CN104297485B - The monoclonal antibody of specific bond Actinobacillus pleuropneumoniae serum 7-type antigen and test kit - Google Patents
The monoclonal antibody of specific bond Actinobacillus pleuropneumoniae serum 7-type antigen and test kit Download PDFInfo
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Abstract
The invention discloses the monoclonal antibody of a kind of specific bond Actinobacillus pleuropneumoniae serum 7-type antigen and secrete its hybridoma cell line, this hybridoma cell line is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCC No.9051。The preparation method that the invention also discloses this monoclonal antibody。The invention also discloses this monoclonal antibody that a kind of chemical labeling in vitro processes。The invention also discloses competitive ELISA detection Actinobacillus pleuropneumoniae serum 7-type antibody kit。The competitive ELISA kit detection sample of the present invention can complete in 1h, 0.5h faster than current commercialization indirect ELISA testing kit, 1.5h faster than the blocking-up ELISA that inspection and quarantine industry standard uses, can complete multiple batches of sample detection, meet the requirement of epidemic disease quick diagnosis in one day。
Description
Technical field
The invention belongs to biotechnology and animal quarantine technical field, it is specifically related to the monoclonal antibody of a kind of specific bond Actinobacillus pleuropneumoniae serum 7-type antigen and secretes its hybridoma cell line, moreover, it relates to a kind of competitive ELISA detection Actinobacillus pleuropneumoniae serum 7-type antibody kit。
Background technology
Porcine contagious pleuropneumonia (Porcinecontagiouspleuropneumoniae) is by Actinobacillus pleuropneumoniae (Actinobacilluspleuropneumoniae, it is called for short App) a kind of respiratory infectious disease of pig of causing, there is hyperinfection, how lethal rapidly in the most acute or acute course of disease, its M & M is all more than 50%, the mortality rate of most acute may be up to 80-100%, chronic type may result in pig poor growth, become cad pig, this disease is one of Compact Develop Prevention of Common Occurrence Porcine Disease, it it is one of the swine diseases of China import boar quarantine。China once repeatedly detected porcine contagious pleuropneumonia from the import boars such as the U.S., Denmark, Britain。Difference according to Actinobacillus pleuropneumoniae soluble antigen capsular polysaccharide and boivin antigen, can be classified as 15 serotypes。Serology antibody test is one of most important diagnostic method。
Due to App various between not cross protection completely, the virulence of each serotype of App is different, and each serotype is also different in the distribution of every country, and therefore, it is prevention and the basis controlling this disease that Accurate Diagnosis infects serotype。App serum 7-type is important pathogenic serotypes, and China is desirable that this serotype of quarantine from the U.S., Canada import boar。Being linked to be etc. the App Strain Virulence separated from China Changchun to measure according to Jilin University's thunder, serum 7-type is significantly high to the fatality rate of mice, reaches 66.5%。
ELISA detection method easily and fast, sensitive, special, be antibody test most common method。
This detection method does not need expensive instrument, and almost all of detection unit is equipped with ELISA instrument, and ELISA kit is easy to popularization and application。For App, domestic and international commercialization ELISA detection kit is but without the test kit only detecting serum 7-type antibody at present。Also without the test kit adopting competitive ELISA detection App antibody。
Summary of the invention
One of the technical problem to be solved in the present invention is to provide the monoclonal antibody being combined with App serum 7-type antigen-specific。
The two of the technical problem to be solved in the present invention are to provide secretion and produce the hybridoma cell line of said monoclonal antibody。
The preparation method that the three of the technical problem to be solved in the present invention are to provide the monoclonal antibody of the present invention。
The four of the technical problem to be solved in the present invention are to provide the monoclonal antibody that a kind of chemical labeling in vitro is crossed。
The five of the technical problem to be solved in the present invention are to provide the competitive ELISA detection Actinobacillus pleuropneumoniae serum 7-type antibody kit developed based on said monoclonal antibody。
The present invention is achieved by the following technical solutions:
In one aspect of the invention, the hybridoma monoclonal cell of a kind of monoclonal antibody secreting specific bond Actinobacillus pleuropneumoniae serum 7-type antigen is provided, code name is SHCIQ-S7-8D12 hybridoma cell strain, detect through indirect ELISA and competitive ELISA, it was demonstrated that its secreted monoclonal antibody can specific recognition in conjunction with Actinobacillus pleuropneumoniae serum 7-type antigen。This hybridoma cell strain is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (referred to as CGMCC) on April 10th, 2014, and deposit number is CGMCCNo.9051, preservation place: China, Beijing。
