be played, improve positive rate, beneficially early diagnosis；Decrease unnecessary failing to pinpoint a disease in diagnosis and Misdiagnosis, energy
Direct basis is provided for understanding Infected with Brucella situation, therapeutic scheme, selection and Index for diagnosis etc..Dual-antigen sandwich method relatively passes
The indirect method of system can improve susceptiveness and the specificity of detection.The sample negative for Detection of antigen, antibody test is positive can
It is classified as suspicious specimen further to be detected.Height suspicious specimen should be classified as the sample that Detection of antigen is positive, and take
Effective measures prevent transmission of disease.In a word, the Infected with Brucella test strip that the present invention provides can be brucella
Sick prevention and control provide one to have the technical advantages such as accurate, easy, quick, and the detection means of low cost, reduce inspection
The requirement of survey personnel's level of skill.
Detailed description of the invention
Below in conjunction with embodiment, the present invention will be described in detail, and embodiment is only the preferred embodiment of the present invention,
It it not limitation of the invention.
Embodiment 1
1, the preparation of nanoparticle label detection antibody: (time resolution is glimmering to take the polystyrene fluorescent microsphere of 1mg particle diameter 110nm
Light, excites: 360nm, launches: 615nm), washs granule 2 times with PBS, with the resuspended granule of 1ml PBS, adds EDC and NHS and makes it
Final concentration is respectively 20mg/ml, 5mg/ml；Room temperature reaction 20min；Centrifugal, abandon supernatant, and wash granule 2 times with PBS；Resuspended
Granule, adds 300 μ g brucella lipopolysaccharide monoclonal antibody (C epi-position)；Room temperature reaction 2h；Add BSA to final concentration 1%(W/
V), room temperature reaction 2h is continued；Centrifugal, abandon supernatant, and wash granule 3 times with PBS；Liquid (10mM Tris-HCl is redissolved with 10ml
(pH8.0), 0.5%BSA) resuspended granule, standby.
The preparation of nanoparticle label OMP28 albumen: take the polystyrene fluorescent microsphere (time resolution of 1mg particle diameter 110nm
Fluorescence, excites: 360nm, launches: 615nm), washs granule 2 times with PBS, with the resuspended granule of 1ml PBS, adds EDC and NHS and makes
Its final concentration is respectively 20mg/ml, 5mg/ml；Room temperature reaction 20min；Centrifugal, abandon supernatant, and wash granule 2 times with PBS；Weight
Outstanding granule, adds 500 μ g OMP28 albumen (Xin T, Yang H, Wang N, Wang F, Zhao P, Wang H, Mao
K, Zhu H, Ding J. Limitations of the BP26 protein-based indirect enzyme-
linked immunosorbent assay for diagnosis of Brucellosis. Clin Vaccine
Immunol. 2013,20 (9): 1410-7.)；Room temperature reaction 2h；Add BSA to final concentration 1%(W/V), continue room temperature reaction
2h；Centrifugal, abandon supernatant, and wash granule 3 times with PBS；Liquid (10mM Tris-HCl(pH8.0), 0.5%BSA is redissolved) with 10ml
Resuspended granule, standby.
2, the preparation of chromatographic film: spray concentration successively according to the amount of 1 μ l/cm on Sai Duolisi CN95 nitrocellulose filter
For the OMP28 albumen of the rabbit anti-brucella lipopolysaccharide polyclonal antibody (Detection of antigen district) of 0.5mg/ml, 2mg/ml, (palace is known
Bright, Jing Tao, kingdom controls, Li Kemei, Wang Bingxiang, Fu Linfeng. the expression and purification of brucella BP26 albumen and antigenic grind
Study carefully. China Amphixenosis journal .2009,11:1085-1088, antibody test district), 0.5mg/ml sheep anti-mouse igg (Quality Control
District), it is placed in 37 DEG C of dry 2h, standby.
3, the preparation of pad: by detection antibody and the detection antigen equal-volume of nanoparticle label of nanoparticle label
Mixing；It is sprayed on 8964 glass fibre according to 2 μ l/cm, is dried in 37 DEG C, standby.
4, the preparation of sample pad: use 20mM Tris-HCl(pH8.0), 1%BSA, 0.5%Tween 20 soak non-woven fabrics
10min；It is placed in 37 DEG C to be dried, standby.
5, the preparation of test strips: sample pad, pad, chromatographic film (are followed successively by adsorptive pads direction by sample pad: rabbit resists
Brucella lipopolysaccharide polyclonal antibody, OMP28 albumen, sheep anti-mouse igg), adsorptive pads overlap successively and paste PVC base plate, cut out
Become 3.5mm width, be placed in 4 DEG C of kept dry.
