CN103217524A - Cystic echinococcosis cyst fluid degreasing antigen for detecting alveolar echinococcosis antibody, and preparation method thereof - Google Patents
Cystic echinococcosis cyst fluid degreasing antigen for detecting alveolar echinococcosis antibody, and preparation method thereof Download PDFInfo
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- CN103217524A CN103217524A CN2012105826860A CN201210582686A CN103217524A CN 103217524 A CN103217524 A CN 103217524A CN 2012105826860 A CN2012105826860 A CN 2012105826860A CN 201210582686 A CN201210582686 A CN 201210582686A CN 103217524 A CN103217524 A CN 103217524A
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Abstract
The invention relates to the technical field of human and animal alveolar echinococcosis (AE) antibody detections, specifically to cystic echinococcosis (CE) cyst fluid degreasing antigen for detecting AE antibody, and a preparation method thereof. According to the present invention, the CE cyst fluid degreasing antigen is used for detecting AE antibody by using an enzyme-linked immunosorbent test double antigen sandwich method so as to effectively increase quality of the detection reagent, and provide characteristics of strong specificity, high sensitivity, stable performance and wide uses; the CE cyst fluid degreasing antigen can be used for detecting human and animal AE antibodies so as to conveniently compare antibody contents of different animal varieties; and according to the immunological reaction principle, the CE cyst fluid degreasing antigen further can be used for preparing colloidal gold immunochromatography reagents and other rapid detection reagents for detecting AE antibodies so as to create favorable conditions for echinococcosis identification diagnosis, prevalence surveys and epidemic situation surveillance works.
Description
Technical field
The present invention relates to humans and animals alveolar echinococcosis antibody test technical field, is that a kind of cystic echinococcosis capsule liquid that detects alveolar echinococcosis antibody that is applicable to takes off ester antigen and preparation method thereof.
Background technology
Alveolar echinococcosis (Alveolar echinococcosis, AE) be by Echinococcus multilocularis (Echinococcus multilocularis, Em) the parasitogenic Amphixenosis of larva, early stage non-evident sympton, how focus spreads during discovery, focus is gradual infiltration and increases, and conventional diagnostic method such as ultrasound wave and CT examination is difficult for and the tumor focus difference, and serology detects and helps making a definite diagnosis of AE.Detect the important indicator that echinococcosis antibody is the diagnosis echinococcosis, antigen is the key reagents that influences detection specificity and susceptibility.Saltout, method such as ion chromatography, affinity chromatography, preparation electrophoresis, materials such as Echinococcus hydatid cyst protoscolex, cyst wall, capsule liquid all once were used to extract the antigen with purification assays echinococcosis antibody; In recent years, technique for gene engineering and artificial synthetic polypeptide are also used in the antigen in preparation, and the antigen type that once is used to detect the research of echinococcosis antibody is various, all fails to significantly improve specificity and the susceptibility that detects echinococcosis antibody.Cystic echinococcosis (Cystic echinococcosis, CE) be the parasitogenic Amphixenosis of larva by Echinococcus granulosus (Echinococcus granulosur.Eg), detect the method for CE antibody, positive reaction also appears in AE antibody, there is same or similar reactive antigenic component in the protoscolex of prompting Em, Eg or the capsule liquid, has the antigen that is applicable to detection alveolar echinococcosis antibody in the CE capsule liquid.Conventional method is extracted the CE cyst fluid antigen, program complexity not only, and harvest yield is very few, and with multiple parasite cross reaction is arranged.A kind of enzyme mark of enzyme linked immunosorbent assay indirect method second antibody can only detect the antibody of a kind of animal, detect multiple animal echinococcosis antibody, the enzyme mark second antibody of the multiple animal blood serum immunoglobulin (Ig) of essential preparation, not only workload is big, and be subjected to the tire influence of difference of enzyme mark second antibody, be difficult to animal's antibody content more not of the same race, dual-antigen sandwich method detects antibody, enzyme-labelled antigen substitutes second antibody, can detect the antibody of multiple animal, be Amphixenosis investigation and the epidemic monitoring condition that facilitates.
Summary of the invention
The invention provides a kind of cystic echinococcosis capsule liquid that detects alveolar echinococcosis antibody that is applicable to and take off ester antigen and preparation method thereof, overcome the deficiency of above-mentioned prior art, it can improve specificity, susceptibility and the stability that detects alveolar echinococcosis antibody, and purposes is wide.
