CN106896228A - A kind of clostridium perfringens alpha toxin Anti-HBV permanence detection method - Google Patents
A kind of clostridium perfringens alpha toxin Anti-HBV permanence detection method Download PDFInfo
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Abstract
The present invention relates to animal bacteria field, there is provided the capture ELISA detection method of clostridium perfringens alpha toxin antibody, the method with the A type C.perfringens exotoxins for preparing be antigen, one is to prepare hyper-immune serum for rabbit is immunized, and two is concentration, after purification as the combination antigen for capturing ELISA；Detection method is comprised the following steps：(1) coating (2) closing (3) plus antigen (4) plus measuring samples (5) add secondary antibody (6) colour developing (7) to terminate (8) result judgement.The method has that specificity is high, sensitivity is strong, favorable repeatability, low cost and other advantages, and the method can be with mass detection sample, for the detection and research of the disease are provided effectively, the serological diagnostic method of simplicity in addition.
Description
Technical field
The present invention relates to animal bacteria field, the invention provides a kind of clostridium perfringens alpha toxin antibody capture
ELISA detection method.
Background technology
C.perfringens (C.perfringens) is widely present in natural environment also known as clostridieum welchii (C.welchii)
In, and see in the alimentary canal of nearly all warm-blooded animal, belong to the member of normal flora in humans and animals enteron aisle.Perfringens
Clostridium can cause the disease such as lamb dysentery and lamb, calf, piglet, rabbit, the necrotic enteritis of chick, enterotoxemia, morbidity
The anxious, death rate is high, is the important diseases of serious harm aquaculture, is the main pathogen of China domestic animal " Sudden Death Syndrome " in recent years, gives
Various countries' animal husbandry development brings huge economic losses.Various C.perfringens produces alpha toxin (CPA), and this bacterium can cause people
The enterotoxemia and necrotic enteritis of class emphysematous gangrene and many animals, it produces alpha toxin antibody in vivo after domestic animal is infected,
Therefore alpha toxin antibody is to diagnose one of sick important evidence of C.perfringens.
Small white mouse toxin neutralization test is to detect the classical way of clostridium perfringens toxoid antibody in the prior art, although
Accurately and reliably, but it is time-consuming, take a lot of work.It is cumbersome, it is difficult to promote and hydrolyzed lecithin suppresses experiment and owes sensitivity, and poor repeatability
Using.Recently as the development of biotechnology, have there are the commodity of various detection clostridium perfringens toxoid antibody in foreign countries
Detection kit, but it is expensive, therefore in order to make up the blank of domestic clostridium perfringens alpha toxin antibody test, need in a hurry
Quick, sensitive, special and energy mass detection ELISA method is set up, is that C.perfringens disease is diagnosed, research is provided with
Effect instrument.
The patent that the present inventor once applied：" a kind of A types clostridium perfringens toxoid antibody it is indirect
ELISA detection method " although disclosing the indirect ELISA detection method of A type C.perfringens exotoxin antibody, due to
What it was detected is all exotoxins of A type C.perfringens, poor specificity, it is impossible to qualitatively detect perfringens alpha poison
Element, the blank therefore, it is difficult to fill up prior art.
The content of the invention
The above-mentioned situation for prior art is confirmed, the present inventor provides a kind of C.perfringens
The capture ELISA detection method of alpha toxin antibody, the method is antigen with the A type C.perfringens exotoxins for preparing, and one is to use
Hyper-immune serum is prepared rabbit is immunized, two be concentration, after purification as the combination antigen for capturing ELISA；Detection method includes following
Step：(1) coating (2) closing (3) plus antigen (4) plus measuring samples (5) add secondary antibody (6) colour developing (7) to terminate (8) result and sentence
It is fixed.The method has that specificity is high, sensitivity is strong, favorable repeatability, low cost and other advantages, and the method can be examined in high volume in addition
Test sample product, for the detection and research of the disease are provided effectively, easy serological diagnostic method.
Indirect ELISA detection method from the A type clostridium perfringens toxoid antibody of previous application is different, side of the invention
Method can specifically detect clostridium perfringens alpha toxin, and accuracy rate and specificity are significantly improved than prior art, most
The capture ELISA detection clostridium perfringens alpha toxin antibody methods set up eventually can be used for batch samples detection, be livestock and poultry product
The diagnosis and research of gas capsular clostridium disease provide more rapidly, effective instrument, and be vaccine quality and food safety monitoring
Solid foundation has been established in research.
The A types C.perfringens that inventor is used in the present invention uses existing strain, in particular selected from strain
NCTC528 (National Collection of Type Cultures United Kingdom National Type Tissue Collections are preserved in,
Preserving number:NCTC528), can be directly obtained by existing channel, so the biomaterial belongs to can directly obtain in the prior art
Material, be not required to carry out single biological deposits.
