CN109709338A - Detect ox or the enzyme linked immunological kit of ovine skeletal muscle Troponin I and the preparation method and application thereof - Google Patents
Detect ox or the enzyme linked immunological kit of ovine skeletal muscle Troponin I and the preparation method and application thereof Download PDFInfo
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Abstract
The present invention relates to a kind of detection oxen or the enzyme-linked immunologic detecting kit of ovine skeletal muscle Troponin I and the preparation method and application thereof, belong to immunological technique field and Food Safety Analysis technical field.The enzyme linked immunological kit, which contains, is coated with the capture ELISA Plate of antibody, the detection antibody of horseradish peroxidase-labeled, ox or ovine skeletal muscle Troponin I standard solution, substrate developing solution, terminate liquid, concentrated cleaning solution.The capture antibody is secreted to obtain by the hybridoma cell strain 3A8 that deposit number is CCTCC NO:C2018217, and the detection antibody is secreted to obtain by the hybridoma cell strain 3D3 that deposit number is CCTCC NO:C2018218.The advantages that enzyme linked immunological kit has sensitivity, precision, accuracy high, and cross reacting rate is low, is suitble to batch samples detection.
Description
Technical field
The present invention relates to enzyme linked immunological kit and preparation method thereof of a kind of detection ox or ovine skeletal muscle Troponin I with
Using belonging to immunological technique field and Food Safety Analysis technical field.
Background technique
In meat product, due to price, religion, health etc., it is true that many countries formulate laws and regulations requirement food labelling
It is real clearly to mark meat sources, forbid adulterated behavior, to protect the interests of consumer, but obscures meat kind in the market
Phenomenon is still very universal, and adulterated mode includes the means such as mixing, being mixed into, extracting, palming off, especially with beef or mutton product
Doping, adulterated behavior are most commonly seen, these adulterated behaviors greatly compromise the interests of consumer.
Currently, many laboratories all use the derived component in Protocols in Molecular Biology means detection meat products both at home and abroad,
DNA detection method means multiplicity, predominantly nucleic acid probe hybridization, DNA fingerprint analysis, PCR specific amplified, PCR-RFLP etc..This
A little methods have certain sensitivity, however, there are also it is at high cost, height, heavy workload, Bu Nengxian are required to test object
, there is very big uncertainty in the disadvantages of field detecting especially for cooked meat product detection, because this is largely dependent upon cold cuts
The treatment process of product, the diversity of production process result in the different Degradation Levels of genetic stew.Furthermore this method can only be examined
Survey the possibility source of animal DNA, it is necessary to the moment prevents cross contamination, milk, blood, fat be likely to be DNA source.Cause
This, needs other methods mutually to confirm come its testing result with him.Immunological method have high sensitivity, specificity it is good,
The features such as at low cost, easy to operate, is suitable for batch samples and screens, and wherein enzyme linked immunosorbent assay and colloidal gold strip are most
Common method.
However cooked meat product will generally pass through high temperature, HIGH PRESSURE TREATMENT, many albumen are all become in this process
Property, antigenicity and water solubility are lost, this just brings difficulty to the foundation of immunological method.To Immunological Method is applied to
The detection of animal derived materials, it is necessary to find a specific heat-staple protein as marker antigen, then develop
Out for the monoclonal antibody of the specificity of this antigen.Troponin I (TnI) in animals skeletal muscle has kind specificity,
It can be used as a kind of heat-resisting type marker protein, distinguish the meat type source of different genera life, cooked meat product.With ox or sheep bone bone
Flesh Troponin I (skTnI) is target detection thing, is able to achieve building for ox or mutton derived component immunology detection authentication technique
It is vertical.Therefore, the preparation of ox or ovine skeletal muscle Troponin I enzyme-linked immunologic detecting kit for carry out beef or mutton meat source at
The immunological test identification work divided has important practical significance and social effect.
