CN111220809B - Colloidal gold immunochromatography test strip for detecting chicken or duck skeletal muscle troponin I and preparation method and application thereof - Google Patents
Colloidal gold immunochromatography test strip for detecting chicken or duck skeletal muscle troponin I and preparation method and application thereof Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
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- G01N2333/4712—Muscle proteins, e.g. myosin, actin, protein
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- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to a colloidal gold chromatography test strip 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. The test strip structure comprises a bottom plate, a sample pad, a colloidal gold pad, a coating film and a water absorption pad, wherein protective films are arranged at two ends of the test strip, and the sample pad, the colloidal gold pad, the coating film and the water absorption pad are sequentially adhered to the bottom plate; the colloidal gold pad is coated with a capture antibody marked by colloidal gold; the coating film is provided with a invisible detection line printed by a detection antibody solution and a invisible quality control line printed by a goat anti-mouse IgG solution. The capture antibody is secreted by a hybridoma cell strain skTnI-3E7 with the preservation number of CCTCC NO: C202003, and the detection antibody is a chicken skeletal muscle troponin I rabbit-derived polyclonal antibody. The colloidal gold chromatography detection test strip has the characteristics of convenience, rapidness, intuitiveness, strong timeliness and the like, and is wide in application range, low in cost and convenient to popularize and apply.
Description
Technical Field
The invention relates to a colloidal gold immunochromatography test strip 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.
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. 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 methods have certain sensitivity, but have the defects 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 method is greatly dependent on the treatment process of the cooked meat products, and the different degradation levels of genetic substances are caused by the diversity of the production process. Furthermore, the method can only detect possible sources of animal DNA, and cross contamination must be prevented at all times, and milk, blood and fat are all possible sources of DNA. 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 colloidal gold immunochromatographic test strip for chicken or duck skeletal troponin I has important practical significance and social significance for carrying out the immunological detection and identification of chicken or duck meat source components.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide the colloidal gold immunochromatographic test strip for detecting the chicken or duck skeletal troponin I, which has the advantages of simple and quick operation and suitability for primary screening of a large number of samples. In addition, the invention further provides a preparation method and application of the colloidal gold immunochromatographic test strip for detecting chicken or duck skeletal muscle troponin I.
The technical problems are realized by the following technical scheme.
A colloidal gold immunochromatographic test strip for detecting chicken or duck skeletal muscle troponin I comprises a bottom plate, a sample pad, a colloidal gold pad, a coating film and a water absorption pad, wherein protective films are arranged at two ends of the test strip; the sample pad, the colloidal gold pad, the coating film and the water absorption pad are sequentially adhered to the bottom plate; the colloidal gold pad is a capture antibody adsorbed with a colloidal gold mark, the coating film contains a detection line T line pre-coated with a detection antibody, a quality control line C line pre-coated with a goat anti or rabbit anti-mouse IgG solution, and the two lines are vertically arranged in parallel.
The colloidal gold immunochromatographic test strip is obtained by secreting a hybridoma cell strain skTnI-3E7 with a preservation number of CCTCC NO: C202003, and the cell strain is delivered to the China Center for Type Culture Collection (CCTCC) of Chinese Wuhan in 12 months 10 of 2019 for preservation; the detection antibody is an anti-chicken skeletal muscle troponin I polyclonal antibody.
The colloidal gold immunochromatographic test strip is characterized in that 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 bottom plate of the colloidal gold immunochromatographic test strip is a PVC strip or other hard materials which do not absorb water; the sample pad and the colloidal gold pad are made of glass fiber cotton; the water absorption pad is made of water absorption filter paper; the coating film can be a nitrocellulose film or a cellulose acetate film; the protective film is an opaque adhesive film.
The preparation method of the colloidal gold chromatographic test strip provided by the invention comprises the following steps:
(1) Preparing a colloidal gold-labeled capture antibody:
centrifuging 10000r/min of a capture antibody (3E 7) solution to be marked for 30min; taking 10mL of colloidal gold solution, using 0.1mol/LK 2 CO 3 The pH value of the colloidal gold solution is regulated to 8.2; mixing the same amount of capture antibody solution with colloidal gold solution under magnetic stirring, incubating at room temperature for 15min, centrifuging at 10000r/min for 30min, discarding supernatant, and collecting precipitate with Na containing 10g BSA, 0.5g sodium azide and 0.02mol/L per liter 2 B 4 O 7 Diluting the solution to obtain a colloidal gold-labeled capture antibody, and preserving at 4 ℃ for later use;
(2) Preparing a colloidal gold pad: cutting glass fiber cotton according to the specification, uniformly spraying a capture antibody marked by colloidal gold on the glass fiber cotton by a gold spraying instrument, drying at 37 ℃ for 1h, sealing, and preserving at 4 ℃ for later use;
(3) Preparation of detection lines and quality control lines: placing detection antibody (chicken skeletal troponin I polyclonal antibody) in a gold spraying instrument storage tank A, placing goat anti-rabbit IgG solution in a storage tank B, starting up and respectively spot-shooting at the center of a membrane to form a linear invisible detection line and an invisible quality control line with a spacing of 0.5cm, naturally drying, sealing, and preserving at 4 ℃ for later use;
(4) Assembling a test strip: sequentially adhering a sample pad, a colloidal gold pad, a coating film and a water absorption pad on a bottom plate, adhering protective films on the surfaces of two ends of a test strip, and cutting the test strip into test strips on a grooving machine.