In another aspect of this invention, it is provided that the monoclonal antibody of a kind of specific bond Actinobacillus pleuropneumoniae serum 7-type antigen, the hybridoma cell line secretion that above-mentioned deposit number is CGMCCNo.9051 produce。
Terminology employed herein " monoclonal antibody (monoclonal antibody) " refers to be sheerly, from one, the immunoglobulin that cell obtains, and has identical structure and chemical characteristic, single antigenic determinat is had specificity。Monoclonal antibody is different from conventional polyclonal antibody preparation (usually having the different antibodies for different determinants), and each monoclonal antibody is for the single determinant on antigen。Except their specificity, it is cultivated by hybridoma or recombined engineering cell to obtain that the benefit of monoclonal antibody also resides in them, will not be contaminated with other immunoglobulin。Modifier " monoclonal " illustrates the characteristic of antibody, is obtain from homogeneous antibody population, and this is not construed as needing to produce antibody with any specific process。
Terminology employed herein " hybridoma cell line " (English name: hybridomacellline), refers to the cell line that screening and culturing goes out from hybridoma。
In another aspect of this invention, it is provided that the preparation method of a kind of said monoclonal antibody, comprise the steps:
Step 1, preparation immunogen: preparation chocolate culture-medium, recover Actinobacillus pleuropneumoniae serum 7-type reference culture, mass propgation, bacterium is washed with normal saline, centrifugal, abandon supernatant, bacterial precipitation normal saline washes suspension, centrifugal, and the S7 antibacterial holoantigen that normal saline suspends is as immunogen；
Step 2, mouse immune: by the mice subcutaneous inoculation of repeated multiple times low dose, select the mice of the anti-App serum 7-type polyclonal antibody that can secrete high-titer；
Step 3, cell fusion: take immune mouse spleen cell, merged with murine myeloma cell by external；
Step 4, positive hybridoma cell screen: with the positive hybridoma cell of the anti-Actinobacillus pleuropneumoniae serum 7-type antigen of indirect ELISA experiment sieving；
Step 5, positive hybridoma cell acquisition: select strong positive reaction hybridoma, cloning continuously with limiting dilution assay 3 times, after last clone, the hole gained cell strain of test positive is the hybridoma cell strain of stably excreting, special anti-Actinobacillus pleuropneumoniae serum 7-type antibody；
Step 6, the cultivation of hybridoma and preservation: by positive hybridoma cell amplification cultivation, collect cell；A part is used for producing mouse ascites antibody, another part Liquid nitrogen storage。
As currently preferred technical scheme, step 1 is particularly as follows: prepare chocolate culture-medium, Actinobacillus pleuropneumoniae serum 7-type reference culture is recovered, mass propgation, washes down bacterium with normal saline, and 8000r/min is centrifuged 10min, abandon supernatant, bacterial precipitation normal saline washes suspension, centrifugal, so washes bacterium 3 times。Bacterial precipitation suspends with containing 0.5% formaldehyde normal saline, places 4 DEG C and overnight inactivates, and the centrifugal 10min of 8000r/min removes formaldehyde, washes 3 times with normal saline。The S7 antibacterial holoantigen that normal saline suspends is as immunogen；
As currently preferred technical scheme, step 2 is particularly as follows: by above-mentioned immunogen and isopyknic Freund's complete adjuvant mixing and emulsifying, the female Mus of BALB/C of 8 week old is cooked lumbar injection, it is emulsified in freund 's incomplete adjuvant by same immunogen after 3-4 week, make second time lumbar injection, with the last lumbar injection of immune original work of doubling dosage after 2-3 week, take mouse spleen after three days for cell fusion。
As currently preferred technical scheme, step 3, particularly as follows: take immune mouse spleen cell, is merged under 50%PEG in the ratio of 5-10:1 with murine myeloma cell SP2/0 by external, uses HAT Screening of Media；Fused cell changes liquid once again by HAT culture medium after cultivating 5d, and 10d HT culture medium changes liquid, until the low 5%-30% of fused cell coverage hole, takes supernatant indirect ELISA method and screens。
As currently preferred technical scheme, in step 4, during screening, envelope antigen is the antigen that AppS7 type processes for 3 times through multigelation, and described antigen dilutes with 1:5000, and with the App of other serotype for negative control。
Additionally, in another aspect of this invention, the monoclonal antibody of the above-mentioned anti-Actinobacillus pleuropneumoniae serum 7-type antigen that a kind of chemical labeling in vitro processes is also provided for。
The step that described chemical labeling in vitro processes is as follows:
1) ascites monoclonal antibody ammonium sulfate is purified；
2) with improvement Over-voltage protection by antibody and HRP coupling, salting out method removes unconjugated HRP in sample, and end product to PBS overnight, namely obtains the monoclonal antibody of the horseradish peroxidase-labeled that chemical labeling in vitro is crossed。
