CN109810191B - A kind of anti-sheep skeletal muscle troponin I monoclonal antibody and its application - Google Patents
A kind of anti-sheep skeletal muscle troponin I monoclonal antibody and its application Download PDFInfo
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Abstract
本发明涉及一种抗羊骨骼肌肌钙蛋白I单克隆抗体及其应用，属于免疫学技术领域和食品安全分析技术领域。所述抗单克隆抗体由保藏号为CCTCC NO:C2018218的杂交瘤细胞株分泌产生，该抗体效价为1:2.56×105，亚型为IgM，亲和力常数Ka＝7.6×108L/mol。所述的单克隆抗体可用于制备检测生鲜肉及其制品中羊骨骼肌肌钙蛋白I的酶联免疫试剂盒和胶体金试层析试纸条，以达到快速且灵敏地检测动物源性食品中羊肉源性成分检测的目的。
The invention relates to an anti-sheep skeletal muscle troponin I monoclonal antibody and an application thereof, belonging to the technical field of immunology and the technical field of food safety analysis. The anti-monoclonal antibody is secreted and produced by the hybridoma cell line whose deposit number is CCTCC NO: C2018218, the antibody titer is 1:2.56×10 5 , the subtype is IgM, and the affinity constant Ka=7.6×10 8 L/mol . The monoclonal antibody can be used to prepare an enzyme-linked immunosorbent assay kit and a colloidal gold test chromatographic test strip for detecting sheep skeletal troponin I in raw meat and its products, so as to achieve rapid and sensitive detection of animal origin. The purpose of detection of mutton-derived components in food.
Description
Technical Field
The invention relates to a monoclonal antibody for resisting sheep skeletal muscle troponin I, a hybridoma cell strain for producing the monoclonal antibody and application of the antibody in detecting sheep skeletal muscle troponin I, belonging to the technical fields of immunology and 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 must be 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 sheep skeletal muscle troponin I (skTnI) is taken as a target detection object, and the establishment of the mutton-derived component immunological detection and identification technology can be realized. Therefore, the preparation of the monoclonal antibody for resisting the sheep skeletal muscle troponin I has important practical significance and social significance for carrying out the immunological test and identification work of mutton meat components.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the technical defects of the existing meat-derived detection, and provide a monoclonal antibody against sheep skeletal muscle troponin I, wherein the monoclonal antibody has specificity to sheep skeletal muscle troponin I, and is generated by a hybridoma cell strain skTnI-3D3 with the preservation number of CCTCC NO: C2018218. Furthermore, the monoclonal antibody is applied to detecting the sheep skeletal muscle troponin I.
The technical problem of the invention is realized by the following technical scheme.
A monoclonal antibody for resisting sheep skeletal muscle troponin I is prepared from the hybridoma cell strain whose preservation number is CCTCC NO: C2018218. The hybridoma cell strain is named as a hybridoma cell strain skTnI-3D3 and is delivered to a China Center for Type Culture Collection (CCTCC) for collection in 2018, 12 months and 20 days.
The monoclonal antibody for resisting sheep skeletal muscle troponin I can be specifically combined with sheep skeletal muscle troponin I, and the titer is 1:2.56 × 105The subtype is IgM, and the affinity constant Ka is 7.6X 108L/mol。
The application of the sheep skeletal muscle troponin I monoclonal antibody in preparing a non-diagnosis purpose detection product for detecting sheep skeletal muscle troponin I in fresh meat and meat products.
In the application, the non-diagnosis-purpose detection product is an enzyme-linked immunosorbent assay kit or a colloidal gold chromatography test strip.
Further, the enzyme linked immunosorbent assay kit for detecting the sheep skeletal muscle troponin I contains the monoclonal antibody for resisting the sheep skeletal muscle troponin I.
A colloidal gold chromatography test strip for detecting sheep skeletal muscle troponin I contains the monoclonal antibody of the sheep skeletal muscle troponin I.
A method for preparing the sheep skeletal muscle troponin I monoclonal antibody comprises the following steps:
(1) preparation of antigens
Taking sheep skeletal muscle, removing fat and connective tissue, grinding, mixing, 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. After SDS-PAGE electrophoresis identification, the SkTnI electrophoresis bands are ground and diluted by normal saline to be respectively used as a detection antigen and an immune antigen.
