CN101387641B - Liver fluke rapid immune detection kit for fresh water fishes and detection method - Google Patents
Liver fluke rapid immune detection kit for fresh water fishes and detection method Download PDFInfo
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- CN101387641B CN101387641B CN2007100769767A CN200710076976A CN101387641B CN 101387641 B CN101387641 B CN 101387641B CN 2007100769767 A CN2007100769767 A CN 2007100769767A CN 200710076976 A CN200710076976 A CN 200710076976A CN 101387641 B CN101387641 B CN 101387641B
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Abstract
The invention discloses a fresh water fish liver fluke fast immunity test kit and a test method. The kit box comprises an enzyme linked reaction board, an enzyme conjugate, washing liquid, a substrate solution, a color developer, a diluent and an end solution, wherei the enzyme linked reaction board is made by extracting and purifying soluble antigen from liver fluke and enveloping the soluble antigen; the enzyme conjugate is made by labeling anti-fish immunoglobulin via horseradish peroxidase. The invention utilizes the sensitivity, the specificity, the practicality and the economy of the fast immunity test technique to establish a fresh water fish liver fluke fast immunity test method, is practical and simple while the kit can be opened and used instantly, without preparation or dilution, with simple operation; the result can be judged quantitatively by instruments and can be judged by eyes, thus is suitable of the on-site detection of different conditions; the invention is safe and environment friend, which adopts the harmless enzyme linked immunity color developer as tetramethylbenzidine to avoid the harm and pollution of general methods.
Description
Technical field
The present invention relates to technical field of biological, in particular, a kind of liver fluke rapid immune detection kit for fresh water fishes and detection method.
Background technology
Liver fluke is a kind of common parasite of human, infects by eating the freshwater fish that contains metacercaria of liver flukes.The liver fluke adult parasitizes in human liver's bile duct, causes liver rot, causes a series of liver and gall infringement, comprises bile duct inflammation, calculus, biliary cirrhosis, finally can induced hepatocellular carcinoma.
At present a lot of to the diagnostic method of Human clonorchiasis, comprise that the aetology method checks that faecal egg and immunological method detect liver fluke antibody in the patients serum.Accurately and reliably, but worm's ovum is little, ovulates irregular for the aetology method, easily fail to pinpoint a disease in diagnosis, and to gather Feces of Patients, and by the clear water repeated precipitation, also eluriate repeatedly, get sediment, by the professional and technical personnel, checked with high-power microscope afterwards, process is longer, generally needs several hours or more, operates loaded down with trivial details inconvenience, labor intensive, be difficult to promote generaI investigation; The immunological method sensitivity is special, and fast and easy detects serum and gets final product, and therefore, obtains applying comparatively widely.
In immunological method, the application of enzyme linked immunosorbent detection technology is the most general, domestic in the nineties in 20th century with regard to develop for detection of human body in the rapid immune detecting kit (Hu Shaoliang etc. of liver fluke, the foundation of clonorchiasis Fast ELISA for Diagnosis kit, China's preventing and treating verminosis magazine, 84 phases of volume of nineteen ninety-five, the start-stop page number: 295-296), susceptibility reaches more than 92%, and specificity reaches more than 98%.
Yet, really cut off liver fluke popular, ensure people ' s health, must, from source, the fresh-water fishes meat being carried out to safety supervision, prevent that the sick fish that contains metacercaria of liver flukes from coming into the market.But at present the fresh-water fishes liver fluke is detected and also lacks effective means.Traditional fish liver fluke checks, be to adopt microscope to observe the metacercaria of liver flukes in the flesh of fish, but this method will be in the flesh of fish of 1000 grams that generally weighs, the bladder worm that is observed visually 1 millimeter of less than is very difficult, inefficiency not only, and very easily omit.Therefore, the microexamination flesh of fish, look into liver fluke, except scientific research, almost is not applied in practice, and this is also to cause the fresh-water fishes liver fluke to lack the major reason of safety supervision.And fresh-water fishes liver fluke immunologic detection method is not set up so far yet.
Therefore, prior art does not detect the effective tool of freshwater fish liver fluke.
Summary of the invention
The object of the present invention is to provide a kind of liver fluke rapid immune detection kit for fresh water fishes and detection method, thereby a kind of testing tool and method that detects the freshwater fish liver fluke is provided.
Technical scheme of the present invention is as follows:
A kind of liver fluke rapid immune detection kit for fresh water fishes, arrange enzyme-linked reaction plate, enzyme conjugates, cleansing solution, substrate solution, developer, dilution, stop buffer in its box body; Wherein, described enzyme-linked reaction plate is from liver fluke, extracting soluble antigen purifying, then being coated with the enzyme-linked reaction plate that described soluble antigen makes; Described enzyme conjugates is the enzyme conjugates of the anti-fish immunoglobulin (Ig) of horseradish peroxidase mark.
