CN104749362A - Deer brucellosis colloidal gold antibody detection test paper strip and preparation method thereof - Google Patents
Deer brucellosis colloidal gold antibody detection test paper strip and preparation method thereof Download PDFInfo
- Publication number
- CN104749362A CN104749362A CN201510100964.8A CN201510100964A CN104749362A CN 104749362 A CN104749362 A CN 104749362A CN 201510100964 A CN201510100964 A CN 201510100964A CN 104749362 A CN104749362 A CN 104749362A
- Authority
- CN
- China
- Prior art keywords
- deer
- antibody
- brucellosis
- colloidal gold
- pad
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
-
- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/23—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Brucella (G)
Abstract
The invention provides a deer brucellosis colloidal gold antibody detection test paper strip and a preparation method thereof, relates to the field of detection of pathogens of people and livestock zoonotic diseases, and aims at solving the problem that an existing detection method cannot rapidly detect deer brucellosis. The deer brucellosis colloidal gold antibody detection test paper strip comprises a plastic card, a PVC (Polyvinyl Chloride) bottom plate arranged in the plastic card, and a sample cushion, a gold label binding cushion, an NC membrane with a detection line and a quality control line, and paper absorption paper, which are sequentially stuck on the PVC bottom plate; an antigen and a second antibody are spotted on the detection line and the quality control line of the NC membrane respectively; and the antigen is 1.50mg/mL recombinant brucella BCSP31 protein and the second antibody is 0.75mg/mL rabbit anti-deer IgG; and after colloidal gold is marked with an antibody protein (rabbit anti-deer IgG), an obtained purified gold label antibody solution is dripped onto the binding cushion to form the gold label binding cushion. The deer brucellosis colloidal gold antibody detection test paper strip can be used for rapidly screening the brucellosis antibody in deer blood serum or deer whole blood, and rapidly diagnosing the deer brucellosis, and is convenient for field operation of farm technicists and veterinarians.
Description
Technical field
The present invention relates to the detection technique field of infectious diseases common to human beings and animals pathogen, be specifically related to a kind of deer brucellosis colloidal gold antibody test strip and preparation method thereof.
Background technology
Brucella surface protein 31 (Brucella cellsurface protein 31ku, BCSP31) gene was cloned order-checking and vivoexpression in 1988, it encodes size is that 31ku has immunogenic soluble cell surface protein, except sheep epididymis brucella, in other 6 kinds of brucella, the expression of BCSP31 albumen all can be detected.Brucella BCSP31 gene does not belong to homology in the brucella bacterial strain in source together in 8 strains and reaches 99.3%, is brucella detection, has well-conserved.
At present, existing scholar utilizes the method for eukaryotic expression to have expressed BCSP31 albumen, and utilizes its eukaryotic expression product immune mouse, studies the ability of its anti-Infected with Brucella in Mice Body; Also there is scholar to utilize prokaryotic expression carrier pGEX4p-1 to have expressed BCSP31 albumen, and have developed the monoclonal antibody of BCSP31 albumen.
Collaurum is a kind of conventional labelling technique, is a kind of novel immunolabelling technique being applied to antigen-antibody using collaurum as tracer label thing, has the advantage of its uniqueness.In recent years widely use in various biological study.It is nearly all used to mark at the immunoblot assay of Clinical practice.All may example use in biochip at streaming, Electronic Speculum, immunity, molecular biology simultaneously.
At present, the development adopting brucella BCSP31 albumen to be used for deer brucellosis colloidal gold antibody test strip as antigen have not been reported.
Summary of the invention
The object of this invention is to provide a kind of deer brucellosis colloidal gold antibody test strip and preparation method thereof, for Brucella antibody in rapid screening deer serum or deer whole blood.
The technical scheme that the present invention adopts for technical solution problem is as follows:
A kind of deer brucellosis colloidal gold antibody test strip provided by the invention, comprise plastic clip, the PVC base plate be contained in plastic clip, the successively sticky sample pad be posted on PVC base plate, gold-marking binding pad, NC film, thieving paper with detection line and nature controlling line, on the detection line of described NC film and nature controlling line, point sample has antigen and two to resist respectively; Described antigen is the restructuring brucella BCSP31 albumen of 1.50mg/mL, and described two resist the anti-deer IgG of rabbit for 0.75mg/mL.
Further, described gold-marking binding pad is prepared by the following method:
(1) with the borate buffer solution of pH9.0, the antibody protein of initial concentration 2.45mg/mL is diluted to final concentration 1.0mg/mL, with the K of 0.1M
2cO
3solution regulates the pH of collaurum to be 9.0, and collaurum particle size is 30nm; Add the ratio of 40 μ g antibody proteins with every 1mL collaurum, dropwise add antibody protein, after stirring, static 30min under normal temperature; Adding 10%BSA to final concentration is 1%, continues to stir 15min; Adding 3%PEG2000 to final concentration is 0.1%, continues to stir 15min, obtains golden labeling antibody solution;
(2) golden labeling antibody solution step (1) obtained is at 4 DEG C, and the centrifugal 20min of 1500rpm, removes precipitation, obtain supernatant; By supernatant at 4 DEG C, the centrifugal 30min of 8000rpm, abandons supernatant, gets the kermesinus precipitation of flowing, is resuspended in the storage liquid of supernatant initial volume 1/10, obtains the golden labeling antibody solution of purifying, 4 DEG C of preservations;
(3) immersed by pad in the PBS damping fluid of 0.01M, take out after 10min, room temperature is dried, 4 DEG C of preservations; The golden labeling antibody solution of purifying step (2) obtained is added drop-wise on above-mentioned pad, until pad is saturated, dry 1h at 37 DEG C, obtains gold-marking binding pad.
