CN104877027A - Anti-swine SC protein monoclonal antibody and application of monoclonal antibody in preparing mycoplasma hyopneumoniae SIgA antibody ELISA detection kit - Google Patents

Abstract

The invention provides an anti-swine SC protein monoclonal antibody and an application of the monoclonal antibody in preparing a mycoplasma hyopneumoniae SIgA antibody ELISA detection kit, and relates to the technical field of animal virological and epizootiological detection. The anti-swine SC protein monoclonal antibody is secreted by a hybridoma cell strain 4H11 and the collection number of the anti-swine SC protein monoclonal antibody is CCTCC NO: C201526. The invention also discloses the monoclonal antibody and the application of the monoclonal antibody in preparing the mycoplasma hyopneumoniae SIgA antibody ELISA detection kit. The kit has high specificity, high stability and high sensitivity; a detection sample can be sampled conveniently; and the kit is capable of distinguishing porcine mycoplasma pneumonia inactivated vaccine immunization and natural infection, and can be applied to the early diagnosis of pneumonic porcine mycoplasma infection and the evaluation of the immune effect after attenuated live vaccine immunization.

Description

Anti-pig SC protein monoclonal antibody and preparing the application in mycoplasma hyopneumoniae SIgA antibody ELISA detection kit

Technical field

The present invention relates to animal virology and epizootiology detection technique field, be specifically related to anti-pig SC protein monoclonal antibody and applying.

Background technology

Mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae, Mhp) causes porcine mycoplasmal pneumonia (MPS), is commonly called as the cause of disease of swine enzootic pneumonia.Swine enzootic pneumonia be in swinery popular the widest, propagate the soonest, one of the epidemic disease of the most difficult purification.Mycoplasma hyopneumoniae passes through respiratory infectious, cause the atrophy of respiratory tract cilium after infecting airway epithelial, come off, damage, epithelial cell is downright bad, reduces the immunologic function of respiratory mucosa, cause lung functions to damage, easily produce the secondary infection of other respiratory pathogens.

Research shows, to the prevention of MPS mainly through the cellular immunization of respiratory tract local and mucosa-immune.SIgA (secretory immunoglobulin A) is the main effects factor participating in mucosa-immune reaction.They are the important component parts in body immune system, play an important role respectively in immunne response.SIgA can antiviral, neutralize a toxin and other biological activity antigen, there is immanoprotection action widely, but topmost provide protection be stop bacterium surface epithelial cell is adhered to, and remove invasion cause of disease.It is also the immune response that produces at first after pathogenic agent invades, therefore, often according to the detection of the specificity SIgA early diagnosis as disease.SIgA by secreting composition (SC albumen), IgA and J chain forms.SC albumen is the extracellular fragment part of the many immunoglobulin receptors (PlgR) on epithelial cell, is the first defence line of mucosa-immune, can stops the invasion of the objectionable impuritiess such as the virus in respiratory tract and enteric cavity, bacterium, toxin and alien material.SC albumen enhances the best protection effect of SIgA, and SIgA is declined to the susceptibility of proteolytic enzyme, and mucus is more viscous, enhances adhesion and defence capability.SC albumen is the important component of mucosal immune system, when SC can not normally produce, slgA can be caused normally to synthesize and to cause various diseases.SC albumen still distinguishes the mark of serotype IgA and secretory IgA.

Anti-mycoplasma hyopneumoniae SIgA is the specificity marker that Mhp infects or first produces at respiratory tract after immunity, also reflects the protected effect after vaccine immunity simultaneously.

The diagnostic method of mycoplasma hyopneumoniae comprises Pathogen test and Serum Antibody Detection.The Methods of Detection of Pathogens comprises the Isolation and ldentification of cause of disease, polymerase chain reaction (PCR), real-time quantitative PCR and hybridization in situ, but these methods all exist Sensitivity and Specificity compared with low, consuming time compared with long, detection difficulty is large, detected result is uncertain, required equipment condition and be not suitable for the shortcomings such as In vivo detection.Serum Antibody Detection method comprises: Flame atomic absorptionspectrometry (IHA) and enzyme linked immunosorbent assay (ELISA) etc.But also exist in the detection of IHA and detect that low, the result of titre is low with the dependency of infection, the problem such as aggegation terminal point determining subjective and technological deficiency inherently, therefore IHA actual application prospect in the Mhp detection of commodity swinery is limited.The Sensitivity and Specificity of the ELISA method of existing detection serum antibody can not meet diagnosis requirement.In addition, ELISA method used at present is all used to detect Mhp IgG antibody.After mycoplasma hyopneumoniae infection, IgG antibody produces relatively slow; And research shows, the immanoprotection action of humoral immunization to this cause of disease of whole body is little.On the contrary, respiratory tract local produce mucosa-immune and cellular immunization can produce good immune protective effect.Therefore, the ELISA of existing serum antibody is not suitable for the monitoring of immune indexes after the early diagnosis of this disease and active immunity.

R1 district is a repeating unit of Mhp cilium adhesion protein (being also called adhesion factor or adhesin) P97, and being present in the C end of cilium adhesion protein P97, is also the main antigenic determinant of this albumen.The R1 district of Mhp cilium adhesion protein P97 is called P97R1 albumen.Mhp, by adhering to infected pigs's segmental bronchus ciliated epithelial cell, produces follow-up pathogenic effects then.Body produces rapidly the reaction of corresponding mucosa-immune for the adhesion energy of mycoplasma.If utilize P97R1 albumen as coating antigen can the very first time detects that corresponding SIgA reacts after infection, be suitable for the early diagnosis of this disease.And P97 albumen has species specificity, there is not serology cross interference with other porcine mycoplasmal.

Summary of the invention

The object of the present invention is to provide the hybridoma cell strain secreting anti-pig SC protein monoclonal antibody.

Another object of the present invention is to the anti-pig SC protein monoclonal antibody providing described hybridoma cell strain to secrete, this monoclonal antibody has higher specificity.

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

Another object of the present invention is to provide mycoplasma hyopneumoniae SIgA antibody ELISA detection kit, has higher specificity, sensitivity and stability, can distinguish the immunity of porcine mycoplasmal pneumonia inactivated vaccine and natural infection, for early diagnosis; Also can be used for the assessment of immune effect after the immunity of porcine mycoplasmal pneumonia attenuated live vaccines.

For achieving the above object, the technical solution used in the present invention is as follows:

Secrete a hybridoma cell strain 4H11 for anti-pig SC protein monoclonal antibody, preserving number is: CCTCCNO:C201526.

The present invention also claimed described hybridoma cell strain secretion anti-pig SC protein monoclonal antibody and preparing the application in mycoplasma hyopneumoniae SIgA antibody ELISA detection kit.

Mycoplasma hyopneumoniae SIgA antibody ELISA detection kit, contain the enzyme conjugates working fluid of the check-out console being coated with mycoplasma hyopneumoniae P 97 R 1 albumen prepared by antibodycapture and the anti-pig SC protein monoclonal antibody containing horseradish peroxidase-labeled, described anti-pig SC protein monoclonal antibody is secreted by hybridoma cell strain described in claim 1.

