CN103941004A - Clostridium perfringens alpha toxin double-antibody sandwich ELIS quantitative determination method - Google Patents

Clostridium perfringens alpha toxin double-antibody sandwich ELIS quantitative determination method Download PDF

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CN103941004A
CN103941004A CN201410036188.5A CN201410036188A CN103941004A CN 103941004 A CN103941004 A CN 103941004A CN 201410036188 A CN201410036188 A CN 201410036188A CN 103941004 A CN103941004 A CN 103941004A
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alpha toxin
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王海荣
孙佳芝
柴同杰
李课
陈勇
逄伟
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Shandong Agricultural University
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Abstract

The invention relates to a clostridium perfringens alpha toxin double-antibody sandwich ELIS quantitative determination method. According to the clostridium perfringens alpha toxin double-antibody sandwich ELIS quantitative determination method, alpha toxin protein obtained via prokaryotic expression is taken as an immunogen; monoclonal antibodies obtained via hybridoma technique are taken as detection antibodies and capture antibodies; reaction conditions are optimized via experiments; alpha toxin protein samples with a series of concentration are used for construction of a standard curve; the double-antibody sandwich ELIS method is established; and indexes of the double-antibody sandwich ELIS are verified. The clostridium perfringens alpha toxin double-antibody sandwich ELIS quantitative determination method specifically comprises following steps: (1) prokaryotic expression of alpha toxin; (2) preparation of anti-alpha toxin monoclonal antibodies; (3) establishment of the double-antibody sandwich ELIS method; (4) establishment of the standard curve; and (5) performance evaluation on the double-antibody sandwich ELIS method. The clostridium perfringens alpha toxin double-antibody sandwich ELIS quantitative determination method is excellent in specificity, and high in sensitivity and stability, is fast and convenient, and can be used for effective quantitative determination of alpha toxin in A-E type clostridium perfringens cultural supernatants; and the high-efficient detection method is provided for alpha toxin determination.

Description

Clostridium perfringens alpha toxin double-antibody sandwich elisa quantitative detecting method
(1) technical field
The present invention relates to a kind of clostridium perfringens alpha toxin double-antibody sandwich elisa quantitative detecting method.
(2) background of invention
C.perfringens can cause the diseases such as the necrotic enteritis, enterotoxemia of lamb dysentery and lamb, calf, piglet, rabbit, chick etc., morbidity is anxious, mortality ratio is high, be the important diseases of serious harm aquaculture, bring tremendous economic loss to various countries' animal husbandry development.Various C.perfringens all produces alpha toxin (CPA), so alpha toxin is the important evidence of diagnosis clostridium welchii disease, is also the important indicator of judging food security.
The classical way that detects alpha toxin comprises animal experiment, toxin neutralization test, lecithin decomposition run etc., but complex operation, susceptibility are lower.In recent years along with the development of biotechnology, to the existing remarkable progress of the detection method of alpha toxin, Naylor etc. have set up the ELISA method on how anti-basis both at home and abroad, and Hale etc. have set up capture antibody ELISA, and McCourt etc. detect alpha toxin with two sandwich ELISAs.But these method sensitivity are not high especially, and operability is short of at home, also cannot be quantitatively and mass detection and the detection kit that abroad can buy is expensive, be difficult to promote at home, therefore in order to make up the method blank of alpha toxin in detecting at home, need to set up in a hurry quick, sensitive, specially and can be quantitatively and the ELISA method of mass detection, the foundation of this method be also for C.perfringens disease early diagnosis, follow-up study provide effective tool.
(3) summary of the invention
The present invention has set up that specificity is good, highly sensitive, good stability, quick, easy, the double-antibodies sandwich ELISA of efficient detection alpha toxin.The method detection speed is fast, simple to operate, can quantitatively detect and mass detection, can greatly shorten sense cycle, for the early detection of alpha toxin and the propagation of clostridieum welchii prevention provide effective testing tool.
For achieving the above object, technical scheme of the present invention is:
A clostridium perfringens alpha toxin double-antibody sandwich elisa quantitative detecting method, concrete operation step is:
The prokaryotic expression of 1 clostridium perfringens alpha toxin, obtains the recombinant alpha toxin protein of purifying;
The preparation of 2 anti-clostridium perfringens alpha toxin monoclonal antibodies:
Select with the female BALB/c mouse of pure lines (4-6 age in week) of myeloma cell Sp2/0 homology as immune animal, immunization protocol: head exempts from as the recombinant alpha toxin protein (obtaining in step 1) after purifying and equal-volume are not after the complete emulsification of formula Freund's complete adjuvant, a 100 μ g/ lumbar injection; Two exempt from, three exempt to exempt from respectively at head after two weeks and five weeks by recombinant alpha toxin protein and equal-volume not after the complete emulsification of formula Freund's incomplete adjuvant, a 100 μ g/ lumbar injection; Three exempt from after three weeks lumbar injections do not add adjuvant recombinant alpha toxin protein 50～100 μ g/ only, after injection, within 3-5 days, gets the highest mouse boosting cell of tiring for Fusion of Cells.
