CN104807992A - Blocking ELISA method for detecting antibody of NetB toxin of clostridium perfringens - Google Patents
Blocking ELISA method for detecting antibody of NetB toxin of clostridium perfringens Download PDFInfo
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- CN104807992A CN104807992A CN201510201521.8A CN201510201521A CN104807992A CN 104807992 A CN104807992 A CN 104807992A CN 201510201521 A CN201510201521 A CN 201510201521A CN 104807992 A CN104807992 A CN 104807992A
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Abstract
The invention discloses a blocking ELISA method for detecting an antibody of NetB toxin (Necrotic enteritis B-like toxin) of clostridium perfringens. The blocking ELISA method is used for early stage detection of NetB toxin clostridium perfringens infection, and provides an effective detection tool for specific diagnosis, comprehensive control and follow-up scientific research of clostridium perfringens. The blocking ELISA method comprises the following two steps: 1, establishing the blocking ELISA detection method, and 2, determining a result judgment standard. The quick, sensitive and specific locking ELISA method is established for detecting the specific antibody of the NetB toxin in clostridium perfringens infected serum, is expected to be an important tool for clinical clostridium perfringens quick diagnosis and immunodetection, can certainly play an important role in diagnosis, prevention and control of clostridium perfringens, and has a very good application value.
Description
Technical field
The invention belongs to animal's antibody detection technique field, relate to a kind of ELISA method detecting C.perfringens NetB toxin antibody.
Background technology
C.perfringens (Clostridium perfringens, C.perfringens) be a kind of important infecting both domestic animals and human encephalapthy agent, be distributed widely in nature, cause emphysematous gangrene and the food poisoning of necrosing property of many animals enteritis (Necrotic enteritis, NE) and enterotoxemia and the mankind.The multiple toxin of C.perfringens secretion has vital role in its disease caused.
Recent two decades comes, and alpha toxin is identified the main pathogenic of aviculture necrotic enteritis, and the culture supernatant of A type C.perfringens can make broiler chicken occur gangrenosum acne pathology, and anti alpha toxin antibody has protective effect.But the mechanism of causing a disease of research to this disease in the recent period about NetB toxin (Necroticenteritis B-like toxin) turn increases new explanation.NetB toxin finds in the A type C.perfringens be separated in the sick chicken body of NE first.The a large amount of bacterial strains be separated in chicken necrotizing enterocolitis infected animal body carry NetB gene, and the necrotic enteritis of the cardinal principle pathology that animal disease model occurs and clinical report is in a disguised form same.Therefore, think that NetB may be a kind of key and important elucidate pathogenic factor in the pathogenesis of bird necrotic enteritis.Whether stop band restrain method detects in C.perfringens serum is vital containing NetB toxin.Therefore the diagnosis of detection to C.perfringens disease of NetB toxin is significant.
Summary of the invention
The object of this invention is to provide a kind of stop band restrain method detecting C.perfringens NetB toxin antibody, for the early detection of NetB toxin, for the early diagnosis of C.perfringens and integrated control and follow-up scientific research provide effective testing tool.The method only needs a small amount of blood serum sample, and the detection for disease is more convenient.
Object of the present invention is achieved through the following technical solutions:
Detect a stop band restrain method for C.perfringens NetB toxin antibody, comprise the steps:
One, the foundation of stop band restrain detection method
(1) with coating buffer, C.perfringens NetB toxin recombinant protein is diluted to 5 μ g/mL, 100 Bao Bei96 hole, μ L/ hole ELISA Plate, 4 DEG C are spent the night;
(2) PBST solution washing 3 times, add with PBS solution dilution 1%BSA as confining liquid 200 μ L/ hole, hatch 2h for 37 DEG C;
(3) add measuring samples after PBST solution washing 3 times, arrange feminine gender and positive control, positive control is NetB toxin polyclonal antibody, and negative control is negative serum simultaneously, and 100 μ L/ holes, hatch 1.5h for 37 DEG C;
After (4) 3 PBST solution washings, the F9G3 monoclonal antibody PBS damping fluid of anti-C.perfringens NetB toxin is diluted to 0.635 μ g/mL, 100 μ L/ holes, 37 DEG C of reaction 1h;
(5) PBST solution washing 3 times, adds the sheep anti-mouse igg antibody of the HRP mark with the dilution of PBS damping fluid 1: 5000 volume ratio, hatches 1h for 37 DEG C, PBST solution washing 3 times;
(6) finally add nitrite ion 100 μ L/ hole, room temperature condition lucifuge reaction 10min, uses 2M H
2sO
4solution cessation reaction, 490
nmplace reads light absorption value.