In the present invention, preparation with the following method adopted by the check-out console being coated with mycoplasma hyopneumoniae P 97 R 1 albumen: check-out console is first adopted anti-mycoplasma hyopneumoniae P 97 R 1 protein monoclonal antibody bag quilt, then adopts mycoplasma hyopneumoniae P 97 R 1 albumen bag quilt.

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

In the present invention, described test kit also comprises positive control solution, negative controls, washings, nitrite ion and stop buffer.

In preferred technical scheme, positive control solution is the pig bronchoalveolar lavage fluid of artificial infected pigs mycoplasma pneumoniae, and negative controls is the pig bronchoalveolar lavage fluid of non-infected pigs mycoplasma pneumoniae; Washings compound method: get 40gNaCl, 1.0g KCl, 14.5g Na ₂hPO ₄12H ₂o, 1.2g KH ₂pO ₄, 2.5ml tween 20 is dissolved in deionized water, is settled to 500ml; Nitrite ion comprises nitrite ion A and nitrite ion B, and wherein nitrite ion A is by 21mg3,3', and 5,5'-tetramethyl benzidine obtains after being dissolved in 5ml dehydrated alcohol; Described nitrite ion B obtains after 33mg urea peroxide element is dissolved in 200ml phosphate buffered saline buffer; Described stop buffer is the sulphuric acid soln of 2mol/L.

Beneficial effect:

1. high specific: this test kit guarantees the high specific detected by following three aspects.(1) catch by monoclonal antibody envelope antigen P97R1 albumen to be guaranteed by bag pure property by method, to avoid in the P97R1 proteantigen prepared by escherichia coli expression foreign protein to the impact of detection sensitivity; (2) specific proteins that the envelope antigen P97R1 albumen that check-out console uses is mycoplasma hyopneumoniae, avoids the cross interference of other porcine mycoplasmal or cause of disease in measuring samples; (3) in enzyme conjugates working fluid, activeconstituents is the anti-pig SC protein monoclonal antibody of HRP mark, therefore, only has mycoplasma hyopneumoniae SIgA antibody to be detected, avoids the interference of IgG and IgM antibody in measuring samples.Experimental result also show test kit of the present invention can not with swinery common virus pig breathe with reproductive syndrome virus, pseudorabies, H1 type swine influenza virus, pig pleuropneumonia, mycoplasma hyorhinis and intestinal bacteria SIgA antibody positive nose swab sample produce cross reaction.

2. high stability and hypersensitivity: the antigen-reactive ability of the envelope antigen prepared due to different batches is different, be ensure that the high stability of different batches product by the check-out console that legal system is standby by antibody capture bag, and ensure that the hyperergy of envelope antigen in antibody test plate, substantially increase the hypersensitivity of test kit.

3. sample is convenient: measuring samples is nose swab, but not serum, achieves needlelessization sampling, reduce to pig stress.

4. can distinguish the immunity of porcine mycoplasmal pneumonia inactivated vaccine and natural infection: after intramuscular injection immune swine mycoplasma pneumonia inactivated vaccine, only produce the specific IgG antibodies in serum, and specificity SIgA antibody cannot be produced on respiratory tract surface.The natural infection of mycoplasma hyopneumoniae directly occurs in respiratory tract target organ pipe, both can produce specific IgG antibodies in serum, can produce very strong specificity SIgA antibody (mycoplasma pneumoniae SIgA antibody) again on respiratory tract surface.Apply test kit provided by the invention and can monitor infection conditions in porcine mycoplasmal pneumonia inactivated vaccine immunity field, and mycoplasma hyopneumoniae antibody assay kit in the market cannot be accomplished.

5. can be used for the early diagnosis that pneumonia porcine mycoplasmal infects: the adhesins such as mycoplasma hyopneumoniae P 97 R 1 albumen are adsorbed onto the first step that Pig bronchial epithelial cell is mycoplasma hyopneumoniae infection host, the earliest a collection of in the anti-mycoplasma hyopneumoniae P 97 R 1 protein antibodies antibody that to be also host produce for the different albumen of mycoplasma hyopneumoniae; It is the mucosa-immune of respiratory tract that host response respiratory pathogens infects the immune response produced at first, is then only the general immunity in blood.SIgA antibody is topmost immune molecule in mucosa-immune.This test kit for envelope antigen, with respiratory tract SIgA antibody for detected object, achieves the early diagnosis to mycoplasma hyopneumoniae infection from pathogen infection step and host immune response order two aspects with P97R1 albumen respectively.

6. the assessment of immune effect after can be used for the immunity of porcine mycoplasmal pneumonia attenuated live vaccines: the more difficult specific IgG antibodies reaction producing high titre in serum after the immunity of porcine mycoplasmal pneumonia attenuated live vaccines, cannot be assessed Attenuate vaccine immune effect by existing commercial mycoplasma hyopneumoniae antibody assay kit temporarily.But attenuated vaccine immunity can produce the specificity SIgA antibody of higher titre at respiratory tract, therefore, carry out indirect assessment by this test kit to the immune effect of attenuated vaccine.

Accompanying drawing explanation

Fig. 1 is the checking electrophorogram of pig SC protein gene fragment, wherein M-DNA marker, 1-SC albumen PCR primer

Fig. 2: (A) Recombinant Swine SC protein induced expression SDS-PAGE schemes the front thalline of .M: albumen maker.1:BL21-pCold I induction; Thalline after 2:BL21-pCold I induces; Thalline before 3:BL21-pCold I/SC induces; Thalline after 4:BL21-pCold I/SC induces. lysate supernatant after (B) Recombinant Swine SC albumen existence form qualification .M: albumen maker.1:BL21-pCold I/SC induction; Lysate precipitation after 2:BL21-pCold I/SC induces. (C) pig restructuring SC protein purification qualification figure .M: albumen maker.1: SDS-PAGE figure before purifying; 2: SDS-PAGE figure after purifying. the Western blotting of (D) pig restructuring SC albumen identifies figure.

Fig. 3 is the specificity identification of the anti-pig SC protein monoclonal antibody that hybridoma cell strain 4H11 secretes, wherein M: pre-dyed albumen maker, 1: pig restructuring SC albumen, 2: the natural SC albumen of pig, 3: pig IgA albumen, 4: pig IgG albumen.

Fig. 4. anti-pig SC protein monoclonal antibody prepared by the present invention compares with the specificity of commercialization monoclonal antibody.A shows the specificity of anti-pig SC protein monoclonal antibody prepared by the present invention; The specificity of B display of commodity pig SC monoclonal antibody; M: albumen Marker; 1: restructuring SC albumen, 2: pig IgA albumen; 3: pig IgG albumen.

The detected result of specificity SIgA antibody after Fig. 5 porcine mycoplasmal pneumonia attenuated vaccine immunity.