The colloidal gold chromatographic test strip is applied to the chicken or duck skeletal muscle troponin I in fresh meat and products thereof.
The application of the kit for detecting chicken or duck skeletal muscle troponin I 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 by colloidal gold chromatographic test strip
Taking out the colloidal gold immunochromatographic test strip for detecting the chicken or duck skeletal muscle troponin I from the package, inserting a sample end into a sample liquid to be detected, taking out the test strip after about 10-20 seconds, horizontally placing, observing and judging a detection result for 3-5 minutes, and invalidating the result after 10 minutes;
(3) Result determination
(a) Positive: if the position of the corresponding quality control area C on the coating film shows a brownish red line, the position of the detection area T shows a brownish red line, which indicates that the detection result is positive, and the sample to be detected contains chicken or duck skeletal troponin I;
(b) Negative: if the T position does not develop color on the coating film, a brownish red line is displayed at the C position, and the result is negative, which indicates that the sample to be tested does not contain chicken or duck skeletal troponin I;
(c) Failure: when the quality control area C does not show a brownish red band, the test paper is judged to be invalid whether the detection area T shows a brownish red band or not.
The sensitivity of the colloidal gold chromatography test strip to the skeletal muscle troponin I of the chicken or the duck is 30ug/kg, and the cross reaction rate with the skeletal muscle troponin I extract of the cattle, the sheep and the pigs is lower than 10 percent. The method for detecting the skeletal muscle troponin I of the chicken or duck by using the colloidal gold chromatographic test strip is simple, convenient, quick, visual, accurate, wide in application range, low in cost and easy to popularize and use.
Drawings
FIG. 1 is a schematic diagram of a cross-sectional structure of a test paper for detecting chicken or duck skeletal troponin I colloidal gold chromatography test strips according to the present invention
FIG. 2 is a schematic diagram showing the structure of a colloidal gold chromatography test strip for detecting chicken or duck skeletal troponin I according to the present invention in a top view
FIG. 3 is a graph showing the result of detecting chicken or duck skeletal muscle troponin I colloidal gold chromatography test paper
FIG. 4 is a graph showing the sensitivity of the colloidal gold chromatography test strip for detecting chicken or duck skeletal muscle troponin I according to the present invention
Detailed Description
The invention is described in further detail below in connection with specific embodiments, which are set forth without limiting the scope of the invention in any way.
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
Diluting the detection antigen to 5 mug/mL by using carbonate buffer solution with pH of 9.6, coating the detection plate, diluting the purified monoclonal antibody by 1:2000,1:4000,1:8000, … …, 1:1024000, adding into an enzyme-labeled plate hole, adding HRP-labeled goat anti-mouse secondary antibody after reaction, and finally developing by using TMB, wherein the result shows that the titer of the purified duck skeletal troponin I monoclonal antibody at the concentration of 1mg/mL reaches 1:10 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 the colloidal gold chromatography test strip of the invention
1. Preparing colloidal gold solution: taking 100mL of distilled water, boiling for 5min, adding 1mL of chloroauric acid with the mass concentration of 1% and 1.5mL of trisodium citrate solution with the mass concentration of 1%, continuously stirring and heating, finally carrying out grey and wine red treatment on the solution until the solution is transparent red, cooling to room temperature, respectively carrying out quality identification by a visual inspection method and an ultraviolet spectrophotometry, and storing the qualified colloidal gold solution in a dark place at 4 ℃;
2. preparation of colloidal gold-labeled capture antibody (3E 7): centrifuging the capture antibody solution to be marked for 30min at 10000 r/min; taking 10mL of colloidal gold solution, using 0.1mol/L K 2 CO 3 The pH value of the colloidal gold solution is regulated to 8.2; mixing the same amount of capture antibody solution with colloidal gold solution under magnetic stirring, incubating at room temperature for 15min, centrifuging at 10000r/min for 30min, discarding supernatant, and collecting precipitate with Na containing 10g BSA, 0.5g sodium azide and 0.02mol/L per liter 2 B 4 O 7 Diluting the solution to obtain a colloidal gold-labeled capture antibody, and preserving at 4 ℃ for later use;
3. preparing a colloidal gold pad: cutting glass fiber cotton according to the specification, uniformly spraying a gold-labeled antibody on the glass fiber cotton by a gold spraying instrument, drying at 37 ℃ for 1h, sealing, and preserving at 4 ℃ for later use;
4. preparation of detection lines and quality control lines: placing the detection antibody in a gold spraying instrument storage tank A, placing goat anti-mouse IgG solution in a storage tank B, starting up and respectively spot-shooting at the center of a membrane to form a linear invisible detection line T line and an invisible quality control line C line with the interval of 0.5cm, naturally drying, sealing, and preserving at 4 ℃ for later use;
5. assembling a test strip: sequentially adhering a sample pad, a colloidal gold pad, a coating film and a water absorption pad on a bottom plate, adhering protective films on the surfaces of two ends of a test strip, and cutting the test strip into test strips on a grooving machine.