6, detection sample: sample pad is lain against on table top；Take serum/milk 80 μ l in sample pad；After 15min in time
Between sentence read result on resolved fluorometric test strips interpretation equipment: 1) only quality control region has signal: do not infected brucella；2) only
There is a signal in quality control region and antibody test district: infected but rehabilitation or intracellular have been carried disease germs；3) three lines all have a signal: carry disease germs and
Produce antibody；4) quality control region and Detection of antigen district is only had to have a signal: to carry disease germs, initial infection；5) quality control region no signal: detection nothing
Effect, need to detect again.Actual sample testing result is shown in Table 1.This programme test strips is capable of detecting when that all bacterium cultivate positive sample,
And cultivate patients with negative serum sample from 1098 parts of bacterium and detect 15 parts of positive sample；There is no false positive sample.
Table 1 uses 1725 parts of human serum samples of the present embodiment ELISA test strip
Embodiment 2
1, the preparation of nanoparticle label detection antibody: take the nanogold particle of 10ml particle diameter 40nm, add 90 μ g brucella
Lipopolysaccharide monoclonal antibody (C epi-position)；Room temperature reaction 15min；Add BSA to final concentration 1%(W/V), continue room temperature reaction
15min；Centrifugal, redissolve liquid (10mM Tris-HCl(pH8.0), 0.5%BSA with 1ml) resuspended granule, standby.
2, the preparation of nanoparticle label OMP28 albumen: take the nanogold particle of 10ml particle diameter 40nm, adds 140 μ
GOMP28 albumen (1-75AA with 135-225AA is spliced, pET-32a plasmid, and e. coli bl21 is expressed)；Room temperature reaction
15min；Add BSA to final concentration 1%(W/V), continue room temperature reaction 15min；Centrifugal, redissolve liquid (10mM Tris-with 1ml
HCl(pH8.0), 0.5%BSA) resuspended granule, standby.
3, the preparation of chromatographic film: spray dense successively according to the amount of 0.8 μ l/cm on Sai Duolisi CN90 nitrocellulose filter
Degree is the brucella lipopolysaccharide monoclonal antibody (C epi-position) of 2mg/ml, OMP28 albumen (Gong Xiaowei, Jing Tao, the king of 2mg/ml
State controls, Li Kemei, Wang Bingxiang, Fu Linfeng. the expression and purification of brucella BP26 albumen and antigenic research thereof. and Chinese beast
Altogether ill journal .2009,11:1085-1088., antibody test district), 0.02mg/ml sheep anti-mouse igg (quality control region), be placed in 37
DEG C dry 2h, standby.
4, the preparation of micropore is reacted: by bodies such as the detection antigens detecting antibody and nanoparticle label of nanoparticle label
Volume blending；It is dispensed in micropore according to 20 μ l/ holes；-80 DEG C of freezing 24h；It is placed in freeze dryer lyophilizing, seals, kept dry, standby
With.
5, sample pad: 8964 glass fibre.
6, the preparation of test strips: sample pad, chromatographic film (are followed successively by adsorptive pads direction by sample pad: brucella fat
Monoclonal Antibody against Polysaccharides, OMP28 albumen, sheep anti-mouse igg), adsorptive pads overlap successively and paste PVC base plate, be cut into 4.5mm width,
It is placed in 4 DEG C of kept dry.
7, detection sample: take sample 200 μ l in micropore, reacts 3min in 37 DEG C；Test strips is inserted in micropore,
Sentence read result after 10min: 1) only have quality control region to have signal: do not infected brucella；2) only have quality control region and antibody test district:
Infected but rehabilitation or intracellular have been carried disease germs；3) three lines all have signal: carry disease germs and produced antibody；4) only have quality control region and resist
Former detection zone has a signal: carry disease germs, initial infection；5) quality control region no signal: it is invalid to detect, need to detect again.Actual sample detects
The results are shown in Table 2.This programme test strips is capable of detecting when that all bacterium cultivate positive sample, and cultivates patients with negative from 1098 parts of bacterium
Serum sample detects 15 parts of positive sample；There is no false positive sample.
Table 2 uses 1725 parts of human serum samples of the present embodiment ELISA test strip
Embodiment 3
1, the preparation of nanoparticle label detection antibody: take the nanogold particle of 10ml particle diameter 40nm, add 90 μ g brucella
Lipopolysaccharide monoclonal antibody (C epi-position)；Room temperature reaction 15min；Add BSA to final concentration 1%(W/V), continue room temperature reaction
15min；Centrifugal, redissolve liquid (10mM Tris-HCl(pH8.0), 0.5%BSA with 1ml) resuspended granule, standby.
2, the preparation of nanoparticle label OMP28 albumen: take the nanogold particle of 10ml particle diameter 40nm, adds 140 μ
GOMP28 albumen (1-75AA with 135-225AA is spliced, pET-32a carrier, and e. coli bl21 is expressed)；Room temperature reaction
15min；Add BSA to final concentration 1%(W/V), continue room temperature reaction 15min；Centrifugal, redissolve liquid (10mM Tris-with 1ml
HCl(pH8.0), 0.5%BSA) resuspended granule, standby.
