CN103217524B - 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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- CN103217524B CN103217524B CN201210582686.0A CN201210582686A CN103217524B CN 103217524 B CN103217524 B CN 103217524B CN 201210582686 A CN201210582686 A CN 201210582686A CN 103217524 B CN103217524 B CN 103217524B
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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 being applicable to detect alveolar echinococcosis antibody takes off ester antigen and preparation method thereof.
Background technology
Alveolar echinococcosis (Alveolar echinococcosis, AE) be by Echinococcus multilocularis (Echinococcus multilocularis, Em) Amphixenosis that parasitized larvae causes, early stage non-evident sympton, during discovery, focus is many spreads, focus is that gradual infiltration increases, and the diagnostic method of the routine such as ultrasound wave and CT examination is not easily distinguished with tumor focus, and Serologic detection contributes to making a definite diagnosis of AE.Detect the important indicator that echinococcosis antibody is Diagnosis of Human Hydatidosis, antigen is the key reagents affecting detection specificity and susceptibility.Saltout, the method such as ion chromatography, affinity chromatography, PAGE production, the materials such as Protoscolex, cyst wall, capsule liquid, all once detect the antigen of echinococcosis antibody for Isolation and purification; In recent years, technique for gene engineering and artificial synthetic polypeptide are also applied preparing in antigen, and the antigen type once for detecting the research of echinococcosis antibody is various, all fail to significantly improve the specificity and susceptibility that detect echinococcosis antibody.Cystic echinococcosis (Cystic echinococcosis, CE) be the Amphixenosis caused by the parasitized larvae of Echinococcus granulosus (Echinococcus granulosur.Eg), detect the method for CE antibody, also there is positive reaction in AE antibody, there is same or similar reactive antigenic component in the protoscolex of prompting Em, Eg or capsule liquid, the antigen being applicable to detect alveolar echinococcosis antibody in CE capsule liquid, may be had.Conventional method extracts CE cyst fluid antigen, and not only program is complicated, and harvest yield is very few, and has cross reaction with multiple parasite.A kind of enzyme of indirect enzyme linked immunosorbent assay mark second antibody can only detect the antibody of a kind of animal, detect many animals echinococcosis antibody, the enzyme mark second antibody of many animals serum immune globulin must be prepared, not only workload is large, and be subject to the impact of enzyme mark second antibody potency differences, be difficult to compare different animal species antibody content, dual-antigen sandwich method detects antibody, enzyme-labelled antigen substitutes second antibody, the antibody of many animals can be detected, for Amphixenosis's investigation and epidemic monitoring provide convenience.
Summary of the invention
The invention provides a kind of cystic echinococcosis capsule liquid being applicable to detect alveolar echinococcosis antibody and take off ester antigen and preparation method thereof, overcome the deficiency of above-mentioned prior art, it can improve the specificity, susceptibility and the stability that detect alveolar echinococcosis antibody, and purposes is wide.
One of technical scheme of the present invention is realized by following measures: a kind of cystic echinococcosis capsule liquid being applicable to detect alveolar echinococcosis antibody takes off ester antigen, obtains in the steps below:
The first step, gather sheep cystic echinococcosis hydatid cyst fluid, in hydatid cyst fluid, add solid ammonium sulfate dissolve, the concentration of ammonium sulfate in hydatid cyst fluid is reached capacity 30% of concentration, pH 7.2 is adjusted to NaOH or ammonium hydroxide, then chloroform or sherwood oil is added by the amount of regulate liquid volume after pH 30% to 50%, thermal agitation becomes suspension, suspension leaves standstill to chloroform or sherwood oil and hydatid cyst fluid layering, thermal agitation stratification 2 times again, then after the centrifugal 20min of 4000g, abandon precipitation and chloroform or sherwood oil, collect the hydatid cyst fluid after processing;
Second step, add solid ammonium sulfate in the hydatid cyst fluid after first step process and dissolve, ammonium sulfate concentrations in the hydatid cyst fluid after process is reached capacity 50% to 70% of concentration, and being adjusted to pH 7.2 with NaOH or ammonium hydroxide, is 4 in temperature
0c to 10
0place 4h in the refrigerator of C, then collecting precipitation thing after the centrifugal 10min of 4000g, abandons supernatant;
3rd step, the sediment that second step is collected is joined in the ammonium sulfate of 20 times of sediment volume amounts, the pH of ammonium sulfate is 7.2, the concentration of ammonium sulfate is 50% to 70% of saturation concentration, vibration becomes suspension, then, after the centrifugal 10min of 4000g, collecting precipitation thing, abandons supernatant;
4th step, the sediment that 3rd step is collected is joined in the ammonium sulfate of 20 times of sediment volume amounts, the pH of ammonium sulfate is 7.2, the concentration of ammonium sulfate is 50% to 70% of saturation concentration, vibration becomes suspension, then collecting precipitation after the centrifugal 10min of 4000g, abandon supernatant, the distilled water of 10 times of sediment volume amounts is added in the sediment collected, after vibration makes precipitate dissolves, add with precipitate dissolves after the chloroform of solution equal volume amounts or sherwood oil, thermal agitation becomes suspension, leave standstill to chloroform or sherwood oil and solution layering, thermal agitation stratification 2 times again, collect aqueous solution,
5th step, the aqueous solution of the 4th step being collected loads in bag filter, and bag filter is put into physiological saline, and the volume of physiological saline is 20 times of aqueous solution volume in bag filter, is 4 in temperature
0c to 10
0dialysis 12h to 24h is carried out in the refrigerator of C, change physiological saline therebetween 3 times, each replacing physiological saline volume is aqueous solution volume 20 times in bag filter, after having dialysed, the centrifugal 20min of solution 4000g in bag filter, abandon precipitation, collect supernatant, supernatant is the cystic echinococcosis capsule liquid being applicable to detect alveolar echinococcosis antibody and takes off ester antigen.
