CN104877027B - Anti- pig SC protein monoclonal antibodies and its application in terms of mycoplasma hyopneumoniae SIgA antibody ELISA detection kit is prepared - Google Patents

Abstract

The present invention provides anti-pig SC protein monoclonal antibodies and its application in terms of mycoplasma hyopneumoniae SIgA antibody ELISA detection kit is prepared, and is related to animal virology and epizootiology detection technique field.The anti-pig SC protein monoclonal antibodies are secreted by hybridoma cell strain 4H11, and preserving number is：CCTCC NO：C201526.The monoclonal antibody and its application in terms of mycoplasma hyopneumoniae SIgA antibody ELISA detection kit is prepared is also claimed in the present invention.Kit of the present invention has high specific, high stability, hypersensitivity, it is convenient to detect sample, it can distinguish that porcine mycoplasmal pneumonia inactivated vaccine is immune and natural infection, the assessment of immune effect after the early diagnosis and attenuated live vaccines available for the infection of pneumonia porcine mycoplasmal are immune.

Description

Anti- pig SC protein monoclonal antibodies and its preparing mycoplasma hyopneumoniae SIgA antibody Application in terms of ELISA detection kit

Technical field

The present invention relates to animal virology and epizootiology detection technique field, and in particular to anti-pig SC albumen Dan Ke Grand antibody and its applying.

Background technology

Mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae, Mhp) is to cause porcine mycoplasmal pneumonia (MPS), custom Claim the cause of disease of swine enzootic pneumonia.Swine enzootic pneumonia is that prevalence is most wide in swinery, propagates one of most fast, to be most difficult to purification epidemic disease.Pig pneumonia Mycoplasma is by respiratory infectious, and infecting causes the atrophy of respiratory tract cilium after airway epithelial, come off, damage, and epithelial cell is bad Extremely, the immunologic function of respiratory mucosa is reduced, causes lung functions to be damaged, easily produces the secondary sense of other respiratory pathogenses Dye.

Research shows that the prevention to MPS mainly passes through the local cellular immunity of respiratory tract and mucosa-immune.SIgA (secretions Type immunoglobulin A) it is the main effects factor for participating in mucosa-immune reaction.They are the important compositions in body immune system Part, played an important role respectively in immune response.SIgA can it is antiviral, neutralize a toxin and other bioactivity antigens, With extensive immanoprotection action, but most important protective effect is to prevent bacterium from adhering to surface epithelial cell, and is removed The cause of disease of invasion.It is also caused immune response at first after pathogen intrusion, therefore, is often made according to specific SIgA detection For the early diagnosis of disease.SIgA is made up of secretion composition (SC albumen), IgA and J chains.SC albumen is that on epithelial cell exempt from more The extracellular fragment part of epidemic disease Ig receptor (PlgR), it is the first defence line of mucosa-immune, can prevents in respiratory tract and enteric cavity The invasion of the harmful substances such as virus, bacterium, toxin and alien material.SC albumen enhances SIgA best protection effect, makes SIgA declines to the sensitiveness of protease, and mucus is more viscous, enhances adhesion and defence capability.SC albumen is mucosa-immune The important component of system, when SC can not be produced normally, slgA can be caused can not normally to synthesize and trigger a variety of diseases.SC albumen Also it is to discriminate between the mark of serotype IgA and secretory IgA.

Anti-mycoplasma hyopneumoniae SIgA be Mhp infection or be immune after in respiratory tract specificity marker caused first, simultaneously It also reflects the protecting effect after vaccine immunity.

The diagnostic method of mycoplasma hyopneumoniae includes Pathogen test and Serum Antibody Detection.The Methods of Detection of Pathogens includes cause of disease Separation and identification, PCR (PCR), real-time quantitative PCR and hybridization in situ, but these methods exist it is quick Perception and specificity it is relatively low, it is time-consuming compared with it is long, detection difficulty is big, testing result is uncertain, required appointed condition and is not suitable for living The shortcomings of physical examination is surveyed.Serum Antibody Detection method includes：Flame atomic absorptionspectrometry (IHA) and EUSA (ELISA) etc..But there is detection titre is low, result is low with the correlation of infection, aggegation terminal point determining in IHA detection Subjective and technological deficiency inherently the problems such as, therefore IHA practical applications in the Mhp detections of commodity swinery Limited promise.The Sensitivity and Specificity of the ELISA method of existing detection serum antibody can not meet that diagnosis requires.In addition, mesh Preceding ELISA method used is for detecting Mhp IgG antibodies.After mycoplasma hyopneumoniae infection, IgG antibody produces phase To slower；And research shows that the humoral immunity of whole body is little to the immanoprotection action of the cause of disease.On the contrary, respiratory tract locally produces Raw mucosa-immune and cellular immunity can produce preferable immune protective effect.Therefore, the ELISA of existing serum antibody is not applied to The monitoring of immune indexes after the sick early diagnosis and active immunity.

R1 areas are Mhp ciliums adhesion protein (also known as adhesion factor or adhesin) P97 repeat units, are present in Cilium adhesion protein P97 C-terminal, and the antigenic determinant that the albumen is main.Mhp cilium adhesion proteins P97 R1 areas are referred to as P97R1 albumen.Mhp then produces follow-up pathogenic effects by adhering to infected pigs' bronchus ciliated epithelial cell.Body is directed to The adhesion energy of mycoplasma produces rapidly corresponding mucosa-immune reaction.If by the use of P97R1 albumen as coating proper energy after infection The very first time detects corresponding SIgA reactions, is suitable for the sick early diagnosis.And P97 albumen has species specificity, with Serology cross jamming is not present in other porcine mycoplasmals.

The content of the invention

It is an object of the invention to provide the hybridoma cell strain for secreting anti-pig SC protein monoclonal antibodies.

, should another object of the present invention is to provide the anti-pig SC protein monoclonal antibodies of the hybridoma cell strain secretion Monoclonal antibody has higher specificity.

Another object of the present invention is to provide the anti-pig SC protein monoclonal antibodies and is preparing mycoplasma hyopneumoniae SIgA Application in terms of antibody ELISA detection kit.

It is yet another object of the invention to provide mycoplasma hyopneumoniae SIgA antibody ELISA detection kit, has higher Specificity, sensitivity and stability, it is possible to distinguish porcine mycoplasmal pneumonia inactivated vaccine is immunized and natural infection, for examining in early days It is disconnected；It can be additionally used in the assessment of the immune rear immune effect of porcine mycoplasmal pneumonia attenuated live vaccines.

To achieve the above object, the technical solution adopted by the present invention is as follows：

A kind of hybridoma cell strain 4H11 for secreting anti-pig SC protein monoclonal antibodies, preserving number are：CCTCC NO： C201526。

The present invention is also claimed the anti-pig SC protein monoclonal antibodies of the hybridoma cell strain secretion and its prepared Application in terms of mycoplasma hyopneumoniae SIgA antibody ELISA detection kit.

Mycoplasma hyopneumoniae SIgA antibody ELISA detection kit, pig is coated with containing what is prepared by antibodycapture The enzyme of the detection plate of mycoplasma pneumoniae P97R1 albumen and anti-pig SC protein monoclonal antibodies containing horseradish peroxidase-labeled Conjugate working solution, the anti-pig SC protein monoclonal antibodies are that hybridoma cell strain is secreted as described in claim 1.

In the present invention, the detection plate for being coated with mycoplasma hyopneumoniae P 97 R 1 albumen is prepared with the following method：Will detection Plate is first coated with using anti-mycoplasma hyopneumoniae P 97 R 1 protein monoclonal antibody, then using mycoplasma hyopneumoniae P 97 R 1 albumen bag Quilt.

In preferable technical scheme, the coating concentration of the anti-mycoplasma hyopneumoniae P 97 R 1 protein monoclonal antibody is 1.5-2.5 μ g/mL, the coating concentration 1.5-2.5 μ g/mL of the P97R1 albumen.

In the present invention, the kit also includes positive control solution, negative controls, cleaning solution, nitrite ion and termination Liquid.

