CN109507434A - Detect the ELISA kit of brucella antibody - Google Patents
Detect the ELISA kit of brucella antibody Download PDFInfo
- Publication number
- CN109507434A CN109507434A CN201811363909.8A CN201811363909A CN109507434A CN 109507434 A CN109507434 A CN 109507434A CN 201811363909 A CN201811363909 A CN 201811363909A CN 109507434 A CN109507434 A CN 109507434A
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- Prior art keywords
- antibody
- omp31
- serum
- plate
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/26—Infectious diseases, e.g. generalised sepsis
Abstract
The invention discloses a kind of ELISA kits for detecting brucella antibody, utilize the gene constructed recombination engineering of Omp31, expression Omp31 recombinant protein prepares albumen coating plate as coating antigen, establish ELISA antibody assay kit, this kit specificity is good, sensibility is high, testing cost is cheap, is suitable for promoting.Important method is provided in terms of the detection of the antibody level of this disease, epidemiological survey, class disease identify and take effective anti-measure processed.This kit Main Components: Omp31 albumen is coated with plate, positive control serum, negative control sera, enzyme label rabbit-anti polyclonal antibody, substrate solution, terminate liquid, dilution, cleaning solution, serum and dilutes plate.
Description
Technical field
The present invention relates to ELISA kit, especially a kind of ELISA kit for detecting brucella antibody.
Background technique
Brucella is a kind of Gram-negative, intracellular parasitic bacteria, is small rod bacterium, can be in a variety of domestic animal bodies
Survival, the animals such as ox, sheep, pig, dog and camel, deer may all be infected, by being infected by contact animal or infected food
Object and laboratory contact can be broadcast to the mankind.To artificial mainly Maltese brucella (" Malta at infection
Heat "), bacillus abortus, ox, sheep brucella, Brucella suis and dog brucella, wherein with sheep cloth Lu Shi
Bacillosis is the most common.
Britain medical officer Bruce in 1886 isolates " Bu Lu from the soldier's spleen for dying of " Malta fever " on Malta island
Salmonella " specifies the pathogen of the disease for the first time.After people's illness, the symptom occurred first is fever, and body temperature is held up to 38-40 DEG C
The continuous time is long, is in prolonged low grade fever state；Somebody's body temperature is wave-shaped, i.e. several days of high fever, and body temperature lowers several days, and starts
Height, it is repeated multiple times, so cloth disease is also known as wave-like heat.
Brucellosis so far, is continued to bring out about this disease diagnostic method with research achievement from appearance.Traditional cloth Lu Shi
Bacillus detection method, since the disadvantages of its detection cycle is long, program is complicated, required reagent is various has been far from satisfying modern inspection
It surveys and requires.There are some problems in practical applications by the etiology nucleic acid detection technique of representative of polymerase chain reaction technology,
As Common Polymerase Chain Reaction technology needs special instrument, and the spy cumbersome in the presence of easy cross contamination and operating process
Point.Immunological detection method is a series of measurement antigens, antibody, immunocyte and its secretion of applied immunology Theoretical Design
The experimental method of cell factor.Antigen meets with corresponding antibodies and can specifically bind, and is under the influence of external condition
Certain existing reacting phenomenon, is such as aggregated or precipitates, and can detect unknown antibody (or antigen) with known antigens (or antibody) whereby.
Summary of the invention
The technical problem to be solved by the invention is to provide a kind of quick, easy, sensitive, accurate detection cloth Lu Shi bars
The ELISA kit of bacteria antibody.
In order to solve the above-mentioned technical problem, the technical solution adopted by the present invention is that: a kind of detection brucella antibody
ELISA kit, including Omp31 recombinant protein are coated with plate, and the Omp31 recombinant protein is using part Omp31 gene order
And the synthesis of the nucleotide sequence full genome as shown in the SEQ ID NO.1 in sequence table is obtained after codon optimization, after digestion
31 gene of Omp is connected in pET-28a (+) plasmid, e. coli bl21 competent cell is converted after identified, induces table
It reaches, affinity chromatography isolates and purifies acquisition.
The method for coating of the albumen coating plate are as follows: Omp31 recombinant protein is diluted with 0.5M carbonate buffer solution, to 96 holes
0.1mL, 2~8 DEG C of coating 15h are added in each hole of ELISA Plate；Every hole adds 250 μ L confining liquids, and room temperature closes 2h, after discarding confining liquid
Drying at room temperature 2h；Coating plate and desiccant are put into aluminium foil bag, vacuum sealing.
