CN111077320B - ELISA kit for detecting chicken or duck skeletal muscle troponin I and preparation method and application thereof - Google Patents
ELISA kit for detecting chicken or duck skeletal muscle troponin I and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a double-antibody sandwich enzyme-linked immunosorbent assay kit for detecting chicken or duck skeletal muscle troponin I, and a preparation method and application thereof, belonging to the technical field of immunology and the technical field of food safety analysis. The ELISA kit comprises an ELISA plate coated with a capture antibody, a detection antibody marked by horseradish peroxidase, a chicken or duck skeletal troponin I standard solution, a substrate chromogenic solution, a stop solution and a concentrated washing solution. The capture antibody is secreted by a hybridoma cell strain skTnI-3E7 with a preservation number of CCTCC NO: C202003, and the detection antibody is an anti-chicken skeletal muscle troponin I polyclonal antibody. The ELISA kit has the advantages of high sensitivity, high precision, high accuracy, low cross reaction rate, suitability for mass sample detection and the like.
Description
Technical Field
The invention relates to an enzyme-linked immunosorbent assay kit for detecting chicken or duck skeletal muscle troponin I, and a preparation method and application thereof, belonging to the technical field of immunology and food safety analysis.
Background
In meat products, due to price, religion, health and other reasons, many countries make regulations that require food labels to truly and clearly mark meat sources, inhibit adulteration, so as to protect consumer benefits, but the phenomenon of confusing meat varieties in the market is still very common, adulteration modes include means of blending, mixing, extracting, counterfeiting and the like, especially the adulteration and adulteration of beef and mutton products are most common, and the adulteration actions greatly damage consumer benefits. In meat products, due to price, religion, health and other reasons, many countries make regulations that require food labels to truly and clearly mark meat sources, inhibit adulteration, so as to protect consumer benefits, but the phenomenon of confusing meat varieties in the market is still very common, adulteration modes include means of blending, mixing, extracting, counterfeiting and the like, especially the adulteration and adulteration of beef and mutton products are most common, and the adulteration actions greatly damage consumer benefits. Duck, chicken and other birds are short in raising period and low in price, meanwhile, the textures of duck meat and beef and mutton are highly similar, a large amount of duck meat and a small amount of beef and mutton fat are mixed, and the degree of 'false and spurious' can be achieved after processing, so that the duck meat is often used as a raw material for making the beef and mutton. This adulteration compromises the consumer's benefits, disturbing the marketing rules. Therefore, it is important to establish a rapid identification method of duck meat source components.
At present, a plurality of laboratories at home and abroad use molecular biology technology to detect the source components in meat products, and DNA detection methods are various, mainly nucleic acid probe hybridization, DNA fingerprint analysis, PCR specific amplification, PCR-RFLP and the like. The method has certain sensitivity, but has the defects of high cost, high requirement on detection objects, large workload, incapability of on-site detection and the like, and particularly has great uncertainty on detection of cooked meat products, because the cooked meat products can degrade genetic substances at different levels in the treatment process. Therefore, other methods are needed to mutually verify his detection results. The immunological method has the characteristics of high sensitivity, good specificity, low cost, convenient operation and the like, and is suitable for large-batch sample screening, wherein an enzyme-linked immunosorbent assay and a colloidal gold test strip are the most commonly used methods.
Cooked meat products are generally subjected to high temperature and high pressure, and many proteins are denatured in the process, so that antigenicity and water solubility are lost, which makes the establishment of an immunological method 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 skeletal muscle of animals has species specificity and can be used as a heat-resistant type marker protein to distinguish meat species sources of different species of raw and cooked meat products. The chicken or duck skeletal muscle troponin I is taken as a target detection object, so that the establishment of an immunological detection and identification technology of chicken or duck meat-derived components can be realized. Therefore, the preparation of the chicken or duck skeletal muscle troponin I ELISA kit has important significance for carrying out the immunological detection and identification work of chicken or duck meat source components.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide the ELISA kit for detecting chicken or duck skeletal troponin I, which has the advantages of high sensitivity, precision and accuracy, low cross reaction rate, suitability for mass sample detection and the like. In addition, the invention further provides a preparation method and application of the ELISA kit for detecting chicken or duck skeletal muscle troponin I.
The technical problems are realized by the following technical scheme.