In preferable technical scheme, positive control solution is the pig bronchoalveolar lavage fluid of artificial infected pigs' mycoplasma pneumoniae, negative Comparison liquid is to be uninfected by the pig bronchoalveolar lavage fluid of mycoplasma hyopneumoniae；Wash liquid making method：Take 40g NaCl, 1.0g KCl, 14.5g Na₂HPO₄·12H₂O、1.2g KH₂PO₄, 2.5ml Tween-20s be dissolved in deionized water, be settled to 500ml；Nitrite ion bag Include nitrite ion A and nitrite ion B, wherein nitrite ion A is by 21mg3,3', 5, after 5'- tetramethyl benzidines are dissolved in 5ml absolute ethyl alcohols Obtain；The nitrite ion B is obtained after 33mg urea peroxide elements are dissolved in into 200ml phosphate buffers；The terminate liquid is 2mol/L sulfuric acid solution.

Beneficial effect：

1. high specific：This kit ensures the high specific of detection by following three aspects.(1) captured by monoclonal antibody Coating method ensures the pure property of envelope antigen P97R1 albumen, avoids in the P97R1 proteantigens prepared by Bacillus coli expression Influence of the foreign protein to detection sensitivity；(2) the envelope antigen P97R1 albumen that detection plate uses is the special of mycoplasma hyopneumoniae Property albumen, avoids other porcine mycoplasmals or the cross jamming of cause of disease in measuring samples；(3) in enzyme conjugates working solution activity into It is divided into the anti-pig SC protein monoclonal antibodies of HRP marks, therefore, only mycoplasma hyopneumoniae SIgA antibody could be detected, and be kept away The interference of IgG and IgM antibody in measuring samples is exempted from.Experimental result displays that kit of the present invention will not be with swinery common virus Pig breathes and reproductive syndrome virus, pseudorabies, H1 types swine influenza virus, pig pleuropneumonia, mycoplasma hyorhinis and large intestine bar Bacterium SIgA antibody positive nose swabs sample produces cross reaction.

2. high stability and hypersensitivity：Due to the antigen-reactive ability difference of envelope antigen prepared by different batches, lead to Cross detection plate prepared by antibody capture coating method and ensure that the high stability of different batches of product, and ensure that antibody test plate The high response of middle envelope antigen, substantially increase the hypersensitivity of kit.

3. sample is convenient：Measuring samples are nose swab, rather than serum, realize needlelessization sampling, reduce to pig Stress.

4. can distinguish, porcine mycoplasmal pneumonia inactivated vaccine is immune and natural infection：Intramuscular injection is immunized porcine mycoplasmal pneumonia and gone out After live vaccine, the specific IgG antibodies in serum are only produced, and specific SIgA antibody can not be produced on respiratory tract surface.Pig The natural infection of mycoplasma pneumoniae occurs directly in respiratory tract target device pipe, and specific IgG antibodies can be both produced in serum, again may be used Very strong specific SIgA antibody (mycoplasma pneumoniae SIgA antibody) is produced on respiratory tract surface.Using reagent provided by the invention Field monitoring infection conditions can be immunized in porcine mycoplasmal pneumonia inactivated vaccine in box, and mycoplasma hyopneumoniae antibody in the market is examined Test agent box can not be accomplished.

5. the early diagnosis available for the infection of pneumonia porcine mycoplasmal：The adhesins such as mycoplasma hyopneumoniae P 97 R 1 albumen adsorb It is the first step of mycoplasma hyopneumoniae infection host to Pig bronchial epithelial cell, anti-mycoplasma hyopneumoniae P 97 R 1 albumen resists Body be also host be directed to it is earliest a collection of in antibody caused by mycoplasma hyopneumoniae difference albumen；Host response respiratory pathogenses sense The mucosa-immune that caused immune response at first is respiratory tract is contaminated, followed by the general immunity in blood.SIgA antibody is glutinous Most important immune molecule during film is immune.The kit is using P97R1 albumen as envelope antigen, using respiratory tract SIgA antibody as detection Object, the early stage to mycoplasma hyopneumoniae infection is realized in terms of pathogen infection step and host immune response order two respectively Diagnosis.

6. the assessment available for porcine mycoplasmal pneumonia attenuated live vaccines immune effect after immune：The weak poison of porcine mycoplasmal pneumonia is living The more difficult specific IgG antibodies reaction that high titre is produced in serum, can not temporarily pass through existing commercial pig after vaccine immunity Mycoplasma pneumiae anti-body detection reagent box is assessed Attenuate vaccine immune effect.But attenuated vaccine immunity can be in respiratory tract The specific SIgA antibody compared with high titre is produced, therefore, the immune effect of attenuated vaccine can be carried out by this kit indirect Assess.

Brief description of the drawings

Fig. 1 is the checking electrophoretogram of pig SC GFP fragments, wherein M-DNA marker, 1-SC albumen PCR primers

Fig. 2：(A) the protein induced expression SDS-PAGE figures .M of Recombinant Swine SC:Albumen maker.1:BL21-pCold I are induced Preceding thalline；2:Thalline after BL21-pCold I inductions；3:Thalline before BL21-pCold I/SC inductions；4:BL21-pCold I/SC Thalline (B) Recombinant Swine SC albumen existence form identifies .M after induction:Albumen maker.1:Split after BL21-pCold I/SC inductions Solve liquid supernatant；2:Liquid precipitate (C) pig restructuring SC protein purification qualification figures .M is cracked after BL21-pCold I/SC inductions:Albumen maker.1:SDS-PAGE schemes before purification；2:SDS-PAGE schemes the Western blotting of (D) pig restructuring SC albumen after purification Qualification figure.

Fig. 3 be hybridoma cell strain 4H11 secretion anti-pig SC protein monoclonal antibodies specificity identification, wherein M:In advance Contaminate albumen maker, 1:Pig restructuring SC albumen, 2:The natural SC albumen of pig, 3:Pig IgA albumen, 4:Pig IgG albumen.

Anti- pig SC protein monoclonal antibodies prepared by Fig. 4 present invention are compared with the specificity of commercialization monoclonal antibody.A The specificity of anti-pig SC protein monoclonal antibodies prepared by the display present invention；B display of commodity pig SC monoclonal antibodies it is special Property；M:Albumen Marker；1：Restructuring SC albumen, 2：Pig IgA albumen；3：Pig IgG albumen.

The testing result of specific SIgA antibody after Fig. 5 porcine mycoplasmal pneumonia attenuated vaccine immunities.

Detection of specific antibody in serum after Fig. 6 porcine mycoplasmal pneumonia attenuated vaccine immunities.

Embodiment

Embodiment 1 prepares pig restructuring SC albumen

1. the RNA extractions of pig SC albumen

Sample is intestinal mucosa epithelial cell, tracheal epithelial cell or lymphocyte, or the mixture of three.

The processing of intestinal mucosa epithelium (tracheal epithelium)：Careful scraping intestinal mucosa epithelium (tracheal epithelium), shreds, The PBS (0.01M, pH7.2-7.4) added after 500 μ l autoclaving is homogenized afterwards；

The processing of lymphocyte：500 μ l pig bloods are taken, add 500 μ l lymphocyte separation mediums (purchased from Shanghai China essence biology High Seience Technology Co., Ltd.), centrifuge (20min under the conditions of 2000rpm), PBS of the precipitation after 500 μ l autoclavings (0.01M, pH7.2-7.4) is resuspended；

By the sample after processing using conventional method extraction cell total rna

2. expand pig SC GFP fragments

(1) RT-PCR (agents useful for same is purchased from Dalian treasured bioengineering Co., Ltd)：

Response procedures are：42 DEG C, 60min；95 DEG C, 5min.

(2) PCR (agents useful for same is purchased from Dalian treasured bioengineering Co., Ltd)

RT-PCR products cDNA is taken to enter performing PCR reaction.PCR reaction systems are as follows：

(the SEQ ID NO of primer 1:2)：5’-GCGGAATTCAAGAGTCCCATATTCG-3’；

Primer 2 (SEQ ID NO:3):5’-TTAAGCTTTTTGGAGCCCCC-3’.