Two, the determination of result criterion
Calculate CP negative sample mean value
with standard deviation (S), try to achieve
for positive critical value,
for negative critical value; When sample blocking-up rate
then blood serum sample is judged to the positive; When
time be negative; For at marginal blood serum sample, be judged to be suspicious, need duplicate detection once, as still suspicious, be judged to be the positive.
In the present invention, described coating buffer is that 0.05mol/L carbonate bag is buffered liquid, and its compound method is as follows: take Na
2cO
31.5g, NaHCO
32.93g, regulate pH to 9.6, adding distil water to 1000mL, 4 DEG C of preservations.
In the present invention, described PBST solution is the PBS solution containing 0.05%Tween-20, and its compound method is: take NaCl 8g, KCl 0.2g, Na
2hPO
412H
2o 3.58g, KH
2pO
40.27g, adding distil water, to 1000mL, is adjusted to pH7.4; Add 0.5mLTween-20 again.
In the present invention, described confining liquid is the 1%BSA of PBS solution dilution, and compound method is: add 1g BSA in every 100ml PBS solution.
Tool of the present invention has the following advantages:
1, the present invention establishes a kind of quick, responsive, special stop band restrain method to detect the NetB toxin specific antibody in C.perfringens serum on the basis utilizing monoclonal antibody.
2, this method is expected to become the clinical quick diagnosis of C.perfringens disease and the important tool of immune detection, plays a significant role, have good using value in the Diagnosis and treatment of C.perfringens disease.
3, the present invention determines the package amount of best antigen, the dilutability of antibody, the bag of antigen by time, best confining liquid and off-period, best sample incubation time, the incubation time of optimum monoclonal antibody, the concentration of enzyme labelled antibody and the condition such as action time, best developing time.Determine critical value by negative sample, minimum antigen detected level determines its sensitivity, and it is good that specific test determines its specificity, and between criticizing in batch, test determines that the repeatability of the method is good.
4, the present invention only needs a small amount of blood serum sample, and the NetB toxin blocks ELISA detection method that application is set up can detect more easily to disease.
5, the present invention is simple to operate, detection speed is fast, can quantitatively detect and mass detection.
Accompanying drawing explanation
Fig. 1 is C.perfringens NetB toxin expression of recombinant proteins, 1: empty carrier, and 2:marker, 3:0 hr-control, 4:1 hour, 5:2 hour, 6:3 hour, 7:4 hour, 8:5 hour, 9: supernatant, 10: precipitation;
Fig. 2 is F9G3 strain monoclonal antibody ascites purifying, 0: before purifying, 1: after purifying, 2:Marker;
Fig. 3 is optimum protein, the many anti-and monoclonal antibody concentration that Three factors method is determined;
Fig. 4 is the selection of antigen coated time;
Fig. 5 is the selection of best confining liquid;
Fig. 6 is the selection of best off-period;
Fig. 7 is the selection of best measuring samples incubation time;
Fig. 8 is the selection of optimum monoclonal antibody incubation time;
Fig. 9 is the selection of best enzyme labelled antibody dilute concentration;
Figure 10 is the selection of best enzyme labelled antibody incubation time;
Figure 11 is the selection of best nitrite ion action time;
Figure 12 is stop band restrain specific test.
Embodiment
Below in conjunction with accompanying drawing, technical scheme of the present invention is further described; but be not limited thereto; everyly technical solution of the present invention modified or equivalent to replace, and not departing from the spirit and scope of technical solution of the present invention, all should be encompassed in protection scope of the present invention.