One of technical scheme of the present invention realizes by following measure: a kind of cystic echinococcosis capsule liquid that detects alveolar echinococcosis antibody that is applicable to takes off ester antigen, obtains by following step:
The first step, gather sheep cystic echinococcosis packing liquid, in packing liquid, add the solid ammonium sulfate dissolving, make the concentration of ammonium sulfate in the packing liquid 30% of the concentration that reaches capacity, be adjusted to pH 7.2 with NaOH or ammonium hydroxide, add chloroform or sherwood oil by 30% to 50% the amount of regulating liquid volume behind the pH then, thermal agitation becomes suspension, suspension leaves standstill to chloroform or sherwood oil and the layering of packing liquid, thermal agitation and standing demix are 2 times again, behind the centrifugal 20min of 4000g, abandon precipitation and chloroform or sherwood oil then, the packing liquid after the collection and treatment;
Second step added the solid ammonium sulfate dissolving in the packing liquid after the first step is handled, make in the packing liquid after the processing ammonium sulfate concentrations 50% to 70% of the concentration that reaches capacity, and was adjusted to pH 7.2 with NaOH or ammonium hydroxide, was 4 in temperature
0C to 10
0Place 4h in the refrigerator of C, collecting precipitation thing behind the centrifugal 10min of 4000g is abandoned supernatant then;
The 3rd step, the sediment that second step was collected joins in the ammonium sulfate of 20 times of sediment volumes, the pH of ammonium sulfate is 7.2, the concentration of ammonium sulfate is 50% to 70% of saturation concentration, vibration makes it become suspension, behind the centrifugal 10min of 4000g, the collecting precipitation thing is abandoned supernatant then;
The 4th step, the sediment that the 3rd step was collected joins in the ammonium sulfate of 20 times of sediment volumes, the pH of ammonium sulfate is 7.2, the concentration of ammonium sulfate is 50% to 70% of saturation concentration, vibration makes it become suspension, collecting precipitation behind the centrifugal 10min of 4000g then, abandon supernatant, after the distilled water that adds 10 times of sediment volumes in the sediment of collecting, vibration make the sediment dissolving, add chloroform or sherwood oil with sediment dissolving back solution equal volume amounts, thermal agitation becomes suspension, leave standstill to chloroform or sherwood oil and solution layering, thermal agitation and standing demix are 2 times again, collect aqueous solution;
In the 5th step, the aqueous solution that the 4th step was collected is packed in the bag filter, and bag filter is put into physiological saline, and the volume of physiological saline is 20 times of aqueous solution volume in the bag filter, is 4 in temperature
0C to 10
012h to 24h dialyses in the refrigerator of C, change physiological saline therebetween 3 times, each physiological saline volume of changing is 20 times of aqueous solution volumes in the bag filter, after dialysis is finished, the centrifugal 20min of solution 4000g in the bag filter, abandon precipitation, collect supernatant, supernatant is and is applicable to that the cystic echinococcosis capsule liquid that detects alveolar echinococcosis antibody takes off ester antigen.
Two of technical scheme of the present invention realizes by following measure: a kind of preparation method who is applicable to that the cystic echinococcosis capsule liquid that detects alveolar echinococcosis antibody takes off ester antigen, undertaken by following step:
The first step, gather sheep cystic echinococcosis packing liquid, in packing liquid, add the solid ammonium sulfate dissolving, make the concentration of ammonium sulfate in the packing liquid 30% of the concentration that reaches capacity, be adjusted to pH 7.2 with NaOH or ammonium hydroxide, add chloroform or sherwood oil by 30% to 50% the amount of regulating liquid volume behind the pH then, thermal agitation becomes suspension, suspension leaves standstill to chloroform or sherwood oil and the layering of packing liquid, thermal agitation and standing demix are 2 times again, behind the centrifugal 20min of 4000g, abandon precipitation and chloroform or sherwood oil then, the packing liquid after the collection and treatment;
Second step added the solid ammonium sulfate dissolving in the packing liquid after the first step is handled, make in the packing liquid after the processing ammonium sulfate concentrations 50% to 70% of the concentration that reaches capacity, and was adjusted to pH 7.2 with NaOH or ammonium hydroxide, was 4 in temperature
0C to 10
0Place 4h in the refrigerator of C, collecting precipitation thing behind the centrifugal 10min of 4000g is abandoned supernatant then;
The 3rd step, the sediment that second step was collected joins in the ammonium sulfate of 20 times of sediment volumes, the pH of ammonium sulfate is 7.2, the concentration of ammonium sulfate is 50% to 70% of saturation concentration, vibration makes it become suspension, behind the centrifugal 10min of 4000g, the collecting precipitation thing is abandoned supernatant then;
The 4th step, the sediment that the 3rd step was collected joins in the ammonium sulfate of 20 times of sediment volumes, the pH of ammonium sulfate is 7.2, the concentration of ammonium sulfate is 50% to 70% of saturation concentration, vibration makes it become suspension, collecting precipitation behind the centrifugal 10min of 4000g then, abandon supernatant, after the distilled water that adds 10 times of sediment volumes in the sediment of collecting, vibration make the sediment dissolving, add chloroform or sherwood oil with sediment dissolving back solution equal volume amounts, thermal agitation becomes suspension, leave standstill to chloroform or sherwood oil and solution layering, thermal agitation and standing demix are 2 times again, collect aqueous solution;
In the 5th step, the aqueous solution that the 4th step was collected is packed in the bag filter, and bag filter is put into physiological saline, and the volume of physiological saline is 20 times of aqueous solution volume in the bag filter, is 4 in temperature
0C to 10
012h to 24h dialyses in the refrigerator of C, change physiological saline therebetween 3 times, each physiological saline volume of changing is 20 times of aqueous solution volumes in the bag filter, after dialysis is finished, the centrifugal 20min of solution 4000g in the bag filter, abandon precipitation, collect supernatant, supernatant is and is applicable to that the cystic echinococcosis capsule liquid that detects alveolar echinococcosis antibody takes off ester antigen.