Response procedures：95℃5min；95 DEG C of 1min 30s, 52-54 DEG C of 1min 30s, 72 DEG C of 2min, 30cycles；72℃ 10min, 4 DEG C of 30s.PCR primer is subjected to 1% agarose gel electrophoresis, Successful amplification goes out pig SC GFP fragments, and size is about For 1857bp (containing primer sequence) as shown by the arrows in Figure 1.By purpose fragment with glue reclaim kit (being purchased from Qiagen companies) Recovery.

(3) pig SC GFPs fragment and its sequence are obtained

By DNA fragmentation and the carrier pMD 18-T carriers of step (2) recovery (18-T Vector, the precious biology in Dalian Engineering Co., Ltd) connection.

Whole operation process in ice bath on carry out.(reagent in connection procedure is purchased from Dalian to carrier coupled reaction system Precious bioengineering Co., Ltd) it is as follows：

After linked system mixes, connected overnight under the conditions of being placed in 4 DEG C

The conversion of connection product：

1. connection liquid 10 μ l full doses are taken to convert 50 μ l competent cell DH5 α (Dalian treasured bioengineering Co., Ltd), in ice Middle placement 30min；

2. after 42 DEG C of heating 45s, then 1min is placed in ice；

3. often pipe adds the LB fluid nutrient mediums (being free of antibiotic) of 500 μ l preheatings (37 DEG C), 37 DEG C are placed in after mixing, 200r/min vibrates 60min, makes bacteria resuscitation；

4. taking out, centrifuge (5000rpm, 3min), often the remaining 100 μ l bacterium solutions of pipe are laid on containing 100 μ g/ml ammonia benzyl mycins (Amp) LB flat boards, after room temperature places 20-30min, 37 DEG C are inverted culture 12-16h, and picking white single bacterium colony, is obtained from flat board Obtain positive restructuring bacterium.

Recombinant plasmid extraction, identification and the sequencing of target gene：By the single bacterium colony of positive restructuring bacterium, 5ml is inoculated in containing 100 In the LB culture mediums of μ g/ml ampicillins, 12-16h is cultivated under the conditions of 37 DEG C of 200r/min.The extraction of 2ml bacterium solutions is taken to insert The recombinant plasmid of pig SC GFP fragments, with restriction enzyme EcoR I and Hind III digestion recombinant plasmids, reactant It is 10 μ l, each composition is as follows：

After mixing, 37 DEG C of water-bath digestion 2h are placed in.Digestion products are subjected to 1% agarose gel electrophoresis identification, as a result shown Show and pig SC GFPs are successively inserted into selected recombinant plasmid.It is that positive plasmid is named as pMD 18-T by digestion identification Vector/SC, deliver to the sequencing of Nanjing Si Pujin biologies Co., Ltd, the sequence such as SEQ ID NO of pig SC GFPs:Shown in 1.

3. structure prepares the recombinant bacterium of pig SC albumen

(1) recombinant expression plasmid pCold I/SC structure

Enter by pCold I Vector and recombinant plasmid pMD 18-T Vector/SC, while with EcoR I and Hind III Row double digestion.Digestion system is as follows：

After digestion system is incubated into 3h in 37 DEG C of water-baths, is detected with 1% agarose electrophoresis and reclaim pig SC GFPs Endonuclease bamhi, carrier pCold I endonuclease bamhis.Digestion recovery fragment is connected using T4 ligases, linked system is as follows：

Mentioned reagent is mixed after 4 DEG C of connections overnight.Connection product is converted using above-mentioned same procedure, selected Recombinant plasmid, and digestion is identified, positive recombinant plasmid is named as pCold I/SC.

Take the μ L of pCold I/SC 10 to use in conventional method transformed competence colibacillus cell BL21 (DE3), reflected through PCR and sequence It is fixed, positive restructuring bacterium is obtained, is named as BL21-pCold I/SC.Meanwhile by pCold I plasmid transformed competence colibacillus cells BL21 (DE3) in, obtain compareing bacterium BL21-pCold I.

4. pig recombinates the preparation of SC albumen

BL21-pCold I/SC expression pig restructuring SC albumen is induced, using BL21-pCold I as control, specific method is such as Under：

(1) by BL21-pCold I/SC bacterium solutions with 2% LB of the ratio inoculation containing 100 μ g/ml ampicillins (AMP) Fluid nutrient medium, final concentration of 1m M IPTG is added when culture reaches 0.6-0.8 to OD600 values under the conditions of 37 DEG C, 200rpm；

(2) bacterium solution is placed into 30min at 15 DEG C；

(3) by bacterium solution under the conditions of 15 DEG C, 200rpm induced expression 24h, collect bacterium solution.

(4) expression product is used into SDS-PAGE electrophoresis detections according to a conventional method, as a result confirm pig restructuring SC albumen by into Work(is expressed, molecular weight of albumen about 70kDa (Fig. 2A).

(5) expression product existence is analyzed：

The bacterial precipitation after expression is taken, is resuspended with pH7.2 PBS, ultrasonic disruption 2min, until bacterium solution becomes clear It is clear bright, cellular lysate liquid supernatant cellular lysate liquid precipitate is collected respectively after 9000r/min centrifugations 15min carries out SDS-PAGE Electroresis appraisal (Fig. 2 B), as a result prove, Recombinant Swine SC albumen mainly exists with inclusion bodies.

(6) purifying of pig restructuring SC albumen：Using German Macherey-nagel companies Ni-TED 2000 ni-sepharose purification kits pigs recombinate SC albumen, and concrete operations are carried out according to the specification of kit.

Pig restructuring SC albumen after purification is through SDS-PAGE electroresis appraisals, as a result such as Fig. 2 C.It can be seen that pig after purification SC purity of protein is recombinated up to 96%.

(7) the Western blot identifications of pig restructuring SC albumen：

With goat-anti people SC albumen more anti-(Santa Cruz companies of the U.S.) for primary antibody, expression is produced using Western blot Thing identified, as a result as shown in Figure 2 D, it was demonstrated that pig restructuring SC albumen has preferable antigen reactivity.

Embodiment 2 prepares anti-pig SC protein monoclonal antibodies

1. mouse immune

The pig that embodiment 1 is purified recombinates the body such as SC albumen and Freund's complete adjuvant (Sigma-Aldrich) Product mixing, and it is fully emulsified, and 58 week old BALB/c mouses (Yangzhou University's comparative medicine center) are immunized in subcutaneous multiple spot, and inoculation is anti- Former dosage is 100 μ g/.After head exempts from 2 weeks, same dose pig restructuring SC albumen and incomplete Freund's adjuvant (U.S. Sigma- Aldrich) with approach mouse is immunized after emulsification, mouse is immunized in every 2 weeks same ways afterwards, is repeated 3 times, until serum is imitated Valency reaches 1:More than 6400.3-5 days before cell fusion, the antigen booster immunization without adjuvant of doubled amount is injected intraperitoneally once, Cell fusion can be carried out.

The preparation of the myeloma cells of 2.sp 2/0

20d recoveries freeze the cells of sp 2/0 in liquid nitrogen before fusion, and supernatant is abandoned after 1000r/min centrifugations 5min, with containing The DMEM culture mediums (being purchased from Gibco companies of the U.S.) of 20% hyclone are resuspended, and are transferred in cell bottle, be positioned over 37 DEG C, 5% CO₂, Secondary Culture in saturated humidity incubator.The fusion same day is taken in exponential phase, and cellular morphology is homogeneous, sharpness of border, The good cells of sp 2/0 of growth conditions, suspend again after being washed with plasma-free DMEM medium, and cell counting count board counts, will be thin Born of the same parents are diluted to 10⁶Individual/mL, volume are 10mL or so, place 37 DEG C, 5%CO₂, merge in saturated humidity incubator it is standby.

3. cell fusion

The splenocyte of the BALB/c mouse in title 1 after booster immunization is taken, fusion agent is made with PEG4000, immune spleen is thin Born of the same parents and SP2/0 myeloma cell carry out cell fusion according to a conventional method.

