CN104297485A - Monoclonal antibody capable of specifically combining actinobacillus pleuropneumoniae serum 7-type antigen and kit - Google Patents
Monoclonal antibody capable of specifically combining actinobacillus pleuropneumoniae serum 7-type antigen and kit Download PDFInfo
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Abstract
The invention discloses a monoclonal antibody capable of specifically combining an actinobacillus pleuropneumoniae serum 7-type antigen and a hybridoma cell line for secreting a monoclonal antibody. The hybridoma cell line is preserved in China General Microbiological Culture Collection Center with the preservation number of CGMCC No.9051. The invention also discloses a preparation method of the monoclonal antibody. The invention also discloses the monoclonal antibody subjected to in-vitro chemical marking treatment. The invention also discloses a kit for detecting the actinobacillus pleuropneumoniae serum 7-type antigen by adopting competitive ELISA. The competitive ELISA kit can be used for detecting a sample within one hour, is higher than a present commercial indirect ELISA detection kit by 0.5 hour in detection speed, is higher than blocking ELISA used in the inspection and quarantine industry standard by 1.5 hours in detection speed, can detect multiple batches of samples within one day, and can meet the rapid epidemic disease diagnosis requirement.
Description
Technical field
The invention belongs to biotechnology and animal quarantine technical field, be specifically related to a kind of monoclonal antibody of specific bond Actinobacillus pleuropneumoniae serum 7-type antigen and secrete its hybridoma cell line, in addition, the invention still further relates to a kind of competitive ELISA and detect Actinobacillus pleuropneumoniae serum 7-type antibody kit.
Background technology
Porcine contagious pleuropneumonia (Porcine contagious pleuropneumoniae) is by Actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae, be 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 ratio of most acute can up to 80-100%, chronic type can cause pig poor growth, become cad pig, this disease is one of Compact Develop Prevention of Common Occurrence Porcine Disease, it is one of swine disease of China's import boar quarantine.China once repeatedly detected porcine contagious pleuropneumonia from the import boars such as the U.S., Denmark, Britain.According to the difference of Actinobacillus pleuropneumoniae soluble antigen capsular polysaccharide and boivin antigen, 15 serotypes can be divided into.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, Accurate Diagnosis infects the basis that serotype is this disease of prevention and corntrol.App serum 7-type is important pathogenic serotypes, and China all requires from the U.S., Canadian import boar this serotype of quarantining.The App Strain Virulence be separated from China Changchun such as to be linked to be according to Jilin University's thunder to measure, serum 7-type is very high to the fatal rate of mouse, reaches 66.5%.
ELISA detection method easily and fast, responsive, special, be the most frequently used method of antibody test.
This detection method does not need expensive instrument, and nearly all detection unit all has ELISA instrument, and ELISA kit is easy to apply.With regard to App, commercialization ELISA detection kit does not also only detect the kit of serum 7-type antibody both at home and abroad at present.Competitive ELISA is not adopted to detect the kit of App antibody yet.
Summary of the invention
One of the technical problem to be solved in the present invention is to provide the monoclonal antibody that can be combined with App serum 7-type antigen-specific.
Two of the technical problem to be solved in the present invention is to provide the hybridoma cell line that secretion produces said monoclonal antibody.
Three of the technical problem to be solved in the present invention is to provide the preparation method of monoclonal antibody of the present invention.
Four of the technical problem to be solved in the present invention is to provide the monoclonal antibody that a kind of chemical labeling in vitro is crossed.
The competitive ELISA that five of the technical problem to be solved in the present invention is to provide based on said monoclonal antibody development detects Actinobacillus pleuropneumoniae serum 7-type antibody kit.
The present invention is achieved by the following technical solutions:
In one aspect of the invention, a kind of hybridoma monoclonal cell secreting the monoclonal antibody of 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, confirm that monoclonal antibody secreted by it 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 CGMCC No.9051, preservation place: China, Beijing.
In another aspect of this invention, provide a kind of monoclonal antibody of specific bond Actinobacillus pleuropneumoniae serum 7-type antigen, the hybridoma cell line being CGMCC No.9051 by above-mentioned deposit number is secreted and is produced.
The term " monoclonal antibody (monoclonal antibody) " adopted herein refers to the immunoglobulin (Ig) obtained from a pure lines cell, has identical structure and chemical characteristic, has specificity to single antigenic determinat.Monoclonal antibody is different from conventional polyclonal antibody preparation (normally having the different antibodies for different determinant), and each monoclonal antibody is for the single determinant on antigen.Except their specificity, the benefit of monoclonal antibody is also that they are obtained by hybridoma or recombined engineering cell chulture, can not mix and have other immunoglobulin (Ig).Modifier " monoclonal " illustrates the characteristic of antibody, is to obtain from homogeneous antibody population, and this should not be construed as needing to produce antibody with any specific process.