" improvement Over-voltage protection " is referred to the pathogenetic bacteria inspection technology (the 1st edition p204 in Jilin science tech publishing house July in 1992) of the chief editors such as the Korean fine jade, and key step is as follows:
(1) being dissolved in by 5mgHRP in 0.5ml distilled water, add the 0.06mol/LNaIO4 aqueous solution 0.5ml of new preparation, 30min in refrigerator is put in mixing。
(2) add 0.16mol/L ethanol water 0.5ml, put room temperature and place 30min。
(3) adding aqueous solution 1ml containing 5mg antibody purification, load bag filter after mixing, with 0.05mol/L, pH value 9.5 carbonate buffer solution is slowly stirred dialysis 6 hours or overnight。
(4), after sucking-off, add NaBH4 solution (5mg/ml) 0.2ml, put refrigerator 2 hours。
(5) above-mentioned conjugate mixed liquor is added equal-volume saturated ammonium sulfate solution, puts refrigerator 30 minutes, centrifugal, gained precipitate is dissolved in a little 0.02mol/L, in pH value 7.4PBS, and to dialysed overnight。
(6) it is centrifuged off insoluble matter, namely obtains the monoclonal antibody of horseradish peroxidase (HRP) labelling that chemical labeling in vitro is crossed。
Additionally, in another aspect of this invention, also provide for a kind of competitive ELISA detection Actinobacillus pleuropneumoniae serum 7-type antibody kit, specifically include that Actinobacillus pleuropneumoniae serum 7-type antigen；What chemical labeling in vitro as claimed in claim 3 processed can the monoclonal antibody of specific bond Actinobacillus pleuropneumoniae serum 7-type antigen；Actinobacillus pleuropneumoniae serum 7-type positive control serum；Negative control sera。
The using method of this test kit, comprises the steps: 1) envelope antigen；2) plate is washed；3) close；4) plate is washed；5) add tested serum, be simultaneously introduced the monoclonal antibody of the anti-App serum 7-type antigen of horseradish peroxidase-labeled；6) plate is washed；7) substrate is added；8) reaction is terminated；9) detection light absorption value (OD value)；10) also result of determination is calculated。Step 10) in, described result of determination is specific as follows: (1) negative control sera OD value is more than 0.8；(2) the OD positive/OD feminine gender≤20%；Comparison is set up just can carry out sample judgement；(3) blood serum sample 1:16 dilution, its S/N value (OD sample/feminine gender OD)≤20% is positive, and S/N > 40% is negative, and 40% >=S/N > 20% is suspicious。
Compared with prior art, the beneficial effects of the present invention is: for accurately detecting App serum 7-type, developing on the basis of App serum 7-type monoclonal antibody, have developed competitive ELISA detection Actinobacillus pleuropneumoniae serum 7-type antibody kit。Compared with using polyclonal antibody, using monoclonal antibody, detection kit is easily produced in batches, and the quality of test kit is easily controlled。Compared with indirect ELISA, competitive ELISA detects without kind Idiotype, except detection pig antibody, moreover it is possible to the laboratory animal antibody such as detection Mus, it is possible to be used for setting up porcine contagious pleuropneumonia experimental animal model recruitment evaluation。And the competitive ELISA method detection sample that the present invention sets up can complete in 1h, 0.5h faster than current commercialization indirect ELISA testing kit, 1.5h faster than the blocking-up ELISA that inspection and quarantine industry standard uses, multiple batches of sample detection can be completed in one day, meet the requirement of epidemic disease quick diagnosis。
Accompanying drawing explanation
Fig. 1 is 12%SDS-PAGEIgM electrophoretogram in the embodiment of the present invention 2；Wherein, M represents Protein Marker (170kD, 130kD, 95kD, 72kD (red), 55kD, 43kD, 34kD, 26kD, 17kD, 11kD)；1 represents 2ugBSA；2 represent 4ugBSA；3 represent 8ugBSA；4 represent 16ugBSA；5 represent 1ulIgM (diluting 10 times)；6 represent 2ulIgM (diluting 10 times)；7 represent 4ulIgM (diluting 10 times)。
Fig. 2 is the result schematic diagram of the different dilution factor antigen of enzyme concentration detection of 1:1 ten thousand dilution in the embodiment of the present invention 3；
Fig. 3 is the result schematic diagram of the different dilution factor antigen of enzyme concentration detection of 1:2 ten thousand dilution in the embodiment of the present invention 3；
Fig. 4 is the scatterplot of the positives serum ratio of the embodiment of the present invention 3；
Fig. 5 is the scatterplot of negative serum ratio in the embodiment of the present invention 3；
Fig. 6 is the negative change schematic diagram with positive serum light absorption value of different time in the embodiment of the present invention 3；
Code name is the mouse hybridoma cell strain (deriving from mice Musmusculus) of SHCIQ-S7-8D12, detect through indirect ELISA and competitive ELISA, it was demonstrated that its secreted monoclonal antibody can specific recognition in conjunction with Actinobacillus pleuropneumoniae serum 7-type antigen。This mouse hybridoma cell strain is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (referred to as CGMCC) on April 10th, 2014, deposit number is CGMCCNo.9051, preservation place: North Star West Road 1, Chaoyang District, city of BeiJing, China institute 3, Institute of Microorganism, Academia Sinica。