4. the screening of positive hybridoma cell

3d is seen whether in fusion after fusion, 4d HAT nutrient solutions (Sigma-Aldrich) half after fusion Amount changes liquid, 7d or so and uses HT nutrient solutions (Sigma-Aldrich) instead, while carries out colony count.Treat hybridoma When cell covers with bottom hole 1/10, i.e. desirable Hybridoma Cell Culture liquid supernatant, supernatant is detected using indirect ELISA after liquid 2d is changed The potency of the monoclonal antibody of moderate resistance pig SC albumen, while using the cell culture supernatants of sp 2/0 as negative control, immune swine The mice serum after SC albumen is recombinated as positive control.For positive hole, then with pig recombinant influenza HA albumen, (preparation method is same Recombinate SC albumen, only change the SC GFP fragments in recombinant bacterium into swine flu HA GFPs) coating 96 orifice plates after, carry out Indirect ELISA detects.Select and be positive with pig restructuring SC albumen reactions, the hybridization being negative with the reaction of pig recombinant influenza HA albumen Oncocyte hole is as positive cell strain.

Indirect ELISA detection method：The pig restructuring SC albumen (prepared by embodiment 1) of purifying is coated with 96 orifice plates, hybridoma Cell culture supernatant PBST buffer solutions make doubling dilution, and SP2/0 cell culture supernatants are as negative control, pig restructuring SC The serum of protein immunization mouse detects the potency of monoclonal antibody in Hybridoma Cell Culture liquid supernatant as positive control.

Reagent in indirect ELISA detection method：Phosphate buffer (pH7.2~7.4)：NaCl 8g, KCl0.2g, Na₂HPO₄1.44g KH₂PO₄0.24g is dissolved in water, adjusts pH to 7.2~7.4, is settled to 1L.PBST buffer solutions：Phosphate-buffered Contain 0.05% tween in liquid (pH7.2~7.4).Coating buffer is 0.05M, pH9.6 sodium carbonate buffer：Na₂CO₃1.59g NaHCO₃2.93g, add distilled water to dissolve, be settled to 1000mL.Nitrite ion：Nitrite ion A in embodiment 3 and nitrite ion B is pressed It is 1 according to volume ratio:40 mix and produce.Terminate liquid：With in embodiment 3.

Indirect ELISA detection method is specific as follows：

1. coating：Antigen (pig after purification recombinates SC albumen) is diluted to 1 μ g/ml by coating buffer, adds ELISA Plate, 100 μ L/ holes, 37 DEG C of effect 1h, are then stayed overnight for 4 DEG C；

2. washing：PBST is washed, 300 μ l/ holes, each 5min, is washed 3 times, is patted dry；

3. closing：PBST solution closing containing 1% casein, 100 μ l/ holes, 37 DEG C of effect 2h；

3. washing：With 2；

4. primary antibody：

1. dilute：Diluted in 1.5ml EP pipes, the first pipe carries out 1:100 dilutions (10 μ l cell line nutrient solutions supernatants+ 1ml PBST), later each doubling dilution of pipe 2, i.e., 500 μ l are drawn from the first pipe, added in the second pipe (containing 500 μ l PBST), mix, then 500 μ l are drawn from the second pipe, add in the 3rd pipe, mix, by that analogy, until the 12nd pipe；Simultaneously Gathered by the use of PBST as blank control, SP2/0 cell culture supernatants as negative control, pig after recombinating SC protein immunization mouse Positive serum (i.e. collect the present embodiment step 1 in immunized mice serum) be used as positive control；

2. it is incubated：The good sample of above-mentioned dilution is separately added into corresponding aperture, 100 μ l/ holes, 37 DEG C of incubation 1h；

5. washing：With 2；

6. secondary antibody：Using PBST, by sheep anti-mouse igg-HRP, (HRPO marks goat anti-mouse IgG antibody, is purchased from Wuhan doctor's moral company) carry out 1:20000 dilutions, are added in each hole, 100 μ l/ holes, 37 DEG C of incubation 30min；

7. washing：PBST is washed, 300 μ l/ holes, each 5min, is washed 5 times, is patted dry altogether；

8. colour developing：Add nitrite ion, 100 μ l/ holes, lucifuge colour developing 5min；

9. terminate：Add terminate liquid, 50 μ l/ holes；

10. detect the light absorption value OD at 450nm_450nm.With sample OD_450nm>=2.1 × negative control OD_450nm, it is determined as sun Property, potency of the positive dilution factor maximum using hybridoma cell strain supernatant as monoclonal antibody.

5. subclone

Carried out by limiting dilution assay.The hole that selection antibody titer is high, clone's number is few carries out subclone 3-5 times, until all Cloning cell hole Positive rate is 100%.Final to have screened hybridoma cell strain 4H11, nutrient solution supernatant is using indirect The potency of ELISA method detection is 1:3200, the hybridoma cell strain nutrient solution supernatant is positive with pig restructuring SC albumen reactions, It is negative with the reaction of pig recombinant influenza HA albumen.

Hybridoma cell strain 4H11 preservation information is as follows：

Classification And Nomenclature is：Secrete the hybridoma cell strain 4H11 of anti-pig SC protein monoclonal antibodies, preservation date 2015 On March 24, depositary institution's full name are China typical culture collection center, abbreviation CCTCC, depositary institution address：Wuhan is big Learn, deposit number is：CCTCC NO：C201526.

6. the expansion culture of hybridoma is with freezing

By hybridoma cell strain 4H11, culture is enlarged in 24 porocyte plates, after the cell in 24 orifice plates covers with, Cell is transferred in 6 orifice plates and cultivated, is then transferred in cell bottle and cultivates, the Secondary Culture after cell covers with, while freeze hybridization Tumor cell strain.

7. monoclonal antibody repeated pruning

The hybridoma cell strain 4H11 recoveries of 3 months will be frozen, the potency of nutrient solution supernatant in 24 orifice plates are detected, as commenting Whether valency cell line has the index of secretory antibody ability.As a result the hybridoma cell strain 4H11 for confirming to freeze remains to stably excreting and resisted The monoclonal antibody of pig SC albumen, potency are up to 1:3200.

8. monoclonal antibody specificity identification

The natural SC albumen of pig and pig IgA are extracted from from pig colostrum using conventional method, pig IgG is extracted from Swine serum. After pig restructuring SC albumen, the natural SC albumen of pig, pig IgA and pig IgG albumen are carried out into SDS-PAGE electrophoresis, pvdf membrane is transferred to On, stayed overnight using containing 3%BSA solution in 4 DEG C of closings.TBST solution (takes 1M, pH8.0 Tris-HCL 10ml, NaCl 4.4g and Tween-20 0.5ml are mixed, and 500ml is settled to using deionized water) washing 3 times, each 5min, add hybridoma Cell line 4H11 nutrient solution supernatant, 37 DEG C of incubation 2h.TBST solution washs 3 times, each 5min, adds 1:10 000 times of dilutions Sheep anti-mouse igg-HRP (source is same as above), 37 DEG C incubation 1h.TBST solution washs 5 times, each 5min, Enhanced chemiluminescence (ECL) expose, take pictures, as a result as shown in Figure 3.It can be seen that the anti-pig SC albumen Dan Ke of hybridoma cell strain 4H11 secretions Grand antibody can recombinate SC albumen with pig and specific binding reaction occurs for the natural SC albumen of pig, and with pig IgA and pig IgG albumen without Cross reaction, specificity is preferably.