Described kit, wherein, described enzyme-linked reaction plate is coated soluble antigen on the general purpose polystyrene microwell plate, then in room temperature standing 12～24 hours, after vacuum freezedrying, the pack sealing was made; Wherein, described soluble antigen is from infection animal, obtaining the liver fluke adult, and extraction obtains its soluble antigen; Coating buffer is the damping fluid that contains 0.01%～1% soluble antigen, and described damping fluid is phosphate, borate or the carbonate buffer solution of 0.01mol/L～0.15mol/L, pH7.5～pH9.5.
Described kit, wherein, described enzyme conjugates, for adopting the fish immunoglobulin (Ig) immune animal of purifying, is isolated anti-fish immunoglobulin (Ig), and purified rear employing horseradish peroxidase is carried out immune labeled making.
Described kit, wherein, described enzyme conjugates is the 0.01mol/L that contains 0.05%～1% the anti-fish immunoglobulin (Ig) of horseradish peroxidase-labeled～0.05 mol/L phosphate buffer, pH6～8; And add the antiseptic that content is enough to bacteria growing inhibiting.
Described kit, wherein, its also comprise at least negative control product, positive reference substance one of them.
Described kit, wherein, described cleansing solution is the 0.01mol/L that includes 0.1%～1% surfactant～0.5mol/L phosphate buffer, pH6～8; Substrate solution is 0.1%～0.5% superoxol; Developer is TMB; Dilution is 0.01mol/L～0.5mol/L phosphate buffer, pH6～8; Stop buffer is sulfuric acid or the NaOH of 1mol/L～2mol/L.
A kind of detection method for kit claimed in claim 1, it comprises step:
A1, from infection animal, obtaining the liver fluke adult, and extract and to obtain its soluble antigen, coated described soluble antigen on the general purpose polystyrene microwell plate, then in room temperature standing 12～24 hours, after vacuum freezedrying, make enzyme-linked reaction plate, and the pack sealing is preserved; Wherein, coating buffer is the damping fluid that contains 0.01%～1% described soluble antigen, and described damping fluid is phosphate, borate or the carbonate buffer solution of 0.01mol/L～0.15mol/L of pH7.5～pH9.5;
A2, the fish immunoglobulin (Ig) immune animal of adopting purifying, isolate anti-fish immunoglobulin (Ig), and purified rear employing horseradish peroxidase is carried out the immune labeled enzyme conjugates of making;
A3, the every hole of described enzyme-linked reaction plate add respectively 1 of dilution, add sample 50ul to be checked, mix; Wherein, described dilution is 0.01mol/L～0.5mol/L phosphate buffer, pH6～8;
A4, fully after reaction, get rid of liquid hole in, pure water is filled with after adding 1 of cleansing solution immediately in every hole, after standing 30 seconds, gets rid of, then washs at least one times with pure water, gets rid of, and pats dry; Wherein, described cleansing solution is the 0.01mol/L that includes 0.1%～1% surfactant～0.5mol/L phosphate buffer, pH6～8;
A5, every hole add 2 of enzyme conjugates, fully after the reaction, get rid of liquid hole in, and pure water is filled with after adding 1 of cleansing solution immediately in every hole, after standing 30 seconds, gets rid of, then washs at least one times with pure water, gets rid of, and pats dry;
A6, add each 1 of substrate solution and developer, mix, fully after chromogenic reaction, add 1 of stop buffer, mix cessation reaction; Wherein, described substrate solution is 0.1%～0.5% superoxol; Described developer is TMB; Described stop buffer is sulfuric acid or the NaOH of 1mol/L～2mol/L.
Described detection method, wherein, in steps A 3, also comprise step: negative hole, positive hole and blank hole are set, negative hole, positive hole add respectively 1 of dilution, then add respectively negative control product, each 50ul of positive reference substance, and the blank hole adds 2 of dilutions.
Described detection method, wherein, described abundant reaction is 37 ℃ of lucifuge reactions 30 minutes, described abundant chromogenic reaction is 37 ℃ of lucifuge reactions 10 minutes.
Described detection method wherein, in steps A 1, after standing 12～24 hours of room temperature, also comprises step: adopt 0.1%～5% ovalbumin or bovine serum albumin(BSA) sealing 2 hours, after vacuum freezedrying, make described enzyme-linked reaction plate.
Adopt such scheme, the present invention, by susceptibility, specificity and practicality, the economy of utilizing the tachysynthesis detection technique, has set up freshwater fish liver fluke tachysynthesis detection method, is applicable to simultaneously live fish and dead fish; Convenient and practical, kit is model of instant use once to turn it on, and the user needn't prepare separately or dilute, and operation is simple; Not only can, by the quantitative judged result of instrument, also can directly judge by naked eyes the Site Detection that the condition that especially is suitable for is limited; Safety and environmental protection, adopted harmless enzyme linked immunological developer tetramethyl benzidine, harm and the pollution of having avoided conventional method to bring.