The cystic echinococcosis capsule liquid that is applicable to detection alveolar echinococcosis antibody of gained of the present invention takes off ester antigen and is used for enzyme linked immunosorbent assay dual-antigen sandwich method detection alveolar echinococcosis antibody, can effectively improve the specificity and the susceptibility of detectable, have high specificity, susceptibility height, the stable and wide characteristics of purposes, can detect people and various animal alveolar echinococcosis antibody, be applicable to animal's antibody content more not of the same race; According to the immunological response principle, the cystic echinococcosis capsule liquid that detects alveolar echinococcosis antibody that is applicable to of gained of the present invention takes off ester antigen, also can be used for preparing the reagent for quickly examining of multiple detection alveolar echinococcosis antibody such as colloidal gold immunochromatographimethod reagent, for convenience has been created in echinococcosis antidiastole, prevalence study and epidemic monitoring work.
Embodiment
The present invention is not subjected to the restriction of following embodiment, can determine concrete embodiment according to technical scheme of the present invention and actual conditions.
Embodiment 1, and a kind of cystic echinococcosis capsule liquid that detects alveolar echinococcosis antibody that is applicable to takes off ester antigen, obtains by following step:
The first step, gather sheep cystic echinococcosis packing liquid, in packing liquid, add the solid ammonium sulfate dissolving, make the concentration of ammonium sulfate in the packing liquid 30% of the concentration that reaches capacity, be adjusted to pH 7.2 with NaOH or ammonium hydroxide, add chloroform or sherwood oil by 30% to 50% the amount of regulating liquid volume behind the pH then, thermal agitation becomes suspension, suspension leaves standstill to chloroform or sherwood oil and the layering of packing liquid, thermal agitation and standing demix are 2 times again, behind the centrifugal 20min of 4000g, abandon precipitation and chloroform or sherwood oil then, the packing liquid after the collection and treatment;
Second step added the solid ammonium sulfate dissolving in the packing liquid after the first step is handled, make in the packing liquid after the processing ammonium sulfate concentrations 50% to 70% of the concentration that reaches capacity, and was adjusted to pH 7.2 with NaOH or ammonium hydroxide, was 4 in temperature
0C to 10
0Place 4h in the refrigerator of C, collecting precipitation thing behind the centrifugal 10min of 4000g is abandoned supernatant then;
The 3rd step, the sediment that second step was collected joins in the ammonium sulfate of 20 times of sediment volumes, the pH of ammonium sulfate is 7.2, the concentration of ammonium sulfate is 50% to 70% of saturation concentration, vibration makes it become suspension, behind the centrifugal 10min of 4000g, the collecting precipitation thing is abandoned supernatant then;
The 4th step, the sediment that the 3rd step was collected joins in the ammonium sulfate of 20 times of sediment volumes, the pH of ammonium sulfate is 7.2, the concentration of ammonium sulfate is 50% to 70% of saturation concentration, vibration makes it become suspension, collecting precipitation behind the centrifugal 10min of 4000g then, abandon supernatant, after the distilled water that adds 10 times of sediment volumes in the sediment of collecting, vibration make the sediment dissolving, add chloroform or sherwood oil with sediment dissolving back solution equal volume amounts, thermal agitation becomes suspension, leave standstill to chloroform or sherwood oil and solution layering, thermal agitation and standing demix are 2 times again, collect aqueous solution;
In the 5th step, the aqueous solution that the 4th step was collected is packed in the bag filter, and bag filter is put into physiological saline, and the volume of physiological saline is 20 times of aqueous solution volume in the bag filter, is 4 in temperature
0C to 10
012h to 24h dialyses in the refrigerator of C, change physiological saline therebetween 3 times, each physiological saline volume of changing is 20 times of aqueous solution volumes in the bag filter, after dialysis is finished, the centrifugal 20min of solution 4000g in the bag filter, abandon precipitation, collect supernatant, supernatant is and is applicable to that the cystic echinococcosis capsule liquid that detects alveolar echinococcosis antibody takes off ester antigen.The cystic echinococcosis capsule liquid that detects alveolar echinococcosis antibody being applicable to of gained of the present invention takes off in the ester antigen and adds thimerosal, makes thimerosal be applicable to that the mass percent that the cystic echinococcosis capsule liquid that detects alveolar echinococcosis antibody takes off in the ester antigen is 0.1% ,-20
0C preserves standby.
Embodiment 2, and the cystic echinococcosis capsule liquid that detects alveolar echinococcosis antibody that is applicable to of the foregoing description 1 gained takes off the application of ester antigen in the alveolar echinococcosis antibody test; Its application is as follows:
One, enzyme linked immunological sandwich method kit of detection alveolar echinococcosis antibody and preparation method thereof
Detect the enzyme linked immunological sandwich method kit of alveolar echinococcosis antibody, comprise specification be the cystic echinococcosis capsule liquid of * 12 of 8 pipes take off 1 of ester antigen coated microplate, horseradish peroxidase-labeled cystic echinococcosis capsule liquid take off ester antigen 1 0ml, sample diluent 10ml, 20 times of concentrated washing fluid 50ml, alveolar echinococcosis antibody positive contrast liquid 3ml, alveolar echinococcosis negative antibodies contrast liquid 3ml, suppress with cystic echinococcosis capsule liquid take off ester antigen 6ml, colour developing liquid A liquid 6ml, colour developing liquid B liquid 6ml, stop buffer 6ml develops the color.