Two of technical scheme of the present invention is realized by following measures: a kind of cystic echinococcosis capsule liquid being applicable to detect alveolar echinococcosis antibody takes off the preparation method of ester antigen, carries out in the steps below:
The first step, gather sheep cystic echinococcosis hydatid cyst fluid, in hydatid cyst fluid, add solid ammonium sulfate dissolve, the concentration of ammonium sulfate in hydatid cyst fluid is reached capacity 30% of concentration, pH 7.2 is adjusted to NaOH or ammonium hydroxide, then chloroform or sherwood oil is added by the amount of regulate liquid volume after pH 30% to 50%, thermal agitation becomes suspension, suspension leaves standstill to chloroform or sherwood oil and hydatid cyst fluid layering, thermal agitation stratification 2 times again, then after the centrifugal 20min of 4000g, abandon precipitation and chloroform or sherwood oil, collect the hydatid cyst fluid after processing;
Second step, add solid ammonium sulfate in the hydatid cyst fluid after first step process and dissolve, ammonium sulfate concentrations in the hydatid cyst fluid after process is reached capacity 50% to 70% of concentration, and being adjusted to pH 7.2 with NaOH or ammonium hydroxide, is 4 in temperature
0c to 10
0place 4h in the refrigerator of C, then collecting precipitation thing after the centrifugal 10min of 4000g, abandons supernatant;
3rd step, the sediment that second step is collected is joined in the ammonium sulfate of 20 times of sediment volume amounts, the pH of ammonium sulfate is 7.2, the concentration of ammonium sulfate is 50% to 70% of saturation concentration, vibration becomes suspension, then, after the centrifugal 10min of 4000g, collecting precipitation thing, abandons supernatant;
4th step, the sediment that 3rd step is collected is joined in the ammonium sulfate of 20 times of sediment volume amounts, the pH of ammonium sulfate is 7.2, the concentration of ammonium sulfate is 50% to 70% of saturation concentration, vibration becomes suspension, then collecting precipitation after the centrifugal 10min of 4000g, abandon supernatant, the distilled water of 10 times of sediment volume amounts is added in the sediment collected, after vibration makes precipitate dissolves, add with precipitate dissolves after the chloroform of solution equal volume amounts or sherwood oil, thermal agitation becomes suspension, leave standstill to chloroform or sherwood oil and solution layering, thermal agitation stratification 2 times again, collect aqueous solution,
5th step, the aqueous solution of the 4th step being collected loads in bag filter, and bag filter is put into physiological saline, and the volume of physiological saline is 20 times of aqueous solution volume in bag filter, is 4 in temperature
0c to 10
0dialysis 12h to 24h is carried out in the refrigerator of C, change physiological saline therebetween 3 times, each replacing physiological saline volume is aqueous solution volume 20 times in bag filter, after having dialysed, the centrifugal 20min of solution 4000g in bag filter, abandon precipitation, collect supernatant, supernatant is the cystic echinococcosis capsule liquid being applicable to detect alveolar echinococcosis antibody and takes off ester antigen.
The cystic echinococcosis capsule liquid being applicable to detect alveolar echinococcosis antibody of gained of the present invention takes off ester antigen and detects alveolar echinococcosis antibody for enzyme linked immunosorbent assay dual-antigen sandwich method, effectively can improve the specificity and susceptibility that detect reagent, have the advantages that high specificity, susceptibility are high, stable and purposes is wide, people and various animal alveolar echinococcosis antibody can be detected, be applicable to compare different animal species antibody content; According to immunological response principle, the cystic echinococcosis capsule liquid being applicable to detect alveolar echinococcosis antibody of gained of the present invention takes off ester antigen, also can be used for the reagent for quickly examining preparing the multiple detection alveolar echinococcosis antibody such as colloidal gold immunochromatographimethod reagent, for echinococcosis antidiastole, prevalence study and epidemic monitoring work create convenience.