Embodiment 1
The abduction delivering of C.perfringens NetB toxin recombinant protein:
PCR is identified the recombinant expressed bacterium of the pGEX-NetB positive is inoculated in 20mLAmp+LB fluid nutrient medium by 1: 100 (v/v), in 37 DEG C of shaken cultivation 2-3h.As the OD of culture
600when value reaches 0.4-0.6, take out 1mL bacterium liquid as (0h) contrast before induction, be placed in 4 DEG C and temporarily preserve.In residue bacterium liquid, add derivant IPTG to final concentration is 1mmol/L, continues to cultivate.1mL bacterium liquid is taken out every 1h (i.e. induction rear 1st, 2,3,4 and 5h).Each hour bacterium liquid is carried out the centrifugal 5min of 10000r/min and collects bacterial precipitation, add after 20 μ L PBS have hanged precipitation and add equivalent 2 × SDS sample-loading buffer (containing 200mmol/L DTT) mixing, boil 10min, analyze the expression of recombinant protein for polyacrylamide gel electrophoresis (SDS-PAGE).Recombinant protein molecular size range choice for use concentration according to prediction is that the separation gel of 12% and the compression glue of 5% carry out SDS-PAGE analysis (see Fig. 1), through cutting NetB toxin recombinant protein (see Fig. 2) prepared by glue purification renaturation; Immunoblotting (Western-blot) verifies purity and the immunogenicity of the NetB toxin recombinant protein after purifying, for follow-up animal immune experiment and test experience.
Embodiment 2
The preparation of anti-C.perfringens NetB toxin polyclonal antibody:
Choose 2-3Kg new zealand white rabbit, head exempts from NetB toxin recombinant protein into purifying and the equal-volume not subcutaneous multi-point injection of formula Freund's complete adjuvant complete emulsification back part, dosage is 2mg/, head exempts from the complete emulsification of NetB toxin recombinant protein of 2 Zhou Houyong not formula Freund's incomplete adjuvant and equal-volume purifying, dosage is 1mg/, immunity 1 time weekly, 3 exempt from after 1 week use do not add the NetB toxin recombinant protein booster immunization 1 time of the purifying of adjuvant, after 7d, rabbit heart blood sampling obtains serum, obtain anti-C.perfringens NetB toxin polyclonal antibody, antibody titer is detected by indirect elisa method, immunoblotting is used for how anti-CHARACTERISTICS IDENTIFICATION.
Embodiment 3
The preparation of anti-C.perfringens NetB toxin monoclone antibody:
A, C.perfringens NetB toxin recombinant protein immunity BALB/c mouse
Select with the pure lines female BAl BIc/c mouse of myeloma cell SP2/0 homology as immune animal, with the NetB toxin recombinant protein after purifying and renaturation for immunizing antigen, get 50 μ g purifying proteins during first immunisation and equal-volume complete Freund's adjuvant carries out emulsification, adopt lumbar injection mode to carry out immunity to female BAl BIc/c mouse in 6 week age; Get 50 μ g purifying proteins and the emulsification of equal-volume incomplete Freund's adjuvant after 3 weeks and carry out second time immunity; After this carried out primary immune response every 2 weeks, immunizing dose is identical with second time immunity with immunization route, within 3-5 days, gets the highest mouse boosting cell of tiring for Fusion of Cells after last immunity;
The screening of b, Fusion of Cells and hybridoma cell strain
Get growth conditions good myeloma cell SP2/0 and above-mentioned ELISA to detect the highest mouse boosting cell of tiring and utilize PEG 3350 to carry out chemical method fusion, use indirect ELISA method screening positive hybridoma cell, and adopt limiting dilution assay to carry out 3-5 subclone, obtain the hybridoma cell strain F9G3 secreting anti-C.perfringens NetB toxin;
The preparation of c, monoclonal antibody ascites
Select 8 week age, health, female BAl BIc/c mouse, lumbar injection sterilized liquid paraffin sensitization, 0.5mL/ only, injects hybridoma after 7d.Collect exponential phase hybridoma, the centrifugal 10min of 1000r/min, supernatant discarded, adds the incomplete RPMI-1640 of 5mL, is hanged by cell in cell precipitation, the centrifugal 10min of 1000r/min.Supernatant discarded, the full RPMI-1640 that cannots be used up has hanged cell, carries out cell count after cell suspension 100 times dilution, and adjustment cell density is 10
6individual/mL, the cell suspension of every this density of mouse peritoneal injection 0.5mL.After injection, 7d starts to observe mouse web portion state, until mouse be in flat sleeping state lower abdomen both sides obviously expand time, use cotton ball soaked in alcohol sterilization skin of abdomen, thrust skin of abdomen with 10mL syringe needle, sterile centrifugation tube collects the faint yellow or dark red solution flowed out from syringe needle.4 DEG C, the centrifugal 15min of 3000r/min, discard the fat on upper strata, the weak yellow liquid in syringe collecting middle layer is ascites, and-20 DEG C save backup.