Cystic echinococcosis capsule liquid takes off the ester antigen coated microplate and obtains by following step:
Bag reagent, coating buffer: at 0.05mol/L pH is to add gained of the present invention in 6.4 to 9.6 phosphate or the carbonate buffer solution to be applicable to that the cystic echinococcosis capsule liquid that detects alveolar echinococcosis antibody takes off ester antigen and obtains coating buffer, and gained of the present invention is applicable to that it is 0.1 μ g/m1 to 1 μ g/m1 that the cystic echinococcosis capsule liquid that detects alveolar echinococcosis antibody takes off the concentration of ester antigen in coating buffer;
Saturated coating buffer: newborn calf serum or bovine serum albumin(BSA) are joined 0.05mol/L pH obtain saturated coating buffer in 6.4 to 9.6 phosphate or the carbonate buffer solution, the percent by volume of newborn calf serum in saturated coating buffer be 2% or the mass percent of bovine serum albumin(BSA) in saturated coating buffer be 0.05%;
Bag is by reaction plate: adding coating buffer 100 μ 1 in every pipe of reaction plate, is 28 in temperature
0C to 37
0C places 3h to 4h down, adds saturated coating buffer 100 μ 1 then in every pipe of reaction plate, is 28 in temperature
0C to 37
0C places 3h down, get rid of the liquid in pipe of abandoning reaction plate, remove the globule in the pipe with behind the distilled water flushing reaction plate 3 times at the thieving paper arsis, 20 times of concentrated washing fluids are obtained embathing liquid with behind 20 times of the distilled water dilutings, embathe contain in the liquid newborn calf serum volume ratio be 2% or the bovine serum albumin(BSA) mass percent be 0.05%, in every pipe of reaction plate, add then and embathe liquid 200 μ 1 and embathe, 28
0C to 37
0Place 3h under the temperature of C, get rid of the liquid in pipe of abandoning reaction plate, use distilled water flushing reaction plate 5 times, firmly patting reaction plate no visible globule inside and outside the pipe of reaction plate on the thieving paper; It is 37 that reaction plate after handling is uprightly put into temperature
OThe incubator of C, the every 30min air blast or ventilation l time of opening the door are taken out behind the dry 8h to 12h, obtain cystic echinococcosis capsule liquid and take off ester antigen coated microplate (abbreviation: the antigen plate), seal in the polybag of packing into and preserve.
Horseradish peroxidase-labeled cystic echinococcosis capsule liquid takes off ester antigen and obtains by following step:
The first step, activation horseradish peroxidase: take by weighing horseradish peroxidase (RZ 〉=3.0) the 5mg test tube of packing into, add distilled water 1m1 and make the horseradish peroxidase dissolving in test tube, the freshly prepared mass percent of adding is 1.3% sodium periodate aqueous solution 0.5m1 mixing in test tube, 4
0C to 10
0Place 30 min under the temperature of C, in test tube, add percent by volume again and be 1% glycol water 0.5m1, in room temperature 20
0C to 37
0C places 30min, the horseradish peroxidase that obtains activating;
Second step, to contain 5mg/m1 gained of the present invention and be applicable to that the cystic echinococcosis capsule liquid that detects alveolar echinococcosis antibody takes off the horseradish peroxidase mixing of the solution 1m1 of ester antigen and the activation that the first step obtains, the bag filter of packing into is that 9.6 carbonate buffer solution 200m1 is 4 in temperature with 0.05mol/L pH
0C to 10
0C is dialysis 12h to 16h down, and dialysis back freshly prepared mass percent of adding in the solution of bag filter is 0.5% sodium borohydride solution 0.2m1, is 4 in temperature
0C to 10
0Place 2h in the refrigerator of C, with the 0.01mol/L pH of 20,0m1 7.2 phosphate buffer dialysis 8h to 12h, change phosphate buffer therebetween 3 times, each 200ml, the centrifugal 10min of solution 4000g in the bag filter of dialysis back gets supernatant, and supernatant is horseradish peroxidase-labeled cystic echinococcosis capsule liquid and takes off ester antigen (abbreviation: enzyme-labelled antigen);
The 3rd step, measuring horseradish peroxidase-labeled cystic echinococcosis capsule liquid takes off ester antigen and use concentration: with the 0.1mol/L pH that contains volume ratio 2% calf serum and 2% glycerine is that 7.2 phosphate buffer dilution horseradish peroxidase-labeled cystic echinococcosis capsule liquid takes off ester antigen, by the enzyme linked immunological sandwich method procedure operation that detects alveolar echinococcosis antibody, detect alveolar echinococcosis antibody positive contrast OD＞2.0, the horseradish peroxidase-labeled cystic echinococcosis capsule liquid of negative control OD＜0.1 takes off the ester antigen concentration for using concentration, and it is that 7.2 phosphate buffer is diluted to and uses the concentration packing with the 0.1mol/L pH that contains volume ratio 2% calf serum and 50% glycerine that horseradish peroxidase-labeled cystic echinococcosis capsule liquid is taken off ester antigen.
Sample diluent: the 0.01mol/L pH that contains percent by volume and be 0.05% Tween-20, percent by volume and be 2% newborn calf serum, mass percent and be 0.1% thimerosal is 7.2 phosphate buffer.
