CN105388295A - Blocking ELISA method for detecting cysticercosis pisiformis cyst fluid antibody of rabbit - Google Patents
Blocking ELISA method for detecting cysticercosis pisiformis cyst fluid antibody of rabbit Download PDFInfo
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Abstract
A blocking ELISA method for detecting a cysticercosis pisiformis cyst fluid antibody of a rabbit is used for early detection and clinical diagnosis of cysticercosis pisiformis of the rabbit, solves the problem that at present, the cysticercosis pisiformis of the rabbit cannot be diagnosed by using a serological method, and is an effective detection method for specificity diagnosis and comprehensive prevention and control of the cysticercosis pisiformis of the rabbit. The blocking ELISA method includes the following three steps of purification of a cysticercosis pisiformis cyst fluid, antigenicity detection of purified protein and establishment of the blocking ELISA detection method. The rapid, sensitive and specific blocking ELISA method is used for detecting the specific cyst fluid antibody in the serum of the rabbit suffering from the cysticercosis pisiformis, is good in result stability, can be commercialized and automatic and can be also used for determination with naked eyes. Therefore, the blocking ELISA method is expected to become an important tool clinically for rapid diagnosis and immune detection of the cysticercosis pisiformis of the rabbit, will certainly play an important role in diagnosis and prevention and control of the cysticercosis pisiformis of the rabbit and has good application prospect.
Description
Technical field:
The present invention relates to animal immune to detect, a kind of particularly stop band restrain method detecting rabbit cysticercus pisiformis capsule humoral antibody.
Background technology:
Rabbit cysticercus pisiformis disease parasitizes by the larva cysticercus pisiformis (Cysticercuspisiformis) of Taenia Pisiformis a kind of tapeworm larva disease caused in the liver of rabbit, omentum majus, mesenterium and abdominal cavity.This disease is worldwide distribution, and all there is generation China various places, and are in a bad way.It is reported, from Heilungkiang, Liaoning of northeast, to Qinghai and the Xinjiang of the northwestward, from Sichuan in southwest, Chongqing and Guizhou, to the Fujian of the southeast and the Guangxi in south China, arrive the Henan in Central China again,, all there is the generation of this disease in the provinces such as the Zhejiang in East China and Shandong, and disease of falling ill rate is not from 5% to 100% etc., average rate reaches about 40%, and mortality ratio is between 4%-20%.This sick infection rate is high, to the equal easy infection of rabbit of all ages and classes and kind.Warren is once break out this disease, and infection rate is almost 100%.The sick rabbit performance digestive disorders of low-grade infection, young rabbit growth retardation, adult rabbits is become thin, and the price of deed reduces, easy secondary other diseases, and rabbit can be caused during a large amount of parasitism only dead, and the development of rabbit keeping in serious threat.Epidemic Scope at home constantly expanded in recent years, and the state of an illness is more and more heavier, has the trend of deterioration, has a strong impact on the development of rabbit keeping.
At present, the serodiagnosis problem of China to this disease does not also solve, and effective serological diagnostic method is not also set up.It is reported.The capsule liquid of capsule liquid fraction and pig cysticercus tenuicollis that Zhang Shoufa etc. (1994) apply rabbit cysticercus pisiformis first at home makes antigen, with capsule liquid and cephalomere, the Capsule wall antigens of rabbit cysticercus pisiformis, positive serum and the negative serum of rabbit cysticercus pisiformis disease have detected, analyzes, compare the effect of various diagnostic antigen rabbit cysticercus pisiformis disease with Carbon agglutination Test.Result shows, rabbit cysticercus pisiformis capsule liquid crosses post by sephadex G 1, and gained second peak egg is the strongest from antigenicity, and specificity is best, and positive coincidence rate is the highest; Next is the cyst fluid antigen of rabbit cysticercus pisiformis; And the cephalomere of rabbit cysticercus pisiformis, Capsule wall antigens are little, the diagnosis of this disease can not be used for.After this, Wang Xiaoxia etc. (2009) Crude protein of cysticercus pisiformis establishes the Elisa method for laboratory diagnosis, but recall rate is lower, can not be used for clinical detection.Up to now, the serological method for clinical detection rabbit cysticercus pisiformis disease is not also set up.