Detailed description of the invention
The following examples can make those skilled in the art more fully understand the present invention, but does not limit the present invention in any way。
The preparation of the preparation of embodiment 1 hybridoma cell strain and monoclonal antibody
Step 1, immunogenic preparation: preparation chocolate culture-medium, chocolate culture-medium is rule by Actinobacillus pleuropneumoniae serum 7-type reference culture (AppS7) recovery, mass propgation, with normal saline, bacterium is washed down, 8000r/min is centrifuged 10min, abandon supernatant, bacterial precipitation normal saline suspends, centrifugal, so washes bacterium 3 times。Bacterial precipitation suspends with containing 0.5% formaldehyde normal saline, places 4 DEG C and overnight inactivates, and the centrifugal 10min of 8000r/min removes formaldehyde, washes 3 times with normal saline。The AppS7 antibacterial holoantigen that normal saline suspends is as immunogen；Step 2, BALB/C mice immunity: by above-mentioned immunogen (containing 104Actinobacillus pleuropneumoniae) and isopyknic Freund's complete adjuvant mixing and emulsifying, the female Mus of BALB/C of 8 week old is cooked lumbar injection immunity, it is emulsified in freund 's incomplete adjuvant by same immunogen after 3-4 week, do second time lumbar injection immunity, carry out lumbar injection booster immunization by the immunogen of doubling dosage after 2-3 week, take mouse spleen after three days for cell fusion。Step 3, cell fusion: take above-mentioned immune mouse spleen cell with murine myeloma cell (SP2/0) in the ratio of 5-10:1, RPMI-1640 (Gibco) culture medium of serum-free mixes, 1500rpm is centrifuged 5min, remove culture medium, with 50%PEG (Sigma, molecular weight 1500) as fusion agent, 1ml is added under water-bath at 37 DEG C, it is made to merge 2min, after terminating fusion by the RPMI-1640 culture medium of serum-free, 1500rpm is centrifuged 5min, precipitation HAT culture medium suspends, it is dispensed in the cell plates that feeder cells are contained in 96 holes, 37 DEG C, 5%CO2Cell culture incubator in cultivate。Step 4, positive hybridoma cell screen: after cultivating 5 days in cell culture incubator, liquid is changed once by HAT culture medium, within 10th day, change liquid by HT culture medium, secrete the positive hybridoma cell of anti-Actinobacillus pleuropneumoniae serum 7-type antigen-antibody with indirect ELISA experiment sieving until 5%-30% at the bottom of fused cell coverage hole, during screening, envelope antigen is antigen (the 1:5000 dilution that AppS7 type processes for 3 times through multigelation, weight/volume, g/ml), and with the App of other serotype for negative control。Step 5, positive hybridoma cell acquisition: select indirect ELISA strong positive reaction hybridoma wells, with the continuous clone cell of limiting dilution assay 3 times, after last clone, the hole gained cell strain of test positive is the hybridoma cell strain of secrete monoclonal antibody。Positive hybridoma cell is the hybridoma monoclonal cell of stably excreting, special anti-Actinobacillus pleuropneumoniae serum 7-type；
Step 6, the cultivation of hybridoma and preservation: by positive hybridoma cell amplification cultivation, collect cell；A part is used for producing mouse ascites antibody, suspends with frozen stock solution and preserves in liquid nitrogen after being sub-packed in cryopreservation tube after another part cell。
Prepared by step 7, ascites: take 8 week old health BALB/C mice, the liquid paraffin of 0.5ml/ sterilizing of Intraperitoneal injection, and only (concentration is 10 to inject hybridoma 0.5ml/ after 10 days6Individual/ml), treat that mouse web portion expands, during moving difficulty, thrust mouse web portion with large size syringe needle and collect ascites, 4000g is centrifuged 3min, and supernatant is ascites monoclonal antibody。
Embodiment 2 chemical labeling in vitro monoclonal antibody
Have main steps that:
1, the hypotype of monoclonal antibody is identified: using the Holland HyCultBiotechnology mouse monoclonal antibody detection kit (MouseMabIsotyingtestkit) produced, step is as follows:
A) cell conditioned medium or ascites pH7.4PBS are suitably diluted, standby to antibody concentration 1-50 μ g/mL；
B) 500 μ L diluents are added to the detection pipe containing a test strips；
C) add 500 μ La again) in the sample that dilute extremely detect in pipe；
D) test strips it is completely immersed in and is gently agitated for；
E) jog bottle mixing rabbit against murine κ chain micelle, takes 1mL and adds to detection pipe；
F) vibration about 30min sentence read result in 18-25 DEG C of environment, test strips remains leaching in the solution in oscillatory process。
After testing, the monoclonal antibody that hybridoma monoclonal cell system (preservation code name is SHCIQ-S7-8D12) of the anti-Actinobacillus pleuropneumoniae serum 7-type antibody of stably excreting that the present invention sets up is secreted is IgM hypotype。
2, the purification of monoclonal antibody