Following several special characters are mainly contained in concrete technical scheme of the invention：
The preparation of 1A type clostridium perfringens toxoid antigens:
A type C.perfringens strains NCTC528 is inoculated on blood plate culture medium and is recovered, 37 DEG C of Anaerobic culturels
36h, chooses single colony inoculation liquid sulfur ethanol culture medium and increases bacterium 12 hours, and enrichment liquid then is pressed into percent by volume 5%
It is inoculated in the Gordon soup of improvement, 45 DEG C of culture 5h produce poison, and 0.22um seitz filter filtration sterilizations are used after culture is centrifuged,
Obtain it is degerming after A type C.perfringens exotoxins；One part formalin-inactivated immunizing rabbit is taken, it is outer that residue is obtained
Through ammonium sulfate precipitation, then through bag filter desalination, Sephadex-G25 column chromatographies carry out desalting purifying concentration to exotoxin to toxin, make
To capture the capture antigen of ELISA；
Obtain after above-mentioned exotoxin, inventor utilizes LD50Ectotoxic activity is produced in measuring.
As described above, inventor has carried out following concrete application using it：
(1) formalin-inactivated exotoxin, obtains toxoid, is emulsified with Freund's adjuvant, as the antigen of immunizing rabbit；
(2) ammonium sulfate precipitation, then through bag filter desalination, it is dense that Sephadex-G25 column chromatographies carry out desalting purifying to exotoxin
Contracting, as the capture antigen of capture ELISA.
2. the recovery of anti-clostridium perfringens alpha toxin hybridoma and the preparation of monoclonal antibody is purified；
By the hybridoma cell strain F12 of clostridium perfringens alpha toxin, (Chinese microorganism strain collection guarantor is preserved in
Hide, deposit number：CGMCC No.8870) carry out conventional recovery, culture；Selection 6-8 week old female BAl BIc/c mouse 10, abdomen
Inject not 0.5mL/ sensitization of formula Freund's incomplete adjuvant, week post injection positive hybridoma cell strain F12, dosage 0.5-1 × 10 in chamber6
It is individual/only, mouse ascites are collected after 7-10 days, obtain a large amount of clostridium perfringens alpha toxin monoclonal antibodies, ascites through caprylic acid-
Ammonium sulfate method is purified, SDS-PAGE detection purification effects.
3. the preparation of anti-clostridium perfringens alpha toxin polyclonal antibody：
The rabbit 5 of the 1.0-1.5Kg not inoculated vaccines of health is chosen, the above-mentioned exotoxin 2mL and Freund that will be inactivated are complete
Full adjuvant is according to volume 1:1 mixing and emulsifying, skin of back multi-point injection 1mg/ is only；After head exempts from two weeks, the exotoxin 2mL that will be inactivated
With incomplete Freund's adjuvant according to volume 1:1 mixing and emulsifying, injection dosage is with method ibid；Method of the same race carries out three and exempts from after 2 weeks；
Three exempt from latter 2 weeks with without adjuvant antigen booster immunization, and with method ibid, gained hyper-immune serum is right as the capture ELISA positives for dosage
According to serum；Control group injecting normal saline, and ensure to take identical immune programme for children with experimental group, synchronously tested, gained
Serum is used as capture ELISA negative control seras.
After above three important technology key element is obtained, inventor further provides capture ELISA detection method
It is as follows：
(1) it is coated with：The clostridium perfringens alpha toxin monoclonal antibody antigen coat liquid of above-mentioned acquisition is diluted to 0.3 μ
G/ml, per the μ L of hole 100, is added to 96 hole elisa Plates, and 4 DEG C of sealing is overnight；
(2) close：Above-mentioned ELISA Plate is washed with PBST cleaning solutions 3 times, each 3min after the completion of washing, is added and contained 5wt%
, used as confining liquid, per the μ L of hole 200,4 DEG C of closings are overnight for the PBST solution of skimmed milk power；
(3) antigen is added：3 each 3min of above-mentioned ELISA Plate are washed with PBST solution and is patted dry, added per hole and diluted with PBS
To capture the antigen 1 00ul, 37 DEG C of incubation 1h of 2ug/ml；
(4) serum to be checked is added：3 each 3min are washed with PBST solution and is patted dry, per hole addition PBS with 1:
The tested serum of 160 volume ratios dilution, 100 μ L/ holes, while setting yin and yang attribute and blank, PBS is used as blank pair
According to 37 DEG C of incubation 1h；
(5) secondary antibody is added：3 each 3min are washed with PBST solution and is patted dry, addition presses 1 with PBS:8000 volumes
Than the goat anti-rabbit igg that the HRP for diluting is marked, 37 DEG C of incubation 1h；
(6) develop the color：3 each 3min are washed with PBST solution and is patted dry, be eventually adding soluble T MB substrate nitrite ions
100 μ L/ holes, 37 DEG C of condition lucifuges react 15min；
(7) terminate：Add 100ul2M H per hole2SO4Solution terminating reaction, reads light absorption value at 450nm.
5. the determination of result judgement standard
Calculate negative sample average valueWith standard deviation (SD), try to achieveIt is positive critical value,It is the moon
Property critical value.OD is determined with the capture ELISA method set up450nm,It is the positive,It is the moon
Property,It is suspicious specimen.
