CN106556701A - Brucella melitensis indirect ELISA antibody assay kit - Google Patents
Brucella melitensis indirect ELISA antibody assay kit Download PDFInfo
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- CN106556701A CN106556701A CN201611023727.7A CN201611023727A CN106556701A CN 106556701 A CN106556701 A CN 106556701A CN 201611023727 A CN201611023727 A CN 201611023727A CN 106556701 A CN106556701 A CN 106556701A
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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
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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
- G01N33/531—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
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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
Abstract
The present invention relates to Brucella melitensis indirect ELISA antibody assay kit.The brucella lipopolysaccharide (LPS) of purification is coated with 96 hole elisa Plates Jing after quantitative by the present invention, positive serum is prepared with artificial immunity health sheep, negative serum is made with healthy nonimmune sheep serum, assembled with components such as HRP labelling rabbit-anti sheep Fc ends antibody, substrate solution, terminate liquid, sample diluting liquid, HRP labelling rabbit-anti sheep IgG diluents and 20 × cleaning mixture, for detecting the smooth type Brucella antibody in sheep blood serum.The present invention in traditional extraction LPS increased protease K digesting and phenol extraction step, effectively increase LPS purity；Screen and the monoclonal antibody of good affinity has been respectively provided with to goat and sheep as ELIAS secondary antibody.The present invention shortens the time needed for high throughput testing by the improvement and optimization to main agents formula, the reaction background of effective control test kit.
Description
Technical field
The present invention relates to a kind of diagnostic method of animal brucellosis --- Brucella melitensis indirect ELISA antibody test
Test kit, belongs to biological product detection technique field.
Technical background
Brucellosis (brucellosis) are a kind of serious worldwide important zoonosiss for threatening human health, can be infected
People, various domestic animals and wild animal, are mainly shown as heating, miscarriage and infertility, chronic arthritiss and nerve injury etc..It is complete at present
It is to slaughter to combine with immunity that the sick main method is eliminated in world wide, thus set up fast and accurately diagnostic method to anti-
Control and to remove brucellosis very necessary.
International trade in Brucella antibody detection specifies test mainly to have rose bengal precipitation test (RBT), complement
Binding tests (CFT) and elisa (ELISA).Fluorescence polarization assay (FPA) is Selection experiment in international trade.
Current China《Brucellosis Prevention Technique specification》Specified in Brucella abortus antibody detection method be：The brave red coagulations of first Jing
(RBPT) Preliminary detection is tested, then Jing tube agglutination tests (SAT) or complement fixation test are made a definite diagnosis.Tube agglutination test is not only grasped
Make complicated, time-consuming, and there is also the not high problem of specificity；Complement fixation test is generally acknowledged diagnosis method, but examination
Complex operation is tested, need to be had good test facilities and well-trained personnel to make accurate component titration, reagent and be preserved and result
Judge.Fluorescence polarization assay (FPA) requires all higher to experiment condition and operating technology.
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, and both animal population quarantine can be applied to as screening test, and be 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.Niu Bulu
Salmonella disease ELISA detection method has been one of detection method for specifying in international trade, this method solves conventional method and takes
Laborious deficiency, improves specificity, sensitivity and the convenience of detection.
Because of the sheep brucellosis more low reason of sickness rate in the world, up to the present, commercialization is there is no in the world
It is specifically designed for the indirect ELISA diagnostic reagent kit of sheep brucellosis.And the cultivation scale of China sheep is very big, brucellosis sickness rate is again very
Height, therefore exploitation is specifically designed for the brucellosis indirect ELISA diagnostic reagent kit of sheep and has its realistic meaning.Cattle brucellosis are being developed successfully
On the basis of indirect ELISA, we have succeeded in developing sheep brucellosis indirect ELISA antibody assay kit again.By to monoclonal antibody system
The optimization of many-sided condition such as standby and screening, envelope antigen, reaction condition, result criterion, the particularly sheep to extract
IgG Fc sections carry out monoclonal antibody screening simultaneously using sheep IgG and goat IgG, choose to same concentration goat IgG as immunogen
There is the monoclonal antibody of similar response strength as the traget antibody of horseradish peroxidase with sheep IgG, test kit be made on detection mountain
With equal sensitivity when sheep blood serum and sheep serum；In addition, having carried out accurate mark to the potency of positive serum in the present invention
It is fixed, specificity and sensitive question in Brucella melitensis indirect ELISA antibody diagnosis method is solved more satisfactoryly.
Present disclosure
The purpose of the present invention is to set up a kind of Brucella melitensis antibody high throughput testing technology, and its core is by the cloth of purification
Shandong Salmonella lipopolysaccharide (LPS) is coated with 96 hole elisa Plates Jing after quantitative, prepares positive serum with artificial immunity health sheep, with health
Nonimmune sheep serum makees negative serum, with the anti-sheep Fc section monoclonal antibodies of HRP labelling Mus, substrate solution, terminate liquid, sample diluting liquid,
The component such as the anti-sheep IgG diluents of HRP labelling Mus and 20 × cleaning mixture assembles, for detecting the light in sheep and goat serum
Slip Brucella antibody.