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

Embodiment

Embodiment 1 prepares pig restructuring SC albumen

1. the RNA of pig SC albumen extracts

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

The process of intestinal mucosa epithelium (tracheal epithelium): careful scraping intestinal mucosa epithelium (tracheal epithelium), shreds, add PBS damping fluid (0.01M, the pH7.2-7.4) homogenate afterwards after the autoclaving of 500 μ l;

Lymphocytic process: get 500 μ l pig bloods, add 500 μ l lymphocyte separation mediums (purchased from Shanghai smart biological High Seience Technology Co., Ltd. of China), centrifugal (under 2000rpm condition 20min), precipitate resuspended with the PBS damping fluid (0.01M, pH7.2-7.4) after 500 μ l autoclavings;

Ordinary method is adopted to extract cell total rna in the sample after process

2. increase pig SC protein gene fragment

(1) RT-PCR (agents useful for same is all purchased from the precious biotechnology company limited in Dalian):

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

(2) PCR (agents useful for same is all purchased from the precious biotechnology company limited in Dalian)

Get RT-PCR product cDNA and carry out PCR reaction.PCR reaction system is as follows:

Primer 1 (SEQ ID NO:2): 5 '-GCGGAATTCAAGAGTCCCATATTCG-3 ';

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

Response procedures: 95 DEG C of 5min; 95 DEG C of 1min 30s, 52-54 DEG C of 1min 30s, 72 DEG C of 2min, 30cycles; 72 DEG C of 10min, 4 DEG C of 30s.PCR primer is carried out 1% agarose gel electrophoresis, and Successful amplification goes out pig SC protein gene fragment, and size is about 1857bp (containing primer sequence) as shown by the arrows in Figure 1.Object fragment glue is reclaimed test kit (purchased from Qiagen company) to reclaim.

(3) pig SC protein gene fragment and sequence thereof is obtained

The DNA fragmentation that step (2) is reclaimed and carrier pMD 18-T carrier ( 18-T Vector, the precious biotechnology company limited in Dalian) connect.

Whole operating process in ice bath on carry out.Carrier ligation system (reagent in connection procedure is purchased from the precious biotechnology company limited in Dalian) is as follows:

After linked system mixing, connection of spending the night under being placed in 4 DEG C of conditions

Connect the conversion of product:

1. get connecting fluid 10 μ l full dose and transform 50 μ l competent cell DH5 α (the precious biotechnology company limited in Dalian), in ice, place 30min;

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

3. often pipe adds the LB liquid nutrient medium (not containing microbiotic) that 500 μ l preheat (37 DEG C), and mixing is placed on 37 DEG C, and 200r/min vibrates 60min, makes bacteria resuscitation;

4. take out, centrifugal (5000rpm, 3min), often to remain the LB that 100 μ l bacterium liquid are laid on containing 100 μ g/ml ammonia benzyl mycin (Amp) dull and stereotyped for pipe, after room temperature places 20-30min, is inverted for 37 DEG C and cultivates 12-16h, from flat board, the single bacterium colony of picking white, obtains positive recombinant bacterium.

Recombinant plasmid extracts, qualification and the order-checking of target gene: by single bacterium colony of positive recombinant bacterium, be inoculated in 5ml containing in the LB substratum of 100 μ g/ml penbritins, cultivate 12-16h under 37 DEG C of 200r/min conditions.Get 2ml bacterium liquid and extract the recombinant plasmid inserting pig SC protein gene fragment, cut recombinant plasmid with restriction enzyme EcoR I and HindIII enzyme, reaction system 10 μ l, each composition is as follows:

After mixing, be placed in 37 DEG C of water-bath digestion 2h.Digestion products is carried out 1% agarose gel electrophoresis qualification, result display is selected in recombinant plasmid and is successfully inserted pig SC protein gene.Enzyme is cut qualification and be positive plasmid called after pMD 18-T Vector/SC, deliver to the order-checking of Nanjing Si Pujin biological company limited, the sequence of pig SC protein gene is as shown in SEQ ID NO:1.

3. build the recombinant bacterium of preparation pig SC albumen

(1) structure of recombinant expression plasmid pCold I/SC

By pCold I Vector and recombinant plasmid pMD 18-T Vector/SC, carry out double digestion with EcoR I and Hind III simultaneously.It is as follows that enzyme cuts system:

After system of being cut by enzyme is incubated 3h in 37 DEG C of water-baths, detects by 1% agarose electrophoresis and reclaim pig SC protein gene endonuclease bamhi, carrier pCold I endonuclease bamhi.Enzyme being cut back to close fragment adopts T4 ligase enzyme to connect, and linked system is as follows:

In 4 DEG C of connections of spending the night after mentioned reagent is mixed.Adopt above-mentioned same procedure to transform connection product, select recombinant plasmid, and enzyme cuts qualification, positive recombinant plasmid called after pCold I/SC.

Get pCold I/SC 10 μ L to adopt in ordinary method transformed competence colibacillus cell BL21 (DE3), through PCR and Sequence Identification, obtain positive recombinant bacterium, called after BL21-pCold I/SC.Meanwhile, by pCold I Plastid transformation competent cell BL21 (DE3), obtain contrast bacterium BL21-pCold I.

4. the preparation of pig restructuring SC albumen

Induction BL21-pCold I/SC expresses pig restructuring SC albumen, and with BL21-pCold I in contrast, concrete grammar is as follows:

(1) by BL21-pCold I/SC bacterium liquid with 2% ratio inoculation containing the LB liquid nutrient medium of 100 μ g/ml penbritins (AMP), 37 DEG C, be cultured to when OD600 value reaches 0.6-0.8 under 200rpm condition and add the IPTG that final concentration is 1m M;

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

(3) by bacterium liquid at 15 DEG C, abduction delivering 24h under 200rpm condition, collect bacterium liquid.

(4) according to a conventional method expression product is adopted SDS-PAGE electrophoresis detection, result confirms that pig restructuring SC albumen is by successful expression, and molecular weight of albumen is about 70kDa (Fig. 2 A).

(5) expression product existence is analyzed:

Get the bacterial precipitation after expression, resuspended with the PBS damping fluid of pH7.2, ultrasonic disruption 2min, until bacterium liquid change clarification is bright, collect cleer and peaceful cellular lysate liquid precipitate on cellular lysate liquid after the centrifugal 15min of 9000r/min respectively and carry out SDS-PAGE electroresis appraisal (Fig. 2 B), result proves, Recombinant Swine SC albumen mainly exists with inclusion bodies.

(6) purifying of pig restructuring SC albumen: adopt German Macherey-nagel company ni-TED 2000 ni-sepharose purification kits pig restructuring SC albumen, concrete operations are carried out according to the specification sheets of test kit.

Pig restructuring SC albumen after purifying is through SDS-PAGE electroresis appraisal, and result is as Fig. 2 C.Can see, the pig restructuring SC purity of protein after purifying reaches 96%.

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

With goat-anti people SC albumen many anti-(Santa Cruz company of the U.S.) for primary antibodie, adopt Western blot to identify expression product, result as shown in Figure 2 D, proves that pig restructuring SC albumen has good antigen reactivity.

Embodiment 2 prepares anti-pig SC protein monoclonal antibody

1. mouse immune

The pig of embodiment 1 purifying restructuring SC albumen is mixed with Freund's complete adjuvant (Sigma-Aldrich) equal-volume, and it is fully emulsified, subcutaneous multiple spot immunity 5 BALB/c mouse in 8 week ages (Yangzhou University's comparative medicine center), antigen inoculation dosage is 100 μ g/.After head exempts from 2 weeks, same dose pig restructuring SC albumen and Freund's incomplete adjuvant (Sigma-Aldrich) emulsification after with approach immune mouse, every 2 weeks afterwards same way immune mouses, repeat 3 times, until serum titer reaches more than 1:6400.3-5 days before cytogamy, abdominal injection doubled amount not containing adjuvant antigen booster immunization once, can cytogamy be carried out.