Summary of the invention:
The object of this invention is to provide a kind of stop band restrain method that detection efficiency is high, testing result detects rabbit cysticercus pisiformis capsule humoral antibody accurately.
Technical scheme of the present invention is, (claim elements).
The invention has the beneficial effects as follows: a kind of sick rabbit to suffering from cysticercus pisiformis can be provided to carry out the method for clinical diagnosis, namely can be clinical treatment service, can be again generaI investigation epidemic prevention service, make cysticercus pisiformis disease wait until timely Diagnosis and treatment, thus improve the economic benefit of aquaculture.The present invention stablizes Yang Tu colony (middle-size and small-size warren) to city and has vital role.Find in the investigation of rabbit warren, many medium and small rabbit warrens are all that peasant does, popular due to cysticercus pisiformis disease, and cause a large amount of young rabbit dead, closed down in a lot of warren, social layabout increases.If energy Timeliness coverage and this disease of control, increase foster rabbit and take in, the enthusiasm of rabbit raiser person will be made to increase, and the layabout absorbing rural area enters rabbit keeping.This both can increase the income of a people, can promote again the stable and united of society.In addition, the present invention contributes to blocking the pernicious biological cycle of cysticercus pisiformis, has environment purification, ensures the effect of rabbit, dog and human health.
Tool of the present invention has the following advantages: (1) the present invention by the white separation of cysticercus pisiformis capsule liquid eggs, purifying and detection of antigenicity, establish a kind of fast, responsive, special stop band restrain method detects the specificity capsule humoral antibody of suffering from the sick rabbit anteserum of beans shape cercaria.(2) this method is expected to, to the sick quick diagnosis of carrying out before death of rabbit cysticercus pisiformis, play a significant role, have good economic worth and social value by the clinical diagnosis and control of rabbit beans shape larva of a tapeworm or the cercaria of a schistosome cercaria disease.(3) the present invention determines the package amount of best antigen, the dilutability of antibody, the bag of antigen by conditions such as time, best confining liquid and off-period, best sample incubation time and best developing times, the stable performance of detection, reproducible.(4) the present invention only needs a small amount of blood serum sample, the stop band restrain method can applying the detection rabbit cysticercus pisiformis capsule humoral antibody of foundation to rabbit beans shape capsule disease carry out convenient, detect fast.(5) simple to operate, the detection speed of the present invention can detect case soon, both, can quarantine in enormous quantities again.
Accompanying drawing illustrates:
Fig. 1 be the difference bag of cysticercus pisiformis capsule liquid of the present invention by the reaction result curve map of time,
Fig. 2 is the response curve figure of the different off-periods of cysticercus pisiformis capsule liquid of the present invention,
Fig. 3 is the response curve figure of two anti-different dilution ratios of hyper-immune serum of the present invention,
Fig. 4 is the reaction result curve map of difference two anti-action time of hyper-immune serum of the present invention,
Fig. 5 is the specificity analyses curve map of stop band restrain.
Embodiment:
Step 1
With micropore ultra-filtration centrifuge tube, cysticercus pisiformis capsule liquid eggs is carried out in vain to the method for separation and purification:
(1) get each two of the ultra-filtration centrifuge tube of the micropore ultra-filtration centrifuge tube of 100kDa, 50kDa, after using ultrapure water rinse clean respectively, fill ultrapure water, be placed in 4 DEG C of refrigerators, precooling 5-10min;
(2) get after 1200 μ L full capsule liquid eggs mixes with isopyknic ultrapure water in vain, obtain 2400 μ L samples;
(3) open hydro-extractor in advance and be cooled to 4 DEG C, the ultra-filtration centrifuge tube of two good for pre-service 100kDa is taken out, discards ultrapure water in pipe, add 1200 μ L samples separately, centrifugal treating 30-35min;
(4) after centrifugal, take out ultra-filtration centrifuge tube, draw 500 μ L ultrapure water piping and druming 20-30 time, by the collecting protein being greater than 100kDa that is positioned in micropore ultra-filtration centrifuge tube pipe in clean little centrifuge tube,-80 DEG C save backup, and the albumen being less than 100kDa being positioned at micropore ultra-filtration centrifuge tube outer tube is directly collected in clean 10mL centrifuge tube;
(5) open hydro-extractor in advance and be cooled to 4 DEG C, the ultra-filtration centrifuge tube of two good for pre-service 50kDa is taken out, discard ultrapure water in pipe, the albumen being less than 100kDa that the step (4) adding equivalent is respectively collected, after centrifugal treating 30-35min, with the sample of 500 μ L ultrapure water 30-40 piping and druming 50-100kDa, be collected in little centrifuge tube,-80 DEG C save backup, and are directly collected in 10mL centrifuge tube by the albumen being less than 50kDa, and-80 DEG C save backup.