Technical scheme
1. a kind of Brucella melitensis indirect ELISA antibody assay kit, it is characterised in that the test kit is mainly contained：With
The brucella lipopolysaccharide of preparation prepares positive serum with artificial immunity health sheep as envelope antigen, non-to gather health
Immune sheep serum makees negative serum, with the anti-sheep IgG Fc antibody of HRP labelling Mus, substrate solution, terminate liquid, sample diluting liquid,
The component such as the anti-sheep IgG diluents of HRP labelling Mus and 20 × cleaning mixture assembles, for detecting the light in sheep and goat serum
Slip Brucella antibody.
2. Brucella melitensis indirect ELISA antibody assay kit of the present invention, it is characterised in that the system of positive serum
It is standby with scaling method to be：
With brucella rose bengal precipitation test, tube agglutination test and complement fixation test to clinical manifestation health
15 monthly ages, adult sheep was detected, screened brucellosis negative antibody sheep；Negative sheep to screening refers to virulent strain M28 with brucella
Inactivation antigen is with 5 × 1010CFU/ sheep cervical region subcutaneous inoculations, doubling dosage booster immunization 1 time after 3 months.Adopt within 2 weeks after booster immunization
Blood, it is 800IU/ml to determine agglutination titer with tube agglutination test.Venesection, separate serum, with sheep brucellosis negative serum with
Potency is diluted for the positive serum equal-volume of 800IU, and its potency is adjusted to 400IU/mL, with 0.45 μm of filter filtration sterilization,
As brucellosis positive serum in test kit.
3. Brucella melitensis indirect ELISA antibody assay kit of the present invention, it is characterised in that the anti-sheep of HRP labelling Mus
IgG Fc antibody is prepared by being named as anti-sheep IgG Fc section Monoclonal Antibody Cell 6F9 strains, and the cell strain is in 2016
On November 14, in delivers China Microbiological in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica
The common micro-organisms center preservation of culture presevation administration committee, deposit number is CGMCCNo.13282.
4. the application of Brucella melitensis indirect ELISA antibody assay kit of the present invention, it is characterised in that using should
Test kit can carry out high throughput testing to goat and Xinjiang Brucella ovis disease.
The specific embodiment of the invention
1. antigen prepares the identification with strain (brucella melitensis M28 strains, B.Melitensis M28).
(1) colonial morphology colony edge is neat, mellow and full, reveals drop-wise, skew ray irradiation, backlight observation micro-strip blue-opalescent.
(2) it is the coccobacilluss to dye form, single to be dispersed in, and does not form spore and pod membrane.Size is between 0.3～0.6 μm.Leather
Blue Albert'stain Albert is feminine gender, and Ke's Albert'stain Albert is redness.
(3) purely inspection is by existing《Chinese veterinary pharmacopoeia》(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 carries out, should be pure.
(4) specific assay
1) serological specificity makes antigen with culture, coagulation should occurs with smooth type brucella positive serum, with
Rough type serum occurs without coagulation.
2) with M28 genomic DNAs template, using following 4 primers, size can be amplified and is respectively 178bp and 733bp
Specific PCR band.
Feri：5 '-GCGCCGCGAAGAACTTATCAA-3 ' (sequence 1)
Reri:5 '-CGCCATGTTAGCGGCGGTGA-3 ' (sequence 2)
Fmelitensis:5 '-AAATCGCGTCCTTGCTGGTCTGA-3 ' (sequence 3)
RIS711:5 '-TGCCGATCACTTAAGGGCCTTCAT-3 ' (sequence 4)
PCR reaction systems：In the reaction system of 50 μ L, containing 5 μ 10 × Buffer of L, 8 μ L2.5mMdNTPs, mix primer
2 μ L of storing liquid, Taq enzyme 2U, 1 μ L (or picking colony is a little) of template DNA.
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% agarose gel, 2 specific PCR bands should occurs, greatly
It is little to be respectively 178bp and 733bp (see accompanying drawing 1).
2. the purification of envelope antigen LPS and quantitative
(1) bacteria suspension is prepared and for qualified B.Melitensis M28 strain secondary seeds to be inoculated in pancreasThe flat bottle of agar
In, after 37 DEG C are cultivated 48 hours, per bottle adds immersion more than the phage surface 10min of the normal saline 20ml containing 0.5% phenol,
Lower culture is washed, is collected in sterile glass vials.
(2) bacteria suspension for gathering is heated to 80 DEG C by bacterium solution inactivation and inactivation inspection, is maintained 120min, is treated which is naturally cold
But, after, 4 DEG C are stored in.Inactivation inspection should be carried out to inactivated bacterial liquid.Take inactivated bacterial liquid 0.1ml coating pancreasAgar or tryptone are big
Bean agar (TSA) flat board, 37 DEG C are cultivated 7 days, do not answer asepsis growth.
