CN109709338B - Enzyme linked immunosorbent assay kit for detecting skeletal muscle troponin I of cattle or sheep and preparation method and application thereof - Google Patents
Enzyme linked immunosorbent assay kit for detecting skeletal muscle troponin I of cattle or sheep and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to an enzyme-linked immunoassay kit for detecting skeletal muscle troponin I of cattle or sheep, a preparation method and application thereof, belonging to the technical field of immunology and the technical field of food safety analysis. The enzyme linked immunosorbent assay kit comprises an enzyme label plate coated with a capture antibody, a detection antibody marked by horseradish peroxidase, a bovine or sheep skeletal muscle troponin I standard solution, a substrate color development solution, a stop solution and a concentrated washing solution. The capture antibody is obtained by secreting a hybridoma cell strain skTnI-3A8 with the preservation number of CCTCC NO: C2018217, and the detection antibody is obtained by secreting a hybridoma cell strain skTnI-3D3 with the preservation number of CCTCC NO: C2018218. The enzyme linked immunosorbent assay kit has the advantages of high sensitivity, precision and accuracy, low cross reaction rate, suitability for large-batch sample detection and the like.
Description
Technical Field
The invention relates to an enzyme linked immunosorbent assay kit for detecting skeletal muscle troponin I of cattle or sheep, and a preparation method and application thereof, and belongs to the technical field of immunology and the technical field of food safety analysis.
Background
In meat products, due to reasons of price, religion, health and the like, regulations established by many countries require food labels to truly and definitely mark meat sources and prohibit adulteration behaviors so as to protect the benefits of consumers, but phenomena of confusing meat varieties in the market are still very common, adulteration modes comprise means of adulteration, mixing, extraction, counterfeiting and the like, particularly the most common behaviors of adulteration and adulteration of beef and mutton products, and the adulteration behaviors greatly damage the benefits of the consumers.
At present, many laboratories at home and abroad use molecular biology technical means to detect source components in meat products, and DNA detection methods are various and mainly comprise nucleic acid probe hybridization, DNA fingerprint analysis, PCR specific amplification, PCR-RFLP and the like. These methods have a certain sensitivity, but also have the disadvantages of high cost, high requirement on detection objects, large workload, incapability of on-site detection and the like, and particularly have great uncertainty on detection of cooked meat products, because the methods depend on the processing process of the cooked meat products to a great extent, and the diversity of the production process causes different degradation levels of gene substances. Furthermore, this method only detects possible sources of animal DNA, and must be done at all times to prevent cross-contamination, and milk, blood, and fat are all possible sources of DNA. Therefore, other methods are needed to mutually corroborate with other test results. The immunological method has the characteristics of high sensitivity, good specificity, low cost, convenient operation and the like, and is suitable for screening large-batch samples, wherein an enzyme-linked immunosorbent assay and a colloidal gold test strip are the most common methods.
However, cooked meat products are generally subjected to high temperature and high pressure treatment, and many proteins are denatured in the process, so that antigenicity and water solubility are lost, and the establishment of an immunological method is difficult. In order to apply the immunological method to the detection of animal-derived components, a specific thermostable protein must be found as a marker antigen, and then a specific monoclonal antibody against the antigen is developed. Troponin I (TnI) in animal skeletal muscle has species specificity, and can be used as a heat-resistant species marker protein for distinguishing meat species sources of different species of raw and cooked meat products. The bovine or sheep skeletal muscle troponin I (skTnI) is taken as a target detection object, and the establishment of the immunological detection and identification technology of bovine or sheep meat-derived components can be realized. Therefore, the preparation of the bovine or sheep skeletal muscle troponin I enzyme-linked immunoassay kit has important practical significance and social significance for carrying out the immunological detection and identification work of beef or mutton meat components.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defects of the prior art and provide the enzyme-linked immunoassay kit for detecting the skeletal muscle troponin I of the cattle or sheep, and the enzyme-linked immunoassay kit has the advantages of high sensitivity, precision and accuracy, low cross reaction rate, suitability for large-batch sample detection and the like. In addition, the invention further provides a preparation method and application of the enzyme linked immunosorbent assay kit for detecting the skeletal muscle troponin I of the cattle or sheep.
The technical problem of the invention is realized by the following technical scheme.