It further include enzyme label rabbit-anti ox polyclonal antibody, enzyme marks rabbit-anti ox preparation method of polyclonal antibody as follows: using
Ox polyclonal antibody rabbit tests detection antibody titer, horseradish mostly anti-through Protein A column purification using AGP test
Peroxidase HRP label.
The beneficial effects of the present invention are: being used for the detection of brucella antibody, kit specificity is good, sensibility is high,
Testing cost is cheap, is suitable for promoting.Identify and take effectively anti-in the antibody level detection, epidemiological survey, class disease of this disease
Important method is provided in terms of measure processed.
Specific embodiment
Invention is further described in detail With reference to embodiment:
The ELISA kit of detection brucella antibody of the invention, including Omp31 recombinant protein are coated with plate, described
Omp31 recombinant protein is obtained such as the SEQ ID in sequence table using part Omp31 gene order and after codon optimization
31 gene of Omp, is connected in pET-28a (+) plasmid, through reflecting by the synthesis of nucleotide sequence full genome shown in NO.1 after digestion
E. coli bl21 competent cell, inducing expression are converted after fixed, affinity chromatography isolates and purifies acquisition.
The method for coating of the albumen coating plate are as follows: Omp31 recombinant protein is diluted with 0.5M carbonate buffer solution, to 96 holes
0.1mL, 2~8 DEG C of coating 15h are added in each hole of ELISA Plate；Every hole adds 250 μ L confining liquids, and room temperature closes 2h, after discarding confining liquid
Drying at room temperature 2h；Coating plate and desiccant are put into aluminium foil bag, vacuum sealing.
It further include enzyme label rabbit-anti ox polyclonal antibody, enzyme marks rabbit-anti ox preparation method of polyclonal antibody as follows: using
Ox polyclonal antibody rabbit tests detection antibody titer, horseradish mostly anti-through Protein A column purification using AGP test
Peroxidase HRP label.
The preparation of 1 component
The preparation of 1.1 antigens
With Brucella melitensis strain 152Omp31 gene order object, full genome synthesizes this sequence,
31 gene of Omp is connected in pET-28a (+) plasmid after EcoRI and XhoI digestion, converts Escherichia coli after identified
BL21 competent cell, IPTG inducing expression, affinity chromatography isolate and purify acquisition.
1.1.1 bacterial strain, plasmid
E.coli DH5 α, E.coli BL21 (DE3) are purchased from TaKaRa company；Expression vector pET-28a is purchased from Promega
Company.
1.1.2 reagent
Restriction enzyme, nucleic acid and protein standard marker Marker are purchased from Fennentas company；Plasmid extraction
Kit is purchased from TIANGEN Biotech (Beijing) Co., Ltd.；Rabbit-anti ox IgG-HRP, rabbit-anti sheep IgG-HRP are purchased from KPL company；
Protein purification His.BindKits is purchased from Merk company.
According to listed Brucella melitensis strain 152Omp31 complete genome sequence, selection wherein can
Enough express the gene of 231 amino acid and through codon optimization, upstream introducing EcoRI restriction enzyme site, downstream introducing XhoI digestion
Site carries out full genome synthesis and is subsequently introduced in plasmid (Nanjing Genscript Biotechnology Co., Ltd.).Full genome synthesizes sequence
Column are as shown in the SEQ ID NO.1 in sequence table.
1.1.3 the building of expression vector
Plasmid containing complete genome sequence is transformed into DH5 α bacterial strain, plasmid is extracted with plasmid extraction kit, through EcoRI
With XhoI digestion, it is cloned in construction recombination plasmid pet28a-Omp 31 in expression vector pet28a.