The preparation of 2.sp 2/0 myeloma cell

Before merging, frozen sp 2/0 cell in liquid nitrogen of 20d recovery, abandons supernatant after the centrifugal 5min of 1000r/min, resuspended with the DMEM substratum (purchased from American Gibco company) containing 20% foetal calf serum, proceeds in cell bottle, is positioned over 37 DEG C, 5%CO ₂, Secondary Culture in saturated humidity incubator.Merge to get the same day and be in logarithmic phase, cellular form is homogeneous, sharpness of border, sp 2/0 cell that growth conditions is good, and with Eddy diffusion after plasma-free DMEM medium washing, cell counting count board counts, and is 10 by cell dilution ⁶individual/mL, volume is about 10mL, places 37 DEG C, 5%CO ₂, merge in saturated humidity incubator for subsequent use.

3. cytogamy

The splenocyte of the BALB/c mouse in label taking topic 1 after booster immunization, makes fusogen with PEG4000, immune spleen cell and SP2/0 myeloma cell is carried out cytogamy according to a conventional method.

4. the screening of positive hybridoma cell

On after merging, whether 3d observes and merge, after merging, 4d HAT nutrient solution (Sigma-Aldrich) half amount changes liquid, and about 7d uses HT nutrient solution (Sigma-Aldrich) instead, carries out colony count simultaneously.When hybridoma to cover with at the bottom of hole 1/10, it is desirable Hybridoma Cell Culture liquid supernatant after changing liquid 2d, indirect ELISA is adopted to detect tiring of the monoclonal antibody of anti-pig SC albumen in supernatant, simultaneously using sp 2/0 cell culture supernatant as negative control, the mice serum after immune swine restructuring SC albumen is as positive control.For positive hole, then pig recombinant influenza HA albumen (preparation method, with restructuring SC albumen, only changes the SC protein gene fragment in recombinant bacterium into porcine influenza HA protein gene) bag is used by after 96 orifice plates, to carry out indirect ELISA detection.Select SC albumen test of recombinating with pig to be positive, the hybridoma wells be negative with pig recombinant influenza HA albumen test is as positive cell strain.

Indirect ELISA detection method: by the pig of purifying restructuring SC albumen (prepared by embodiment 1) bag by 96 orifice plates, doubling dilution made by Hybridoma Cell Culture liquid supernatant PBST damping fluid, SP2/0 cell culture supernatant is as negative control, the serum of pig restructuring SC protein immunization mouse is as positive control, and in detection Hybridoma Cell Culture liquid supernatant, monoclonal antibody tires.

Reagent in indirect ELISA detection method: phosphate buffered saline buffer (pH7.2 ~ 7.4): NaCl 8g, KCl0.2g, Na ₂hPO ₄1.44g, KH ₂pO ₄0.24g is water-soluble, adjusts pH to 7.2 ~ 7.4, is settled to 1L.PBST damping fluid: containing 0.05% tween in phosphate buffered saline buffer (pH7.2 ~ 7.4).Coating buffer is the sodium carbonate buffer of 0.05M, pH9.6: Na ₂cO ₃1.59g, NaHCO ₃2.93g, adding distil water dissolves, and is settled to 1000mL.Nitrite ion: be that 1:40 mixes and get final product with nitrite ion B according to volume ratio by the nitrite ion A in embodiment 3.Stop buffer: with in embodiment 3.

Indirect ELISA detection method is specific as follows:

1. bag quilt: antigen (the pig restructuring SC albumen after purifying) dilution is 1 μ g/ml by coating buffer, and add enzyme plate, 100 μ l/ holes, 37 DEG C of effect 1h, then 4 DEG C are spent the night;

2. wash: PBST washs, 300 μ l/ holes, and each 5min, washs 3 times, pat dry;

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

3. wash: with 2;

4. primary antibodie:

1. dilute: dilute in 1.5ml EP pipe, first pipe carries out 1:100 dilution (10 μ l cell strain nutrient solution supernatant+1ml PBST), later each pipe 2 doubling dilution, namely draws 500 μ l from the first pipe, adds (containing 500 μ l PBST) in the second pipe, mixing, from the second pipe, draw 500 μ l again, add in the 3rd pipe, mixing, by that analogy, until the 12 pipe; Use PBST as blank, SP2/0 cell culture supernatant is as negative control, and the positive serum (namely collecting the serum of immunized mice in the present embodiment step 1) gathered after pig restructuring SC protein immunization mouse is as positive control simultaneously;

2. hatch: added respectively in corresponding aperture by sample good for above-mentioned dilution, 100 μ l/ holes, hatch 1h for 37 DEG C;

5. wash: with 2;

6. two resist: adopt PBST that sheep anti-mouse igg-HRP (horseradish peroxidase mark goat anti-mouse IgG antibody, purchased from Wuhan doctor's moral company) is carried out 1:20000 dilution, add in each hole, 100 μ l/ holes, hatch 30min for 37 DEG C;

7. wash: PBST washs, 300 μ l/ holes, each 5min, wash 5 times altogether, pat dry;

8. develop the color: add nitrite ion, 100 μ l/ holes, lucifuge colour developing 5min;

9. stop: add stop buffer, 50 μ l/ holes;

10. detect the light absorption value OD at 450nm place _450nm.With sample OD _450nm>=2.1 × negative control OD _450nm, be judged to be the positive, using positive extent of dilution the tiring as monoclonal antibody that hybridoma cell strain supernatant is maximum.

5. subclone

Undertaken by limiting dilution assay.The hole that antibody titers is high, clone's number is few is selected to carry out subclone 3-5 time, until all cloning cell hole Positive rates are 100%.Finishing screen has chosen hybridoma cell strain 4H11, and what nutrient solution supernatant adopted indirect ELISA method to detect tires as 1:3200, and the SC albumen test of recombinating of this hybridoma cell strain nutrient solution supernatant and pig is positive, and is negative with pig recombinant influenza HA albumen test.

Hybridoma cell strain 4H11 preservation information is as follows:

Classification And Nomenclature is: the hybridoma cell strain 4H11 secreting anti-pig SC protein monoclonal antibody, preservation date is on March 24th, 2015, depositary institution's full name is China typical culture collection center, be called for short CCTCC, depositary institution address: Wuhan University, deposit number is: CCTCC NO:C201526.

6. the enlarged culturing of hybridoma and frozen

By hybridoma cell strain 4H11, in 24 porocyte plates, carry out enlarged culturing, after the cell in 24 orifice plates covers with, cell is proceeded in 6 orifice plates and cultivate, then proceed in cell bottle and cultivate, Secondary Culture after cell covers with, simultaneously frozen hybridoma cell strain.

7. monoclonal antibody repeated pruning

The hybridoma cell strain 4H11 of frozen 3 months being recovered, detects tiring of nutrient solution supernatant in 24 orifice plates, whether having the index of secretory antibody ability as evaluating cell strain.Result confirms that frozen hybridoma cell strain 4H11 still can the monoclonal antibody of stably excreting anti-pig SC albumen, tires and is up to 1:3200.