Further, in step (1), described antibody protein is the anti-deer IgG of rabbit.
Further described antigen and the two point sample consumptions resisted are 2 μ L/1cm.
Present invention also offers the preparation method of above-mentioned deer brucellosis colloidal gold antibody test strip, the method comprises the following steps:
(1) with the borate buffer solution of pH9.0, the antibody protein of initial concentration 2.45mg/mL is diluted to final concentration 1.0mg/mL, with the K of 0.1M
2cO
3solution regulates the pH of collaurum to be 9.0, and collaurum particle size is 30nm; Add the ratio of 40 μ g antibody proteins with every 1mL collaurum, dropwise add antibody protein, after stirring, static 30min under normal temperature; Adding 10%BSA to final concentration is 1%, continues to stir 15min; Adding 3%PEG2000 to final concentration is 0.1%, continues to stir 15min, obtains golden labeling antibody solution;
(2) golden labeling antibody solution step (1) obtained is at 4 DEG C, and the centrifugal 20min of 1500rpm, removes precipitation, obtain supernatant; By supernatant at 4 DEG C, the centrifugal 30min of 8000rpm, abandons supernatant, gets the kermesinus precipitation of flowing, is resuspended in the storage liquid of supernatant initial volume 1/10, obtains the golden labeling antibody solution of purifying, 4 DEG C of preservations;
(3) immersed by pad in the PBS damping fluid of 0.01M, take out after 10min, room temperature is dried, 4 DEG C of preservations; The golden labeling antibody solution of purifying step (2) obtained is added drop-wise on above-mentioned pad, until pad is saturated, dry 1h at 37 DEG C, obtains gold-marking binding pad;
(4) with the restructuring brucella BCSP31 albumen after 1.50mg/mL purifying for antigen, be two to resist with the anti-deer IgG of 0.75mg/mL rabbit, with 2 μ L/1cm, respectively point samples are on the detection line and nature controlling line of NC film, and room temperature fixes 2h; NC film after point sample is immersed in the PBS damping fluid of 0.01M, after closing 30min at 37 DEG C, then uses the PBS damping fluid rinsing NC film surface of 0.01M, dry at 37 DEG C, obtain the NC film with restructuring brucella BCSP31 albumen and the anti-deer IgG of rabbit;
(5) the NC film with restructuring brucella BCSP31 albumen and the anti-deer IgG of rabbit step (4) obtained is pasted on PVC base plate, and gold-marking binding pad step (3) obtained is sticky is posted on NC film left end, superposition 2mm; Gold-marking binding pad left end is posted on, superposition 2mm by sticky for sample pad; NC film right-hand member is posted on, superposition 2mm by sticky for thieving paper; Namely colloidal gold colloidal gold detection test paper strip is obtained after fixing with plastic clip.
Further, in step (1), described antibody protein is the anti-deer IgG of rabbit.
Further, in step (2), described storage liquid is the PBS damping fluid containing 1%BSA and 0.05%PEG2000, and concentration is 0.01M, pH is 8.5 ~ 9.0.
Further, in step (3), containing 5% sucrose and 0.05% Tween-20 in described PBS damping fluid.
Further, in step (4), containing 1%BSA in described PBS damping fluid.
Further, described gold-marking binding pad is glass film, and model is GL0194, length × wide=6mm × 5mm.
Further, the model of described NC film is Vivid170, length × wide=25mm × 5mm.
Further, the length × wide=60mm × 5mm of described PVC base plate.
Further, described sample pad is glass film, and model is GL0194, length × wide=20mm × 5mm.
Further, the model of described thieving paper is H5076, length × wide=15mm × 5mm.
The invention has the beneficial effects as follows: deer brucellosis colloidal gold antibody test strip of the present invention is made up of sample pad, gold-marking binding pad, NC film, thieving paper, PVC base plate and plastic clip, sample pad, gold-marking binding pad, NC film and thieving paper are fixed on successively on PVC base plate, then integral installation is in plastic clip.Gold-marking binding pad is formed by being added drop-wise on pad by the golden labeling antibody solution of the purifying obtained after colloidal gold labeled monoclonal antibody albumen (rabbit anti-deer IgG); With brucella BCSP31 albumen for antigen, with the anti-deer IgG of rabbit be two resist, the anti-deer IgG of rabbit of to be the brucella BCSP31 albumen of 1.50mg/mL and concentration by concentration be 0.75mg/mL respectively with 2 μ L/1cm point samples on NC film, be sprayed on detection line (T line) by brucella BCSP31 albumen, anti-for rabbit deer IgG be sprayed on nature controlling line (C line).
Deer brucellosis colloidal gold antibody test strip of the present invention realizes based on indirect colloidal gold immunochromatographimethod technology, during detection, Brucella antibody in sample is combined with the antibody protein of collaurum bag quilt and forms antibody complex, and the other end of NC film is flowed to along test paper, when flowing to the T district on NC film when this antibody complex, be fixed on specific antigen on film and brucella BCSP31 albumen is caught this compound and is aggregated into a visible detection line (T line) gradually, nature controlling line (C line) occurs then showing that immunochromatography occurs, namely test paper is effective, then showing in sample containing Brucella antibody appears in detection line (T line).