9. a large amount of preparations of anti-pig SC protein monoclonal antibodies

Breeding method in Mice Body is taken largely to prepare SC monoclonal antibody ascites.12 week old BALB/c are taken to breed dams, Every dams Intraperitoneal injection 0.5mL sterilized liquid paraffin.After 7 days, the hybridoma that growth conditions are good is taken, uses autoclaving PBS (pH 7.4) afterwards adjusts hybridoma concentration to (4-6) × 10⁶Individual/ml, Mice Inoculated is injected intraperitoneally, 0.5ml/ is only.After 1 week, mouse web portion substantially swells, and has touched fluctuation, now collects ascites.Typically after 2-3 days, mouse Belly can swell again, can continue to collect ascites, every mouse collects 1-3 times.The ascites 1500r/min being collected into is centrifuged 10min, take intermediate layer (grease for being careful not to be drawn onto the superiors), centrifuging and taking supernatant, with Protein A affinity columns (U.S. GE Healthcare companies of state) purifying, the anti-pig SC protein monoclonal antibodies purified, -20 DEG C freeze in.

10. horseradish peroxidase (HRP) mark of anti-pig SC protein monoclonal antibodies

The anti-pig SC albumen Dan Ke that hybridoma cell strain 4H11 secretes in commission Nanjing Genscript Biotechnology Co., Ltd. Grand antibody is marked using horseradish peroxidase (HRP), uses BCA methods to detect enzyme labelled antibody concentration as 1.357mg/mL.

11. the comparison with commercialization monoclonal antibody specificity

The anti-pig SC protein monoclonal antibodies of commercialization mouse (are purchased from AbD Serotec companies of Britain, article No. respectively MCA634) carried out with the anti-pig SC protein monoclonal antibodies of the invention prepared with pig SC albumen, pig IgA albumen and pig IgG albumen Western blot are tested, to compare the specificity of two kinds of monoclonal antibodies.As a result it is as shown in Figure 4.Anti- pig prepared by the present invention SC protein monoclonal antibodies only react with pig SC albumen, and are not reacted (Fig. 4 A) with pig IgA and IgG.The anti-pig SC of commercialization mouse Monoclonal antibody not only reacts with pig SC albumen, while can be reacted with pig IgA and IgG, and with pig IgG reaction more (Fig. 4 B) strongly.

The mycoplasma hyopneumoniae SIgA antibody ELISA detection kit of embodiment 3

It is provided with mycoplasma hyopneumoniae SIgA antibody ELISA detection kit：

1. the antibody test plate for being coated with mycoplasma hyopneumoniae P 97 R 1 albumen is prepared by antibodycapture

(1) preparation of mycoplasma hyopneumoniae P 97 R 1 albumen：

By document, (Liu Maojun, Shao Guoqing, Zhang Ying, Nie is to front yard mycoplasma hyopneumoniae P97 gene antigen determinant R1 areas Cloning and expression Jiangsu's agriculture journal .2005.21 (3):207-11) structure carries mycoplasma hyopneumoniae P97 gene antigens and determined The recombinant bacterium of cluster R1 areas genetic fragment.The recombinant bacterium is seeded to the LB fluid nutrient mediums containing ampicillin, in 37 DEG C, Cultivated in 180rpm shaking tables, OD to be reached₆₀₀The IPTG that final concentration of 0.1mmol/L is added when=0.6~0.8 carries out induced expression, Thalline is collected after continuing 1~5h of culture, is washed 1 time with PBS (pH7.2), by thalline suspension lysozyme and ultrasonic treatment, 5 000rpm centrifuge 30min at 4 DEG C, take lysate supernatant, are separated with His-Tag affinity chromatographic columns (MERCK Products) pure Change, obtain recombinating mycoplasma hyopneumoniae P 97 R 1 albumen, protein concentration is detected using BCA methods.- 20 DEG C save backup.

(2) preparation of the monoclonal antibody of anti-mycoplasma hyopneumoniae P 97 R 1 albumen

By document (Feng Zhixin, Liu Maojun, Wang Haiyan, Gan Yuan, Wu Xusu, Shao state green grass or young crops mycoplasma hyopneumoniae adhesion factors P97R1 areas monoclonal antibody prepares Jiangsu's agriculture journal .2009.25 (6):1438-1441.), prepared using hybridoma cell strain 6C5 anti- The monoclonal antibody of mycoplasma hyopneumoniae P 97 R 1 albumen, and it is pure with ProteinA affinity columns (GE Healthcare companies of the U.S.) Change, antibody concentration is detected using BCA methods.

(3) preparation of antibody test plate

By square formation titration, the optimal coating concentration for determining anti-mycoplasma hyopneumoniae P 97 R 1 protein monoclonal antibody is 2.0 μ g/mL, the optimal coating concentration of capture antigen restructuring P97R1 albumen is 2 μ g/mL.With 0.05mol/L, the carbonate of pH value 9.6 Buffer solution (Na₂CO₃1.59g NaHCO₃2.93g, dissolved with distilled water, add distilled water to be settled to 1000mL) resisting purifying Mycoplasma hyopneumoniae P 97 R 1 protein monoclonal antibody is diluted to 2.0 μ g/mL, and detection plate, 2-8 DEG C of effect are coated with by every μ L of hole 100 12-18h.The coating buffer in hole is discarded, is washed 3 times, each 3-5min with PBST (10 × concentrated cleaning solution dilutes 10 times).Per hole Add the PBS solution that 200 μ L contain 10g/L caseins and (take 8g NaCl, 0.2g KCl, 2.9g Na₂HPO₄·12H₂O and 0.24g KH₂PO₄Distilled water is dissolved in, adds distilled water to be settled to 1000mL, pH7.4) closed, discarded after putting 37 DEG C of incubation 2h. Washed 3 times, dried with PBST (10 × concentrated cleaning solution dilutes 10 times).The Recombinant Swine lung of 2 μ g/mL purifying is added by 100 μ L/ holes Scorching mycoplasma P97R1 protein solutions, discarded after putting 37 DEG C of incubation 1h, 3 are washed with PBST (10 × concentrated cleaning solution dilutes 10 times) It is secondary, each 3-5min, pat dry.150 μ L 50g/L sucrose solutions are added per hole, are discarded after putting 37 DEG C of incubation 1h.With PBST (10 × concentrated cleaning solution dilutes 10 times) wash 3 times, sealing is vacuumized after drying, puts 2-8 DEG C of preservation.

2.10 × enzyme conjugates working solution

The anti-pig SC protein monoclonal antibodies of horseradish peroxidase (HRP) mark prepared by embodiment 2, using BCA Method detects enzyme labelled antibody concentration, and it is 1mg/mL to adjust concentration.Then made with PBST (10 × concentrated cleaning solution dilutes 10 times) 1：1000 dilutions, add final concentration of 25 μ g/ml gentamicins, degerming with 0.22 μm of membrane filtration, obtain 10 × enzyme conjugates work Make liquid, aseptic subpackaged, 1.2ml/ branch, 2~8 DEG C of preservations.

3. positive control solution

It is derived from the test pig bronchoalveolar lavage fluid of artificial challenge's mycoplasma hyopneumoniae Js strains.The serum of infected pigs is public with IDEXX Mycoplasma hyopneumoniae antibody assay kit (the i.e. HerdChek Mycoplasma hyopneumoniae ELISA antibody of department Detection kit, for detecting serum IgG antibody, similarly hereinafter) test positive, alveolar wass liquid precipitate is through mycoplasma hyopneumoniae set (Lu knows quick, Feng Zhixin, Liu Maojun, Wu Xusu, Gan Yuan, Zhang Ying, green grass or young crops mycoplasma hyopneumoniae sleeve type PCRs detection side of Shao state to formula PCR The foundation of method and application Jiangsu's agriculture journal .2010.26 (1):91-95.) test positive, lungs cut open inspection point leaf, lobus cardiacus, in Between leaf and lobus diaphragmaticus leading edge be in " pancreas sample " or " shrimp sample " asthma typical cytopathic, be judged to infecting successfully.Bronchoalveolar lavage fluid is passed through 10,000rpm centrifugation 30min, take supernatant, adding final concentration of 0.01% after 0.22 μm of filter filtration sterilization, (quality percentage is dense Degree) thimerosal, as positive control solution, aseptic subpackaged 1ml/ pipe, 2~8 DEG C of preservations.