6. The structure of the colloidal gold chromatographic test strip is shown in fig. 1 and 2. In the figure, a bottom plate 1 is made of a PVC plate, a sample pad 2 is made of glass fiber cotton, a capture antibody is adsorbed on a colloidal gold pad 3, a coating film 4 is a nitrocellulose film, a water absorption pad 7 is made of water absorption filter paper, and layers of numbers 2, 3, 4 and 7 are sequentially stuck and fixed on the bottom plate 1 from right to left, and fibers at the junction of the two layers are mutually crossed and permeated. The coating film 4 is provided with a invisible detection line 5 and an invisible quality control line 6, wherein the invisible detection line is printed by a detection antibody, the invisible quality control line is printed by a goat anti-mouse IgG antibody solution, and the two blots are arranged in parallel to form an 'I'. The sample end protective film 8 covers the sample pad 2 and the colloidal gold pad 3, the handle end protective film 9 covers the water absorbing pad 7, and the arrow and MAX characters are printed on the protective film on the right side of the sample marking line.
Embodiment five the colloidal gold chromatography test strip of the invention detects chicken or duck skeletal muscle troponin I 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, 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 by colloidal gold chromatographic test strip
And taking out the colloidal gold immunochromatographic test strip for detecting the chicken or duck skeletal muscle troponin I from the package, inserting the sample end into the sample liquid to be detected, taking out the test strip after about 10-20 seconds, horizontally placing, observing and judging the detection result for 3-5 minutes, and invalidating the result after 10 minutes.
3. Result determination
As shown in fig. 3, a is negative, a brown red line is displayed at the position of a corresponding quality control area C on the coating film, a brown red line is not displayed at the position of a detection area T, the detection result is negative, and the fact that the sample to be detected does not contain chicken or duck skeletal troponin I is indicated; b is positive, a two-day brownish red line is displayed at a T, C position on the coating film, and the result is positive, which indicates that the sample to be detected contains chicken or duck skeletal troponin I; c. d is failure, and when the quality control area C does not show a brownish red band, whether the detection area T shows a brownish red band or not is judged to be invalid.
Example six Performance detection of the colloidal gold chromatography test strip of the invention
1. Sensitivity of
Chicken or duck skeletal troponin I is diluted into a series of standard solutions of 100g/kg, 50g/kg, 30g/kg, 20g/kg and 10g/kg respectively by PBS (phosphate buffer solution), the standard solutions are respectively marked as 1, 2, 3, 4 and 5, 90 mu L of the standard solutions are respectively dripped on a sample pad of a test strip, and the color development condition of the test strip is observed after 3 min.
As a result, as shown in FIG. 4, when the addition concentration was 30g/kg, the T line started to develop, indicating that the sensitivity of the colloidal gold test strip was 20ug/kg.
2. Specificity (specificity)
Selecting bovine, sheep and pig skeletal muscle troponin extracts, diluting the extracts to 30g/kg with PBS for testing, observing the color development effect of the test strip, and displaying the detection result that the prepared colloidal gold test strip has no cross reaction with the bovine, sheep and pig skeletal muscle extracts.