(6) result, has three kinds: one to be the albumen that molecular weight is greater than 100kDa with the albumen of ultra-filtration centrifuge tube separation and purification; Two is the albumen of molecular weight between 50-100kDa; Three is the albumen that molecular weight is less than 50kDa, is represented respectively by kind of the albumen of three after separation and purification with sequence number 1.2.3., has carried out concentration determination, the results are shown in Table 1 with ultramicron protein nucleic acid analyser.
Table 1 purifying protein concentration
| Albumen sequence number | Concentration (μ g/mL) |
| Non-purifying capsule liquid eggs is white | 5150.8 |
| 1 | 459.4 |
| 2 | 1018.0 |
| 3 | 382.2 |
As can be seen from Table 1, with unpurified capsule liquid eggs in vain compared with, the protein concentration after separation and purification obviously reduces.The albumen of molecular weight between 50-100kDa is maximum, and secondly for molecular weight is greater than the albumen of 100kDa, and the albumen that molecular weight is less than 50kDa is minimum.
Step 2
The white antigenicity of the capsule liquid eggs of purifying is detected by PVC-Dot-ELISA method:
Adopt PVC-Dot-ELISA detection method, screen and detect the antigenicity of the purified albumen of above-mentioned enforcement.The purifying protein of the different molecular weight be separated through ultra-filtration centrifuge tube is carried out wrapper sheet as coating antigen, and the antiserum of primary antibodie prepared by the white immune mouse of full capsule liquid eggs, two resist the mountain sheep anti-mouse igg for horseradish peroxidase (HRP) marks.
Basic operation steps is:
(1) by the concentration bag of 10 μ g/mL by 96 hole PVC board, every hole 50 μ L, often kind of purifying protein wraps separately by a line (12 hole), and 4 DEG C are spent the night, and PBST washs 3 times, every minor tick 5min;
(2) close with the PBST confining liquid containing 5% skimmed milk power, every hole 200 μ L, 37 DEG C, close 2h, PBST and wash 3 times, every minor tick 5min;
(3) add primary antibodie respectively, often row stays four holes, and three skies are negative control, and a sky is blank, and the first sky starts according to 1:100, carries out doubling dilution successively to 1:12800,37 DEG C, and reaction 30min, PBST wash 3 times, every minor tick 5min;
(4) sheep anti mouse two adding horseradish peroxidase-labeled resists, and dilution ratio presses 1:5000, every hole 50 μ L, 37 DEG C, and reaction 30min, PBST wash 3 times, every minor tick 5min;
(5) substrate nitrite ion is added, every hole 50 μ L, reaction 5min;
(6) stop buffer is added, every hole 50 μ L, reading immediately;
(7) measure the OD value in the 96 each holes of PVC reaction plate, hole by microplate reader at 450 nm, and preserve.
(8) result, carries out detection of antigenicity by PVC-Dot-ELISA detection method to three of separation and purification kinds of albumen, the results are shown in Table 2.
Table 2 PVC-Dot-ELISA method detects the antigenicity of purifying protein
As can be seen from this table, when purifying protein 1 and 2 reacts as coating antigen, can effectively detect tiring of primary antibodie, and when purifying protein 3 is as coating antigen, OD value and negative hole close, cannot detect primary antibodie, the OD value reading of purifying protein 1 and 2 is very close.
Step 3
The preparation of hyper-immune serum (primary antibodie):
(1) immune animal: the Healthy female BALB/c mouse in 56 week ages.
(2) immunizing antigen: molecular weight is that 50-100kDa capsule liquid eggs is white.
(3) immunologic adjuvant: Freund's complete adjuvant and incomplete Freund's adjuvant.
(4) immune position: 4, back hypodermic injection.
(5) immunizing dose: the immunizing dose of the immunity every mouse is 50 μ g
. 200 μ L.