4. negative controls

It is taken from the bronchoalveolar lavage fluid of mycoplasma hyopneumoniae negative healthy pig.Test pig lung of the Swine serum through IDEXX companies Scorching mycoplasma antibody assay kit is detected as feminine gender, cuts open and kills rear lungs without asthma typical cytopathic, alveolar is detected with sleeve type PCR Mycoplasma hyopneumoniae antigen is feminine gender in lavation liquid precipitate, is determined as mycoplasma hyopneumoniae negative healthy pig.By bronchoalveolar lavage fluid 30min is centrifuged through 10,000rpm, takes supernatant, adding final concentration of 0.01% after 0.22 μm of filter filtration sterilization, (quality percentage is dense Degree) thimerosal, as negative controls, aseptic subpackaged 1ml/ pipe, 2~8 DEG C of preservations.

5.10 × concentrated cleaning solution

Take 40g NaCl, 1.0g KCl, 14.5g Na₂HPO₄·12H₂O、1.2g KH₂PO₄, 2.5ml Tween-20s are dissolved in Ionized water, it is settled to 500ml.Add the thimerosal of final concentration of 0.01% (mass percentage concentration), removed with 0.22 μm of membrane filtration Bacterium, aseptic subpackaged as 10 × concentrated cleaning solution, 30ml/ bottles.

6. nitrite ion A

21mg TMB (3,3', 5,5'- tetramethyl benzidines, purchased from BIOSHARP companies) are dissolved in 5ml absolute ethyl alcohols, mix Packing, 1ml/ branch, as nitrite ion A.

7. nitrite ion B

33mg UHP (urea peroxide element, purchased from Shanghai Jing Chun biochemical technologies limited company) are dissolved in 200ml, pH and are 5.2 phosphate buffer (takes 2.7g Na₂HPO₄·12H₂O、13.2g KH₂PO₄Deionized water is dissolved in, is settled to 500ml), Add final concentration of 25 μ g/ml gentamicin, aseptic subpackaged, 15ml/ bottle, as nitrite ion degerming with 0.22 μm of membrane filtration B。

8. terminate liquid

Terminate liquid is 2mol/L sulfuric acid solution.

The sample of kit detection of the present invention is hog snout swab samples to be checked.Kit containment of the present invention is in 4-7 DEG C of condition Under.

The detection operation sequence of the mycoplasma hyopneumoniae SIgA antibody ELISA detection kit of embodiment 4

This example demonstrates that the detection behaviour of mycoplasma hyopneumoniae SIgA antibody ELISA detection kit prepared by embodiment 3 Make program.

1. preparation of reagents

(1) before detecting, all reagents in mycoplasma hyopneumoniae SIgA antibody ELISA detection kit are placed at room temperature 30 minutes, all reagents are made to recover to room temperature.

(2) 1 × cleaning solutions are prepared.Recover to 10 × concentrated cleaning solution of room temperature, can be in 37 DEG C of warm bath 5~10 if any precipitation Minute, 10 times are diluted with sterilizing pure water, as 1 × cleaning solution, 2~8 DEG C preserve 7.

(3) 1 × enzyme conjugates working solutions.It is standby by 10 × enzyme conjugates working solution, 10 times of 1 × cleaning solution dilution.

(4) substrate nitrite ion is prepared.Nitrite ion A and nitrite ion B is 1 according to volume ratio:40 are mixed, and are obtained substrate and are shown Color liquid, it is standby.

2. detect operation sequence

(1) antibody test plate is taken, negative control hole, Positive control wells and sample detection hole are set, a repetition is done per hole. Negative controls are added in negative control hole, positive control solution is added in Positive control wells, are added in sample detection hole Nose swab sample to be checked, addition are 100 μ L/ holes.

(2) the antibody test plate after sample-adding is put into 37 DEG C to incubate 120 minutes.

(3) liquid in antibody test plate hole is discarded, each hole adds the μ L of 1 × cleaning solution 250 and washed, and washs 5 times.Note Meaning, after last time is washed, antibody test plate is dried, firmly patted dry on absorbent material, remove remaining liquid.

(4) the μ L of 1 × enzyme conjugates working solution 100 are added per hole, 37 DEG C is put and incubates 60 minutes.

(5) repeat step (3).

(6) 100 μ L substrate nitrite ions are added per hole, 37 DEG C incubate lucifuge and develop the color 7 minutes.

(7) 50 μ L terminate liquids, terminating reaction are added per hole.

(8) light absorption value (OD of sample detection hole and control wells at 450nm is measured and recorded with ELIASA₄₅₀Value).

3. result judgement

As 1.2 ＜ Positive control wells OD₄₅₀Value ＜ 2.0, and 0.05 ＜ negative control holes OD₄₅₀During value ＜ 0.25, experiment knot Fruit is reliable.

According to sample S/P=(sample detection hole OD₄₅₀Value-negative control hole OD₄₅₀Value)/(Positive control wells OD₄₅₀Value-the moon Property control wells OD₄₅₀Value), calculate sample S/P.When sample S/P >=0.17 is judged to the positive, illustrate that pig-pig infection pneumonia branch to be checked is former The wild poison of body or vaccine virus；When sample S/P ＜ 0.17 are judged to feminine gender, illustrate that pig to be checked is uninfected by mycoplasma hyopneumoniae.

The specificity of the mycoplasma hyopneumoniae SIgA antibody ELISA detection kit of embodiment 5

1. nose swab sample

Nose swab sample includes 150 parts of mycoplasma hyopneumoniae SIgA negative antibody nose swab samples and 7 parts of other respiratory tracts The positive nose swab sample of cause of disease.

150 parts of mycoplasma hyopneumoniae SIgA negative antibody nose swab samples：Collection is from without immune any pig pneumonia branch original Body vaccine and nearly 2 years pig farms that porcine mycoplasmal pneumonia does not occur, its corresponding serum antibody use the pig pneumonia branch of IDEXX companies The detection of mycoplasma antibody detection kit is mycoplasma hyopneumoniae negative antibody.

The Antigen positive hybridomas nose swab sample of 7 parts of other respiratory diseases：Including pig breathing and reproductive syndrome virus (PRRSV) SIgA antibody positive hog snout swabs, pseudorabies (PRV) SIgA antibody positive hog snout swabs, H1 types swine influenza virus (SIV) SIgA antibody positive hog snout swabs, pig pleuropneumonia (APP) SIgA antibody positive hog snout swabs, mycoplasma hyorhinis (MHR) SIgA Antibody positive hog snout swab, Escherichia coli (E.coli) SIgA antibody positive nose swabs, mycoplasma hyopneumoniae (MHP) SIgA antibody Positive nose swab.

2. detection method

Above-mentioned 157 parts of samples are detected using method A and B simultaneously.

Method A：Using in embodiment 3 method prepare mycoplasma hyopneumoniae SIgA antibody ELISA detection kit according to Method detects whether above-mentioned each sample with the kit occurs nonspecific reaction in embodiment 4, calculates negative recall rate.

Method B：Detected using Application No. 2009100257043, entitled anti-mycoplasma hyopneumoniae SIgA indirect ELISA Method disclosed in the application for a patent for invention of method, detect above-mentioned each sample whether with this method occur nonspecific reaction, calculate Negative recall rate.

3. result

As a result show：Kit of the present invention is to 7 parts of other equal no cross reactions of pathogenic autoantibody, to 150 parts of MHP feminine gender product examines Extracting rate is up to 97.3% (146/150), is shown in Table 1.Application No. 2009100257043, entitled anti-mycoplasma hyopneumoniae SIgA Method disclosed in the application for a patent for invention of indirect ELISA detection method, only only have to 150 parts of MHP feminine gender product examine extracting rates 92.7% (139/150), it is maximum the defects of be that this method has cross reaction to Escherichia coli antibody, be shown in Table 2.

The method A of table 1 specificity

Note：S/P value >=0.17 is the positive, and S/P values ＜ 0.17 is feminine gender."-" represents negative, and "+" represents positive.

The method B of table 2 specificity

Note：S/P value >=0.15 is the positive, and S/P values ＜ 0.1 is feminine gender, as 0.1≤S/P value ＜ 0.15 to be suspicious.“-” Represent negative, "+" represents positive.