The accompanying drawing explanation
Fig. 1 is the structural representation of product of the present invention;
Fig. 2 is the schematic flow sheet of the inventive method.
Embodiment
Below preferred embodiment of the present invention is described in detail.
As shown in Figure 1, the invention provides a kind of liver fluke rapid immune detection kit for fresh water fishes, enzyme-linked reaction plate 101 and various reagent 102 are set in its box body, and various reagent specifically comprises enzyme conjugates, cleansing solution, substrate solution, developer, dilution, stop buffer etc.; Wherein, described enzyme-linked reaction plate is from liver fluke, extracting soluble antigen purifying, then being coated with the enzyme-linked reaction plate that described soluble antigen makes; Described enzyme conjugates is the enzyme conjugates of the anti-fish immunoglobulin (Ig) of horseradish peroxidase mark.
Below enzyme-linked reaction plate and the various reagent of kit of the present invention is elaborated.
Described enzyme-linked reaction plate is coated soluble antigen on the general purpose polystyrene microwell plate, and then in room temperature standing 12～24 hours, after vacuum freezedrying, the pack sealing was made.After standing 12～24 hours of room temperature, can also adopt 0.1%～5% ovalbumin or bovine serum albumin(BSA) sealing 2 hours, and then after vacuum freezedrying, described enzyme-linked reaction plate is made in the pack sealing.Wherein, the general purpose polystyrene microwell plate, can be 48 orifice plates or 96 orifice plates, or other plates; Described soluble antigen is from infection animal, obtaining the liver fluke adult, and extraction obtains its soluble antigen, the i.e. antigen of the liver fluke adult of water soluble; Coating buffer is the damping fluid that contains 0.01%～1% soluble antigen, and described damping fluid is phosphate, borate or the carbonate buffer solution of 0.01mol/L～0.15mol/L, pH7.5～pH9.5.
Described enzyme conjugates, for adopting the fish immunoglobulin (Ig) immune animal of purifying, is isolated anti-fish immunoglobulin (Ig), and purified rear employing horseradish peroxidase is carried out immune labeled making.For example, described enzyme conjugates is the 0.01mol/L～0.05mol/L phosphate buffer of pH6～8, wherein contains the anti-fish immunoglobulin (Ig) of 0.05%～1% process horseradish peroxidase-labeled, i.e. antiantibody; And add the antiseptic that content is enough to bacteria growing inhibiting.For example, described antiseptic is 0.01%～0.1% thimerosal (Thimerosal also claims Sodium Mercurothiolate, merthiolate).The present invention does not have extra restriction to kind and the consumption of antiseptic, as long as can bacteria growing inhibiting.Wherein, phosphate buffer is preferably 0.05mol/L, pH7.0.
Described kit can also comprise negative control product (negative control) and/or positive reference substance (positive control), uses for ease of the user, preferably possesses simultaneously negative control product and positive reference substance.Described negative control product are normal fish serum sample, and described positive reference substance is the fish serum sample that contains liver fluke antibody.In general, positive reference substance and negative control product are control product of check test validity, and simultaneously also as the contrast of judged result, so the basic composition of reference substance, particularly positive reference substance should be as far as possible consistent with the composition that detects sample.The negative control product must detect in advance, determine and wherein do not contain test substance.Positive reference substance is matrix mainly with the damping fluid that contains protein protective agent, wherein adds a certain amount of material to be checked, and this amount is preferably in the reagent instructions and indicates; The amount added should match with the susceptibility of reagent, the light absorption value obtained in mensuration with examined the sample light absorption value relatively, a "ball-park" estimate can be arranged the amount of tested substance in sample.In reference substance, generally add microbiotic and antiseptic, in order to preservation.
In described kit, described cleansing solution is the 0.01mol/L that includes 0.1%～1% surfactant～0.5mol/L phosphate buffer, pH6～8; Substrate solution is 0.1%～0.5% superoxol; Developer is TMB; Dilution is 0.01mol/L～0.5mol/L phosphate buffer, pH6～8; Stop buffer is sulfuric acid or the NaOH of 1mol/L～2mol/L.
And, as shown in Figure 2, the invention allows for a kind of detection method for liver fluke rapid immune detection kit for fresh water fishes, it comprises the following steps.
A1, from infection animal, obtaining the liver fluke adult, and extract and to obtain its soluble antigen, coated described soluble antigen on the general purpose polystyrene microwell plate, then in room temperature standing 12～24 hours, after vacuum freezedrying, make enzyme-linked reaction plate, and the pack sealing is preserved; Wherein, coating buffer is the damping fluid that contains 0.01%～1% described soluble antigen, and described damping fluid is phosphate, borate or the carbonate buffer solution of 0.01mol/L～0.15mol/L of pH7.5～pH9.5.
