CN101387641B - A freshwater fish liver fluke rapid immunoassay kit and detection method - Google Patents

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

A freshwater fish liver fluke rapid immunoassay kit and detection method

technical field

The invention relates to the technical field of biological detection, in particular to a rapid immunological detection kit and detection method for freshwater fish liver fluke.

Background technique

Liver fluke is a common human parasite that is acquired by ingesting freshwater fish that contain liver fluke metacercariae. Liver fluke adults parasitize in the bile ducts of the human liver, leading to liver fluke disease, causing a series of liver and gallbladder damage, including bile duct inflammation, stones, biliary cirrhosis, and finally can induce liver cancer.

Currently, there are many methods for diagnosing human liver fluke disease, including pathogenic methods to detect fecal eggs and immunological methods to detect liver fluke antibodies in patients' serum. The etiology method is accurate and reliable, but the eggs are small, ovulation is irregular, and it is easy to miss the diagnosis. In addition, it is necessary to collect the patient's feces, repeatedly precipitate with clean water and wash it several times, take the sediment, and then check it with a high-powered microscope by professional technicians. The process is long. Generally, it takes several hours or more, and the operation is cumbersome and inconvenient, labor-intensive, and it is difficult to popularize the census; the immunological method is sensitive and specific, convenient and fast, and only needs to detect serum, so it has been widely used.

Among immunological methods, enzyme-linked immunoassay technology is the most common application. In the 1990s, a rapid immunoassay kit for detecting liver flukes in humans was successfully developed in China (Hu Shaoliang et al., Rapid ELISA Diagnosis of Clonorchiasis sinensis The establishment of the kit, Chinese Journal of Parasitic Disease Control, Volume 8, Issue 4, 1995, page number: 295-296), the sensitivity reaches over 92%, and the specificity reaches over 98%.

However, in order to truly cut off the prevalence of liver fluke and protect people's health, it is necessary to carry out safety supervision on freshwater fish meat from the source to prevent diseased fish containing liver fluke metacercaria from entering the market. However, there is still a lack of effective means for the detection of liver flukes in freshwater fish. The traditional fish liver fluke inspection is to use a microscope to observe the liver fluke metacercariae in the fish meat, but it is very difficult to observe the metacercariae less than 1 mm in the fish meat with a weight of 1000 grams in this method. , not only inefficient, but also prone to omissions. Therefore, except for scientific research, microscopic examination of fish meat and liver fluke has hardly been applied in practice, which is also an important reason for the lack of safety supervision of freshwater fish liver fluke. The immunological detection method of freshwater fish liver fluke has not been established so far.

Therefore, the prior art does not have effective tools for the detection of liver flukes in freshwater fish.

Contents of the invention

The purpose of the present invention is to provide a freshwater fish liver fluke rapid immunological detection kit and detection method, thereby providing a detection tool and method for detecting freshwater fish liver fluke.

Technical scheme of the present invention is as follows:

A quick immunoassay kit for freshwater fish liver fluke, in which an enzyme-linked reaction plate, an enzyme conjugate, a washing solution, a substrate solution, a color developing agent, a diluent, and a stop solution are arranged in the box; wherein, the enzyme-linked reaction The plate is an enzyme-linked reaction plate prepared by extracting and purifying soluble antigens from liver flukes, and then coating the soluble antigens; the enzyme conjugate is an enzyme conjugate of horseradish peroxidase-labeled anti-fish immunoglobulin.

The kit, wherein, the enzyme-linked reaction plate is made by coating a soluble antigen on a general-purpose polystyrene microwell plate, then standing at room temperature for 12 to 24 hours, and then bagging and sealing after lyophilization and vacuum drying; wherein , the soluble antigen is obtained from the adult liver fluke from infected animals, and extracted to obtain its soluble antigen; the coating solution is a buffer containing 0.01% to 1% soluble antigen, and the buffer is 0.01mol/L to 0.15mol /L of phosphate, borate or carbonate buffer solution, pH7.5~pH9.5.