Getting myeloma cell SP2/0 that growth conditions is good and above-mentioned ELISA detects the highest mouse boosting cell of tiring and utilizes PEG1500 to carry out chemical method fusion, use indirect ELISA method screening positive hybridoma cell, and adopt limiting dilution assay to carry out subclone 3～5 times, obtain anti-clostridium perfringens alpha toxin hybridoma cell strain CP f12; Utilize CP f12 to prepare monoclonal antibody F12.
The preparation of 3 anti-clostridium perfringens alpha toxin polyclonal antibodies:
Choose new zealand white rabbit as immune animal, head exempts from that not the subcutaneous multi-point injection 1mg/ of the complete emulsification back part of formula Freund's complete adjuvant is only into the recombinant alpha toxin protein of purifying and equal-volume, the not complete emulsification of recombinant alpha toxin protein of formula Freund's incomplete adjuvant and equal-volume purifying after head exempts from, interval immunity in two weeks once, three exempt from the recombinant alpha toxin protein that does not add the purifying of adjuvant, to strengthen exempting from for latter two weeks, after 15d, rabbit heart blood sampling obtains serum, utilize saturated ammonium sulphate method antibody purification, obtain polyclonal antibody.
The foundation of 4 double-antibody sandwich elisa quantitative detecting methods and typical curve
(1) with antigen coated liquid, (0.1M carbonate buffer solution pH9.6) is diluted to 3 μ g/mL by polyclonal antibody (coated antibody).100 μ L/ holes are coated with 96 hole ELISA Plate, and 4 ℃ are spent the night;
(2) with PBST solution (the PBS solution that contains 0.05%Tween-20: NacL8g/L, KCL0.2g/L, Na 2hPO 412H 2o3.58g/L, KH 2pO 40.27g/L, pH7.4) wash 3 times, add the PBST solution that contains 5% skimmed milk power as confining liquid 200 μ L/ holes, 4 ℃ of sealings are spent the night;
(3) with after PBST solution washing 3 times, add sample to be checked, 100 μ L/ holes, yin and yang attribute and blank are set simultaneously, positive control is the purification of Recombinant alpha toxin albumen that step 1 obtains, and Escherichia coli culture supernatant and PBS damping fluid that negative control is respectively preparation according to a conventional method (contain NacL8g/L, KCL0.2g/L, Na 2hPO 4.12H 2o3.58g/L, KH 2pO 40.27g/L), blank, for not adding any sample, is hatched 1h for 37 ℃;
After (4) 3 PBST solution washings, monoclonal antibody F12 prepared by hybridoma cell strain CP f12 is diluted to 0.2 μ g/mL with PBS damping fluid, 100 μ L/ holes, 37 ℃ of reaction 1h
(5) use PBST solution washing 3 times, add the sheep anti-mouse igg with the HRP mark of PBS damping fluid 1:10000 volume ratio dilution, hatch 1h for 37 ℃, PBST solution washing 3 times;
(6) finally add soluble T MB substrate nitrite ion 100 μ L/ holes, room temperature condition lucifuge reaction 15min, uses 2M H 2sO 4solution cessation reaction, 450nm reads light absorption value in place.
(7) take alpha toxin albumen dilute concentration is horizontal ordinate, OD 450nmvalue is ordinate curve plotting figure; According to curve map, select optimum linear scope, the natural logarithm LN (x) of concentration of take is horizontal ordinate, OD 450nmvalue, for ordinate drawing standard curve, is calculated alpha toxin concentration in testing sample by typical curve.
Determining of 4 result criterion
Calculate negative sample mean value with standard deviation (SD), try to achieve positive critical value, negative critical value.Use the DAS-ELISA setting up to measure OD 450nm, it is positive, it is negative, X &OverBar; + 2 SD < OD 450 nm < X &OverBar; + 3 SD For suspicious specimen.
The anti-clostridium perfringens alpha toxin hybridoma cell strain that the present invention prepares, called after CP f12, Classification And Nomenclature: anti-clostridium perfringens alpha toxin hybridoma cell strain; Chinese common micro-organisms culture presevation administrative center, preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences, preserving number: CGMCC8770 on January 14th, 2014, have been preserved in.
Beneficial effect of the present invention is mainly reflected in:
(1) high specificity: have stronger reactivity with recombinant alpha toxin protein (prokaryotic expression protein in step 1) and natural alpha toxin, β toxin, Escherichia coli culture supernatant, other irrelevant albumen etc., for antigen carries out ELISA detection, show no cross reaction.
(2) highly sensitive: by test and typical curve, learn, the range of linearity 5.86～375ng/mL is the valid analysing range of the method; Mean value and 2 times of standard deviation sum (OD 450nm), concentration corresponding in typical curve is 4.57ng/mL, is the method lowest detection line.