3, the preparation of nanoparticle label OMP25 albumen: take the nanogold particle of 10ml particle diameter 40nm, adds 210 μ
GOMP25 albumen (prokaryotic expression)；Room temperature reaction 15min；Add BSA to final concentration 1%(W/V), continue room temperature reaction 15min；
Centrifugal, redissolve liquid (10mM Tris-HCl(pH8.0), 0.5%BSA with 1ml) resuspended granule, standby.
4, the preparation of nanoparticle label OMP16 albumen: take the nanogold particle of 10ml particle diameter 40nm, adds 140 μ
GOMP16 albumen (20-125AA, pET-32a plasmid, e. coli bl21 is expressed)；Room temperature reaction 15min；Add BSA to the denseest
Degree 1%(W/V), continue room temperature reaction 15min；Centrifugal, redissolve liquid (10mM Tris-HCl(pH8.0), 0.5%BSA with 1ml) weight
Outstanding granule, standby.
5, the preparation of chromatographic film: spray concentration successively according to the amount of 0.8 μ l/cm on Mi Libo CN95 nitrocellulose filter
For the OMP16 albumen (total length, prokaryotic expression) of 2.5mg/ml, the brucella lipopolysaccharide monoclonal antibody (M epi-position) of 2mg/ml,
The OMP25 albumen of 1mg/ml, 2mg/ml OMP28 albumen (Gong Xiaowei, Jing Tao, kingdom controls, Li Kemei, Wang Bingxiang, Fu Linfeng.
The expression and purification of brucella BP26 albumen and antigenic research thereof. China Amphixenosis journal .2009,11:1085-
1088), 0.5mg/ml sheep anti-mouse igg (quality control region), be placed in 37 DEG C of dry 2h, standby.
6, the preparation of pad: by detection antibody and the detection antigen equal-volume of nanoparticle label of nanoparticle label
Mixing；It is sprayed on 8964 glass fibre according to 5 μ l/cm, is dried in 37 DEG C, standby.
7, the preparation of sample pad: use 20mM Tris-HCl(pH8.0), 1%BSA, 0.5%Tween 20 soak non-woven fabrics
10min；It is placed in 37 DEG C to be dried, standby.
8, the preparation of test strips: by sample pad, pad, chromatographic film (be followed successively by adsorptive pads direction by sample pad:
OMP16 albumen, brucella lipopolysaccharide monoclonal antibody, OMP25 albumen, OMP28 albumen, sheep anti-mouse igg (quality control region), water suction
Pad overlap joint successively pastes PVC base plate, is cut into 3.5mm width, is placed in 4 DEG C of kept dry.
9, detection sample: sample pad is lain against on table top；Take serum/milk 80 μ l in sample pad；Interpretation after 15min
Result: 1) only have quality control region to have signal: do not infected brucella；2) quality control region and antibody test district is only had to have signal: to infect
Crossing but rehabilitation or intracellular have been carried disease germs, the probability that the most explanations of bar number were infected/infected is the highest；3) five lines all have a signal: carry disease germs,
And produced antibody；4) quality control region and Detection of antigen district is only had to have a signal: to carry disease germs, initial infection；5) detect invalid, need to again examine
Survey.Actual sample testing result is shown in Table 3.This programme test strips is capable of detecting when that all bacterium cultivate positive sample, and from 1098
Part bacterium is cultivated in patients with negative serum sample and detects 15 parts of positive sample；There is no false positive sample.
Table 3 uses 1725 parts of human serum samples of the present embodiment ELISA test strip
Embodiment 4
1, the preparation of nanoparticle label detection antibody: take the nanogold particle of 10ml particle diameter 40nm, add 90 μ g brucella
Lipopolysaccharide polyclonal antibody；Room temperature reaction 15min；Add BSA to final concentration 1%(W/V), continue room temperature reaction 15min；It is centrifugal,
Liquid (10mM Tris-HCl(pH8.0), 0.5%BSA is redissolved with 1ml) resuspended granule, standby.
2, the preparation of nanoparticle label OMP28 albumen: take the nanogold particle of 10ml particle diameter 40nm, adds 140 μ
GOMP28 albumen (1-75AA with 135-225AA is spliced, PET-32a plasmid, and e. coli bl21 is expressed)；Room temperature reaction
15min；Add BSA to final concentration 1%(W/V), continue room temperature reaction 15min；Centrifugal, redissolve liquid (10mM Tris-with 1ml
HCl(pH8.0), 0.5%BSA) resuspended granule, standby.
3, the preparation of nanoparticle label OMP19 albumen: take the nanogold particle of 10ml particle diameter 40nm, adds 90 μ g
OMP19 albumen (prokaryotic expression)；Room temperature reaction 15min；Add BSA to final concentration 1%(W/V), continue room temperature reaction 15min；From
The heart, redissolves liquid (10mM Tris-HCl(pH8.0), 0.5%BSA with 1ml) resuspended granule, standby.