After standing 12～24 hours of room temperature, can also comprise step: adopt 0.1%～5% ovalbumin or bovine serum albumin(BSA) sealing 2 hours, after vacuum freezedrying, make described enzyme-linked reaction plate.
A2, the fish immunoglobulin (Ig) immune animal of adopting purifying, isolate anti-fish immunoglobulin (Ig), and purified rear employing horseradish peroxidase is carried out the immune labeled enzyme conjugates of making.Usually, described enzyme conjugates is the 0.01mol/L that contains 0.05%～1% the anti-fish immunoglobulin (Ig) of horseradish peroxidase-labeled～0.05mol/L phosphate buffer, pH6～8; And add the antiseptic that content is enough to bacteria growing inhibiting.For example, described antiseptic is 0.01% thimerosal.
A3, the every hole of described enzyme-linked reaction plate add respectively 1 of dilution, add sample 50ul to be checked, mix; Wherein, described cleansing solution is the 0.01mol/L that includes 0.1%～1% surfactant～0.5mol/L phosphate buffer, pH6～8; Be more preferably, described dilution is the 0.1mol/L phosphate buffer, pH7.0.
And in steps A 3, can also comprise step: negative hole, positive hole and blank hole are set, and negative hole, positive hole add respectively 1 of dilution, then add respectively negative control product, each 50ul of positive reference substance, the blank hole adds 2 of dilutions.
A4, fully after reaction, get rid of liquid hole in, pure water is filled with after adding 1 of cleansing solution immediately in every hole, after standing 30 seconds, gets rid of, then washs at least one times with pure water, gets rid of, and pats dry; Wherein, described cleansing solution is the 0.01mol/L that includes 0.1%～1% surfactant～0.5mol/L phosphate buffer, pH6～8.General, adopt the pure water washing at least four times or more than, better effects if some.Wherein, fully reaction is generally 37 ℃ of lucifuge reactions 30 minutes.
A5, every hole add 2 of enzyme conjugates, fully after the reaction, get rid of liquid hole in, and pure water is filled with after adding 1 of cleansing solution immediately in every hole, after standing 30 seconds, gets rid of, then washs at least one times with pure water, gets rid of, and pats dry.General, adopt the pure water washing at least four times or more than, better effects if some.Wherein, fully reaction is generally 37 ℃ of lucifuge reactions 30 minutes.
A6, add each 1 of substrate solution and developer, mix, fully after chromogenic reaction, add 1 of stop buffer, mix cessation reaction; Wherein, described substrate solution is 0.1%～0.5% superoxol; Described developer is TMB; Described stop buffer is sulfuric acid or the NaOH of 1mol/L～2mol/L.For example, described stop buffer is the sulfuric acid of 1.5mol/L.Described abundant chromogenic reaction is generally 37 ℃ of lucifuge reactions 10 minutes.
Then, judge in the hole that adds sample to be checked, whether liquid develops the color.
Below kit of the present invention and detection method are specifically described.For achieving the above object, the present invention has adopted following technical scheme.
One, liver fluke antigen prepare Purification, specifically comprise: obtain the liver fluke adult from infection animal; Extract liver fluke Soluble Adult antigen; Antigen is carried out to purifying, such as methods such as adopting chromatography, antigen is carried out to purifying; The antigen of purification Identification.Authentication method can comprise: measure protein content; Protein electrophoresis is observed the antigen composition; ELISA measures antigen active.By by the coated reaction plate of purifying antigen, with known antibodies content liver fluke immune serum, react, whether observing response intensity reaches previous level, thus the judgement antigen active.
Two, the fresh-water fishes immunoglobulin (Ig) prepare the Purification mark, specifically comprise the following steps.
2.1, fish can produce immune response to antigenic stimulus, form antibody.Research to various fish such as bony fish eel, trout, carps shows, the fish immunoglobulin (Ig) is mainly the 19S type, has structure and the performance of antibody.
2.2, to the extraction and purification of fish immunoglobulin (Ig).At first, adopt the globulin in salting out method primary extraction fish serum; Secondly, adopt chromatography to be further purified and extract its immunoglobulin (Ig).