(3) reproducible: in the range of linearity, select 375,93.75, a 11.72ng/mL3 concentration point, in single test, each concentration is surveyed 10 holes, calculates variation within batch number of times; Survey 10 batches continuously, calculate interassay coefficient of variation.The method variation within batch coefficient is 2.59%～5.02%, and interassay coefficient of variation is 2.67%～5.86%, and both are all less than 10%, illustrates that same sample degree of variation in same batch and different batches is tested is very little, shows that the method has good precision.
(4) determination of recovery rates: in 10 tests, the recovery that the protein sample of 3 concentration point is measured, in 97.22%～102.85% scope, proves that the method accuracy is higher.
(4) Figure of description:
Fig. 1 clostridium perfringens alpha toxin double-antibody sandwich elisa quantitative detecting method curve map.By figure, learnt, the alpha toxin albumen dilute concentration of take is horizontal ordinate, OD 450nmvalue is depicted as curve map for ordinate, and known alpha toxin albumen has good linear relationship in 5.86～375ng/mL concentration range.
Fig. 2 clostridium perfringens alpha toxin double-antibody sandwich elisa quantitative detecting method typical curve.The natural logarithm LN of concentration (x) and OD 450nmvalue is remarkable linear relationship, and regression equation is y=0.2533x-0.2047, coefficient R 2=0.9935.
(5) embodiment:
If do not specialize, the conventional means that in embodiment, technological means used is well known to those skilled in the art.
Reagent and source thereof used in embodiment: expression vector pET28a is purchased from German Novagen company; PMD18-T is purchased from the precious biotech firm in Dalian; Bacterial genomes DNA extracts kit purchased from Tian Gen biochemical technology company limited; DNA purifying reclaims kit purchased from Tian Gen biochemical technology company limited; PCR product reclaims kit purchased from U.S. OMEGA Bio-Tek company; Restriction enzyme Bam HI and Hind III are purchased from Fermentas company; High Affinity Ni-IDA Resin is purchased from Nanjing Jin Sirui company; Not formula Freund's complete adjuvant with not formula Freund's incomplete adjuvant, IPTG, Tween20 all purchased from U.S. Sigma company; Anti-His Tag rabbit how anti-, goat anti-rabbit igg-HRP and goat anti-mouse igg-HRP all purchased from Hangzhou Huaan Bio-Tech. Co., Ltd.; 96 hole ELISA Plate are purchased from Solarbio company; Anaerobism liver bouillon, thioglycollate medium are purchased from the rich biology in sea, Qingdao; Fusion agent PEG1500 is purchased from German Roche company; BL21 (DE3) competent cell is purchased from Beijing Quan Shi King Company; Sp2/0 myeloma cell strain is purchased from Chinese Typical Representative culture collection center (Wuhan); Soluble T MB substrate nitrite ion is purchased from Tian Gen biochemical technology company limited; It is pure that other chemical reagent used are analysis.
The prokaryotic expression of embodiment 1 clostridium perfringens alpha toxin
According to the specific primer of primmer5 Software for Design:
Upstream primer: 5'-GCGGAATTCATGAAAAGAAAGATTTGT-3', as shown in SEQ No.1;
Downstream primer: 5'-GCGGCGAAGCTTTTATTTTATATTATAAGTT-3', as shown in SEQ No.2;
Selection standard bacterial classification NCTC528 increases after bacterium, with bacterial genomes DNA, extract kit and extract after DNA, utilize pcr amplification clostridium perfringens alpha toxin genetic fragment, amplification condition: 94 ℃ of 5min, 94 ℃ of 1min, 54 ℃ of 1min, 72 ℃ of 70s, carry out altogether 32 circulations, 72 ℃ are extended 7min.The object product that reclaims kit purifying through DNA purifying is connected to order-checking with pMD18-T.Check order correct recombinant plasmid and carrier pET-28a respectively with EcoR I and the processing of Xho I double digestion, after connection, proceed to and express in competent cell BL21, bacterium liquid is coated to solid LB nutrient culture media (Nacl1g, peptone 1g, yeast extract 0.5g, agar powder 1.5g, with autoclaving after 100mL deionized water dissolving), picking list colony inoculation is (kanamycins final concentration is 100 μ g/mL) in the liquid LB nutrient culture media that contains kanamycins, cultivates 3h to OD for 37 ℃ 600nmduring for 0.4-0.6, add IPTG(isopropylthiogalactoside, final concentration 1mmol/L) abduction delivering 6h, after thalline ultrasonication, through the alpha toxin recombinant protein of High Affinity Ni-changed Resin purification; Utilize purity and the immunogenicity of the alpha toxin recombinant protein after polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting (Western-blot) checking purifying, for follow-up animal immune experiment and test experience.