Said kit, wherein said enzyme conjugate is prepared by immunizing animals with purified fish immunoglobulin, isolating anti-fish immunoglobulin, and performing immunolabeling with horseradish peroxidase after purification.

Said kit, wherein said enzyme conjugate is 0.01mol/L～0.05mol/L phosphate buffer containing 0.05%～1% horseradish peroxidase-labeled anti-fish immunoglobulin, pH 6～ 8; and add preservatives in an amount sufficient to inhibit bacterial growth.

Said kit, wherein, it also includes at least one of a negative control substance and a positive control substance.

The kit, wherein, the washing solution is 0.01mol/L～0.5mol/L phosphate buffer solution containing 0.1%～1% surfactant, pH 6～8; the substrate solution is 0.1%～0.5 % hydrogen peroxide solution; the chromogen is 3,3',5,5'-tetramethylbenzidine; the diluent is 0.01mol/L～0.5mol/L phosphate buffer, pH6～8; the stop solution is 1mol/L～2mol/L sulfuric acid or sodium hydroxide.

A detection method for the kit according to claim 1, comprising the steps of:

A1. Obtain liver fluke adult worms from infected animals, and extract their soluble antigens, coat the soluble antigens on a general-purpose polystyrene microporous plate, then stand at room temperature for 12 to 24 hours, freeze and dry in a vacuum The enzyme-linked reaction plate is stored in a sealed bag; wherein, the coating solution is a buffer solution containing 0.01% to 1% of the soluble antigen, and the buffer solution is 0.01mol/L to 0.15 of pH7.5 to pH9.5 mol/L phosphate, borate or carbonate buffer;

A2. Using purified fish immunoglobulin to immunize animals, isolate anti-fish immunoglobulin, and use horseradish peroxidase to immunolabel after purification to make enzyme conjugates;

A3. Add 1 drop of diluent to each well of the enzyme-linked reaction plate, add 50ul of the sample to be tested, and mix well; wherein, the diluent is 0.01mol/L～0.5mol/L phosphate buffer, pH6～ 8;

A4. After fully reacting, shake off the liquid in the well, add 1 drop of washing solution to each well, and immediately fill it with pure water, let it stand for 30 seconds, shake it off, wash it with pure water at least once, shake it off, and pat dry; among them, all The washing solution is 0.01mol/L-0.5mol/L phosphate buffer containing 0.1%-1% surfactant, pH 6-8;

A5. Add 2 drops of enzyme conjugates to each well, shake off the liquid in the wells after fully reacting, add 1 drop of washing solution to each well, immediately fill with pure water, let it stand for 30 seconds, shake off, and then wash with pure water at least once, shake off, pat dry;

A6. Add 1 drop of substrate solution and 1 drop of chromogenic agent, mix well, add 1 drop of stop solution after sufficient color reaction, mix well, stop reaction; wherein, the substrate solution is 0.1%～0.5% hydrogen peroxide solution; the developer is 3,3',5,5'-tetramethylbenzidine; the stop solution is 1mol/L-2mol/L sulfuric acid or sodium hydroxide.

The detection method, wherein, in step A3, also includes the step of: setting negative wells, positive wells and blank control wells, adding 1 drop of diluent to the negative wells and positive wells respectively, and then adding each of the negative control substance and the positive control substance. 50ul, add 2 drops of diluent to the blank control well.

The detection method, wherein, the sufficient reaction is 30 minutes at 37° C. in the dark, and the sufficient color reaction is 10 minutes at 37° C. in the dark.

The detection method, wherein, in step A1, after standing at room temperature for 12 to 24 hours, it also includes the step of: using 0.1% to 5% ovalbumin or bovine serum albumin to seal for 2 hours, and then freeze and vacuum dry it to prepare into the enzyme-linked reaction plate.