(3) inactivation inspection qualified bacteria suspension is centrifuged 20min with 10000g by slightly carrying for LPS, collects precipitation.Weigh and count
Thalline weight in wet base is calculated, thalline weight in wet base is pressed into 1:During 3 (W/W) ratio adds sterile purified water, after fully mixing, 66 DEG C are heated to, so
90% (V/V) phenol solution of 66 DEG C of preheatings is added afterwards.(66 DEG C) persistently stir 15min at this temperature, put room temperature naturally cold
But after, in 4 DEG C of 10000g centrifugation 15min.With the henna phenol phase of a long tube Xi Qi lower floors, by water mutually with No. 1 filter of Whatman
Paper is filtered and removes big bacterial chip.Phenol phase volume is measured with graduated cylinder, the methanol for being subsequently adding pre-cooling below -15 DEG C of 3 times of volumes (contains
The sodium acetate of 1% methanol saturation), 4 DEG C are incubated 2 hours, and 4 DEG C of 10000g are centrifuged 10min, abandon supernatant.
(4) protease digestion and phenol extraction are resuspended with 1/2 volume distilled water of raw water phase by precipitation, add a certain amount of egg
White enzyme K so as to which final concentration is up to 50 μ g/ml, and adjusts pH value to 8.0 with 1N NaOH, puts 56 DEG C of water-baths and digests 2 hours.Add etc.
After volume of phenol solution extracts 2 times, 4 DEG C of 10000g are centrifuged 10min.Collect supernatant to preserve in 4 DEG C, add eventually in supernatant
Concentration is 5% trichloroacetic acid, and after 15min is stirred at room temperature, 10000g centrifugation 15min discard precipitation, and supernatant is saturating with distilled water
Analysis overnight, changes liquid 2 times (at least 4000ml each time), collects bag filter content, this LPS for purifying.
(5) LPS purity testings will be weighed after the LPS lyophilizing of preparation, record LPS net weight M.Will be commercialization LPS standard substance dilute
It is interpreted into the difference dilution factor such as 1mg/ml, 100 μ g/ml, 10 μ g/ml, 1 μ g/ml, 100ng/ml, 10ng/ml；Basis is weighed simultaneously
As a result LPS is configured to the initial concentration of 1mg/ml, and is diluted to the concentration of 100 μ g/ml.Will be above-mentioned each dilution LPS molten
Liquid takes 100 μ l/ samples and adds in 96 hole elisa Plates, under 529nm wavelength reads absorbance.Set up each dilution factor of LPS standard substance
OD529 values, and the standard curve set up between concentration and absorbance.According to standard curve and brucella LPS absorbances,
Calculate the concentration of LPS.LPS purity is LPS concentration × unit volume/unit mass.
3. the preparation of negative control sera and positive control serum and inspection in test kit
(1) preparation of negative control sera
1) animal rose bengal precipitation test, tube agglutination test and complement fixation test be detected as brucella resist
Or so 15 negative monthly ages of body, sexually matured healthy sheep.
2) serum prepares jugular vein blood collection, separates serum, with 0.45 μm of filter filtration sterilization, the serum addition of collection
0.05%proclin300 anti-corrosions, aseptic subpackaged, after the assay was approved, -20 DEG C save backup.
(2) inspection of negative control sera
1) character is orange or faint yellow supernatant liquid.
2) steriling test is by existing《Chinese veterinary pharmacopoeia》Annex is tested, and answers asepsis growth.
3) specific assay Jing rose bengal precipitation tests, tube agglutination test and complement fixation test inspection should be the moon
Property.Using the negative control sera for preparing as serum to be checked, indirect ELISA detection is carried out, its OD450nm<0.1, and P%<
10%.
(3) preparation of positive control serum and inspection
(1) preparation of positive control serum
1) animal screening brucella rose bengal precipitation test, tube agglutination test and complement fixation test are to clinic
Or so 15 monthly ages of performance health, adult sheep was detected, screened brucellosis negative antibody sheep.
2) serum is prepared and for brucella to make bacteria suspension with reference to virulent strain M28 inactivation antigen, with 5 × 1010CFU/ sheep necks
Portion's subcutaneous inoculation, doubling dosage booster immunization 1 time after 3 months.Take a blood sample within 2 weeks after booster immunization.Determine solidifying with tube agglutination test
Collect potency, now potency is generally 1:800(800IU/ml).When potency is more than 800IU/ml, venesection separates serum.
According to the potency of practical measurement, its potency is diluted to into 400IU/mL with sheep brucellosis negative serum, is crossed with 0.45 μm of filter and filtered
Bacterium, adds Proclin 300 to final concentration of 0.05%, and -20 DEG C save backup, used as brucellosis positive serum in test kit.
(2) inspection of positive control serum
1. character is orange or faint yellow supernatant liquid.
2. steriling test is by existing《Chinese veterinary pharmacopoeia》Annex is tested, and answers asepsis growth.
3. titration determines the potency of positive control serum using tube agglutination test, should be 1:400.The sun that will be prepared
Property control serum carries out indirect ELISA detection, its OD450nm >=0.8 as serum to be checked.Positive control serum is negative
Control serum carries out 1:32 dilutions carry out indirect ELISA detection as serum to be checked, calculate P%, should be not less than 15%.