4, the preparation of nanoparticle label OMP31 albumen: take the nanogold particle of 10ml particle diameter 40nm, adds 160 μ
GOMP31 albumen (Zheng WY, Wang Y, Zhang ZC, Yan F.Immunological characteristics of
outer membrane protein omp31 of goat Brucella and its monoclonal
Antibody.Genet Mol Res. 2,015 4 (4): 11965-74.)；Room temperature reaction 15min；Add BSA to final concentration 1%
(W/V), room temperature reaction 15min is continued；Centrifugal, redissolve liquid (10mM Tris-HCl(pH8.0), 0.5%BSA with 1ml) resuspended
Grain, standby.
5, the preparation of chromatographic film: spraying concentration successively according to the amount of 0.8 μ l/cm on Sai Duolisi CN140 film is
OMP31 albumen (Zheng WY, Wang Y, Zhang ZC, the Yan F.Immunological of 2.5mg/ml
characteristics of outer membrane protein omp31 of goat Brucella and its
Monoclonal antibody.Genet Mol Res. 2,015 4 (4): 11965-74.), the brucella fat of 2mg/ml many
Sugar monoclonal antibody (C epi-position), the OMP19 albumen of 1mg/ml, (Gong Xiaowei, Jing Tao, kingdom controls the OMP28 albumen of 2mg/ml, Lee
Scrupulously and respectfully prunus mume (sieb.) sieb.et zucc., Wang Bingxiang, Fu Linfeng. the expression and purification of brucella BP26 albumen and antigenic research thereof. China Amphixenosis
Journal .2009,11:1085-1088), 0.5mg/ml sheep anti-mouse igg (quality control region), be placed in 37 DEG C of dry 2h, standby.
6, the preparation of micropore is reacted: by bodies such as the detection antigens detecting antibody and nanoparticle label of nanoparticle label
Volume blending；It is dispensed in micropore according to 45 μ l/ holes；It is placed in-80 DEG C of freezing 24h；Lyophilizing；Kept dry, standby.
7, the preparation of sample pad: use 80mM Tris-HCl(pH8.0), 1%BSA, 0.5%Tween 20 soak non-woven fabrics
10min；It is placed in 37 DEG C to be dried, standby.
8, the preparation of test strips: by sample pad, chromatographic film (be followed successively by adsorptive pads direction by sample pad: OMP31 albumen,
Brucella lipopolysaccharide monoclonal antibody, OMP19 albumen, OMP28 albumen, sheep anti-mouse igg (quality control region), adsorptive pads overlaps successively
Paste PVC base plate, be cut into 4.5mm width, be placed in 4 DEG C of kept dry.
9, detection sample: take serum/milk 200 μ l in reaction micropore in, in 40 DEG C react 3min；Insert test strips,
Sentence read result after 15min: 1) only have quality control region to have signal: do not infected brucella；2) only have quality control region and antibody test district
Having a signal: infected but rehabilitation or intracellular have been carried disease germs, the probability that the most explanations of bar number were infected/infected is the highest；3) five lines are equal
There is signal: carry disease germs and produced antibody；4) quality control region and Detection of antigen district is only had to have a signal: to carry disease germs, initial infection；5) detection
Invalid, need to again detect.Actual sample testing result is shown in Table 4.This programme test strips is capable of detecting when that all bacterium cultivate positive sample
This, and cultivate patients with negative serum sample from 1098 parts of bacterium and detect 15 parts of positive sample；There is no false positive sample.
Table 4 uses 1725 parts of human serum samples of the present embodiment ELISA test strip
Embodiment 5
1, the preparation of nanoparticle label detection antibody: take the nanogold particle of 10ml particle diameter 40nm, add 90 μ g brucella
Lipopolysaccharide monoclonal antibody (C epi-position)；Room temperature reaction 15min；Add BSA to final concentration 1%(W/V), continue room temperature reaction
15min；Centrifugal, redissolve liquid (10mM Tris-HCl(pH8.0), 0.5%BSA with 1ml) resuspended granule, standby.
2, the preparation of nanoparticle label OMP28 albumen: take the nanogold particle of 10ml particle diameter 40nm, adds 140 μ
GOMP28 albumen (1-75AA with 135-225AA is spliced, pET-32a plasmid, and e. coli bl21 is expressed)；Room temperature reaction
15min；Add BSA to final concentration 1%(W/V), continue room temperature reaction 15min；Centrifugal, redissolve liquid (10mM Tris-with 1ml
HCl(pH8.0), 0.5%BSA) resuspended granule, standby.
3, the preparation of nanoparticle label OMP25 albumen: take the nanogold particle of 10ml particle diameter 40nm, adds 170 μ g
OMP25 albumen (pET-32a plasmid, e. coli bl21 is expressed)；Room temperature reaction 15min；Add BSA to final concentration 1%(W/V),
Continue room temperature reaction 15min；Centrifugal, redissolve liquid (10mM Tris-HCl(pH8.0), 0.5%BSA with 1ml) resuspended granule, standby
With.