Ascites monoclonal antibody ammonium sulfate is purified
A) 2ml ascites adds 2mlPBS solution dilution, there are 4ml solution, adds saturated ammonium sulfate solution 4ml (being slowly added to, add and mix), places overnight for 4 DEG C；
B) 4 DEG C of centrifugal (10000rpm, 10 minutes) supernatant discarded, precipitation adds 4mlPBS solution, resuspended, adds saturated ammonium sulfate solution 2ml (being slowly added to, add and mix), places overnight for 4 DEG C；
C) 4 DEG C of centrifugal (10000rpm, 10 minutes) supernatant discarded, precipitation adds 4mlPBS solution, resuspended, adds saturated ammonium sulfate solution 2ml (being slowly added to, add and mix), places overnight for 4 DEG C；
D) 4 DEG C of centrifugal (10000rpm, 10 minutes) supernatant discarded, precipitation adds 2mlPBS solution, 24 hours (changing 2 dialysis solution) that 1LPBS is dialysed。
3, the electrophoresis detection of monoclonal antibody content
Preparation 12%SDS-PAGE glue, after being diluted by the IgM monoclonal antibody 10 times of purification, takes 1ul, 2ul, 4ul electrophoresis respectively, with BSA as qualitative reference, sees Fig. 1。According to electrophoresis detection result, after purification, the concentration of IgM (namely before labelling) is about 10mg/ml。
4, the labelling of monoclonal antibody
With reference to the pathogenetic bacteria inspection technology (the 1st edition p204 in Jilin science tech publishing house July in 1992) of the chief editors such as the Korean fine jade, by Shanghai Youke Biological Technology Co., Ltd. with improvement Over-voltage protection by antibody and HRP coupling, key step is as follows:
(1) being dissolved in by 5mgHRP in 0.5ml distilled water, add the 0.06mol/LNaIO4 aqueous solution 0.5ml of new preparation, 30min in refrigerator is put in mixing。
(2) add 0.16mol/L ethanol water 0.5ml, put room temperature and place 30min。
(3) adding aqueous solution 1ml containing 5mg antibody purification, load bag filter after mixing, with 0.05mol/L, pH value 9.5 carbonate buffer solution is slowly stirred dialysis 6 hours or overnight。
(4), after sucking-off, add NaBH4 solution (5mg/ml) 0.2ml, put refrigerator 2 hours。
(5) above-mentioned conjugate mixed liquor is added equal-volume saturated ammonium sulfate solution, puts refrigerator 30 minutes, centrifugal, gained precipitate is dissolved in a little 0.02mol/L, in pH value 7.4PBS, and to dialysed overnight。
(6) it is centrifuged off insoluble matter, namely obtains the monoclonal antibody of horseradish peroxidase (HRP) labelling that chemical labeling in vitro is crossed。
5, the Preliminary Determination of enzyme mark titer
Using Salmonella, envelope antigen is AppS7 and AppS6, and equal 1:50 times is diluted, and the latter is as negative control。Being diluted according to 1:625-1:40000 by enzyme mark monoclonal antibody, titration enzyme labelled antibody titer, in Table 1, initial option enzyme concentration 1:1 ten thousand-1:2 ten thousand。
Table 1 enzyme labelled antibody titer titration
The detection Actinobacillus pleuropneumoniae serum 7-type antibody kit test of embodiment 3 competitive ELISA
The test procedure of competitive ELISA kit, including following content: 1) antigen prepares 2) antigen valence titration 3) determination 4 of Sample serum the best diluted concentration) determination 5 of CUTOFF value) competitive ELISA test procedure a) envelope antigen, 4 DEG C of refrigerator overnight；B) plate is washed 3 times；C) 1h is closed for 37 DEG C；D) plate is washed 3 times；E) add tested serum, be simultaneously introduced the monoclonal antibody of the anti-App serum 7-type antigen of horseradish peroxidase-labeled, 37 DEG C of reaction 30min；F) plate is washed 3 times；G) substrate is added, 37 DEG C of reaction 10min；H) reaction is terminated；I) detection light absorption value (OD value)；J) also result of determination is calculated。Specific as follows:
1) preparation of antigen
At the upper bacterium colony cultivating 6h of TSCA culture medium (containing 0.1%NAD), elute with the normal saline containing 0.5% formalin (formaldehyde) and be collected in a bottle, be positioned over 4 DEG C, overnight；Next day, 8000r/min, centrifugal within 15 minutes, remove supernatant, precipitation normal saline is resuspended and to adjust bacterial concentration be 1010CFU/mL。The bacterium solution of suspendible being again positioned in 100 DEG C of water-baths and act on 1h, after cooling, 20000r is centrifuged 20min, retains supernatant。With the membrane filtration supernatant of 0.22 μm, filtrate is polysaccharide antigen。Subpackage, puts-20 DEG C and saves backup。
2) titration of antigen valence
By antigen 1: 200 to 1:3200 dilutes, enzyme concentration uses 1:1 ten thousand and 1:2 ten thousand, competitive ELISA is used to detect porcine blood serum positive, negative and blank, result is in Table 2, Fig. 2 is the enzyme concentration of 1:1 ten thousand dilution, Fig. 3 is the enzyme concentration of 1:2 ten thousand dilution, when antigen is below 1:800 dilution factor, the uncontested effect of negative control, therefore, select antigen 1: 800 is work antigen, at this antigen, during the concentration 1:1 ten thousand of enzyme, its OD value is for being about 2, and during the concentration 1:2 ten thousand of enzyme, OD value is about 1, therefore, selecting enzyme concentration is that 1:2 ten thousand is as work enzyme concentration。