Embodiment
The present invention by the restriction of following embodiment, can not determine concrete embodiment according to technical scheme of the present invention and actual conditions.
Embodiment 1, a kind of cystic echinococcosis capsule liquid being applicable to detect alveolar echinococcosis antibody takes off ester antigen, obtains in the steps below:
The first step, gather sheep cystic echinococcosis hydatid cyst fluid, in hydatid cyst fluid, add solid ammonium sulfate dissolve, the concentration of ammonium sulfate in hydatid cyst fluid is reached capacity 30% of concentration, pH 7.2 is adjusted to NaOH or ammonium hydroxide, then chloroform or sherwood oil is added by the amount of regulate liquid volume after pH 30% to 50%, thermal agitation becomes suspension, suspension leaves standstill to chloroform or sherwood oil and hydatid cyst fluid layering, thermal agitation stratification 2 times again, then after the centrifugal 20min of 4000g, abandon precipitation and chloroform or sherwood oil, collect the hydatid cyst fluid after processing;
Second step, add solid ammonium sulfate in the hydatid cyst fluid after first step process and dissolve, ammonium sulfate concentrations in the hydatid cyst fluid after process is reached capacity 50% to 70% of concentration, and being adjusted to pH 7.2 with NaOH or ammonium hydroxide, is 4 in temperature
0c to 10
0place 4h in the refrigerator of C, then collecting precipitation thing after the centrifugal 10min of 4000g, abandons supernatant;
3rd step, the sediment that second step is collected is joined in the ammonium sulfate of 20 times of sediment volume amounts, the pH of ammonium sulfate is 7.2, the concentration of ammonium sulfate is 50% to 70% of saturation concentration, vibration becomes suspension, then, after the centrifugal 10min of 4000g, collecting precipitation thing, abandons supernatant;
4th step, the sediment that 3rd step is collected is joined in the ammonium sulfate of 20 times of sediment volume amounts, the pH of ammonium sulfate is 7.2, the concentration of ammonium sulfate is 50% to 70% of saturation concentration, vibration becomes suspension, then collecting precipitation after the centrifugal 10min of 4000g, abandon supernatant, the distilled water of 10 times of sediment volume amounts is added in the sediment collected, after vibration makes precipitate dissolves, add with precipitate dissolves after the chloroform of solution equal volume amounts or sherwood oil, thermal agitation becomes suspension, leave standstill to chloroform or sherwood oil and solution layering, thermal agitation stratification 2 times again, collect aqueous solution,
5th step, the aqueous solution of the 4th step being collected loads in bag filter, and bag filter is put into physiological saline, and the volume of physiological saline is 20 times of aqueous solution volume in bag filter, is 4 in temperature
0c to 10
0dialysis 12h to 24h is carried out in the refrigerator of C, change physiological saline therebetween 3 times, each replacing physiological saline volume is aqueous solution volume 20 times in bag filter, after having dialysed, the centrifugal 20min of solution 4000g in bag filter, abandon precipitation, collect supernatant, supernatant is the cystic echinococcosis capsule liquid being applicable to detect alveolar echinococcosis antibody and takes off ester antigen.Take off in ester antigen at the cystic echinococcosis capsule liquid being applicable to detect alveolar echinococcosis antibody of gained of the present invention and add thimerosal, make the cystic echinococcosis capsule liquid mass percent taken off in ester antigen that thimerosal is being applicable to detect alveolar echinococcosis antibody be 0.1% ,-20
0c saves backup.
Embodiment 2, the cystic echinococcosis capsule liquid being applicable to detect alveolar echinococcosis antibody of above-described embodiment 1 gained takes off the application of ester antigen in alveolar echinococcosis antibody test; Its application is as follows:
One, enzyme linked immunological sandwich method kit detecting alveolar echinococcosis antibody and preparation method thereof
Detect the enzyme linked immunological sandwich method kit of alveolar echinococcosis antibody, comprise specification be 8 pipe × 12 cystic echinococcosis capsule liquid takes off ester antigen coated microplate 1 piece, horseradish peroxidase-labeled cystic echinococcosis capsule liquid takes off ester antigen 1 0ml, sample diluent 10ml, 20 times of concentrated washing fluid 50ml, alveolar echinococcosis antibody positive contrast liquid 3ml, alveolar echinococcosis antibody negative controls liquid 3ml, suppression cystic echinococcosis capsule liquid take off ester antigen 6ml, nitrite ion A liquid 6ml, nitrite ion B liquid 6ml, colour developing stop buffer 6ml.