4, the preparation of chromatographic film: spraying concentration successively according to the amount of 0.8 μ l/cm on Mi Libo 139 film is 2.5mg/ml
OMP25 albumen (pET-32a plasmid, e. coli bl21 express), the brucella lipopolysaccharide monoclonal antibody (M of 2mg/ml
Epi-position and A epi-position monoclonal antibody mixed in equal amounts), (Gong Xiaowei, Jing Tao, kingdom controls the OMP28 albumen of 2mg/ml, Li Kemei, king
Grasp Xiang, Fu Linfeng. the expression and purification of brucella BP26 albumen and antigenic research thereof. China's Amphixenosis's journal
.2009,11:1085-1088), 0.5mg/ml sheep anti-mouse igg (quality control region), be placed in 37 DEG C of dry 2h, standby.
5, the preparation of pad: by detection antibody and the detection antigen equal-volume of nanoparticle label of nanoparticle label
Mixing；It is sprayed on 8964 glass fibre according to 5 μ l/cm, is dried in 37 DEG C, standby.
6, the preparation of sample pad: use 20mM Tris-HCl(pH8.0), 1%BSA, 0.5%Tween 20 soak non-woven fabrics
10min；It is placed in 37 DEG C to be dried, standby.
7, the preparation of test strips: by sample pad, pad, chromatographic film (be followed successively by adsorptive pads direction by sample pad:
OMP25 albumen, brucella lipopolysaccharide monoclonal antibody, OMP28 albumen, 0.5mg/ml sheep anti-mouse igg (quality control region), adsorptive pads
Overlap joint pastes PVC base plate successively, is cut into 3.5mm width, is placed in 4 DEG C of kept dry.
8, detection sample: sample pad is lain against on table top；Take serum/milk 80 μ l in sample pad；Interpretation after 15min
Result: 1) only have quality control region to have signal: do not infected brucella；2) quality control region and antibody test district is only had to have signal: to infect
Crossing but rehabilitation or intracellular have been carried disease germs, the probability that the most explanations of bar number were infected/infected is the highest；3) four lines all have a signal: carry disease germs,
And produced antibody；4) quality control region and Detection of antigen district is only had to have a signal: to carry disease germs, initial infection；5) detect invalid, need to again examine
Survey.Actual sample testing result is shown in Table 5.This programme test strips is capable of detecting when that all bacterium cultivate positive sample, and from 1098
Part bacterium is cultivated in patients with negative serum sample and detects 15 parts of positive sample；There is no false positive sample.
Table 5 uses 1725 parts of human serum samples of the present embodiment ELISA test strip
Embodiment 6
1, the preparation of nanoparticle label detection antibody: take the nanogold particle of 10ml particle diameter 40nm, add 90 μ g brucella
Lipopolysaccharide monoclonal antibody (C epi-position)；Room temperature reaction 15min；Add BSA to final concentration 1%(W/V), continue room temperature reaction
15min；Centrifugal, redissolve liquid (10mM Tris-HCl(pH8.0), 0.5%BSA with 1ml) resuspended granule, standby.
2, the preparation of nanoparticle label OMP28 albumen: take the nanogold particle of 10ml particle diameter 40nm, adds 140 μ g
OMP28 albumen (1-75AA with 135-225AA is spliced, pET-32a carrier, and e. coli bl21 is expressed)；Room temperature reaction
15min；Add BSA to final concentration 1%(W/V), continue room temperature reaction 15min；Centrifugal, redissolve liquid (10mM Tris-with 1ml
HCl(pH8.0), 0.5%BSA) resuspended granule, standby.
3, the preparation of nanoparticle label OMP10 albumen: take the nanogold particle of 10ml particle diameter 40nm, adds 120 μ g
OMP10 albumen (Gong Xiaowei, Zhou Jizhang. Bacillus brucellae outer membrane protein omp10 is expressed and antigenic research. and animal medicine is in progress.
2009,30 (12): 25-29.)；Room temperature reaction 15min；Add BSA to final concentration 1%(W/V), continue room temperature reaction 15min；
Centrifugal, redissolve liquid (10mM Tris-HCl(pH8.0), 0.5%BSA with 1ml) resuspended granule, standby.
4, the preparation of nanoparticle label OMP31 albumen: take the nanogold particle of 10ml particle diameter 40nm, adds 160 μ g
OMP31 albumen (Zheng WY, Wang Y, Zhang ZC, Yan F.Immunological characteristics of
outer membrane protein omp31 of goat Brucella and its monoclonal
Antibody.Genet Mol Res. 2,015 4 (4): 11965-74.)；Room temperature reaction 15min；Add BSA to final concentration 1%
(W/V), room temperature reaction 15min is continued；Centrifugal, redissolve liquid (10mM Tris-HCl(pH8.0), 0.5%BSA with 1ml) resuspended
Grain, standby.
5, the preparation of chromatographic film: spray concentration successively according to the amount of 0.8 μ l/cm on Mi Libo CN95 nitrocellulose filter
For the brucella lipopolysaccharide monoclonal antibody (C epi-position) of 2mg/ml, 2.5mg/ml OMP10 albumen (Gong Xiaowei, Zhou Jizhang.