2.3, purified is carried out to Analysis and Identification.Comprise and measure protein content and carry out electrophoretic analysis etc., for example, take BRADFORD the quantition of microgram quantities of protein method to measure protein concentration; Take SDS-PAGE to carry out constituent analysis to the fish immunoglobulin (Ig) of purifying, the immunoglobulin (Ig) after purifying only has 55KD left and right heavy chain and 23KD left and right light chain two strip-types, without obvious other assorted band;
2.4, the preparation of anti-fish immune globulin antibody: with the fish immunoglobulin (Ig) immune animal of purifying; The anti-fish immunoglobulin (Ig) of separation and purification; Enzyme is immune labeled, for example adopts improvement sodium periodate method to carry out horseradish peroxidase-labeled, then separation and purification identifying.Improvement sodium periodate method, be to get the acetate buffer 0.5ml that 5mgHRP is dissolved in 0.2mol/L pH5.6, then add the sodium periodate of 0.1mol/L 0.25ml to mix.Add the about 5mg of anti-fish immune globulin antibody to mix, adjust pH to be 9.0,4 ℃ and spend the night, add sodium borohydride 0.5mg to mix, dialysed overnight.In above solution, slowly add isopyknic saturated ammonium sulfate solution, mix, 4 ℃ of 30min, centrifugal, remove supernatant, precipitation is dissolved with a little 0.02mol/L pH7.4 PBS liquid, and the bag filter of packing into spends the night at 4 ℃ of dialysis desalinations with same liquid; Take out centrifugal next day, to remove insolubles, obtains enzyme-antibody conjugates, adds to 5ml with 0.02mol/L pH7.4PBS liquid; After titration is qualified, add equivalent high-quality glycerine, the packing bottle, low temperature is preserved.Certainly, also can adopt the anti-fish immune globulin antibodies of mark such as alkaline phosphatase (alkaline pHosohatase, AP), glucose oxidase, beta-D-galactosidase and urase.The present invention to concrete improvement sodium periodate method, horseradish peroxidase-labeled method etc. all without additional limits.
Three, set up liver fluke rapid immune detection kit for fresh water fishes
3.1, antigen coated microplate: adopt Fast Packet by technique, in advance envelope antigen on the general purpose polystyrene microwell plate.Coating buffer is phosphate, borate or the carbonate buffer solution that contains the 0.01-0.15mol/L of appropriate antigen, pH7.5-9.5; The standing 12-24 hour of room temperature; With 0.1-5% ovalbumin or bovine serum albumin(BSA), sealed 2 hours; Vacuum freezedrying; The vacuum bagging sealing, and indicate outside the plate bag.
Package amount is generally 0.01%～1% of damping fluid, under the practical measurement condition, generally carries out " drip and join ", usings and selects can reach high sensitive greatest dilution as the working concentration in kit.For example, in actual selection, can measure the suitableeest package amount by the square formation titrimetry; Be about to antigen and carry out a series of dilutions with coated damping fluid, as 1: 100,1: 200,1: 400......1: 25600, with certain density enzyme conjugates, react, select OD (Optical Delnsity, optical density, also claim absorbance) value is coated optimum concentration at the dilutability of 1.2 left and right.
3.2, enzyme conjugates liquid: phosphate buffer, pH7.0,0.05mol/L, adding content is enough to the appropriate antiseptic of bacteria growing inhibiting, as percentage by weight, be 0.01% thimerosal, and percentage by weight is 0.05%～1% anti-fish immune globulin antibody-horseradish peroxidase-labeled bond.For example, as mentioned above, label adopts the sodium periodate method that horseradish peroxidase is resisted to fish immunoglobulin (Ig) antiantibody mark.
3.3, washing agent: adopt the phosphate buffer (pH7.0) of 0.5%TRITON-X100, can also add appropriate antiseptic, for example 0.01%～1% Sodium Mercurothiolate.
3.4, substrate solution and developer: substrate solution is superoxol; Developer is harmless TMB (3,3 ', 5,5 '-tetramethyl benzidine) solution, also can be ABTS (2,2 '-Bian nitrogen base-bis-(3-ethyl benzo thiophene pyrrolin-6 sulfonic acid)) or o-phenylenediamine (O-phenylenediamine, OPD) etc.
3.5, the sample dilution: phosphate buffer, pH7.0,0.1mol/L.
3.6, stop buffer: 1.0-2mol/L sulfuric acid or NaOH.
3.7, above component comprised the needed whole reagent of detection except pure water, wherein the equal adjusted of liquid component, in working concentration the plastic dropping bottle of packing into, need not be diluted and can directly use; Plastic tube with cover is put in the negative and positive contrast, can directly use; Coated plate also can directly be used after sealing off.
Liver fluke rapid immune detection kit for fresh water fishes of the present invention, select the liver fluke soluble antigen, and substep makes refining antigen by sad method and DEAE (Diethylaminoethyl, diethylaminoethanol) 52 ion chromatography column chromatography purifications.Carry out trial test, measure and select suitable antigen coated concentration.With the antigen coated 96 hole polystyrene microwell plates of carbonate buffer solution dilution, 100 microlitres/hole; Ambient temperature overnight; Get rid of liquid in hole, with same dosage, contain phosphate buffer (pH7.0,0.05mol/L) the room temperature sealing 2 hours of 1% ovalbumin; Vacuum freezedrying; Under vacuum condition, pack, on the plate bag, paste corresponding sign.