The preparation of the anti-clostridium perfringens alpha toxin monoclonal antibody of embodiment 2
Select with the female BALB/c mouse of pure lines (4-6 age in week) of myeloma cell Sp2/0 homology as immune animal, immunization protocol: head exempts from as the recombinant alpha toxin protein of preparation in step 1 and equal-volume are not after the complete emulsification of formula Freund's complete adjuvant, a 100 μ g/ lumbar injection; Two exempt from, three exempt to exempt from respectively at head after two weeks and five weeks by recombinant alpha toxin protein and equal-volume not after the complete emulsification of formula Freund's incomplete adjuvant, a 100 μ g/ lumbar injection; Three exempt from after three weeks lumbar injections do not add adjuvant recombinant alpha toxin protein 50～100 μ g/ only, after injection, within 3-5 days, gets the highest mouse boosting cell of tiring for lower step Fusion of Cells.After each immunity, 7d blood sampling detects antibody horizontal by indirect ELISA during this time, to detect antibody horizontal constantly, selects to tire optimum mouse for follow-up test.
Getting myeloma cell SP2/0 that growth conditions is good and above-mentioned ELISA detects the highest mouse boosting cell of tiring and utilizes PEG1500 to carry out chemical method fusion, use indirect ELISA method screening positive hybridoma cell, and adopt limiting dilution assay to carry out after 3～5 subclones, the hybridoma cell strain CP f12(that obtains the anti-clostridium perfringens alpha toxin of 2 strain has submitted Chinese microorganism strain preservation center preservation to), CP b12.Selection 6-8 age in week, female BALB/c mouse was 20, was divided into 2 groups, and lumbar injection is a formula Freund's incomplete adjuvant 0.5mL/ sensitization not, first group of injection positive hybridoma cell strain CP f12 after a week, second group of injection positive hybridoma cell strain CP b12,0.5-1 * 10 6individual/only, after 7-10 days, to collect respectively 2 groups of mouse ascites, obtain respectively a large amount of monoclonal antibody F12, B12, through indirect ELISA method, detect titer of ascites.Ascites is after caprylic acid-ammonium sulfate method purifying, and SDS-PAGE detects purification effect; Immunoblotting (Western-blot) is for monoclonal antibody CHARACTERISTICS IDENTIFICATION: the specificity of checking monoclonal antibody, identify the reactivity of monoclonal antibody and alpha toxin.
The preparation of the anti-clostridium perfringens alpha toxin polyclonal antibody of embodiment 3:
Choose 5 2-3Kg new zealand white rabbits, head exempts from that not the subcutaneous multi-point injection 1mg/ of the complete emulsification back part of formula Freund's complete adjuvant is only into the recombinant alpha toxin of purifying and equal-volume, the not complete emulsification of recombinant alpha toxin of formula Freund's incomplete adjuvant and equal-volume purifying later, interval immunity in 2 weeks once, 3 exempt from the recombinant alpha toxin that does not add the purifying of adjuvant, to strengthen exempting from for latter 2 weeks, and after 15d, rabbit heart blood sampling obtains serum.Utilize saturated ammonium sulphate method antibody purification, obtain anti-clostridium perfringens alpha toxin polyclonal antibody, by indirect elisa method, detect antibody titer, SDS-PAGE electrophoresis detection purity.
The foundation of embodiment 4 double antibodies sandwich ELISA quantitative detecting methods
By square formation titration experiments, determine the best pairing of coated antibody and detection antibody (monoclonal antibody F12, B12), and the best dilute concentration of the coated concentration of the best of coated antibody and detection antibody, and ELISA condition is groped and optimized, finally determine following system:
(1) selection of pairing antibody and optium concentration are determined
The anti-clostridium perfringens alpha toxin polyclonal antibody of initial option embodiment 3 preparations is as capture antibody, monoclonal antibody F12, the B12 being prepared by hybridoma cell strain CP f12, CP b12 as detecting antibody, tests the selection of carrying out optimum antibody pairing respectively by square formation.Capture antibody is added to 96 hole ELISA Plate the first rows with the 1:100 dilution by volume of antigen coated liquid, and with the downward 1:2 of antigen coated liquid doubling dilution to the eight row, 100 μ L/ holes, 4 ℃ of coated spending the night, PBST washing 3 times, 3min, pats dry at every turn.Every hole adds confining liquid 200 μ L, and 4 ℃ of sealings are spent the night, PBST washing 3 times, and each 3min, pats dry.Every hole adds recombinant alpha toxin protein 100 μ L/ holes (with PBS dilution toxin protein concentration to 4 μ g/mL), hatches 1h for 37 ℃, PBST washing 3 times, and each 3min, pats dry.Detection antibody is added to 96 hole ELISA Plate first rows, secondary series with the 1:100 dilution by volume of PBS damping fluid, and do 1,3,5,7,9,11 row 1:2 doubling dilutions with PBS damping fluid, 2,4,6,8,10,12 row are in like manner done 1:2 doubling dilution, 100 μ L/ holes, hatch 1h for 37 ℃, PBST solution washing 3 times, each 3min, pats dry.Add soluble T MB substrate nitrite ion, lucifuge colour developing 15min, every hole adds 50 μ L2M H 2sO 4cessation reaction, measures every hole OD value at 450nm place by microplate reader.Using P/N value as the foundation of selecting best pairing antibody and best dilute concentration.