The term " hybridoma cell line " (English name: hybridoma cell line) adopted herein, refers to the clone that screening and culturing goes out from hybridoma.
In another aspect of this invention, a kind of preparation method of said monoclonal antibody is provided, comprises the steps:
Step 1, preparation immunogene: preparation chocolate culture-medium, recover Actinobacillus pleuropneumoniae serum 7-type reference culture, mass propgation, bacterium is washed with physiological saline, centrifugal, abandon supernatant, suspension washed by bacterial precipitation physiological saline, centrifugal, and the S7 bacterium holoantigen that physiological saline suspends is as immunogene;
Step 2, mouse immune: by the mouse subcutaneous inoculation of repeated multiple times low dose, select the mouse of the anti-App serum 7-type polyclonal antibody can secreting high-titer;
Step 3, Fusion of Cells: get immune mouse spleen cell, merged with murine myeloma cell by external;
Step 4, positive hybridoma cell screen: with the positive hybridoma cell of indirect ELISA experiment sieving anti-Actinobacillus pleuropneumoniae serum 7-type antigen;
The acquisition of step 5, positive hybridoma cell: the hybridoma selecting strong positive reaction, clone 3 times continuously with limiting dilution assay, after last clone, the hole gained cell line of test positive is the hybridoma cell strain of stably excreting, special anti-Actinobacillus pleuropneumoniae serum 7-type antibody;
The cultivation of step 6, hybridoma and preservation: by positive hybridoma cell amplification cultivation, collecting cell; A part of for the production of mouse ascites antibody, another part Liquid nitrogen storage.
As the preferred technical scheme of the present invention, step 1 is specially: preparation chocolate culture-medium, Actinobacillus pleuropneumoniae serum 7-type reference culture is recovered, mass propgation, washes down bacterium with physiological saline, the centrifugal 10min of 8000r/min, abandon supernatant, suspension washed by bacterial precipitation physiological saline, centrifugal, so washes bacterium 3 times.Bacterial precipitation suspends with containing 0.5% formaldehyde physiological saline, places 4 DEG C of deactivations of spending the night, and the centrifugal 10min of 8000r/min removes formaldehyde, washes 3 times with physiological saline.The S7 bacterium holoantigen that physiological saline suspends is as immunogene;
As the preferred technical scheme of the present invention, step 2 is specially: by above-mentioned immunogene and isopyknic Freund's complete adjuvant mixing and emulsifying, lumbar injection is done to the female mouse of the BALB/C in 8 week age, 3-4 week is emulsified in freund 's incomplete adjuvant by same immunogene afterwards, make second time lumbar injection, use the last lumbar injection of immune original work of doubling dose 2-3 week afterwards, get mouse spleen after three days for Fusion of Cells.
As the preferred technical scheme of the present invention, step 3 is specially: get immune mouse spleen cell, is merged, use HAT Screening of Media by external with murine myeloma cell SP2/0 in the ratio of 5-10:1 under 50%PEG; Fused cell is cultivated after 5d, and change liquid more once with HAT nutrient culture media, 10d HT nutrient culture media changes liquid, by the time during the low 5%-30% of fused cell coverage hole, gets supernatant indirect ELISA method and screens.
As the preferred technical scheme of the present invention, in step 4, during screening, envelope antigen is the antigen of App S7 type through multigelation 3 process, and described antigen dilutes with 1:5000, and with the App of other serotype for negative control.
In addition, in another aspect of this invention, a kind of monoclonal antibody of above-mentioned anti-Actinobacillus pleuropneumoniae serum 7-type antigen of chemical labeling in vitro process is also provided.
The step of described chemical labeling in vitro process is as follows:
1) ascites monoclonal antibody ammonium sulfate is purified;
2) with improveing Over-voltage protection by antibody and HRP coupling, salting out method removes unconjugated HRP in sample, and final product, to PBS dialysed overnight, namely obtains the monoclonal antibody of the horseradish peroxidase-labeled that chemical labeling in vitro is crossed.