1.1.4 the expression and purity of recombinant protein Omp 31
Recombinant plasmid pet28a-Omp 31 is converted in e. coli bl21 (DE3), picking positive colony bacterium is inoculated in
LB culture medium.It stays overnight in 37 DEG C of shaking table 200r/min shaken cultivations, is seeded in 5ml LB culture medium in the ratio of 1:100.Training
Support to OD600 be 0.6-0.8 when, IPTG to final concentration 1mmol/L is added, receives bacterium after inducing expression 6h.By 1.5ml bacterium solution from
The heart takes precipitating, 80 μ L1%SDS and 20 μ L albumen sample-loading buffers is added, after oscillation mixes, boiling water boiling 5min is slightly centrifuged, takes
10 μ L of supernatant carries out 12%SDS-PAGE analysis.According to Ni-NTA protein purification kit operating instruction purifying protein.Use electricity
Instrument is transferred under the conditions of 60mA, 4 DEG C of transfers are stayed overnight, by SDS glue surface protein delivery to PVDF nitrocellulose filter.5% defatted milk
4 DEG C of powder PBST confining liquid closing overnight, then by film be packed into there is His to exempt from serum primary antibody (1:1000 dilution) sterile small plastics
In bag, hatching combination 2h is decolourized on TBST room temperature shaker, is washed 3 times, each 5min.Add secondary antibody (1:10000 dilution, sheep
Anti-rabbit IgG-HRP is purchased from Novagen company), incubation at room temperature combines 2h, is then washed on shaking table with TBST 3 times, each 5min.
DAB colour developing.The experimental results showed that the Escherichia coli containing positive recombinant vector are expressed under IPTG induction, different induction times
Product detected through SDS-PAGE, the visible band of expression at about 29KD is consistent with expected albumen size, and non-induction bacterium
After the induction of empty carrier bacterium, the albumen for not occurring the purifying of this protein band is destination protein to be expressed.
The preparation of 1.2 enzyme mark polyclonal antibodies (enzyme marker)
Conventionally immunizing rabbit is taken a blood sample after being repeatedly immunized, with AGP test measuring antibody titer, is made
It is purified with how anti-antigen column is to rabbit-anti ox.Made polyclonal antibody is marked using horseradish peroxidase (HRP),
Enzyme mark polyclonal antibody is made.
1.3 albumen are coated with plate preparation
Aseptically, the albumen of purifying is diluted with carbonate buffer solution, is added into each hole of 96 hole elisa Plates
0.1mL, 2~8 DEG C of 15h.Add confining liquid in each hole of ELISA Plate being coated with, every 250 μ L of hole, room temperature closes 2h, discards envelope
Close liquid.Coating plate is put into aluminium foil bag, vacuum sealing.
1.4 positive controls and negative control
It purchases from Chinese veterinary microorganism culture presevation administrative center.
The assembling of 2 kits
Operation instructions part
Substrate A liquid 30mL (main component: carbamide peroxide, PEG-2000, disodium hydrogen phosphate dodecahydrate, a hydration lemon
Lemon acid)
Substrate B liquid 30mL (main component: monohydrate potassium, sodium thiosulfate, light stabilizer 292, DMF)
25 times of concentrated solution for washing 60mL
Terminate liquid 60mL
Dilution 200mL
Serum dilutes 96 hole of plate
3 usages and judgement
3.1 preparation of samples
Animal's whole blood is taken, after waiting for blood to solidify, 10min is centrifuged with 4000r/min, collects supernatant.Blood can also be acquired, to
Naturally serum is precipitated after solidification.Serum answers limpid, no haemolysis.
The preparation of 3.2 cleaning solutions
Before use, restoring the cleaning solution of concentration to room temperature (20~25 DEG C), and shake, dissolves crystallization, then spend
Ionized water makees 1:25 dilution, mixing.
The dilution of 3.3 serum and control serum to be checked
It is dilute that serum to be checked, standard negative control serum and standard positive control serum made into 1:50 in serum dilution plate
It releases, Plays positive and negative control serum respectively adds 2 holes.It is mixed well after serum is added.
3.4 operating procedure
3.4.1 negative control sera, positive control serum, sample diluting liquid and measuring samples respectively take 50 μ L to add to corresponding micro-
Then 50 μ L monoclonal antibody working solutions are added in Kong Zhong in all holes.Cover board film is covered, ELISA Plate l0s is gently vibrated, it is sufficiently mixed
It is even, at room temperature (25 ± 2 DEG C), it is protected from light 30min.
3.4.2 the solution in hole is got rid of, the 300 μ L of cleaning solution diluted is added in every hole, and concussion washing 4 times, each 30s is left
The right side pats on clean blotting paper and removes remaining cleaning solution；100 μ L ELIAS secondary antibody working solutions are added in every hole, cover cover board film, gently
Light oscillation ELISA Plate 10s, mixes well, (25 ± 2 DEG C) are protected from light 30min at room temperature.