By adopting the above scheme, the present invention establishes a rapid immunological detection method for liver fluke in freshwater fish by utilizing the sensitivity, specificity, practicability and economy of the rapid immunological detection technology, which is applicable to both live fish and dead fish; it is convenient and practical, and the reagent The box is ready-to-use, the user does not need to prepare or dilute it, and the operation is simple and easy; not only can the quantitative judgment result be used by the instrument, but also can be judged directly by the naked eye, which is especially suitable for on-site detection with limited conditions; it is safe and environmentally friendly. The harmful enzyme-linked immunosorbent reagent tetramethylbenzidine avoids the harm and pollution caused by conventional methods.

Description of drawings

Fig. 1 is the structural representation of product of the present invention;

Fig. 2 is a schematic flow chart of the method of the present invention.

Detailed ways

Preferred embodiments of the present invention are described in detail below.

As shown in Figure 1, the present invention provides a freshwater fish liver fluke rapid immunoassay kit, in which an enzyme-linked reaction plate 101 and various reagents 102 are arranged in the box, and various reagents specifically include enzyme conjugates, washing liquid, Substrate solution, chromogen, diluent, stop solution, etc.; wherein, the enzyme-linked reaction plate is an enzyme-linked reaction plate prepared by extracting and purifying soluble antigens from liver flukes, and then coating the soluble antigens; The enzyme conjugate is an enzyme conjugate of horseradish peroxidase labeled anti-fish immunoglobulin.

The enzyme-linked reaction plate and various reagents of the kit of the present invention are described in detail below.

The enzyme-linked reaction plate is made by coating the soluble antigen on a general-purpose polystyrene microwell plate, then standing at room temperature for 12 to 24 hours, freeze-dried in a vacuum, and then bagging and sealing. After standing still at room temperature for 12 to 24 hours, 0.1% to 5% ovalbumin or bovine serum albumin can be used to block for 2 hours, and then freeze and vacuum dry, then bag and seal to form the enzyme-linked reaction plate. Wherein, the general-purpose polystyrene microwell plate can be a 48-well plate or a 96-well plate, or other plates; the soluble antigen is obtained from infected animals with liver fluke adults and extracted to obtain its soluble antigen, which can be dissolved in water Antigens of liver fluke adult worms; the coating solution is a buffer containing 0.01% to 1% soluble antigen, and the buffer is 0.01mol/L to 0.15mol/L phosphate, borate or carbonate buffer , pH7.5~pH9.5.

The enzyme conjugate is prepared by adopting purified fish immunoglobulin to immunize animals, isolating anti-fish immunoglobulin, and adopting horseradish peroxidase to carry out immune labeling after purification. For example, the enzyme conjugate is 0.01mol/L-0.05mol/L phosphate buffer solution with pH 6-8, which contains 0.05%-1% of anti-fish immunoglobulin labeled with horseradish peroxidase, that is anti-antibodies; and added preservatives in amounts sufficient to inhibit bacterial growth. For example, the preservative is 0.01%-0.1% Thimerosal (Thimerosal, also known as Thimerosal, Ethylmercur). The present invention has no additional restrictions on the type and amount of the preservative, as long as it can inhibit the growth of bacteria. Among them, the best phosphate buffer is 0.05mol/L, pH7.0.

The kit can also include a negative control and/or a positive control. For the convenience of users, it is best to have both the negative control and the positive control. The negative control substance is a normal fish serum sample, and the positive control substance is a fish serum sample containing liver fluke antibody. Generally speaking, the positive control substance and the negative control substance are the control substances for testing the validity of the test, and also serve as the control for judging the results. Therefore, the basic composition of the control substance, especially the positive control substance, should be as consistent as possible with the composition of the test specimen. The negative control substance must be tested first to ensure that it does not contain the substance to be tested. The positive control substance is usually based on a buffer solution containing a protein protective agent, and a certain amount of the substance to be tested is added to it, which is best indicated in the reagent instruction manual; Comparing the absorbance value with the absorbance value of the tested sample can give a rough estimate of the amount of the tested substance in the sample. Antibiotics and preservatives are generally added to the reference substance to facilitate preservation.