Cystic echinococcosis capsule liquid takes off ester antigen coated microplate and obtains in the steps below:
Wrap by with reagent, coating buffer: 0.05mol/L pH be 6.4 to 9.6 phosphate or carbonate buffer solution in add cystic echinococcosis capsule liquid that gained of the present invention is applicable to detect alveolar echinococcosis antibody and take off ester antigen and obtain coating buffer, it is 0.1 μ g/m1 to 1 μ g/m1 that the cystic echinococcosis capsule liquid that gained of the present invention is applicable to detect 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 be 6.4 to 9.6 phosphate or carbonate buffer solution in obtain saturated coating buffer, 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: in the often pipe of reaction plate, add coating buffer 100 μ 1, be 28 in temperature
0c to 37
0place 3h to 4h under C, then in the often pipe of reaction plate, adding saturated coating buffer 100 μ 1, is 28 in temperature
0c to 37
03h is placed under C, get rid of the liquid in pipe abandoning reaction plate, the globule in pipe is removed at thieving paper arsis with after distilled water flushing reaction plate 3 times, obtain embathing liquid by after 20 times of concentrated washing fluid distilled water dilutings 20 times, embathe in liquid containing newborn calf serum volume ratio be 2% or protein amount number percent be 0.05%, then add in the often pipe of reaction plate and embathe liquid 200 μ 1 and embathe, 28
0c to 37
0place 3h at the temperature of C, get rid of the liquid in pipe abandoning reaction plate, with distilled water flushing reaction plate 5 times, thieving paper firmly pats reaction plate until reaction plate pipe inside and outside without the visible globule; It is 37 that reaction plate after process is uprightly put into temperature
othe incubator of C, every 30min air blast or enabling ventilation l time, take out after dry 8h to 12h, obtains cystic echinococcosis capsule liquid and take off ester antigen coated microplate (being called for short: antigen plate), load sealing in polybag and preserve.
Horseradish peroxidase-labeled cystic echinococcosis capsule liquid takes off ester antigen and obtains in the steps below:
The first step, activation horseradish peroxidase: take horseradish peroxidase (RZ>=3.0) 5mg and load test tube, in test tube, add distilled water 1m1 makes horseradish peroxidase dissolve, add in test tube freshly prepared mass percent be 1.3% sodium periodate aqueous solution 0.5m1 mix, 4
0c to 10
0place 30 min at the temperature of C, then in test tube, add the glycol water 0.5m1 that percent by volume is 1%, in room temperature 20
0c to 37
0c places 30min, obtains the horseradish peroxidase activated;
Second step, the horseradish peroxidase cystic echinococcosis capsule liquid being applicable to detect alveolar echinococcosis antibody containing 5mg/m1 gained of the present invention being taken off the activation that the solution 1m1 of ester antigen and the first step obtain mixes, loading bag filter, is that the carbonate buffer solution 200m1 of 9.6 is 4 in temperature with 0.05mol/L pH
0c to 10
0dialyse under C 12h to 16h, and in the solution of bag filter, add the sodium borohydride solution 0.2m1 that freshly prepared mass percent is 0.5% after dialysis, be 4 in temperature
0c to 10
02h is placed in the refrigerator of C, with the phosphate buffer dialysis 8h to 12h that the 0.01mol/L pH of 200m1 is 7.2, change phosphate buffer therebetween 3 times, each 200ml, the centrifugal 10min of solution 4000g after dialysis in bag filter gets supernatant, and supernatant is horseradish peroxidase-labeled cystic echinococcosis capsule liquid and takes off ester antigen (being called for short: enzyme-labelled antigen);
3rd step, measure horseradish peroxidase-labeled cystic echinococcosis capsule liquid and take off ester antigen application concentration: take off ester antigen with the phosphate buffer dilution horseradish peroxidase-labeled cystic echinococcosis capsule liquid that the 0.1mol/L pH containing volume ratio 2% calf serum and 2% glycerine is 7.2, by the enzyme linked immunological sandwich method procedure operation detecting 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 ester antigen concentration for application concentration, horseradish peroxidase-labeled cystic echinococcosis capsule liquid is taken off the ester antigen phosphate buffer that the 0.1mol/L pH containing volume ratio 2% calf serum and 50% glycerine is 7.2 and be diluted to the packing of application concentration.
Sample diluent: the 0.01mol/L pH being the thimerosal of 0.1% containing percent by volume be 0.05% Tween-20, percent by volume be 2% newborn calf serum, mass percent is the phosphate buffer of 7.2.