The pathogenetic bacteria inspection technology (the 1st edition p204 in Jilin science tech publishing house July in 1992) that " improvement Over-voltage protection " can be edited with reference to the Korean fine jade etc., key step is as follows:
(1) be dissolved in 0.5ml distilled water by 5mgHRP, add the 0.06mol/L NaIO4 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) add the aqueous solution 1ml containing 5mg antibody purification, load bag filter after mixing, with 0.05mol/L, pH value 9.5 carbonate buffer solution slowly stirs dialysis 6 hours or spends the night.
(4) after sucking-off, add NaBH4 solution (5mg/ml) 0.2ml, put refrigerator 2 hours.
(5) above-mentioned bond mixed liquor is added equal-volume saturated ammonium sulfate solution, puts refrigerator 30 minutes, centrifugal, gained sediment is dissolved in a little 0.02mol/L, in pH value 7.4PBS, and to dialysed overnight.
(6) centrifugal removing insolubles, namely obtains the monoclonal antibody that horseradish peroxidase (HRP) that chemical labeling in vitro crosses marks.
In addition, in another aspect of this invention, also provide a kind of competitive ELISA to detect Actinobacillus pleuropneumoniae serum 7-type antibody kit, mainly comprise: Actinobacillus pleuropneumoniae serum 7-type antigen; Chemical labeling in vitro process as claimed in claim 3 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 kit, comprises the steps: 1) envelope antigen; 2) plate is washed; 3) close; 4) plate is washed; 5) add tested serum, add the monoclonal antibody of the anti-App serum 7-type antigen of horseradish peroxidase-labeled simultaneously; 6) plate is washed; 7) substrate is added; 8) cessation reaction; 9) light absorption value (OD value) is detected; 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 greater than 0.8; (2) the OD positive/OD feminine gender≤20%; Contrast is set up just can carry out sample judgement; (3) blood serum sample 1:16 dilutes, and its S/N value (OD sample/negative OD)≤20% is positive, and S/N > 40% is negative, and 40% >=S/N > 20% is suspicious.
Compared with prior art, beneficial effect of the present invention is: for accurately detecting App serum 7-type, on the basis of development App serum 7-type monoclonal antibody, have developed competitive ELISA and detects Actinobacillus pleuropneumoniae serum 7-type antibody kit.Compared with use polyclonal antibody, use monoclonal antibody, detection kit is easily produced in batches, and the quality of kit easily controls.Compared with indirect ELISA, competitive ELISA detects without kind Idiotype, except detection pig antibody, can also detect the animal used as test antibody such as mouse, may 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 stop band restrain of inspection and quarantine industry standard use, 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-PAGE IgM 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 2ug BSA; 2 represent 4ug BSA; 3 represent 8ug BSA; 4 represent 16ug BSA; 5 represent 1ul IgM (diluting 10 times); 6 represent 2ul IgM (diluting 10 times); 7 represent 4ul IgM (diluting 10 times).
Fig. 2 is that the enzyme concentration that in the embodiment of the present invention 3,1:1 ten thousand dilutes detects the result schematic diagram of different dilutability antigen;
Fig. 3 is that the enzyme concentration that in the embodiment of the present invention 3,1:2 ten thousand dilutes detects the result schematic diagram of different dilutability antigen;
Fig. 4 is the scatter diagram of the positives serum ratio of the embodiment of the present invention 3;
Fig. 5 is the scatter diagram of negative serum ratio in the embodiment of the present invention 3;
Fig. 6 is the change schematic diagram of different time feminine gender and positive serum light absorption value in the embodiment of the present invention 3;
Code name is the mouse hybridoma cell strain (deriving from mouse Mus musculus) of SHCIQ-S7-8D12, detect through indirect ELISA and competitive ELISA, confirm that monoclonal antibody secreted by it 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 CGMCC No.9051, preservation place: No. 3, No. 1, North Star West Road, Chaoyang District, city of BeiJing, China institute, Institute of Microorganism, Academia Sinica.