Final definite capture antibody is anti-clostridium perfringens alpha toxin polyclonal antibody, and best effort concentration is 3 μ g/mL; Detecting antibody is monoclonal antibody F12 prepared by hybridoma cell strain CP f12, and best effort concentration is 0.2 μ g/mL.
(2) best coated condition determines
The coated capture antibody of 0.01mol/L carbonate buffer solution for first and second row of 96 hole ELISA Plate, third and fourth row with 0.05mol/L carbonate buffer solution be coated with catch anti-, five, the coated capture antibody of 0.1mol/L carbonate buffer solution for six row, the coated time of first and second row is 37 ℃ of 2h, third and fourth row is coated with time 4 ℃ of 12h, five, the coated time of six row is 37 ℃ of 1h+4 ℃ of 12h, carries out square formation titration experiments, according to P/N value, determines best coated condition.
Final definite best coated condition is the coated capture antibody of 0.1mol/L carbonate buffer solution, and 4 ℃ of 12h conditions are best.
(3) best sealing condition determines
With the coated 96 hole ELISA Plate of the best coated concentration of capture antibody, with best effort concentration dilution, detect antibody, use respectively 5% skimmed milk power, 5% hyclone, 1%BSA as confining liquid, choose 5 parts of recombinant alpha toxin protein antigens, 5 parts of negative antigens, measure it in the OD at 450nm place value, according to P/N value, determine suitable confining liquid.
The final best sealing condition of determining is 5% skimmed milk power.
(4) detect determining of antibody the best use of time
Under best coated condition, add each 10 parts of recombinant alpha toxin protein and negative samples, to detect the 0.2 μ g/mL dilution of antibody best effort concentration, detect antibody, put 37 ℃ and react respectively 30min, 60min, 90min, 120min, carry out ELISA test, according to P/N value, determine the best use of time of detecting antibody.
Final definite the best use of time of detecting antibody is 1h.
(5) utilize square formation titration experiments to be optimized ELISA testing conditions, finally determine following reaction system:
1. (0.1M carbonate buffer solution pH9.6) is diluted to the polyclonal antibody as capture antibody 2 μ g/mL to use antigen coated liquid.100 μ L/ holes are coated with 96 hole ELISA Plate, and 4 ℃ are spent the night;
2. use PBST solution (the PBS solution that contains 0.05%Tween-20: NacL8g/L, KCL0.2g/L, Na 2hPO 412H 2o3.58g/L, KH 2pO 40.27g/L, pH7.4) wash 3 times, add the PBST solution that contains 5% skimmed milk power as confining liquid 200 μ L/ holes, 4 ℃ of sealings are spent the night;
3. use after PBST solution washing 3 times, front 5 row add sample to be checked, the 6th row adds purification of Recombinant alpha toxin albumen as positive control, and the 7th row adds the Escherichia coli culture supernatant of preparation according to a conventional method, and eighth row adds PBS damping fluid (containing NacL8g/L, KCL0.2g/L, Na 2hPO 4.12H 2o3.58g/L, KH 2pO 40.27g/L), the 7th row and eighth row be as negative control, 100 μ L/ holes; Blank, for not adding any sample, is hatched 1h for 37 ℃;
4. after 3 PBST solution washings, monoclonal antibody F12 prepared by hybridoma cell strain CP f12 is diluted to 0.2 μ g/mL with PBS damping fluid, 100 μ L/ holes, 37 ℃ of reaction 1h;
5. use PBST solution washing 3 times, add the sheep anti-mouse igg with the HRP mark of PBS damping fluid 1:10000 dilution, hatch 1h for 37 ℃, PBST cleansing solution washing 3 times;
6. finally add soluble T MB substrate nitrite ion 100 μ L/ holes, room temperature condition lucifuge reaction 15min, uses 2M H 2sO 4cessation reaction, 450nm reads light absorption value in place.
Embodiment 5: the foundation of double-antibodies sandwich ELISA typical curve
According to the two sandwich ELISA quantitative detecting method running programs that established, alpha toxin protein standard substance twice serial dilution to 0.73ng/mL, negative sample and blank well from 1500ng/mL of restructuring detected respectively, measure after completion of the reaction OD 450nmvalue.The alpha toxin albumen dilute concentration of take is horizontal ordinate, OD 450nmvalue is ordinate curve plotting figure; According to curve map, select optimum linear scope, the natural logarithm LN (x) of concentration of take is horizontal ordinate, OD 450nmvalue is ordinate drawing standard curve.This curve linear scope is more satisfactory, can calculate alpha toxin concentration in testing sample according to typical curve.