The preparation of the anti-sheep IgG Fc section monoclonal antibodies of 4.HRP labellings
(1) preparation of goat IgG Fc section
Pepsin (sigma company, article No. 77161-100G) hydrolysis sheep IgG antibody of the concentration for 10mg/ml.
After 37 DEG C are stirred overnight, Tris buffer terminating reactions are added.By SPA affinity columns, the IgG Fc sections that eluting has digested.
BCA test kits and spectrophotometric determination its protein content are used respectively.
(2) preparation of monoclonal antibody
1) sheep IgG Fc sections after purification are added into Freund adjuvant immune mouse, immunizing dose is 10ng/, immunity 3～4
Secondary rear extracting spleen cell is merged with myeloma cell.
2), after cell fusion, lowlenthal serum and sheep serum carbonate buffer solution are diluted to 10% coating respectively
Liquid, 100 μ l/ holes are coated with 96 hole elisa Plates, and above-mentioned antibody first makees 1:10 dilutions, after then making 2 times of gradient dilutions, 100 μ l/ holes add
Enter each hole as serum to be checked, carry out ELISA detections according to a conventional method.
3) cell strain for having sound response with goat and sheep serum, the cell strain are selected after 3～4 sub-clones
(this plant of cell delivers Beijing on November 14th, 2016 to be named as anti-sheep IgG Fc sections Monoclonal Antibody Cell 6F9 strains
No. 3 China Committee for Culture Collection of Microorganisms of Institute of Microorganism, Academia Sinica of institute of Chaoyang District North Star West Road 1 are common
Microorganism center CGMCC No.13282), identify its hypotype and chromosome number after amplification culture, it is qualified after by cell cryopreservation simultaneously
Prepare ascites (105Individual cell/only, lumbar injection).
5) mouse ascites for preparing carry out purification using Protein A affinity columns, and the antibody purification for obtaining is using peppery
Root peroxidase (HRP) is marked.Briefly step is 4 DEG C after the antibody mixing by horseradish peroxidase solution with purification
Dialyzed overnight, adds ammonium sulfate postprecipitation, the antibody after precipitation is dissolved in PBS solution and removes ammonium ion after dialysis, from
The heart takes the enzyme labelled antibody that supernatant is horseradish peroxidase-labeled.
6) enzyme labelled antibody of preparation is identified into its reactivity by ELISA method, adds 50% -20 DEG C of glycerol frozen.
5. the preparation and inspection of test kit each component
(1) preparation and inspection of antigen coated microplate
1) preparation of primordial covering plate is being coated with buffer (0.05M carbonate buffer solutions, pH9.6) by antigen diluent to 10 μ
G/ml, is added in ELISA Plate, and per 100 μ l of hole, 2～8 DEG C are coated with 16 hours.Washed 1 time with 1 × cleaning mixture, envelope is added per hole
Liquid (PBS of the 0.01M pH7.4 containing 3% gelatin) 200 μ l are closed, 2～8 DEG C are closed 24 hours.Washed 1 time with 1 × cleaning mixture,
Pat dry in absorbent paper, natural air drying, be vacuum-packed in aluminium foil bag.
2) the test package bag closing of primordial covering plate is good, bottom hole clean transparent, foreign.
(2) 20 × wash the preparation and inspection of liquid
1) prepare and weigh Na2HPO4·12H2O 5.0g, NaH2PO4·2H2O 70.0g, NaCl 170.0g, Tween-20
10.0ml, plus deionized water is to 800ml, adjusts pH value to 7.2～7.6, is settled to 1000ml.Subpackage is standby.
2) check
1. character colourless transparent liquid, it is possible that white crystals during cryopreservation, 37 DEG C of water-baths dissolve can which.
2. pH value should be 7.2～7.6.
(3) preparation and inspection of sample diluting liquid
1) prepare and weigh Na2HPO4·12H2O 0.25g, NaH2PO4·2H2O 3.5g, NaCl 8.5g, Tween-20
0.5ml, sucrose 5g, plus deionized water is to 800ml, adjusts pH value 7.2～7.6, is settled to 1000ml.Add proclin 300 molten
Liquid make its final concentration of 0.05%, it is after filtration sterilization, aseptic subpackaged.
2) check
1. character is colourless transparent liquid.
2. steriling test is by existing《Chinese veterinary pharmacopoeia》Annex is tested, and answers asepsis growth.
3. pH value should be 7.2～7.6.
(4) preparation and inspection of the anti-sheep Fc monoclonal antibodies of HRP- Mus
1) HRP- rabbit-antis sheep Fc section monoclonal antibodies (6F9) prepared by this laboratory is prepared, is protected with ELIAS secondary antibody
Liquid makees 1:100 dilution, add 300 solution of proclin make its final concentration of 0.05%, it is aseptic subpackaged.
2) check
1. the orange-yellow clear liquid of character.
2. steriling test is pressed《Chinese veterinary pharmacopoeia》Annex is tested, and answers asepsis growth.