D, sad-saturated ammonium sulfate method purifying ascites
Two volumes 0.06mol/L acetate buffer (pH4.5) is added, uniform stirring in pretreated ascites.Add the long-pending caprylic acid (it is sad dropwise to add under ice bath) of triploid in every part of ascites and stir 30min, 4 DEG C more than standing 2h hour.Above-mentioned solution is taken out the centrifugal 30min of 11000rpm, abandons precipitation, supernatant 2mol/LNaOH adjusts pH to 7.4.Saturated ammonium sulfate to 50% saturation degree is added, ice bath beating action 30min, 4 DEG C of standing more than 2h under 4 DEG C of conditions.The centrifugal 30min of 11000rpm, abandons supernatant, and precipitation is dissolved in the PBS (pH 7.4) of 5ml 0.1M.Above-mentioned solution adds appropriate saturated ammonium sulfate solution while stirring, makes concentration in ammonium sulfate be 30-40%, ice bath beating action 30min, 4 DEG C of static more than 2h.The centrifugal 30min of 11000rpm, gets precipitation, and it is in the PBS of 7.4 that precipitation is incorporated 2mL 0.1M pH.Loaded by above-mentioned suspending liquid in dialysis band, with PBS (pH 7.4) the 4 DEG C dialysis of 0.1M, 2h changes liquid once, dialysis 2d.Survey the concentration of ascites after purifying, and get 10 μ L and carry out SDS-PAGE identification and analysis purifying situation (see Fig. 3).After purifying, ascites is the F9G3 monoclonal antibody of this detection method anti-C.perfringens NetB toxin used, and-80 DEG C save backup.
Embodiment 4
The foundation of stop band restrain detection method:
One, the determination of monoclonal antibody and polyvalent antibody working concentration:
(1) according to Three factors method, the NetB albumen after antigen and purifying is pressed 0.125ug/100ul, the amount bag in 0.25ug/100ul, 0.5ug/100ul, 1ug/100ul/ hole is spent the night by 4 DEG C.Anti-for rabbit NetB polyclonal antibody (2.4mg/ml) is pressed 1: 40 (60ug/ml), 1: 80 (30ug/ml), 1: 160 (15ug/ml), 1: 320 (7.5ug/ml), 1: 640 (3.75ug/ml) doubling dilution as inspection antibody with PBS, and rabbit negative serum carries out identical dilution as negative control.Mouse-anti NetB monoclonal antibody F9G3 strain (1.20mg/ml) after purifying PBS dilution is pressed 1: 240 (5ug/ml), 1: 480 (2.5ug/ml), 1: 960 (1.25ug/ml), 1: 1920 (0.625ug/ml) and 1: 3840 (1.25ug/ml) as binding antibody.HRP-sheep anti-mouse igg enzyme labelled antibody 1: 5000 times dilution resists as two.Add nitrite ion, 100ul/ hole, room temperature effect 10min; Add 2mol/L H
2sO
450 μ l/ hole cessation reactions.Running program is carried out as stated above, and microplate reader measures OD
490nmvalue post analysis.Calculate the positive serum of working concentration and negative serum to the blocking-up rate of every strain monoclonal antibody.Descend the tested serum of formulae discovery to the blocking-up rate of monoclonal antibody according to this:
(% blocking-up rate=100-100 × tested serum OD value/negative control mean OD value).