The ELISA kit for detecting chicken or duck skeletal muscle troponin I adopts a double-antibody sandwich method, and consists of an ELISA plate coated with a capture antibody, an enzyme-labeled detection antibody, a substrate color development solution, a stop solution and a concentrated washing solution.
In the ELISA kit, the capture antibody is secreted by a hybridoma cell strain skTnI-3E7 with a preservation number of CCTCC NO: C202003, and the cell strain is delivered to China Center for Type Culture Collection (CCTCC) for preservation in 12 months and 10 days in 2019; the detection antibody is an anti-chicken skeletal muscle troponin I polyclonal antibody.
In the enzyme-linked immunosorbent assay kit, the capture antibody is a murine, equine, ovine, rabbit or guinea pig monoclonal antibody, preferably a murine monoclonal antibody; the detection antibody is a polyclonal antibody of murine, equine, ovine, rabbit or guinea pig, preferably a polyclonal antibody of rabbit; both the capture antibody and the detection antibody are obtained by extracting immune antigen from chicken or duck skeletal muscle troponin I for immunization.
The enzyme-linked immunosorbent assay kit adopts an enzyme labeling scheme which is conventional in the art, and for example, a sodium iodate method can be adopted to couple the labeled enzyme with the detection antibody.
In the ELISA kit, the enzyme-labeled detection antibody is labeled with horseradish peroxidase or alkaline phosphatase, when the enzyme-labeled detection antibody is horseradish peroxidase, the chromogenic substrates comprise chromogenic substrate A and chromogenic substrate B, the chromogenic substrate A is hydrogen peroxide or carbamide peroxide, the chromogenic substrate B is o-phenylenediamine or tetramethyl benzidine, and the stop solution is sulfuric acid or hydrochloric acid buffer solution with the concentration of 1-2 mol/L; when the marking enzyme is alkaline phosphatase, the chromogenic substrate is p-nitrophosphate buffer solution, and the stop solution is 1-2mol/L sodium hydroxide.
In order to facilitate on-site monitoring and screening of a large number of samples, the chicken or duck skeletal troponin I standard solution, the substrate chromogenic solution, the stop solution and the concentrated washing solution in the ELISA kit are provided in the form of working solutions.
Through the technical scheme, the method can realize rapid detection of chicken or duck skeletal muscle troponin I in fresh meat and products thereof by a double antibody sandwich ELISA detection method, has detection sensitivity of 20ug/kg for chicken or duck skeletal muscle troponin I, has a cross reaction rate of 100% with chicken skeletal muscle extracts, has a cross reaction rate of 82.2% with duck skeletal muscle extracts, has a cross reaction rate of 10% or less with skeletal muscle extracts of cattle, sheep and pigs, and has higher sensitivity and specificity.
The preparation method of the ELISA kit provided by the invention comprises the following steps:
(1) Preparing an ELISA plate coated with a capture antibody:
(a) Diluting the capture antibody to 1:4000 with coating buffer, and adding 100 ul/well into the enzyme-labeled plate well;
(b) Incubating overnight at 4deg.C or 37deg.C for 2 hr, removing liquid in the hole, washing with washing liquid for 4-5 times, and drying;
(c) Adding a blocking solution buffer solution, 200 ul/hole, and incubating for 2 hours at 37 ℃;
(d) Pouring out the liquid in the hole, beating to dry, pumping the liquid in a vacuum drying oven at room temperature for 5 hours, and vacuum-molding the liquid in an aluminum foil bag to obtain the ELISA plate coated with the capture antibody;
wherein the coating buffer is 0.05mol/L carbonate buffer with pH of 9.6, and the blocking buffer is 1% gelatin;
(2) Preparing an enzyme-labeled detection antibody working solution:
coupling horseradish peroxidase and a detection antibody by adopting a sodium iodate method;
(3) An ELISA kit for detecting chicken or duck skeletal muscle troponin I is formed, which comprises the following components:
(a) An ELISA plate coated with a capture antibody;
(b) Enzyme labeling detection antibody working solution;
(c) The substrate color development solution A is carbamide peroxide solution, and the substrate color development solution B is tetramethyl benzidine (TMB) solution;
(d) The stop solution is sulfuric acid solution with the concentration of 1-2 mol/L;
(e) The concentrated washes were phosphate buffer at 0.2mol/L pH7.4 containing 1% Tween-20.