The light absorption value of the different antigen concentration of table 2
3) determination of Sample serum the best diluted concentration
Use S7 antigen determined above and enzyme concentration, S7 pig hyper-immune serum, S7 rabbit hyper-immune serum and pig negative serum and rabbit negative serum are respectively according to 1:4-1:512 doubling dilution, detect with competitive ELISA, set up blank (replacing serum with diluent) simultaneously, during result serum 1:16, negative serum is almost without competition, in Table 3, therefore, the best diluted concentration selecting serum 1:16 to be tested serum。
Table 3 competitive ELISA measures the OD value of different serum dilution
4) determination of competitive ELISA detection Sample Negative and positive marginal value (Cutoff value)
For determining the serum 235 parts of cutoff value, wherein for determining the positive 32 parts of cutoff value, positive for App serum 7-type antibacterial counteracting toxic substances pig or clinical quarantine, it is used that two kinds of commercial kits are defined as the porcine blood serum of the positive。The serum 203 part negative for determining cutoff value, respectively from 90 parts of Canada, 22 parts of France, 55 parts of import boars of Denmark and Shanghai for 36 parts of pig farm, port, is all defined as the porcine blood serum of feminine gender through two kinds of commercial kit detections。The equal 1:16 dilution of serum, detects according to competitive ELISA kit regulated procedure。The percentage ratio of the light absorption value of positive serum and negative control light absorption value, in Table 4, does scatterplot according to its ratio, sees that the percentage ratio of Fig. 4, negative serum light absorption value and negative control light absorption value is in Table 5, does scatterplot according to its ratio and sees Fig. 5。Analyze the positive serum ratio overwhelming majority below 20% (only 1 part of serum ratio is more than 20%, and probability is only 3.13%) by scatter diagram, therefore positive ratios is set for being positive marginal value less than or equal to 20%。Analyze the negative ratio of major part all more than 40% (only 3 parts of negative serums are below 40%, and its probability is 1.48%) by scatter diagram, therefore negative ratio is set for being negative marginal value during more than 40%。
The ratio of table 4 positive serum absorbance
The ratio of table 5 negative serum absorbance
5) competitive ELISA test concrete steps and result judge, specific as follows:
A) envelope antigen: dilute antigen to working concentration with the carbonic acid buffer (being namely coated liquid) of pH9.5, take 96 hole ELISA Plate, every hole adds 100ul, 4 DEG C of refrigerator overnight。
B) taking-up is coated plate, and with containing 0.5% tween 20, the phosphate buffer (PBS-T) of pH7.4 washes plate 3 times。
C) every hole adds the confining liquid (adding 10% defatted milk powder in PBS-T) of 200 microlitres, after sticking shrouding film, places 37 DEG C of calorstats, 1 hour。
D) plate is washed 3 times。
E) Sample serum dilution plate in diluent (adding 1% defatted milk powder in PBS-T) 1:16 dilution (as, take 10ul serum and add 150ul diluent), take 100ul and add in reacting hole, it is simultaneously introduced the monoclonal antibody of the horseradish peroxidase-labeled of 100ul working concentration, each sample does a hole, arranges positive serum controls, positive serum controls simultaneously。Feminine gender does 2 holes with negative control is each, after sticking shrouding film, 37 DEG C of calorstats, react 30 minutes。Sample is recommended to do diplopore。
F) plate is washed 3 times。
G) every hole adds 100 microlitres reaction substrate TMB (tetramethyl benzidine), 37 DEG C of calorstats, 10min。
H) every hole adds 50 microlitre 2Mol/L sulphuric acid termination reactions。
I) light absorption value is measured at 450nm。
J) result of calculation: negative control sera mean OD value is more than 0.8；2) positive/average OD feminine gender≤20% of average OD；Comparison is set up just can carry out sample judgement。
K) sample judges: blood serum sample 1:16 dilutes, and its S/N value (OD sample/mean negative OD)≤20% is positive, and S/N > 40% is negative, and 40% >=S/N > 20% is suspicious。Suspicious specimen does diplopore repeated trials, if being the positive or suspicious positive that is judged to, if being feminine gender, it is judged to feminine gender。
6) stability test of competitive ELISA kit
A) Sptting plate is prepared
With being coated liquid dilution antigen to working concentration, taking 96 hole ELISA Plate, every hole adds 100ul, 4 DEG C of refrigerator overnight；Taking-up is coated plate, washes plate 3 times with PBS-T；Every hole add 200 microlitres containing protein protective agent confining liquid (protein protective agent is purchased from Shanghai Xi Bao Bioisystech Co., Ltd), after sticking shrouding film, place 37 DEG C of constant temperature 1h；Wash plate 3 times；Vacuum preserves, and 4 DEG C of Refrigerator stores are standby。
B) serum is according to after adding preservative, and 4 DEG C of Refrigerator stores are standby。
C) enzyme labelled antibody
