CN105717299A - Indirect ELISA kit based on Mo P113 protein and use method - Google Patents

Indirect ELISA kit based on Mo P113 protein and use method Download PDF

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CN105717299A
CN105717299A CN201610065704.6A CN201610065704A CN105717299A CN 105717299 A CN105717299 A CN 105717299A CN 201610065704 A CN201610065704 A CN 201610065704A CN 105717299 A CN105717299 A CN 105717299A
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serum
mop113
elisa
indirect elisa
reagent kit
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程振涛
吴燕
岳筠
文明
周碧君
王开功
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Guizhou University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

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Abstract

The invention discloses an indirect ELISA kit based on Mo P113 protein and a use method. The indirect ELISA kit based on Mo P113 protein is prepared from an elisa plate coated with Mo P113 protein, Mo standard positive serum, Mo standard negative serum, bethyl, a 50*PBST buffer solution, a substrate solution A, a substrate solution B and a stop solution. The indirect ELISA kit can detect sheep serum Mo infection antibodies and vaccine immunity antibodies, a key technology is provided for earlier monitoring of goat and sheep mycoplasmal pneumonia caused by Mo, vaccine immunity effect evaluation and correlational research work, and great significance is achieved in early finding and early prevention and control of epidemic diseases caused by the pathogeny.