Deer brucellosis colloidal gold antibody test strip of the present invention is used for Brucella antibody in rapid screening deer serum or deer whole blood, quick diagnosis is made to deer brucellosis, be convenient to plant technician and animal doctor personnel on site operation, if naked-eye observation all shows dark bands to detection line (T line) and nature controlling line (C line), explanation is brucella animals showing positive, if only have C line to show dark bands, explanation is brucella negative animal, if detection line (T line) and nature controlling line (C line) all occur without obvious dark bands, illustrate that test strips lost efficacy or operation failure.Compare existing rose bengal precipitation test and indirect ELISA method detection deer brucellosis, the positive rate that deer brucellosis colloidal gold antibody test strip of the present invention detects is higher than the positive rate 5.48% higher than rose bengal precipitation test, positive coincidence rate is 100%, higher than the positive coincidence rate 91% of rose bengal precipitation test, simultaneously also higher than the positive coincidence rate 95% of indirect ELISA method.
Accompanying drawing explanation
Fig. 1 is the pcr amplification result figure of brucella BCSP31 gene, in figure: M5 is the genomic DNA of brucella melitensis vaccine strain M5 (B.melitensis M5), 544A is the genomic DNA of B. abortus reference culture 544A (B.abortus 544A), 16 is the genomic DNA of brucella melitensis reference culture 16M (B.melitensis 16M), and M is DNA marker.
Fig. 2 is the abduction delivering result figure of SUMO-BCSP31 recombinant plasmid in e. coli bl21, in figure: M is albumen Marker, 1,2, the 3 and 4 restructuring BCSP31 albumen being abduction delivering.
Fig. 3 is the expression of results figure of SUMO-BCSP31 recombinant protein in cell conditioned medium of induction, in figure: 1 and 2 are cell conditioned medium product, and 3 and 4 are cell precipitation product.
Fig. 4 is the results of immunoblot analysis figure of brucella BCSP31 albumen and HIS label monoclonal antibody.
Fig. 5 be recombinant protein SUMO-BCSP31 through ni-sepharose purification result figure, in figure: M is albumen Marker, 1,2 and 3 are nickel post eluted product, 4 be nickel post combine after resultant product.
Fig. 6 is the results of immunoblot analysis figure of brucella BCSP31 albumen and Bovine brucellosis positive serum, in figure: M is albumen Marker, and 1 is the anti-ox IgG of goat, and 2 is Bovine brucellosis positive serum.
Fig. 7 is mice serum antibody titer testing result figure.
Fig. 8 is the results of immunoblot analysis figure of brucella BCSP31 albumen and immune serum, in figure: M is albumen Marker, and 1 is brucella BCSP31 solubility expression of protein product, and 2 is brucella BCSP31 protein purification products.
Fig. 9 is mice serum hypotype IgG1 and IgG2a ELISA testing result figure.
Figure 10 is the ELISA testing result figure of mice serum cell factor IFN-γ.
The ELISA testing result figure that Figure 11 is mice serum cell factor IL-2.
Figure 12 is the result figure adopting deer brucellosis colloidal gold antibody test strip of the present invention to detect testing sample.In Figure 12 a, testing sample is PBS damping fluid; In Figure 12 b, testing sample is negative sample; In Figure 12 c, testing sample is positive.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail.
Experiment material: brucella melitensis reference culture 16M (B.melitensis 16M), brucella melitensis vaccine strain M5 (B.melitensis M5) and B. abortus reference culture 544A (B.abortus 544A) are all purchased from China Veterinary Drugs Supervisory Inst.; IPTG available from Sigma, article No. is 16758; BSA (bovine serum albumin(BSA)) is purchased from sigma company; Blunt carrier and T4 ligase are all purchased from NEB company; SUMO carrier is purchased from Invitrogen company; Bacillus coli DH 5 alpha competent cell and e. coli bl21, purchased from Beijing Quan Shi King Company, are kept in-80 DEG C of refrigerators; Goat anti-ox IgG, the anti-deer IgG of rabbit, collaurum are all purchased from Shanghai outstanding person one.
The expression and purification of embodiment 1 brucella BCSP31 albumen
(1) cloning and sequencing of brucella BCSP31 gene
1. according to the B. abortus BCSP31 protein gene sequence design primer that GenBank announces:
Upstream primer P1:5 '-TGACCTGCTAGTAGGTATGAAATTCGGAAGCA-3 ',
Downstream primer P2:5 '-TGTCTAGATTATTTCAGCACGCCCGCT-3 ',
Above-mentioned upstream and downstream primer synthesizes by gene portion of Invitrogen Bioisystech Co., Ltd.
2. respectively with the genomic DNA of brucella melitensis reference culture 16M (B.melitensis 16M), brucella melitensis vaccine strain M5 (B.melitensis M5) and B. abortus reference culture 544A (B.abortus 544A) for template, pcr amplification BCSP31 genetic fragment; Reaction conditions is: 94 degree, sex change 5min, 94 degree of sex change 30s, 60 degree of annealing 1min, and 72 degree extend 1min, 35 circulations, and last 72 degree extend 10min.
