CN109239361A - A kind of detection kit of cardiac muscle troponin I and preparation method thereof - Google Patents
A kind of detection kit of cardiac muscle troponin I and preparation method thereof Download PDFInfo
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- CN109239361A CN109239361A CN201811150480.4A CN201811150480A CN109239361A CN 109239361 A CN109239361 A CN 109239361A CN 201811150480 A CN201811150480 A CN 201811150480A CN 109239361 A CN109239361 A CN 109239361A
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Abstract
The present invention relates to detection kits of a kind of cardiac muscle troponin I and preparation method thereof.The detection kit of cardiac muscle troponin I disclosed by the invention includes detecting the capture antibody of antibody and tracer-labelling, the capture antibody is two kinds of monoclonal antibodies respectively in connection with cardiac muscle troponin I different loci, and the detection antibody is the polyclonal antibody of cardiac muscle troponin I.The present invention is by screening the capture antibody that suitable cardiac muscle troponin I immunochromatography detects, detection antibody and combinations thereof, optimize the dilution component of kit simultaneously, it has been obviously improved the sensitivity and linear measurement range of detection, it ensure that accuracy and precision simultaneously, can be used for the efficient detection of cardiac muscle troponin I.
Description
Technical field
The present invention relates to field of biological detection, and in particular to a kind of detection kit and its preparation of cardiac muscle troponin I
Method.
Background technique
Cardiac muscle troponin I (Cardiac Troponin I, cTnI) is the subunit of cardiac troponin, is present in the heart
It is the distinctive structural proteins of cardiac muscle cell on flesh muscle fibril actin filament.Under normal circumstances, the cTnI content in blood is very
Low, when myocardial damage occurs, cTnI is discharged rapidly in blood, and the cTnI content in blood is caused to increase.Cardiac muscle cell's damage
, there is cTnI raising in wound 3 hours~5 hours in peripheral blood, 12 hours~24 hours reach to peak values, the duration is long, 5 days reachable~
It 8 days, returns to normal within 10 days or so.Cardiac muscle troponin I can be used as the clinical assistant diagnosis of acute myocardial infarction AMI (AMI),
The index of acute coronary syndrome (ACS) classification of risks, prognosis evaluation and dynamic monitoring in the recent period.
The common detection method of cTnI has ELISA method, chemoluminescence method, immunochromatographic method etc..ELISA method detection cycle
It is long；Chemoluminescence method is expensive, need to there is specialized equipment and professional's operation, is only applicable to central laboratory's use, detection
Period is longer, is not able to satisfy the clinical needs quickly detected, is not also suitable for middle and small hospital particular without central laboratory
Community hospital and township hospital；And fluorescence immune chromatography method can realize rapid quantitative detection to emergency treatment sample.
Currently, in the prior art that there are sensitivity is lower about cardiac muscle troponin I immunochromatographic measurement method, detection
Therefore the problems such as concentration range is smaller develops efficient cardiac troponin detection reagent and detection method is of great significance.
Summary of the invention
To solve problems of the prior art, the object of the present invention is to provide a kind of detections of cardiac muscle troponin I
Kit and preparation method thereof.
The present invention provides a kind of detection kit of cardiac muscle troponin I, and the kit includes detection antibody and shows
The capture antibody of track substance markers, the capture antibody are anti-for two kinds of monoclonals respectively in connection with cardiac muscle troponin I different loci
Body, the detection antibody are the polyclonal antibody of cardiac muscle troponin I.
Specifically, it is respectively such as SEQ ID that capture antibody of the present invention, which is with the binding site of cardiac muscle troponin I,
Any two kinds of monoclonal antibodies in the position 41-49 of amino acid sequence shown in NO.1,83-93 and 190-196.
Preferably, two kinds of monoclonal antibodies of the capture antibody and the binding site of cardiac muscle troponin I are respectively such as
The position 41-49 of amino acid sequence shown in SEQ ID NO.1 and 83-93.
The amino acid sequence of cardiac muscle troponin I of the present invention is as shown in SEQ ID NO.1, list of the present invention
The binding site of clonal antibody and cardiac muscle troponin I is according to calculating with the amino acid sequence as shown in SEQ ID NO.1.
Monoclonal antibody of the present invention and polyclonal antibody can be for commercially available antibody or using ability domain antibodies system
The monoclonal antibody or polyclonal antibody that standby conventional means are prepared.
Tracer of the present invention is fluorescent microsphere.
The fluorescent material of the fluorescent microsphere includes but is not limited to rare earth element, fluorescein, semiconductor-quantum-point, carbon quantum
Point.
Preferably, one of rare earth doped lanthanide series of the fluorescent microsphere or a variety of, the partial size of the fluorescent microsphere
For 100~300nm.