3.3 get rid of the solution in hole, and the 300 μ L of cleaning solution diluted is added in every hole, and concussion washing 4 times, each 30s is left
The right side pats on clean blotting paper and removes remaining cleaning solution；Then as early as possible by substrate solution A and substrate solution B mixed in equal amounts, after mixing
100 μ L are added in every hole, cover cover board film, under room temperature (25 ± 2 DEG C), are protected from light 15min.
50 μ L of terminate liquid is added in 3.4 every holes, and each hole OD is read in microplate reader450nmValue.
3.5 result judgement
Calculate the mean OD value of Sample dilution.It is 0 that Sample dilution, which is considered suppression percentage (I%), i.e., does not press down
System.The suppression percentage of other controls and sample calculates according to the following formula:
I%=100- sample OD value × 100/ Sample dilution mean OD value.
If I% >=70, it is judged to the Brucella antibody positive；If I% < 70, it is judged to Brucella antibody feminine gender.
The verifying of 4 kits
4.1 sensibility
Test result can be divided into positive or negative according to critical value, every batch of sets the control of 6 hole bacterial antigens, bacterial antigens
Any serum is not added in control, is directly diluted to working concentration with PBST, addition ELISA Plate synchronous with serum/bacterial antigen complex
Hole, 50 holes μ L/.It surveys bacterial antigens and compares OD450nm value.It is critical value with bacterial antigens control OD450nm value 50%, indicates resistance
The control OD450nm value of disconnected 50% reaction.Compareing the 50% of average OD450nm value with bacterial antigens is critical value, is detected serum
OD450nm value is greater than the negative hole of critical value, is positive hole less than or equal to the hole of critical value.
It is indicated with standard serum positive rate, with 50 parts of standard positive serum samples and 50 parts of standard female serum inspections
The comparison for surveying result determines its sensibility, while to determine the limit of identification of kit.
Sensibility=positive sample/gross sample × 100%
Specificity=negative sample/gross sample × 100%
4.2 cross reaction
With the O-shaped positive serum of aftosa, foot-and-mouth disease a type positive serum, aftosa Asia I type positive serum, ox paratyphoid
Sick positive serum is as detection antibody, using brucella positive sample as control, using PBS as blank control, with three batches
Kit, every kind of virus-4 part positive serum do cross reaction.
4.3 precision
Precision usually indicates that three batch kits are to feminine gender always, weakly positive, robust positive control sample with the coefficient of variation
It carries out 4 repetitions to detect, calculates the coefficient of variation.The coefficient of variation, i.e., its corresponding mean value of standard deviation of a series of detection datas
Ratio.
4.4 result
4.4.1 sensibility
Negative, the positive critical value measurement result of 1 kit of table
Negative, the positive critical value of the kit is that value≤1.08 OD450nm are the positive, and OD450nm value > 1.08 is feminine gender.
2 susceptibility testing result of table
| Serum type | Sample number | Positive detection sample number | Sensibility (%) | Specific (%) |
| Standard positive serum | 50 | 48 | 96.0 | - |
| Standard female serum | 50 | 3 | - | 94.0 |
Brucella antibody detection kit is 96.0% to standard positive serum sensibility, and specificity is 94.0%.
4.4.2 cross reaction
3 kit cross reaction testing result of table
Brucella antibody detection kit and the O-shaped positive serum of aftosa, foot-and-mouth disease a type positive serum, aftosa are sub-
I type positive serum of continent, ox paratyphoid disease positive serum do not have cross reaction.
4.4.3 precision
4 three batches of kit precision testing results of table
To the precision testing result of three batch kits, less than 5.5%, weak positive serum becomes the negative serum coefficient of variation
Different coefficient is less than 7.3%, and for the strong positive coefficient of variation less than 7.0%, which is respectively less than 10%.
4.5 conclusion
The Brucella antibody detection kit that the present invention develops is 96.0% to standard positive serum sensibility, specificity
It is 94.0%.Kit and the O-shaped positive serum of aftosa, foot-and-mouth disease a type positive serum, aftosa Asia I type positive serum, ox
Paratyphoid disease positive serum does not have cross reaction.To the precision testing result of three batch kits, the negative serum coefficient of variation
Less than 5.5%, the weak positive serum coefficient of variation is less than 7.3%, and the strong positive coefficient of variation is less than 7.0%, the kit variation lines
Number is respectively less than 10%.
In conclusion the contents of the present invention are not limited in the above embodiments, the knowledgeable people in same area can
Can propose other embodiments easily within technological guidance's thought of the invention, but this embodiment is included in this hair
Within the scope of bright.