The application of the ELISA kit in chicken or duck skeletal muscle troponin I 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, centrifuging for 30min 2000 g; the precipitate was removed and the supernatant was filtered through Whatman No. 1 filter paper and the filtrate was collected for detection.
(2) Detection Using a kit
Taking out the ELISA kit, recovering the reagent to room temperature (20-25 ℃), and placing in the room for more than 30min; taking out the battens according to the requirement, and placing the battens on the ELISA plate; sequentially adding 100ul of standard substances or samples to be tested into the holes, gently shaking and uniformly mixing, and incubating for 30min at 37 ℃ in a dark place; spin-drying the liquid in the holes, diluting 20 times of concentrated washing liquid with deionized water for 20 times, washing for 4 times at 250 ul/hole, and beating to dry; adding 100ul of diluted enzyme-labeled detection antibody working solution, gently shaking and 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 100 ul/hole for color development, and incubating for 15min at 25 ℃ in a dark place; the reaction was stopped by adding 50ul of stop solution, and the reaction was measured at 450nm by an ELISA reader.
(3) Analyzing the detection result
(a) Quantitative analysis: respectively calculating average absorbance values of a standard substance and a sample to be detected, wherein the absorbance value (B) of the standard substance or the sample to be detected is an ordinate, the standard concentration is an abscissa, and a standard curve is drawn; substituting the absorbance value of the sample to be measured into a standard curve to obtain the corresponding concentration, and multiplying the concentration by the dilution multiple to obtain the content of the sample.
(b) 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 using professional computer software, and the test range is 20 g/kg-400 g/kg.
The detection principle of the kit in the invention is a double-antibody sandwich ELISA method. Coating a micropore lath with an antibody (3E 7) to prepare a solid-phase capture antibody, sequentially adding a standard substance or a sample into micropores coated with monoclonal antibody, combining with a detection antibody marked by horseradish peroxidase to form an antibody-antigen-enzyme-labeled antibody compound, adding TMB to develop a color, and finally forming a yellow color under the action of acid, wherein the color depth is positively correlated with the content of chicken or duck skeletal troponin I in the sample.
The curve range of the ELISA kit is 20 g/kg-400 g/kg; the detection sensitivity to chicken or duck skeletal muscle troponin I is 20g/kg; the variation coefficient in the batch is below 10.4%, and the variation coefficient between batches is below 11.7%; the cross reaction rate with the skeletal muscle extract of chicken is 100%, the cross reaction rate with the skeletal muscle extract of duck is more than 82.2%, and the cross reaction rate with the skeletal muscle extract of cattle, sheep and pigs is less than 10%; the kit is placed at the temperature of between 2 and 8 ℃ for 12 months, the parameters such as ODmax, the coefficient of variation and the like of the kit are detected every 1 month during the preservation period, the results show that the parameters are in the normal range, meanwhile, the kit is placed at the temperature of between 37 and 20 ℃ for 6 days, the detection is carried out once a day, and the parameters such as ODmax, the coefficient of variation and the like of the kit are in the normal range, so that the kit can be preserved at the temperature of between 2 and 8 ℃ for at least 12 months.
Drawings
FIG. 1 is a standard curve chart of chicken skeletal muscle troponin I detection by the ELISA kit of the invention
FIG. 2 is a standard graph of the ELISA kit for detecting Duck skeletal muscle troponin I
Detailed Description
The invention is further illustrated below in conjunction with specific examples. It should be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example one preparation of the skeletal muscle troponin I antigen of chickens or ducks according to the invention
Taking skeletal muscle of chicken (or duck) to remove fat and connective tissue, grinding and mixing uniformly, weighing 20g, and adding 0.15M NaCL solution (1:2w/v); further mixing, ultrasonic extracting for 5min (50W, 20 KHz), heating with boiling water for 20min, and centrifuging for 30min at 2000 g; the precipitate was removed, and half of the supernatant was filtered to give treatment solution 1. Centrifuging the other half of the supernatant at 121deg.C for 30min at 5000g for 30min, filtering the supernatant with Whatman No. 1 filter paper, adding 90% ethanol (1:3.74 v/v) into the filtrate, centrifuging the mixed solution at 7000g for 20min, oven drying the precipitate at 37deg.C, and redissolving with physiological saline to obtain treatment solution 2. Treatment fluid 1 and treatment fluid 2 were identified by SDS-PAGE, and the identification results showed that the chicken and duck skeletal troponin I immunogens and the detection precursors had protein bands at 24kD and 37kD, which are consistent with the skeletal troponin T, I subunits reported in the literature. The corresponding electrophoresis strips were ground and diluted with physiological saline to serve as detection antigen and immune antigen, respectively.