In above-mentioned indirect ELISA detection method, described blood plate medium component is：100ml deionized waters, 3.7g
The de- fibre Sheep Blood of bean powder agar, 1g glucose and 5% volume ratio；
The Gordon soup toxin producing medium compositions of the improvement are：Peptone 2g, dextrin 1g, yeast extract 2g, l- essence
Propylhomoserin 1.2g, glucose 1g, last PBS are settled to 100ml, and concentrated hydrochloric acid regulation pH is 7.5, and high-temperature sterilization is obtained final product；
Described antigen coat liquid is：0.1M carbonate buffer solutions, (effect of the coating buffer is for of the invention for pH 9.6
Antigen coat best results)；
Described PBS composition is：1L deionized waters, potassium dihydrogen phosphate (KH2PO4)：0.27g, disodium hydrogen phosphate
(Na2HPO4)：1.42g, sodium chloride (NaCl)：8g, potassium chloride (KCl) 0.2g, pH 7.4；
The PBST cleaning solutions composition is：500 μ L Tween-20 are dissolved in the PBS of 1L；
The confining liquid composition is：5g skimmed milk powers are dissolved in 100mlPBST；
Terminate liquid (the 2M H2SO4) composition is：Measure dense H2SO427.6ml is added in 450ml deionized waters, is mixed
1L is settled to after even.
Using present invention detection method disclosed above, have the advantages that：
(1) high specificity：There is stronger reactivity with natural alpha toxin serum antibody, and with Escherichia coli, salmonella,
Riemerella anatipestifer antibody serum testing result is feminine gender, shows no cross reaction, and specificity is stronger；Compared with indirect method
It is indirectly to survey various toxin antibodies, the method in the present invention only surveys alpha toxin antibody, high specificity；
(2) sensitiveness is high：Above-mentioned obtained positive serum is surveyed in its serum titer and classics mouse and compared with experiment.Will
Positive serum presses 1：10,1：20,1：40,1：80,1：160,1：320,1：After 640 continuous doubling dilutions, ELISA detections are carried out,
The serum titer of detection is 1:640.Measure out 0.1ml serum and can neutralize 400 toxin to mouse with experimental result in mouse
Minimum lethal dose, it is higher that ELISA method sensitiveness is built in as a result explanation.
(3) it is reproducible：1 piece is randomly selected from 3 coated 96 hole elisa Plates respectively, clinically known background is detected
10 parts of serum.Every part of serum repeats to do 3 times, sets 1 hole yin and yang attribute control respectively every time.Compare the OD values of yin and yang attribute serum, by
Detect that the repeated result of sample shows in batch and between criticizing, variation within batch coefficient is 1.21%-6.53%, interassay coefficient of variation is
1.78%-7.31%, both of which is less than 10%, illustrates same sample with batch, explanation small with degree of variation in different batches of experiments
The method has repeatability well.Because the individual difference without zoopery, the good stability of experiment, and be conducive to animal
Welfare is protected.
Brief description of the drawings：
Fig. 1 slightly carries exotoxin protein SDS-PAGE electrophoretic analysis figure for A types C.perfringens；
1,2 Lane Samples have the obvious band of the main exotoxin of C.perfringens (alpha toxin) at 43KD in figure.
Fig. 2 is mouse ascites SDS-PAGE analysis charts after purification；
1,2 Lane Samples can see two apparent protein bands in figure：Heavy chain and 23kD of 53kD or so or so
Light chain, and have no other miscellaneous bands, as a result show the effect of its purifying preferably, its purity reaches more than 90%.
Specific embodiment
It is explained further the present invention with reference to embodiments, but embodiment does not do any type of limit to the present invention
It is fixed.The technological means such as all culture mediums and molecular biology that are related in embodiment are well known to those skilled in the art.
Reagent used and its source in embodiment：Not formula Freund's complete adjuvant is purchased with not formula Freund's incomplete adjuvant, Tween 20
From Sigma Co., USA；Goat anti-rabbit igg-HRP and goat anti-mouse igg-HRP are purchased from Hangzhou Huaan Bio-Tech. Co., Ltd.；
96 hole elisa Plates are purchased from Solarbio companies；Anaerobism liver bouillon, thioglycollate medium are purchased from the rich biology in Qingdao sea；Can
Dissolubility tmb substrate nitrite ion is purchased from Tiangeng biochemical technology Co., Ltd；Anti- clostridium perfringens alpha toxin monoclonal antibody F12's
Hybridoma cell strain is preserved in Chinese microorganism strain collection (preserving number：CGMCCNo.8870)；Other chemistry examinations used
It is pure that agent is analysis.