In the kit, the washing solution is 0.01mol/L～0.5mol/L phosphate buffer solution containing 0.1%～1% surfactant, pH 6～8; the substrate solution is 0.1%～0.5% Hydrogen oxide solution; color reagent is 3,3',5,5'-tetramethylbenzidine; diluent is 0.01mol/L～0.5mol/L phosphate buffer, pH6～8; stop solution is 1mol/L L～2mol/L sulfuric acid or sodium hydroxide.

Moreover, as shown in FIG. 2 , the present invention also proposes a detection method for a rapid immunological detection kit for liver fluke in freshwater fish, which includes the following steps.

A1. Obtain liver fluke adult worms from infected animals, and extract their soluble antigens, coat the soluble antigens on a general-purpose polystyrene microporous plate, then stand at room temperature for 12 to 24 hours, freeze and dry in a vacuum The enzyme-linked reaction plate is stored in a sealed bag; wherein, the coating solution is a buffer solution containing 0.01% to 1% of the soluble antigen, and the buffer solution is 0.01mol/L to 0.15 of pH7.5 to pH9.5 mol/L phosphate, borate or carbonate buffer.

After standing at room temperature for 12 to 24 hours, it may also include the step of: using 0.1% to 5% ovalbumin or bovine serum albumin to block for 2 hours, freeze and vacuum dry to prepare the enzyme-linked reaction plate.

A2. Animals are immunized with purified fish immunoglobulin, and anti-fish immunoglobulin is isolated. After purification, horseradish peroxidase is used for immunolabeling to prepare an enzyme conjugate. Generally, the enzyme conjugate is 0.01mol/L-0.05mol/L phosphate buffer solution containing 0.05%-1% horseradish peroxidase-labeled anti-fish immunoglobulin, pH 6-8; and the added content Preservative sufficient to inhibit bacterial growth. For example, the preservative is 0.01% thimerosal.

A3. Add 1 drop of diluent to each well of the enzyme-linked reaction plate, add 50ul of the sample to be tested, and mix well; wherein, the washing solution is 0.01mol/L-1% surfactant containing 0.1%-1%. 0.5 mol/L phosphate buffer, pH 6-8; more preferably, the diluent is 0.1 mol/L phosphate buffer, pH 7.0.

Moreover, in step A3, it may also include the steps of: setting negative wells, positive wells and blank control wells, adding 1 drop of diluent to the negative wells and positive wells respectively, and then adding 50ul each of the negative control substance and the positive control substance respectively, and adding 50ul each to the blank control wells. Add 2 drops of diluent.

A4. After fully reacting, shake off the liquid in the well, add 1 drop of washing solution to each well, and immediately fill it with pure water, let it stand for 30 seconds, shake it off, wash it with pure water at least once, shake it off, and pat dry; among them, all The washing liquid is 0.01mol/L-0.5mol/L phosphate buffer solution containing 0.1%-1% surfactant, pH6-8. Generally, it is better to wash with pure water at least four times or more. Among them, the full reaction is generally 30 minutes at 37° C. in the dark.

A5. Add 2 drops of enzyme conjugates to each well, shake off the liquid in the wells after fully reacting, add 1 drop of washing solution to each well, immediately fill with pure water, let it stand for 30 seconds, shake off, and then wash with pure water at least once, Toss and pat dry. Generally, it is better to wash with pure water at least four times or more. Among them, the full reaction is generally 30 minutes at 37° C. in the dark.

A6. Add 1 drop of substrate solution and 1 drop of chromogenic agent, mix well, add 1 drop of stop solution after sufficient color reaction, mix well, stop reaction; wherein, the substrate solution is 0.1%～0.5% hydrogen peroxide solution; the developer is 3,3',5,5'-tetramethylbenzidine; the stop solution is 1mol/L-2mol/L sulfuric acid or sodium hydroxide. For example, the stop solution is 1.5 mol/L sulfuric acid. The full color reaction is generally carried out at 37° C. for 10 minutes in the dark.