(2) Preparing a sheep skeletal muscle troponin I monoclonal antibody:
(a) animal immunization: selecting extracted immune antigen, immunizing female Balb/c mice of 6-8 weeks old, immunizing for 1 time at intervals of 2 weeks, after 3 times of immunization, cutting tail and taking blood to measure titer and inhibition rate, and selecting the mice with the best immune result for fusion;
(b) cell fusion: fusing splenocytes of the mouse selected in the step (a) with myeloma SP2/0 cells of the mouse, determining supernatant by an indirect ELISA method, selecting positive high holes, and subcloning the positive holes by a limiting dilution method until a hybridoma cell strain generating a single monoclonal antibody against sheep skeletal muscle troponin I is established;
(c) 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 at-20 ℃ to obtain the goat skeletal muscle troponin I monoclonal antibody.
A method for identifying the characteristics of the sheep skeletal muscle troponin I monoclonal antibody comprises the following steps:
(a) 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 goat skeletal muscle troponin I monoclonal antibody reaches 1:2.56 multiplied by 10 when the concentration of the purified goat skeletal muscle troponin I monoclonal antibody is 1mg/mL5。
(b) Subtype determination
Subtype determination is carried out by adopting a murine monoclonal antibody subtype identification kit purchased from Sigma company, and the result shows that the subtype of the goat skeletal muscle troponin I monoclonal antibody is IgM.
(c) Affinity assay
The affinity constant of the monoclonal antibody against sheep skeletal muscle troponin I was determined by indirect ELISA, and the result showed that the affinity constant Ka was 7.6X 108L/mol。
(d) Specific assay
The cross-reactivity of the monoclonal antibody with skeletal muscle extracts of sheep, cattle, chicken, duck and pig was determined by indirect ELISA. The result shows that the potency of the sheep sktnI-3D3 and the skeletal muscle extract of ruminants (sheep and cattle) is 1:100 ten thousand, the potency of the sheep sktnI-3D3 and the skeletal muscle extract of chickens, ducks and pigs is lower than 1:1 ten thousand, and the specificity is better.
The monoclonal antibody provided by the invention can be applied to detection of sheep skeletal muscle troponin I in a sample, and is mainly applied to preparation of an enzyme-linked immunosorbent assay kit and colloidal gold test strip paper for detection of sheep skeletal muscle troponin I. The quality of the monoclonal antibody for resisting the sheep skeletal muscle troponin I provided by the invention has strong controllability and repeatability, and experimental basis is provided for further establishment and application of a specific and sensitive immunoassay method through preliminary analysis and identification of the characteristics of the obtained antibody.
Drawings
FIG. 1 is an ISDS-PAGE identification chart of sheep skeletal muscle troponin extracted according to the invention, wherein FIG. 1a is a color chart, and FIG. 1b is a black-and-white chart
FIG. 2 is a graph showing the measurement of the titer of the monoclonal antibody against troponin I from skeletal muscle of sheep
FIG. 3 is a graph showing the measurement of the subtype of the monoclonal antibody against troponin I of skeletal muscle of sheep
FIG. 4 is a diagram showing the specific assay of the sheep skeletal muscle troponin I monoclonal antibody of the present invention
The hybridoma cell strain skTnI-3D3 of the monoclonal antibody for resisting the sheep 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: C2018218.
Detailed Description
The present invention is further described in detail with reference to the following specific embodiments, which 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 sheep skeletal muscle troponin I antigen according to the invention
Taking sheep skeletal muscle, removing fat and connective tissue, grinding, mixing, 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. After the treatment fluid 1 and the treatment fluid 2 are identified by SDS-PAGE electrophoresis, the identification result is shown in figure 1, and the result shows that the treatment fluid 1 and the treatment fluid 2 have protein bands in 21-22 kD, and the protein bands are consistent with the molecular weight of skTnI in 21-24 kD 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 sheep skeletal muscle troponin I according to the invention
(1) Animal immunization: immunizing female Balb/c mice of 6-8 weeks old with the prepared immune antigen for 1 time at intervals of 2 weeks, wherein the immune process is shown in Table 1, cutting off the tail after three-immunization, taking blood, measuring titer and inhibition rate, and selecting the mice with the best immune result for fusion;
TABLE 1 immunization scheme
(2) Cell fusion: fused mice are bled by eyes, serum is used as positive control, spleens are taken out under aseptic condition after cervical removal and sacrifice, splenocytes are prepared and fused with SP2/0 cells according to the proportion of 5:1 by PEG, fused cell suspension is added into a 96-well plate which is paved with feeder cells, the 96-well plate is placed at 37 ℃ and 5% CO2Culturing in an incubator;
(3) screening positive hybridoma cell strains: the fused cells were checked for contamination the next day, and were replaced with HT medium 10 days after fusion. And (3) after liquid replacement, screening positive holes by using indirect ELISA (enzyme-linked immunosorbent assay), selecting holes with strong positive, single clone and good cell state as much as possible, carrying out subcloning by using a limiting dilution method, and simultaneously carrying out amplification culture and cryopreservation until a monoclonal cell strain with single secretory antibody is established.