Summary of the invention
The technical problem to be solved by the present invention is to overcome prior art defect, a kind of detection ox or ovine skeletal muscle flesh are provided
The enzyme-linked immunologic detecting kit of calcium protein I, the enzyme linked immunological kit have sensitivity, precision, accuracy high, intersect
The advantages that reactivity is low, is suitble to batch samples detection.In addition, the present invention further provides the detection ox or ovine skeletal muscle flesh calcium
The preparation method and application of the enzyme linked immunological kit of protein I.
Technical problem of the present invention is realized by the following technical solutions.
A kind of enzyme linked immunological kit detecting ox or ovine skeletal muscle Troponin I, the enzyme linked immunological kit is using double
Anti- sandwich method, by being coated with the ELISA Plate of capture antibody, detection antibody, ox or the ovine skeletal muscle Troponin I standard of enzyme label
Product solution, substrate developing solution, terminate liquid, concentrated cleaning solution composition.
Above-mentioned enzyme linked immunological kit, the hybridoma that the capture antibody is CCTCC NO:C2018217 by deposit number
Cell strain 3A8 secretes to obtain, the hybridoma cell strain 3D3 that the detection antibody is CCTCC NO:C2018218 by deposit number
Secretion obtains；Two plants of cell strains deliver China typical culture collection center (CCTCC) preservation on December 20th, 2018.
Above-mentioned enzyme linked immunological kit, the capture antibody and detection antibody are source of mouse, Ma Yuan, Yang Yuan, rabbit source or cavy
Resource monoclonal antibody, preferably source of mouse monoclonal antibody；It extracts immunizing antigen by ox or ovine skeletal muscle Troponin I and is immunized
It arrives.
Above-mentioned enzyme linked immunological kit, the enzyme labelling assay for determining antibody use the enzyme tagging scheme of this field routine, example
Such as, marker enzyme and detection antibody coupling can be obtained using sodium iodate method.
Above-mentioned enzyme linked immunological kit, marker enzyme is horseradish peroxidase or alkalinity in the detection antibody of enzyme label
Phosphate, when marker enzyme is horseradish peroxidase, the substrate developing solution includes chromogenic substrate A and chromogenic substrate B, institute
Stating chromogenic substrate A is hydrogen peroxide or urea peroxide, and the chromogenic substrate B is o-phenylenediamine or tetramethyl benzidine, the end
Only liquid is the sulfuric acid or hydrochloride buffer of 1-2mol/L；When marker enzyme is alkaline phosphatase, the substrate developing solution is to nitre
Based phosphates buffer, the terminate liquid are 1-2mol/L sodium hydroxide.
The concentration of above-mentioned enzyme linked immunological kit, the ox or ovine skeletal muscle Troponin I standard solution is respectively
4.875mg/L, 9.75mg/L,19.5mg/L,39mg/L,78.125mg/L,156.25mg/L,312.5mg/L；The concentration
Cleaning solution is the phosphate buffer of the 0.2mol/L PH7.4 containing 1% Tween-20.
In order to facilitate on-site supervision and great amount of samples screening, ox or ovine skeletal muscle flesh calcium egg in the enzyme linked immunological kit
White I standard solution, substrate developing solution, terminate liquid, concentrated cleaning solution provide in the form of working solution.
Through the above technical solutions, the present invention can by double antibodies sandwich enzyme-linked immune detection method, to fresh meat and its
Ox or ovine skeletal muscle Troponin I realize quickly detection in product, are limited to the lowest detection of ox or ovine skeletal muscle Troponin I
4.8mg/L is 1% to the detection sensitivity of beef and mutton, to chicken, duck, Animal muscles Troponin I extract no cross reaction,
Sensitivity and specificity with higher.