Embodiment
The following examples can make the present invention of those skilled in the art's comprehend, but do 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 (App S7) recovery, mass propgation, with physiological saline, bacterium is washed down, the centrifugal 10min of 8000r/min, abandon supernatant, bacterial precipitation physiological saline suspends, centrifugal, so washes bacterium 3 times.Bacterial precipitation suspends with containing 0.5% formaldehyde physiological saline, places 4 DEG C of deactivations of spending the night, and the centrifugal 10min of 8000r/min removes formaldehyde, washes 3 times with physiological saline.The App S7 bacterium holoantigen that physiological saline suspends is as immunogene; Step 2, BALB/C mice immunity: by above-mentioned immunogene (containing 10
4actinobacillus pleuropneumoniae) and isopyknic Freund's complete adjuvant mixing and emulsifying, lumbar injection immunity is done to the female mouse of the BALB/C in 8 week age, 3-4 week is emulsified in freund 's incomplete adjuvant by same immunogene afterwards, do the immunity of second time lumbar injection, 2-3 week carries out lumbar injection booster immunization by the immunogene of doubling dose afterwards, gets mouse spleen for Fusion of Cells after three days.Step 3, Fusion of Cells: get above-mentioned immune mouse spleen cell and murine myeloma cell (SP2/0) ratio in 5-10:1, mix in RPMI-1640 (Gibco) nutrient culture media of serum-free, the centrifugal 5min of 1500rpm, remove nutrient culture media, 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, stop merging the rear centrifugal 5min of 1500rpm with the RPMI-1640 nutrient culture media of serum-free, precipitation HAT nutrient culture media suspends, being dispensed into 96 holes contains in the cell plates of feeder cells, 37 DEG C, 5%CO
2cell culture incubator in cultivate.Step 4, positive hybridoma cell screen: cultivate after 5 days in cell culture incubator, liquid is changed once with HAT nutrient culture media, within 10th day, change liquid with HT nutrient culture media, by the time the positive hybridoma cell of anti-Actinobacillus pleuropneumoniae serum 7-type antigen-antibody is secreted at the bottom of fused cell coverage hole during 5%-30% with indirect ELISA experiment sieving, during screening, envelope antigen is that through the antigen of multigelation 3 process, (1:5000 dilutes App S7 type, weight/volume, g/ml), and with the App of other serotype for negative control.The acquisition of step 5, positive hybridoma cell: the hybridoma wells selecting indirect ELISA strong positive reaction, with the continuous clone cell of limiting dilution assay 3 times, after last clone, the hole gained cell line 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;
The cultivation of step 6, hybridoma and preservation: by positive hybridoma cell amplification cultivation, collecting cell; A part for the production of mouse ascites antibody, with cryopreserving liquid suspend be sub-packed in cryopreservation tube after another part cell after preserve in liquid nitrogen.
Prepared by step 7, ascites: get healthy BALB/C mice in 8 week age, the whiteruss of an Intraperitoneal injection 0.5ml/ sterilizing, and only (concentration is 10 within 10 days, to inject hybridoma 0.5ml/ afterwards
6individual/ml), treat that mouse web portion expands, during moving difficulty, thrust mouse web portion with large size syringe needle and collect ascites, the centrifugal 3min of 4000g, supernatant is ascites monoclonal antibody.
Embodiment 2 chemical labeling in vitro monoclonal antibody
Key step is:
1, the hypotype qualification of monoclonal antibody: the mouse monoclonal antibody detection kit (Mouse Mab Isotying test kit) using Dutch HyCult Biotechnology to produce, step is as follows:
A) cell conditioned medium or ascites pH7.4PBS are suitably diluted, for subsequent use to antibody concentration 1-50 μ g/mL;
B) 500 μ L dilutions are added in the detector tube containing a test strips;
C) add again 500 μ L a) in the sample that diluted in detector tube;
D) test strips immersed completely and stir gently;
E) jog bottle mixing rabbit against murine κ chain micelle, gets 1mL and is added in detector tube;
F) vibrate in 18-25 DEG C of environment about 30min sentence read result, test strips remains leaching in the solution in oscillatory process.
After testing, the monoclonal antibody that the hybridoma monoclonal cell system (preservation code name is SHCIQ-S7-8D12) of the stably excreting anti-Actinobacillus pleuropneumoniae serum 7-type antibody of the present invention's foundation secretes is IgM hypotype.
2, the purifying of monoclonal antibody
Ascites monoclonal antibody ammonium sulfate is purified
A) 2ml ascites adds the dilution of 2ml PBS solution, and obtain 4ml solution altogether, add saturated ammonium sulfate solution 4ml (slowly add, add and mix), 4 DEG C of placements are spent the night;
B) 4 DEG C of centrifugal (10000rpm, 10 minutes) supernatant discarded, precipitation adds 4ml PBS solution, and resuspended, add saturated ammonium sulfate solution 2ml (slowly add, add and mix), 4 DEG C of placements are spent the night;
C) 4 DEG C of centrifugal (10000rpm, 10 minutes) supernatant discarded, precipitation adds 4ml PBS solution, and resuspended, add saturated ammonium sulfate solution 2ml (slowly add, add and mix), 4 DEG C of placements are spent the night;
D) 4 DEG C of centrifugal (10000rpm, 10 minutes) supernatant discarded, precipitation adds 2ml PBS solution, dialyses 24 hours (changing 2 dislysates) to 1LPBS.