(4) Large-scale preparation of monoclonal antibodies: adopting a method of inducing ascites in mice, taking healthy Balb/c female mice, injecting 0.5mL of paraffin oil into each mouse, adjusting 10 g of clone positive hybridoma after 7 days6and/mL, injecting 1mL into the abdominal cavity of each mouse, taking ascites after 7-9 days, centrifuging, removing fat, purifying by using caprylic acid-ammonium sulfate, freeze-drying, and storing at-20 ℃ to obtain the sheep skeletal muscle troponin I monoclonal antibody.
EXAMPLE III characterization of monoclonal antibodies to sheep skeletal muscle troponin I according to the invention
(1) The titer measurement adopts an indirect ELISA method, and comprises the following specific steps:
coating: diluting the coated antigen with carbonate buffer solution until the concentration is 5 mug/mL, and keeping the concentration of the coated antigen in a 96-well enzyme label plate at 100 mug/well for overnight at 4 ℃;
washing: recovering the coated plate to room temperature, decanting off coating solution, adding 300 μ L of washing solution into each well, standing for L min each time, washing for 3 times, and patting to dry for the last time;
and (3) sealing: adding 200 μ L of lotion containing 10% calf serum into each well, and keeping the temperature at 37 deg.C for 1 h; pouring off the confining liquid, washing for 3 times, and patting dry;
adding a primary antibody: diluting the monoclonal antibody with washing solution at a ratio of 1:2000 times, adding 100 μ L per well, setting blank control well (PBS) and negative control (negative serum), and standing at 37 deg.C for 45 min; pouring out primary antibody, washing for 3 times, and patting dry;
adding an enzyme-labeled secondary antibody: adding 100 μ L of HRP enzyme-labeled goat anti-mouse IgG diluted by 10000 times into each well, and standing at 37 deg.C for 30 min; pouring out the secondary antibody, washing for 3 times, and patting dry; color development: adding 100 mu L of substrate developing solution into each hole, and reacting for 15min at 37 ℃ in a dark place;
and (4) terminating: adding 50 mu L of stop solution into each hole to stop the reaction;
and (3) detection: the absorbance value at a wavelength of 450nm was measured (A450 nm).
And (4) judging a result: and (A sample well-A blank)/(A negative control-A blank) is not less than 2.1, and when A450nm of the negative control is less than 0.2, the dilution factor of the antibody is the antibody titer. As shown in FIG. 2, the titer of the monoclonal antibody 3D3 against sheep skeletal muscle troponin I at a concentration of 1mg/ml was 1: 2.56X 105。
(2) Subtype determination
Subtype determination was carried out using a murine monoclonal antibody subtype identification kit purchased from Sigma. The monoclonal antibody secreted by the hybridoma cell is obviously different from the secondary antibodies of different subclasses in color development, wherein the A450nm value of the secondary antibody with the Ig M is the highest, the color development of the secondary antibody with the Ig G1 is weak, the secondary antibodies with the Ig G2a, the Ig G2b, the Ig G3 and the Ig A are hardly developed, and as a result, the type of the antibody secreted by the 3D3 is mainly IgM as shown in figure 3.