Enzyme linked immunological kit preparation method of the present invention, includes the following steps:
(1) preparation is coated with the ELISA Plate of capture antibody:
(a) it is diluted to 1:4000 by antibody is captured with coating buffer, the hole 100ul/ is added in ELISA Plate hole；
(b) 4 DEG C overnight or 37 DEG C of incubations 2h, liquid in hole of inclining, cleaning solution washing 4-5 times pat dry；
(c) confining liquid buffer, the hole 200ul/, 37 DEG C of incubation 2h are added；
(d) incline liquid in hole, pats dry, room temperature drains 5h in a vacuum drying oven, with aluminium foil bag vacuum plastic sealing to get packet
The ELISA Plate for being had capture antibody；
Wherein, the carbonate buffer solution for the 0.05mol/L that the coating buffer is pH9.6, Block buffer is 1%
Gelatin；
(2) enzyme labelling assay for determining antibody working solution is prepared:
Horseradish peroxidase is coupled with detection antibody using sodium iodate method；
(3) enzyme linked immunological kit for detecting ox or ovine skeletal muscle Troponin I, including following each components are set up:
(a) it is coated with the ELISA Plate of capture antibody；
(b) enzyme labelling assay for determining antibody working solution；
(c) standard solution, 1ml/ ox or ovine skeletal muscle Troponin I standard solution: are prepared using gradient dilution method
Bottle, concentration be respectively 4.875mg/L, 9.75mg/L, 19.5mg/L, 39mg/L, 78.125mg/L, 156.25mg/L,
312.5mg/L；
(d) substrate developing solution A liquid is urea peroxide solution, and substrate developing solution B liquid is tetramethyl benzidine (TMB) solution；
(e) terminate liquid is the sulfuric acid solution of 1-2mol/L；
(f) concentrated cleaning solution is the phosphate buffer of the 0.2mol/L PH7.4 containing 1% Tween-20.
The application of above-mentioned enzyme linked immunological kit ox or ovine skeletal muscle Troponin I in for fresh meat and its product,
Include the following steps:
(1) sample pre-treatments
Fresh meat removal fat and connective tissue, meat processing food remove casing, weigh the NaCL that 0.15M is added in 1g
Solution 2ml (1:2w/v), heats 20min with boiling water after homogenate, and 2000g is centrifuged 30min；Removal precipitating, supernatant Whatman 1
The filtering of number filter paper collects filtrate for detecting；
(2) it is detected using kit
Enzyme linked immunological kit is taken out, reagent restores to room temperature (20-25 DEG C), places 30min or more indoors；As needed
Lath is taken out, is placed on ELISA Plate；Standard items or sample to be tested 100ul are sequentially added into hole, gently oscillation mixes, and 37 DEG C
It is protected from light and is incubated for 30min；Liquid in hole is dried, wash operating solution deionized water is diluted 20 times of 20 × concentrated cleaning solution
Afterwards, the hole 250ul/ is washed 4 times, is patted dry；Diluted enzyme labelling assay for determining antibody working solution 100ul is added, gently oscillation mixes, and 37
It DEG C is protected from light and to be incubated for 30min；By developing solution A and developing solution B, 1:1 ratio is mixed by volume, and colour developing is added in the hole 100ul/, and 25 DEG C are kept away
Light is incubated for 15min；Add terminate liquid 50ul to terminate reaction, is measured under microplate reader 450nm using dual wavelength 450nm/630nm, according to
Standard curve carries out quantitative or qualitative；
(3) analysis detection result
(a) quantitative analysis: calculating separately the mean absorbance values of standard items and sample to be tested, standard items or sample to be tested
Absorbance value (B) is ordinate, and the logarithm of normal concentration is abscissa, draws standard curve；By the absorbance value of sample to be tested
Standard curve is substituted into, corresponding concentration can be found out, be the content of sample multiplied by extension rate；
(b) it qualitative analysis: is compared with the mean absorbance values of sample to be tested with the absorbance value of standard items, you can get it
The concentration range of sample to be tested.
Testing result can also be calculated with the computer software of profession, and test scope is 4.8mg/L~312.5mg/
L。
The testing principle of kit is double crush syndrome method in the present invention.It is coated with microplate with antibody (3D3),
Solid-phase capture antibody is made, sequentially adds standard items or sample into the micropore of coating monoclonal antibody, then with horseradish peroxidase mark
The detection antibody (3A8) of note combines, and forms antibody-antigene-hrp-antibody complex, TMB is added to develop the color, blue, in the work of acid
With lower finally in yellow, shade is positively correlated with bovine muscle Troponin I content in sample.