3, the electrophoresis detection of monoclonal antibody content
Preparation 12%SDS-PAGE glue, after being diluted by the IgM monoclonal antibody 10 times of purifying, gets 1ul, 2ul, 4ul electrophoresis respectively, with BSA as qualitative reference, sees Fig. 1.According to electrophoresis detection result, after purifying, the concentration of the IgM of (namely before mark) is about 10mg/ml.
4, the mark 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 improveing Over-voltage protection by antibody and HRP coupling, key step is as follows:
(1) be dissolved in 0.5ml distilled water by 5mgHRP, add the 0.06mol/L NaIO4 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) add the aqueous solution 1ml containing 5mg antibody purification, load bag filter after mixing, with 0.05mol/L, pH value 9.5 carbonate buffer solution slowly stirs dialysis 6 hours or spends the night.
(4) after sucking-off, add NaBH4 solution (5mg/ml) 0.2ml, put refrigerator 2 hours.
(5) above-mentioned bond mixed liquor is added equal-volume saturated ammonium sulfate solution, puts refrigerator 30 minutes, centrifugal, gained sediment is dissolved in a little 0.02mol/L, in pH value 7.4PBS, and to dialysed overnight.
(6) centrifugal removing insolubles, namely obtains the monoclonal antibody that horseradish peroxidase (HRP) that chemical labeling in vitro crosses marks.
5, the enzyme mark Preliminary Determination of tiring
Use Salmonella, envelope antigen is App S7 and App S6, and equal 1:50 doubly dilutes, and the latter is as negative control.Diluted according to 1:625-1:40000 by enzyme mark monoclonal antibody, titration enzyme labelled antibody is tired, in table 1, and initial option enzyme concentration 1:1 ten thousand-1:2 ten thousand.
Table 1 enzyme labelled antibody is tired titration
Embodiment 3 competitive ELISA detects the test of Actinobacillus pleuropneumoniae serum 7-type antibody kit
The test routine of competitive ELISA kit, comprise following content: 1) antigen prepares 2) antigen valence titration 3) determination 4 of the best dilute concentration of Sample serum) 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) 37 DEG C of closed 1h; D) plate is washed 3 times; E) add tested serum, add the monoclonal antibody of the anti-App serum 7-type antigen of horseradish peroxidase-labeled simultaneously, 37 DEG C of reaction 30min; F) plate is washed 3 times; G) substrate is added, 37 DEG C of reaction 10min; H) cessation reaction; I) light absorption value (OD value) is detected; J) also result of determination is calculated.Specific as follows:
1) preparation of antigen
At the upper bacterium colony cultivating 6h of TSCA nutrient culture media (containing 0.1%NAD), elute with the physiological saline containing 0.5% formalin (formaldehyde) and be collected in a bottle, be positioned over 4 DEG C, spend the night; Next day, 8000r/min, removes supernatant in centrifugal 15 minutes, and precipitation physiological saline is resuspended and to adjust bacterial concentration be 10
10cFU/mL.Again the bacterium liquid of suspendible be positioned in 100 DEG C of water-baths and act on 1h, after cooling, the centrifugal 20min of 20000r, retains supernatant.With the membrane filtration supernatant of 0.22 μm, filtrate is polysaccharide antigen.Packing, puts-20 DEG C and saves backup.
2) titration of antigen valence
Antigen 1: 200 to 1:3200 is diluted, enzyme concentration uses 1:1 ten thousand and 1:2 ten thousand, competitive ELISA is used to detect the positive, negative Swine serum and blank, the results are shown in Table 2, Fig. 2 is the enzyme concentration that 1:1 ten thousand dilutes, Fig. 3 is the enzyme concentration that 1:2 ten thousand dilutes, when antigen is below 1:800 dilutability, 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, enzyme concentration is selected to be 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 the best dilute concentration of Sample serum
Use the above S7 antigen determined 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 dilution) simultaneously, during result serum 1:16, negative serum is not almost competed, in table 3, therefore, selection serum 1:16 is the best dilute concentration of tested serum.