The mycoplasma hyopneumoniae SIgA antibody ELISA detection kit of embodiment 6 is distinguished porcine mycoplasmal pneumonia inactivated vaccine and exempted from Epidemic disease and natural infection

1. sample

260 pigs from 4 pig farms, the nose swab sample and blood serum sample of every pig are gathered respectively, respectively altogether 260 Part.Wherein, any i (mycoplasma hyopneumoniae) vaccine is not immunized in A pig farms, porcine mycoplasmal pneumonia does not occur within nearly 2 years；B pig farms immune swine Mycoplasma pneumoniae inactivated vaccine, porcine mycoplasmal pneumonia clinical symptoms are not found；The not immune any pig pneumonia branch in C and D pig farms is former The porcine mycoplasmal pneumonia clinical symptoms such as cough, asthma, abdominal respiration be present in body vaccine, more than 70% pig.

2. detection method

(1) serum IgG antibody detects：Test method is tried in strict accordance with the mycoplasma hyopneumoniae antibody test of IDEXX companies The specification operating procedure of agent box is carried out.

(2) mycoplasma hyopneumoniae SIgA antibody test, the mycoplasma hyopneumoniae SIgA prepared using method in embodiment 3 are resisted Body ELISA detection kit detects according to method in embodiment 4.

(3) mycoplasma hyopneumoniae antigen detects：By document, (Lu knows the mycoplasma hyopneumoniae sets such as quick, Feng Zhixin, Liu Maojun The foundation of formula PCR detection method and application Jiangsu's agriculture journal .2010.26 (1):91-95) method is using sleeve type PCR detection pig Mycoplasma pneumoniae.

3. result

As a result as shown in table 3, what three kinds of detection method result differences were maximum in four pig farms appears in B pig farms.For B pigs , mycoplasma hyopneumoniae SIgA antibody and the positive rate of mycoplasma hyopneumoniae antigen testing result are relatively low, and coincidence rate is higher；And The serum IgG antibody positive rate of the ELISA antibody assay kits detection of IDEXX companies is very high, with other two kinds of Indexs measures As a result coincidence rate is very low.Generally, after mycoplasma hyopneumoniae inactivated vaccine is immune, pig produces higher serum IgG Antibody, it is more difficult to produce or only produce low-level mucous membrane SIgA antibody；The pig of natural infection mycoplasma hyopneumoniae, it can produce higher Serum IgG antibody and mucous membrane SIgA antibody.Therefore the mycoplasma hyopneumoniae SIgA antibody positive rate on B pig farms is relatively low, C, D pig farm The positive rate of mycoplasma hyopneumoniae SIgA antibody is higher.Analysis of test results by sleeve type PCR to mycoplasma hyopneumoniae antigen Prove, kit of the invention can detect the pig of natural infection mycoplasma hyopneumoniae, it is impossible to detect to be inoculated with porcine mycoplasmal The pig of pneumonia inactivated vaccine, i.e., kit of the present invention can distinguish that porcine mycoplasmal pneumonia inactivated vaccine is immune and natural infection.

The mycoplasma hyopneumoniae antibody/antigen Positive rate of table 3. counts

The mycoplasma hyopneumoniae SIgA antibody ELISA detection kit of embodiment 7 is used for porcine mycoplasmal pneumonia attenuated live vaccines The assessment of immune effect

1. sample

The pig that porcine mycoplasmal pneumonia attenuated live vaccines (Nanjing Tianbang Bio-industry Co., Ltd.) are immunized from intrapulmonary exists The nose swab sample and blood serum sample of different time points collection.

2. detection method

(1) Serum Antibody Detection：Test method in strict accordance with IDEXX companies mycoplasma hyopneumoniae antibody assay kit Specification operating procedure carry out.

(2) mycoplasma hyopneumoniae SIgA antibody test：Detected using kit in embodiment 3 according to method in embodiment 4.

3. result

As shown in figure 5, using kit in embodiment 3 can effective detection to porcine mycoplasmal pneumonia attenuated live vaccines it is immune after Caused specific SIgA antibody-secretings in porcine respiratory, and there is certain rise and the rule declined after immune in antibody titer Rule.It is more difficult after porcine mycoplasmal pneumonia attenuated live vaccines are immune to detect that antibody turns sun (S/P from serum and Fig. 6 results show> 0.4 is the positive).This embodiment result proves that kit of the present invention can be to porcine mycoplasmal pneumonia attenuated live vaccines immune effect Indirectly assessed.

The sensitiveness of the mycoplasma hyopneumoniae SIgA antibody ELISA detection kit of embodiment 8

1. nose swab sample

135 parts of clinical hog snout swabs, collection are from the pig farm without immune any i (mycoplasma hyopneumoniae) vaccine, and more than 70% Pig cough be present, asthma, the porcine mycoplasmal pneumonia clinical symptoms such as abdominal respiration, the serum antibody of its corresponding pig is public through IDEXX The mycoplasma hyopneumoniae antibody assay kit detection of department is the mycoplasma hyopneumoniae IgG antibody positive.Another 61 parts of positive noses are wiped Son is the hog snout swab samples of collection in 28 days after artificial infected pigs' mycoplasma pneumoniae Js strains.

2. detection method

Preparing three batches of kits according to embodiment 3, (lot number is respectively 001,002,003, reagent hereinafter referred to as of the present invention Box), and detect whether contain mycoplasma hyopneumoniae SIgA antibody in all samples, calculate positive rate.It is alternative to select artificial sense 5 parts of positive nose swab samples that titre is higher in sample are contaminated, detect total protein concentration, and do 1:2~1:64 continuous 2 doubling dilutions Use kit in embodiment 3 to detect afterwards, calculate limit of identification.

Use (the entitled anti-mycoplasma hyopneumoniae SIgA indirect ELISA detection side of Application No. 2009100257043 simultaneously Method) application for a patent for invention disclosed in method detection mycoplasma hyopneumoniae SIgA antibody, calculate positive rate and minimum detection Amount.

3. result

Using 196 parts of mycoplasma hyopneumoniae positive clinical nose swab samples of three batches kit detection clinical acquisitions of the present invention Product, its positive rate are respectively 192/196 (98.0%), 191/196 (97.4%), 191/196 (97.4%).Using Application No. Disclosed in the application for a patent for invention of 2009100257043 (entitled anti-mycoplasma hyopneumoniae SIgA indirect ELISA detection methods) Method detects, number positive 180/196, and positive rate only only has 91.8%, is shown in Table 4.

5 parts of artificial challenge's positive nose swab sample (Ps1~5) measure protein concentrations of selection, and after 2 doubling dilutions, respectively Method disclosed in application for a patent for invention with kit of the present invention (lot number 002) and Application No. 2009100257043 detects pig Mycoplasma pneumoniae SIgA antibody.As a result show, kit of the present invention is to mycoplasma hyopneumoniae SIgA antibody positive nose swab sample Minimum concentrations are 42.50~48.75 μ g/ml, and maximum detection extension rate is 1:16 (being shown in Table 5).Using Application No. Method disclosed in 2009100257043 applications for a patent for invention detects, to mycoplasma hyopneumoniae SIgA antibody positive nose swab sample Minimum concentrations are 170.00~390.00 μ g/ml, and maximum detection extension rate is 1:2~1:4 (being shown in Table 6).

The kit of the present invention of table 4 faces with the detection of published anti-mycoplasma hyopneumoniae SIgA indirect ELISA detection method Bed sample statistics result

Concentration limit of 5 kit of the present invention of table to artificial challenge's positive nose swab sample

Note：S/P value >=0.17 is the positive, and S/P values ＜ 0.17 is feminine gender.

Minimum detection of the application for a patent for invention method of table 6 2009100257043 to artificial challenge's positive nose swab sample is dense Degree

Note：S/P value >=0.15 is the positive, and S/P values ＜ 0.1 is feminine gender, as 0.1≤S/P value ＜ 0.15 to be suspicious.