The reagent being related in embodiment is as follows：
Described blood plate medium component is：100ml deionized waters, 3.7g bean powderes agar, 1g glucose and 5% volume
Than de- fibre Sheep Blood；
The Gordon soup toxin producing medium compositions of the improvement are：Peptone 2g, dextrin 1g, yeast extract 2g, l- essence
Propylhomoserin 1.2g, glucose 1g, last PBS are settled to 100ml, and concentrated hydrochloric acid regulation pH is 7.5, and high-temperature sterilization is obtained final product；
Described antigen coat liquid is：0.1M carbonate buffer solutions, pH 9.6；
Described PBS composition is：1L deionized waters, potassium dihydrogen phosphate (KH2PO4)：0.27g, disodium hydrogen phosphate
(Na2HPO4)：1.42g, sodium chloride (NaCl)：8g, potassium chloride (KCl) 0.2g, pH 7.4；
The PBST cleaning solutions composition is：500 μ L Tween-20 are dissolved in the PBS of 1L；
The confining liquid composition is：5g skimmed milk powers are dissolved in 100mlPBST；
Terminate liquid (the 2M H2SO4) composition is：Measure dense H2SO427.6ml is added in 450ml deionized waters, is mixed
1L is settled to after even.
The embodiment 1A ectotoxic preparation and purifications of type C.perfringens
A type C.perfringens strain (National Collection of Type Cultures- Britain microbial bacteria
Plant collection preservation preserving number:NCTC528) it is inoculated on blood plate culture medium and is recovered, 37 DEG C of Anaerobic culturel 36h chooses
Single bacterium colony is taken in THIOGLYCOLLIC ACID salt nutrient broth enriched medium, is 88%N in gas concentration2, 7%H2, 5%CO2's
38 DEG C of Anaerobic culturel 12h under the conditions of anaerobic environment.5mL A type C.perfringens enrichment liquids are inoculated into 100ml pH7.5's
In toxin producing medium (per 100mL PBSs dissolving 2g peptones, 1g dextrin, 2g yeast extracts and 1.2gL- arginine,
1g glucose) in, the shaken cultivation under anaerobic environment, 43 DEG C of culture 5h efficiently produce poison, then in 4 DEG C of 8000r/min centrifugations
15min, then with filtration sterilization in the seitz filter in 0.22 μm of aperture, that is, obtain it is degerming after A type C.perfringens outer poison
Element.Finally measure ectotoxic LD50=24.25, will exotoxin dilute 19.03 times, intraperitoneal injection 1ml can make half mouse
It is dead.
Toxin soiutions after will be degerming is slowly added to saturated ammonium sulfate solution, finally makes the volume of ammonium sulfate in mixed solution
Concentration reaches 50%, and 4 DEG C stand overnight；Next day, solution 8000r/min is centrifuged 20min, discards supernatant, precipitation is with right amount
0.05M Tris-HCL buffer solutions, are slowly added to saturated ammonium sulfate solution, the ultimate density of ammonium sulfate is reached 40%, weight
Multiple aforesaid operations, will finally be precipitated and dissolved in Tris-HCL；Then exotoxin is removed with Sephadex-G25 column chromatographies
Salt purifying concentration；By toxin protein after purification through SDS-PAGE electrophoresis, as a result analyzing proteins purity is shown at molecular weight 43KD
There is obvious band (as shown in Figure 1), be principal causative exotoxin alpha toxin；With under spectrophotometric determination 260nm and 280nm
Absorbance, determines that toxin protein concentration is：(1.45×A280- 0.74 × A260) × extension rate.
The recovery of the anti-clostridium perfringens alpha toxin hybridoma of embodiment 2 and the preparation purifying of monoclonal antibody；
By the hybridoma cell strain F12 of clostridium perfringens alpha toxin, (Chinese microorganism strain collection guarantor is preserved in
Hide, deposit number：CGMCC No.8870) carry out conventional recovery, culture；Selection 6-8 week old female BAl BIc/c mouse 10, abdomen
Inject not 0.5mL/ sensitization of formula Freund's incomplete adjuvant, week post injection positive hybridoma cell strain F12, dosage 0.5-1 × 10 in chamber6
It is individual/only, mouse ascites are collected after 7-10 days, obtain a large amount of clostridium perfringens alpha toxin monoclonal antibodies, ascites through caprylic acid-
Ammonium sulfate method is purified, SDS-PAGE detection purification effects, as a result as shown in Figure 2:1,2 Lane Samples can see two in figure
Apparent protein band：The light chain of the heavy chain and 23kD of 53kD or so or so, and other miscellaneous bands are had no, as a result show that it is purified
Effect preferably, its purity reaches more than 90%, with spectrophotometric determination monoclonal antibody purification 260nm and 280nm
Absorbance, determine that toxin protein concentration is：(1.45×A280- 0.74 × A260) × extension rate.