(3) Affinity assay
The affinity constant (Ka) was determined by a non-competitive ELISA method. The method comprises the following specific steps:
coating: diluting the coated antigen with carbonate buffer solution until the concentration is 1, 0.5, 0.1 and 0.05 mu g/mL, respectively coating the 96-well ELISA plate with 100 mu L/well, and standing overnight at 4 ℃; pouring out the coating solution, washing for 3 times, and patting dry;
and (3) sealing: adding 200 μ L of lotion containing 10% calf serum into each well, and keeping the temperature at 37 deg.C for 1 h; pouring off the confining liquid, washing for 3 times, and patting dry;
adding a monoclonal antibody: diluting the monoclonal antibody with washing solution at a ratio of 100 μ g/mL, adding 100 μ L per well, and keeping the temperature and humidity at 37 deg.C for 45 min; pouring out primary antibody, washing for 3 times, and patting dry;
adding an enzyme-labeled secondary antibody: adding 100uL of goat anti-mouse IgG labeled with HRP enzyme and diluted by 10000 times into each hole, and standing at 37 ℃ for 30 min; pouring out the secondary antibody, washing for 3 times, and patting dry;
color development: adding 100 mu L of substrate developing solution into each hole, and reacting for 15min at 37 ℃ in a dark place;
and (4) terminating: adding 50 mu L of stop solution into each hole to stop the reaction;
and (3) detection: the absorbance value at a wavelength of 450nm was measured (A450 nm). Calculating Ka according to the following formula
Ka＝(n-1)/2(n Ab’-Ab)
In the formula: ab is the antibody concentration which generates a half-absorbance value when the antigen concentration is Ag; ab 'is the concentration of the antibody which generates a half-absorbance value when the antigen concentration is Ag'; n is the dilution multiple between Ag and Ag', the affinity constant of the antibody is determined by a non-competitive enzyme-linked immunosorbent assay, and the affinity constant Ka of the monoclonal antibody of the sheep skeletal muscle troponin I is determined to be 7.6 multiplied by 108L/mol。
(4) Specific assay
The indirect competitive ELISA method is adopted, and the specific steps are as follows:
coating: diluting the coating antigen (extract of skeletal muscle troponin I of sheep, cattle, chicken, duck and pig) with carbonate buffer solution to the concentration of 5 μ g/mL, and standing overnight at 4 deg.C with 96-well enzyme labeling plate of 100 μ L/well; pouring out the coating solution, washing for 3 times, and patting dry;
and (3) sealing: adding 200 μ L of lotion containing 10% calf serum into each well, and keeping the temperature at 37 deg.C for 1 h; pouring off the confining liquid, washing for 3 times, and patting dry;
adding a primary antibody: diluting the monoclonal antibody with washing solution at a ratio of 1:2000 times, adding 100 μ L per well, setting blank control well (PBS) and negative control (negative serum), and standing at 37 deg.C for 45 min; pouring out primary antibody, washing for 3 times, and patting dry;
adding an enzyme-labeled secondary antibody: adding 100 μ L of HRP enzyme-labeled goat anti-mouse IgG diluted by 10000 times into each well, and standing at 37 deg.C for 30 min; pouring out the secondary antibody, washing for 3 times, and patting dry;
color development: adding 100 mu L of substrate developing solution into each hole, and reacting for 15min at 37 ℃ in a dark place;
and (4) terminating: adding 50 mu L of stop solution into each hole to stop the reaction;
and (3) detection: the absorbance value at a wavelength of 450nm was measured (A450 nm).
And (4) judging a result: and (A sample well-A blank)/(A negative control-A blank) is not less than 2.1, and when A450nm of the negative control is less than 0.2, the dilution factor of the antibody is the antibody titer. The results are shown in figure 4, the potency of sheep SkTnI-3D3 to the ruminant sheep and cattle skeletal muscle extract is 1:25 ten thousand, the potency to chicken, duck and pig skeletal muscle extract is less than 1:1 ten thousand, and the specificity is better
Example four applications of the monoclonal antibody against sheep skeletal muscle troponin I of the invention
The embodiment is an application example of the sheep skeletal muscle troponin I monoclonal antibody in establishing an enzyme-linked immunosorbent assay kit or a colloidal gold chromatography test strip method for detecting sheep skeletal muscle troponin I, and can be used for detecting sheep skeletal muscle troponin I in fresh meat and products thereof.
(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; the precipitate was removed, the supernatant was filtered through Whatman No. 1 filter paper, and the filtrate was collected for detection.
(2) The antibody is applied to the detection of an enzyme-linked immunoassay kit method
The detection principle of the kit is a double-antibody sandwich ELISA method. Coating a microporous plate strip with an antibody (3D3) to prepare a solid-phase capture antibody, sequentially adding a standard substance or a sample into the micropores coated with the monoclonal antibody, combining with a detection antibody (3A8) marked by horseradish peroxidase to form an antibody-antigen-enzyme-labeled antibody compound, adding TMB for developing color, wherein the compound is blue and finally yellow under the action of acid, and the color depth is in positive correlation with the content of sheep skeletal muscle troponin I in the sample.