Being diluted according to 1:100 by enzyme labelled antibody with stabilizer (being purchased from Shanghai Xi Bao Bioisystech Co., Ltd), put 37 DEG C and save backup, during use, 1:200 is diluted to working concentration again。
D) detecting program according to competitive ELISA, different time period detections is negative, the light absorption value of positive serum, is specifically shown in table 6 and Fig. 6。Result enzyme is stored in 37 DEG C the 7th day, and negative serum OD value does not significantly change, and within the 9th day, OD value is remarkably decreased, but the ratio positive and negative by the 16th day still is below 20%, and positive control is still set up。Conventionally enzyme often store 37 DEG C within one day, be equivalent to store 2-8 DEG C 1.5 months, according to 7 days calculate, this test kit was minimum 10 months of 4 DEG C of shelf-lifves。
In table 6ELISA test kit, enzyme is stored in 37 DEG C of different time testing results
7) replica test of competitive ELISA kit
Detecting program according to competitive ELISA, to 4 parts of serum duplicate detection 7 times, result is completely the same, is consistent with expection, is specifically shown in table 7。
Table 7 competitive ELISA repeated trials result
| Sample 1 | Sample 2 | Sample 3 | Sample 4 | |
| 2014-8-8 | + | + | ± | - |
| 2014-8-9 | + | + | ± | - |
| 2014-8-11 | + | + | ± | - |
| 2014-8-13 | + | + | ± | - |
| 2014-8-15 | + | + | ± | - |
| 2014-8-16 | + | + | ± | - |
| 2014-8-18 | + | + | ± | - |
Embodiment 4 uses indirect ELISA to identify monoclonal antibody specificity
Indirect ELISA identifies App monoclonal antibody step:
1. comparison antigen includes the App reference culture of other 14 types except S7 type, the same S7 of production of these bacterial strains。Other related strain 28 strain, these bacterial strains produce according to respective requirement。
2. it is coated liquid and is coated various antigen, be coated concentration and be about 1x107, after sealer, 4 DEG C of refrigerator overnight。
3.PBS-T washes plate 3 times。
4. every hole adds the confining liquid of 200ul, after pad pasting, and 37 DEG C of isothermal reaction 60min。
5. wash plate 3 times。
6. every hole adds 100ul monoclonal antibody (1:5000 dilution), after sealer, and 37 DEG C of reaction 30min。
7. wash plate 3 times。
8. every hole adds the enzyme labelled antibody of the against murine of 100ul alkali phosphatase enzyme mark, after shrouding, and 37 DEG C of reaction 30min。
9. wash plate 3 times。
10. every hole adds 100ul reaction substrate PNPP (p-nitrophenyl phosphate), 37 DEG C of reaction 60min。
11. every hole adds 50 microlitre 2Mol/L sodium hydroxide solutions terminates reaction。
12. measure light absorption value at 405nm
After testing, 8D12-C10 monoclonal antibody only can specific detection S7 antigen, it is impossible to detecting antigen and the related strain antigen of other serotype of App, concrete testing result is in Table 8, table 9。
Table 88D12-C10 detects App reference culture result
Table 98D12-C10 detects control strain result
The specific detection of embodiment 5 competitive ELISA detection Actinobacillus pleuropneumoniae serum 7-type antibody kit
Specific test use control serum be, the pig hyper-immune serum of App serum 1-12 type, App serum 4 type, 7 types rabbit hyper-immune serum, the positive serum of 11 parts of relevant swine diseasess, and the Lin Shi Actinobacillus rabbit hyper-immune serum nearest with App homology。Detecting through competitive ELISA, detection App serum 7-type serum that can only be special, specificity is 100%。Concrete testing result is in Table 10。
Table 10 competitive ELISA specific detection result
Note: Cutoff2.053x20%=0.41；2.053x40%=0.82
Embodiment 6 competitive ELISA detection Actinobacillus pleuropneumoniae serum 7-type counteracting toxic substances pig different time antibody level of serum
Use App No. 1 pig of serum 7-type reference culture counteracting toxic substances and No. 2 pigs, before counteracting toxic substances and after counteracting toxic substances 56 days, take a blood sample weekly once, and blood sampling in 134 days and 143 days, detect with competitive ELISA, 7 days antibody is negative before result counteracting toxic substances and after counteracting toxic substances, counteracting toxic substances after 14 days antibody be the positive (except after No. 1 pig counteracting toxic substances 28 days for suspicious)。It is specifically shown in table 11, within after App serum 7-type counteracting toxic substances is described 14 days, produces detectable antibody。
Table 11 competitive ELISA detection App serum 7-type reference culture counteracting toxic substances pig antibody horizontal
Remarks: cutoff value 1.395X20%=0.2791.395X40%=0.558
Embodiment 7 competitive ELISA detection counteracting toxic substances porcine blood serum antibody dynamic regularity