The preparation of the anti-clostridium perfringens alpha toxin polyclonal antibody of embodiment 3
To be obtained in embodiment 1 it is degerming after A type C.perfringens exotoxin in add formaldehyde make its final concentration of
0.3vt%, after fully mixing, 37 DEG C of inactivation 96h, period rocks once every 5-6h vibrations；
The rabbit 5 of the 1.0-1.5Kg not inoculated vaccines of health is chosen, the above-mentioned alpha toxin 2mL and Freund that will be inactivated are complete
Adjuvant is according to volume 1:1 mixing and emulsifying, skin of back multi-point injection 1mg/ is only；After head exempts from two weeks, will inactivate exotoxin 2mL and
Incomplete Freund's adjuvant is according to volume 1:1 mixing and emulsifying, injection dosage is with method ibid；Method of the same race carries out three and exempts from after 2 weeks；Three
With without adjuvant antigen booster immunization, with method ibid, gained hyper-immune serum is used as capture ELISA positive controls for dosage within 2 weeks after exempting from
Serum；Control group injecting normal saline, and ensure to take identical immune programme for children with experimental group, synchronously tested, gained blood
Clear conduct capture ELISA negative control seras.
Embodiment 3 captures the foundation of ELISA detection method
The best pairing of coated antibody (obtained by embodiment 2) and antigen (gained of embodiment 1) is determined as square formation titration
Diluted concentration, and tested antibody optimal diluted concentration and the optimal diluted concentration of enzyme labelled antibody, and ELISA conditions are carried out
Grope and optimize, be finally defined below system：
(1) determination with antagonist and antigen optium concentration
The anti-clostridium perfringens alpha toxin monoclonal antibody F12 that initial option embodiment 2 is recovered and purified is real as capture antibody
1 exotoxin of extraction purification of example is applied as combining antigen.In order to determine coated antibody and antigen optium concentration, first determine to be checked
In the case of serum optimum diluting multiple and ELIAS secondary antibody optimum diluting multiple, being tested by square formation carries out optimum antibody pairing
Selection.Will capture antibody coating buffer by volume 1:100 dilutions add 96 hole elisa Plates first rows, and with coating buffer downward 1:
2 doubling dilutions to the tenth row, 100 μ L/ holes, overnight, PBST is washed 3 times 4 DEG C of coatings, and each 3min is patted dry.Add confining liquid per hole
200 μ L, overnight, PBST is washed 3 times, and each 3min is patted dry for 4 DEG C of closings.Alpha toxin albumen is pressed into volume 1 with PBS:250 dilutions add
Enter 96 hole elisa Plates the first rows and with PBS downward 1:Per the μ L/ holes of hole 100,37 DEG C are incubated 1h, PBST to 2 doubling dilutions to the 8th row
Washing 3 times, each 3min is patted dry.By positive antibody serum PBS by volume 1：160 dilutions add 96 hole enzyme marks
Plate is per the μ L of hole 100 and sets negative control.37 DEG C of incubation 1h, PBST solution is washed 3 times, and each 3min is patted dry.Addition PBS
Buffer solution presses 1:The goat anti-rabbit igg of the HRP marks of 8000 volume ratios dilution, 37 DEG C of incubation 1h.Add the colour developing of soluble T MB substrates
Liquid, 37 DEG C of colour developing 15min of lucifuge, 100 μ L 2M H2SO4 terminating reactions are added per hole, are determined per hole with ELIASA at 450nm
OD values.With the foundation of P/N values alternatively antibody and antigen best pairing concentration.
It is final to determine capture antibody：Anti- clostridium perfringens alpha toxin monoclonal antibody optimum diluting multiple is 1:6400 are
0.3ug/ml；Antigen best effort concentration is 2ug/mL.
(2) selection of optimal coating buffer
The row 0.01mol/L carbonate buffer solutions of 96 hole elisa Plates the one or two, the three or four row are delayed with 0.05mol/L carbonate
Fliud flushing, the five or six uses 0.1mol/L carbonate buffer solutions, and the seven or eight distinguishes coated antibody with 0.2mol/L carbonate buffer solutions.1
35 rows add positive, and 246 rows add negative sample control.Optimal coating condition is determined according to p/N values.
It is final determine can handy 0.1mol/L carbonate buffer solution as coating buffer best results.
(3) determination of the optimal incubation time of coated antibody
Antibody coating buffer is diluted to working concentration, while setting negative control, 37 DEG C of 1h+4 DEG C of 12h, 4 is respectively placed in
DEG C 12h, is incubated under the conditions of 37 DEG C of 2h.Determine that antibody is most preferably coated with condition according to p/N values.
Can be 4 DEG C of 12h in the optimal incubation time of coated antibody through comparing and analyzing.
(4) determination of optimal sealing condition
96 hole elisa Plates the one or two, 5% skimmed milk power of row, the three or four row use 5% hyclone, and the five or six uses 1%BSA,
1st, 3,5 rows add positive control, 2,4,6 rows to add negative serum control.Optimal sealing condition is determined according to p/N values.
5% skimmed milk power is used as confining liquid best results.
(5) determination of optimal off-period
Three ELISA Plates are coated with respectively, and preceding four row of each ELISA Plate adds positive control, and four rows add negative control afterwards,
4 DEG C of 12h, 37 DEG C of 2h are respectively placed in, are incubated under the conditions of 37 DEG C of 1h+4 DEG C of 12h.When determining that the optimal encapsulating of antibody is closed according to p/N values
Between.
Finally determine that optimal off-period is 4 DEG C of 12h.