Then with the anti-fish immunoglobulin (Ig) horseradish peroxidase-labeled bond of phosphate buffer dilution.Preparation contains the cleansing solution of 0.5%TRITON-X100.The preparation hydrogen peroxide is as substrate, and preparation TMB solution is as developer.The sample dilution is phosphate buffer.Stop buffer is the 2mol/L sulfuric acid solution.Above liquid component, in the plastic dropping bottle of packing into.
Liver fluke antigen coated microplate and presser for liquid reagent bottle are packed in kit successively, the parallel placement of standing; After checking, after content, capping, be liver fluke rapid immune detection kit for fresh water fishes.
Kit of the present invention adopts the tachysynthesis detection technique to detect liver fluke in freshwater fish.This reagent sensitivity is high, and high specificity is reproducible, easy to operate quick, can use several times.
For example, the reagent of kit is composed as follows: 1. pre-coated plate, 48T/96T; 2. enzyme conjugates (No. 1 liquid) is 1; 3. cleansing solution (No. 2 liquid) is 1; 4. substrate (No. 3 liquid) is 1; 5. developer (No. 4 liquid) is 1; 6. sample dilution (No. 5 liquid) is 1; 7. stop buffer (No. 6 liquid) is 1; 8. positive reference substance is 1; 9. the negative control product are 1; Can also comprise 1 part of 10. operation instructions.
Running program is described as follows:
1. application of sample reaction: take out the reaction lath of aequum, every hole adds 1 of sample dilution (No. 5 liquid), then adds respectively sample 50ul to be checked, mixes.Establish simultaneously each hole of feminine gender, the positive and blank, negative, positive control is synchronizeed and is processed with sample to be checked, and the blank hole only adds 2 of sample dilutions (No. 5 liquid).37 ℃ of lucifuge reactions were got rid of liquid in hole after 30 minutes, and every hole adds 1 of cleansing solution (No. 2 liquid), with distilled water, fills with immediately, after standing 30 seconds, got rid of, then, directly with distilled water washing four times, got rid of, and patted dry.
2. add enzyme reaction: all the other every holes add 2 of enzyme conjugates (No. 1 liquid) except the blank hole, and 37 ℃ of lucifuge reactions were got rid of liquid in hole after 30 minutes, and as above washing, pat dry.
3. chromogenic reaction: add each 1 of substrate (No. 3 liquid) and developer (No. 4 liquid), mix, 37 ℃ of lower lucifuges colour developings 10 minutes.Add 1 of stop buffer (No. 6 liquid), mix, cessation reaction (after adding stop buffer, blueness can become yellow).TMB solution is during as developer, can the lucifuge chromogenic reaction, and the time of general chromogenic reaction also can extend a little, for example 10～30 minutes.
Then, relatively add in sample aperture to be checked the shade of liquid in liquid and negative control product hole by naked eyes or microplate reader, carry out judgement as a result, be described as follows.
1. visual inspection: negative control is near colourless, and positive control is obvious yellow, means that test effectively.Treat that verify is negative near this sample of colourless expression, treat that verify is obvious yellow this sample of expression positive; Perhaps sample aperture color to be checked obviously is deeper than negative control product hole, and sample to be checked is the liver fluke positive.
2. instrument judgement: read O.D value by microplate reader in 450nm (620nm makes reference wavelength) with the blank zeroing, treat that verify O.D value (absorbance) is greater than 2.1 times of persons of negative control (containing 2.1 times) positive.Otherwise sample to be checked is the liver fluke feminine gender.When negative control O.D value is calculated by 0.05 lower than 0.05 the time.
It should be noted that
1. kit is preserved under 2-8 ℃, and while taking out, first balance is used to room temperature at every turn.Drop bottle at every turn with after lid must be tightened, between each bottle cap, can't use with.Between each kit, can not use with.
2., when contrast is dried frozen aquatic products as negative and positive, please first by sign, add distilled water to dissolve rear the use.
3. while washing, distilled water is filled it up with in hole, parked for 30 seconds at every turn, get rid of liquid in hole.Other water sources such as forbidding tap water.
4. for improving the comparability of test, the instrument sentence read result is used in suggestion.
5. if had ready conditions, preferably use water bath to hatch.
Adopt fresh-water fishes liver fluke tachysynthesis of the present invention to detect box, open up the new way that the fresh-water fishes liver fluke detects, utilize susceptibility, specificity and practicality, the economy of tachysynthesis detection technique, set up fresh-water fishes liver fluke tachysynthesis detection method; Realize the commercialization of fresh-water fishes liver fluke detection technique, promoted effectively comprehensive raising of fresh-water fishes liver fluke level monitoring, and for the food safety Regulation of fish meat provides one to be worth the thinking of using for reference, thereby the liver fluke preventing and controlling promoted.This not only greatly facilitates user's operation, but also has improved the consistance that detects index, and practicality and reliability all improve.