App serum 7-type reference culture counteracting toxic substances hands over big No. 2 pigs, and immune programme for children is: AppS7 cultivates 18h, and normal saline is washed down, intranasal inoculation 4.5x108Individual bacterium；Within 6th day, take 6h cultivation App, 3ml and be about 8.5x109Individual bacterium adds equivalent incomplete Freund's adjuvant and divides three position intramuscular injection；Within 7th day, take 6h and cultivate App, about 7.5x109Individual bacterium (0.2% formalin normal saline dilution, 4 DEG C of refrigerator overnight) intravenous injection；Within 24th day, ditto it is repeated once, bacterium amount 2.5x109Individual bacterium；Within 28th day, take 6h and cultivate App, bacterium amount 2.5x109Individual bacterium (normal saline dilution) intravenous injection；Within 32nd day, ditto it is repeated once。Taking a blood sample weekly once before counteracting toxic substances and after counteracting toxic substances, separate serum ,-20 DEG C save backup。
The serum gathered by different time is with after normal saline doubling dilution, according to the competitive ELISA method detection set up, result counteracting toxic substances can detect antibody after 14 days, through immunity several times repeatedly, after 35 days, titer rises to 1:64 (final concentration 1:1024), after 143 days, antibody titer remains within higher level, is specifically shown in table 12。
Antibody dynamic regularity after big No. 2 pig S7 counteracting toxic substances handed over by table 12
Embodiment 8 competitive ELISA detection Actinobacillus pleuropneumoniae serum 7-type antibody kit compares with other commercialization detection kit。
1, competitive ELISA detection Actinobacillus pleuropneumoniae serum 7-type antibody kit detection counteracting toxic substances pig antibody compares with Idvet company of France test kit (detection App4/7 type) and Canada's Biovet test kit (detection App4/5/7 type), and result detection three kinds of test kits of No. 2 pigs are consistent completely。If the suspicious positive of can be regarded as, detecting No. 1 pig, have a sample not to be inconsistent, all the other are all consistent, and concrete outcome is in Table 13。
Table 13 competitive ELISA detection counteracting toxic substances pig antibody compares with IDvet and biovet。
2, competitive ELISA detection Actinobacillus pleuropneumoniae serum 7-type antibody kit detects from Denmark, France, Canada, imported from America boar, and Shanghai is for 259 parts of clinical samples of porcine blood serum on pig farm, port and Chongqing censorship, with Idvet company of France test kit (detection App4/7 type), Canada's Biovet test kit (detection App4/5/7 type) compares, and result is in Table 14。
14 3 kinds of test kit detection clinical sample results of table
3, competitive ELISA detects the rate that is consistent of Actinobacillus pleuropneumoniae serum 7-type antibody kit and other commercialization detection kit
Comparing after the result detected above is collected (see table 15), if by the suspicious positive of can be regarded as, the rate that is consistent of competitive ELISA and Idvet is 97.2%, is 98.2% with the rate that is consistent of Biovet；If can be regarded as feminine gender by suspicious, then competitive ELISA is 97.2% with the rate that is consistent of Idvet, is 97.9% with the rate that is consistent of Biovet, calculates under seeing。Illustrate that the competitive ELISA kit that the present invention sets up has good compatibility with commercialization detection kit, can be used for the detection of clinical sample。
15 3 kinds of test kit detection clinical sample results of table
Three kinds of test kits rate that is consistent is calculated as follows:
By suspicious positive phase competitive ELISA of can be regarded as with the rate that is consistent of Idvet it is: (281-8)/281=97.2%
By suspicious positive phase competitive ELISA of can be regarded as with the rate that is consistent of Biovet it is: (281-5)/281=98.2%
By suspicious feminine gender phase competitive ELISA of can be regarded as with the rate that is consistent of Idvet it is: (281-8)/281=97.2%
By suspicious feminine gender phase competitive ELISA of can be regarded as with the rate that is consistent of Biovet it is: (281-6)/281=97.9%
Claims (5)
1. secreting a hybridoma cell line for the monoclonal antibody of specific bond Actinobacillus pleuropneumoniae serum 7-type antigen, its deposit number is CGMCCNo.9051。
2. a monoclonal antibody for specific bond Actinobacillus pleuropneumoniae serum 7-type antigen, the hybridoma cell line secretion described in claim 1 produces。
3. the monoclonal antibody of the specific bond Actinobacillus pleuropneumoniae serum 7-type antigen as claimed in claim 2 of a chemical labeling in vitro process。
4. a monoclonal antibody as claimed in claim 3, it is characterised in that the step that described chemical labeling in vitro processes is as follows:
1) ascites monoclonal antibody ammonium sulfate is purified；
2) with improvement Over-voltage protection by antibody and HRP coupling, salting out method removes unconjugated HRP in sample, and end product to PBS overnight, namely obtains the monoclonal antibody that chemical labeling in vitro is crossed。
5. a competitive ELISA detection Actinobacillus pleuropneumoniae serum 7-type antibody kit, it is characterised in that specifically include that Actinobacillus pleuropneumoniae serum 7-type antigen；What chemical labeling in vitro as claimed in claim 3 processed can the monoclonal antibody of specific bond Actinobacillus pleuropneumoniae serum 7-type antigen；Actinobacillus pleuropneumoniae serum 7-type positive control serum；Negative control sera。
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