(6) immunization method:
S1. during first time immunity, dilute capsule liquid eggs with the PBS of sterilizing white, then the amount of 1:1 by volume mixes with Freund's complete adjuvant, carries out immunity after fully emulsified.
S2. carry out secondary immunity after two weeks, mix by the amount of incomplete Freund's adjuvant with the white 1:1 by volume of the capsule liquid eggs after dilution, after fully emulsified, carry out immunity.Carried out primary immune response every two weeks, altogether immunity four times later.
S3., from second time immunity, latter 9th day of each immunity, detects the antibody titer of serum by PVC-Dot-ELISA method from tail vein blood.
S4. after the 4th immunity the 9th day, again mice serum antibody is detected, when serum antibody titer is 1:6400, be hyper-immune serum.
S5. every other day, implement to extract eyeball blood sampling to immune mouse, by whole blood collection in centrifuge tube, place 2-3h for 37 DEG C, 4 DEG C are spent the night, centrifugal 1000g, collect hyper-immune serum.
S6. a part of hyper-immune serum is sub-packed in the centrifuge tube of 0.5mL, be placed on-20 DEG C for subsequent use, remaining hyper-immune serum is put in-80 DEG C and saves backup.
Embodiment 4
Detect the foundation of the stop band restrain method of rabbit cysticercus pisiformis capsule humoral antibody:
(1) antigen the best wraps the determination by concentration and serum dilution
S1. wrapper sheet: by the order of 96 hole PVC reaction plates according to row × row=12 × 8, be that 1.0,2.0,3.0,4.0,5.0,6.0 μ g/mL carry out wrapper sheet by concentration successively, every two row press same concentration bag quilt, every hole 50 μ L, 4 DEG C are spent the night, and PBST washs 3 times, every minor tick 5min;
S2. close: close with the PBST confining liquid containing 5% skimmed milk power, every hole 200 μ L, 37 DEG C, close 2-4h, PBST and wash 3 times, every minor tick 5min;
S3. primary antibodie reaction: removing first row, all the other every holes first add 50 μ LPBS, then odd-numbered line first hole adds the diluted mouse positive serum of 100 μ L, and even number line first hole adds the diluted mouse negative serum of 100 μ L, and the first hole doubly starts according to 1:100, then in turn doubling dilution to octal, discard 50 μ L liquid, 37 DEG C, reaction 30min, PBST washs 3 times, every minor tick 5min;
S4. two anti-reflective are answered: according to the dilution ratio of 1:5000, and by the goat anti-mouse IgG of the PBST confining liquid dilution HRP mark containing 5% skimmed milk power, after mixing, then often hole adds 50 μ L, 37 DEG C, and reaction 30min, PBST wash 3 times, every minor tick 5min;
S5. substrate colour developing: with 1000 μ L sample loading guns respectively No. 3 liquid of draws equal amounts and No. 4 liquid mix with in reactive tank, every hole 50 μ L, reacts 5min;
S6. reading: add stop buffer rapidly after chromogenic reaction, every hole 50 μ L, puts into microplate reader immediately, carries out reading according to the program set.
S7. criterion: when OD value more close to 1.0 time, and measured value/feminine gender value > 2.1, the bag corresponding to the OD value of this hole is the best by concentration and serum dilution.
S8. result: application PVC-Dot-ELISA methods combining square formation titrimetry carries out the best determination of wrapping by concentration and serum dilution of antigen.Experimental result shows, and antigen the best bag is 5 μ g/mL by concentration, and best serum dilution is that 1:800(is in table 3).
The best antigen coated concentration of table 3 and best serum dilution measurement result
(2) determination of best wrapper sheet time:
The best bag determined according to (1) is by concentration wrapper sheet, 5 experimental group are set altogether, often group arranges 4 holes as parallel laboratory test (positive and each 4 holes of feminine gender), undertaken by condition and time according to different bags respectively: 4 DEG C spend the night, 37 DEG C of 1h, 37 DEG C of 2h, 37 DEG C of 3h, 37 DEG C of 4h, other experiment conditions are identical with the time, carry out the experiment of PVC-Dot-ELISA detection method, measure each group of each hole OD value at 450 nm.Criterion is: when P/N is maximum, and the corresponding time is best bag by the time.
Tested by PVC-Dot-ELISA, result show, antigen at 4 DEG C of bags spent the night by effect under condition best (see table 4 and Fig. 1).