Embodiment 6: double-antibodies sandwich ELISA performance evaluation
(1) specific test uses the double-antibodies sandwich ELISA establishing, and detects 30 parts of clostridium perfringens alpha toxin positive, 20 parts of the negative samples such as Escherichia coli culture supernatant, and other irrelevant protein 10 parts, and blank is set.Result shows that other are all negative, show no cross reaction except reacting with recombinant alpha toxin protein and alpha toxin and being positive.
(2) sensitivity analysis is according to obtaining in above-mentioned test value, from typical curve, find out corresponding concentration, be the detectability of the method.According to the range of linearity of typical curve, determine valid analysing range.
(3) precision analysis in the range of linearity, select 375,93.75, a 11.72ng/mL3 concentration point, in single test, each concentration is surveyed 10 holes, calculates variation within batch number of times; Survey 10 batches continuously, calculate interassay coefficient of variation.The method variation within batch coefficient is 2.59%～5.02%, and interassay coefficient of variation is 2.67%～5.86%, and both are all less than 10%, illustrates that same sample degree of variation in same batch and different batches is tested is very little, shows that the method has good precision.
(4) determination of recovery rates adds variable concentrations (375,93.75,11.72ng/mL) recombinant alpha toxin protein in blank detects matrix, by standard detection program determination, according to measured value/theoretical value * 100%, tries to achieve average recovery rate.In 10 tests, the recovery that the protein sample of 3 concentration point is measured, in 97.22%～102.85% scope, proves that the method accuracy is higher.
Embodiment 7: take the recombinant alpha toxin protein of expressing and the natural alpha toxin of extraction is test material
(1) preparation of the natural alpha toxin of recombinant alpha toxin protein and extraction
1. alpha toxin prokaryotic expression method is prepared recombinant alpha toxin protein
According to the specific primer of primmer5 Software for Design:
Upstream primer: 5'-GCGGAATTCATGAAAAGAAAGATTTGT-3', as shown in SEQ No.1;
Downstream primer: 5'-GCGGCGAAGCTTTTATTTTATATTATAAGTT-3', as shown in SEQ No.2;
Selection standard bacterial classification NCTC528 increases after bacterium, with bacterial genomes DNA, extract kit and extract after DNA, utilize pcr amplification clostridium perfringens alpha toxin genetic fragment, amplification condition: 94 ℃ of 5min, 94 ℃ of 1min, 54 ℃ of 1min, 72 ℃ of 70s, carry out altogether 32 circulations, 72 ℃ are extended 7min.The object product that reclaims kit purifying through DNA purifying is connected to order-checking with pMD18-T.Check order correct recombinant plasmid and carrier pET-28a respectively with EcoR I and the processing of Xho I double digestion, after connection, proceed to and express in competent cell BL21, coat solid LB nutrient culture media (Nacl1g, peptone 1g, yeast extract 0.5g, agar powder 1.5g, with autoclaving after 100mL deionized water dissolving), picking list colony inoculation is (kanamycins final concentration is 100 μ g/mL) in the liquid LB nutrient culture media that contains kanamycins, cultivates 3h to OD for 37 ℃ 600nmduring for 0.4-0.6, add IPTG(isopropylthiogalactoside, final concentration 1mmol/L) abduction delivering 6h, after thalline ultrasonication, through the alpha toxin recombinant protein of High Affinity Ni-changed Resin purification; Utilize purity and the immunogenicity of the alpha toxin recombinant protein after polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting (Western-blot) checking purifying, for follow-up animal immune experiment and test experience.
2. A type C.perfringens reference culture (NCTC756) inoculation blood plate nutrient culture media anaerobism is cultivated after 36-48h, access thioglycollate medium (FTG) increases after bacterium cultivation, and 6h is cultivated in 40 ℃ of anaerobism concussions of inoculation anaerobism liver bouillon;
3. 8000r, 15min centrifuging and taking supernatant;
4. supernatant, through saturated ammonium sulfate method purifying, obtains natural alpha toxin; Escherichia coli (ATCC11775) culture supernatant and do not inoculate anaerobism liver bouillon all as negative control
2, double-antibodies sandwich ELISA detects
1. (0.1M carbonate buffer solution, pH9.6) tests by square formation the polyclonal antibody of determining as capture antibody and is diluted to 3 μ g/mL, and 100 μ L/ holes are coated with 96 hole ELISA Plate, and 4 ℃ are spent the night to use antigen coated liquid.