Example II preparation of monoclonal antibodies to chicken or Duck skeletal muscle troponin I according to the present invention
1. Animal immunization: selecting the extracted immune antigen, immunizing female Balb/c mice with the age of 6-8 weeks, immunizing 1 time at intervals of 2 weeks, performing tail breaking and blood sampling after 3 times of immunization to determine the titer and the specificity, and selecting the mice with the best immune results to prepare fusion;
2. cell fusion: fusing the spleen cells of the mice selected in the step 1 with the myeloma SP2/0 cells of the mice, measuring the supernatant by an indirect ELISA method, selecting a hole with high positive, subcloning the positive hole by a limiting dilution method until a hybridoma cell strain skTnI-3E7 for generating a monoclonal antibody of single anti-chicken or duck skeletal muscle troponin I is established;
3. preparation of monoclonal antibodies in large quantities: selecting a female Balb/c mouse with a larger individual, preparing a large amount of ascites by adopting an in-vivo induced abdominal water method, purifying the ascites by caprylic acid-ammonium sulfate precipitation, dividing the ascites into small tubes, and preserving the small tubes at the temperature of minus 20 ℃ to obtain the chicken or duck skeletal troponin I monoclonal antibody.
EXAMPLE III preparation of polyclonal antibodies to chicken or Duck skeletal muscle troponin I according to the invention
1. Animal immunization: selecting the extracted immune antigen, immunizing New Zealand white rabbits, immunizing 1 time at intervals of 2 weeks, and measuring titer and specificity of blood sampling of the ear margin vein after 3 times of immunization;
2. mass production of polyclonal antibodies: the jugular vein of the immunized rabbit is sampled, serum is separated and the volume is measured, ammonium sulfate (0.313 g is added to each milliliter of serum) is added, stirring is carried out for 30min, standing is carried out for 2h, the mass is centrifuged for 20min at 8000g, the sediment is taken and dissolved by 0.01M PBS, and dialysis is carried out for 1d at-20 ℃ for storage, thus obtaining the polyclonal antibody of chicken or duck skeletal troponin I.
Example IV characterization of Capture antibodies and detection antibodies according to the invention
1. Characterization of Capture antibody (anti-Duck skeletal troponin I monoclonal antibody 3E 7)
(a) Potency determination
The detection antigen was diluted to 5. Mu.g with carbonate buffer pH9.6Per mL of coated assay plate, the purified monoclonal antibodies were diluted 1:2000,1:4000,1:8000, … … 1:1024000, added into the wells of the enzyme-labeled plate, HRP-labeled goat anti-mouse secondary antibody was added after reaction, and finally developed with TMB, and the results showed that the titer of 1 at 1mg/mL of purified duck skeletal troponin I monoclonal antibody reached: 10 5 。
(b) Subtype determination
Subtype determination was performed using a murine monoclonal antibody subtype identification kit purchased from Sigma, which showed that the duck skeletal troponin I monoclonal antibody subtype was IgG1.
(c) Affinity assay
The affinity constant of the duck skeletal muscle troponin I monoclonal antibody was measured by indirect ELISA method, and the result shows that the affinity constant Ka=5.9X10 5 L/mol。
(d) Specificity assay
The cross-reactivity of the monoclonal antibodies with skeletal muscle extracts of cattle, sheep, chickens, ducks, fish was determined by indirect ELISA. The result shows that the cross reaction rate of 3E7 and the skeletal muscle extract of the duck is 100 percent, the cross reaction rate of the 3E7 and the skeletal muscle extract of the cattle, sheep, chicken and pigs is lower than 12.5 percent, and the specificity is better.