The curve ranges of enzyme linked immunological kit of the present invention are within the scope of 4.8mg/L~312.5mg/L；To ox or
The lowest detection of ovine skeletal muscle Troponin I is limited to 4.8mg/L, and the detection sensitivity to beef and mutton is 1%；Variation within batch coefficient
13.6% hereinafter, interassay coefficient of variation is below 14.1%；Intersect instead with ox, ovine skeletal muscle Troponin I extract
Should rate be 100%, with chicken, duck, Animal muscles Troponin I extract cross reacting rate be lower than 0.1%；Kit is placed on 2
DEG C -8 DEG C save 12 months, during which primary every detection in 1 month, the parameters such as ODmax, coefficient of variation of assay kit,
Kit in the normal range, while being placed on 37 DEG C and -20 DEG C and placed 6 days by each parameter as the result is shown, detects one daily
Secondary, the parameters such as ODmax, coefficient of variation of assay kit are within normal range (NR), and therefore, this kit can be 2
DEG C -8 DEG C save at least 12 months.
Detailed description of the invention
The canonical plotting of Fig. 1 enzyme-linked immunologic detecting kit detection ox skTnI of the present invention
The canonical plotting of Fig. 2 enzyme-linked immunologic detecting kit detection sheep skTnI of the present invention
Fig. 3 enzyme-linked immunologic detecting kit specific assay figure of the present invention
Fig. 4 enzyme-linked immunologic detecting kit actual sample measurement chart of the present invention
The hybridoma cell strain skTnI-3A8 of anti-bovine muscle Troponin I monoclonal antibody of the present invention, in
Deliver China typical culture collection center (abbreviation CCTCC, address: Wuhan University, Chinese Typical Representative training on December 20th, 2018
Support object collection, postcode: 430072) preservation, deposit number CCTCC NO:C2018217.
The hybridoma cell strain skTnI-3D3 of anti-ovine skeletal muscle Troponin I monoclonal antibody of the present invention, in
Deliver China typical culture collection center (abbreviation CCTCC, address: Wuhan University, Chinese Typical Representative training on December 20th, 2018
Support object collection, postcode: 430072) preservation, deposit number CCTCC NO:C2018218.
Specific embodiment
Below with reference to specific example, the present invention is further explained.It should be understood that these examples are merely to illustrate the present invention,
And it is not intended to limit the scope of the invention.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
The preparation of the ox of the present invention of embodiment one or ovine skeletal muscle Troponin I antigen
The skeletal muscle of ox or sheep removal fat and connective tissue are taken, is ground, the NaCL for weighing 40g addition 0.15M is molten
Liquid (1:2w/v)；Further mix after, ultrasonic extraction 5min (50W, 20KHz), then with boiling water heat 20min after, 2000g from
Heart 30min；Removal precipitating takes after half supernatant liquid filtering as treatment fluid 1.121 DEG C of high pressure 30min of the other half supernatant,
After 5000g is centrifuged 30min, with No. 1 filter paper filtering of Whatman, filtrate is added 90% ethyl alcohol (1:3.74v/v), takes mixed liquor
7000g is centrifuged 20min, is treatment fluid 2 after taking 37 DEG C of drying of precipitating, physiological saline to redissolve.Treatment fluid 1 and treatment fluid 2 are through SDS-
PAGE electroresis appraisal, qualification result show that ox and ovine skeletal muscle Troponin I immunogene and detection original have albumen in 21~22kD
Band is consistent with 21~24kD of molecular weight of skTnI reported in the literature.SkTnI electrophoretic band is ground, normal saline dilution is used
Afterwards respectively as detection antigen and immunizing antigen.