8. monoclonal antibody specificity identification

Adopt ordinary method just to extract the natural SC albumen of pig and pig IgA in Ruzhong from from pig, from porcine blood serum, extract pig IgG.Pig is recombinated after SC albumen, the natural SC albumen of pig, pig IgA and pig IgG albumen carries out SDS-PAGE electrophoresis, be transferred on pvdf membrane, adopt to close at 4 DEG C containing 3%BSA solution and spend the night.TBST solution (gets the Tris-HCL 10ml of 1M, pH8.0, NaCl 4.4g and Tween-20 0.5ml mixes, and adopts deionized water to be settled to 500ml) wash 3 times, each 5min, add the nutrient solution supernatant of hybridoma cell strain 4H11, hatch 2h for 37 DEG C.TBST solution washing 3 times, each 5min, adds the sheep anti-mouse igg-HRP (originating the same) of 1:10 000 times dilution, hatches 1h for 37 DEG C.TBST solution washing 5 times, each 5min, Enhanced chemiluminescence (ECL) exposes, and take pictures, result as shown in Figure 3.As seen from the figure, the anti-pig SC protein monoclonal antibody that hybridoma cell strain 4H11 secretes can react with recombinate SC albumen and pig natural SC albumen generation specific binding of pig, and with pig IgA and pig IgG albumen no cross reaction, specificity is better.

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

Breeding method in Mice Body is taked to prepare SC monoclonal antibody ascites in a large number.Get 12 week age BALB/c and bred female mouse, often only female mouse Intraperitoneal injection 0.5mL sterilising liq paraffin.After 7 days, get the hybridoma that growth conditions is good, adjust hybridoma concentration to (4-6) × 10 with the PBS damping fluid (pH 7.4) after autoclaving ⁶individual/ml, abdominal injection Mice Inoculated, 0.5ml/ only.After 1 week, mouse web portion obviously swells, and has touched fluctuation, now can collect ascites.After general 2-3 days, mouse web portion can swell again, can continue to collect ascites, and every mouse can be collected 1-3 time.By the centrifugal 10min of ascites 1500r/min collected, get middle layer (noting not being drawn onto the oily matter of the superiors), centrifuging and taking supernatant liquor, with Protein A affinity post (GE Healthcare company of the U.S.) purifying, obtain the anti-pig SC protein monoclonal antibody of purifying ,-20 DEG C frozen in.

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

The anti-pig SC protein monoclonal antibody of entrusting Nanjing Genscript Biotechnology Co., Ltd. to be secreted by hybridoma cell strain 4H11 adopts horseradish peroxidase (HRP) mark, and adopting BCA method to detect enzyme labelled antibody concentration is 1.357mg/mL.

11. with the comparing of commercialization monoclonal antibody specificity

Respectively by commercialization mouse-anti pig SC protein monoclonal antibody (purchased from AbD Serotec company of Britain, article No. MCA634) and the anti-pig SC protein monoclonal antibody prepared of the present invention and pig SC albumen, pig IgA albumen and pig IgG albumen carry out Western blot experiment, to compare the specificity of two kinds of monoclonal antibodies.Result as shown in Figure 4.Anti-pig SC protein monoclonal antibody prepared by the present invention only with pig SC albumen test, and all not react (Fig. 4 A) with pig IgA and IgG.Commercialization mouse-anti pig SC monoclonal antibody not only reacts with pig SC albumen, all can react with pig IgA and IgG simultaneously, and reacts stronger (Fig. 4 B) with pig IgG.

Embodiment 3 mycoplasma hyopneumoniae SIgA antibody ELISA detection kit

Be provided with in mycoplasma hyopneumoniae SIgA antibody ELISA detection kit:

1. wrapped by the antibody test plate of mycoplasma hyopneumoniae P 97 R 1 albumen by antibodycapture preparation

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

By document (Liu Maojun, Shao Guoqing, Zhang Ying, Nie Xiangting. the cloning and expression in mycoplasma hyopneumoniae P97 gene antigen determinant R1 district. Jiangsu's agriculture journal .2005.21 (3): 207-11) build the recombinant bacterium carrying mycoplasma hyopneumoniae P97 gene antigen determinant R1 district gene fragment.This recombinant bacterium is seeded to the LB liquid nutrient medium containing penbritin, in 37 DEG C, cultivate in 180rpm shaking table, OD to be reached ₆₀₀adding final concentration when=0.6 ~ 0.8 is that the IPTG of 0.1mmol/L carries out abduction delivering, thalline is collected after continuing cultivation 1 ~ 5h, 1 time is washed with PBS (pH7.2), by thalline suspension N,O-Diacetylmuramidase and ultrasonic treatment, at 4 DEG C, the centrifugal 30min of 5 000rpm, gets lysate supernatant, with His-Tag affinity chromatographic column (MERCK Products) separation and purification, obtain restructuring mycoplasma hyopneumoniae P 97 R 1 albumen, adopt BCA method to detect protein concentration.-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 Guoqing. prepared by mycoplasma hyopneumoniae adhesion factor P97R1 district monoclonal antibody. and Jiangsu's agriculture journal .2009.25 (6): 1438-1441.), adopt hybridoma cell strain 6C5 to prepare the monoclonal antibody of anti-mycoplasma hyopneumoniae P 97 R 1 albumen, and with ProteinA affinity post (GE Healthcare company of the U.S.) purifying, adopt BCA method to detect antibody concentration.

(3) preparation of antibody test plate

By square formation volumetry, determine that the best bag of anti-mycoplasma hyopneumoniae P 97 R 1 protein monoclonal antibody is 2.0 μ g/mL by concentration, the best bag of capture antigen restructuring P97R1 albumen is 2 μ g/mL by concentration.With the carbonate buffer solution (Na of 0.05mol/L, pH value 9.6 ₂cO ₃1.59g, NaHCO ₃2.93g, dissolve with distilled water, adding distil water is settled to 1000mL) the anti-mycoplasma hyopneumoniae P 97 R 1 protein monoclonal antibody of purifying is diluted to 2.0 μ g/mL, wrap tested drafting board by every hole 100 μ L, 2-8 DEG C of effect 12-18h.Discard the coating buffer in hole, with PBST (10 × concentrated cleaning solution dilutes 10 times) washing 3 times, each 3-5min.Every hole adds 200 μ L and contains the caseic PBS solution of 10g/L and (get 8g NaCl, 0.2g KCl, 2.9gNa ₂hPO ₄12H ₂o and 0.24g KH ₂pO ₄be dissolved in distilled water, adding distil water is settled to 1000mL, pH7.4) close, put 37 DEG C hatch 2h after discard.With PBST (10 × concentrated cleaning solution dilutes 10 times) washing 3 times, dry.Add the restructuring mycoplasma hyopneumoniae P 97 R 1 protein solution of 2 μ g/mL purifying by 100 μ L/ holes, put 37 DEG C hatch 1h after discard, with PBST (10 × concentrated cleaning solution dilutes 10 times) washing 3 times, each 3-5min, pats dry.Every hole adds the 50g/L sucrose solution of 150 μ L, put 37 DEG C hatch 1h after discard.With PBST (10 × concentrated cleaning solution dilutes 10 times) washing 3 times, vacuumize sealing after drying, put 2-8 DEG C of preservation.