(6) determination of the optimal incubation time of antigen
Three ELISA Plates are coated with respectively, and preceding four row of each ELISA Plate adds positive control, and four rows add negative control afterwards,
37 DEG C of 30min, 37 DEG C of 1h are respectively placed in, are incubated under the conditions of 37 DEG C of 2h.The optimal incubation time of antigen is determined according to p/N values.
Finally determine that 37 DEG C of 1h are its optimal incubation times.
(7) it is detected the determination of serum optimum dilution degree
In order to determine the optium concentration of serum, positive serum and negative serum are pressed 1 respectively:20,1:40,1:80,1:
160,1:320,1:640,1:1280,1:2560,1:5120,1:10240,1:20480,1:40560 are diluted to two 96 respectively
Orifice plate, adds 96 orifice plate 1-12 row, each dilution factor to add the hole of a row 8 respectively.OD values are surveyed, tested serum is determined according to P/N values.
Be can determine that when tested serum diluting multiple is 1 by yin and yang attribute ratio:P/N is maximum when 160, then 1：160 are
Its optimum diluting multiple.
(8) it is detected the determination of serum optimum reacting time
Positive serum and negative serum are pressed after optimum dilution degree dilution in 96 hole elisa Plates that have been coated with of addition, is put respectively
In 37 DEG C of 30min, 37 DEG C of 2h, it is incubated under the conditions of 37 DEG C of 1h.Determine that antibody most preferably wraps off-period according to p/N values.
Can determine that the optimal incubation time of tested serum is 37 DEG C of 1h. eventually through yin and yang attribute ratio
(9) determination of ELIAS secondary antibody optimum diluting multiple
Wrapper sheet is carried out by the optimum condition groped respectively, positive serum and negative serum are added to by optimum dilution degree
After 96 hole elisa Plates being coated with are incubated, ELIAS secondary antibody presses 1 respectively:5000,1:8000,1:10000 are diluted survey OD values, root
Determine that antibody most preferably wraps off-period according to p/N values.
Can determine that ELIAS secondary antibody optimum diluting multiple is 1 eventually through yin and yang attribute ratio:8000；
(10) determination of ELIAS secondary antibody incubation time
Wrapper sheet is carried out by the optimum condition groped respectively, positive serum and negative serum are added to by optimum dilution degree
After 96 hole elisa Plates being coated with are incubated, the ELIAS secondary antibody for adding optimum diluting multiple is respectively placed in 37 DEG C of 30min, 37 DEG C of 1h,
It is incubated under the conditions of 37 DEG C of 2h.The optimal incubation time of antibody is determined according to p/N values.
Can determine that the optimal incubation time of ELIAS secondary antibody is 37 DEG C of 1h. by yin and yang attribute ratio
(11) determination of optimal developing time
Respectively the processes such as wrapper sheet incubation, plus the lower incubation at room temperature of nitrite ion lucifuge are carried out by the optimum condition groped
10min, 15min, 20min are incubated under the conditions of 10min, 15min, 20min, 37 DEG C.And negative control is set up, according to p/N values really
Determine the optimal incubation time of antibody.
Can determine that optimal developing time is 37 DEG C of 15min. by yin and yang attribute ratio
(12) capture ELISA testing conditions are optimized using square formation titration experiments, is finally defined below reaction system：
(1) it is coated with：The clostridium perfringens alpha toxin monoclonal antibody antigen coat liquid of above-mentioned acquisition is diluted to 0.3 μ
G/ml, per the μ L of hole 100, is added to 96 hole elisa Plates, and 4 DEG C of sealing is overnight；
(2) close：Above-mentioned ELISA Plate is washed with PBST cleaning solutions 3 times, each 3min after the completion of washing, is added and contained 5wt%
, used as confining liquid, per the μ L of hole 200,4 DEG C of closings are overnight for the PBST solution of skimmed milk power；
(3) antigen is added：3 each 3min of above-mentioned ELISA Plate are washed with PBST solution and is patted dry, added per hole and diluted with PBS
To capture the antigen 1 00ul, 37 DEG C of incubation 1h of 2ug/ml；
(4) serum to be checked is added：3 each 3min are washed with PBST solution and is patted dry, per hole addition PBS with 1:
The tested serum of 160 volume ratios dilution, 100 μ L/ holes, while setting yin and yang attribute and blank, PBS is used as blank pair
According to 37 DEG C of incubation 1h；
(5) secondary antibody is added：3 each 3min are washed with PBST solution and is patted dry, addition presses 1 with PBS:8000 volumes
Than the goat anti-rabbit igg that the HRP for diluting is marked, 37 DEG C of incubation 1h；
(6) develop the color：3 each 3min are washed with PBST solution and is patted dry, be eventually adding soluble T MB substrate nitrite ions
100 μ L/ holes, 37 DEG C of condition lucifuges react 15min；
(7) terminate：Add 100ul2M H per hole2SO4Solution terminating reaction, reads light absorption value at 450nm.
Embodiment 4 captures the determination of ELISA detection method critical value
33 parts of negative serums are surveyed with 96 orifice plates being coated with, is operated by the condition after optimization, carry out ELISA detections.