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 (4)
1. A colloidal gold immunochromatographic test strip for detecting chicken or duck skeletal muscle troponin I comprises a bottom plate, a sample pad, a colloidal gold pad, a coating film and a water absorption pad, wherein protective films are arranged at two ends of the test strip; the sample pad, the colloidal gold pad, the coating film and the water absorption pad are sequentially adhered to the bottom plate; the colloidal gold pad is a capture antibody adsorbed with a colloidal gold mark, a detection line T line pre-coated with a detection antibody is contained on the coating film, a quality control line C line pre-coated with a goat anti-rabbit IgG solution is contained, and the two lines are vertically and parallelly arranged; the bottom plate is a PVC strip or other hard materials which do not absorb water; the sample pad and the colloidal gold pad are made of glass fiber cotton; the water absorption pad is made of water absorption filter paper; the coating film can be a nitrocellulose film or a cellulose acetate film; the protective film is an opaque adhesive film; the capture antibody is a murine monoclonal antibody; the detection antibody is a rabbit polyclonal antibody; 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 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.
2. The method for preparing the colloidal gold immunochromatographic test strip for detecting chicken or duck skeletal muscle troponin I according to claim 1, which is characterized by comprising the following steps:
(1) Preparing a colloidal gold-labeled capture antibody:
centrifuging the capture antibody solution to be marked for 30min at 10000 r/min; taking 10mL of colloidal gold solution, using 0.1mol/L K 2 CO 3 The pH value of the colloidal gold solution is regulated to 8.2; mixing the same amount of capture antibody solution with colloidal gold solution under magnetic stirring, incubating at room temperature for 15min, centrifuging at 10000r/min for 30min, discarding supernatant, and collecting precipitate with Na containing 10g BSA, 0.5g sodium azide and 0.02mol/L per liter 2 B 4 O 7 Diluting the solution to obtain a colloidal gold-labeled capture antibody, and preserving at 4 ℃ for later use;
(2) Preparing a colloidal gold pad: cutting glass fiber cotton according to the specification, uniformly spraying a capture antibody marked by colloidal gold on the glass fiber cotton by a gold spraying instrument, drying at 37 ℃ for 1h, sealing, and preserving at 4 ℃ for later use;
(3) Preparation of detection lines and quality control lines: placing the detection antibody in a gold spraying instrument storage tank A, placing goat anti-rabbit IgG solution in a storage tank B, starting up and respectively spot-shooting at the center of the membrane to form a linear invisible detection line and an invisible quality control line with a spacing of 0.5cm, naturally drying, sealing and preserving at 4 ℃ for later use;
(4) Assembling a test strip: sequentially adhering a sample pad, a colloidal gold pad, a coating film and a water absorption pad on a bottom plate, adhering protective films on the surfaces of two ends of a test strip, and cutting the test strip into test strips on a grooving machine.
3. Use of the colloidal gold chromatography test strip according to claim 1 for detecting chicken or duck skeletal troponin I.
4. The use according to claim 3, characterized by 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 (1:2 w/v), 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 by colloidal gold chromatographic test strip
Taking out the colloidal gold immunochromatographic test strip for detecting the chicken or duck skeletal muscle troponin I from the package, inserting a sample end into a sample liquid to be detected, taking out the test strip after 10-20 seconds, horizontally placing, observing and judging a detection result after 3-5 minutes, and invalidating the result after 10 minutes;
(3) Result determination
(a) Positive: if the position of the corresponding quality control area C on the coating film shows a brownish red line, the position of the detection area T shows a brownish red line, the detection result is positive, and the fact that the sample to be detected contains chicken or duck skeletal troponin I is indicated;
(b) Negative: if the T position does not develop color on the coating film, a brownish red line is displayed at the C position, and the result is negative, which indicates that the sample to be tested does not contain chicken or duck skeletal troponin I;
(c) Failure: when the quality control area C does not show a brownish red band, the test paper is judged to be invalid whether the detection area T shows a brownish red band or not.
Priority Applications (1)
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| CN104502595A (en) * | 2014-11-10 | 2015-04-08 | 江苏宏泰格尔生物医学工程有限公司 | Troponin detection method and kit thereof |
| 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 |
| CN109709339A (en) * | 2018-12-28 | 2019-05-03 | 河北省科学院生物研究所 | Detect colloidal gold immuno-chromatography test paper strip and the application of ox or ovine skeletal muscle Troponin I |
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| CN104502595A (en) * | 2014-11-10 | 2015-04-08 | 江苏宏泰格尔生物医学工程有限公司 | Troponin detection method and kit thereof |
| 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 |
| CN109709339A (en) * | 2018-12-28 | 2019-05-03 | 河北省科学院生物研究所 | Detect colloidal gold immuno-chromatography test paper strip and the application of ox or ovine skeletal muscle Troponin I |
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