Bacillus brucellae outer membrane protein omp10 is expressed and antigenic research. and animal medicine is in progress. and 2009,30 (12): 25-29.),
OMP31 albumen (Zheng WY, Wang Y, Zhang ZC, the Yan F.Immunological of 2mg/ml
characteristics of outer membrane protein omp31 of goat Brucella and its
Monoclonal antibody.Genet Mol Res. 2,015 4 (4): 11965-74.), the OMP28 albumen (palace of 2mg/ml
Knowing bright, Jing Tao, kingdom controls, Li Kemei, Wang Bingxiang, Fu Linfeng. the expression and purification of brucella BP26 albumen and antigenic
Research. China Amphixenosis journal .2009,11:1085-1088), 0.5mg/ml sheep anti-mouse igg (quality control region), be placed in 37
DEG C dry 2h, standby.
6, the preparation of pad: by detection antibody and the detection antigen equal-volume of nanoparticle label of nanoparticle label
Mixing；It is sprayed on 8964 glass fibre according to 5 μ l/cm, is dried in 37 DEG C, standby.
7, the preparation of sample pad: use 20mM Tris-HCl(pH8.0), 1%BSA, 0.5%Tween 20 soak non-woven fabrics
10min；It is placed in 37 DEG C to be dried, standby.
8, the preparation of test strips: sample pad, pad, chromatographic film (are followed successively by adsorptive pads direction by sample pad: Bu Lu
Salmonella lipopolysaccharide monoclonal antibody, OMP10 albumen, OMP31 albumen, OMP28 albumen, 0.5mg/ml sheep anti-mouse igg (quality control region),
Adsorptive pads overlaps successively and pastes PVC base plate, is cut into 3.5mm width, is placed in 4 DEG C of kept dry.
9, detection sample: sample pad is lain against on table top；Take serum/milk 80 μ l in sample pad；Interpretation after 15min
Result: 1) only have quality control region to have signal: do not infected brucella；2) quality control region and antibody test district is only had to have signal: to infect
Crossing but rehabilitation or intracellular have been carried disease germs, the probability that the most explanations of bar number were infected/infected is the highest；3) five lines all have a signal: carry disease germs,
And produced antibody；4) quality control region and Detection of antigen district is only had to have a signal: to carry disease germs, initial infection；5) detect invalid, need to again examine
Survey.Actual sample testing result is shown in Table 6.This programme test strips is capable of detecting when that all bacterium cultivate positive sample, and from 1098
Part bacterium is cultivated in patients with negative serum sample and detects 15 parts of positive sample；There is no false positive sample.
Table 6 uses 1725 parts of human serum samples of the present embodiment ELISA test strip
Embodiment 7
1, the preparation of nanoparticle label detection antibody: take the nanogold particle of 10ml particle diameter 40nm, add 90 μ g brucella
Lipopolysaccharide monoclonal antibody (C epi-position)；Room temperature reaction 15min；Add BSA to final concentration 1%(W/V), continue room temperature reaction
15min；Centrifugal, redissolve liquid (10mM Tris-HCl(pH8.0), 0.5%BSA with 1ml) resuspended granule, standby.
2, the preparation of nanoparticle label OMP28-OMP31 fusion protein: take the nanogold particle of 10ml particle diameter 40nm, add
Enter 140 μ g brucella fat OMP28-OMP31 fusion protein；Room temperature reaction 15min；Add BSA to final concentration 1%(W/V), continue
Continuous room temperature reaction 15min；Centrifugal, redissolve liquid (10mM Tris-HCl(pH8.0), 0.5%BSA with 1ml) resuspended granule, standby.
3, the preparation of chromatographic film: spray dense successively according to the amount of 0.8 μ l/cm on Sai Duolisi CN90 nitrocellulose filter
Degree is the brucella lipopolysaccharide monoclonal antibody (C epi-position) of 2mg/ml, (peptide sequence is such as the polypeptide-BSA conjugate of 2mg/ml
Shown in table 7, antibody test district, is the covalent coupling thing of the mixture of polypeptide listed by table 7 and BSA herein), 0.02mg/ml goat-anti
Mus IgG(quality control region), it is placed in 37 DEG C of dry 2h, standby.
Table 7 peptide sequence
| Numbering | Sequence |
| OMP28-1 | LGVNQGGDLNLVNDNP |
| OMP28-2 | TLADAAGVGLGRVVE |
| OMP28-3 | KKAGIEDRDLQTGGI |
| OMP31-1 | SISAGASGLEGKAET |
| OMP31-2 | GVVLGAETDFQGSSV |
4, the preparation of micropore is reacted: the detection antibody of nanoparticle label and the detection antigen equal-volume of nanoparticle label are mixed
Even；It is dispensed in micropore according to 20 μ l/ holes；-80 DEG C of freezing 24h；It is placed in freeze dryer lyophilizing, seals, kept dry, standby.