OD values are surveyed, average value and standard deviation is calculated, tried to achieveIt is positive critical value,It is negative critical value.With foundation
Capture ELISA method determine OD450nm,It is the positive,It is feminine gender, It is suspicious specimen.
Calculate its average valueWith standard deviation SD=0.015, try to achieveIt is 0.273 to be worth,It is worth and is
0.288, so as tested serum OD450nm<It is feminine gender, OD450nm when 0.273>It is the positive, 0.273 when 0.288<OD450nm
<It is suspicious when 0.288.
Embodiment 5 captures ELISA method performance evaluation
(1) specific test:With the capture ELISA method for establishing, detection clostridium perfringens alpha toxin antibody have compared with
Strong reactivity, with immune rabbit gained serum is captured respectively with Escherichia coli, salmonella, riemerella anatipestifer
ELISA detects no cross reaction.
(2) sensitivity experiment：Above-mentioned obtained positive serum surveys its serum titer, with classical mouse in and experiment do ratio
Compared with.Positive serum is pressed 1：10,1：20,1：40,1：80,1：160,1：320,1：After 640 continuous doubling dilutions, ELISA is carried out
Detection, the serum titer of detection is 1：640.
The physiological saline doubling dilution, intraperitoneal injection 0.5mL is used to make whole respectively the above-mentioned α-exotoxin through desalting purifying
The content of toxin is the minimum lethal dose of mouse during the maximum dilution multiple of dead mouse.The toxin of preparation is measured to mouse
MLD is 2uL.
2 times of lethal dose toxin and the dilutions of 0.2mL 1: 50,1: 100,1: 200,1: 400,1: 600,1: 800,1: 1000
Hyper-immune serum mixes, and with physiological saline constant volume to 1mL, is put in 37 DEG C of incubator 2h, takes 0.5mL intraperitoneal injection of mice, observes mouse
Survival condition.Result show that 0.1mL serum can neutralize minimum lethal dose of 400 toxin to mouse.
(3) repeated experiment：1 piece is randomly selected from 3 coated 96 hole elisa Plates respectively, the clinically known back of the body is detected
10 parts of the serum of scape.Every part of serum repeats to do 3 times, sets 1 hole yin and yang attribute control respectively every time.Compare the OD values of yin and yang attribute serum,
Detect that the repeated result of sample shows in by criticizing and between criticizing, variation within batch coefficient is 1.21%-6.53%, interassay coefficient of variation
It is 1.78%-7.31%, both of which is less than 10%, illustrates that same sample degree of variation in same batch and different batches are tested is small, says
Bright the method has repeatability well.
The concrete application of embodiment 7
1. with the capture ELISA method for establishing to from 100 parts of Virus monitory aerogenesis pods in Taian Shandong warren
Film perfringens alpha toxin antibody, using indirect ELISA detection C.perfringens exotoxin antibody method compare (Sun Jiazhi,
2014).Testing result shows：Indirect ELISA detects that the blood serum sample of 90 rabbits is detected as feminine gender, and 10 is the positive, positive rate
It is 10%；Capture ELISA method detection is feminine gender to 92 blood serum samples of rabbit, and 8 is the positive, and positive rate is 8%.
2. take 8 parts of positive serums of above-mentioned utilization indirect ELISA method gained and survey in its serum titer and classical mouse and real
Test and compare.Positive serum is pressed 1：10,1：20,1：40,1：80,1：160,1：320,1：After 640 continuous doubling dilutions, carry out
ELISA detects that the serum titer of detection is respectively 1：320,1:320,1:160,1:160,1:160,1:320,1:320,1:
320。
The physiological saline doubling dilution, intraperitoneal injection 0.5mL is used to make whole respectively the above-mentioned α-exotoxin through desalting purifying
The content of toxin is the minimum lethal dose of mouse during the maximum dilution multiple of dead mouse.(MLD is above-mentioned for 2 times of lethal dose toxin
It is measured as 2ul) mixed with the hyper-immune serum of the dilutions of 0.2mL 1: 50,1: 100,1: 200,1: 400,1: 600,1: 800,1: 1000
Close, with physiological saline constant volume to 1mL, be put in 37 DEG C of incubator 2h, take 0.5mL intraperitoneal injection of mice, observe mouse survival situation.Knot
Fruit show that 0.1mL serum can neutralize 200,200,100,50,100,200,100,200 toxin pair respectively
The minimum lethal dose of mouse.
To sum up explanation the method its Positive rate compared with indirect ELISA method is slightly lower, but its specific detection is produced
Gas capsular clostridium alpha toxin antibody, its positive rate is relatively low to be true to life, and illustrates its high specificity, to other exotoxins without
Reaction, and indirect ELISA detection C.perfringens exotoxin antibody, and targeted object is the mixing of Multiple Antibodies
Thing, but its positive rate poor specificity high, therefore result is rational；It is compared with experiment with classical mouse, it is sensitive
Property is higher；Result explanation：The capture ELISA detection clostridium perfringens alpha toxin antibody methods set up can be used for high-volume sample
Product examine survey, for livestock and poultry C.perfringens disease diagnosis and research provide more rapidly, effective instrument, and for vaccine quality with
Solid foundation has been established in the research of food safety monitoring.