Kit of the present invention is convenient and practical, safety and environmental protection, and kit not only has been equipped with and has detected necessary reagent component, and is model of instant use once to turn it on, and the user needn't prepare separately or dilute, and makes that operation is simple.Not only can, by the quantitative judged result of instrument, also can directly judge by naked eyes the Site Detection that the condition that especially is suitable for is limited.The present invention has adopted harmless TMB reagent, harm and the pollution of having avoided conventional method to bring.
Should be understood that, for those of ordinary skills, can be improved according to the above description or conversion, and all these improve and conversion all should belong to the protection domain of claims of the present invention.
Claims (3)
1. a liver fluke rapid immune detection kit for fresh water fishes, arrange enzyme-linked reaction plate, enzyme conjugates, cleansing solution, substrate solution, developer, dilution, stop buffer in its box body; It is characterized in that, described enzyme-linked reaction plate is from liver fluke, extracting soluble antigen purifying, then being coated with the enzyme-linked reaction plate that described soluble antigen makes; Described enzyme conjugates is the enzyme conjugates of the anti-fish immunoglobulin (Ig) of horseradish peroxidase mark, described soluble antigen is the antigen of water-soluble liver fluke adult, and substep makes refining described soluble antigen by sad method and DEAE52 ion chromatography column chromatography purification;
Described enzyme-linked reaction plate is coated soluble antigen on the general purpose polystyrene microwell plate, then after standing 12～24 hours of room temperature, adopt 0.1%～5% ovalbumin or bovine serum albumin(BSA) sealing 2 hours, then after vacuum freezedrying, the pack sealing is made; Wherein, described soluble antigen is from infection animal, obtaining the liver fluke adult, and extraction obtains its soluble antigen; Coating buffer is the damping fluid that contains 0.01%～1% soluble antigen, and described damping fluid is phosphate, borate or the carbonate buffer solution of 0.01mol/L～0.15mol/L, pH7.5～pH9.5;
Described enzyme conjugates, for adopting the fish immunoglobulin (Ig) immune animal of purifying, is isolated anti-fish immunoglobulin (Ig), and purified rear employing horseradish peroxidase is carried out immune labeled making;
The Purification mark for preparing to the fresh-water fishes immunoglobulin (Ig) comprises step:
At first, adopt the globulin in salting out method primary extraction fish serum; Secondly, adopt chromatography to be further purified and extract its immunoglobulin (Ig); Purified is carried out to Analysis and Identification, comprise and measure protein content and carry out electrophoretic analysis; . the preparation of anti-fish immune globulin antibody: with the fish immunoglobulin (Ig) immune animal of purifying; The anti-fish immunoglobulin (Ig) of separation and purification; Enzyme is immune labeled, adopts improvement sodium periodate method to carry out horseradish peroxidase-labeled, then separation and purification identifying; Improvement sodium periodate method, to get the acetate buffer 0.5ml that 5mgHRP is dissolved in 0.2mol/L pH5.6, add again the sodium periodate of 0.1mol/L0.25ml to mix, add the about 5mg of anti-fish immune globulin antibody to mix, adjusting pH is 9.0,4 ℃ are spent the night, and add sodium borohydride 0.5mg to mix, dialysed overnight, in above solution, slowly add isopyknic saturated ammonium sulfate solution, mix, 4 ℃ of 30min are centrifugal, remove supernatant, precipitation is dissolved with a little 0.02mol/L pH7.4PBS liquid, and the bag filter of packing into spends the night at 4 ℃ of dialysis desalinations with same liquid; Take out centrifugal next day, to remove insolubles, obtains enzyme-antibody conjugates, adds to 5ml with 0.02mol/L pH7.4PBS liquid; After titration is qualified, add equivalent high-quality glycerine, the packing bottle, low temperature is preserved;
Described enzyme conjugates is the 0.05mol/L phosphate buffer that contains 0.05%～1% the anti-fish immunoglobulin (Ig) of horseradish peroxidase-labeled, pH7.0; And add the antiseptic that content is enough to bacteria growing inhibiting, described antiseptic is 0.01%～0.1% thimerosal;
Described kit also comprises negative control product and positive reference substance;
Described cleansing solution is the 0.01mol/L that includes 0.1%～1% surfactant～0.5m0l/L phosphate buffer, pH6～8; Substrate solution is 0.1%～0.5% superoxol; Developer is TMB; Dilution is the 0.1mol/L phosphate buffer, pH7.0; Stop buffer is sulfuric acid or the NaOH of 1mol/L～2mol/L.