Sequence table
<110>Animal-Plant and food Detecting Center, Tianjin Exit-Entery Inspection & Quarant
<120>ELISA kit of brucella antibody is detected
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 651
<212> DNA
<213>composition sequence (Artificial Sequence)
<400> 1
ccggaattca tgttcgccac gtccgctatg gctgccgacg tggttgtttc tgaaccttcc 60
gcccccactg ctgctcctgt tgacaccttc tcgtggaccg gcggctatat cggtatcaac 120
gccggttacg caggcggcaa gttcaagcat ccattttcta gctttgacaa ggaagacaac 180
gaacaggttt ccggttcgct cgacgtaaca gctggcggct tcgtcggtgg tgttcaggcc 240
ggttacaact ggcagctcga caacggcgtc gtgctcggcg cggaaaccga cttccaggga 300
tcgagcgtta cgggttcgat ttcagccggt gccagcggtc tcgaaggcaa agctgaaacc 360
aaggtcgagt ggttcggcac agttcgtgcc cgtcttggct acacggctac cgaacgcctc 420
atggtttatg gtaccggcgg tctggcctat ggtaaggtca agtctgcgtt caacctgggt 480
gatgatgcaa gtgccctgca cacgtggtcc gacaagacga aagctggctg gaccctcggc 540
gctggtgctg aatatgccat caacaacaac tggacgctca agtcggaata cctctacacc 600
gacctcggca agcgcaacct cgtcgacgtt gacaatagct tcctcgagcg g 651
Claims (3)
1. a kind of ELISA kit for detecting brucella antibody, which is characterized in that it is coated with plate including Omp31 recombinant protein,
The Omp31 recombinant protein is using part Omp31 gene order and to obtain after codon optimization such as the SEQ in sequence table
31 gene of Omp, is connected in pET-28a (+) plasmid by the synthesis of nucleotide sequence full genome shown in ID NO.1 after digestion, passes through
E. coli bl21 competent cell, inducing expression are converted after identification, affinity chromatography isolates and purifies acquisition.
2. detecting the ELISA kit of brucella antibody according to claim 1, which is characterized in that the albumen packet
By the method for coating of plate are as follows: Omp31 recombinant protein is diluted with 0.5M carbonate buffer solution, is added into each hole of 96 hole elisa Plates
0.1mL, 2~8 DEG C of coating 15h；Every hole adds 250 μ L confining liquids, and room temperature closes 2h, discards drying at room temperature 2h after confining liquid；It will packet
It is put into aluminium foil bag by plate and desiccant, vacuum sealing.
3. detecting the ELISA kit of brucella antibody according to claim 1, which is characterized in that further include enzyme mark
Remember that rabbit-anti ox polyclonal antibody, enzyme mark rabbit-anti ox preparation method of polyclonal antibody as follows: using ox polyclonal antibody man
Rabbit tests detection antibody titer using AGP test, horseradish peroxidase HRP label mostly anti-through Protein A column purification.
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| CN201811363909.8A CN109507434A (en) | 2018-11-16 | 2018-11-16 | Detect the ELISA kit of brucella antibody |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201811363909.8A CN109507434A (en) | 2018-11-16 | 2018-11-16 | Detect the ELISA kit of brucella antibody |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106432440A (en) * | 2016-07-21 | 2017-02-22 | 山东绿都生物科技有限公司 | Brucella melitensis antibody PPA-ELISA detection kit and preparation method thereof |
| KR20170023554A (en) * | 2015-08-24 | 2017-03-06 | 대한민국(농림축산식품부 농림축산검역본부장) | Method for Brucella canis antigen |
-
2018
- 2018-11-16 CN CN201811363909.8A patent/CN109507434A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20170023554A (en) * | 2015-08-24 | 2017-03-06 | 대한민국(농림축산식품부 농림축산검역본부장) | Method for Brucella canis antigen |
| CN106432440A (en) * | 2016-07-21 | 2017-02-22 | 山东绿都生物科技有限公司 | Brucella melitensis antibody PPA-ELISA detection kit and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| ASHRAFZADEH,S.P等: "登录号GQ184729.1", 《GENBANK》 * |
| 赵桂炎: "鹿源布鲁氏菌外膜蛋白Omp31基因的克隆与原核表达", 《中国优秀硕士学位论文全文数据库农业科技辑》 * |
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Application publication date: 20190322 |