The preparation of the ox of the present invention of embodiment two or ovine skeletal muscle Troponin I monoclonal antibody
1, animal immune: the female Balb/c mouse of 6-8 week old is immunized in the immunizing antigen of selective extraction, is spaced 2 weeks and is immunized
1 time, 3 times it is immune after docking take hematometry potency and inhibiting rate, select the optimal mouse of immune result to prepare fusion；
2, cell fusion: the splenocyte and mouse myeloma SP2/0 cell for the mouse for taking step 1 selected are merged,
It connects ELISA method measurement supernatant and chooses positive high hole, positive hole is subcloned by limiting dilution assay, until establishing
Generate the hybridoma cell strain of the monoclonal antibody of single anti-ox or ovine skeletal muscle Troponin I；
3, a large amount of preparations of monoclonal antibody: choosing the biggish female Balb/c mouse of individual, induces ascites using internal
Method largely prepares ascites, and purifies ascites by octanoic acid-ammonium sulfate precipitation, is divided into tubule, and -20 DEG C of preservations obtain ox or sheep bone
Bone flesh Troponin I monoclonal antibody.
The capture antibody of the present invention of embodiment three and detection antibody characteristic identification
1, titration
Will test antigen diluent with the carbonate buffer solution of pH9.6 is that 5 μ g/ml are coated with detection plate, by Dan Ke after purification
Grand antibody carries out 1:2000, and 1:4000,1:8000 ... ... 1:1024000 dilution are added in ELISA Plate hole, are added after reaction
The sheep anti mouse secondary antibody of HRP label, is finally developed the color with TMB, the results show that bovine muscle Troponin I monoclonal after purification is anti-
Potency when bulk concentration is 1mg/mL reaches 1:106, ovine skeletal muscle Troponin I MAb concentration after purification is 1mg/mL
When potency reach 1:2.56 × 105。
2, hypotype measures
Hypotype measurement is carried out using the mouse source monoclonal antibody hypotype identification kit purchased from Sigma company, the results show that ox bone bone
Flesh Troponin I monoclonal antibody hypotype is IgG1, and ovine skeletal muscle Troponin I monoclonal antibody hypotype is IgM.
3, affinity determination
Using the affinity costant of Dot-ELISA measurement monoclonal antibody, the results show that anti-bovine muscle flesh calcium egg
White I monoclonal affinity costant is Ka=8.1 × 108L/mol, anti-ovine skeletal muscle Troponin I monoclonal affinity costant are Ka=
7.6×108 L/mol。
The preparation of the example IV ELISA Plate of the present invention for being coated with capture antibody
Antibody 3D3 will be captured with the carbonate buffer solution of PH9.6 to dilute by 1:4000,100 holes μ L/ are added to ELISA Plate hole
On, 4 DEG C of coatings are overnight；Washing 4-5 times, pats dry；It is closed with the PBS containing 1% gelatin, 200 holes μ L/, 37 DEG C of incubation 2h；
Room temperature drains 5h in a vacuum drying oven, with aluminium foil bag vacuum plastic sealing.
The preparation of the enzyme labelling assay for determining antibody working solution of the present invention of embodiment five
The preparation of enzyme labelling assay for determining antibody: horseradish peroxidase and detection antibody 3A8 are carried out occasionally using sodium iodate method
Connection, antibody concentration is 7.7mg/ml after coupling, and potency is 1:2.0 × 106。
Embodiment six is of the present invention for detecting the establishment of ox or ovine skeletal muscle Troponin I enzyme linked immunological kit
Set up ox or ovine skeletal muscle Troponin I enzyme-linked immunologic detecting kit, including following each components:
(1) it is coated with the ELISA Plate of capture antibody；
(2) enzyme labelling assay for determining antibody working solution；
(3) standard solution, 1ml/ ox or ovine skeletal muscle Troponin I standard solution: are prepared using gradient dilution method
Bottle, concentration be respectively 4.875mg/L, 9.75mg/L, 19.5mg/L, 39mg/L, 78.125mg/L, 156.25mg/L,
312.5mg/L；
(4) substrate developing solution A liquid is urea peroxide solution, and substrate developing solution B liquid is tetramethyl benzidine (TMB) solution；
(5) terminate liquid is the sulfuric acid solution of 1-2mol/L；
(6) concentrated cleaning solution is the phosphate buffer of the 0.2mol/L PH7.4 containing 1% Tween-20.