Description

A kind of indirect ELISA reagent kit based on Mo P113 albumen and using method
Technical field
The present invention relates to a kind of indirect ELISA reagent kit based on MoP113 albumen and using method.
Background technology
Mycoplasma ovine pneumoniae is to cause one of goat and sheep generation chronic respiratory road transmission disease and the main pathogenic fungi of important epidemic disease affecting sheep husbandry development, often harm 1 ~ 3 monthly age lamb, flock of sheep infect after performance respiratory disorder, rhinorrhea, gradual become thin, the chronic severe acute respiratory syndrome symptom (EnginBalikci, 2008) such as growth retardation and interstitial pneumonia.Mackay in 1963 etc. separate in sheep body first and obtain mycoplasma ovine pneumoniae, Cottew is separated to identical pathogen in the Australian sheep body suffered from an inflammation of the lungs subsequently, Carmicheal is separated to has pathogenic this novel mycoplasma, and called after mycoplasma ovine pneumoniae (MackayJMK, 1963；GS.Cottew, 971；CarmichaelLE, 1972).China in 1978 is separated to this cause of disease first in the kind sheep body introduced by New Zealand and other places, hereafter multiple provinces and cities such as China Guizhou, Yunnan, Gansu, Hunan have this sick report (Hu Jingshao, 1982；Li Ai, 2007).The early stage mycoplasma pneumonia case prevalence that mycoplasma ovine pneumoniae causes is high, mortality rate is low, not caused the attention of people for a long time, but in recent years investigated discovery, this disease mortality rate is continuous ascendant trend, serious economic loss (Zhang Shuanxiang, 2013) is carried out to sheep raising industrial belt.
Prevention and control to mycoplasma ovine pneumoniae, currently mainly rely on chemoprophylaxis, still lack reliable vaccine, trace it to its cause waste time and energy with Mo separation and Culture and genome research slowly relevant (wait Xiang Shan etc., 2006；Zhao Ping etc., 2008).Clinical vaccine (infectious pleuropneumonia in sheep inactivated vaccine) application displays that the Mo popular case vaccine immunity control effect of infection not good, carrys out serious economic loss (Zhang Shuanxiang, 2012) to sheep raising industrial belt.And lack at present the rapid serological detection means needed for epidemic disease detection and prevention and control, especially can high throughput testing, ELISA method that sensitivity is high.MoP113 albumen is a kind of important adhesion molecule and film surface immunogen, and adhesion molecule and cause of disease pathogenicity have dependency, and the target protein that can study as subunit vaccine and serodiagnosis (Zhang Yaning, 2013；SimionattoS, 2012；Zhang Yue, 2013；Wei Yanna, 2013).And P113 albumen is relatively big, full genome is expressed and is easily formed inclusion body, affects expression efficiency, selects it relatively to stablize C-terminal gene and can effectively detect the feature such as antigenicity of this albumen.
Summary of the invention
The technical problem to be solved in the present invention is in that, choose the Mo Guizhou separation strain strain that nucleotide homology is higher, construction and expression by MoP113 prokaryotic expression vector, set up the indirect ELISA method based on expression product MoP113 albumen, and carry out condition optimizing, prepare into commercialization indirect ELISA reagent kit.The invention of this indirect ELISA reagent kit, provides key technology by the goat caused for Mo and the early monitoring of sheep interstitial pneumonia epidemic disease, Efficacy evaluation, and the morning of epidemic disease caused by this cause of disease being found, early prevention and control are significant.
The technical scheme is that a kind of indirect ELISA reagent kit based on MoP113 albumen, the composition of raw materials of indirect ELISA reagent kit is: coated elisa plate 1 ~ 5, Mo standard positive serum 0.5-1.5mL, Mo standard female serum 0.5-1.5mL, ELIAS secondary antibody 300 ~ 500 μ L, 50 × PBST buffer 20 ~ 25mL, substrate solution A10mL ~ 15mL, substrate solution B10mL ~ 15mL and stop buffer 10mL ~ 15mL.
Coated elisa plate is that recombiant protein is coated, recombiant protein is that MoP113 gene is by escherichia coli Rosseta(DE3) expression system acquisition P113 recombiant protein, and carry out recombinant protein purification by nickel post method protein purification test kit, it is 1.5 μ g ~ 3.5 μ g/100 μ L, room temperature coated elisa plate 12h that use is coated buffer dilution.The BSA solution of 3g/100mL, is coated elisa plate after 37 DEG C of closing ELISA Sptting plate 1.5h.
The hyper-immune serum that Mo standard positive serum application mycoplasma ovine pneumoniae GZ-QX1 strain immune goat obtains, dilutes by 1:100.
Mo standard female serum is gather the lowlenthal serum from Mo serum antibody feminine gender, dilutes by 1:100.
Described ELIAS secondary antibody is the anti-sheep IgG-HRP of rabbit, uses by 1:2000 dilution.
Described 50 × PBST buffer is: NaCl400.0g, KCl10.0g, KH2PO410.0g, Na2HPO4 12H2O145g, Tween-2025mL are dissolved in 400 ~ 500mL distilled water, and adjusting pH is 7.2～7.6, is settled to 1000mL.
Described substrate solution A is Na2HPO414.60g, citric acid 9.33g, carbamide peroxide 0.52g, is dissolved in tri-distilled water, and is settled to 1000mL, is adjusted to pH5.0～5.4.Described substrate solution B is TMB20mg, dehydrated alcohol 8 ~ 10mL, adds distilled water to 1000mL, and filtration sterilization is aseptic subpackaged.
Described stop buffer is 0.2%～0.3%HF solution.
The using method of a kind of indirect ELISA reagent kit based on MoP113 albumen, comprises the steps of
First, take coated elisa plate, application PBST washs ELISA Sptting plate 5 times；
Second, positive and negative control serum and serum to be checked are done 1: 100 times of dilution, add in ELISA Plate reacting holes by 100 μ L, 37 DEG C hatch 1h after, application PBS washs each reacting hole of ELISA ELISA Plate 5 times；
3rd, anti-for rabbit sheep IgG-HRP PBST is 1:2000 times and dilutes, add ELISA Plate reacting hole by 100 μ L, after 37 DEG C of effect 1h, application PBST detersive enzyme target reacting hole 5 times；
4th, each for substrate solution A and B 50 μ L are added ELISA Sptting plate, lucifuge color development at room temperature 10 ~ 15min, adds 50 μ L stop buffers, under microplate reader 630nm wavelength, read OD value immediately.