20 times of concentrated washing fluids: the 0.1mol/L pH being the sodium chloride of 17% containing percent by volume be 1% Tween-20, mass percent is the phosphate buffer of 7.2.
Alveolar echinococcosis antibody positive contrast liquid: the newborn calf serum, the mass percent that are 2% containing percent by volume are the thimerosal of 0.05%, 0.01 mol/L pH of 2 μ g/ml alveolar echinococcosis antibody is the phosphate buffer of 7.2.
Alveolar echinococcosis antibody negative controls liquid: the 0.01 mol/L pH being the thimerosal of 0.05% containing percent by volume be 2% newborn calf serum, mass percent is 7.2 phosphate buffers.
Suppression cystic echinococcosis capsule liquid takes off ester antigen: the 0.01mol/L pH that the cystic echinococcosis capsule liquid being applicable to detect alveolar echinococcosis antibody containing percent by volume be 0.05% Tween-20, the percent by volume newborn calf serum that is 2%, the mass percent thimerosal that is 0.05%, 5 μ g/ml gained of the present invention takes off ester antigen is the phosphate buffer of 7.2.
TMB nitrite ion A liquid: by 0.2 mol/L NaH
2pO
4mix with 0.1 mol/L citric acid equal-volume, add by 0.05% of liquor capacity after mixing the hydrogen peroxide that concentration of volume percent is 30%, be TMB nitrite ion A liquid.
TMB nitrite ion B liquid: absolute ethyl alcohol 10ml and METHYLPYRROLIDONE 10ml is mixed, add 3.3-5.5-tetramethyl benzidine (TMB) 50mg, dissolve completely to 3.3-5.5-tetramethyl benzidine, then adding distil water is to 70ml, add the citric acid 10ml of 0.1mol/L, after adding the EDTA 1ml mixing of 0.1mol/L, be TMB nitrite ion B liquid.
Colour developing stop buffer: percent by volume is the sulfuric acid solution of 10%.
Mentioned reagent and antigen plate load the enzyme-linked immunosorbent assay sandwich kit detecting alveolar echinococcosis antibody, 4
0c to 8
0c preserves, the term of validity 1 year.
Two, enzyme linked immunological sandwich method detects echinococcosis Antibody Screening (qualitative) test
1. add sample diluent: according to sample and quantity that is positive, negative control, assembling cystic echinococcosis capsule liquid takes off ester antigen coated microplate (hereinafter referred to as antigen plate), and antigen plate often pipe adds sample diluent 50 μ l.
2. add sample: in the often pipe of antigen plate, add 1 part of sample 50 μ l by accession designation number position, be placed in wet box or polybag, 37
0c leaves standstill reaction 90min.
3. rinse antigen plate: after 20 times of concentrated washing fluid distilled water dilutings 20 times, rinsing antigen plate with washing trigger, repeatedly rinsing 5 times; The mouth of pipe is downward, removes the globule inside and outside antigen plate pipe at thieving paper arsis.
4. add horseradish peroxidase-labeled cystic echinococcosis capsule liquid and take off ester antigen (hereinafter referred to as enzyme-labelled antigen): in the often pipe of antigen plate enzyme-added mark antigen 1 00 μ l, add a cover, be placed in wet box or polybag; 37
0c reaction time 45min, after 20 times of concentrated washing fluid distilled water dilutings 20 times, rinsing antigen plate with washing trigger, repeatedly rinsing 5 times; The mouth of pipe is downward, removes the globule inside and outside antigen plate pipe at thieving paper, filter paper arsis.
5. develop the color: in the often pipe of antigen plate, add TMB nitrite ion A liquid and each 50 μ l of B 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 the often pipe of antigen plate, positive nitrite ion becomes yellow from blue look, feminine gender is still colourless or color is very light.
7. observational record test findings: to add after stop buffer observed result in 10min.
Range estimation: flat hold antigen plate, apart from white background 10 cm to 15cm, from directly over observe downwards;
Tintmeter colorimetric: TMB nitrite ion (A liquid and B liquid) 450nm and 630nm dual wavelength colorimetric, negative: (-) is colourless or color is very light, OD < 0.20; Positive: (+) color is obviously visible, OD 0.20 to 0.39; (++) is light yellow, OD 0.4 to 0.99; (+++) is identical with 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, positive reaction sample arranges respectively by (+), (++), (+++), (++++); (+) with 2 tubules, (++) with 4 tubules, (+++) with 6 to 8 tubules, (++++), with 8 to 12 tubules, adds sample diluent 100u1 in the often pipe of antigen plate.