Embodiment seven uses ox or ovine skeletal muscle troponin in enzyme linked immunological kit of the present invention detection sample
I
1, the pre-treatment of sample
Fresh meat removal fat and connective tissue, meat processing food remove casing, and the NaCL for weighing 1g addition 0.15M is molten
Liquid 2ml (1:2w/v), heats 20min with boiling water after homogenate, and 2000g is centrifuged 30min；Removal precipitating, supernatant Whatman 1
The filtering of number filter paper collects filtrate for detecting.
2, detection method
(1) enzyme linked immunological kit is taken out, reagent restores to room temperature (20-25 DEG C), at least places 30min indoors；
(2) lath is taken out as needed, is placed on ELISA Plate；
(3) 100 μ L of standard items (or sample to be tested) is sequentially added into hole, gently oscillation mixes, and 37 DEG C are protected from light incubation
30min；
(4) liquid in hole is dried, with wash operating solution (being diluted 20 times of 20 × concentrated cleaning solution with deionized water) 250
μ L/hole is washed 4 times, is patted dry；
(5) monoclonal antibody of diluted HRP label, 100 holes μ L/, 37 DEG C of incubation 30min are added；
(6) liquid in hole is dried, with wash operating solution (being diluted 20 times of 20 × concentrated cleaning solution with deionized water) 250
μ L/hole is washed 4 times, is patted dry；
(7) developing solution A and developing solution B is mixed in 1:1 ratio, colour developing is added in the hole 100ul/, and 25 DEG C are protected from light incubation
15min；
(8) plus 50 μ L of terminate liquid terminates reaction, measurement (being proposed with the measurement of dual wavelength 450/630) under microplate reader 450nm,
It is carried out according to standard curve quantitative or qualitative.
3, testing result
(a) quantitative analysis: calculating separately the mean absorbance values of standard items and sample to be tested, standard items or sample to be tested
Absorbance value (B) is ordinate, and the logarithm of normal concentration is abscissa, draws standard curve；By the absorbance value of sample to be tested
Standard curve is substituted into, corresponding concentration can be found out, be the content of sample multiplied by extension rate.
(b) it qualitative analysis: is compared with the mean absorbance values of sample to be tested with the absorbance value of standard items, you can get it
The concentration range of sample to be tested.
Testing result can also be calculated with the computer software of profession, and test scope is 4.8mg/L~312.5mg/
L。
The enzyme linked immunological kit sensitivity of the present invention of embodiment eight, specificity, accuracy and shelf-life experiment
1, the foundation of kit standard curve
The drafting of ox skTnI standard curve is detected as shown in Figure 1, standard curve is within the scope of 4.8mg/L~312.5mg/L
In good linear relationship, linear equation y=0.4902x-0.2297 (R2=0.9903), bovine muscle Troponin I is most
Low detection is limited to 4.8mg/L.Detect sheep skTnI standard curve drafting as shown in Fig. 2, standard curve 4.8mg/L~
It is in good linear relationship, linear equation y=0.6626x-0.312 (R within the scope of 312.5mg/L2=0.9934), ovine skeletal muscle
The lowest detection of Troponin I is limited to 4.8mg/L.
2, kit accuracy measures
Precision is the repetition degree that reaction assay method repeatedly measures acquired results to a certain specific sample, commonly uses variation
Coefficient indicates.The ELISA Plate that same batch and different batches are chosen in the kit qualified from detection, the beef and mutton boiled is mentioned
It takes object to be used as 1:10,1:20,1:50 times to dilute, as positive sample, same batch, different batches are repeated 4 times respectively, measurement
Criticize interior, interassay coefficient of variation and the rate of recovery.It the results are shown in Table 1, variation within batch coefficient is 13.6% hereinafter, interassay coefficient of variation is equal
Below 14.1%.
1 sample of table criticizes interior, interassay coefficient of variation measurement
3, cross reaction measures
The extract of the cattle and sheep chicken and duck meat boiled and BSA (negative control) are 1:20 and 1:40 with PBS to dilute, with this
It invents the kit to be detected, to determine the specificity of this method.From the figure 3, it may be seen that the sandwich ELISA method established has
Cattle and sheep specificity, does not react with chicken, duck, Animal muscles extract.