Table 3 competitive ELISA measures the OD value of different serum dilution
4) competitive ELISA detects the determination of Sample Negative and positive critical value (Cutoff value)
For determining the serum 235 parts of cutoff value, wherein for determining the positive 32 parts of cutoff value, for App serum 7-type bacterium attacks malicious pig or the clinical quarantine positive, be all defined as positive Swine serum with two kinds of commercial kits.For determining the serum 203 parts that cutoff value is negative, supplying 36 parts, pig farm, port from 90 parts, Canada, 22 parts, France, Denmark 55 parts of import boars and Shanghai respectively, all detecting through two kinds of commercial kits and being defined as negative Swine serum.The equal 1:16 dilution of serum, detects according to competitive ELISA kit regulated procedure.The light absorption value of positive serum and the number percent of negative control light absorption value, in table 4, do scatter diagram according to its ratio, see Fig. 4, and the number percent of negative serum light absorption value and negative control light absorption value, in table 5, does scatter diagram 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 is positive critical value for being less than or equal to 20%.Analyze most of negative ratio 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 critical value when being greater 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 (i.e. coating buffer) of pH9.5, get 96 hole ELISA Plate, every hole adds 100ul, 4 DEG C of refrigerator overnight.
B) take out bag by plate, 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% skimmed milk power in PBS-T) of 200 microlitres, after sticking shrouding film, places 37 DEG C of constant temperature ovens, 1 hour.
D) plate is washed 3 times.
E) Sample serum dilution plate in dilution (adding 1% skimmed milk power in PBS-T) 1:16 dilution (as, get 10ul serum and add 150ul dilution), getting 100ul adds in reacting hole, add the monoclonal antibody of the horseradish peroxidase-labeled of 100ul working concentration simultaneously, each sample does a hole, arranges positive serum controls, positive serum controls simultaneously.Negative and negative control is each does 2 holes, after sticking shrouding film, 37 DEG C of constant temperature ovens, react 30 minutes.Sample is recommended to do diplopore.
F) plate is washed 3 times.
G) every hole adds 100 microlitre reaction substrate TMB (tetramethyl benzidine), 37 DEG C of constant temperature ovens, 10min.
H) every hole adds 50 microlitre 2Mol/L sulfuric acid cessation reactions.
I) light absorption value is measured at 450nm.
J) result of calculation: negative control sera mean OD value is greater than 0.8; 2) positive/average OD feminine gender≤20% of average OD; Contrast 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 revision test, if be positive or the suspicious positive that is judged to, if be feminine gender, be judged to feminine gender.
6) stability test of competitive ELISA kit
A) preparation feedback plate
With coating buffer dilution antigen to working concentration, get 96 hole ELISA Plate, every hole adds 100ul, 4 DEG C of refrigerator overnight; Take out bag by plate, wash 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 is preserved, and 4 DEG C of Refrigerator stores are for subsequent use.
B) serum is according to after adding antiseptic, and 4 DEG C of Refrigerator stores are for subsequent use.
C) enzyme labelled antibody
Diluted according to 1:100 by enzyme labelled antibody with stabilizing agent (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) according to competitive ELISA trace routine, the different time periods detects light absorption value that is negative, positive serum, specifically in table 6, and Fig. 6.Result enzyme be stored in 37 DEG C the 7th day, negative serum OD value does not significantly change, and the 9th day OD value significantly declines, but the ratio positive and negative by the 16th day is still lower than 20%, and positive control is still set up.Conveniently 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 kit was minimum 10 months of 4 DEG C of shelf-lifves.
In table 6ELISA kit, enzyme is stored in 37 DEG C of different time testing results
7) replica test of competitive ELISA kit
According to competitive ELISA trace routine, to 4 parts of serum duplicate detection 7 times, result is completely the same, conforms to expection, specifically in table 7.
Table 7 competitive ELISA revision test 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 qualification App monoclonal antibody step:
1. contrast the App reference culture that antigen comprises other 14 types except S7 type, the same S7 of production of these bacterial strains.Other related strain 28 strain, these bacterial strains are produced according to respective requirement.
2. coating buffer bag is by various antigen, wraps and is about 1x10 by concentration
7, 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 holes add 50 microlitre 2Mol/L sodium hydroxide solution cessation reactions.
12. measure light absorption value at 405nm
After testing, 8D12-C10 monoclonal antibody only can specific detection S7 antigen, and can not detect 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
Embodiment 5 competitive ELISA detects the specific detection of Actinobacillus pleuropneumoniae serum 7-type antibody kit
The control serum that specific test uses is, the pig hyper-immune serum of App serum 1-12 type, the rabbit hyper-immune serum of App serum 4 type, 7 types, the positive serum of 11 parts of relevant swine diseases, and the Lin Shi Actinobacillus rabbit hyper-immune serum nearest with App homology.Detect 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 detects Actinobacillus pleuropneumoniae serum 7-type and attacks malicious pig different time antibody level of serum
App serum 7-type reference culture is used to attack poison No. 1 pig and No. 2 pigs, attack poison before and attack poison latter 56 days, take a blood sample weekly once, and 134 days and blood sampling in 143 days, detect with competitive ELISA, before result attacks poison and attack poison latter 7 days antibody for negative, attack poison after 14 days antibody be the positive (except No. 1 pig attacks latter 28 days of poison for suspicious).Specifically in table 11, illustrate that App serum 7-type is attacked poison and produced detectable antibody in latter 14 days.