20 times of concentrated washing fluids: the 0.1mol/L pH that contains percent by volume and be 1% Tween-20, mass percent and be 17% sodium chloride is 7.2 phosphate buffer.
Alveolar echinococcosis antibody positive contrast liquid: the 0.01 mol/L pH that contains percent by volume and be 2% newborn calf serum, mass percent and be 0.05% thimerosal, 2 μ g/ml alveolar echinococcosis antibody is 7.2 phosphate buffer.
Alveolar echinococcosis negative antibody contrast liquid: the 0.01 mol/L pH that contains percent by volume and be 2% newborn calf serum, mass percent and be 0.05% thimerosal is 7.2 phosphate buffers.
Suppress to take off ester antigen: contain percent by volume and be 0.05% Tween-20, percent by volume and be 2% newborn calf serum, mass percent and be 0.01mol/L pH that 0.05% thimerosal, 5 μ g/ml gained of the present invention be applicable to that the cystic echinococcosis capsule liquid of detection alveolar echinococcosis antibody takes off ester antigen and be 7.2 phosphate buffer with cystic echinococcosis capsule liquid.
TMB colour developing liquid A liquid: with 0.2 mol/L NaH
2PO
4Mix with 0.1 mol/L citric acid equal-volume, by mix the back liquor capacity 0.05% to add concentration of volume percent be 30% hydrogen peroxide, be TMB colour developing liquid A liquid.
TMB colour developing liquid B liquid: absolute ethyl alcohol 10ml and N-N-methyl-2-2-pyrrolidone N-10ml are mixed, add 3.3-5.5-tetramethyl benzidine (TMB) 50mg, dissolve fully to the 3.3-5.5-tetramethyl benzidine, adding distil water is to 70ml then, the citric acid 10ml that adds 0.1mol/L is TMB colour developing liquid B liquid after the EDTA 1ml of adding 0.1mol/L mixes.
The colour developing stop buffer: percent by volume is 10% sulfuric acid solution.
Mentioned reagent and antigen plate are packed into and are detected the enzyme-linked immunosorbent assay sandwich kit of alveolar echinococcosis antibody, 4
0C to 8
0C preserves, the term of validity 1 year.
Two, the enzyme linked immunological sandwich method detects echinococcosis antibody primary dcreening operation (qualitative) test
1. add the sample dilution: according to sample and quantity positive, negative control, assembling cystic echinococcosis capsule liquid take off the ester antigen coated microplate (hereinafter to be referred as: the antigen plate), the every pipe of antigen plate adds sample dilution 50 μ l.
2. add sample: in every pipe of antigen plate, add 1 part of sample, 50 μ l by the accession designation number position, place in wet box or the polybag 37
0C leaves standstill reaction 90min.
3. flushing antigen plate: 20 times of concentrated washing fluids with 20 times of distilled water dilutings after, wash the antigen plate with washing the plate machine, wash repeatedly 5 times; The mouth of pipe is downward, the globule inside and outside the thieving paper arsis removes antigen plate pipe.
4. add horseradish peroxidase-labeled cystic echinococcosis capsule liquid take off ester antigen (hereinafter to be referred as: enzyme-labelled antigen): enzyme-added mark antigen 1 00 μ l in every pipe of antigen plate, add a cover, place in wet box or the polybag; 37
0C reaction time 45min, 20 times of concentrated washing fluids with 20 times of distilled water dilutings after, wash the antigen plate with washing the plate machine, wash repeatedly 5 times; The mouth of pipe is downward, the globule inside and outside thieving paper, filter paper arsis remove antigen plate pipe.
5. colour developing: in every pipe of antigen plate, add A liquid and each 50 μ l of B liquid of TMB colour developing liquid, 37
0C chromogenic reaction 15min, positive reaction is blue look; Negative reaction is colourless or develops the color very light.
6. color development stopping: add stop buffer 50 μ l in every pipe of antigen plate, positive colour developing liquid becomes yellow by blue look, and feminine gender still is colourless or color is very light.
7. observational record test findings: add behind the stop buffer observed result in the 10min.
Range estimation: flat hold the antigen plate, apart from white background 10 cm to 15cm, from directly over observe downwards;
Tintmeter colorimetric: TMB colour developing liquid (A liquid and B liquid) 450nm and 630nm dual wavelength colorimetric, feminine gender: (-) is colourless or color is very light, OD＜0.20; Positive: (+) color obviously as seen, OD 0.20 to 0.39; (++) is light yellow, and OD 0.4 to 0.99; (+++) identical with the positive control color, OD 1.0 to 2.0; (++ ++) buff is darker than positive control, OD＞2.0.
Three, demonstration test
1. detection reaction titre test:
(1) according to positive sample number and the primary dcreening operation colour developing depth, the positive reaction sample is arranged respectively by (+), (++), (+++), (++ ++); (+) with 2 tubules, and (++) with 4 tubules, and (+++) with 6 to 8 tubules, (++ ++) with 8 to 12 tubules, in every pipe of antigen plate, add sample dilution 100u1.
(2) add 1 part of sample 100u1 to be checked in the first pipe of every row of antigen plate, pressure-vaccum 3 times moves in 100u1 to the 2 pipes, and so continuous 2 times are diluted to the most last pipe; The positive control sample dilutes synchronously; Add a cover, place in wet box or the polybag, 37
0C reaction time 90min; For measuring the titre terminal point, can detect titre again with after 16 times to 64 times of the sample dilutions of (++ ++).