And (3) detection: diluting the goat skeletal muscle troponin I monoclonal antibody with a carbonate buffer solution at a ratio of 1:1000, coating the diluted monoclonal antibody with 100 mu L/hole at 4 ℃ overnight; washing and drying; blocking with 1% gelatin in PBS, 200. mu.L/well, incubating at 37 ℃ for 2 h; washing and drying; adding a sample to be detected, and incubating for 1h at 37 ℃; washing and drying; adding diluted HRP-marked bovine skTnI-3A8 monoclonal antibody, 100 mu L/hole, and incubating for 1h at 37 ℃; washing and drying; adding 100 μ L/well of color developing solution, developing for 15min, adding 50 μ L/well of stop solution, and measuring.
And (4) judging a result:
(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.
(3) Method for detecting colloidal gold chromatography test paper strip by using antibody
The reaction principle adopts a double-antibody sandwich method to qualitatively detect the sheep skeletal muscle troponin I, the sheep skeletal muscle troponin I existing in a sample is firstly combined with an antibody marked by gold particles in the process of moving up along a test strip to form a gold-marked antibody-sheep skeletal muscle troponin I compound, a capture antibody fixed on an NC membrane is combined with the sheep skeletal muscle troponin I in the compound, and the color development strength of a T position (a detection line) is in direct proportion to the content of the sheep skeletal muscle troponin I in the sample.
And (3) detection: and taking out the colloidal gold chromatography test strip of the sheep skeletal muscle troponin I, inserting the sample end into the sample liquid to be detected, wherein the insertion depth is not more than the mark line, taking out the test strip in about 10-20 seconds, horizontally placing, observing for 3-5 minutes to judge the detection result, and after 10 minutes, the result is invalid.
And (4) judging a result: a brownish red line is displayed at the position (quality control line) of a corresponding quality control area C on the envelope, and a brownish red line is not displayed at the position T of the detection area, which indicates that the detection result is negative, and indicates that the sample to be detected does not contain sheep skeletal muscle troponin I; two brown-red lines are displayed at the position T, C on the coating film, the result is positive, and the result indicates that the sample to be detected contains sheep skeletal muscle troponin I; and when the quality control area C does not show a brownish red strip, the test paper is judged to be invalid whether the detection area T shows a brownish red strip or not.
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. A monoclonal antibody for resisting sheep skeletal muscle troponin I is characterized in that the monoclonal antibody is generated by a hybridoma cell strain skTnI-3D3 with the preservation number of CCTCC NO: C2018218; the antibody titer is 1:2.56 × 105Subtype IgM with an affinity constant Ka = 7.6X 108 L/mol。
2. A hybridoma cell strain skTnI-3D3 producing the monoclonal antibody against sheep skeletal muscle troponin I as claimed in claim 1, which is deposited in China center for type culture Collection with the preservation number of CCTCC NO: C2018218, and the preservation date is 2018, 12 months and 20 days.
3. Use of the monoclonal antibody against sheep skeletal muscle troponin I according to claim 1 for the preparation of a non-diagnostic test product for the detection of sheep skeletal muscle troponin I in a biological sample.
4. The use of claim 3, wherein the non-diagnostic detection product is an enzyme linked immunosorbent assay kit or a colloidal gold chromatography strip.
5. An enzyme linked immunosorbent assay kit for detecting sheep skeletal muscle troponin I, which is characterized by comprising the monoclonal antibody against sheep skeletal muscle troponin I as claimed in claim 1.
6. A colloidal gold chromatography test strip for detecting sheep skeletal muscle troponin I, which comprises the monoclonal antibody against sheep skeletal muscle troponin I according to claim 1.
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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 bovine or sheep skeletal muscle troponin I and its application |
| CN109709338B (en) * | 2018-12-28 | 2022-03-15 | 河北省科学院生物研究所 | Enzyme linked immunosorbent assay kit for detecting skeletal muscle troponin I of cattle or sheep and preparation method and application thereof |
| CN113402608B (en) * | 2021-06-24 | 2022-02-22 | 河北省科学院生物研究所 | Hybridoma cell strain, monoclonal antibody secreted by hybridoma cell strain and application of monoclonal antibody |
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