An enzyme linked immunosorbent assay kit for detecting skeletal muscle troponin I of cattle or sheep adopts a double-antibody sandwich method and consists of an enzyme label plate coated with a capture antibody, an enzyme-labeled detection antibody, a cattle or sheep skeletal muscle troponin I standard solution, a substrate chromogenic solution, a stop solution and a concentrated washing solution.
In the enzyme linked immunosorbent assay kit, the capture antibody is secreted by a hybridoma cell strain skTnI-3A8 with the preservation number of CCTCC NO: C2018217, and the detection antibody is secreted by a hybridoma cell strain skTnI-3D3 with the preservation number of CCTCC NO: C2018218; the two cell strains are delivered to China Center for Type Culture Collection (CCTCC) for preservation in 2018, 12 months and 20 days.
In the enzyme-linked immunoassay kit, the capture antibody and the detection antibody are murine, equine, ovine, rabbit or guinea pig monoclonal antibodies, preferably murine monoclonal antibodies; it is obtained by immunizing bovine or ovine skeletal muscle troponin I with an immunizing antigen.
The enzyme-linked immunosorbent assay kit is characterized in that the enzyme-labeled detection antibody is obtained by adopting an enzyme labeling scheme which is conventional in the field, and for example, the enzyme-labeled detection antibody can be obtained by coupling a labeled enzyme with the detection antibody by adopting a sodium iodate method.
According to the enzyme-linked immunosorbent assay kit, the labeled enzyme in the enzyme-labeled detection antibody is horseradish peroxidase or alkaline phosphatase, when the labeled enzyme is horseradish peroxidase, the substrate chromogenic solution comprises a chromogenic substrate A and a chromogenic substrate B, the chromogenic substrate A is hydrogen peroxide or carbamide peroxide, the chromogenic substrate B is o-phenylenediamine or tetramethylbenzidine, and the stop solution is 1-2mol/L sulfuric acid or hydrochloric acid buffer solution; when the marker enzyme is alkaline phosphatase, the substrate color development liquid is a para-nitrophosphate buffer solution, and the stop solution is 1-2mol/L sodium hydroxide.
In the enzyme linked immunosorbent assay kit, the concentrations of the bovine or sheep skeletal muscle troponin I standard solution are respectively 4.875mg/L, 9.75mg/L, 19.5mg/L, 39mg/L, 78.125mg/L, 156.25mg/L and 312.5 mg/L; the concentrated washing solution is 0.2mol/L phosphate buffer solution with pH7.4 containing 1% Tween-20.
In order to facilitate field monitoring and large-scale sample screening, a bovine or sheep skeletal muscle troponin I standard solution, a substrate chromogenic solution, a stop solution and a concentrated washing solution in the enzyme-linked immunosorbent assay kit are all provided in the form of working solutions.
Through the technical scheme, the invention can realize rapid detection of the bovine or sheep skeletal muscle troponin I in fresh meat and fresh meat products by a double-antibody sandwich enzyme-linked immunoassay method, the minimum detection limit of the bovine or sheep skeletal muscle troponin I is 4.8mg/L, the detection sensitivity of the bovine or sheep meat is 1%, and the double-antibody sandwich enzyme-linked immunoassay method has no cross reaction on chicken, duck and pig skeletal muscle troponin I extracts and has higher sensitivity and specificity.
The preparation method of the enzyme linked immunosorbent assay kit comprises the following steps:
(1) preparing an enzyme label plate coated with a capture antibody:
(a) diluting the capture antibody to 1:4000 with a coating buffer solution, and adding 100ul of capture antibody into the holes of the ELISA plate;
(b) incubating at 4 deg.C overnight or 37 deg.C for 2h, pouring out the liquid in the well, washing with washing solution for 4-5 times, and patting to dry;
(c) adding a sealing solution buffer solution, incubating at 37 ℃ for 2h at 200 ul/well;
(d) pouring out liquid in the holes, drying the holes, pumping the liquid in a vacuum drying oven at room temperature for 5 hours, and carrying out vacuum plastic packaging by using an aluminum foil bag to obtain an ELISA plate coated with a capture antibody;
wherein the coating buffer solution is 0.05mol/L carbonate buffer solution with pH9.6, and the blocking buffer solution is 1% gelatin;
(2) preparing an enzyme labeling detection antibody working solution:
horseradish peroxidase is coupled with a detection antibody by a sodium iodate method;
(3) the enzyme linked immunosorbent assay kit for detecting skeletal muscle troponin I of cattle or sheep is constructed, and comprises the following components:
(a) an ELISA plate coated with a capture antibody;
(b) enzyme labeling detection antibody working solution;
(c) bovine or sheep skeletal muscle troponin I standard solution: preparing a standard solution by a gradient dilution method, wherein the concentration of the standard solution is 4.875mg/L, 9.75mg/L, 19.5mg/L, 39mg/L, 78.125mg/L, 156.25mg/L and 312.5mg/L respectively in 1 ml/bottle;
(d) the substrate color developing solution A is a carbamide peroxide solution, and the substrate color developing solution B is a Tetramethylbenzidine (TMB) solution;
(e) the stop solution is 1-2mol/L sulfuric acid solution;
(f) the washing solution was concentrated to 0.2mol/L of phosphate buffer pH7.4 containing 1% Tween-20.