4, kit storage life is tested
Kit is placed on 2 DEG C -8 DEG C to save 12 months, it is during which primary every detection in 1 month, assay kit
The parameters such as ODmax, the coefficient of variation are in the normal range.Kit 37 DEG C and -20 DEG C are placed on simultaneously to place 6 days,
Detection is primary daily, and the parameters such as ODmax, coefficient of variation of assay kit are within normal range (NR).This kit can
To be saved at least 12 months at 2 DEG C -8 DEG C.
5, sample test
By fresh beef and mutton, chicken and duck meat (1:1, w/ are added in 1%, 5%, 10%, 20%, 50% ratio respectively
W) it in, is tested with kit of the present invention, as a result as shown in figure 4, the sandwich ELISA method established detects chicken and duck meat
In beef and mutton, when its content reaches 1%, absorbance value is higher than negative 2 times or more, therefore the sensitivity of this method can reach
To 1%.
The technical concept and advantage of above-described embodiment only to illustrate the invention, the present invention also can have other forms and become
Change, as well known to the skilled person, above-described embodiment is functioned only as to the exemplary role in foregoing invention protection scope, right
For those of ordinary skill in the art, there are also many conventional deformations and other implementations in the protection scope defined by the present invention
Example, these deformations and embodiment all will be within the pending protection scopes of the present invention.
Claims (8)
1. the enzyme linked immunological kit of a kind of detection ox or ovine skeletal muscle Troponin I, which is characterized in that the ELISA reagent
Box by be coated with capture antibody ELISA Plate, enzyme label detection antibody, ox or ovine skeletal muscle Troponin I standard solution,
Substrate developing solution, terminate liquid, concentrated cleaning solution composition.
2. enzyme linked immunological kit according to claim 1, which is characterized in that the capture antibody is by deposit number
The hybridoma cell strain 3A8 of CCTCC NO:C2018217 secretes to obtain, and the detection antibody is CCTCC NO by deposit number:
The hybridoma cell strain 3D3 of C2018218 secretes to obtain；Two plants of cell strains deliver Chinese Typical Representative culture on December 20th, 2018
Object collection (CCTCC) preservation.
3. enzyme linked immunological kit according to claim 1, which is characterized in that the capture antibody is mouse with detection antibody
Source, Ma Yuan, Yang Yuan, rabbit source or cavy resource monoclonal antibody, preferably source of mouse monoclonal antibody；It is by ox or ovine skeletal muscle flesh calcium egg
White I extraction immunizing antigen is immune to be obtained.
4. enzyme linked immunological kit according to claim 1, which is characterized in that the enzyme labelling assay for determining antibody uses acid iodide
Sodium method obtains marker enzyme and detection antibody coupling.
5. enzyme linked immunological kit according to claim 1, which is characterized in that marked in the detection antibody of the enzyme label
Enzyme is horseradish peroxidase or alkaline phosphatase, when marker enzyme is horseradish peroxidase, the substrate developing solution packet
It includes chromogenic substrate A and chromogenic substrate B, the chromogenic substrate A is hydrogen peroxide or urea peroxide, the chromogenic substrate B is adjacent benzene
Diamines or tetramethyl benzidine, the terminate liquid are the sulfuric acid or hydrochloride buffer of 1-2mol/L；When marker enzyme is alkaline phosphatase
When enzyme, the substrate developing solution is to nitro phosphate buffer, and the terminate liquid is 1-2mol/L sodium hydroxide.
6. enzyme linked immunological kit according to claim 1, which is characterized in that the ox or ovine skeletal muscle Troponin I
The concentration of standard solution is respectively 4.875mg/L, 9.75mg/L, 19.5mg/L, 39mg/L, 78.125mg/L, 156.25mg/
L,312.5mg/L；The concentrated cleaning solution is the phosphate buffer of the 0.2mol/L PH7.4 containing 1% Tween-20.