3. carry out Cloning Transformation by being connected in after PCR primer purifying on Blunt carrier, obtain BCSP31 cloned plasmids, by Jilin, biotech company of provincial treasury U.S. checks order.Result shows: as shown in Figure 1, brucella BCSP31 albumen all obtains specific amplification at brucella melitensis reference culture 16M (B.melitensis 16M), brucella melitensis vaccine strain M5 (B.melitensis M5) and B. abortus reference culture 544A (B.abortus544A), object clip size is 990bp, and B. abortus BCSP31 protein gene (GenBank:M20404.1) homology that the BCSP31 cloned plasmids of acquisition and GenBank announce is 100%.
(2) structure of SUMO-BCSP31 recombinant expression plasmid and abduction delivering
1. SUMO carrier is selected to be expression vector, with BfuAI and XbaI respectively enzyme cut the correct BCSP31 cloned plasmids of order-checking comparison and SUMO empty carrier plasmid, SUMO empty carrier plasmid can be expressed by inducing soluble efficiently, and increase expression, the albumen after expression is all the albumen of solubility.
2. reclaim digestion products, connect with T4 ligase, 16 DEG C are spent the night, and build SUMO-BCSP31 recombinant expression plasmid.
3. the SUMO-BCSP31 recombinant expression plasmid of acquisition is proceeded to bacillus coli DH 5 alpha competent cell, screening positive clone, obtain positive colony plasmid.
4. positive colony plasmid is proceeded to e. coli bl21 and carry out abduction delivering, express SUMO empty carrier plasmid as negative control simultaneously, when bacterium to be reorganized is cultured to A600nm=0.4 ~ 0.6, add the IPTG (isopropyl-beta D-thio galactopyranoside) of final concentration 1mmol/L, abduction delivering 6h at 30 DEG C, collected by centrifugation thalline after ice bath 30min, thalline is washed 2 times with ice PBS, 5000rpm collected by centrifugation thalline, add the resuspended thalline of 20ml Binding Buffer, add 1%Triton-100 and 100mg/ml lysozyme respectively, 37 DEG C of effect 30min, add protease inhibitors 1, ice-bath ultrasonic 15min (2s/2s), the centrifugal 30min of 12000rpm, collect cleer and peaceful precipitation, carry out SDS-PAGE analysis (polyacrylamide gel electrophoresis) respectively, detect the expression of soluble protein, SDS-PAGE analysis result as shown in Figure 2, 1, 2, 3 and the 4 restructuring BCSP31 albumen being abduction delivering, the expression of results of soluble protein as shown in Figure 3, expressed BCSP31 albumen is in cell conditioned medium.
(3) the western-blotting checking of restructuring brucella BCSP31 albumen
Utilize half-dried transfer printing to be transferred on pvdf membrane by BCSP31 albumen after SDS-PAGE is separated, transfer printing condition is 20V, 15min; Use HIS label monoclonal antibody as primary antibodie (1:8000) after transfer printing completes respectively, goat anti-mouse igg carries out immunoblotting assay as two anti-(1:10000), detects the expression correctness of BCSP31 albumen.As shown in Figure 4, there is specific band at about 53kDa in result, the BCSP31 albumen correction with HIS label is described.
(4) purifying of restructuring brucella BCSP31 albumen
By above-mentioned cell conditioned medium through nickel post (GE-Healthcare 1ml His trap PP) purifying, post is rinsed with 5ml bindingbuffer, cell conditioned medium solution is added with the flow velocity of 1ml/min, with 5ml elution buffer eluted protein, SDS-PAGE detects the purification result of albumen, cell conditioned medium through ni-sepharose purification result as shown in Figure 5, single goal band is obtained at 53kDa place, restructuring brucella BCSP31 albumen after purifying is adopted 0.45 μm of membrane filtration, and-80 DEG C of freeze-drying save backup.
Embodiment 2 is recombinated the detection of brucella BCSP31 protein immunogenic and analysis
(1) BCSP31 albumen and Bovine brucellosis positive serum immunoblotting assay
Using the Bovine brucellosis positive serum detected clinically (positive can be defined as by brave red flat board and test tube agglutination) as primary antibodie, pvdf membrane is hatched according to the concentration of 1:400, resist as two with the anti-ox IgG of goat, pvdf membrane is hatched with the concentration of 1:5000, through TMB colour developing, detect the specific reaction of BCSP31 albumen and Bovine brucellosis positive serum.Result is as shown in Figure 6: brucella BCSP31 albumen can with Bovine brucellosis positive serum generation specific reaction, illustrate that the BCSP31 albumen of this restructuring can produce specific antibody in brucella animals showing positive serum.
(2) BCSP31 protein immunization mouse
20 male BALB/c (albino lab mouse) are divided into 4 groups, often organize 5, namely first group every mouse immune 5*107SUMO-BCSP31 expresses bacterium BL21, second group of every mouse immunity 5*107 transforms SUMO empty carrier BL21,3rd group every mouse immune 5*105 brucella melitensis vaccine strain M5, the 4th group every mouse immune 200ul PBS in contrast.All take lumbar injection, altogether immunity 3 times, every minor tick 21 days.Last immunity draws neck to put to death mouse for latter 21 days, plucks eyeball blood sampling, draws serum, carry out western-blotting respectively, the detection of serum titer and cell factor.