The mycoplasma hyopneumoniae SIgA antibody ELISA detection kit stability of embodiment 9

Investigate the stability of mycoplasma hyopneumoniae SIgA antibody ELISA detection kit prepared by embodiment 3.

1. replica test in batch

With with a batch of 5 kits (Kit1-Kit5) to 10 parts of mycoplasma hyopneumoniae SIgA negative antibody nose swabs Sample (1~10) and 10 parts of mycoplasma hyopneumoniae SIgA antibody positive nose swab samples (11~20) carry out parallel test.

2. replica test between batch

With 3 batch kits (001,002,003 batch) to 10 parts of mycoplasma hyopneumoniae SIgA negative antibody nose swab samples Product (1~10) and 10 parts of parallel contrast tests of mycoplasma hyopneumoniae SIgA antibody positive nose swab sample (11~20) progress.

Sample source and numbering are identical in the present embodiment title 2 and title 1.

3. result

Experimental result is shown, 10 parts of negative nose swabs and 10 parts of positive nose swabs are detected with a batch of 5 kits Variation within batch coefficient C.V.% respectively in 1.99%~9.99% (being shown in Table 7)；10 parts of feminine genders are detected with the kit of 3 batches The interassay coefficient of variation of nose swab and 10 parts of positive nose swabs is in 1.17%~9.90% (being shown in Table 8).Tried between testing and criticize in batch Testing result proves that the kit of the present invention has good stability.

The result of the test of the interior different kit detection mycoplasma hyopneumoniae SIgA antibody of 7 batches, table

The different batches kit of table 8 detects the result of the test of mycoplasma hyopneumoniae SIgA antibody

SEQUENCE LISTING

<110>Jiangsu Province Agriculture Science Institute

<120>Anti- pig SC protein monoclonal antibodies and its preparing mycoplasma hyopneumoniae SIgA antibody ELISA detection examination Agent box

The application of aspect

<130> 20150512

<160> 3

<170> PatentIn version 3.3

<210> 1

<211> 1842

<212> DNA

<213>Pig

<400> 1

aagagtccca tattcggtcc ccaggatgtg agcagcgtgg aaggcagctc ggtgtccatc 60

agatgctact acccagccac ctccgtcaac cggcattctc ggaagtactg gtgccgaata 120

ggagccaagg gccgctgcac aaccctcatc tcctcggagg gctacatctc caaggactac 180

aagggcagag ccaacctcac caacttccca gagaacggca ccttcgtgat ggacattggc 240

cacctgaccc gcggtgactc tgggctctac aagtgtggtc tgggcattag cagccgaggc 300

ctgtcttttg acgtgagcct ggaggtcagc caaggtcctg gacagatagg tgatgtccac 360

gtctacacag cagacctggg cagcacagta accatcaact gccctttcaa gtctgagaat 420

gctcagaagc cgaaatccgt gtacaagaaa ctgggccaga tccgcgtcct ggtcatcgac 480

tccaatgggt atttgaacaa caactttacc aacagagcac atctcagtat tcagggtacc 540

aaccaattag tattcagctt tgtcatcaac cgaatccagc tccgcgatgc tgggatatac 600

atctgccagg ctggggacga agagagcagc gctgacctcc aagtgctgaa gcccgagcct 660

gagctgattt atggagacct gaggggctcg gtgacatttg actgcgccct gggccaggag 720

atggcaaatg tggccaaatt tctgtgccag ctaaaaaatg ggaaaacctg caatgtggtc 780

atcaacaccc tggggaagaa ggctcaggac tttgagggca ggatcctgct cactcccaag 840

gaaaacagcc acttcagcgt gcacatcacc gggctgcgga aagaggacgc aggacactac 900

ctgtgcggag cccaccctga cggtgagcct aaggaaggct ggccggtcca ggcttggcag 960

ctcttcatca atgaagatac tatgattccc ccaagatcct ccgtggtgag aggtgtggtg 1020

ggaggctccg tggccgtgac ctgcccctac aacccgaagg aaacaaacag cctgaagtac 1080

tggtgtcgct gggaagagaa tgaaaacggc cgctgcccgc agctggtgga aagcagcggg 1140

ctagtgaacg agcaatacga gggccggctc gcgctgtacg aggagccagg caatggcacc 1200

ttcacggtca tactcaacca gctcaccaac cgggacgccg gcttctactg gtgtttgacc 1260

aacgaggact ctcgctggag gtccaccgta gagctcaaga ttgttgaagg agaaccgaac 1320

ctcaagctac ccgagaatgt caccgcttgg gtgggagaga ccctcaagct ctcctgccac 1380

ttcccatgca aattctactc ctaccagaag tactggtgta agtggagcaa caccggctgc 1440

agggccctgc ccagccagga cgaaggccag agccaggcct ttgtgaactg tgacaagaag 1500

agccagatca tctccctgaa cctgaaccct gtcagaaagg aggatgaagg ctggtactgg 1560

tgcggggtga aggacggact ccactacgga gagactgggg ctgtctacgt ggcagtggaa 1620

cagaaggcaa aggggtcggg agatgcccgt ctagcaaatg ctgctcctgc tcctgctgaa 1680

gacgcaatag agccaagggc cagggagact gagaacgagg tcctcctgga tcccagcctc 1740

attgcagagg acagagcagt gaaggatggt gaaggcccag ctagtgggag tggagtacct 1800

gcagcccctg gcagctctgt gggccaaggc gggggctcca aa 1842

<210> 2

<211> 25

<212> DNA

<213> artificial

<220>

<223>Primer 1

<400> 2

gcggaattca agagtcccat attcg 25

<210> 3

<211> 20

<212> DNA

<213> artificial

<220>

<223>Primer 2

<400> 3

ttaagctttt tggagccccc 20

Claims (4)

1. mycoplasma hyopneumoniae SIgA antibody ELISA detection kit, the kit contains what is prepared by antibodycapture It is coated with the detection plate of mycoplasma hyopneumoniae P 97 R 1 albumen and the anti-pig SC protein monoclonals containing horseradish peroxidase-labeled The enzyme conjugates working solution of antibody, the anti-pig SC protein monoclonal antibodies are as secreted by hybridoma cell strain 4H11, and it is protected Tibetan number is CCTCC NO：C201526；

The detection plate for being coated with mycoplasma hyopneumoniae P 97 R 1 albumen is prepared with the following method：Detection plate is first used into hybridoma Anti- mycoplasma hyopneumoniae P 97 R 1 protein monoclonal antibody coating caused by cell line 6C5, then using mycoplasma hyopneumoniae P97R1 albumen is coated with.

2. mycoplasma hyopneumoniae SIgA antibody ELISA detection kit according to claim 1, it is characterised in that the anti-pig The coating concentration of mycoplasma pneumoniae P97R1 protein monoclonal antibodies is 1.5-2.5 μ g/mL, the coating concentration of the P97R1 albumen 1.5-2.5µg/mL。

3. according to one of the claim 1-2 mycoplasma hyopneumoniae SIgA antibody ELISA detection kits, it is characterised in that institute Stating kit also includes positive control solution, negative controls, cleaning solution, nitrite ion and terminate liquid.

4. mycoplasma hyopneumoniae SIgA antibody ELISA detection kit according to claim 3, it is characterised in that positive control Liquid is the pig bronchoalveolar lavage fluid of artificial infected pigs' mycoplasma pneumoniae, and negative controls are to be uninfected by the pig alveolar of mycoplasma hyopneumoniae Irrigating solution；Wash liquid making method：Take 40g NaCl, 1.0g KCl, 14.5g Na₂HPO₄•12H₂O、1.2g KH₂PO₄、2.5ml Tween-20 is dissolved in deionized water, is settled to 500ml；Nitrite ion includes nitrite ion A and nitrite ion B, wherein nitrite ion A be by 21mg 3,3', 5,5'- tetramethyl benzidines obtain after being dissolved in 5ml absolute ethyl alcohols；The nitrite ion B is by 33mg urea peroxides Element obtains after being dissolved in 200ml phosphate buffers；The terminate liquid is 2mol/L sulfuric acid solution.