(2) in the first pipe of the often row of antigen plate, add 1 part of sample 100u1 to be checked, pressure-vaccum 3 times, move 100u1 in the 2nd pipe, so continuous 2 times are diluted to most end pipe; Positive control sample synchronously dilutes; Add a cover, be placed in wet box or polybag, 37
0c reaction time 90min; For titre terminal can be measured, titre can be detected again by after sample dilutions 16 doubly to 64 of (++++) times.
(3) antigen plate is rinsed: after 20 times of concentrated washing fluid distilled water dilutings 20 times, rinsing antigen plate with washing trigger, repeatedly rinsing 5 times; The mouth of pipe is downward, removes the globule inside and outside antigen plate pipe at thieving paper, filter paper arsis.
(4) to label antigen: in the often pipe of antigen plate, add enzyme-labelled antigen 100 μ l, add a cover, be placed in wet box or polybag; 37
0c reaction time 45min, after 20 times of concentrated washing fluid distilled water dilutings 20 times, rinsing antigen plate with washing trigger, repeatedly rinsing 5 times; The mouth of pipe is downward, removes the globule inside and outside antigen plate pipe at thieving paper, filter paper arsis.
(5) develop the color: in the often pipe of antigen plate, add TMB nitrite ion A liquid and each 50 μ l of B 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 the often pipe of antigen plate, nitrite ion becomes yellow from blue look, feminine gender is still colourless or color is very light.
(7) observational record test findings: to add after stop buffer observed result in 10min.
Range estimation: flat hold antigen plate, apart from white background 10 Cm to 15Cm, from directly over observe downwards;
Tintmeter colorimetric: TMB nitrite ion (A liquid and B liquid) 450nm and 630nm dual wavelength colorimetric, negative: (-) is colourless or color is very light, OD < 0.20; Positive: (+) color is obviously visible, OD 0.20 to 0.39; (++) is light yellow, OD 0.4 to 0.99; (+++) is identical with 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 synchronously carried out.Inhibition test and detection reaction titre test difference for replacing sample diluent to dilute sample with suppression antigen liquid, and often pipe adds suppression antigen liquid 100u1.
(2) in the first pipe of the often row of antigen plate, add 1 part of sample 100u1 to be checked, pressure-vaccum 3 times, move 100u1 in the 2nd pipe, so continuous 2 times are diluted to most end pipe; Positive control sample synchronously dilutes; Add a cover, be placed in reaction in wet box or polybag, 37
0c reaction time 90min.For titre terminal can be measured, titre can be detected again by after sample dilutions 16 doubly to 64 of (++++) times.
(3) antigen plate is rinsed: after 20 times of concentrated washing fluid distilled water dilutings 20 times, rinsing antigen plate with washing trigger, repeatedly rinsing 5 times; The mouth of pipe is downward, removes the globule inside and outside antigen plate pipe at thieving paper, filter paper arsis.
(4) enzyme-labelled antigen is added: in the often pipe of antigen plate, add enzyme-labelled antigen 100 μ l, add a cover, be placed in wet box or polybag; 37
0c reaction time 45min, after 20 times of concentrated washing fluid distilled water dilutings 20 times, rinsing antigen plate with washing trigger, repeatedly rinsing 5 times; The mouth of pipe is downward, removes the globule inside and outside antigen plate pipe at thieving paper, filter paper arsis.
(5) develop the color: in the often pipe of antigen plate, add TMB nitrite ion A liquid and each 50 μ l of B 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 the often pipe of antigen plate, nitrite ion becomes yellow from blue look, feminine gender is still colourless or color is very light.
(7) observational record test findings: to add after stop buffer observed result in 10min.
Range estimation: flat hold antigen plate, apart from white background 10 cm to 15cm, from directly over observe downwards;
Tintmeter colorimetric: TMB nitrite ion (A liquid and B liquid) 450nm and 630nm dual wavelength colorimetric, negative: (-) is colourless or color is very light, OD < 0.20; Positive: (+) color is obviously visible, OD 0.20 to 0.39; (++) is light yellow, OD 0.4 to 0.99; (+++) is identical with positive control color, OD 1.0 to 2.0; (++++) buff is darker than positive control, OD > 2.0.
Three, observation registration assay
Range estimation: positive (+) color obviously visible most high dilution is reaction titre terminal.Tintmeter colorimetric: negative control pipe adjusts the most high dilution of 0, sample OD > 0.20 for reaction titre terminal.Reaction titre 1:2
nrepresent, n is the pipe number of positive reaction.Inhibition test is that inhibition test is positive than low more than 2 or 2 titres of the titre detecting titre test; Inhibition test is positive, just can judge to detect alveolar echinococcosis antibody test positive.