1. the detection of mice serum ELISA detection specificity antibody
Use the BCSP31 albumen bag of purifying by 96 orifice plates respectively, adopt indirect ELISA method to detect immune serum to be measured, add goat anti-mouse igg-HRP (1:5000) reaction, add substrate OPD and develop the color, put microplate reader and survey 490nm absorbance (A) value, measure the titre of antibody; By immunized mice every the blood sampling of 7 days tail points, until immune latter 8th week, collect serum, indirect ELISA method detects immunized mice serum antibody titer, as shown in Figure 7, express and have the e. coli bl21 of immune recombinant bacterium SUMO-BCSP31 from after immunity the 7th day, serum titer rises result gradually, until within the 8th week, also do not decline after immunity, it is 1 ︰ 5600 that highest serum is tired.
2. mice serum Western-blotting analyzes
In order to detect the specific antibody that can produce BCSP31 albumen after SUMO-BCSP31 expresses bacterium BL21 immune mouse in serum, immune SUMO-BCSP31 is expressed the mice serum of bacterium BL21 as primary antibodie, Western-blotting analysis is carried out respectively with brucella BCSP31 solubility expression of protein product and purified product, result as shown in Figure 8, can there is obvious immune response with BCSP31 albumen at 53bp place in immune serum, the specific antibody obtaining BCSP31 albumen after this SUMO-BCSP31 expresses bacterium BL21 immune mouse is described.
3. the detection of mice serum IgG hypotype
Existing ELISA detection kit is utilized to detect the expression of mice serum IgG1 and IgG2a, result as shown in Figure 9, the IgG hypotype that the mouse induction of immunity recombinant bacterium SUMO-BCSP31 produces is based on IgG1, illustrate that this recombinant bacterium SUMO-BCSP31 is induction of the immune response of TH2 type, based on serum antibody immunity, brucella melitensis vaccine strain M5 then induction simultaneously produces IgG1 and IgG2a, illustrate that brucella attenuated vaccine strain can stimulate body to produce cell immune response and humoral immune reaction simultaneously, PBS control group then produces without obvious IgG1 and IgG2a.
4. the detection of mice serum IFN-γ and IL-2
Utilize existing mouse IFN-γ and IL-2ELISA detection kit to detect the expression of specificity IFN-γ and IL-2 in mice serum, as shown in Figure 10 and Figure 11, immune SUMO-BCSP31 recombinant bacterium mice serum produces without obvious IFN-γ and IL-2 result.
The preparation of embodiment 3 gold medal labeling antibody solution
(1) colloidal gold labeled monoclonal antibody albumen
Adopt pH be 9.0 borate buffer solution be that to be diluted to final concentration be 1.0mg/mL for the antibody protein of 2.45mg/mL by initial concentration; Concentration is adopted to be the K of 0.1M
2cO
3solution regulates the pH of collaurum to be 9.0, and commercial collaurum particle size is 30nm; Add the ratio of 40 μ g antibody proteins with every 1mL collaurum, dropwise add antibody protein, after stirring, static 30min under normal temperature; Adding 10%BSA to final concentration is 1%, continues to stir 15min; Adding 3%PEG2000 to final concentration is 0.1%, continues to stir 15min, obtains golden labeling antibody solution.
Above-mentioned antibody protein is the anti-deer IgG of rabbit, and the corresponding golden labeling antibody obtained is the anti-deer IgG of colloid gold label rabbit.
(2) purifying of golden labeling antibody solution
By the golden labeling antibody solution of above-mentioned acquisition at 4 DEG C, with the centrifugal 20min of the rotating speed of 1500rpm, remove colloid gold particle and assemble the precipitation produced, obtain supernatant; By supernatant at 4 DEG C, with the centrifugal 30min of the rotating speed of 8000rpm, abandon supernatant, get flowable kermesinus precipitation at the bottom of pipe, be resuspended in the storage liquid of initial volume 1/10 that (that is initial volume refers to the volume of supernatant, the volume ratio of supernatant and storage liquid is 10:1), obtain the golden labeling antibody solution of purifying, save backup at 4 DEG C.
Above-mentioned storage liquid is the PBS damping fluid containing 1%BSA and 0.05%PEG2000, and concentration is 0.01M, pH is 8.5 ~ 9.0.
The preparation of embodiment 4 gold-marking binding pad
Immersed by pad in the PBS damping fluid (containing 5% sucrose and 0.05% Tween-20) that concentration is 0.01M, take out after 10min, room temperature stores for future use at drying latter 4 DEG C; Be added drop-wise on the pad of above-mentioned process by the golden labeling antibody solution after above-mentioned purifying, until pad is saturated, dry 1h at 37 DEG C, obtains gold-marking binding pad (glass film, model GL0194, length × wide=6mm × 5mm).
The pre-service of embodiment 5 NC film
Restructuring brucella BCSP31 albumen after the purifying obtained with embodiment 1 is for antigen, with the anti-deer IgG of rabbit be two resist, with 2 μ L/1cm point samples, in NC film, (model is for Vivid170 respectively for the anti-deer IgG of rabbit of to be the brucella BCSP31 albumen of 1.50mg/mL and concentration by concentration be 0.75mg/mL, length × wide=25mm × 5mm) on, particularly brucella BCSP31 albumen is sprayed on detection line (T line), be sprayed on by anti-for rabbit deer IgG on nature controlling line (C line), room temperature fixes 2h; NC film after point sample being immersed in concentration is in the PBS damping fluid (containing 1%BSA) of 0.01M, closes 30min at 37 DEG C; After taking-up, then with concentration be 0.01M PBS damping fluid (containing 1%BSA) rinsing NC film surface, at 37 DEG C dry, obtain be antigen with brucella BCSP31 albumen of recombinate, with the anti-deer IgG of rabbit be two resist NC films.