2. detection method for kit claimed in claim 1 for non-diagnostic purpose, it comprises step:
A1, the every hole of enzyme-linked reaction plate add respectively 1 of dilution, add sample 50ul to be checked, mix; Wherein, described dilution is 0.01mol/L～0.5mol/L phosphate buffer, pH6～8;
Negative hole, positive hole and blank hole are set, and negative hole, positive hole add respectively 1 of dilution, then add respectively negative control product, each 50ul of positive reference substance, and the blank hole adds 2 of dilutions;
A2, fully after reaction, get rid of liquid hole in, pure water is filled with after adding 1 of cleansing solution immediately in every hole, after standing 30 seconds, gets rid of, then washs at least one times with pure water, gets rid of, and pats dry; Wherein, described cleansing solution is the 0.01mol/L that includes 0.1%～1% surfactant～0.5mol/L phosphate buffer, pH6～8;
A3, every hole add 2 of enzyme conjugates, fully after the reaction, get rid of liquid hole in, and pure water is filled with after adding 1 of cleansing solution immediately in every hole, after standing 30 seconds, gets rid of, then washs at least one times with pure water, gets rid of, and pats dry;
A4, add each 1 of substrate solution and developer, mix, fully after chromogenic reaction, add 1 of stop buffer, mix cessation reaction; Wherein, described substrate solution is 0.1%～0.5% superoxol; Described developer is TMB; Described stop buffer is sulfuric acid or the NaOH of 1mol/L～2mol/L.
3. detection method according to claim 2, is characterized in that, described abundant reaction is 37 ℃ of lucifuge reactions 30 minutes, and described abundant chromogenic reaction is 37 ℃ of lucifuge reactions 10 minutes.
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| CN101603964A (en) * | 2009-07-10 | 2009-12-16 | 中国海洋大学 | The enzyme-linked immune detection method of Anisakid nematode in the marine product |
| US8067241B2 (en) * | 2009-08-26 | 2011-11-29 | General Electric Company | Method and apparatus for antigen retrieval process |
| CN102146446A (en) * | 2011-01-11 | 2011-08-10 | 中国疾病预防控制中心寄生虫病预防控制所 | Method and kit for quickly detecting clonorchiasis sinensis metacercaria in freshwater fish |
| CN103323585B (en) * | 2013-06-06 | 2015-07-22 | 广东海大畜牧兽医研究院有限公司 | ELISA kit used for detecting fish streptococcus agalactiae IgM antibody, and preparation method thereof |
| CN105277720B (en) * | 2015-10-08 | 2017-03-22 | 深圳华康生物医学工程有限公司 | ELISA kit for detection of liver fluke IgG4 antibody and preparation method thereof |
| CN111308081A (en) * | 2020-03-10 | 2020-06-19 | 中国工程物理研究院职工医院 | Universal enzyme-linked immunoassay test board and preparation method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1424585A (en) * | 2003-01-15 | 2003-06-18 | 胡绍良 | Quick enzyme immuno-determining method and box for food borne parasites |
| CN2604688Y (en) * | 2003-03-24 | 2004-02-25 | 中山大学 | Clonorchis sinensis bladder worm immunocolloidal gold detecting box |
-
2007
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1424585A (en) * | 2003-01-15 | 2003-06-18 | 胡绍良 | Quick enzyme immuno-determining method and box for food borne parasites |
| CN2604688Y (en) * | 2003-03-24 | 2004-02-25 | 中山大学 | Clonorchis sinensis bladder worm immunocolloidal gold detecting box |
Non-Patent Citations (6)
| Title |
|---|
| 刘健.鲜鱼与肝吸虫病.《医学文选》.1990,10. |
| 华支睾吸虫病快速ELISA诊断试剂盒的建立;胡绍良等;《中国寄生虫病防治杂志》;19951231;第8卷(第4期);295-296 * |
| 肝吸虫囊蚴在鱼体内分布及其在流行病学上的意义;谷宗藩等;《昌潍医学院学报》;19830402;35，36，42 * |
| 胡绍良等.华支睾吸虫病快速ELISA诊断试剂盒的建立.《中国寄生虫病防治杂志》.1995,第8卷(第4期),295-296. |
| 谷宗藩等.肝吸虫囊蚴在鱼体内分布及其在流行病学上的意义.《昌潍医学院学报》.1983,35，36，42. |
| 鲜鱼与肝吸虫病;刘健;《医学文选》;19900302;10 * |
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Denomination of invention: A rapid immunoassay kit and detection method for freshwater fish liver fluke Effective date of registration: 20230927 Granted publication date: 20131127 Pledgee: Bank of Jiangsu Limited by Share Ltd. Shenzhen branch Pledgor: SHENZHEN COMBINED BIOTECH Co.,Ltd. Registration number: Y2023980059542 |
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