Table 11 competitive ELISA detects App serum 7-type reference culture and attacks malicious pig antibody horizontal
Remarks: cutoff value 1.395X20%=0.2791.395X40%=0.558
Embodiment 7 competitive ELISA detects and attacks malicious Swine serum antibody dynamic regularity
App serum 7-type reference culture is attacked poison and is handed over large No. 2 pigs, and immune programme for children is: App S7 cultivates 18h, and physiological saline is washed down, intranasal inoculation 4.5x10
8individual bacterium; Within 6th day, get 6h cultivation App, 3ml and be about 8.5x10
9individual bacterium adds equivalent incomplete Freund's adjuvant and divides three position intramuscular injection; Within 7th day, get 6h and cultivate App, about 7.5x10
9individual bacterium (0.2% formalin normal saline dilution, 4 DEG C of refrigerator overnight) intravenous injection; 24th day ditto repeats once, bacterium amount 2.5x10
9individual bacterium; Within 28th day, get 6h and cultivate App, bacterium amount 2.5x10
9individual bacterium (normal saline dilution) intravenous injection; 32nd day ditto repeats once.Attack poison before and take a blood sample weekly once after attacking poison, separation of serum ,-20 DEG C save backup.
The serum gathered by different time is with after physiological saline doubling dilution, detect according to the competitive ELISA method set up, result attacks poison can detect antibody after 14 days, through immunity several times repeatedly, tire after 35 days and rise to 1:64 (final concentration 1:1024), after 143 days, antibody titer still maintains higher level, specifically in table 12.
Table 12 hands over large No. 2 pig S7 to attack the rear antibody dynamic regularity of poison
Embodiment 8 competitive ELISA detects Actinobacillus pleuropneumoniae serum 7-type antibody kit and compares with other commercialization detection kit.
1, competitive ELISA detects Actinobacillus pleuropneumoniae serum 7-type antibody kit and detects and attack malicious pig antibody and compare with French Idvet company kit (detecting App4/7 type) and Canadian Biovet kit (detecting App4/5/7 type), and result detects No. 2 pigs three kinds of kits and conforms to completely.If can be regarded as the positive suspicious, detect No. 1 pig, have a sample not to be inconsistent, all the other all conform to, and concrete outcome is in table 13.
The detection of table 13 competitive ELISA is attacked malicious pig antibody and is compared with IDvet and biovet.
2, competitive ELISA detects the detection of Actinobacillus pleuropneumoniae serum 7-type antibody kit from Denmark, France, Canada, imported from America boar, and Shanghai supplies Swine serum 259 parts of clinical samples of pig farm, port and Chongqing censorship, with French Idvet company kit (detecting App4/7 type), Canada Biovet kit (detecting App4/5/7 type) compares, and the results are shown in Table 14.
Table 14 three kinds of kits detect clinical sample result
3, competitive ELISA detects the rate that conforms to of Actinobacillus pleuropneumoniae serum 7-type antibody kit and other commercialization detection kit
Compare after the result detected above is gathered (see table 15), if can be regarded as the positive by suspicious, competitive ELISA is 97.2% with the rate that conforms to of Idvet, is 98.2% with the rate that conforms to of Biovet; If can be regarded as feminine gender by suspicious, then competitive ELISA is 97.2% with the rate that conforms to of Idvet, is 97.9% with the rate that conforms to of Biovet, calculates under seeing.Illustrate that the competitive ELISA kit that the present invention sets up and commercialization detection kit have good compatibility, can be used for the detection of clinical sample.
Table 15 three kinds of kits detect clinical sample result
Three kinds of kits rate that conforms to is calculated as follows:
Can be regarded as positive phase competitive ELISA by suspicious with the rate that conforms to of Idvet be: (281-8)/281=97.2%
Can be regarded as positive phase competitive ELISA by suspicious with the rate that conforms to of Biovet be: (281-5)/281=98.2%
Can be regarded as negative phase competitive ELISA by suspicious with the rate that conforms to of Idvet be: (281-8)/281=97.2%
Can be regarded as negative phase competitive ELISA by suspicious with the rate that conforms to of Biovet be: (281-6)/281=97.9%
Claims (10)
1. secrete a hybridoma cell line for the monoclonal antibody of specific bond Actinobacillus pleuropneumoniae serum 7-type antigen, its deposit number is CGMCC No.9051.