HRP- rabbit-antis-sheep Fc the antibody-solutions prepared are made 1 with diluent by 3. titration:200 dilutions, as reagent
The ELIAS secondary antibody of box, with test kit to positive control serum, negative control sera, Quality Control positive serum, Quality Control negative serum
Detected.Testing result should meet：Positive control serum OD450nm >=0.8；Negative control sera OD450nm ＜ 0.1 and
P% ＜ 15%；Quality Control positive serum P1:P% >=60%；P2:P% >=40%；P3:P% >=15%；Quality Control negative serum (N1,
N2, N3, N4, N5) the equal ＜ of P% values 15%.
(5) preparation and inspection of the anti-sheep Fc sections monoclonal antibody diluent of HRP-HRP- Mus
1) prepare and weigh Na2HPO4·12H2O 0.25g, NaH2PO4·2H2O 3.5g, NaCl 8.5g, Tween-20
0.5ml, plus deionized water is to 800ml, adds horse serum 5ml, adjusts pH value 7.2～7.6, is settled to 1000ml.Add ProClin
300 solution make its final concentration of 0.05%, it is aseptic subpackaged.
2) check
1. the faint yellow clear liquid of character.
2. steriling test is pressed《Chinese veterinary pharmacopoeia》Annex is tested, and answers asepsis growth.
(6) source of substrate nitrite ion and inspection
1) substrate solution originated for this test kit is divided into substrate solution A (TMB purchased from KPL companies of the U.S.
Peroxidase Substrate) and substrate solution B (Peroxidase Substrate B).
2) character substrate solution A and B is checked to be brown bottle packaging, colourless transparent liquid.
(7) preparation and inspection of terminate liquid
1) 1M HCL, quantitative separating are prepared.
2) check character colourless transparent liquid.
6. the specificity and sensitivity of the reference serum kits for evaluation of enough samples are used
Basic step is as follows：(1) by the serum sample of 273 parts of field sheep of this collection of inventor between 2009～2013 years,
Rose bengal precipitation test and tube agglutination test is used to detect respectively, by the serum sample of two methods equal test positive and
The sample for being detected as feminine gender confirms that three kinds of methods of selection are all the serum of the positive as positive reference blood again with complement fixation test
Clearly, three kinds of methods are all the serum of feminine gender as negative reference serum；(2) it is defined as the brucellosis positive with 110 parts filtered out
Serum (wherein 43 parts of goat, 67 parts of sheep) and 121 parts of brucellosis negative serums are used as serum to be checked, the reliability of kits for evaluation
Property.
Description of the drawings
1 in the PCR identification qualification figure figures of Fig. 1 Brucella melitensis M28:Escherichia coli negative control；2:DNA Marker；3:
The PCR amplifications of M28 bacterial strains.
Fig. 2 difference coating buffers are coated with the comparison of effect.
The effect of Fig. 3 difference confining liquids compares.
The dilution effect of the anti-sheep Fc sections monoclonal antibody diluent of Fig. 4 difference HRP- Mus compares.
Biomaterial resource information of the present invention
Microorganism involved in the present invention is：Brucella melitensis (Brucella melitensis) M28 strains
(CVCC70003), (China Veterinery Drug Inspection Office, Chinese veterinary are asked for an interview micro- from National Veterinary Culture Collection
Biological inoculum preservation administrative center is write, Chinese veterinary's strain catalogue (second edition), Scientia Agricultura Sinica technology publishing house, and 2002
Year, p32), it is brucella melitensis 1 type of biology, is brucella disease live-vaccine effect evaluation inspection poison Reference Strains by force, by north
Veterinary microorganism culture presevation administrative center of China of China Veterinery Drug Inspection Office mirror of Jing Shi Haidian District Zhong Guan-cun South Street 8
Fixed, keeping and supply.Anti- sheep IgG Fc sections Monoclonal Antibody Cell 6F9 strains involved in the present invention, the cell strain is in 2016
On November 14, in delivers China Microbiological in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica
The common micro-organisms center preservation of culture presevation administration committee, deposit number is CGMCCNo.13282.
Advantages of the present invention
The present invention relates to develop a kind of Brucella melitensis indirect ELISA antibody assay kit.Involved in the present invention should
Test kit is compensate for both at home and abroad without the brucellosis indirect ELISA detection method for being specifically designed for sheep and goat
The present invention has successfully screened one plant of monoclonal antibody for sheep Fc sections, the monoclonal antibody and sheep and goat serum
It is respectively provided with good reactivity.The present invention is used as enzyme labelled antibody.This test kit is allow while being used for goat and silk floss
The diagnosis of Brucella melitensis disease；The present invention is by the improvement and optimization to main agents formula, the reaction of effective control test kit
Background, shortens the time needed for high throughput testing；The present invention is provided for China and in the world the brucellosis diagnosis of sheep
New and effective detection method.
Embodiment
Following examples are to further illustrate the present invention, not limiting the invention.