The application of the enzyme linked immunosorbent assay kit in the application of the skeletal muscle troponin I of cattle or sheep in fresh meat and products thereof comprises the following steps:
(1) sample pretreatment
Removing fat and connective tissue from fresh meat, processing meat food, removing casing, weighing 1g, adding 0.15M NaCl solution 2ml (1:2w/v), homogenizing, heating with boiling water for 20min, and centrifuging at 2000g for 30 min; removing precipitate, filtering supernatant with Whatman No. 1 filter paper, and collecting filtrate for detection;
(2) detection Using the kit
Taking out the enzyme linked immunosorbent assay kit, recovering the reagent to room temperature (20-25 ℃), and placing the reagent indoors for more than 30 min; taking out the lath as required and placing the lath on an enzyme label plate; sequentially adding 100ul of standard substance or sample to be detected into the hole, slightly oscillating and mixing uniformly, and incubating for 30min at 37 ℃ in a dark place; spin-drying the liquid in the holes, diluting the washing working solution by 20 times of the concentrated washing solution with deionized water, washing for 4 times at a rate of 250 ul/hole, and patting to dry; adding 100ul of diluted enzyme labeling detection antibody working solution, lightly shaking and uniformly mixing, and incubating for 30min at 37 ℃ in a dark place; mixing the color development liquid A and the color development liquid B according to the volume ratio of 1:1, adding 100ul per hole for color development, and incubating for 15min in a dark place at 25 ℃; adding 50ul of stop solution to stop the reaction, measuring by using dual-wavelength 450nm/630nm under the condition of 450nm of an enzyme-labeling instrument, and carrying out quantification or qualitative determination according to a standard curve;
(3) analyzing the results of the detection
(a) Quantitative analysis: respectively calculating the average absorbance values of the standard substance and the sample to be detected, wherein the absorbance value (B) of the standard substance or the sample to be detected is a vertical coordinate, the logarithm of the standard concentration is a horizontal coordinate, and drawing a standard curve; substituting the absorbance value of the sample to be detected into the standard curve to obtain the corresponding concentration, and multiplying the corresponding concentration by the dilution factor to obtain the content of the sample;
(b) and (3) qualitative analysis: and comparing the average absorbance value of the sample to be detected with the absorbance value of the standard substance to obtain the concentration range of the sample to be detected.
The detection result can also be calculated by professional computer software, and the test range is 4.8 mg/L-312.5 mg/L.
The detection principle of the kit is a double-antibody sandwich ELISA method. Coating a microporous plate strip with a capture antibody skTnI-3A8 to prepare a solid-phase capture antibody, sequentially adding a standard substance or a sample into micropores coated with a monoclonal antibody, combining with a detection antibody skTnI-3D3 marked by horseradish peroxidase to form an antibody-antigen-enzyme-labeled antibody compound, adding TMB for color development, wherein the compound is blue and is finally yellow under the action of acid, and the color depth is in positive correlation with the content of bovine skeletal muscle troponin I in the sample.