Table 4 difference bag is by the reaction result of time
(3) determination of best off-period:
Wrapper sheet is carried out by concentration and best bag by the time according to (1) and (2) determined the best bag, then 4 experimental group are set, often group arranges 4 holes as parallel laboratory test (positive and each 4 holes of feminine gender), respectively according to closing following different off-period: 37 DEG C of 1h, 37 DEG C of 2h, 37 DEG C of 3h, 37 DEG C of 4h, other experiment conditions are identical with the time, carry out the experiment of PVC-Dot-ELISA detection method, measure each group of each hole OD value at 450 nm.Criterion is: when P/N is maximum, and the corresponding time is best bag by the time.
Tested by PVC-Dot-ELISA, result shows, and the best sealing condition of antigen is that 37 DEG C/3h(is shown in Fig. 2, table 5).
The reaction result of table 5 different off-period
The determination of (4) the two best dilution ratios resisted
Carried out wrapper sheet and close by concentration, best bag by time and best off-period according to (1), (2) and (3) determined the best bag, then 4 experimental group are set, often group arranges 4 holes as parallel laboratory test (positive and each 4 holes of feminine gender), dilutes respectively: 1:5000,1:10000,1:15000,1:20000 tetra-gradients according to following two different anti-dilution ratios.Other experiment conditions are identical with the time, carry out the experiment of PVC-Dot-ELISA detection method, measure each group of each hole OD value at 450 nm.Criterion is: when P/N is maximum, and the corresponding time is best bag by the time.
Tested by PVC-Dot-ELISA, result shows, and two anti-best dilution ratios are that 1:5000(is shown in Fig. 3, table 6)
The reaction result of the anti-different dilution ratio of table 6 two
The determination of (5) two anti-optimum reacting times
Tested by time, best off-period, best serum dilution and two anti-best dilution ratios by concentration, best bag according to (1), (2), (3) and (4) determined the best bag, then 3 experimental group are set, often group arranges 4 holes as parallel laboratory test (positive and each 4 holes of feminine gender), carries out respectively: 30min, 45min, 60min according to the following two different anti-reaction time.Other experiment conditions are identical with the time, carry out the experiment of PVC-Dot-ELISA detection method, measure each group of each hole OD value at 450 nm.Criterion is: when P/N is maximum, and the corresponding time is best bag by the time.
Tested by PVC-Dot-ELISA, result shows, and the reaction conditions of two anti-the bests is 37 DEG C, and reaction 30min(is shown in Fig. 4, table 7).
The reaction result of table 7 different two anti-action times
Step 5
Detect the specificity analyses of the stop band restrain method of rabbit cysticercus pisiformis capsule humoral antibody:
Stop band restrain diagnostic method is set up, for clinically for the quick diagnosis of rabbit cysticercus pisiformis disease by the optimum optimization reaction conditions of PVC-Dot-ELISA detection method set up.With the stop band restrain diagnostic method of invention, respectively with common multiple rabbit pest of rabbit clinically, Pasteurella, rabbit appendix B ly-mphocyte, rabbit coccidia and toxoplasmosis carry out serology survey, simultaneously with the rabbit anteserum of the white rabbit anteserum of artificial immunity cysticercus pisiformis capsule liquid eggs and natural infection cysticercus pisiformis for contrast.
S1. wrapper sheet: the best bag determined according to (1) and (2) is carried out wrapper sheet by concentration and time in vain with unpurified cysticercus pisiformis capsule liquid eggs, and PBST washs 3 times, every minor tick 5min;
S2. close: the best off-period determined according to (3), close with the PBST confining liquid containing 5% skimmed milk power, every hole 200 μ L, PBST washs 3 times, every minor tick 5min;
S3. add rabbit anteserum to be measured: every hole 50 μ L, the first hole is from 1:200, and doubling dilution is until octal in turn, discards 50 μ L liquid, 37 DEG C, and reaction 30min, PBST wash 3 times, every minor tick 5min;
S4. primary antibodie reaction: mouse positive serum (i.e. primary antibodie) optimum dilution degree determined according to (1), dilutes BABL/c mouse positive serum clearly, after mixing with PBS in reactive tank, every hole 50 μ L, 37 DEG C, reaction 30min, PBST washs 3 times, every minor tick 5min;
S5. two anti-reflective are answered: the anti-dilution ratio of the best two determined according to (4), by the goat anti-mouse IgG of the PBST confining liquid dilution HRP mark containing 5% skimmed milk power, after mixing, then every hole adds 50 μ L, the best two anti-reaction time determined according to 2.2.5 tests, PBST washs 3 times, every minor tick 5min;
S6. substrate colour developing: with 1000 μ L sample loading guns respectively No. 3 liquid of draws equal amounts and No. 4 liquid mix in reactive tank, every hole 50 μ L, reacts 5min;
S7. reading: add stop buffer rapidly after chromogenic reaction, every hole 50 μ L, puts into microplate reader immediately, carries out reading according to the program set.