5, sample pad: 8964 glass fibre.
6, the preparation of test strips: sample pad, chromatographic film (are followed successively by adsorptive pads direction by sample pad: brucella fat
Monoclonal Antibody against Polysaccharides, brucella OMP28-OMP31 fusion protein, sheep anti-mouse igg), adsorptive pads overlaps successively and pastes PVC
Base plate, is cut into 4.5mm width, is placed in 4 DEG C of kept dry.
7, detection sample: take sample 200 μ l in micropore, reacts 3min in 37 DEG C；Test strips is inserted in micropore,
Sentence read result after 10min: 1) only have quality control region to have signal: do not infected brucella；2) only have quality control region and antibody test district:
Infected but rehabilitation or intracellular have been carried disease germs；3) three lines all have signal: carry disease germs and produced antibody；4) only have quality control region and resist
Former detection zone has a signal: carry disease germs, initial infection；5) quality control region no signal: it is invalid to detect, need to detect again.Actual sample detects
The results are shown in Table 2.This programme test strips is capable of detecting when that all bacterium cultivate positive sample, and cultivates patients with negative from 1098 parts of bacterium
Serum sample detects 15 parts of positive sample；There is no false positive sample.
Table 8 uses 1725 parts of human serum samples of the present embodiment ELISA test strip
Embodiment 8
1, the preparation of nanoparticle label detection antibody: take the nanogold particle of 10ml particle diameter 40nm, add 90 μ g rabbit anti-cloth Shandongs
Salmonella lipopolysaccharide polyclonal antibody；Room temperature reaction 15min；Add BSA to final concentration 1%(W/V), continue room temperature reaction 15min；
Centrifugal, redissolve liquid (10mM Tris-HCl(pH8.0), 0.5%BSA with 1ml) resuspended granule, standby.
2, (protein that the polypeptide fragment of OMP10, OMP28, OMP31 is in series divides nanoparticle label fusion protein
Son, prokaryotic expression) preparation: take the nanogold particle of 10ml particle diameter 40nm, add 120 μ g fusion protein (OMP10, OMP28,
The protein molecule that the polypeptide fragment of OMP31 is in series, prokaryotic expression)；Room temperature reaction 15min；Add BSA to final concentration 1%
(W/V), room temperature reaction 15min is continued；Centrifugal, redissolve liquid (10mM Tris-HCl(pH8.0), 0.5%BSA with 1ml) resuspended
Grain, standby.
Attached: the protein molecule sequence that the polypeptide fragment of OMP10, OMP28, OMP31 is in series:
KRFRIVAPLALMSDPNGIASSFNGGIGVNQGGDLNLVNDNPTLADAAGVGLGRVVEKKAGIEDRDLQTGGISISAGA
SGLEGKAETGVVLGAETDFQGSSV
3, the preparation of chromatographic film: spraying concentration successively according to the amount of 0.8 μ l/cm on Sai Duolisi CN90 nitrocellulose filter is
The rabbit anti-brucella lipopolysaccharide polyclonal antibody of 2mg/ml, the fusion protein (polypeptide of OMP10, OMP28, OMP31 of 2mg/ml
The protein molecule that fragment is in series), 0.02mg/ml sheep anti-mouse igg (quality control region), be placed in 37 DEG C of dry 2h, standby.
Attached: the protein molecule sequence that the polypeptide fragment of OMP10, OMP28, OMP31 is in series:
KRFRIVAPLALMSDPNGIASSFNGGIGVNQGGDLNLVNDNPTLADAAGVGLGRVVEKKAGIEDRDLQTGGISISAGA
SGLEGKAETGVVLGAETDFQGSSV
4, the preparation of micropore is reacted: the detection antibody of nanoparticle label and the fusion protein equal-volume of nanoparticle label are mixed
Even；It is dispensed in micropore according to 20 μ l/ holes；In micropore, Escherichia coli O 157 lipopolysaccharide or small intestinal knot is added according to 1 μ g/ hole
Enteritis yersinia lipopolysaccharide；-80 DEG C of freezing 24h；It is placed in freeze dryer lyophilizing, seals, kept dry, standby.
5, sample pad: 8964 glass fibre.
6, the preparation of test strips: sample pad, chromatographic film (are followed successively by adsorptive pads direction by sample pad: brucella fat
Monoclonal Antibody against Polysaccharides, fusion protein, sheep anti-mouse igg), adsorptive pads overlap successively and paste PVC base plate, be cut into 4.5mm width, put
In 4 DEG C of kept dry.
7, detection sample: take sample 200 μ l in micropore, reacts 3min in 37 DEG C；Test strips is inserted in micropore,
Sentence read result after 10min: 1) only have quality control region to have signal: do not infected brucella；2) only have quality control region and antibody test district:
Infected but rehabilitation or intracellular have been carried disease germs；3) three lines all have signal: carry disease germs and produced antibody；4) only have quality control region and resist
Former detection zone has a signal: carry disease germs, initial infection；5) quality control region no signal: it is invalid to detect, need to detect again.Actual sample detects
The results are shown in Table 2.