Choose the OD of negative control hole
490nmvalue is about 1, and simultaneously corresponding during positive serum blocking-up rate>=50% dilutability is as the best effort concentration of monoclonal antibody and polyvalent antibody.
Finally determine that C.perfringens NetB toxin recombinant protein envelope antigen concentration be 5ug/ml, NetB toxin polyclonal antibody working concentration is 30 μ g/ml, mouse-anti C.perfringens NetB toxin monoclonal antibody F9G3 detectable concentration is 0.625 μ g/ml, sees Fig. 3.
(2) selection of antigen coated time:
Hatched by optium concentration bag by NetB toxin recombinant protein after purifying, wrap and had 37 DEG C of 1h by condition, 37 DEG C of 1.5h, 37 DEG C of 2h and 4 DEG C spend the night, and carry out stop band restrain by the standard program determined.According to OD
490nmvalue calculates, and chooses the OD of negative control hole
490nmvalue is about 1, simultaneously during positive serum blocking-up rate>=50%, the bag with maximum blocking-up rate by condition as the antigen coated condition of the best.
The antigen coated time finally determined is that 4 DEG C of bags are spent the night, and sees Fig. 4.
(3) selection of the suitableeest confining liquid:
By the NetB toxin recombinant protein after purifying by top condition bag quilt, confining liquid select PBS to dilute respectively 10% hyclone, 5% skimmed milk, 3%BSA, 1%BSA, 37 DEG C of closed 2h, program carries out stop band restrain routinely, according to OD
490nmvalue calculates, and chooses the OD of negative control hole
490nmvalue is about 1, and simultaneously during positive serum blocking-up rate>=50%, the confining liquid with maximum blocking-up rate is best confining liquid.
The best confining liquid finally determined is 1%BSA, sees Fig. 5.
(4) selection of best off-period:
By the NetB toxin recombinant protein after purifying by optium concentration bag quilt, use best confining liquid to close, set 37 DEG C of 1h off-period respectively, 37 DEG C of 1.5h, 37 DEG C of 2h and 37 DEG C 3h, follow procedure carries out stop band restrain.According to OD
490nmvalue calculates, and chooses the OD of negative control hole
490nmvalue is about 1, simultaneously during positive serum blocking-up rate>=50%, has the time of maximum blocking-up rate for best off-period.
The best off-period finally determined is 37 DEG C of closed 2h, sees Fig. 6.
(5) selection of best measuring samples incubation time:
By the NetB toxin recombinant protein after purifying by optium concentration bag quilt, best confining liquid closes appropriate time, and measuring samples hatches 30min at 37 DEG C respectively, 60min, 90min, and follow procedure carries out stop band restrain.According to OD
490nmvalue is about 1, and simultaneously during positive serum blocking-up rate>=50%, the time with maximum blocking-up rate is best measuring samples incubation time.
The best measuring samples incubation time finally determined is 60min, sees Fig. 7.
(6) optimum monoclonal antibody incubation time is selected:
By fixed optimum condition bag by, close, measuring samples hatches, after adding the mouse-anti C.perfringens NetB toxin monoclonal antibody F9G3 of optium concentration, hatch 30min at 37 DEG C respectively, 60min, 90min, follow procedure carries out stop band restrain.According to OD
490nmvalue is about 1, and simultaneously during positive serum blocking-up rate>=50%, the time with maximum blocking-up rate is optimum monoclonal antibody incubation time.
The optimum monoclonal antibody incubation time finally determined is 37 DEG C of effect 1h, sees Fig. 8.