Coating buffer described in this step is that 0.05mol/L carbonate bag is buffered liquid, and its compound method is as follows: take Na
2cO
31.5g, NaHCO32.93g, regulate pH to 9.6, adding distil water to 1000mL, 4 DEG C of preservations.
Substrate described in this step develops the color No. 3 liquid for selecting citric acid 3.15g, and it is containing a water of crystallization, MW:210.4; Anhydrous sodium acetate 6.97g, phenacetin 0.08g, urea peroxide 0.5g, deionized water 1000mL.Compound method: first citric acid and phenacetin are dissolved in 50mL deionized water, heating stirring and dissolving, is dissolved in anhydrous sodium acetate in 900mL deionized water simultaneously, then both mixed and be settled to 1000ml, add urea peroxide again to dissolve, be positioned in brown bottle, 4 DEG C of preservations.
Substrate described in this step No. 4 liquid that develop the color are TMB1.27g, methyl alcohol 500mL, glycerine 500mL.Compound method: TMB is put into clean beaker, adds methyl alcohol, heating for dissolving, seals with preservative film during heating for dissolving, prevents methyl alcohol from volatilizing, then adds glycerine, is positioned in brown bottle after mixing, 4 DEG C of preservations.
Adding rapidly stop buffer after chromogenic reaction described in this step is 2MH
2sO
4, i.e. concentrated sulphuric acid 22.2mL, deionized water 177.8mL, mixes, 4 DEG C of preservations.
S8. result: detect the rabbit anteserum suffering from Encephalitozoon disease, rabbit pest, pasteurellosis, toxoplasmosis and global-worm illness respectively with the stop band restrain diagnostic method that the determined optimum optimization reaction conditions of experiment is set up, two positive controls are set simultaneously, the rabbit anteserum that namely artificial immunity cysticercus pisiformis capsule liquid eggs is white and the rabbit anteserum of natural infection cysticercus pisiformis.Result shows, except two positive controls occur significantly blocking phenomenon, all the other five group reaction colour developing degree are basically identical, and these five groups of OD values are also very close, and two groups of positive controls have obvious graded, prove this stop band restrain diagnosis side and Encephalitozoonosis, rabbit pest, Rabbit cells line VX-2, Toxoplasma gondii disease and the equal no cross reaction of global-worm illness (see Fig. 5, table 8)
The specificity analyses result of table 8 stop band restrain
Artificial immunity (AI), natural infection (NI), rabbit appendix B ly-mphocyte (EC), rabbit pest (RVH), Rabbit cells line VX-2 (RP), Toxoplasma gondii disease (RT) and coccidiosis of rabbit (RC)
Step 6
Detect the stop band restrain method of rabbit cysticercus pisiformis capsule humoral antibody to the verification and measurement ratio of clinical sample:
At government official's warren random acquisition 100 parts of rabbit anteserums, and record the sample number of suffering from cysticercus pisiformis disease in these 100 increment product.The stop band restrain diagnostic method set up with this experiment detects every a rabbit anteserum sample, measuring the OD value in each group of each hole at 450 nm after terminating to stop, whether having blocking-up and OD450 whether to there is graded to judge that this sample is feminine gender or positive according to observing.Then the verification and measurement ratio of this stop band restrain diagnostic method is calculated in conjunction with the positive ratio of slaughtering inspection record.