2. use PBST solution (0.05%Tween20, pH7.4PBST) washing 3 times, add confining liquid (containing the PBST solution of 5% skimmed milk power) 200 μ L/ holes, 4 ℃ of sealings are spent the night;
3. with front 3 row after PBST solution washing 3 times, add the above-mentioned recombinant alpha toxin protein preparing, the capable natural alpha toxin that adds preparation of 4-6,100 μ L/ holes, the 7th, 8 row add respectively negative control (PBS, Escherichia coli (ATCC11775) culture supernatant and do not inoculate the anaerobism liver bouillon of bacterium equal) and blank (not adding sample); Hatch 1h for 37 ℃;
4. after 3 PBST solution washings, add the monoclonal antibody F12 (being diluted to 0.2 μ g/mL with PBS damping fluid) of the conduct detection antibody of preparation, 100 μ L/ holes, 37 ℃ of reaction 1h;
5. with PBST washing 3 times, add the sheep anti mouse I with the HRP mark of PBS damping fluid 1:10000 dilution by volume gg, hatches 1h for 37 ℃;
6. finally add soluble T MB substrate nitrite ion 100 μ L/ holes, room temperature condition lucifuge reaction 15min, uses 2M H 2sO 4cessation reaction, 450nm reads light absorption value in place.
3, result
By Fig. 1, learnt, the concentration of the present embodiment standard items with absorbance can be detected and be significant linear relationship, its coefficient R 2=0.9935, linear equation is y=0.2533x-0.2047, the range of linearity 5.86～375ng/mL, and the method lowest detection line is 4.57ng/mL.The recovery that the present embodiment records, in 97.22%～102.85% scope, meets the demands.Presentation of results this method can detect alpha toxin effectively.
Embodiment 8: A～E type C.perfringens culture supernatant of take is test material
1, the preparation of A-E type C.perfringens culture supernatant
1. under same culture conditions, standard bacterial classification A type (NCTC756), Type B (NCTC8533), C type (NCTC3180), D type (NCTC8504), E type (NCTC6719) inoculation blood plate nutrient culture media anaerobism are cultivated after 36-48h, and access thioglycollate medium (FTG) increases after bacterium cultivation;
2. inoculate 40 ℃ of anaerobism concussions of anaerobism liver bouillon and cultivate 6h;
3. 8000r, 15min centrifuging and taking supernatant, Escherichia coli (ATCC11775) culture supernatant and nonvaccinated anaerobism liver bouillon are negative control.
2, double-antibodies sandwich ELISA detects
1. (0.1M carbonate buffer solution, pH9.6) is diluted to 3 μ g/mL using test the polyclonal antibody of determining as capture antibody by square formation, and 100 μ L/ holes are coated with 96 hole ELISA Plate, and 4 ℃ are spent the night to use antigen coated liquid.
2. use PBST solution (containing 0.05%Tween20, the PBS of pH7.4) washing 96 hole ELISA Plate 3 times, add confining liquid (containing the PBST solution of 5% skimmed milk power) 200 μ L/ holes after patting dry, 4 ℃ of sealings are spent the night;
3. use after PBST solution washing 96 hole ELISA Plate plate 3 times, front 5 row add respectively the culture supernatant of the various C.perfringens of above-mentioned preparation, the 6th row adds positive control recombinant alpha toxin protein, the 7th, 8 row to add respectively negative control (Escherichia coli culture supernatant prepared by conventional method, the anaerobism liver bouillon of not inoculating bacterium, PBS damping fluid), 100 μ L/ holes, and blank (not adding sample), hatch 1h for 37 ℃;
4. after 3 PBST solution washings, add the monoclonal antibody F12 (being diluted to 0.2 μ g/mL with PBS damping fluid) of the conduct detection antibody of preparation, 100 μ L/ holes, 37 ℃ of reaction 1h;
5. use PBST solution washing 3 times, add the sheep anti-mouse igg with the HRP mark of PBS damping fluid 1:10000 dilution by volume, hatch 1h for 37 ℃;
6. finally add soluble T MB substrate nitrite ion 100 μ L/ holes, room temperature condition lucifuge reaction 15min, uses 2M H 2sO 4cessation reaction, 450nm reads light absorption value in place.
3, result
Through sample, the repeatedly replicate determination of the positive and negative control, obtain in A～E type C.perfringens culture supernatant alpha toxin detected value all positive, and use regression equation calculation to go out it in typical curve corresponding concentration according to 450nm place light absorption value, the mean value and the standard deviation that obtain the alpha toxin amount of A～E type C.perfringens reference culture generation are respectively 103.29 ± 4.13ng/mL, 29.20 ± 1.21ng/mL, 16.15 ± 0.97ng/mL, 10.46 ± 0.58ng/mL, 7.05 ± 0.47ng/mL, meeting C.perfringens all has the theory of alpha toxin in various, and can effectively quantitatively detect alpha toxin in A～E type C.perfringens culture supernatant.
The double-antibody sandwich elisa that presentation of results this method is set up on preparation monoclonal antibody basis, can be used for the preliminary quantitatively detection of batch samples detection, alpha toxin and the acquisition of earlier results, for propagation and the research of prevention livestock and poultry C.perfringens provides instrument more fast and effectively, and established solid foundation for the research of vaccine quality and food safety monitoring.