(3) flushing antigen plate: 20 times of concentrated washing fluids with 20 times of distilled water dilutings after, wash the antigen plate with washing the plate machine, wash repeatedly 5 times; The mouth of pipe is downward, the globule inside and outside thieving paper, filter paper arsis remove antigen plate pipe.
(4) antigen of labelling: in every pipe of antigen plate, add enzyme-labelled antigen 100 μ l, add a cover, place in wet box or the polybag; 37
0C reaction time 45min, 20 times of concentrated washing fluids with 20 times of distilled water dilutings after, wash the antigen plate with washing the plate machine, wash repeatedly 5 times; The mouth of pipe is downward, the globule inside and outside thieving paper, filter paper arsis remove antigen plate pipe.
(5) colour developing: in every pipe of antigen plate, add A liquid and each 50 μ l of B liquid of TMB colour developing liquid, 37
0C chromogenic reaction 15min, positive reaction is blue look; Negative reaction is colourless or develops the color very light.
(6) color development stopping: add stop buffer 50 μ l in every pipe of antigen plate, colour developing liquid becomes yellow by blue look, and feminine gender still is colourless or color is very light.
(7) observational record test findings: add behind the stop buffer observed result in the 10min.
Range estimation: flat hold the antigen plate, apart from white background 10 Cm to 15Cm, from directly over observe downwards;
Tintmeter colorimetric: TMB colour developing liquid (A liquid and B liquid) 450nm and 630nm dual wavelength colorimetric, feminine gender: (-) is colourless or color is very light, OD＜0.20; Positive: (+) color obviously as seen, OD 0.20 to 0.39; (++) is light yellow, and OD 0.4 to 0.99; (+++) identical with the positive control color, OD 1.0 to 2.0; (++ ++) buff is darker than positive control, OD＞2.0.
2. inhibition test:
(1) test of inhibition test and detection reaction titre is carried out synchronously.Inhibition test and detection reaction titre test difference are for replacing sample diluent dilution sample with inhibition with antigen liquid, and every pipe adds and suppresses to use antigen liquid 100u1.
(2) add 1 part of sample 100u1 to be checked in the first pipe of every row of antigen plate, pressure-vaccum 3 times moves in 100u1 to the 2 pipes, and so continuous 2 times are diluted to the most last pipe; The positive control sample dilutes synchronously; Add a cover, place wet box or polybag internal reaction, 37
0C reaction time 90min.For measuring the titre terminal point, can detect titre again with after 16 times to 64 times of the sample dilutions of (++ ++).
(3) flushing antigen plate: 20 times of concentrated washing fluids with 20 times of distilled water dilutings after, wash the antigen plate with washing the plate machine, wash repeatedly 5 times; The mouth of pipe is downward, the globule inside and outside thieving paper, filter paper arsis remove antigen plate pipe.
(4) add enzyme-labelled antigen: in every pipe of antigen plate, add enzyme-labelled antigen 100 μ l, add a cover, place in wet box or the polybag; 37
0C reaction time 45min, 20 times of concentrated washing fluids with 20 times of distilled water dilutings after, wash the antigen plate with washing the plate machine, wash repeatedly 5 times; The mouth of pipe is downward, the globule inside and outside thieving paper, filter paper arsis remove antigen plate pipe.
(5) colour developing: in every pipe of antigen plate, add A liquid and each 50 μ l of B liquid of TMB colour developing liquid, 37
0C chromogenic reaction 15min, positive reaction is blue look; Negative reaction is colourless or develops the color very light.
(6) color development stopping: add stop buffer 50 μ l in every pipe of antigen plate, colour developing liquid becomes yellow by blue look, and feminine gender still is colourless or color is very light.
(7) observational record test findings: add behind the stop buffer observed result in the 10min.
Range estimation: flat hold the antigen plate, apart from white background 10 cm to 15cm, from directly over observe downwards;
Tintmeter colorimetric: TMB colour developing liquid (A liquid and B liquid) 450nm and 630nm dual wavelength colorimetric, feminine gender: (-) is colourless or color is very light, OD＜0.20; Positive: (+) color obviously as seen, OD 0.20 to 0.39; (++) is light yellow, and OD 0.4 to 0.99; (+++) identical with the positive control color, OD 1.0 to 2.0; (++ ++) buff is darker than positive control, OD＞2.0.
Three, observation registration assay
Range estimation: the obviously visible high dilution of positive (+) color is reaction titre terminal point.The tintmeter colorimetric: the negative control pipe transfers 0, and the high dilution of sample OD＞0.20 is reaction titre terminal point.Reaction titre 1:2
nExpression, the pipe number of the positive reaction of n.Inhibition test is the inhibition test positive than the titre that detects the titre test low 2 or 2 above titres; The inhibition test positive, just decidable detects the alveolar echinococcosis antibody test positive.