7. a kind of method for preparing the enzyme linked immunological kit as described in claim 1 to 6 any claim, feature exist
In including the following steps:
(1) preparation is coated with the ELISA Plate of capture antibody:
(a) it is diluted to 1:4000 by antibody is captured with coating buffer, the hole 100ul/ is added in ELISA Plate hole；
(b) 4 DEG C overnight or 37 DEG C of incubations 2h, liquid in hole of inclining, cleaning solution washing 4-5 times pat dry；
(c) confining liquid buffer, the hole 200ul/, 37 DEG C of incubation 2h are added；
(d) incline liquid in hole, pats dry, room temperature drains 5h in a vacuum drying oven, is coated with aluminium foil bag vacuum plastic sealing
Capture the ELISA Plate of antibody；
Wherein, it is described coating buffer be pH9.6 0.05mol/L carbonate buffer solution, Block buffer be 1% it is bright
Glue；
(2) enzyme labelling assay for determining antibody working solution is prepared:
Horseradish peroxidase is coupled with detection antibody using sodium iodate method；
(3) enzyme linked immunological kit for detecting ox or ovine skeletal muscle Troponin I, including following each components are set up:
(a) it is coated with the ELISA Plate of capture antibody；
(b) enzyme labelling assay for determining antibody working solution；
(c) ox or ovine skeletal muscle Troponin I standard solution: preparing standard solution using gradient dilution method, 1ml/ bottles, dense
Degree is respectively 4.875mg/L, 9.75mg/L, 19.5mg/L, 39mg/L, 78.125mg/L, 156.25mg/L, 312.5mg/L；
(d) substrate developing solution A liquid is urea peroxide solution, and substrate developing solution B liquid is tetramethyl benzidine (TMB) solution；
(e) terminate liquid is the sulfuric acid solution of 1-2mol/L；
(f) concentrated cleaning solution is the phosphate buffer of the 0.2mol/L PH7.4 containing 1% Tween-20.
8. enzyme linked immunological kit described in claim 1 is for ox or ovine skeletal muscle Troponin I in fresh meat and its product
Application, include the following steps:
(1) sample pre-treatments
Fresh meat removal fat and connective tissue, meat processing food remove casing, weigh the NaCL solution that 0.15M is added in 1g
2ml (1:2w/v), heats 20min with boiling water after homogenate, and 2000g is centrifuged 30min；Removal precipitating, supernatant are filtered with No. Whatman1
Paper filtering collects filtrate for detecting；
(2) it is detected using kit
Enzyme linked immunological kit is taken out, reagent restores to room temperature (20-25 DEG C), places 30min or more indoors；It takes out as needed
Lath is placed on ELISA Plate；Standard items or sample to be tested 100ul are sequentially added into hole, gently oscillation mixes, and 37 DEG C are protected from light
It is incubated for 30min；Liquid in hole is dried, after wash operating solution deionized water is diluted 20 times of 20 × concentrated cleaning solution,
The hole 250ul/ is washed 4 times, is patted dry；Diluted enzyme labelling assay for determining antibody working solution 100ul is added, gently oscillation mixes, and 37 DEG C are kept away
Light is incubated for 30min；By developing solution A and developing solution B, 1:1 ratio is mixed by volume, and colour developing is added in the hole 100ul/, and 25 DEG C are protected from light and incubate
Educate 15min；Add terminate liquid 50ul to terminate reaction, is measured under microplate reader 450nm using dual wavelength 450nm/630nm, according to standard
Curve carries out quantitative or qualitative；
(3) analysis detection result
(a) mean absorbance values of standard items and sample to be tested, the extinction of standard items or sample to be tested quantitative analysis: are calculated separately
Angle value (B) is ordinate, and the logarithm of normal concentration is abscissa, draws standard curve；The absorbance value of sample to be tested is substituted into
Standard curve can find out corresponding concentration, be the content of sample multiplied by extension rate；
(b) qualitative analysis: being compared with the mean absorbance values of sample to be tested with the absorbance value of standard items, and it is to be measured that you can get it
The concentration range of sample.
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