Claims (1)

1. a clostridium perfringens alpha toxin double-antibody sandwich elisa quantitative detecting method, is characterized in that comprising the following steps:
1) prokaryotic expression of clostridium perfringens alpha toxin, obtains the recombinant alpha toxin protein of purifying;
2) preparation of anti-clostridium perfringens alpha toxin monoclonal antibody:
Select as immune animal: head, to exempt from the recombinant alpha toxin protein of the purifying for obtaining in step 1) and equal-volume not after the complete emulsification of formula Freund's complete adjuvant, a 100 μ g/ lumbar injection with the female BALB/c mouse of pure lines of myeloma cell Sp2/0 homology; Two exempt from, three exempt to exempt from respectively at head after two weeks and five weeks by recombinant alpha toxin protein and equal-volume not after the complete emulsification of formula Freund's incomplete adjuvant, a 100 μ g/ lumbar injection; Three exempt from after three weeks lumbar injections do not add adjuvant recombinant alpha toxin protein 50～100 μ g/ only, after injection, within 3-5 days, gets the highest mouse boosting cell of tiring for Fusion of Cells;
Getting myeloma cell SP2/0 that growth conditions is good and above-mentioned ELISA detects the highest mouse boosting cell of tiring and utilizes PEG1500 to carry out chemical method fusion, use indirect ELISA method screening positive hybridoma cell, and adopt limiting dilution assay to carry out subclone 3～5 times, obtain anti-clostridium perfringens alpha toxin hybridoma cell strain CP f12; Utilize CP f12 to prepare monoclonal antibody F12;
3) preparation of anti-clostridium perfringens alpha toxin polyclonal antibody:
Choose new zealand white rabbit as immune animal, head exempts from that not the subcutaneous multi-point injection 1mg/ of the complete emulsification back part of formula Freund's complete adjuvant is only into the recombinant alpha toxin protein of purifying and equal-volume; After head exempts from by the complete emulsification of recombinant alpha toxin protein of not formula Freund's incomplete adjuvant and equal-volume purifying at interval of immunity in two weeks once, the recombinant alpha toxin reinforcement of three purifying that obtain by the step 1) that does not add adjuvant for two weeks after exempting from is exempted from, after 15d, gather rabbit anteserum, utilize saturated ammonium sulphate method antibody purification, obtain polyclonal antibody;
4) foundation of double-antibody sandwich elisa quantitative detecting method and typical curve
(1) with antigen coated liquid, by polyclonal antibody, be that coated antibody is diluted to 3 μ g/mL; 100 μ L/ holes are coated with 96 hole ELISA Plate, and 4 ℃ are spent the night; Described antigen coated fluid component is 0.1M carbonate buffer solution, its pH9.6;
(2) use PBST solution washing 3 times, add the PBST solution that contains 5% skimmed milk power as confining liquid 200 μ L/ holes, 4 ℃ of sealings are spent the night; Described PBST solution component is: the PBS solution that contains 0.05%Tween-20: NacL8g/L, KCL0.2g/L, Na 2hPO 412H 2o3.58g/L, KH 2pO 40.27g/L, pH7.4;
(3) with after PBST solution washing 3 times, add sample to be checked, 100 μ L/ holes, yin and yang attribute and blank are set simultaneously, positive control is step 1) the recombinant alpha toxin protein of the purifying that obtains, negative control is respectively Escherichia coli culture supernatant and the PBS damping fluid of preparation according to a conventional method, and described PBS buffer composition were is: containing NacL8g/L, KCL0.2g/L, Na 2hPO 4.12H 2o3.58g/L, KH 2pO 40.27g/L; Blank, for not adding any sample, is hatched 1h for 37 ℃;
After (4) 3 PBST solution washings, monoclonal antibody F12 prepared by hybridoma cell strain CP f12 is diluted to 0.2 μ g/mL with PBS damping fluid, 100 μ L/ holes, 37 ℃ of reaction 1h;
(5) use PBST solution washing 3 times, add the sheep anti-mouse igg with the HRP mark of PBS damping fluid 1:10000 volume ratio dilution, hatch 1h for 37 ℃, PBST solution washing 3 times;
(6) finally add soluble T MB substrate nitrite ion 100 μ L/ holes, room temperature condition lucifuge reaction 15min, uses 2M H 2sO 4solution cessation reaction, 450nm reads light absorption value in place;
(7) take alpha toxin albumen dilute concentration is horizontal ordinate, OD 450nmvalue is ordinate curve plotting figure; According to curve map, select optimum linear scope, the natural logarithm LN (x) of concentration of take is horizontal ordinate, OD 450nmvalue, for ordinate drawing standard curve, is calculated alpha toxin concentration in testing sample by typical curve;
5) result criterion determines
Calculate negative sample mean value with standard deviation (SD), try to achieve positive critical value, negative critical value; Use the DAS-ELISA setting up to measure it is positive, it is negative, X &OverBar; + 2 SD < OD 450 nm < X &OverBar; + 3 SD For suspicious specimen.
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