Then, it is judged whether the liquid in the hole added to the sample to be tested is colored.

The kit and detection method of the present invention are described in detail below. In order to achieve the above object, the present invention adopts the following technical solutions.

1. Preparation, purification and identification of liver fluke antigens, specifically including: obtaining adult liver flukes from infected animals; extracting soluble antigens of adult liver flukes; purifying the antigens, for example, using chromatography and other methods to purify the antigens; identifying the purified antigens. The identification method may include: measuring protein content; observing antigenic components by protein electrophoresis; and measuring antigenic activity by ELISA. Antigen activity is judged by coating the purified antigen on the reaction plate and reacting with liver fluke immune serum with known antibody content to observe whether the reaction intensity reaches the original level.

2. The preparation, purification and identification marker of the freshwater fish immunoglobulin specifically includes the following steps.

2.1. Fish can produce immune response to antigen stimulation and form antibodies. Studies on bony fish such as eel, trout, carp and other fish have shown that fish immunoglobulins are mainly of the 19S type, which has the structure and properties of antibodies.

2.2. Extraction and purification of fish immunoglobulin. Firstly, the globulin in the fish serum was extracted primarily by salting out; secondly, the immunoglobulin was further purified and extracted by chromatography.

2.3. Analyze and identify the purified product. Including determination of protein content and electrophoresis analysis, etc. For example, BRADFORD trace protein assay method is used to determine protein concentration; SDS-PAGE is used to analyze the composition of purified fish immunoglobulin. The purified immunoglobulin has only about 55KD heavy chain and 23KD around two light chain bands, no other obvious miscellaneous bands;

2.4. Preparation of anti-fish immunoglobulin antibody: immunize animals with purified fish immunoglobulin; separate and purify anti-fish immunoglobulin; enzyme immunolabeling, for example, horseradish peroxidase labeling by improved sodium periodate method, Then isolated, purified and identified. The improved sodium periodate method is to dissolve 5mg of HRP in 0.5ml of 0.2mol/L pH5.6 acetate buffer, then add 0.1mol/L 0.25ml of sodium periodate and mix well. Add about 5 mg of anti-fish immunoglobulin antibody and mix evenly, adjust the pH to 9.0, overnight at 4°C, add 0.5 mg of sodium borohydride, mix evenly, and dialyze overnight. Slowly add an equal volume of saturated ammonium sulfate solution to the above solution, mix well, centrifuge at 4°C for 30 minutes, remove the supernatant, dissolve the precipitate with a little 0.02mol/L pH7.4 PBS solution, put it into a dialysis bag, and use the same solution in Dialyze and desalt overnight at 4°C; take it out and centrifuge the next day to remove insoluble matter, and obtain the enzyme-antibody conjugate, add 0.02mol/L pH7.4 PBS solution to 5ml; after the titer is qualified, add the same amount of high-quality glycerol, Divide into vials and store at low temperature. Of course, anti-fish immunoglobulin antibodies can also be labeled with alkaline phosphatase (alkaline pHosohatase, AP), glucose oxidase, β-D-galactosidase, and urease. The present invention has no additional restrictions on the specific improved sodium periodate method, horseradish peroxidase labeling method and the like.

3. Establishment of rapid immunoassay kit for freshwater fish liver fluke

3.1. Antigen-coated plate: Antigen is pre-coated on a general-purpose polystyrene microwell plate using a rapid coating process. The coating solution is 0.01-0.15mol/L phosphate, borate or carbonate buffer solution containing an appropriate amount of antigen, pH7.5-9.5; stand at room temperature for 12-24 hours; use 0.1-5% ovalbumin or bovine serum albumin for 2 hours; lyophilized and vacuum-dried; vacuum bagged and sealed, and marked on the outside of the bag.