2.10 × enzyme conjugates working fluid

The anti-pig SC protein monoclonal antibody that the horseradish peroxidase (HRP) embodiment 2 prepared marks, adopts BCA method to detect enzyme labelled antibody concentration, and to adjust concentration be 1mg/mL.Then use PBST (10 × concentrated cleaning solution dilutes 10 times) to do 1:1000 dilution, adding final concentration is 25 μ g/ml gentamicins, and degerming with 0.22 μm of membrane filtration, obtain 10 × enzyme conjugates working fluid, aseptic subpackaged, 1.2ml/ props up, 2 ~ 8 DEG C of preservations.

3. positive control solution

Take from the test pig bronchoalveolar lavage fluid of artificial challenge's mycoplasma hyopneumoniae Js strain.The serum of infected pigs mycoplasma hyopneumoniae antibody assay kit (the i.e. HerdChek Mycoplasmahyopneumoniae ELISA antibody assay kit of IDEXX company, for detecting serum IgG antibody, lower same) test positive, (Lu is quick for dawn through mycoplasma hyopneumoniae sleeve type PCR for alveolar wass liquid precipitate, Feng Zhixin, Liu Maojun, Wu Xusu, Gan Yuan, Zhang Ying, Shao Guoqing. the foundation of mycoplasma hyopneumoniae sleeve type PCR detection method and application. Jiangsu's agriculture journal .2010.26 (1): 91-95.) test positive, lungs cut open the sharp leaf of inspection, lobus cardiacus, middle leaf and lobus diaphragmaticus leading edge are in " pancreas sample " or " shrimp sample " asthma typical cytopathic, be judged to infect successfully.By bronchoalveolar lavage fluid through the centrifugal 30min of 10,000rpm, get supernatant, after 0.22 μm of frit is degerming, add the Thiomersalate that final concentration is 0.01% (mass percentage concentration), as positive control solution, aseptic subpackaged 1ml/ pipe, 2 ~ 8 DEG C of preservations.

4. negative controls

Be taken from the bronchoalveolar lavage fluid of mycoplasma hyopneumoniae negative healthy pig.Test pig serum is detected as feminine gender through the mycoplasma hyopneumoniae antibody assay kit of IDEXX company, cut open and kill rear lungs without asthma typical cytopathic, it is negative for detecting mycoplasma hyopneumoniae antigen in alveolar wass liquid precipitate with sleeve type PCR, is judged to be mycoplasma hyopneumoniae negative healthy pig.By bronchoalveolar lavage fluid through the centrifugal 30min of 10,000rpm, get supernatant, after 0.22 μm of frit is degerming, add the Thiomersalate that final concentration is 0.01% (mass percentage concentration), as negative controls, aseptic subpackaged 1ml/ pipe, 2 ~ 8 DEG C of preservations.

5.10 × concentrated cleaning solution

Get 40g NaCl, 1.0g KCl, 14.5g Na ₂hPO ₄12H ₂o, 1.2g KH ₂pO ₄, 2.5ml tween 20 is dissolved in deionized water, is settled to 500ml.Add the Thiomersalate that final concentration is 0.01% (mass percentage concentration), degerming with 0.22 μm of membrane filtration, as 10 × concentrated cleaning solution, aseptic subpackaged, 30ml/ bottle.

6. nitrite ion A

21mg TMB (3,3', 5,5'-tetramethyl benzidine, purchased from BIOSHARP company) is dissolved in 5ml dehydrated alcohol, and mixing packing, 1ml/ props up, as nitrite ion A.

7. nitrite ion B

33mg UHP (urea peroxide element, purchased from Shanghai Jing Chun biochemical technology limited-liability company) be dissolved in 200ml, pH be 5.2 phosphate buffered saline buffer (get 2.7g Na ₂hPO ₄12H ₂o, 13.2g KH ₂pO ₄be dissolved in deionized water, be settled to 500ml), add the gentamicin that final concentration is 25 μ g/ml, degerming, aseptic subpackaged with 0.22 μm of membrane filtration, 15ml/ bottle, as nitrite ion B.

8. stop buffer

Stop buffer is the sulphuric acid soln of 2mol/L.

The sample that test kit of the present invention detects is hog snout swab samples to be checked.Kit containment of the present invention is under 4-7 DEG C of condition.

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

The present embodiment illustrates the detection schedule of operation of mycoplasma hyopneumoniae SIgA antibody ELISA detection kit prepared by embodiment 3.

1. preparation of reagents

(1), before detecting, reagent all in mycoplasma hyopneumoniae SIgA antibody ELISA detection kit is at room temperature placed 30 minutes, makes all reagent return to room temperature.

(2) 1 × washings preparations.Return to 10 × concentrated cleaning solution of room temperature, 37 DEG C of temperature baths 5 ~ 10 minutes, 10 times can be diluted with sterilizing pure water, be 1 × washings, preserve 7 for 2 ~ 8 DEG C if any precipitation.

(3) 1 × enzyme conjugates working fluids.10 × enzyme conjugates working fluid, 1 × washings is diluted 10 times, for subsequent use.

(4) substrate nitrite ion preparation.Nitrite ion A and nitrite ion B are that 1:40 mixes according to volume ratio, obtain substrate nitrite ion, for subsequent use.

2. detect schedule of operation

(1) get antibody test plate, arrange negative control hole, Positive control wells and sample detection hole, a repetition is done in every hole.In negative control hole, add negative controls, in Positive control wells, add positive control solution, add nose swab sample to be checked in sample detection hole, add-on is 100 μ L/ holes.

(2) the antibody test plate after application of sample is put 37 DEG C of incubations 120 minutes.

(3) discard the liquid in antibody test plate hole, each hole adds 1 × washings 250 μ L and washs, and washs 5 times.Note, after last washing, antibody test plate be dried, water-absorbing material firmly pat dry, removes remaining liquid.

(4) every hole adds 1 × enzyme conjugates working fluid 100 μ L, puts 37 DEG C of incubations 60 minutes.

(5) repeating step (3).

(6) every hole adds 100 μ L substrate nitrite ions, and 37 DEG C of incubation lucifuges develop the color 7 minutes.

(7) every hole adds 50 μ L stop buffers, termination reaction.

(8) measure by microplate reader and record sample detection hole and the light absorption value (OD of control wells at 450nm place ₄₅₀value).

3. result judges

As 1.2 < Positive control wells OD ₄₅₀value < 2.0, and 0.05 < negative control hole OD ₄₅₀during value < 0.25, test-results is reliable.

S/P=(sample detection hole OD per sample ₄₅₀value-negative control hole OD ₄₅₀value)/(Positive control wells OD ₄₅₀value-negative control hole OD ₄₅₀value), calculation sample S/P.When sample S/P >=0.17 is judged to the positive, the wild poison of pig-pig infection mycoplasma pneumoniae to be checked or vaccine virus are described; When sample S/P < 0.17 is judged to feminine gender, Zhu Wei infected pigs to be checked mycoplasma pneumoniae is described.

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

1. nose swab sample

Nose swab sample comprises 150 parts of mycoplasma hyopneumoniae SIgA negative antibody nose swab samples and the Antigen positive hybridomas nose swab sample of 7 parts of other respiratory tract disease.