2. Detection antibody (polyclonal antibody against chicken skeletal muscle troponin I)
(a) Potency determination
Diluting the detection antigen to 5 mug/ml with carbonate buffer with pH9.6, coating the detection plate, diluting the purified polyclonal antibody by 1:2000,1:4000,1:8000, … …, 1:1024000, adding into an enzyme-labeled plate hole, adding HRP-labeled goat anti-rabbit secondary antibody after reaction, and finally developing with TMB, wherein the result shows that the titer of the purified polyclonal antibody of the chicken skeletal troponin I is 1: 5X 10 5 。
(b) Specificity assay
The cross-reactivity of polyclonal antibodies with skeletal muscle extracts of cattle, sheep, chickens, ducks, fish was determined by indirect ELISA. The results show that the cross-reaction rate of the antibody with the chicken skeletal muscle extract is 100%, the cross-reaction rate of the antibody with the duck skeletal muscle extract is 50%, the cross-reaction rate of the antibody with the bovine, ovine and porcine skeletal muscle extracts is lower than 12.5%, and the specificity is good.
Example five preparation of the ELISA plate coated with Capture antibody according to the invention
The capture antibody was diluted 1:2000 with pH9.6 carbonate buffer, 100. Mu.L/well was added to the wells of the microplate and coated overnight at 4 ℃; washing for 4-5 times, and beating to dry; blocking with 1% gelatin in PBS, 200. Mu.L/well, incubation at 37℃for 2h; and (5) drying in a vacuum drying oven at room temperature for 5 hours, and vacuum-molding by using an aluminum foil bag.
Example six preparation of working solution for enzyme-labeled detection antibody according to the invention
Preparation of enzyme-labeled detection antibody: coupling horseradish peroxidase with detection antibody by sodium iodate method, wherein the concentration of the coupled antibody is 8.5mg/ml, and the titer is 1:2.0X10 5 。
Example six construction of an ELISA kit for detecting skeletal muscle troponin I in chickens or ducks according to the invention
The kit for detecting the skeletal muscle troponin I of the chicken or the duck by enzyme-linked immunosorbent assay comprises the following components:
(1) An ELISA plate coated with a capture antibody;
(2) Enzyme labeling detection antibody working solution;
(3) Standard solution of chicken or duck skeletal troponin extract: preparing a standard solution by adopting a gradient dilution method, wherein the concentration of the standard solution is 20g/kg, 50g/kg, 100g/kg, 200g/kg and 400g/kg in a 1 ml/bottle;
(4) The substrate color development solution A is carbamide peroxide solution, and the substrate color development solution B is tetramethyl benzidine (TMB) solution;
(5) The stop solution is sulfuric acid solution with the concentration of 1-2 mol/L;
(6) The concentrated washes were phosphate buffer at 0.2mol/L pH7.4 containing 1% Tween-20.
Embodiment seven detection of chicken or Duck skeletal troponin I in samples Using the ELISA kit of the invention
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, centrifuging for 30min 2000 g; the precipitate was removed and the supernatant was filtered through Whatman No. 1 filter paper and the filtrate was collected for detection.
2. Detection method
(1) Taking out the ELISA kit, recovering the reagent to room temperature (20-25deg.C), and standing at least in room for 30min;
(2) Taking out the battens according to the requirement, and placing the battens on the ELISA plate;
(3) Sequentially adding 100 mu L of standard substance (or sample to be detected) into the hole, gently shaking and mixing, and incubating at 37 ℃ in a dark place for 30min;
(4) Spin-drying the liquid in the hole, washing with 250 μl/hole of washing working solution (20×concentrated washing solution 20 times diluted with deionized water) for 4 times, and drying;
(5) Adding diluted HRP-labeled polyclonal antibody, 100 μl/well, and incubating at 37deg.C for 30min;
(6) Spin-drying the liquid in the hole, washing with 250 μl/hole of washing working solution (20×concentrated washing solution 20 times diluted with 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 100 ul/hole into the mixture for color development, and incubating for 15min at 25 ℃ in dark;
(8) The reaction was stopped by adding 50. Mu.L of stop solution, and the reaction was performed quantitatively or qualitatively according to a standard curve by measuring at 450nm using an ELISA reader (measurement using a double wavelength of 450/630 was recommended).
3. Detection result
(a) Quantitative analysis: respectively calculating average absorbance values of a standard substance and a sample to be detected, wherein the absorbance value (B) of the standard substance or the sample to be detected is an ordinate, the standard concentration is an abscissa, and a standard curve is drawn; substituting the absorbance value of the sample to be measured into a standard curve to obtain the corresponding concentration, and multiplying the concentration by the dilution multiple to obtain the content of the sample.