5th, test establishment condition is positive serum OD630 meansigma methods >=2.846, negative serum OD630 meansigma methods < 1.627；Result of the test criterion is sample OD630 value >=2.325 to be checked is the positive, and sample OD630 value < 2.325 to be checked is negative.
The method have the benefit that the inventive method is applicable to detection sheep Mo and infects antibody and immune antibody, Mo infection level or immune effect of vaccine in animal body can be embodied, the early stage of the goat caused for Mo and sheep interstitial pneumonia epidemic disease is detected, Efficacy evaluation and follow-up study work provide key technology, the morning of epidemic disease caused by this cause of disease is found that early prevention and control are significant, changes primary disease immunity prevention and control monitoring and lack the situation of commercial ELISA kit.The present invention compared with prior art, has following technical advantage and good effect:
(1) effective: test kit can embody body Mo infection level or vaccine immunity level preferably；This test kit is coated relative to whole bacterial protein, and its sensitivity, specificity are higher, significant for the Mo goat caused and the early monitoring of sheep interstitial pneumonia, vaccine immunity effect evaluation.
(2) preparation is simple, production cost is low: this test kit is coated relative to whole bacterial protein, solve Mo and cultivate the problems such as difficulty, cultivation cycle length, and ensure that repeatable, concordance between test kit batch, the production in enormous quantities of recombiant protein, purification cost are low, cycle is short, improves marketing and is likely to.
(3) economical, societal benefits are high: the disappearance of commercial kit is effectively filled up in the exploitation of test kit, and wide market, in the Inner Mongol, Xinjiang, the goat culturing area such as Guizhou be widely applied prospect, epidemic disease program caused by Mo can be simplified, improving immune effect of vaccine, the production application of test kit will produce good economic and social benefit.
Accompanying drawing explanation
The indirect ELISA reagent kit that Fig. 1 is the MoP113 albumen of the present invention prepares flow sheet.
Detailed description of the invention
Embodiments of the invention:
Indirect ELISA reagent kit raw material and formula based on MoP113 albumen be:
ELISA Plate；Mo standard positive and standard female serum；Recombiant protein is that MoP113 gene is by escherichia coli Rosseta(DE3) expression system acquisition P113 albumen, and carry out recombinant protein purification by nickel post method protein purification test kit, the coated buffer dilution of purifying protein is 1.5 μ g/100 μ L；It is coated buffer: Na2CO31.59g、NaHCO32.93g is dissolved in deionized water, is settled to 1000mL, is adjusted to pH9.5～9.8；ELIAS secondary antibody: the anti-sheep IgG-HRP of rabbit；1 × PBST buffer: NaCl8.0g, KCl0.2g, KH2PO40.2g、Na2HPO4·12H2O2.9g, Tween-200.5mL are dissolved in 800mL distilled water, and adjusting pH is 7.2～7.6, is settled to 1000mL；Block buffer: 1g ~ 5g gelatin is dissolved in 10mLPBST buffer；Substrate solution A:Na2HPO414.60g, citric acid 9.33g, carbamide peroxide 0.52g, be dissolved in tri-distilled water, and be settled to 1000mL, be adjusted to pH5.0～5.4；Substrate solution B:TMB20mg, dehydrated alcohol 10mL, add distilled water to 1000mL, and filtration sterilization is aseptic subpackaged；Stop buffer: 0.2%～0.3%HF solution；
As schematically shown in Figure 1, the preparation method of the indirect ELISA reagent kit of the present invention is:
First, prokaryotic expression MoP113 albumen, and to obtain protein concentration through affinity chromatography purification be 0.158mg/mL.Application SDS-PAGE analyzes its purification effect.
Second, the purified fusion protein P113 of step one is as envelope antigen, and optimizes indirect ELISA reaction condition, and indirect ELISA method optimum reaction condition is: antigen coated liquid concentration 1.5 μ g/100 μ L；Antigen coated condition room temperature 12h；Confining liquid 1% gelatin, off-period 1h；Serum dilution 1:100 positive, negative and to be checked；ELIAS secondary antibody diluted concentration 1:2000；Chromogenic reaction time 15min.
3rd, take the sheep blood serum of 92 parts of known Mo negative antibodies, by the optimum reaction condition that step 2 is determined, carry out indirect ELISA reaction and determine its criterion.Sample OD630Value >=negative sample OD630Meansigma methods (the X)+3(SD of value)=2.325, the positive can be judged in the level of 99%.Therefore as sample OD630During value >=2.325, it is judged to the positive；As sample OD630During value < 2.325, it is judged to feminine gender；
4th, the best use step based on restructuring MoP113 albumen indirect ELISA reagent kit with method is:
Taking coated elisa plate, application PBST washs ELISA Sptting plate 5 times；Positive serum, negative serum and serum PBST to be checked are done 1: 100 times of dilution, adds ELISA Sptting plate by 100 μ L, after 37 DEG C of effect 1h, wash ELISA Sptting plate 5 times with PBS；Anti-for rabbit sheep IgG-HRP PBST being 1:2000 times dilute, add ELISA Sptting plate with 100 μ L, after 37 DEG C of effect 1h, application PBST washs ELISA Sptting plate 5 times；Each for substrate solution A and B 50 μ L are added ELISA Sptting plate, lucifuge color development at room temperature 15min, adds 50 μ L stop buffers, under microplate reader 630nm wavelength, read OD value immediately.Result of the test criterion is sample OD630Value >=2.325 is positive, OD630Value < 2.325 is negative.
5th, the indirect ELISA method that applying step two ~ tetra-is set up, the OD of detection Mo standard positive serum630Value is 2.846；And detect the OD of the standard positive serums such as Mycoplasma mycoides subsp.capri, toxoplasma, foot and mouth disease, goatpox, bluetongue630Value all shows negative value, illustrates that the indirect ELISA specificity set up is good；
6th, the indirect ELISA method that applying step two ~ tetra-is set up, in batch and batch between the replica test coefficient of variation be all≤15.0%, it is determined that this indirect ELISA criticize interior repeatability and batch between repeatability well；
7th, the indirect ELISA method that applying step two ~ tetra-is set up, detect 184 parts of clinical sheep blood serum samples, positive rate is 39.67%(73/184), it is determined that its clinical applicability is good.
8th, namely step 5, step 6 and step 7 are qualified prepares the indirect ELISA reagent kit based on MoP113 albumen.
Above first to the 7th step carries out according to proportioning raw materials.