Embodiment 1
--- reagent constituents optimizing research
1. the selection of coating buffer
Carbonate buffer solution (pH 9.6), Tris-HCL buffer (pH 8.5), phosphate buffer (pH are selected respectively
7.2) and citrate buffer solution (pH 5.0) as coating buffer, coated antigen concentration be equal 10 μ g/ml, Jing confining liquids closing
Afterwards, OD values soprano is reacted as the best coating buffer of Antigen adsorption effect with positive serum.As a result by 4 kinds of coating buffers
Relatively, show that the carbonate buffer solution of pH9.6 is best (result is shown in accompanying drawing 2) as the coating effect of coating buffer.
2. the selection of confining liquid
The PBS containing 3% gelatin, the PBS containing 10% defatted milk powder, the PBS containing 10% horse serum is respectively adopted and contains 3%
The PBS of BSA is closed as sealer, sealing condition be 2～8 DEG C 24 hours.Compare positive control serum and negative control
The ratio of the OD values of serum, it is determined that optimal confining liquid composition.When as a result using the PBS containing 3% gelatin as sealer, positive blood
Clear OD values and the equal highest of P/N values, therefore, select the PBS containing 3% gelatin as the confining liquid of this method, as a result see accompanying drawing 3.
The selection of the anti-sheep Fc sections monoclonal antibody diluent of 3.HRP- Mus
PBST, the PBST containing 10% defatted milk powder, the PBST containing 5% horse serum are respectively compared as the anti-sheep of HRP labelling Mus
The effect of Fc section monoclonal antibody diluents.Respectively the anti-sheep Fc sections monoclonal antibody of HRP- Mus is carried out with above-mentioned diluent dilute
Release, compare OD values and P/N values, also highest diluent is anti-as HRP- Mus for therefrom selection positive serum OD values height, and P/N values
Sheep Fc section monoclonal antibody diluents.When as a result using the PBST containing 5% horse serum as sample diluting liquid, the OD values of positive serum
It is higher, and P/N value highests, therefore, using the PBST containing 5% horse serum as the anti-sheep Fc sections monoclonal antibody diluent of HRP- Mus,
As a result see accompanying drawing 4.
Embodiment 2
--- the preparation of Brucella melitensis indirect ELISA antibody assay kit positive control serum
With brucella rose bengal precipitation test, tube agglutination test and complement fixation test to clinical manifestation health
Or so 15 monthly ages, adult sheep was detected, screened brucellosis negative antibody sheep.To the negative sheep strong poison of brucella reference for screening
Strain M28 inactivation antigens make bacteria suspension, with 5 × 1010CFU/ sheep cervical region subcutaneous inoculations, doubling dosage booster immunization 1 after 3 months
It is secondary.Take a blood sample within 2 weeks after booster immunization.Agglutination titer is determined with tube agglutination test, now potency is generally 1:800(800IU/
ml).When potency is more than 800IU/ml, venesection separates serum.According to the potency of practical measurement, with sheep brucellosis feminine gender blood
Its potency is diluted to into 400IU/mL clearly, with 0.45 μm of filter filtration sterilization, proclin 300 is added to final concentration of
0.05%, -20 DEG C save backup, used as brucellosis positive serum in test kit.
1. brucellosis feminine gender sheep screening
Clinical trial sheep is examined using rose bengal precipitation test, tube agglutination test and complement fixation test
Survey, select three kinds of methods the sheep of feminine gender to be detected as the candidate sheep for preparing positive serum.As a result volume is have selected in testing
Number for 039,073,104 three parts of Virus monitories, the results are shown in Table 1.3 parts of blood serum samples to be checked are with brave red antigen without agglutinating particle
Occur, in uniform muddiness, be judged to brucellosis negative antibody., with brave red antigen in obvious agglutinating particle, liquid is several for control positive serum
It is fully transparent.3 parts of blood serum samples to be checked, 4 different dilution factors (1 in tube agglutination test:25、1:50、1:100、1:200)
As a result it is feminine gender.In complement test, equal 100% haemolysis of 3 parts of blood serum samples to be checked, is judged to brucellosis negative antibody.
Testing result of the 1 three kinds of distinct methods of table to serum to be checked
Note：(1) rose bengal precipitation test result explanation：
++++：There is big coagulation piece or granule, liquid is fully transparent；+++：There is obvious agglutinating particle, liquid is almost
It is fully transparent；++：There is obvious agglutinating particle, liquid is slightly transparent；+：Coagulation, opaque can slightly be seen；—：Without coagulation,
Liquid is uniformly muddy.
(2) tube agglutination test result explanation：4+：The complete coagulation of thalline and precipitation, liquid 100% are limpid；3+：Thalline is several
Complete coagulation and precipitation, liquid 75% are limpid；2+：There are significant coagulation and precipitation, liquid 50% is limpid；1+：Have and clearly may be used
The coagulation seen and precipitation, liquid 25% are limpid；—：Without coagulation and precipitation, liquid bacterium solution is muddy.
(3) Evaluation of Complement Fixation Test explanation：P：It is positive；N：It is negative.