(b) 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 using professional computer software, and the test range is 20 ug/kg-400 ug/kg.
Example eight sensitivity, specificity, accuracy and shelf-life experiments of the ELISA kit of the invention
1. Establishment of standard curve of kit
The drawing of the standard curve of the detected chicken skeletal muscle troponin I is shown in figure 1, the standard curve has good linear relation in the range of 20 ug/kg-400 ug/kg, and the linear equation y=0.4923x+0.3899 (R) 2 =0.991), the detection sensitivity was 20g/kg; the drawing of the standard curve of the detected duck skeletal troponin I is shown in figure 2, the standard curve has good linear relationship in the range of 20 ug/kg-400 ug/kg, and the linear equation y=0.36780x+0.5822 (R 2 = 0.9943), the detection sensitivity was 20g/kg.
2. Kit accuracy determination
Precision is the degree of repetition of the results obtained by measuring a particular sample multiple times by the reaction measurement method, and is commonly expressed as a coefficient of variation. And selecting the ELISA plates of the same batch and different batches from the qualified detection kit, repeating for 4 times respectively, and measuring the intra-batch and inter-batch variation coefficients and the recovery rate. The results are shown in Table 1, wherein the intra-batch variation coefficients are all 10.4% or less, and the inter-batch variation coefficients are all 11.7% or less.
TABLE 1 determination of the intra-and inter-batch variation coefficients of samples
3. Cross-reaction assay
As shown in Table 2, the cross-reaction rate of the established sandwich ELISA method with the skeletal muscle extract of chicken is 100%, the cross-reaction rate with the skeletal muscle extract of duck is more than 82.2%, and the cross-reaction rate with the skeletal muscle extract of cattle, sheep and pigs is less than 10%, which proves that the method has the species specificity of chicken and duck.
TABLE 2 determination of Cross-reactivity of Sandwich ELISA species
4. Shelf life test of kit
The kit is placed at the temperature of 2-8 ℃ and stored for 12 months, and the parameters such as ODmax, variation coefficient and the like of the kit are measured at intervals of 1 month in the period and are all in the normal range. Meanwhile, the kit is placed at 37 ℃ and-20 ℃ for 6 days, and is detected once a day, and the parameters such as ODmax, variation coefficient and the like of the kit are determined to be within the normal range. The kit can be stored at 2-8deg.C for at least 12 months.
The above-described embodiments are only for illustrating the technical concept and advantages of the present invention, and the present invention may be modified in other forms, which are known to those skilled in the art, and serve only as examples within the scope of the present invention, and many conventional modifications and other embodiments within the scope of the present invention will fall within the scope of the appended claims to those skilled in the art.
Claims (3)
1. An ELISA kit for detecting chicken or duck skeletal muscle troponin I is characterized by comprising an ELISA plate coated with a capture antibody, an enzyme-labeled detection antibody, a substrate color development liquid, a stop solution and a concentrated washing liquid; the capture antibody is secreted by a hybridoma cell strain skTnI-3E7 with a preservation number of CCTCC NO: C202003, and the cell strain is delivered to China Center for Type Culture Collection (CCTCC) for preservation in 12 months and 10 days in 2019; the detection antibody is an anti-chicken skeletal muscle troponin I rabbit-derived polyclonal antibody; the enzyme-labeled detection antibody, which employs enzyme labeling protocols conventional in the art; the enzyme-labeled detection antibody is characterized in that the enzyme label is horseradish peroxidase or alkaline phosphatase, when the enzyme label is horseradish peroxidase, the chromogenic substrates comprise chromogenic substrate A and chromogenic substrate B, the chromogenic substrate A is hydrogen peroxide or carbamide peroxide, the chromogenic substrate B is o-phenylenediamine or tetramethyl benzidine, and the stop solution is sulfuric acid or hydrochloric acid buffer solution with the concentration of 1-2 mol/L; when the marking enzyme is alkaline phosphatase, the chromogenic substrate is p-nitrophosphate buffer solution, and the stop solution is 1-2mol/L sodium hydroxide.