With experiment determine stop band restrain diagnostic method random acquisition 100 rabbit anteserums that optimum optimization reaction conditions is set up, according to observing reaction solution result and whether OD value has graded, determine that having 33 samples is positive, and dissect clinically and confirm that the sample suffering from cysticercus pisiformis disease adds up to 36, therefore the verification and measurement ratio of this stop band restrain method is that 91.66%(is in table 9).Cut open inspection by butchering, the vesica of the cysticercus pisiformis in sick rabbit body, more than 5, all can detect, otherwise not easily detect.NF 3 routine sick rabbits, through observing careful inspection, in its body, the quantity of cysticercus pisiformis vesica is divided is separately 1 (2 example) and 3 (1 example).
Table 9 stop band restrain detects the verification and measurement ratio analysis of clinical sample
| Project | Positive rabbit anteserum sample number | Negative rabbit anteserum sample number | Total number of samples |
| Clinically cut open inspection result | 36 | 64 | 100 |
| This laboratory test results | 33 | 67 | 100 |
| Verification and measurement ratio % | 91.66 |
Above-described embodiment is only for clearly example being described, and the restriction not to embodiment.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here exhaustive without the need to also giving all embodiments, and thus the apparent change of extending out or variation be still within the protection domain of the invention.
Claims (7)
1. one kind is detected the stop band restrain method of rabbit cysticercus pisiformis capsule humoral antibody, it is characterized in that: it is by carrying out abstraction and purification in vain to rabbit cysticercus pisiformis capsule liquid eggs, and detection of antigenicity is carried out to the albumen of each main molecules amount, again with the purifying protein immune mouse that antigenicity is good, prepare and highly exempt from antiserum, and then set up the stop band restrain method detecting rabbit cysticercus pisiformis disease.
2. a kind of stop band restrain method detecting rabbit cysticercus pisiformis capsule humoral antibody according to claim 1, it is characterized in that: the purifying capsule liquid eggs that described antigenicity is good is molecular weight is in vain that 50-100kDa capsule liquid eggs is white, as the Healthy female BALB/c mouse in 56 week ages of antigen immune, after 4 immunity, when serum titer reaches more than 1:6400 times, blood sampling and separation of serum, the height being the stop band restrain method for detecting rabbit cysticercus pisiformis capsule humoral antibody exempts from antiserum.
3. according to claim 1, a kind of stop band restrain method detecting rabbit cysticercus pisiformis capsule humoral antibody, is characterized in that described method specifically comprises the steps:
(1) wrapper sheet: the 50-100kDa capsule liquid eggs of purifying is diluted to 5 μ g/mL in vain with coating buffer, 100 Bao Bei96 hole, μ L/ hole ELISA Plate, 4 DEG C are spent the night; PBST washs 3 times, every minor tick 5min;
(2) close: close with the PBST confining liquid containing 5% skimmed milk power, every hole 200 μ L, PBST washs 3 times, every minor tick 5min;
(3) add rabbit anteserum to be measured: every hole 50 μ L, the first hole is from 1:200, and doubling dilution is until octal in turn, and discard 50 μ L liquid, arrange negative serum and positive serum controls simultaneously, hatch 30min for 37 DEG C, PBST washs 3 times, every minor tick 5min;
(4) primary antibodie reaction: BABL/c mouse positive serum is carried out 1:800 dilution in reactive tank with PBS, after mixing, every hole 50 μ L, hatch 30min for 37 DEG C, PBST washs 3 times, every minor tick 5min;
(5) two anti-reflective are answered: press with the PBST confining liquid containing 5% skimmed milk power the goat anti-mouse IgG that 1:5000 dilutes HRP mark, and after mixing, then often hole adds 50 μ L, and hatch 30min for 37 DEG C, PBST washs 3 times, every minor tick 5min;
(6) substrate colour developing: with 1000 μ L sample loading guns respectively No. 3 liquid of draws equal amounts and No. 4 liquid mix in reactive tank, every hole 50 μ L, hatches 5min;
(7) detecting: add stop buffer rapidly after chromogenic reaction, every hole 50 μ L, puts into microplate reader immediately, measures the OD value in each group of each hole at 450 nm, whether having blocking-up and OD according to observing
450whether there is graded and carry out judged result, have the sample of graded for positive, do not have the sample of graded to be then negative.
4. a kind of stop band restrain method detecting rabbit cysticercus pisiformis capsule humoral antibody according to claim 3, 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 Na2CO31.5g, NaHCO32.93g, regulate pH to 9.6, adding distil water to 1000mL, 4 DEG C of preservations.