(7) selection of enzyme mark sheep anti-mouse igg working concentration:
By fixed optimum condition bag by, close, measuring samples hatches and detects antibody incubation condition and carry out stop band restrain operation, after adding mouse-anti C.perfringens NetB toxin monoclonal antibody, add 1: 1000 respectively, 1: 2500,1: 5000 with 1: 0000 different dilution enzyme mark sheep anti-mouse igg.Result uses microplate reader to measure OD
490nmvalue is about 1, and simultaneously during positive serum blocking-up rate>=50%, the antibody concentration with maximum blocking-up rate is best enzyme labelled antibody working concentration.
The best effort concentration finally determined is 1: 5000, sees Fig. 9.
(8) selection of enzyme mark sheep anti-mouse igg action time:
When two anti-action time of optimization, select to act on 30min respectively, 60min, 90min at 37 DEG C, operate and undertaken by stop band restrain program.Result uses microplate reader to measure OD
490nmvalue is about 1, and simultaneously during positive serum blocking-up rate>=50%, the time with maximum blocking-up rate is the best enzyme labelled antibody working time.
The enzyme labelled antibody finally determined is 37 DEG C and hatches 60min action time, sees Figure 10.
(9) selection of best developing time:
After adding nitrite ion, the colour developing of room temperature lucifuge 5min, 10min, 15min and 20min respectively, microplate reader measures OD
490nmvalue is about 1, and simultaneously during positive serum blocking-up rate>=50%, the time with maximum blocking-up rate is best nitrite ion action time.
The best developing time finally determined is 10min, sees Figure 11.
(10) determination of stop band restrain critical value:
Get the negative chicken serum of 90 parts of C.perfringens, detect by the stop band restrain method set up, the positive and negative control are set simultaneously, measure OD
490nmvalue.Calculate 90 parts of negative serum sample average blocking-up rate mean values
with standard deviation (S)=9.7984%.
Sample blocking-up rate
, then blood serum sample is judged to the positive; When
time be negative; For at marginal blood serum sample, be judged to be suspicious, need duplicate detection once, as still suspicious, be judged to be the positive.So PI >=31.88% is judged to the positive; PI≤22.08% is judged to feminine gender; 22.08% < PI < 31.88% is judged to suspicious.
Embodiment 5
Stop band restrain method performance evaluation:
(1) specific test: adopt the preliminary stop band restrain detection method set up to be that C.perfringens positive serum is as positive control with reference to serum to avian colibacillosis, fowl salmonellosis, avian leukosis, chicken coccidiasis, Bursal Disease, chicken Marek's disease positive serum.Critical value according to having established judges, analyzes and has no cross reaction.Result shows, and only have C.perfringens positive serum test positive, other serum are feminine gender, and statistical result also shows OD when detecting C.perfringens positive serum
490nmvalue and the numerical value detecting other reference serums show difference extremely significantly (P < 0.01), and illustrate that the detection system specificity that this experiment is set up is good, no cross reaction, is shown in Figure 12.
(2) sensitivity test: anti-for rabbit NetB toxin serum PBS damping fluid is done 1: 40,1: 80,1: 160,1: 320,1: 640,1: 1280 and 1: 2560 times of dilution respectively, undertaken by stop band restrain program, set up negative control simultaneously, judge according to positive critical value, the sensitivity detected for monoclonal antibody with most high dilution.The concentration of known batch rabbit anti-C.perfringens NetB toxin polyvalent antibody is 2.4mg/ml.When negative control is set up, according to the positive critical value determined, can detect many anti-most highly diluted multiples is 1: 320, and now antibody concentration is 0.0075mg/mL, the minimum antibody that 0.0075mg/mL can be detected of stop band restrain method is described, in table 1.
(3) replica test: repeat experiment in batch, wraps by with a collection of albumen, chooses 4 parts of serum detect every increment product for continuous 4 days according to the method set up.Repeat experiment between batch, with the albumen wrapper sheet of 2 kinds of different batches, choose 4 parts of serum and for three days on end every increment product are detected according to the method set up.The coefficient of variation of these two replica tests is all less than 10%, proves that the stop band restrain detection method that this experiment is set up has good repeatability, in Table 2-3.