2. A method for preparing the enzyme-linked immunosorbent assay kit as claimed in claim 1, comprising the steps of:
(1) Preparing an ELISA plate coated with a capture antibody:
(a) Diluting the capture antibody to 1:2000 with coating buffer, and adding 100 ul/well into the enzyme-labeled plate well;
(b) Incubating overnight at 4deg.C or 37deg.C for 2 hr, removing liquid in the hole, washing with washing liquid for 4-5 times, and drying;
(c) Adding a blocking solution buffer solution, 200 ul/hole, and incubating for 2 hours at 37 ℃;
(d) Pouring out the liquid in the hole, beating to dry, pumping the liquid in a vacuum drying oven at room temperature for 5 hours, and vacuum-molding the liquid in an aluminum foil bag to obtain the ELISA plate coated with the capture antibody;
wherein the coating buffer is 0.05mol/L carbonate buffer with pH of 9.6, and the blocking buffer is 1% gelatin;
(2) Preparing an enzyme-labeled detection antibody working solution:
coupling horseradish peroxidase and a detection antibody by adopting a sodium iodate method;
(3) An ELISA kit for detecting chicken or duck skeletal muscle troponin I is formed, which comprises the following components:
(a) An ELISA plate coated with a capture antibody;
(b) Enzyme labeling detection antibody working solution;
(c) The substrate color development solution A is carbamide peroxide solution, and the substrate color development solution B is tetramethyl benzidine (TMB) solution;
(d) The stop solution is sulfuric acid solution with the concentration of 1-2 mol/L;
(e) The concentrated washes were phosphate buffer at 0.2mol/L pH7.4 containing 1% Tween-20.
3. Use of the enzyme-linked immunosorbent assay kit as claimed in claim 1 for detecting chicken or duck skeletal troponin I in fresh meat and products thereof, comprising the steps of:
(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 for 30min 2000 g; removing the precipitate, filtering the supernatant with Whatman No. 1 filter paper, and collecting filtrate for detection;
(2) Detection Using a kit
Taking out the ELISA kit, recovering the reagent to room temperature of 20-25 ℃, and placing the kit in a room for more than 30min; taking out the battens according to the requirement, and placing the battens on the ELISA plate; sequentially adding 100ul of standard substances or samples to be tested into the holes, gently shaking and uniformly mixing, and incubating for 30min at 37 ℃ in a dark place; spin-drying the liquid in the holes, diluting 20 times of concentrated washing liquid with deionized water for 20 times, washing for 4 times at 250 ul/hole, and beating to dry; adding diluted enzyme-labeled detection antibody working solution 100ul, gently shaking and mixing, and incubating at 37 ℃ in a dark place for 30min; mixing the color development liquid A and the color development liquid B according to the volume ratio of 1:1, adding 100 ul/hole for color development, and incubating for 15min at 25 ℃ in a dark place; adding 50ul of stop solution to stop the reaction, and measuring at 450nm by an enzyme-labeled instrument;
(3) Analyzing the detection result
And (3) result judgment:
(a) Quantitative analysis: respectively calculating average absorbance values of a standard substance and a sample to be detected, wherein the absorbance value of the standard substance is an ordinate, the addition concentration is an abscissa, and a standard curve is drawn; substituting the absorbance value of the sample to be measured into a standard curve to obtain the corresponding concentration, and multiplying the concentration by the dilution factor to obtain the content of the sample;
(b) 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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| CN112067823A (en) * | 2020-09-14 | 2020-12-11 | 华南农业大学 | Establishment of chicken cardiac troponin I double-antibody sandwich ELISA detection method |
| CN113189325A (en) * | 2021-04-24 | 2021-07-30 | 无锡济煜山禾药业股份有限公司 | Etimicin enzyme-linked immunoassay method |
| CN113533744A (en) * | 2021-07-20 | 2021-10-22 | 郑州大学 | ELISA kit for detecting human sorted tubulin 17 and preparation method thereof |
| CN115927648B (en) * | 2022-07-13 | 2023-08-11 | 江苏省家禽科学研究所 | SNP genetic marker related to chicken bone calcium content and application thereof |
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| CN110272502A (en) * | 2019-07-12 | 2019-09-24 | 深圳市亚辉龙生物科技股份有限公司 | The hybridoma and preparation method, monoclonal antibody and application of immunogene, the anti-cardiac muscle troponin I monoclonal antibody of secretion |
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| CN109709338A (en) * | 2018-12-28 | 2019-05-03 | 河北省科学院生物研究所 | Detect ox or the enzyme linked immunological kit of ovine skeletal muscle Troponin I and the preparation method and application thereof |
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