Experimental result shows, the cystic echinococcosis capsule liquid that being applicable to of gained of the present invention detected alveolar echinococcosis antibody takes off ester antigen and is used for the enzyme linked immunosorbent assay dual-antigen sandwich method and detects alveolar echinococcosis antibody, can effectively improve the specificity and the susceptibility of detectable.High specificity: 416 parts of humans and animals samples that detect no echinococcosis symptom, total negative, wherein non-alveolar echinococcosis epidemic-stricken area does not have 180 parts of the human serums of alveolitoid echinococcosis symptom, slaughters to dissect to confirm 86 parts of no echinococcosis sheep serums, does not inoculate 150 parts of many rooms echinococcus cricetulus griseus serum; 36 parts of the humans and animals samples of cystic echinococcosis, total negative, wherein B ultrasonic diagnosis cystic echinococcosis human serum is 7 parts, 4 parts of the bright and beautiful sheep blood serums that cystic echinococcosis packing diameter 5cm to 15cm is arranged, experiment inoculation capsule type echinococcus scolex and 25 parts of the Meriones meridianus serum of cystic echinococcosis packing occur, serum 1:8 dilution OD value all is lower than 0.2; The susceptibility height: experiment is inoculated the alveolitoid echinococcus scolex and the cricetulus griseus of alveolar echinococcosis packing occurred, begin to occur positive reaction from inoculating the 15th day, by the 22nd day, positive rate reaches more than 60%, positive rate reached 100% in the 80th day, and reaction titre and positive rate and infection time and packing volume are proportionate; Stable: the cystic echinococcosis capsule liquid of gained of the present invention takes off the enzyme-linked immunosorbent assay sandwich kit of the detection of ester antigen foundation to be stablized, 4
0C to 8
0C preserved 1 year, did not influence testing result; Purposes is wide: gained of the present invention is applicable to that the cystic echinococcosis capsule liquid that detects alveolar echinococcosis antibody takes off ester antigen and is used for enzyme-linked immunosorbent assay sandwich kit, can detect people and various animal alveolar echinococcosis antibody, be applicable to animal's antibody content more not of the same race, for convenience has been created in echinococcosis antidiastole, prevalence study and epidemic monitoring work; According to the immunological response principle, gained of the present invention is applicable to that the cystic echinococcosis capsule liquid that detects alveolar echinococcosis antibody takes off ester antigen, also can be used for preparing the reagent for quickly examining of multiple detection alveolar echinococcosis antibody such as colloidal gold immunochromatographimethod reagent.
Be applicable to that by gained of the present invention the cystic echinococcosis capsule liquid that detects alveolar echinococcosis antibody takes off enzyme-linked immunosorbent assay sandwich that ester antigen sets up and detects serum, the internal organs of humans and animals and organize alveolar echinococcosis antibody in the immersion liquid sample; Measured antibody is selected combination through envelope antigen and twice specificity of enzyme-labelled antigen, has improved the specificity that detects; Increase inhibition test procedure, improved the accuracy of testing result; Production of the present invention and the no potential safety hazard of use, simple and efficient to handle, be applicable to that the cystic echinococcosis capsule liquid that detects alveolar echinococcosis antibody takes off in the method that ester antigen sets up and has good sensitivity and stability using gained of the present invention, enzyme labeling cystic echinococcosis capsule liquid takes off ester antigen and substitutes enzyme-labeled secondary antibody, and a kind of enzyme labeling thing can detect the antibody of various animals; Microplate reader detects the OD value and has quantitative meaning, and the range estimation result of determination can be specially adapted to alveolar echinococcosis monitoring and the investigation of epidemic disease source animal in power free on-site field use.
Claims (2)
1. one kind is applicable to that the cystic echinococcosis capsule liquid that detects alveolar echinococcosis antibody takes off ester antigen, is characterized in that obtaining by following step:
The first step, gather sheep cystic echinococcosis packing liquid, in packing liquid, add the solid ammonium sulfate dissolving, make the concentration of ammonium sulfate in the packing liquid 30% of the concentration that reaches capacity, be adjusted to pH 7.2 with NaOH or ammonium hydroxide, add chloroform or sherwood oil by 30% to 50% the amount of regulating liquid volume behind the pH then, thermal agitation becomes suspension, suspension leaves standstill to chloroform or sherwood oil and the layering of packing liquid, thermal agitation and standing demix are 2 times again, behind the centrifugal 20min of 4000g, abandon precipitation and chloroform or sherwood oil then, the packing liquid after the collection and treatment;
Second step added the solid ammonium sulfate dissolving in the packing liquid after the first step is handled, make in the packing liquid after the processing ammonium sulfate concentrations 50% to 70% of the concentration that reaches capacity, and was adjusted to pH 7.2 with NaOH or ammonium hydroxide, was 4 in temperature
0C to 10
0Place 4h in the refrigerator of C, collecting precipitation thing behind the centrifugal 10min of 4000g is abandoned supernatant then;
The 3rd step, the sediment that second step was collected joins in the ammonium sulfate of 20 times of sediment volumes, the pH of ammonium sulfate is 7.2, the concentration of ammonium sulfate is 50% to 70% of saturation concentration, vibration makes it become suspension, behind the centrifugal 10min of 4000g, the collecting precipitation thing is abandoned supernatant then;
The 4th step, the sediment that the 3rd step was collected joins in the ammonium sulfate of 20 times of sediment volumes, the pH of ammonium sulfate is 7.2, the concentration of ammonium sulfate is 50% to 70% of saturation concentration, vibration makes it become suspension, collecting precipitation behind the centrifugal 10min of 4000g then, abandon supernatant, after the distilled water that adds 10 times of sediment volumes in the sediment of collecting, vibration make the sediment dissolving, add chloroform or sherwood oil with sediment dissolving back solution equal volume amounts, thermal agitation becomes suspension, leave standstill to chloroform or sherwood oil and solution layering, thermal agitation and standing demix are 2 times again, collect aqueous solution;
In the 5th step, the aqueous solution that the 4th step was collected is packed in the bag filter, and bag filter is put into physiological saline, and the volume of physiological saline is 20 times of aqueous solution volume in the bag filter, is 4 in temperature
0C to 10
012h to 24h dialyses in the refrigerator of C, change physiological saline therebetween 3 times, each physiological saline volume of changing is 20 times of aqueous solution volumes in the bag filter, after dialysis is finished, the centrifugal 20min of solution 4000g in the bag filter, abandon precipitation, collect supernatant, supernatant is cystic echinococcosis capsule liquid and takes off ester antigen.