2. immune
Immune general need to be carried out twice.Just exempt from：With 2ml brucella M28 inactivation antigens (5 × 1010CFU/ml) cervical region
The negative sheep of subcutaneous inoculation brucellosis.Booster immunization：After just exempting from 3 months, the subcutaneous booster immunization of doubling dosage cervical region 1 time.Strengthen
Take a blood sample within 2 weeks after immunity, agglutination titer is determined with tube agglutination test and should be not less than 1:800.If potency does not reach standard, continue to add
Strong immunity 1～2 time, tries blood again after 15 days, until potency is qualified.After the present invention 039,073 and No. 104 cattle carry out secondary immunity
The potency determined using tube agglutination test is respectively 1:900、1:800、1:1100.
3. prepared by serum
Two exempt from 14 after take a blood sample, determine tube agglutination potency, further with negative control sera by detached positive serum
In proportion (about 3:1) expected potency is diluted to for 1:400, add ProClin 300 (SUPELCO companies) extremely final concentration of
0.05%, it is aseptic subpackaged, as positive control serum to be checked.
4. check
The positive control serum that preparation is completed is tested by following project：
(1) character observation serum product color and luster is yellowish or blush clear liquid.
(2) steriling test is by existing《Chinese veterinary pharmacopoeia》Annex is tested, and is asepsis growth.
(3) serum is made 1 by titration:140,1:150,1:160,1:170,1:180 dilutions carry out tube agglutination test,
Determine its potency.The serum 1 that will be prepared:32 dilutions are entered with Brucella melitensis indirect ELISA testing kit as serum to be checked
Row detection, calculates P%, is not less than 15%.
Embodiment 3
--- test kit sensitivity testss
1. the sensitivity test of the Brucella melitensis antibody positive control serum of pair gradient dilution
Brucella melitensis antibody positive control serum is carried out after 2 times of gradient dilutions, test kit is made by oneself with 3 batches of laboratorys and is entered
Row detection, determines sensitivity of the test kit to the positive control serum of gradient dilution, the results are shown in Table 2.From between 2,3 batches, table
The result for connecing ELISA kit detection gradient dilution positive control serum is consistent, and serum titer is detected to 1:32 times of (P% values
>=15%).
Sensitivity technique of the table 2 to the positive control serum of gradient dilution
Note：The criterion of Brucella melitensis indirect ELISA antibody assay kit is：During sample p% value >=15%, sheep
Brucella abortus antibody positive (is designated as "+")；Sample p% values<When 15%, it is Brucella melitensis negative antibody (being designated as "-").
2. the sensitivity testss of positive sample known to pair
Using 3 batches of Brucella melitensis indirect ELISA antibody assay kits to 110 parts of brucellosis positive serums (wherein goat
43 parts, 67 parts of sheep), 121 parts of brucellosis negative serums are detected, as a result with confirmed 110 portions of sheep brucellosis positive blood final proof
This coincidence rate is 97.3% (107/110), and the coincidence rate with confirmed 121 parts of negative samples is 96.7% (117/121),
Thereby determine that test kit is 97.3% to the sensitivity of field sample, specificity is 96.7%, it is shown that test kit diagnostic result
Reliability.
Note
IELISA method operation instructions
1 usage
1.1 sample treatment take animal's whole blood, after blood coagulation, 10min are centrifuged with 4000r/min, collect supernatant, serum
Should be limpid, without haemolysis.
The preparation of 1.2 1 × cleaning mixture is recovered 20 × cleaning mixture to room temperature (20～25 DEG C), and is shaken, made using front
(5～10min can be heated in 37 DEG C of water-baths) is dissolved in crystallization, and then deionized water makees 1:20 dilutions, fully mix.
1.3 serum to be checked and control serum are diluted in serum to be checked, negative control sera and sun in serum-dilution plate
Property control serum with sample diluting liquid make 1:50 dilutions.
1.4 operating procedure
1.4.1 sample-adding takes antigen coated microplate (how many according to sample, removable gradation use), with 1 × cleaning mixture, 300 μ l/
Hole board-washing 1 time, discards cleaning mixture.The serum to be checked, positive control serum and the negative control sera that have diluted are added separately to
In elisa plate, 100 μ l/ holes, wherein positive control serum and negative control sera respectively add 2 holes (hole 1, hole 2).After sample-adding terminates,
37 DEG C of effect 30min.Sptting plate is taken out, reactant liquor is discarded, 300 μ 1 × cleaning mixture of l is added per hole, is washed 3 times, is got rid of for last 1 time
Do or pat dry.
1.4.2 HRP- Mus anti-sheep Fc sections monoclonal antibody is made 1 with diluent by enzyme-added labeling antibody:After 200 dilutions, add per hole
Enter 100 μ l, 37 DEG C of effect 30min.Wash 3 times by the method in 1.4.1, dry.
Substrate solution A and B are pressed 1 with termination by 1.4.3 colour developing：After 1 (V/V) mixing, ELISA Sptting plates are added to immediately
In, 100 μ l/ holes, after room temperature lucifuge colour developing 15min, add 50 μ l terminate liquid terminating reactions per hole.
After 2 result judgement reaction terminatings, in 15min, OD is determined with microplate reader450nmValue.