The curve range of the enzyme linked immunosorbent assay kit is in the range of 4.8 mg/L-312.5 mg/L; the lowest detection limit of the troponin I of skeletal muscle of cattle or sheep is 4.8mg/L, and the detection sensitivity of the troponin I of cattle or sheep is 1 percent; the intra-batch variation coefficient is below 13.6 percent, and the inter-batch variation coefficient is below 14.1 percent; the cross reaction rate with skeletal muscle troponin I extracts of cattle and sheep is 100 percent, and the cross reaction rate with skeletal muscle troponin I extracts of chickens, ducks and pigs is lower than 0.1 percent; the kit is placed at 2-8 ℃ for 12 months, detection is carried out once every 1 month during the period, all parameters such as ODmax, coefficient of variation and the like of the kit are measured, the result shows that all parameters are in a normal range, meanwhile, the kit is placed at 37 ℃ and-20 ℃ for 6 days, detection is carried out once every day, all parameters such as ODmax, coefficient of variation and the like of the measurement kit are in a normal range, and therefore, the kit can be stored at 2-8 ℃ for at least 12 months.
Drawings
FIG. 1 is a standard curve diagram for detecting cattle skTnI by the ELISA kit of the present invention
FIG. 2 is a standard curve diagram of the enzyme-linked immunoassay kit for detecting sheep skTnI
FIG. 3 is a specific assay diagram of the ELISA kit of the present invention
FIG. 4 is a diagram of the actual sample measurement of the ELISA kit of the present invention
The hybridoma cell strain skTnI-3A8 of the monoclonal antibody for resisting the bovine skeletal muscle troponin I is delivered to a China center for type culture Collection (CCTCC for short, address: Wuhan university, China center for type culture Collection, postal code: 430072) for preservation in 2018, 12 months and 20 days, and the preservation number is CCTCC NO: C2018217.
The hybridoma cell strain skTnI-3D3 of the monoclonal antibody for resisting the sheep skeletal muscle troponin I is delivered to the China center for type culture Collection (CCTCC for short, address: Wuhan university, China center for type culture Collection, zip code: 430072) for preservation in 2018, 12 months and 20 days, and the preservation number is CCTCC NO: C2018218.
Detailed Description
The invention is further illustrated below with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
EXAMPLE A preparation of skeletal muscle troponin I antigen of cattle or sheep according to the invention
Taking cattle or sheep skeletal muscle to remove fat and connective tissue, grinding and mixing evenly, weighing 40g, adding 0.15M NaCL solution (1:2 w/v); further mixing, ultrasonic extracting for 5min (50W, 20KHz), heating with boiling water for 20min, and centrifuging at 2000g for 30 min; removing the precipitate, and filtering half of the supernatant to obtain a treated solution 1. Centrifuging the other half of the supernatant at 121 deg.C under high pressure for 30min, 5000g for 30min, filtering with Whatman No. 1 filter paper, adding 90% ethanol (1:3.74v/v) into the filtrate, centrifuging 7000g of the mixture for 20min, oven drying the precipitate at 37 deg.C, and dissolving in normal saline to obtain treated solution 2. The processing liquid 1 and the processing liquid 2 are identified by SDS-PAGE electrophoresis, and the identification result shows that the bovine and ovine skeletal muscle troponin I immunogen and the detection antigen have protein bands in the range of 21-22 kD, and the molecular weight of the protein bands is consistent with the molecular weight of 21-24 kD of skTnI reported in the literature. And grinding the sktnI electrophoresis strip, and diluting the ground sktnI electrophoresis strip with physiological saline to be respectively used as a detection antigen and an immune antigen.
EXAMPLE two preparation of monoclonal antibody against skeletal muscle troponin I of bovine or ovine of the present invention
1. Animal immunization: selecting the extracted immune antigen, immunizing female Balb/c mice of 6-8 weeks old for 1 time at an interval of 2 weeks, after 3 times of immunization, cutting tail and taking blood to determine titer and inhibition rate, and selecting the mice with the best immune result for fusion;
2. cell fusion: fusing splenocytes of the mouse selected in the step 1 with myeloma SP2/0 cells of the mouse, determining supernatant fluid by an indirect ELISA method, selecting holes with high positive, and subcloning the positive holes by a limiting dilution method until a hybridoma cell strain generating a single monoclonal antibody against bovine or sheep skeletal muscle troponin I is established;
3. large-scale preparation of monoclonal antibodies: selecting a female Balb/c mouse with a larger individual, preparing a large amount of ascites by an in vivo induced ascites method, purifying the ascites by caprylic acid-ammonium sulfate precipitation, dividing the ascites into small tubes, and storing the small tubes at the temperature of minus 20 ℃ to obtain the bovine or sheep skeletal muscle troponin I monoclonal antibody.