2. a monoclonal antibody for specific bond Actinobacillus pleuropneumoniae serum 7-type antigen, is secreted by hybridoma cell line according to claim 1 and produces.
3. a preparation method for monoclonal antibody as claimed in claim 2, comprises the steps:
Step 1, preparation immunogene: preparation chocolate culture-medium, recover Actinobacillus pleuropneumoniae serum 7-type reference culture, mass propgation, bacterium is washed with physiological saline, centrifugal, abandon supernatant, suspension washed by bacterial precipitation physiological saline, centrifugal, and the S7 bacterium holoantigen that physiological saline suspends is as immunogene;
Step 2, mouse immune: by the mouse subcutaneous inoculation of repeated multiple times low dose, select the mouse of the anti-App serum 7-type polyclonal antibody can secreting high-titer;
Step 3, Fusion of Cells: get immune mouse spleen cell, merged with murine myeloma cell by external;
Step 4, positive hybridoma cell screen: with the positive hybridoma cell of indirect ELISA experiment sieving anti-Actinobacillus pleuropneumoniae serum 7-type antigen;
The acquisition of step 5, positive hybridoma cell: the hybridoma selecting strong positive reaction, clone 3 times continuously with limiting dilution assay, after last clone, the hole gained cell line of test positive is the hybridoma cell strain of stably excreting, special anti-Actinobacillus pleuropneumoniae serum 7-type antibody;
The cultivation of step 6, hybridoma and preservation: by positive hybridoma cell amplification cultivation, collecting cell; A part of for the production of mouse ascites antibody, another part Liquid nitrogen storage.
4. preparation method as claimed in claim 3, it is characterized in that, step 1 is specially: preparation chocolate culture-medium, is recovered by Actinobacillus pleuropneumoniae serum 7-type reference culture, mass propgation, with physiological saline, bacterium is washed down, the centrifugal 10min of 8000r/min, abandons supernatant, and suspension washed by bacterial precipitation physiological saline, centrifugal, so wash bacterium 3 times.Bacterial precipitation suspends with containing 0.5% formaldehyde physiological saline, places 4 DEG C of deactivations of spending the night, and the centrifugal 10min of 8000r/min removes formaldehyde, washes 3 times with physiological saline.The S7 bacterium holoantigen that physiological saline suspends is as immunogene.
5. preparation method as claimed in claim 3, it is characterized in that, step 2 is specially: by above-mentioned immunogene and isopyknic Freund's complete adjuvant mixing and emulsifying, lumbar injection is done to the female mouse of the BALB/C in 8 week age, 3-4 week is emulsified in freund 's incomplete adjuvant by same immunogene afterwards, make second time lumbar injection, use the last lumbar injection of immune original work after 2-3 week, get mouse spleen after three days for Fusion of Cells.
6. preparation method as claimed in claim 3, it is characterized in that, step 3 is specially: get immune mouse spleen cell, is merged, use HAT Screening of Media by external with murine myeloma cell SP2/0 in the ratio of 5-10:1 under 50%PEG; Fused cell is cultivated after 5d, and change liquid more once with HAT nutrient culture media, 10d HT nutrient culture media changes liquid, by the time during the low 5%-30% of fused cell coverage hole, gets supernatant indirect ELISA method and screens.
7. preparation method as claimed in claim 3, is characterized in that, in step 4, during screening, envelope antigen is the antigen of App S7 type through multigelation 3 process, and described antigen dilutes with 1:5000, and with the App of other serotype for negative control.
8. 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.
9. a monoclonal antibody as claimed in claim 8, is characterized in that, the step of described chemical labeling in vitro process is as follows:
1) ascites monoclonal antibody ammonium sulfate is purified;
2) with improveing Over-voltage protection by antibody and HRP coupling, salting out method removes unconjugated HRP in sample, and final product, to PBS dialysed overnight, namely obtains the monoclonal antibody of the horseradish peroxidase-labeled that chemical labeling in vitro is crossed.
10. competitive ELISA detects an Actinobacillus pleuropneumoniae serum 7-type antibody kit, it is characterized in that, mainly comprises: Actinobacillus pleuropneumoniae serum 7-type antigen; Chemical labeling in vitro process as claimed in claim 3 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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