The amount of coating is generally 0.01% to 1% of the buffer, and "dropping" is generally performed under actual measurement conditions to select the maximum dilution that can achieve high sensitivity as the working concentration in the kit. For example, in actual selection, square array titration can be used to determine the optimal coating amount; that is, the antigen is diluted serially with coating buffer, such as 1:100, 1:200, 1:400... .1:25600, react with a certain concentration of enzyme conjugates, select the dilution with an OD (Optical Delnsity, optical density, also known as absorbance) value of about 1.2 as the optimal concentration for coating.

3.2. Enzyme conjugate solution: phosphate buffer solution, pH 7.0, 0.05mol/L, plus an appropriate amount of preservatives sufficient to inhibit bacterial growth, such as 0.01% by weight of thimerosal, and 0.05% to 1% by weight Anti-fish immunoglobulin antibody-horseradish peroxidase-labeled conjugate. For example, as described above, a horseradish peroxidase anti-fish immunoglobulin anti-antibody was labeled using the sodium periodate method.

3.3. Detergent: Use 0.5% TRITON-X100 phosphate buffer (pH 7.0), and add an appropriate amount of preservatives, such as 0.01% to 1% thimerosal.

3.4. Substrate solution and color developer: the substrate solution is hydrogen peroxide solution; the color developer is TMB (3,3',5,5'-tetramethylbenzidine) solution which is harmless to human body, and It is ABTS (2,2'-edge nitrogen group-bis(3-ethylbenzothiapyrroline-6 sulfonic acid)) or o-phenylenediamine (O-phenylenediamine, OPD) and so on.

3.5. Sample diluent: phosphate buffer, pH 7.0, 0.1mol/L.

3.6. Stop solution: 1.0-2mol/L sulfuric acid or sodium hydroxide.

3.7. The above components include all the reagents required for the detection except pure water. The liquid components have been adjusted to the working concentration and put into a plastic dropper bottle, which can be used directly without dilution; the positive and negative controls are put in a capped It can be used directly in the plastic tube; the coated board can also be used directly after unpacking.

The freshwater fish liver fluke rapid immunoassay kit of the present invention selects liver fluke soluble antigens, and obtains refined antigens through octanoic acid method and DEAE (Diethylaminoethyl, diethylaminoethanol) 52 ion chromatography column chromatography step by step. Carry out a preliminary test, determine and select the appropriate antigen coating concentration. Dilute the antigen with carbonate buffer solution and coat the 96-well polystyrene microwell plate, 100 microliters/well; overnight at room temperature; shake off the liquid in the well, and use the same dose of phosphate buffer solution containing 1% ovalbumin ( pH7.0, 0.05mol/L) sealed at room temperature for 2 hours; lyophilized and vacuum-dried; bagged under vacuum conditions, and the corresponding label was affixed on the bag.

The anti-fish immunoglobulin horseradish peroxidase-labeled conjugate was then diluted in phosphate buffered saline. Prepare a washing solution containing 0.5% TRITON-X100. Hydrogen peroxide was prepared as a substrate, and TMB solution was prepared as a chromogen. The sample diluent is phosphate buffered saline. The stop solution is 2mol/L sulfuric acid solution. The above liquid ingredients are put into plastic dropper bottles.

Put the liver fluke antigen-coated plate and the liquid reagent bottle into the kit in sequence, and place them in parallel; after checking the contents, seal the cap, and it is the freshwater fish liver fluke rapid immunoassay kit.

The kit of the invention adopts a rapid immunoassay technology to detect liver flukes in freshwater fish. This reagent has high sensitivity, strong specificity, good repeatability, convenient and fast operation, and can be used in multiple times.

For example, the kit’s reagent composition is as follows: 1. Pre-coated plate, 48T/96T; 2. One enzyme conjugate (No. 1 solution); 3. One washing solution (No. 2 solution); 4. Substrate ( No. 3 liquid) 1; 5. Chromogenic reagent (No. 4) 1; 6. Sample diluent (No. 5) 1; 7. Stop solution (No. 6) 1; 8. Positive control substance 1 piece; 9. 1 piece of negative control substance; 10. 1 copy of instruction manual can also be included.