2.1 computational methods
2.2 experiment establishment conditions
As the average OD of positive control450nm>=0.8, the average OD of negative control450nm<0.1 and P%<When 15%, experiment is set up.
2.3 result judgement
When P% value >=15% of serum sample, it is Brucella melitensis disease antibody positive；
When the P% values of serum sample<When 15%, it is Brucella melitensis disease negative antibody.
Sequence table
<110>China Veterinery Drug Inspection Office
<120>Brucella melitensis indirect ELISA antibody assay kit
<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 melitensis
<400> 3
AAATCGCGTC CTTGCTGGTC TGA 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）
Claims (4)
1. a kind of Brucella melitensis indirect ELISA antibody assay kit, it is characterised in that the test kit is mainly contained：To prepare
Brucella lipopolysaccharide as envelope antigen, positive serum is prepared with artificial immunity health sheep, it is nonimmune to gather health
Sheep serum makees negative serum, with the anti-sheep IgG Fc antibody of HRP labelling Mus, substrate solution, terminate liquid, sample diluting liquid, HRP marks
The component such as the anti-sheep IgG diluents of note Mus and 20 × cleaning mixture assembles, for detecting the smooth type in sheep and goat serum
Brucella antibody.
2. Brucella melitensis indirect ELISA antibody assay kit described in claim 1, it is characterised in that the preparation of positive serum
With scaling method it is：
With 15 months of brucella rose bengal precipitation test, tube agglutination test and complement fixation test to clinical manifestation health
Age, adult sheep was detected, screened brucellosis negative antibody sheep；Negative sheep to screening is inactivated with reference to virulent strain M28 with brucella
Antigen is with 5 × 1010CFU/ sheep cervical region subcutaneous inoculations, doubling dosage booster immunization 1 time after 3 months.Take a blood sample within 2 weeks after booster immunization,
It is 800IU/ml that agglutination titer is determined with tube agglutination test.Venesection, separates serum, with sheep brucellosis negative serum and potency
Positive serum equal-volume for 800IU dilutes, and its potency is adjusted to 400IU/mL, with 0.45 μm of filter filtration sterilization, as
Brucellosis positive serum in test kit.
3. Brucella melitensis indirect ELISA antibody assay kit described in claim 1, it is characterised in that the anti-sheep of HRP labelling Mus
IgG Fc antibody is prepared by being named as anti-sheep IgG Fc section Monoclonal Antibody Cell 6F9 strains, and the cell strain is in 2016
On November 14, in delivers China Microbiological in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica
The common micro-organisms center preservation of culture presevation administration committee, deposit number is CGMCCNo.13282.
4. the application of Brucella melitensis indirect ELISA antibody assay kit described in claim 1, it is characterised in that using the examination
Agent box can carry out high throughput testing to goat and Xinjiang Brucella ovis disease.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109490541A (en) * | 2018-09-28 | 2019-03-19 | 河北科技师范学院 | A kind of method for building up for the indirect ELISA detecting Brucella abortus HSP70 |
| CN110028594A (en) * | 2019-03-07 | 2019-07-19 | 中国农业科学院特产研究所 | Process for screening and identifying and the application of the preparation method and its monoclonal antibody of B.melitensis 16M LPS |
| 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 |
| CN115267203A (en) * | 2022-08-02 | 2022-11-01 | 中国农业科学院北京畜牧兽医研究所 | Method for detecting content of specific IgG and IgM of brucellosis in sheep and application |
| CN117051132A (en) * | 2023-10-11 | 2023-11-14 | 中国农业科学院北京畜牧兽医研究所 | Sheep SNP molecular marker and application thereof in sheep brucellosis resistance character detection |
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| CN105067812A (en) * | 2015-08-06 | 2015-11-18 | 中国兽医药品监察所 | Bovine brucella indirect ELISA antibody detection kit |
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| CN105067812A (en) * | 2015-08-06 | 2015-11-18 | 中国兽医药品监察所 | Bovine brucella indirect ELISA antibody detection kit |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109490541A (en) * | 2018-09-28 | 2019-03-19 | 河北科技师范学院 | A kind of method for building up for the indirect ELISA detecting Brucella abortus HSP70 |
| CN110028594A (en) * | 2019-03-07 | 2019-07-19 | 中国农业科学院特产研究所 | Process for screening and identifying and the application of the preparation method and its monoclonal antibody of B.melitensis 16M LPS |
| 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 |
| CN115267203A (en) * | 2022-08-02 | 2022-11-01 | 中国农业科学院北京畜牧兽医研究所 | Method for detecting content of specific IgG and IgM of brucellosis in sheep and application |
| CN117051132A (en) * | 2023-10-11 | 2023-11-14 | 中国农业科学院北京畜牧兽医研究所 | Sheep SNP molecular marker and application thereof in sheep brucellosis resistance character detection |
| CN117051132B (en) * | 2023-10-11 | 2024-01-30 | 中国农业科学院北京畜牧兽医研究所 | Sheep SNP molecular marker and application thereof in sheep brucellosis resistance character detection |
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