150 parts of mycoplasma hyopneumoniae SIgA negative antibody nose swab samples: gather from not having immune any i (mycoplasma hyopneumoniae) vaccine and nearly pig farm that porcine mycoplasmal pneumonia does not occur for 2 years, the serum antibody of its correspondence adopts the mycoplasma hyopneumoniae antibody assay kit of IDEXX company to detect and is mycoplasma hyopneumoniae negative antibody.

The nose swab sample that 7 parts of other respiratory tract disease are Antigen positive hybridomas: comprise pig and breathe and reproductive syndrome virus (PRRSV) SIgA antibody positive hog snout swab, pseudorabies (PRV) SIgA antibody positive hog snout swab, H1 type swine influenza virus (SIV) SIgA antibody positive hog snout swab, pig pleuropneumonia (APP) SIgA antibody positive hog snout swab, mycoplasma hyorhinis (MHR) SIgA antibody positive hog snout swab, intestinal bacteria (E.coli) SIgA antibody positive nose swab, mycoplasma hyopneumoniae (MHP) SIgA antibody positive nose swab.

2. detection method

Adopt method A and B to detect to above-mentioned 157 increment product simultaneously.

Method A: adopt the mycoplasma hyopneumoniae SIgA antibody ELISA detection kit that prepared by method in embodiment 3 according to method in embodiment 4 detect above-mentioned each sample whether with this test kit generation nonspecific reaction, calculate negative recall rate.

Method B: adopt that application number is 2009100257043, name to be called in the application for a patent for invention of anti-mycoplasma hyopneumoniae SIgA indirect ELISA detection method open method, detect above-mentioned each sample whether with the method generation nonspecific reaction, calculate negative recall rate.

3. result

Result shows: test kit of the present invention to 7 parts of equal no cross reactions of other pathogenic autoantibody, to the negative product recall rates of 150 parts of MHP up to 97.3% (146/150), in table 1.Method disclosed in the application for a patent for invention that application number is 2009100257043, name is called anti-mycoplasma hyopneumoniae SIgA indirect ELISA detection method, 92.7% (139/150) is only only had to the negative product recall rate of 150 parts of MHP, maximum defect is that the method exists cross reaction, in table 2 to intestinal bacteria antibody.

The specificity of table 1 method A

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

The specificity of table 2 method B

Note: S/P value >=0.15 is positive, S/P value < 0.1 is negative, is suspicious as 0.1≤S/P value < 0.15."-" represents negative, and "+" represents positive.

Embodiment 6 mycoplasma hyopneumoniae SIgA antibody ELISA detection kit distinguishes the immunity of porcine mycoplasmal pneumonia inactivated vaccine and natural infection

1. sample

From 260 pigs on 4 pig farms, gather nose swab sample and the serum sample of every pig respectively, respectively amount to 260 parts.Wherein, A pig farm does not have immune any i (mycoplasma hyopneumoniae) vaccine, porcine mycoplasmal pneumonia does not occur in nearly 2 years; B pig farm immune swine mycoplasma pneumoniae inactivated vaccine, does not find porcine mycoplasmal pneumonia clinical symptom; C and D pig farm does not have immune any i (mycoplasma hyopneumoniae) vaccine, and the pig of more than 70% exists cough, asthma, the porcine mycoplasmal pneumonia clinical symptom such as ventral breathing.

2. detection method

(1) serum IgG antibody detects: test method is carried out in strict accordance with the specification sheets operation steps of the mycoplasma hyopneumoniae antibody assay kit of IDEXX company.

(2) mycoplasma hyopneumoniae SIgA antibody test, adopts the mycoplasma hyopneumoniae SIgA antibody ELISA detection kit that in embodiment 3 prepared by method to detect according to method in embodiment 4.

(3) mycoplasma hyopneumoniae Detection of antigen: (Lu is quick for dawn by document, Feng Zhixin, Liu Maojun etc. the foundation of mycoplasma hyopneumoniae sleeve type PCR detection method and application. Jiangsu's agriculture journal .2010.26 (1): 91-95) method employing sleeve type PCR detection mycoplasma hyopneumoniae.

3. result

Result is as shown in table 3, and what in four pig farms, three kinds of detection method result differences were maximum appears at B pig farm.For B pig farm, the positive rate of mycoplasma hyopneumoniae SIgA antibody and mycoplasma hyopneumoniae Detection of antigen result is all lower, and coincidence rate is higher; And the serum IgG antibody positive rate that the ELISA antibody assay kit of IDEXX company detects is very high, very low with other two kinds of Indexs measure result coincidence rates.Generally, after the immunity of mycoplasma hyopneumoniae inactivated vaccine, pig produces higher serum IgG antibody, comparatively has difficult labour raw or only produces low-level mucous membrane SIgA antibody; The pig of natural infection mycoplasma hyopneumoniae, can produce higher serum IgG antibody and mucous membrane SIgA antibody.Therefore the mycoplasma hyopneumoniae SIgA antibody positive rate on B pig farm is lower, and the positive rate of C, D pig farm mycoplasma hyopneumoniae SIgA antibody is higher.Proved by the Analysis of test results of sleeve type PCR to mycoplasma hyopneumoniae antigen, test kit of the present invention can detect the pig of natural infection mycoplasma hyopneumoniae, can not detect the pig of Pigs Inoculated mycoplasma pneumonia inactivated vaccine, namely test kit of the present invention can distinguish the immunity of porcine mycoplasmal pneumonia inactivated vaccine and natural infection.

Table 3. mycoplasma hyopneumoniae antibody/antigen Positive rate is added up

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

1. sample

The nose swab sample that the pig deriving from immune swine mycoplasma pneumonia attenuated live vaccines (Nanjing Tianbang Bio-industry Co., Ltd.) in lung gathers in different time points and serum sample.

2. detection method

(1) Serum Antibody Detection: test method is carried out in strict accordance with the specification sheets operation steps of the mycoplasma hyopneumoniae antibody assay kit of IDEXX company.

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

3. result

As shown in Figure 5, adopt the specificity SIgA antibody-secreting produced in porcine respiratory after test kit effectively can detect the immunity of porcine mycoplasmal pneumonia attenuated live vaccines in embodiment 3, and there is certain rising and the rule of decline afterwards in immunity in antibody titers.And Fig. 6 result shows, more difficultly after the immunity of porcine mycoplasmal pneumonia attenuated live vaccines from serum, detect that antibody turns sun (S/P>0.4 for positive).This embodiment result proves, test kit of the present invention indirectly can be assessed porcine mycoplasmal pneumonia attenuated live vaccines immune effect.

The susceptibility of embodiment 8 mycoplasma hyopneumoniae SIgA antibody ELISA detection kit

1. nose swab sample

135 parts of clinical hog snout swabs, all gather the pig farm from not having immune any i (mycoplasma hyopneumoniae) vaccine, there is cough in the pig of more than 70%, asthma, the porcine mycoplasmal pneumonia clinical symptom such as ventral breathing, the serum antibody of its corresponding pig is the mycoplasma hyopneumoniae IgG antibody positive through the mycoplasma hyopneumoniae antibody assay kit detection of IDEXX company.Another 61 parts of positive nose swabs are the hog snout swab samples that after artificial infected pigs mycoplasma pneumoniae Js strain, 28 days gather.