EXAMPLE three characterization of the Capture and detection antibodies of the invention
1. Potency assay
Diluting the detection antigen to 5 mu g/mL by using a carbonate buffer solution with pH9.6 to coat a detection plate, diluting the purified monoclonal antibody by 1:2000, 1:4000, 1:8000, … … 1:1024000, adding the diluted monoclonal antibody into an enzyme label plate hole, adding a goat-anti-mouse secondary antibody marked by HRP after reaction, and finally developing by using TMB (Tetramethylbenzidine), wherein the result shows that the titer of the purified bovine skeletal muscle troponin I monoclonal antibody reaches 1 when the concentration is 1 mg/mL: 106The titer of the purified sheep skeletal muscle troponin I monoclonal antibody reaches 1:2.56 multiplied by 10 when the concentration is 1mg/mL5。
2. Subtype determination
Subtype determination was performed using a murine monoclonal antibody subtype identification kit purchased from Sigma, and the results showed that the bovine skeletal muscle troponin I monoclonal antibody subtype was IgG1 and the ovine skeletal muscle troponin I monoclonal antibody subtype was IgM.
3. Affinity assay
The result of measuring the affinity constant of the monoclonal antibody by indirect enzyme-linked immunoassay shows that the monoclonal affinity constant of anti-bovine skeletal muscle troponin I is Ka-8.1 multiplied by 108L/mol, monoclonal affinity constant Ka 7.6X 10 for anti-sheep skeletal muscle troponin I8L/mol。
EXAMPLE four preparation of the capture antibody-coated microplate according to the invention
Diluting the capture antibody skTnI-3A8 with carbonate buffer solution with pH9.6 at a ratio of 1:4000, adding 100 mu L/well onto a plate hole of an enzyme label plate, and coating overnight at 4 ℃; washing for 4-5 times, and drying; blocking with 1% gelatin in PBS, 200. mu.L/well, incubating at 37 ℃ for 2 h; pumping in a vacuum drying oven for 5h at room temperature, and vacuum packaging with aluminum foil bag.
EXAMPLE V preparation of working solution for enzyme-labeled detection antibody according to the present invention
Preparation of enzyme-labeled detection antibody: adopting sodium iodate method to couple horseradish peroxidase with detection antibody sktnI-3D3, wherein the concentration of the coupled antibody is 7.7mg/ml, and the titer is 1:2.0 × 106。
EXAMPLE sixthly, the construction of the ELISA kit for detecting skeletal muscle troponin I of cattle or sheep according to the present invention
The enzyme-linked immunoassay kit for constructing the skeletal muscle troponin I of the cattle or sheep comprises the following components:
(1) an ELISA plate coated with a capture antibody;
(2) enzyme labeling detection antibody working solution;
(3) bovine or sheep skeletal muscle troponin I standard solution: preparing a standard solution by a gradient dilution method, wherein the concentration of the standard solution is 4.875mg/L, 9.75mg/L, 19.5mg/L, 39mg/L, 78.125mg/L, 156.25mg/L and 312.5mg/L respectively in 1 ml/bottle;
(4) the substrate color developing solution A is a carbamide peroxide solution, and the substrate color developing solution B is a Tetramethylbenzidine (TMB) solution;
(5) the stop solution is 1-2mol/L sulfuric acid solution;
(6) the washing solution was concentrated to 0.2mol/L of phosphate buffer pH7.4 containing 1% Tween-20.
Seventhly, the enzyme linked immunosorbent assay kit is used for detecting the skeletal muscle troponin I of cattle or sheep in a sample
1. Pretreatment of samples
Removing fat and connective tissue from fresh meat, processing meat food, removing casing, weighing 1g, adding 0.15M NaCl solution 2ml (1:2w/v), homogenizing, heating with boiling water for 20min, and centrifuging at 2000g for 30 min; the precipitate was removed, the supernatant was filtered through Whatman No. 1 filter paper, and the filtrate was collected for detection.