Claims (9)

1. the indirect ELISA reagent kit based on MoP113 albumen, it is characterized in that: the composition of raw materials of indirect ELISA reagent kit is: coated elisa plate 1 ~ 5, Mo standard positive serum 0.5-1.5mL, Mo standard female serum 0.5-1.5mL, ELIAS secondary antibody 300 ~ 500 μ L, 50 × PBST buffer 20 ~ 25mL, substrate solution A10mL ~ 15mL, substrate solution B10mL ~ 15mL and stop buffer 10mL ~ 15mL.
2. a kind of indirect ELISA reagent kit based on MoP113 albumen according to claim 1, it is characterized in that: coated elisa plate is that recombiant protein is coated, recombiant protein is that MoP113 gene is by escherichia coli Rosseta(DE3) expression system acquisition P113 recombiant protein, and carry out recombinant protein purification by nickel post method protein purification test kit, it is 1.5 μ g ~ 3.5 μ g/100 μ L, room temperature coated elisa plate 12h that use is coated buffer dilution；The BSA solution of 3g/100mL, is coated elisa plate after 37 DEG C of closing ELISA Sptting plate 1.5h.
3. a kind of indirect ELISA reagent kit based on MoP113 albumen according to claim 1, it is characterised in that: the hyper-immune serum that Mo standard positive serum application mycoplasma ovine pneumoniae GZ-QX1 strain immune goat obtains, dilutes by 1:100.
4. a kind of indirect ELISA reagent kit based on MoP113 albumen according to claim 1, it is characterised in that: Mo standard female serum is gather from the negative lowlenthal serum of Mo serum antibody, dilutes by 1:100.
5. a kind of indirect ELISA reagent kit based on MoP113 albumen according to claim 1, it is characterised in that: described ELIAS secondary antibody is the anti-sheep IgG-HRP of rabbit, uses by 1:2000 dilution.
6. a kind of indirect ELISA reagent kit based on MoP113 albumen according to claim 1, it is characterised in that: described 50 × PBST buffer is: NaCl400.0g, KCl10.0g, KH2PO410.0g、Na2HPO4·12H2O145g, Tween-2025mL are dissolved in 400 ~ 500mL distilled water, and adjusting pH is 7.2～7.6, is settled to 1000mL.
7. a kind of indirect ELISA reagent kit based on MoP113 albumen according to claim 1, it is characterised in that: described substrate solution A is Na2HPO414.60g, citric acid 9.33g, carbamide peroxide 0.52g, be dissolved in tri-distilled water, and be settled to 1000mL, be adjusted to pH5.0～5.4, described substrate solution B is TMB20mg, dehydrated alcohol 8 ~ 10mL, adds distilled water to 1000mL, and filtration sterilization is aseptic subpackaged.
8. a kind of indirect ELISA reagent kit based on MoP113 albumen according to claim 1, it is characterised in that: described stop buffer is 0.2%～0.3%HF solution.
9. the using method of a kind of indirect ELISA reagent kit based on MoP113 albumen as claimed in claim 1, it is characterised in that: comprise the steps of
First, take coated elisa plate, application PBST washs ELISA Sptting plate 5 times；
Second, positive and negative control serum and serum to be checked are done 1: 100 times of dilution, add in ELISA Plate reacting holes by 100 μ L, 37 DEG C hatch 1h after, application PBS washs each reacting hole of ELISA ELISA Plate 5 times；
3rd, anti-for rabbit sheep IgG-HRP PBST is 1:2000 times and dilutes, add ELISA Plate reacting hole by 100 μ L, after 37 DEG C of effect 1h, application PBST detersive enzyme target reacting hole 5 times；
4th, each for substrate solution A and B 50 μ L are added ELISA Sptting plate, lucifuge color development at room temperature 10 ~ 15min, adds 50 μ L stop buffers, under microplate reader 630nm wavelength, read OD value immediately；
5th, test establishment condition is positive serum OD630 meansigma methods >=2.846, negative serum OD630 meansigma methods < 1.627；Result of the test criterion is sample OD to be checked630Value >=2.325 is positive, sample OD to be checked630Value < 2.325 is negative.
CN201610065704.6A 2016-02-01 2016-02-01 Indirect ELISA kit based on Mo P113 protein and use method Pending CN105717299A (en)