2. detection method

Prepare three batches of test kits (lot number is respectively 001,002,003, hereinafter referred to as test kit of the present invention) according to embodiment 3, and detect whether containing mycoplasma hyopneumoniae SIgA antibody in all samples, calculating positive rate.Alternative selects 5 parts of higher positive nose swab samples of titre in artificial challenge's sample, detects total protein concentration, and detects with test kit in embodiment 3 after doing continuous 2 doubling dilutions of 1:2 ~ 1:64, calculates limit of identification.

Be that in the application for a patent for invention of 2009100257043 (name is called anti-mycoplasma hyopneumoniae SIgA indirect ELISA detection method), open method detects mycoplasma hyopneumoniae SIgA antibody simultaneously with application number, calculate positive rate and limit of identification.

3. result

Three batches of test kits of the present invention are adopted to detect 196 parts of mycoplasma hyopneumoniae positive clinical nose swab samples of clinical acquisitions, its positive rate is respectively 192/196 (98.0%), 191/196 (97.4%), 191/196 (97.4%).Adopt application number to be that in the application for a patent for invention of 2009100257043 (name is called anti-mycoplasma hyopneumoniae SIgA indirect ELISA detection method), open method detects, number positive is 180/196, and positive rate only only has 91.8%, in table 4.

Positive nose swab sample (Ps1 ~ 5) of 5 parts of artificial challenges is selected to measure protein concentration, and after 2 doubling dilutions, use test kit of the present invention (lot number 002) and application number to be that in the application for a patent for invention of 2009100257043, open method detects mycoplasma hyopneumoniae SIgA antibody respectively.Result shows, and test kit of the present invention is 42.50 ~ 48.75 μ g/ml to the concentrations that mycoplasma hyopneumoniae SIgA antibody positive nose swab sample is minimum, and the maximum extension rate that detects is 1:16 (see table 5).Application number is adopted to be that in 2009100257043 applications for a patent for invention, open method detects, be 170.00 ~ 390.00 μ g/ml to the concentrations that mycoplasma hyopneumoniae SIgA antibody positive nose swab sample is minimum, the maximum extension rate that detects is 1:2 ~ 1:4 (see table 6).

Table 4 test kit of the present invention and published anti-mycoplasma hyopneumoniae SIgA indirect ELISA detection method detect clinical sample statistics

Table 5 test kit of the present invention is to the minimum detectable concentration of the positive nose swab sample of artificial challenge

Note: S/P value >=0.17 is positive, S/P value < 0.17 is negative.

Table 6 2009100257043 application for a patent for invention method is to the minimum detectable concentration of the positive nose swab sample of artificial challenge

Note: S/P value >=0.15 is positive, S/P value < 0.1 is negative, is suspicious as 0.1≤S/P value < 0.15.

Embodiment 9 mycoplasma hyopneumoniae SIgA antibody ELISA detection kit stability

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

1. replica test batch

With 5 test kits (Kit1-Kit5) of same batch, parallel test is carried out to 10 parts of mycoplasma hyopneumoniae SIgA negative antibody nose swabs sample (1 ~ 10) and 10 parts of mycoplasma hyopneumoniae SIgA antibody positive nose swabs sample (11 ~ 20).

2. replica test batch

With 3 batches of test kits (001,002,003 batch) with 10 parts of mycoplasma hyopneumoniae SIgA antibody positive nose swabs sample (11 ~ 20), parallel simultaneous test is carried out to 10 parts of mycoplasma hyopneumoniae SIgA negative antibody nose swabs sample (1 ~ 10).

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

3. result

Experimental result shows, and detects the variation within batch coefficient C.V.% of 10 parts of negative nose swabs and 10 parts of positive nose swabs respectively in 1.99% ~ 9.99% (see table 7) with 5 test kits of same batch; The interassay coefficient of variation of 10 parts of negative nose swabs and 10 parts of positive nose swabs is detected in 1.17% ~ 9.90% (see table 8) with the test kits of 3 batches.Between testing in batch and criticizing, experiment results proved test kit of the present invention has satisfactory stability.

In table 7 batch, different test kit detects the test-results of mycoplasma hyopneumoniae SIgA antibody

Table 8 different batches test kit detects the test-results of mycoplasma hyopneumoniae SIgA antibody

SEQUENCE LISTING

 

<110> Jiangsu Province Agriculture Science Institute

 

<120> anti-pig SC protein monoclonal antibody and preparing mycoplasma hyopneumoniae SIgA antibody ELISA detection kit

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 (8)

1. secrete a hybridoma cell strain 4H11 for anti-pig SC protein monoclonal antibody, preserving number is: CCTCC NO:C201526.

2. the anti-pig SC protein monoclonal antibody of hybridoma cell strain secretion described in claim 1.

3. described in claim 2, anti-pig SC protein monoclonal antibody is preparing the application in mycoplasma hyopneumoniae SIgA antibody ELISA detection kit.

4. mycoplasma hyopneumoniae SIgA antibody ELISA detection kit, described test kit contains the enzyme conjugates working fluid of the check-out console being coated with mycoplasma hyopneumoniae P 97 R 1 albumen prepared by antibodycapture and the anti-pig SC protein monoclonal antibody containing horseradish peroxidase-labeled, and described anti-pig SC protein monoclonal antibody is secreted by hybridoma cell strain described in claim 1.

5. mycoplasma hyopneumoniae SIgA antibody ELISA detection kit according to claim 4, preparation with the following method adopted by the check-out console that it is characterized in that being coated with mycoplasma hyopneumoniae P 97 R 1 albumen: check-out console is first adopted anti-mycoplasma hyopneumoniae P 97 R 1 protein monoclonal antibody bag quilt, then adopts mycoplasma hyopneumoniae P 97 R 1 albumen bag quilt.

6. mycoplasma hyopneumoniae SIgA antibody ELISA detection kit according to claim 5, it is characterized in that the bag of described anti-mycoplasma hyopneumoniae P 97 R 1 protein monoclonal antibody is 1.5-2.5 μ g/mL by concentration, the bag of described P97R1 albumen is by concentration 1.5-2.5 μ g/mL.

7., according to the described mycoplasma hyopneumoniae SIgA antibody ELISA detection kit of one of claim 4-6, it is characterized in that described test kit also comprises positive control solution, negative controls, washings, nitrite ion and stop buffer.

8. mycoplasma hyopneumoniae SIgA antibody ELISA detection kit according to claim 7, it is characterized in that positive control solution is the pig bronchoalveolar lavage fluid of artificial infected pigs mycoplasma pneumoniae, negative controls is the pig bronchoalveolar lavage fluid of non-infected pigs mycoplasma pneumoniae; Washings compound method: get 40g NaCl, 1.0g KCl, 14.5g Na ₂hPO ₄12H ₂o, 1.2g KH ₂pO ₄, 2.5ml tween 20 is dissolved in deionized water, is settled to 500ml; Nitrite ion comprises nitrite ion A and nitrite ion B, and wherein nitrite ion A obtains after 21mg 3,3', 5,5'-tetramethyl benzidine is dissolved in 5ml dehydrated alcohol; Described nitrite ion B obtains after 33mg urea peroxide element is dissolved in 200ml phosphate buffered saline buffer; Described stop buffer is the sulphuric acid soln of 2mol/L.