2. Detection method
(1) Taking out the enzyme linked immunosorbent assay kit, recovering the reagent to room temperature (20-25 ℃), and placing for at least 30min indoors;
(2) taking out the lath as required and placing the lath on an enzyme label plate;
(3) sequentially adding 100 mu L of standard substance (or sample to be detected) into the holes, lightly shaking and uniformly mixing, and incubating for 30min at 37 ℃ in a dark place;
(4) spin-drying the liquid in the hole, washing with 250 μ L/hole of washing working solution (20 times diluted by deionized water) for 4 times, and drying;
(5) adding diluted monoclonal antibody marked by HRP, 100 mu L/hole, and incubating for 30min at 37 ℃;
(6) spin-drying the liquid in the hole, washing with 250 μ L/hole of washing working solution (20 times diluted by deionized water) for 4 times, and drying;
(7) mixing the color development liquid A and the color development liquid B according to the proportion of 1:1, adding 100ul per hole for color development, and incubating for 15min in a dark place at 25 ℃;
(8) the reaction was stopped by adding 50. mu.L of stop solution, measured with a microplate reader at 450nm (preferably using a dual wavelength 450/630 measurement), and quantified or determined according to a standard curve.
3. The result of the detection
(a) Quantitative analysis: respectively calculating the average absorbance values of the standard substance and the sample to be detected, wherein the absorbance value (B) of the standard substance or the sample to be detected is a vertical coordinate, the logarithm of the standard concentration is a horizontal coordinate, and drawing a standard curve; substituting the absorbance value of the sample to be measured into the standard curve to calculate the corresponding concentration, and multiplying the corresponding concentration by the dilution factor to obtain the content of the sample.
(b) And (3) qualitative analysis: and comparing the average absorbance value of the sample to be detected with the absorbance value of the standard substance to obtain the concentration range of the sample to be detected.
The detection result can also be calculated by professional computer software, and the test range is 4.8 mg/L-312.5 mg/L.
EXAMPLE eighthly, the sensitivity, specificity, accuracy and shelf life of the ELISA kit of the present invention
1. Establishment of standard curve of kit
The tested cattle skTnI standard curve is drawn as shown in fig. 1, the standard curve has a good linear relation in the range of 4.8mg/L to 312.5mg/L, and the linear equation y is 0.4902x-0.2297(R is 0.4902 x-0.2297)20.9903), the minimum detection limit for bovine skeletal muscle troponin I was 4.8 mg/L. The standard curve of the detected sheep skTnI is drawn as shown in fig. 2, the standard curve has a good linear relation in the range of 4.8mg/L to 312.5mg/L, and the linear equation y is 0.6626x-0.312(R is 0.6626 x-0.312)20.9934), the lowest limit of detection of sheep skeletal muscle troponin I was 4.8 mg/L.
2. Kit accuracy determination
Precision is the degree of reproducibility of the results obtained by a reaction measurement method for a particular sample, and is usually expressed by a coefficient of variation. Selecting enzyme-linked immunosorbent assay plates of the same batch and different batches from a qualified detection kit, diluting the boiled beef and mutton extract by 1:10, 1:20 and 1:50 times, using the diluted beef and mutton extract as a positive sample, repeating the steps of the same batch and different batches for 4 times respectively, and determining the intra-batch and inter-batch variation coefficients and the recovery rate. The results are shown in Table 1, and the intra-batch variation coefficient is 13.6% or less, and the inter-batch variation coefficient is 14.1% or less.
TABLE 1 measurement of the Intra-and Inter-batch coefficient of variation of samples
3. Cross reaction assay
Boiled extracts of beef, mutton, chicken and duck meat and BSA (negative control) were mixed with PBS as 1:20 and 1:40 dilutions and detection was performed using the kit of the invention to determine the specificity of the method. As can be seen from FIG. 3, the established sandwich ELISA method has specificity for cattle and sheep, and does not react with the chicken, duck, and pig skeletal muscle extracts.
4. Shelf life test of kit
The kit is placed at 2-8 ℃ for 12 months, detection is carried out once every 1 month during the period, and all parameters such as ODmax, coefficient of variation and the like of the determination kit are in a normal range. Meanwhile, the kit is placed at 37 ℃ and-20 ℃ for 6 days, the detection is carried out once a day, and all parameters of the kit, such as ODmax, coefficient of variation and the like, are determined to be within a normal range. The kit can be stored at 2-8 deg.C for at least 12 months.