The sensitivity tests of table 1 stop band restrain
Table 2 stop band restrain criticizes interior replica test
Table 3 stop band restrain criticizes a replica test
Claims (8)
1. detect a stop band restrain method for C.perfringens NetB toxin antibody, it is characterized in that described detection method step is as follows:
One, the foundation of stop band restrain detection method
(1) with coating buffer, C.perfringens NetB toxin recombinant protein is diluted to 5 μ g/mL, 100 Bao Bei96 hole, μ L/ hole ELISA Plate, 4 DEG C are spent the night;
(2) PBST solution washing 3 times, adds confining liquid 200 μ L/ hole, hatches 2h for 37 DEG C;
(3) add measuring samples after PBST solution washing 3 times, arrange feminine gender and positive control, positive control is NetB toxin polyclonal antibody, and negative control is negative serum simultaneously, and 100 μ L/ holes, hatch 1.5h for 37 DEG C;
After (4) 3 PBST solution washings, the F9G3 monoclonal antibody PBS damping fluid of anti-C.perfringens NetB toxin is diluted to 0.635 μ g/mL, 100 μ L/ holes, 37 DEG C of reaction 1h;
(5) PBST solution washing 3 times, adds the sheep anti-mouse igg antibody of the HRP mark with the dilution of PBS damping fluid 1: 5000 volume ratio, hatches 1h for 37 DEG C, PBST solution washing 3 times;
(6) finally add nitrite ion 100 μ L/ hole, room temperature condition lucifuge reaction 10min, uses 2M H
2sO
4solution cessation reaction, 490
nmplace reads light absorption value;
Two, the determination of result criterion
Calculate CP negative sample mean value
with standard deviation (S), try to achieve
for positive critical value,
for negative critical value; When sample blocking-up rate
then blood serum sample is judged to the positive; When
time be negative; For at marginal blood serum sample, be judged to be suspicious, need duplicate detection once, as still suspicious, be judged to be the positive.
2. the stop band restrain method of detection C.perfringens NetB toxin antibody according to claim 1, it is characterized in that described coating buffer is that 0.05mol/L carbonate bag is buffered liquid, its compound method is as follows: take Na
2cO
31.5g, NaHCO
32.93g, regulate pH to 9.6, adding distil water to 1000mL, 4 DEG C of preservations.
3. the stop band restrain method of detection C.perfringens NetB toxin antibody according to claim 1, it is characterized in that described PBST solution is the PBS solution containing 0.05%Tween-20, its compound method is: take NaCl 8g, KCl 0.2g, Na
2hPO
412H
2o3.58g, KH
2pO
40.27g, adding distil water, to 1000mL, is adjusted to pH7.4; Add 0.5mL Tween-20 again.
4. the stop band restrain method of detection C.perfringens NetB toxin antibody according to claim 1, is characterized in that described confining liquid is the 1%BSA of PBS solution dilution.
5. the stop band restrain method of detection C.perfringens NetB toxin antibody according to claim 4, is characterized in that the compound method of the described PBS solution containing 1%BSA is: add 1g BSA in every 100ml PBS solution.
6. the stop band restrain method of detection C.perfringens NetB toxin antibody according to claim 1, is characterized in that the working concentration of described NetB toxin polyclonal antibody is 30 μ g/ml.
7. the stop band restrain method of the detection C.perfringens NetB toxin antibody according to claim 1 or 6, it is characterized in that the preparation method of described NetB toxin polyclonal antibody is as follows: choose 2-3Kg new zealand white rabbit, head exempts from NetB toxin recombinant protein into purifying and the equal-volume not subcutaneous multi-point injection of formula Freund's complete adjuvant complete emulsification back part, dosage is 2mg/, head exempts from the complete emulsification of NetB toxin recombinant protein of 2 Zhou Houyong not formula Freund's incomplete adjuvant and equal-volume purifying, dosage is 1mg/, immunity 1 time weekly, 3 exempt from after 1 week use do not add the NetB toxin recombinant protein booster immunization 1 time of the purifying of adjuvant, after 7d, rabbit heart blood sampling obtains serum, obtain anti-C.perfringens NetB toxin polyclonal antibody.