2. preparation method who is applicable to that the cystic echinococcosis capsule liquid that detects alveolar echinococcosis antibody takes off ester antigen according to claim 1 and 2 is characterized in that being undertaken by following step:
The first step, gather sheep cystic echinococcosis packing liquid, in packing liquid, add the solid ammonium sulfate dissolving, make the concentration of ammonium sulfate in the packing liquid 30% of the concentration that reaches capacity, be adjusted to pH 7.2 with NaOH or ammonium hydroxide, add chloroform or sherwood oil by 30% to 50% the amount of regulating liquid volume behind the pH then, thermal agitation becomes suspension, suspension leaves standstill to chloroform or sherwood oil and the layering of packing liquid, thermal agitation and standing demix are 2 times again, behind the centrifugal 20min of 4000g, abandon precipitation and chloroform or sherwood oil then, the packing liquid after the collection and treatment;
Second step added the solid ammonium sulfate dissolving in the packing liquid after the first step is handled, make in the packing liquid after the processing ammonium sulfate concentrations 50% to 70% of the concentration that reaches capacity, and was adjusted to pH 7.2 with NaOH or ammonium hydroxide, was 4 in temperature
0C to 10
0Place 4h in the refrigerator of C, collecting precipitation thing behind the centrifugal 10min of 4000g is abandoned supernatant then;
The 3rd step, the sediment that second step was collected joins in the ammonium sulfate of 20 times of sediment volumes, the pH of ammonium sulfate is 7.2, the concentration of ammonium sulfate is 50% to 70% of saturation concentration, vibration makes it become suspension, behind the centrifugal 10min of 4000g, the collecting precipitation thing is abandoned supernatant then;
The 4th step, the sediment that the 3rd step was collected joins in the ammonium sulfate of 20 times of sediment volumes, the pH of ammonium sulfate is 7.2, the concentration of ammonium sulfate is 50% to 70% of saturation concentration, vibration makes it become suspension, collecting precipitation behind the centrifugal 10min of 4000g then, abandon supernatant, after the distilled water that adds 10 times of sediment volumes in the sediment of collecting, vibration make the sediment dissolving, add chloroform or sherwood oil with sediment dissolving back solution equal volume amounts, thermal agitation becomes suspension, leave standstill to chloroform or sherwood oil and solution layering, thermal agitation and standing demix are 2 times again, collect aqueous solution;
In the 5th step, the aqueous solution that the 4th step was collected is packed in the bag filter, and bag filter is put into physiological saline, and the volume of physiological saline is 20 times of aqueous solution volume in the bag filter, is 4 in temperature
0C to 10
012h to 24h dialyses in the refrigerator of C, change physiological saline therebetween 3 times, each physiological saline volume of changing is 20 times of aqueous solution volumes in the bag filter, after dialysis is finished, the centrifugal 20min of solution 4000g in the bag filter, abandon precipitation, collect supernatant, supernatant is and is applicable to that the cystic echinococcosis capsule liquid that detects alveolar echinococcosis antibody takes off ester antigen.
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| CN104101716A (en) * | 2014-07-22 | 2014-10-15 | 重庆澳龙生物制品有限公司 | Sheep echinococcosis ELISA (enzyme-linked immunosorbent assay) antibody detection kit |
| CN112684179A (en) * | 2021-01-20 | 2021-04-20 | 中国水产科学研究院黑龙江水产研究所 | Parasite rapid enzyme immunoassay method and kit for salmon and trout culture |
| CN114088937A (en) * | 2021-11-02 | 2022-02-25 | 新疆医科大学 | Kit for detecting echinococcosis antibody based on chemiluminescence immunoassay technology, and preparation method and use method thereof |
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| CN104101716A (en) * | 2014-07-22 | 2014-10-15 | 重庆澳龙生物制品有限公司 | Sheep echinococcosis ELISA (enzyme-linked immunosorbent assay) antibody detection kit |
| CN112684179A (en) * | 2021-01-20 | 2021-04-20 | 中国水产科学研究院黑龙江水产研究所 | Parasite rapid enzyme immunoassay method and kit for salmon and trout culture |
| CN114088937A (en) * | 2021-11-02 | 2022-02-25 | 新疆医科大学 | Kit for detecting echinococcosis antibody based on chemiluminescence immunoassay technology, and preparation method and use method thereof |
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