Experimental result shows, the cystic echinococcosis capsule liquid being applicable to detect alveolar echinococcosis antibody of gained of the present invention is taken off ester antigen and be used for enzyme linked immunosorbent assay dual-antigen sandwich method detection alveolar echinococcosis antibody, effectively can improve the specificity and susceptibility that detect reagent.High specificity: detect the humans and animals sample 416 parts without echinococcosis symptom, total negative, wherein non-alveolar echinococcosis epidemic-stricken area is without the human serum 180 parts of alveolar echinococcosis symptom, slaughters to dissect to confirm without echinococcosis sheep serum 86 parts, non-Inoculation of Echinococcus multilocularis cricetulus griseus serum 150 parts; The humans and animals sample of cystic echinococcosis 36 parts, total negative, wherein B ultrasonic diagnosis cystic echinococcosis human serum 7 parts, there is the bright and beautiful sheep blood serum 4 parts of cystic echinococcosis packing diameter 5cm to 15cm, experiment is inoculated bladder type echinococcus scolex and is occurred the Meriones meridianus serum 25 parts of cystic echinococcosis packing, and serum 1:8 dilutes OD value all lower than 0.2; Susceptibility is high: experiment is inoculated alveolitoid echinococcus scolex and occurred the cricetulus griseus of alveolar echinococcosis packing, positive reaction is there is 15th day from inoculation, by the 22nd day, positive rate reaches more than 60%, within 80th day, positive rate reaches 100%, and reaction titre and positive rate and infection time and packing volume are proportionate; Stable: the enzyme-linked immunosorbent assay sandwich kit that the cystic echinococcosis capsule liquid of gained of the present invention takes off the detection that ester antigen is set up is stablized, 4
0c to 8
0c preserves 1 year, does not affect testing result; Purposes is wide: the cystic echinococcosis capsule liquid that gained of the present invention is applicable to detect alveolar echinococcosis antibody takes off ester antigen for enzyme-linked immunosorbent assay sandwich kit, people and various animal alveolar echinococcosis antibody can be detected, be applicable to compare different animal species antibody content, for echinococcosis antidiastole, prevalence study and epidemic monitoring work create convenience; According to immunological response principle, the cystic echinococcosis capsule liquid that gained of the present invention is applicable to detect alveolar echinococcosis antibody takes off ester antigen, also can be used for the reagent for quickly examining preparing the multiple detection alveolar echinococcosis antibody such as colloidal gold immunochromatographimethod reagent.
The cystic echinococcosis capsule liquid being applicable to detect alveolar echinococcosis antibody by gained of the present invention takes off enzyme-linked immunosorbent assay sandwich that ester antigen sets up and detects alveolar echinococcosis antibody in the serum of humans and animals, internal organs and tissue extracts sample; Measured antibody is selected to combine through envelope antigen and enzyme-labelled antigen twice specificity, improves the specificity of detection; Add inhibition test procedure, improve the accuracy of testing result; Production of the present invention and use are without potential safety hazard, simple and efficient to handle, the cystic echinococcosis capsule liquid being applicable to detect alveolar echinococcosis antibody at application gained of the present invention takes off in the method that ester antigen sets up and has good susceptibility and stability, enzyme labeling cystic echinococcosis capsule liquid takes off ester antigen and substitutes enzyme-labeled secondary antibody, and a kind of enzyme marker can detect the antibody of various animal; Microplate reader detects OD value and has quantitative meaning, range estimation result of determination, in power free on-site field use, can be specially adapted to alveolar echinococcosis monitoring and the investigation of epidemic disease source animal.