5. a kind of stop band restrain method detecting rabbit cysticercus pisiformis capsule humoral antibody according to claim 3, is characterized in that: described substrate develops the color No. 3 liquid for selecting citric acid 3.15g, and it is containing a water of crystallization, MW:210.4; Anhydrous sodium acetate 6.97g, phenacetin 0.08g, urea peroxide 0.5g, deionized water 1000mL; Compound method: first citric acid and phenacetin are dissolved in 50mL deionized water, heating stirring and dissolving, is dissolved in anhydrous sodium acetate in 900mL deionized water simultaneously, then both mixed and be settled to 1000ml, add urea peroxide again to dissolve, be positioned in brown bottle, 4 DEG C of preservations.
6. a kind of stop band restrain method detecting rabbit cysticercus pisiformis capsule humoral antibody according to claim 3, is characterized in that: described substrate No. 4 liquid that develop the color are TMB1.27g, methyl alcohol 500mL, glycerine 500mL; Compound method: TMB is put into clean beaker, adds methyl alcohol, heating for dissolving, seals with preservative film during heating for dissolving, prevents methyl alcohol from volatilizing, then adds glycerine, is positioned in brown bottle after mixing, 4 DEG C of preservations.
7. a kind of stop band restrain method detecting rabbit cysticercus pisiformis capsule humoral antibody according to claim 3, is characterized in that: adding rapidly stop buffer after described chromogenic reaction is 2MH
2sO
4, i.e. concentrated sulphuric acid 22.2mL, deionized water 177.8mL, mixes, 4 DEG C of preservations.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113862347A (en) * | 2021-09-29 | 2021-12-31 | 中国农业科学院兰州兽医研究所 | Serum circulating miRNA marker related to cysticercosis pisiformis infection and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009099394A1 (en) * | 2008-02-05 | 2009-08-13 | Temasek Life Sciences Laboratory Limited | Binding protein and epitope-blocking elisa for the universal detection of h5-subtype influenza viruses |
| CN103439508A (en) * | 2013-08-25 | 2013-12-11 | 河南科技学院 | Indirect competitive enzyme linked immunosorbent assay kit for detecting chromium ions as well as preparation and detection methods thereof |
| CN103529210A (en) * | 2013-09-27 | 2014-01-22 | 河南科技学院 | Enzyme linked immunosorbent assay kit for detecting content of heavy metal zinc ions in sample |
| US20140099338A1 (en) * | 2008-07-31 | 2014-04-10 | Mao-Xing Biological Technology Co., Ltd. | Yeast expressed classical swine fever virus glycoprotein e2 and use thereof |
| CN104807992A (en) * | 2015-04-25 | 2015-07-29 | 东北农业大学 | Blocking ELISA method for detecting antibody of NetB toxin of clostridium perfringens |
-
2015
- 2015-10-27 CN CN201510704653.2A patent/CN105388295A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009099394A1 (en) * | 2008-02-05 | 2009-08-13 | Temasek Life Sciences Laboratory Limited | Binding protein and epitope-blocking elisa for the universal detection of h5-subtype influenza viruses |
| US20140099338A1 (en) * | 2008-07-31 | 2014-04-10 | Mao-Xing Biological Technology Co., Ltd. | Yeast expressed classical swine fever virus glycoprotein e2 and use thereof |
| CN103439508A (en) * | 2013-08-25 | 2013-12-11 | 河南科技学院 | Indirect competitive enzyme linked immunosorbent assay kit for detecting chromium ions as well as preparation and detection methods thereof |
| CN103529210A (en) * | 2013-09-27 | 2014-01-22 | 河南科技学院 | Enzyme linked immunosorbent assay kit for detecting content of heavy metal zinc ions in sample |
| CN104807992A (en) * | 2015-04-25 | 2015-07-29 | 东北农业大学 | Blocking ELISA method for detecting antibody of NetB toxin of clostridium perfringens |
Non-Patent Citations (1)
| Title |
|---|
| 孙玉倩: "用豆状囊尾蚴囊液免疫动物的形态观察及快速诊断方法的研究", 《中国优秀硕士学位论文全文数据库（电子期刊） 农业科技辑》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113862347A (en) * | 2021-09-29 | 2021-12-31 | 中国农业科学院兰州兽医研究所 | Serum circulating miRNA marker related to cysticercosis pisiformis infection and application thereof |
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