Claims (3)
1. a kind of clostridium perfringens alpha toxin Anti-HBV permanence detection method, it is characterised in that：Concretely comprise the following steps：
(1) it is coated with：The clostridium perfringens alpha toxin monoclonal antibody antigen coat liquid of above-mentioned acquisition is diluted to 0.3 μ g/
Ml, per the μ L of hole 100, is added to 96 hole elisa Plates, and 4 DEG C of sealing is overnight；
(2) close：Above-mentioned ELISA Plate is washed with PBST cleaning solutions 3 times, each 3min after the completion of washing, adds degreasing containing 5wt%
, used as confining liquid, per the μ L of hole 200,4 DEG C of closings are overnight for the PBST solution of milk powder；
(3) antigen is added：3 each 3min of above-mentioned ELISA Plate are washed with PBST solution and is patted dry, added per hole and be diluted to PBS
Capture the antigen 1 00ul, 37 DEG C of incubation 1h of 2ug/ml；
(4) serum to be checked is added：3 each 3min are washed with PBST solution and is patted dry, per hole addition PBS with 1:160
Volume ratio dilution tested serum, 100 μ L/ holes, at the same set yin and yang attribute and blank, PBS as blank,
37 DEG C of incubation 1h；
(5) secondary antibody is added：3 each 3min are washed with PBST solution and is patted dry, addition presses 1 with PBS:8000 volume ratios are dilute
The goat anti-rabbit igg of the HRP marks released, 37 DEG C of incubation 1h；
(6) develop the color：3 each 3min are washed with PBST solution and is patted dry, be eventually adding the μ L/ of soluble T MB substrates nitrite ion 100
Hole, 37 DEG C of condition lucifuges react 15min；
(7) terminate：Add 100ul2M H per hole2SO4Solution terminating reaction, reads light absorption value at 450nm；
Calculate negative sample average valueWith standard deviation (SD), try to achieveIt is positive critical value,For feminine gender is faced
Dividing value.OD is determined with the capture ELISA method set up450nm,It is the positive,It is feminine gender,It is suspicious specimen；
Wherein described clostridium perfringens alpha toxin monoclonal antibody preparation method is as follows：
By the hybridoma cell strain F12 of clostridium perfringens alpha toxin, Chinese microorganism strain collection preservation is preserved in, protected
Hide numbering：CGMCC No.8870, carry out conventional recovery, culture；Selection 6-8 week old female BAl BIc/c mouse 10, intraperitoneal injection
Not 0.5mL/ sensitization of formula Freund's incomplete adjuvant, week post injection positive hybridoma cell strain F12, dosage 0.5-1 × 106Individual/only,
Mouse ascites are collected after 7-10 days, a large amount of clostridium perfringens alpha toxin monoclonal antibodies are obtained；
The capture antigen preparation method is as follows：
A type C.perfringens strains NCTC528 is inoculated on blood plate culture medium and is recovered, 37 DEG C of Anaerobic culturel 36h, choosing
Take single colony inoculation liquid sulfur ethanol culture medium and increase bacterium 12 hours, then enrichment liquid is inoculated in by percent by volume 5%
In the Gordon soup of improvement, 45 DEG C of culture 5h produce poison, and 0.22um seitz filter filtration sterilizations are used after culture is centrifuged, and are removed
A type C.perfringens exotoxins after bacterium；One part formalin-inactivated immunizing rabbit is taken, the exotoxin warp that residue is obtained
Ammonium sulfate precipitation, then through bag filter desalination, Sephadex-G25 column chromatographies carry out desalting purifying concentration to exotoxin, used as capture
The capture antigen of ELISA.
2. clostridium perfringens alpha toxin Anti-HBV permanence detection method according to claim 1, it is characterised in that：It is described
Positive control serum in step (4), preparation method is as follows：
The rabbit 5 of the 1.0-1.5Kg not inoculated vaccines of health is chosen, the exotoxin 2mL and Freund of formalin-inactivated are helped completely
Agent is according to volume 1:1 mixing and emulsifying, skin of back multi-point injection 1mg/ is only；After head exempts from two weeks, the exotoxin 2mL and not that will be inactivated
Family name's Freund's incomplete adjuvant is according to volume 1:1 mixing and emulsifying, injection dosage is with method ibid；Method of the same race carries out three and exempts from after 2 weeks；Three exempt from
With without adjuvant antigen booster immunization, with method ibid, gained hyper-immune serum is used as capture ELISA positive control blood for dosage within 2 weeks afterwards
Clearly.
3. clostridium perfringens alpha toxin Anti-HBV permanence detection method according to claim 1, it is characterised in that：It is described
Negative control sera in step (4) uses the rabbit anteserum that experiment injecting normal saline is synchronized with made hyper-immune serum.
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