Claims (2)
1. the cystic echinococcosis capsule liquid being applicable to detect alveolar echinococcosis antibody takes off an ester antigen, it is characterized in that obtaining in the steps below:
The first step, gather sheep cystic echinococcosis hydatid cyst fluid, in hydatid cyst fluid, add solid ammonium sulfate dissolve, the concentration of ammonium sulfate in hydatid cyst fluid is reached capacity 30% of concentration, pH 7.2 is adjusted to NaOH or ammonium hydroxide, then chloroform or sherwood oil is added by the amount of regulate liquid volume after pH 30% to 50%, thermal agitation becomes suspension, suspension leaves standstill to chloroform or sherwood oil and hydatid cyst fluid layering, thermal agitation stratification 2 times again, then after the centrifugal 20min of 4000g, abandon precipitation and chloroform or sherwood oil, collect the hydatid cyst fluid after processing;
Second step, add solid ammonium sulfate in the hydatid cyst fluid after first step process and dissolve, ammonium sulfate concentrations in the hydatid cyst fluid after process is reached capacity 50% to 70% of concentration, and being adjusted to pH 7.2 with NaOH or ammonium hydroxide, is 4 in temperature
0c to 10
0place 4h in the refrigerator of C, then collecting precipitation thing after the centrifugal 10min of 4000g, abandons supernatant;
3rd step, the sediment that second step is collected is joined in the ammonium sulfate of 20 times of sediment volume amounts, the pH of ammonium sulfate is 7.2, the concentration of ammonium sulfate is 50% to 70% of saturation concentration, vibration becomes suspension, then, after the centrifugal 10min of 4000g, collecting precipitation thing, abandons supernatant;
4th step, the sediment that 3rd step is collected is joined in the ammonium sulfate of 20 times of sediment volume amounts, the pH of ammonium sulfate is 7.2, the concentration of ammonium sulfate is 50% to 70% of saturation concentration, vibration becomes suspension, then collecting precipitation after the centrifugal 10min of 4000g, abandon supernatant, the distilled water of 10 times of sediment volume amounts is added in the sediment collected, after vibration makes precipitate dissolves, add with precipitate dissolves after the chloroform of solution equal volume amounts or sherwood oil, thermal agitation becomes suspension, leave standstill to chloroform or sherwood oil and solution layering, thermal agitation stratification 2 times again, collect aqueous solution,
5th step, the aqueous solution of the 4th step being collected loads in bag filter, and bag filter is put into physiological saline, and the volume of physiological saline is 20 times of aqueous solution volume in bag filter, is 4 in temperature
0c to 10
0dialysis 12h to 24h is carried out in the refrigerator of C, change physiological saline therebetween 3 times, each replacing physiological saline volume is aqueous solution volume 20 times in bag filter, after having dialysed, the centrifugal 20min of solution 4000g in bag filter, abandon precipitation, collect supernatant, supernatant is cystic echinococcosis capsule liquid and takes off ester antigen.
2. the cystic echinococcosis capsule liquid being applicable to detect alveolar echinococcosis antibody according to claim 1 takes off a preparation method for ester antigen, it is characterized in that carrying out in the steps below:
The first step, gather sheep cystic echinococcosis hydatid cyst fluid, in hydatid cyst fluid, add solid ammonium sulfate dissolve, the concentration of ammonium sulfate in hydatid cyst fluid is reached capacity 30% of concentration, pH 7.2 is adjusted to NaOH or ammonium hydroxide, then chloroform or sherwood oil is added by the amount of regulate liquid volume after pH 30% to 50%, thermal agitation becomes suspension, suspension leaves standstill to chloroform or sherwood oil and hydatid cyst fluid layering, thermal agitation stratification 2 times again, then after the centrifugal 20min of 4000g, abandon precipitation and chloroform or sherwood oil, collect the hydatid cyst fluid after processing;
Second step, add solid ammonium sulfate in the hydatid cyst fluid after first step process and dissolve, ammonium sulfate concentrations in the hydatid cyst fluid after process is reached capacity 50% to 70% of concentration, and being adjusted to pH 7.2 with NaOH or ammonium hydroxide, is 4 in temperature
0c to 10
0place 4h in the refrigerator of C, then collecting precipitation thing after the centrifugal 10min of 4000g, abandons supernatant;
3rd step, the sediment that second step is collected is joined in the ammonium sulfate of 20 times of sediment volume amounts, the pH of ammonium sulfate is 7.2, the concentration of ammonium sulfate is 50% to 70% of saturation concentration, vibration becomes suspension, then, after the centrifugal 10min of 4000g, collecting precipitation thing, abandons supernatant;
4th step, the sediment that 3rd step is collected is joined in the ammonium sulfate of 20 times of sediment volume amounts, the pH of ammonium sulfate is 7.2, the concentration of ammonium sulfate is 50% to 70% of saturation concentration, vibration becomes suspension, then collecting precipitation after the centrifugal 10min of 4000g, abandon supernatant, the distilled water of 10 times of sediment volume amounts is added in the sediment collected, after vibration makes precipitate dissolves, add with precipitate dissolves after the chloroform of solution equal volume amounts or sherwood oil, thermal agitation becomes suspension, leave standstill to chloroform or sherwood oil and solution layering, thermal agitation stratification 2 times again, collect aqueous solution,
5th step, the aqueous solution of the 4th step being collected loads in bag filter, and bag filter is put into physiological saline, and the volume of physiological saline is 20 times of aqueous solution volume in bag filter, is 4 in temperature
0c to 10
0dialysis 12h to 24h is carried out in the refrigerator of C, change physiological saline therebetween 3 times, each replacing physiological saline volume is aqueous solution volume 20 times in bag filter, after having dialysed, the centrifugal 20min of solution 4000g in bag filter, abandon precipitation, collect supernatant, supernatant is the cystic echinococcosis capsule liquid being applicable to detect alveolar echinococcosis antibody and 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 |
| CN114088937B (en) * | 2021-11-02 | 2023-08-29 | 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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