The operating procedures are described in detail as follows:

1. Sample addition reaction: Take out the required amount of reaction strips, add 1 drop of sample diluent (No. 5 solution) to each well, then add 50ul of the sample to be tested respectively, and mix well. At the same time, a negative, positive and blank control well were set up, and the negative and positive controls were treated synchronously with the sample to be tested. Only 2 drops of sample diluent (solution No. 5) were added to the blank control well. After reacting at 37°C in the dark for 30 minutes, shake off the liquid in the wells, add 1 drop of washing solution (No. Go, pat dry.

2. Enzyme reaction: Add 2 drops of enzyme conjugate (No. 1 solution) to each well except the blank control well, react in the dark at 37°C for 30 minutes, shake off the liquid in the well, wash as above, and pat dry.

3. Color reaction: Add 1 drop of substrate (No. 3 solution) and 1 drop of color developer (No. 4 solution), mix well, and develop color at 37°C in the dark for 10 minutes. Add 1 drop of stop solution (No. 6 solution), mix well, and stop the reaction (the blue color will turn yellow after adding stop solution). When TMB solution is used as a color developing agent, it is not necessary to protect the color reaction from light, and generally the time of the color reaction can be slightly extended, for example, 10-30 minutes.

Then, use the naked eye or a microplate reader to compare the color depth of the liquid in the well of the sample to be tested and the liquid in the well of the negative control substance, and judge the result. The specific instructions are as follows.

1. Visual observation: the negative control is nearly colorless, and the positive control is obviously yellow, indicating that the test is effective. If the well to be tested is close to colorless, it means that the sample is negative, if the well to be tested is obviously yellow, it means that the sample is positive; or if the color of the well of the sample to be tested is obviously darker than that of the negative control substance, then the sample to be tested is positive for liver fluke.

2. Judgment of the instrument: use a microplate reader to read the O.D value at 450nm (620nm as the reference wavelength) with the blank control, and the O.D value (absorbance) of the well to be tested is greater than 2.1 times (including 2.1 times) of the negative control. Positive. Otherwise, the sample to be tested is negative for liver fluke. When the negative control O.D value is lower than 0.05, it is calculated as 0.05.

have to be aware of is,

1. The kit should be stored at 2-8°C. Every time it is taken out, it should be equilibrated to room temperature before use. The caps of the dropper bottles must be tightened after each use, and the caps of each bottle must not be mixed. The kits cannot be mixed.

2. If the negative and positive control is a freeze-dried product, please add distilled water to dissolve it according to the label before use.

3. When washing, fill the hole with distilled water, stop for 30 seconds each time, and shake off the liquid in the hole. Tap water and other water sources are prohibited.

4. In order to improve the comparability of the test, it is recommended to use an instrument to interpret the results.

5. If possible, it is best to use a water bath for incubation.

The freshwater cod liver fluke rapid immunoassay box of the present invention is used to open up a new way for freshwater cod liver fluke detection, and the rapid immunological detection of freshwater cod liver fluke is established by utilizing the sensitivity, specificity, practicability and economy of the rapid immunoassay technology Methods: Realized the commercialization of freshwater fish liver fluke detection technology, effectively promoted the overall improvement of freshwater fish liver fluke monitoring level, and provided a worthwhile reference for the food safety supervision of fish meat, thus promoting the prevention and control of liver fluke. This not only greatly facilitates the user's operation, but also improves the consistency of the detection index, and the practicability and reliability are both improved.

The kit of the present invention is convenient, practical, safe and environment-friendly. The kit is not only equipped with necessary reagent components for detection, but also is ready-to-use, and the user does not need to prepare or dilute separately, so that the operation is simple and easy. Not only can the results be quantitatively judged by instruments, but also can be judged directly by naked eyes, which is especially suitable for on-site detection with limited conditions. The invention adopts the harmless TMB reagent, avoiding the harm and pollution caused by conventional methods.

It should be understood that those skilled in the art can make improvements or changes based on the above description, and all these improvements and changes should belong to the protection scope of the appended 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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