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Cited By (6)

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CN109781973A (en) * 2019-01-30 2019-05-21 贵州省畜牧兽医研究所 Infectious pleuropneumonia in sheep indirect ELISA checkout and diagnosis preparation method of reagent thereof
CN110045117A (en) * 2019-05-06 2019-07-23 江苏南农高科技股份有限公司 Mycoplasma hyopneumoniae solid phase competitive ELISA kit and its preparation method and application
CN110596402A (en) * 2019-09-12 2019-12-20 广西壮族自治区兽医研究所 ELISA kit for rapidly detecting mycoplasma ovipneumoniae antibody
CN111122877A (en) * 2020-01-14 2020-05-08 西南民族大学 Sheep mycoplasma pneumoniae antibody indirect ELISA detection kit
CN111323602A (en) * 2020-03-15 2020-06-23 中国农业科学院兰州兽医研究所 iELISA method for screening sheep mycoplasma pneumoniae and goat mycoplasma goat pneumonia subspecies serological negative sheep
CN112213484A (en) * 2020-12-10 2021-01-12 北京科牧丰生物制药有限公司 Method for detecting in vitro efficacy of mycoplasma hyopneumoniae inactivated vaccine based on ELISA method

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109781973A (en) * 2019-01-30 2019-05-21 贵州省畜牧兽医研究所 Infectious pleuropneumonia in sheep indirect ELISA checkout and diagnosis preparation method of reagent thereof
CN110045117A (en) * 2019-05-06 2019-07-23 江苏南农高科技股份有限公司 Mycoplasma hyopneumoniae solid phase competitive ELISA kit and its preparation method and application
CN110596402A (en) * 2019-09-12 2019-12-20 广西壮族自治区兽医研究所 ELISA kit for rapidly detecting mycoplasma ovipneumoniae antibody
CN110596402B (en) * 2019-09-12 2022-07-22 广西壮族自治区兽医研究所 ELISA kit for rapidly detecting mycoplasma ovipneumoniae antibody
CN111122877A (en) * 2020-01-14 2020-05-08 西南民族大学 Sheep mycoplasma pneumoniae antibody indirect ELISA detection kit
CN111323602A (en) * 2020-03-15 2020-06-23 中国农业科学院兰州兽医研究所 iELISA method for screening sheep mycoplasma pneumoniae and goat mycoplasma goat pneumonia subspecies serological negative sheep
CN112213484A (en) * 2020-12-10 2021-01-12 北京科牧丰生物制药有限公司 Method for detecting in vitro efficacy of mycoplasma hyopneumoniae inactivated vaccine based on ELISA method

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Application publication date: 20160629