8. the stop band restrain method of detection C.perfringens NetB toxin antibody according to claim 1, is characterized in that the preparation method of described NetB toxin monoclone antibody is as follows:
A, C.perfringens NetB toxin recombinant protein immunity BALB/c mouse:
Select with the pure lines female BAl BIc/c mouse of myeloma cell SP2/0 homology as immune animal, with the NetB toxin recombinant protein after purifying and renaturation for immunizing antigen, get 50 μ g purifying proteins during first immunisation and equal-volume complete Freund's adjuvant carries out emulsification, adopt lumbar injection mode to carry out immunity to female BAl BIc/c mouse in 6 week age; Get 50 μ g purifying proteins and the emulsification of equal-volume incomplete Freund's adjuvant after 3 weeks and carry out second time immunity; After this carried out primary immune response every 2 weeks, immunizing dose is identical with second time immunity with immunization route, within 3-5 days, gets the highest mouse boosting cell of tiring for Fusion of Cells after last immunity;
The screening of b, Fusion of Cells and hybridoma cell strain:
Getting the good myeloma cell SP2/0 of growth conditions and above-mentioned detection the highest mouse boosting cell of tiring utilizes PEG 3350 to carry out chemical method fusion, use indirect ELISA method screening positive hybridoma cell, and adopt limiting dilution assay to carry out 3-5 subclone, obtain the hybridoma cell strain F9G3 secreting anti-C.perfringens NetB toxin;
The preparation of c, monoclonal antibody ascites:
Select 8 week age, health, female BAl BIc/c mouse, lumbar injection sterilized liquid paraffin sensitization, 0.5mL/ only, injects hybridoma after 7d; Collect exponential phase hybridoma, the centrifugal 10min of 1000r/min, supernatant discarded, adds the incomplete RPMI-1640 of 5mL, is hanged by cell in cell precipitation, the centrifugal 10min of 1000r/min; Supernatant discarded, the full RPMI-1640 that cannots be used up has hanged cell, carries out cell count after cell suspension 100 times dilution, and adjustment cell density is 10
6individual/mL, the cell suspension of every this density of mouse peritoneal injection 0.5mL; After injection, 7d starts to observe mouse web portion state, until mouse be in flat sleeping state lower abdomen both sides obviously expand time, use cotton ball soaked in alcohol sterilization skin of abdomen, thrust skin of abdomen with 10mL syringe needle, sterile centrifugation tube collects the faint yellow or dark red solution flowed out from syringe needle; 4 DEG C, the centrifugal 15min of 3000r/min, discard the fat on upper strata, the weak yellow liquid in syringe collecting middle layer is ascites, and-20 DEG C save backup;
D, sad-saturated ammonium sulfate method purifying ascites:
Two volumes 0.06mol/L acetate buffer is added, uniform stirring in pretreated ascites; Add the long-pending caprylic acid of triploid in every part of ascites and stir 30min, 4 DEG C more than standing 2h hour; Above-mentioned solution is taken out the centrifugal 30min of 11000rpm, abandons precipitation, supernatant 2mol/L NaOH adjusts pH to 7.4; Saturated ammonium sulfate to 50% saturation degree is added, ice bath beating action 30min, 4 DEG C of standing more than 2h under 4 DEG C of conditions; The centrifugal 30min of 11000rpm, abandons supernatant, and it is in the PBS of 7.4 that precipitation is dissolved in 5ml 0.1M pH; Above-mentioned solution adds appropriate saturated ammonium sulfate solution while stirring, makes concentration in ammonium sulfate be 30-40%, ice bath beating action 30min, 4 DEG C of static more than 2h; The centrifugal 30min of 11000rpm, gets precipitation, and it is in the PBS of 7.4 that precipitation is incorporated 2mL 0.1M pH; Loaded by above-mentioned suspending liquid in dialysis band, be the PBS4 DEG C of dialysis of 7.4 with 0.1M pH, 2h changes liquid once, and dialysis 2d, after purifying, ascites is the F9G3 monoclonal antibody of anti-C.perfringens NetB toxin, and-80 DEG C save backup.
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