The assembling of embodiment 6 colloidal gold colloidal gold detection test paper strip
Embodiment 5 is obtained with being antigen with brucella BCSP31 albumen of recombinating, being that two anti-NC films are pasted on PVC base plate (length × wide=60mm × 5mm) with rabbit anti-deer IgG, NC film left end Edge Distance PVC base plate left end edge 22mm, NC film right-hand member Edge Distance PVC base plate right-hand member edge 13mm; Gold-marking binding pad embodiment 4 obtained is sticky is posted on NC film left end upper surface, and superposition length is 2mm; Be posted on gold-marking binding pad left end upper surface by sticky for sample pad (glass film, model is GL0194, length × wide=20mm × 5mm), superposition length is 2mm; Be posted on NC film right-hand member upper surface by sticky for thieving paper (model is H5076, length × wide=15mm × 5mm), superposition length is 2mm; Load onto the assembling namely completing colloidal gold colloidal gold detection test paper strip after plastic clip is fixed, the colloidal gold colloidal gold detection test paper strip obtained is of a size of: length × wide=60mm × 5mm.
The using method of embodiment 7 deer brucellosis colloidal gold antibody test strip
(1) gather testing sample, need to detect immediately after testing sample has gathered, as do not detected immediately, at needing to be placed on 4 DEG C, refrigeration or cryogenic freezing are preserved, and testing sample is deer whole blood (anti-freezing) 1ml or deer serum 500ul.
(2) return to room temperature after intact deer brucellosis colloidal gold antibody test strip being taken out, be placed on horizontal level.
(3) adopt suction pipe to draw testing sample 1 slowly to instill in preprepared 2 PBS damping fluids, slowly instillation 2 ~ 3 in the well of the backward test strips of mixing dilution.
(4) 5 minutes judged results, the result after 10 minutes is invalid.
(5) result judges:
As figure 12 a shows, nature controlling line (C line) and detection line (T line) do not show dark bands or nature controlling line (C line) does not show dark bands and detection line (T line) shows dark bands, and it is invalid to be all judged to; As shown in Figure 12b, nature controlling line (C line) shows dark bands, and detection line (T line) does not show dark bands, is judged to feminine gender; As shown in fig. 12 c, nature controlling line (C line) and detection line (T line) all show dark bands, are judged to the positive.
Embodiment 8 clinical sample detects
Deer brucellosis colloidal gold antibody test strip of the present invention, rose bengal precipitation test and indirect ELISA method is adopted to carry out the detection of deer brucellosis to 420 deer clinically respectively.
The positive rate of comparative analysis deer brucellosis colloidal gold antibody test strip, rose bengal precipitation test and indirect ELISA method.Result is as shown in table 1: the positive rate of deer brucellosis colloidal gold antibody test strip of the present invention is 9.05%, higher than the positive rate 5.48% of rose bengal precipitation test, and lower than the positive rate 12% of indirect ELISA method.
The positive coincidence rate of comparative analysis deer brucellosis colloidal gold antibody test strip, rose bengal precipitation test and indirect ELISA method.Result is as shown in table 2, and the positive coincidence rate of deer brucellosis colloidal gold antibody test strip of the present invention is 100%, higher than the positive coincidence rate 91% of rose bengal precipitation test, simultaneously also higher than the positive coincidence rate 95% of indirect ELISA method.
Table 1
| Detection method | Quantity (head) | Positive (head) | Negative (head) | Positive rate (%) |
| Colloidal gold colloidal gold detection test paper strip | 420 | 38 | 382 | 9.05 |
| Rose bengal precipitation test | 420 | 23 | 397 | 5.48 |
| ELISA | 420 | 47 | 373 | 12 |
Table 2
| Detection method | Positive (head) | Meet number (head) | Coincidence rate (%) |
| Colloidal gold colloidal gold detection test paper strip | 38 | 38 | 100 |
| Rose bengal precipitation test | 23 | 21 | 91 |
| ELISA | 47 | 36 | 95 |
Claims (10)
1. deer brucellosis colloidal gold antibody test strip, comprise plastic clip, the PVC base plate be contained in plastic clip, the successively sticky sample pad be posted on PVC base plate, gold-marking binding pad, NC film, thieving paper with detection line and nature controlling line, on the detection line of described NC film and nature controlling line, point sample has antigen and two to resist respectively; It is characterized in that, described antigen is the restructuring brucella BCSP31 albumen of 1.50mg/mL, and described two resist the anti-deer IgG of rabbit for 0.75mg/mL.