5. Sample testing
Fresh beef and mutton are added into chicken and duck meat (1: 1, w/w) according to the proportion of 1%, 5%, 10%, 20% and 50%, and the test is carried out by using the kit disclosed by the invention, the result is shown in figure 4, the built sandwich ELISA method is used for detecting the beef and mutton in the chicken and duck meat, when the content of the beef and mutton reaches 1%, the absorbance value is more than 2 times of negative value, and therefore, the sensitivity of the method can reach 1%.
The above-described embodiments are intended to illustrate the technical idea and advantages of the invention, and the invention may also be subject to other variants, as known to the skilled person, which serve merely as illustrations of the scope of protection of the invention described above, and to the skilled person in the art who is within the scope of protection of the invention defined by the present invention there are many conventional variants and other embodiments, which are all within the scope of protection of the invention covered by the present invention.
Claims (6)
1. An enzyme linked immunosorbent assay kit for detecting skeletal muscle troponin I of cattle or sheep, which consists of an ELISA plate coated with a capture antibody, an enzyme-labeled detection antibody, a standard solution of skeletal muscle troponin I of cattle or sheep, a substrate chromogenic solution, a stop solution and a concentrated washing solution; the method is characterized in that the capture antibody is obtained by secreting a hybridoma cell strain skTnI-3A8 with the preservation number of CCTCC NO: C2018217, and the detection antibody is obtained by secreting a hybridoma cell strain skTnI-3D3 with the preservation number of CCTCC NO: C2018218; the two cell strains are delivered to China Center for Type Culture Collection (CCTCC) for preservation in 2018, 12 months and 20 days.
2. The ELISA kit of claim 1 wherein the enzyme-labeled detection antibody is obtained by coupling a labeled enzyme with a detection antibody by a sodium iodate method.
3. The ELISA kit of claim 1, wherein the labeled enzyme in the enzyme-labeled detection antibody is horseradish peroxidase or alkaline phosphatase, when the labeled enzyme is horseradish peroxidase, the substrate color developing solution comprises a color developing substrate A and a color developing substrate B, the color developing substrate A is hydrogen peroxide or carbamide peroxide, the color developing substrate B is o-phenylenediamine or tetramethylbenzidine, and the stop solution is 1-2mol/L sulfuric acid or hydrochloric acid buffer solution; when the marker enzyme is alkaline phosphatase, the substrate color development liquid is a p-nitrophosphate buffer solution, and the stop solution is 1-2mol/L sodium hydroxide.
4. The ELISA kit of claim 1 wherein the bovine or ovine skeletal muscle troponin I standard solution has a concentration of 4.875mg/L, 9.75mg/L, 19.5mg/L, 39mg/L, 78.125mg/L, 156.25mg/L, 312.5mg/L, respectively; the concentrated washing solution is 0.2mol/L phosphate buffer solution with pH7.4 containing 1% Tween-20.
5. A method for preparing the ELISA kit of any one of claims 1 to 4 comprising the steps of:
(1) preparing an enzyme label plate coated with a capture antibody:
(a) diluting the capture antibody to 1:4000 with a coating buffer solution, and adding 100ul of capture antibody into the holes of the ELISA plate;
(b) incubating at 4 ℃ overnight or 37 ℃ for 2h, pouring out liquid in the holes, washing for 4-5 times by using a washing solution, and patting dry;
(c) adding a sealing solution buffer solution, incubating at 37 ℃ for 2h at 200 ul/well;
(d) pouring out liquid in the holes, drying the holes, pumping the liquid in a vacuum drying oven at room temperature for 5 hours, and carrying out vacuum plastic packaging by using an aluminum foil bag to obtain an ELISA plate coated with a capture antibody;
wherein the coating buffer solution is 0.05mol/L carbonate buffer solution with the pH value of 9.6, and the blocking buffer solution is 1% of gelatin;
(2) preparing an enzyme labeling detection antibody working solution:
horseradish peroxidase is coupled with a detection antibody by a sodium iodate method;
(3) the enzyme linked immunosorbent assay kit for detecting skeletal muscle troponin I of cattle or sheep is constructed, and comprises the following components:
(a) an ELISA plate coated with a capture antibody;
(b) enzyme labeling detection antibody working solution;
(c) bovine or sheep skeletal muscle troponin I standard solution: preparing a standard solution by a gradient dilution method, wherein the concentration of the standard solution is 4.875mg/L, 9.75mg/L, 19.5mg/L, 39mg/L, 78.125mg/L, 156.25mg/L and 312.5mg/L in a 1 ml/bottle respectively;
(d) the substrate color development liquid A is urea peroxide solution, and the substrate color development liquid B is tetramethyl benzidine TMB solution;
(e) the stop solution is 1-2mol/L sulfuric acid solution;
(f) the washing solution was concentrated to 0.2mol/L phosphate buffer pH7.4 containing 1% Tween-20.