Table 9 uses 1725 parts of human serum samples of the present embodiment ELISA test strip
Embodiment described above only have expressed embodiments of the present invention, therefore it describes more concrete and detailed, but can not be
And it is interpreted as the restriction to the scope of the claims of the present invention, as long as using the technical side that the form of equivalent or equivalent transformation is obtained
Case, all should fall within the scope and spirit of the invention.
Claims (10)
1. a detection device for Infected with Brucella, including test strips；The chromatographic film of test strips is provided with brucellergen
Detection zone, Brucella antibody detection zone.
The detection device of Infected with Brucella the most according to claim 1, it is characterised in that described brucellergen is examined
Survey district's brucella lipopolysaccharide in specific detection sample；Described Brucella antibody detection zone is used for detecting in sample
Brucella outer membrane protein antibody.
The detection device of Infected with Brucella the most according to claim 1, it is characterised in that Detection of antigen district is coated with catches
Obtain antibody, the detection antibody of nanoparticle label and the coated antibody sandwich detection brucellergen in Detection of antigen district；Antibody
Detection zone is coated with capture antigen, and the detection antigen of nanoparticle label detects cloth Shandong with the capture antigen sandwich in antibody test district
Salmonella antibody.
The detection device of Infected with Brucella the most according to claim 3, it is characterised in that described detection antibody and capture
In antibody, at least one is the monoclonal antibody specific binding with brucella lipopolysaccharide O chain.
The detection device of Infected with Brucella the most according to claim 3, it is characterised in that described detection antibody and capture
Antibody is polyclonal antibody.
The detection device of Infected with Brucella the most according to claim 1, it is characterised in that described detection antigen and capture
Antigen is brucella outer membrane protein；Described brucella outer membrane protein be OMP10 albumen, OMP19 albumen, OMP25 albumen,
One or more in OMP28 albumen, OMP31 albumen, OMP16 albumen, or two or more above-mentioned protein fusion tables
The protein reached, or the conjugate of the polypeptide fragment of above-mentioned protein and carrier protein, or the difference of above-mentioned protein is many
The protein molecule that fragments of peptides is in series.
The detection device of Infected with Brucella the most according to claim 1, it is characterised in that described capture antigen is cloth Shandong
Salmonella is derived from the polypeptide fragment of OMP28 albumen and the covalent coupling thing of carrier protein.
The detection device of Infected with Brucella the most according to claim 1, it is characterised in that described chromatographic film is by sample pad
The antibody test district be followed successively by, to absorption pad direction, the Detection of antigen district being coated brucella lipopolysaccharide, being coated OMP28 albumen,
Or, the Detection of antigen district that be followed successively by the antibody test district being coated OMP16 albumen, is coated brucella lipopolysaccharide, it is coated OMP25 egg
White antibody test district, it is coated the antibody test district of OMP28 albumen.
The detection device of Infected with Brucella the most according to claim 1, it is characterised in that described chromatographic film is by sample pad
The Detection of antigen district that be followed successively by, to absorption pad direction, the antibody test district being coated OMP31 albumen, is coated brucella lipopolysaccharide, bag
By the antibody test district of OMP19 albumen, the antibody test district that is coated OMP28 albumen, or it is followed successively by the antibody being coated OMP25 albumen
Detection zone, it is coated the Detection of antigen district of brucella lipopolysaccharide, is coated the antibody test district of OMP28 albumen.
The detection device of Infected with Brucella the most according to claim 1, it is characterised in that described chromatographic film is by sample
Pad is followed successively by absorption pad direction and is coated the Detection of antigen district of brucella lipopolysaccharide monoclonal antibody, is coated the anti-of fusion protein
Body detection zone；Described fusion protein be OMP28-OMP31 fusion protein or OMP10, OMP28, OMP31 polypeptide fragment series connection and
The protein molecule become.
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| CN109116035A (en) * | 2018-09-14 | 2019-01-01 | 重庆理工大学 | A kind of Brucella antibody competition AlphaLISA detection kit and its detection method |
| CN109541206A (en) * | 2018-12-28 | 2019-03-29 | 洛阳现代生物技术研究院有限公司 | A kind of Brucella antibody serology antidiastole test paper |
| CN109655614A (en) * | 2019-02-26 | 2019-04-19 | 广州维佰生物科技有限公司 | Ox brucellosis virus fluorescence micro-ball immune chromatography test paper and preparation method thereof and detection method |
| CN110514831A (en) * | 2019-09-07 | 2019-11-29 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | A kind of colloidal gold fast detecting test paper strip of Brucella bp 26 protein epitope fusion protein and its application |
| CN111537730A (en) * | 2020-04-22 | 2020-08-14 | 武汉优恩生物科技有限公司 | Test paper for detecting brucella antibody |
| 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 |
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