2. deer brucellosis colloidal gold antibody test strip according to claim 1, it is characterized in that, described gold-marking binding pad is prepared by the following method:
(1) with the borate buffer solution of pH9.0, the antibody protein of initial concentration 2.45mg/mL is diluted to final concentration 1.0mg/mL, with the K of 0.1M
2cO
3solution regulates the pH of collaurum to be 9.0, and collaurum particle size is 30nm; Add the ratio of 40 μ g antibody proteins with every 1mL collaurum, dropwise add antibody protein, after stirring, static 30min under normal temperature; Adding 10%BSA to final concentration is 1%, continues to stir 15min; Adding 3%PEG2000 to final concentration is 0.1%, continues to stir 15min, obtains golden labeling antibody solution;
(2) golden labeling antibody solution step (1) obtained is at 4 DEG C, and the centrifugal 20min of 1500rpm, removes precipitation, obtain supernatant; By supernatant at 4 DEG C, the centrifugal 30min of 8000rpm, abandons supernatant, gets the kermesinus precipitation of flowing, is resuspended in the storage liquid of supernatant initial volume 1/10, obtains the golden labeling antibody solution of purifying, 4 DEG C of preservations;
(3) immersed by pad in the PBS damping fluid of 0.01M, take out after 10min, room temperature is dried, 4 DEG C of preservations; The golden labeling antibody solution of purifying step (2) obtained is added drop-wise on above-mentioned pad, until pad is saturated, dry 1h at 37 DEG C, obtains gold-marking binding pad.
3. deer brucellosis colloidal gold antibody test strip according to claim 2, is characterized in that, in step (1), described antibody protein is the anti-deer IgG of rabbit.
4. deer brucellosis colloidal gold antibody test strip according to claim 1, is characterized in that, described antigen and the two anti-equal 2 μ L/1cm of point sample consumption.
5. prepare the method for deer brucellosis colloidal gold antibody test strip according to claim 1, it is characterized in that, comprise the following steps:
(1) with the borate buffer solution of pH9.0, the antibody protein of initial concentration 2.45mg/mL is diluted to final concentration 1.0mg/mL, with the K of 0.1M
2cO
3solution regulates the pH of collaurum to be 9.0, and collaurum particle size is 30nm; Add the ratio of 40 μ g antibody proteins with every 1mL collaurum, dropwise add antibody protein, after stirring, static 30min under normal temperature; Adding 10%BSA to final concentration is 1%, continues to stir 15min; Adding 3%PEG2000 to final concentration is 0.1%, continues to stir 15min, obtains golden labeling antibody solution;
(2) golden labeling antibody solution step (1) obtained is at 4 DEG C, and the centrifugal 20min of 1500rpm, removes precipitation, obtain supernatant; By supernatant at 4 DEG C, the centrifugal 30min of 8000rpm, abandons supernatant, gets the kermesinus precipitation of flowing, is resuspended in the storage liquid of supernatant initial volume 1/10, obtains the golden labeling antibody solution of purifying, 4 DEG C of preservations;
(3) immersed by pad in the PBS damping fluid of 0.01M, take out after 10min, room temperature is dried, 4 DEG C of preservations; The golden labeling antibody solution of purifying step (2) obtained is added drop-wise on above-mentioned pad, until pad is saturated, dry 1h at 37 DEG C, obtains gold-marking binding pad;
(4) with the restructuring brucella BCSP31 albumen after 1.50mg/mL purifying for antigen, be two to resist with the anti-deer IgG of 0.75mg/mL rabbit, with 2 μ L/1cm, respectively point samples are on the detection line and nature controlling line of NC film, and room temperature fixes 2h; NC film after point sample is immersed in the PBS damping fluid of 0.01M, after closing 30min at 37 DEG C, then uses the PBS damping fluid rinsing NC film surface of 0.01M, dry at 37 DEG C, obtain the NC film with restructuring brucella BCSP31 albumen and the anti-deer IgG of rabbit;
(5) the NC film with restructuring brucella BCSP31 albumen and the anti-deer IgG of rabbit step (4) obtained is pasted on PVC base plate, and gold-marking binding pad step (3) obtained is sticky is posted on NC film left end, superposition 2mm; Gold-marking binding pad left end is posted on, superposition 2mm by sticky for sample pad; NC film right-hand member is posted on, superposition 2mm by sticky for thieving paper; Namely colloidal gold colloidal gold detection test paper strip is obtained after fixing with plastic clip.
6. the preparation method of deer brucellosis colloidal gold antibody test strip according to claim 5, is characterized in that, in step (1), described antibody protein is the anti-deer IgG of rabbit.
7. the preparation method of deer brucellosis colloidal gold antibody test strip according to claim 5, it is characterized in that, in step (2), described storage liquid is the PBS damping fluid containing 1%BSA and 0.05%PEG2000, concentration is 0.01M, pH is 8.5 ~ 9.0.
8. the preparation method of deer brucellosis colloidal gold antibody test strip according to claim 5, is characterized in that, in step (3), containing 5% sucrose and 0.05% Tween-20 in described PBS damping fluid.
9. the preparation method of deer brucellosis colloidal gold antibody test strip according to claim 5, is characterized in that, in step (4), containing 1%BSA in described PBS damping fluid.