6. The use of the enzyme linked immunosorbent assay kit of claim 3 for detecting bovine or ovine skeletal muscle troponin I in fresh meat and products thereof, comprising the following steps:
(1) sample pretreatment
Removing fat and connective tissue from fresh meat, processing meat food, removing casing, weighing 1g, adding 0.15M NaCl solution 2ml, homogenizing, heating with boiling water for 20min, centrifuging at 2000g for 30 min; removing precipitate, filtering the supernatant with Whatman No. 1 filter paper, and collecting filtrate for detection;
(2) detection Using enzyme-linked immunosorbent assay kit
Taking out the enzyme linked immunosorbent assay kit, recovering the reagent to the room temperature of 20-25 ℃, and placing the reagent indoors for more than 30 min; taking out the lath as required and placing the lath on an enzyme label plate; sequentially adding 100ul of standard substance or sample to be tested into the hole, gently shaking and mixing, and incubating at 37 deg.C in dark for 30 min; spin-drying the liquid in the holes, diluting the washing working solution by 20 times of the 20 multiplied concentrated washing solution by deionized water, washing for 4 times at a rate of 250 ul/hole, and patting dry; adding 100ul of diluted enzyme labeling detection antibody working solution, lightly shaking and uniformly mixing, and incubating for 30min at 37 ℃ in a dark place; mixing the color development liquid A and the color development liquid B according to the volume ratio of 1:1, adding 100ul per hole for color development, and incubating for 15min in a dark place at 25 ℃; adding 50ul of stop solution to stop the reaction, measuring by using dual-wavelength 450nm/630nm under the condition of 450nm of an enzyme-labeling instrument, and carrying out quantification or qualitative determination according to a standard curve;
(3) analyzing the detection result
(a) Quantitative analysis: respectively calculating the average absorbance values of the standard substance and the sample to be detected, wherein the absorbance value of the standard substance is a vertical coordinate, the logarithm of the standard concentration is a horizontal coordinate, and drawing a standard curve; substituting the absorbance value of the sample to be detected into the standard curve to obtain the corresponding concentration, and multiplying the corresponding concentration by the dilution factor to obtain the content of the sample;
(b) and (3) qualitative analysis: and comparing the average absorbance value of the sample to be detected with the absorbance value of the standard substance to obtain the concentration range of the sample to be detected.
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| CN109709339B (en) * | 2018-12-28 | 2022-03-15 | 河北省科学院生物研究所 | Colloidal gold immunochromatographic test strip for detecting skeletal muscle troponin I of cattle or sheep and application thereof |
| CN109705216B (en) * | 2018-12-28 | 2021-09-07 | 河北省科学院生物研究所 | Monoclonal antibody for resisting bovine skeletal muscle troponin I and application thereof |
| CN109810191B (en) * | 2018-12-28 | 2021-09-07 | 河北省科学院生物研究所 | Monoclonal antibody for resisting sheep skeletal muscle troponin I and application thereof |
| CN111220809B (en) * | 2019-12-27 | 2023-06-16 | 河北省科学院生物研究所 | Colloidal gold immunochromatography test strip for detecting chicken or duck skeletal muscle troponin I and preparation method and application thereof |
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| CN111077320B (en) * | 2019-12-27 | 2023-06-16 | 河北省科学院生物研究所 | ELISA kit for detecting chicken or duck skeletal muscle troponin I and preparation method and application thereof |
| CN112129949A (en) * | 2020-08-18 | 2020-12-25 | 兰州百源基因技术有限公司 | Retinol binding protein detection kit, preparation method and use method thereof |
| CN113009152B (en) * | 2021-02-19 | 2022-05-10 | 山东省大健康精准医疗产业技术研究院 | Glycosylated CD59 enzyme-linked immunoassay kit and preparation method and application thereof |
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