10. the preparation method of deer brucellosis colloidal gold antibody test strip according to claim 5, it is characterized in that, described gold-marking binding pad is glass film, and model is GL0194, length × wide=6mm × 5mm; The model of described NC film is Vivid170, length × wide=25mm × 5mm; Length × wide=60mm × the 5mm of described PVC base plate; Described sample pad is glass film, and model is GL0194, length × wide=20mm × 5mm; The model of described thieving paper is H5076, length × wide=15mm × 5mm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510100964.8A CN104749362A (en) | 2015-03-09 | 2015-03-09 | Deer brucellosis colloidal gold antibody detection test paper strip and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510100964.8A CN104749362A (en) | 2015-03-09 | 2015-03-09 | Deer brucellosis colloidal gold antibody detection test paper strip and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104749362A true CN104749362A (en) | 2015-07-01 |
Family
ID=53589353
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510100964.8A Pending CN104749362A (en) | 2015-03-09 | 2015-03-09 | Deer brucellosis colloidal gold antibody detection test paper strip and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104749362A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105137073A (en) * | 2015-08-06 | 2015-12-09 | 中国兽医药品监察所 | Bovine Brucella colloidal gold antibody detection test paper strip |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101270160A (en) * | 2008-05-05 | 2008-09-24 | 中国人民解放军第四军医大学 | Monoclone antibody 1F1, uses thereof and hybridoma cell line BCSP31-1F1 excreting the antibody |
| CN101362800A (en) * | 2008-05-26 | 2009-02-11 | 北京庄笛浩禾生物医学科技有限公司 | Test strip for rapid detection of brucella |
| CN103149357A (en) * | 2013-01-29 | 2013-06-12 | 杭州迪恩科技有限公司 | Test paper card for testing Brucella antibody through competition method |
| CN103543261A (en) * | 2013-09-13 | 2014-01-29 | 中国农业科学院特产研究所 | Cattle and sheep brucellosis indirect enzyme-linked immunosorbent assay antibody detection kit and preparation method thereof |
-
2015
- 2015-03-09 CN CN201510100964.8A patent/CN104749362A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101270160A (en) * | 2008-05-05 | 2008-09-24 | 中国人民解放军第四军医大学 | Monoclone antibody 1F1, uses thereof and hybridoma cell line BCSP31-1F1 excreting the antibody |
| CN101362800A (en) * | 2008-05-26 | 2009-02-11 | 北京庄笛浩禾生物医学科技有限公司 | Test strip for rapid detection of brucella |
| CN103149357A (en) * | 2013-01-29 | 2013-06-12 | 杭州迪恩科技有限公司 | Test paper card for testing Brucella antibody through competition method |
| CN103543261A (en) * | 2013-09-13 | 2014-01-29 | 中国农业科学院特产研究所 | Cattle and sheep brucellosis indirect enzyme-linked immunosorbent assay antibody detection kit and preparation method thereof |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105137073A (en) * | 2015-08-06 | 2015-12-09 | 中国兽医药品监察所 | Bovine Brucella colloidal gold antibody detection test paper strip |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104007261B (en) | Fowl three kinds of breathing problem three quick detection kit and application | |
| CN105527437B (en) | A kind of detection kit and its application | |
| CN103543261B (en) | Cattle and sheep Brucellosis indirect enzyme-linked immunosorbent assay antibody assay kit and preparation method thereof | |
| CN104316703B (en) | A kind of Mycoplasma bovis test strip and its preparation method | |
| CN102539782A (en) | Immune chromatography test strip for detecting cystic echinococcosis and alveolar echinococcosis and preparation method | |
| CN101130765B (en) | Reagent kit for detecting syncytial virus of respiratory passage | |
| CN1880961B (en) | Immunochromatographic assay test paper for detecting staphylococcal enterotoxin B and preparation method thereof | |
| CN106290868A (en) | Fluorescent quantitation detection swine fever virus neutralizing antibody immune chromatography reagent kit | |
| Basha et al. | Poor memory B cell generation contributes to non-protective responses to DTaP vaccine antigens in otitis-prone children | |
| CN105753981B (en) | The immune chromatography reagent kit of anti-human Respiratory Syncytial Virus(RSV) N protein antibody and the application antibody | |
| CN104237521A (en) | Three-in-one colloidal gold chromatographic test strip for detecting thiamphenicol, chloramphenicol and florfenicol and preparation method thereof | |
| CN104062430B (en) | A kind of kit for detecting influenza virus in sample and detection method thereof and application | |
| Winter et al. | Antibodies specific for the high-molecular-weight adhesion proteins of nontypeable Haemophilus influenzae are opsonophagocytic for both homologous and heterologous strains | |
| CN101833003A (en) | Vesicular stomatitis virus antibody colloidal goldtest paper tape and preparation method thereof | |
| CN104749362A (en) | Deer brucellosis colloidal gold antibody detection test paper strip and preparation method thereof | |
| CN101692089A (en) | Immunochromatographic test paper for detecting mycobacterium bovis antibodies and preparation method thereof | |
| CN105861520A (en) | Application of Rv3121 protein for detecting mycobacterium tuberculosis infection | |
| CN102033128B (en) | Edwardsiella tarda rapid detection test paper as well as rapid detection method and application | |
| CN102608330A (en) | Immunochromatographic strip for detecting alveolar echinococcosis and preparation method thereof | |
| CN102532309B (en) | Colloid gold test strip for detecting porcine reproductive and respiratory syndrome antibody | |
| CN101000347B (en) | Immunological chromatographic test paper for testing francisella tularensis and its preparation method | |
| CN105585633B (en) | The immune chromatography reagent kit of anti-human haemophilus influenzae P6 protein antibodies and the application antibody | |
| CN105218648A (en) | Negre antigen, nucleotide sequence and application thereof | |
| CN105753982B (en) | The immune chromatography reagent kit of anti-human streptococcus pneumonia fam1 family PspA protein antibodies and the application antibody | |
| Nielsen et al. | Prototype single step lateral flow technology for detection of avian influenza virus and chicken antibody to avian influenza virus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20150701 |