The rare earth lanthanide includes but is not limited to europium (Eu), samarium (Sm), erbium (Er), neodymium (Nd).
As the preferred embodiment of the present invention, the fluorescent microsphere is used as fluorescence doped with rare earth lanthanide europium (Eu)
Substance；The fluorescent microsphere partial size is 150nm~200nm.
Fluorescent microsphere of the present invention is modified with amino or carboxyl, preferably carboxyl modified.
Fluorescent microsphere of the present invention can be fluorescence commercially available or being prepared using conventional technical means in the art
Microballoon.
Preferably, the fluorescent microsphere is polystyrene fluorescent microsphere, polyphosphazene fluorescent microsphere, melamino-formaldehyde fluorescence
Microballoon, Lauxite fluorescent microsphere, any one in silica fluorescent microballoon；It is highly preferred that the fluorescent microsphere is poly-
Styrene fluorescent microballoon.
Kit of the present invention further includes dilution, and the dilution includes polyvinylpyrrolidone (PVP).
Specifically, the dilution includes polyvinylpyrrolidone and Tween-20.
Preferably, the dilution includes polyvinylpyrrolidone and Tween-20, further include glycine, sodium chloride, BSA,
One of casein, trehalose are a variety of.
It is highly preferred that the dilution includes following component: glycine, sodium chloride, BSA, casein, trehalose, tween-
20, PVP-40 and ProClin-300 (PC-300).
As the preferred embodiment of the present invention, the dilution includes following component: 0.1~0.5mol/L of glycine,
Sodium chloride 0.1~1.0%, BSA1~2%, casein 0.1~0.5%, trehalose 1~3%, Tween-20 0.3~0.8%,
PVP-40 0.3~1.0%, ProClin-3000.1~0.5%, pH 7.0~7.5.
Dilution of the present invention is used for dilute reaction solution, to obtain the working concentration of reaction solution.
As the preferred embodiment of the present invention, kit of the present invention includes test card, reaction solution and dilution；
The test card includes following component: bottom plate, sample pad, nitrocellulose filter and water absorption pad, the cellulose nitrate
The Quality Control that the BSA of the detection line and marked by fluorescein isothiocyanate crossed on plain film containing the detection antibody crosses
Line；
The fluorescein isothiocynate of capture antibody and fluorescent microsphere label containing fluorescent microsphere label in the reaction solution
Antibody；
The dilution includes polyvinylpyrrolidone and Tween-20.
It will be appreciated by those skilled in the art that the capture antibody and fluorescent microsphere mark of fluorescent microsphere label of the present invention
The fluorescein isothiocynate antibody of note can be prepared as component of the different forms as kit, including but not limited to following shape
Formula:
(1) be prepared as the form of the reaction solution in the preferred embodiment for the present invention, when in use can in advance by sample with
Reaction solution carries out mixed processing, then is loaded to test card and is detected；
(2) it is prepared as the form of bonding pad: by the different sulphur cyanogen of the capture antibody of fluorescent microsphere label and fluorescent microsphere label
Even application is prepared into bonding pad on solid phase carrier after sour fluorescein monoclonal antibody mixing.
Although title that both the above mode will lead to reagent constituents is different, but its essence is using fluorescent microsphere
The fluorescein isothiocynate monoclonal antibody that capture antibody and the fluorescent microsphere of label mark is mixture prepared, therefore,
Within the scope of the present invention.
The present invention also provides a kind of preparation method of the detection kit of cardiac muscle troponin I, the preparation including reaction solution
With the preparation of test card；
The preparation method for the capture antibody that fluorescent microsphere marks in the reaction solution includes the following steps:
(1) activation of fluorescent microsphere；
(2) take cardiac muscle troponin I monoclonal antibody 41-49 and 83-93 mixing as capture antibody, the monoclonal is anti-
The mass ratio of body 41-49 and 83-93 are 1:1, capture antibody are added, reaction is marked into the fluorescent microsphere solution of activation；
(3) BSA processing is added in the capture antibody for the fluorescent microsphere label that step (2) obtains, end mark reaction obtains
The capture antibody marked to fluorescent microsphere.
What the preparation method of the fluorescein isothiocynate antibody of the fluorescent microsphere label in reaction solution was marked with fluorescent microsphere
Capture the preparation method of antibody.
Test card in kit of the present invention is to fix the sample pad, nitrocellulose filter and water absorption pad
In being prepared on bottom plate；
The preparation method of the nitrocellulose filter includes the following steps:
(1) preparation of detection line and nature controlling line: the detection antibody is crossed on nitrocellulose filter and obtains detection line；
The BSA of marked by fluorescein isothiocyanate is crossed on nitrocellulose filter and obtains nature controlling line；
(2) the nitrocellulose filter drying for standby for being coated with detection line and nature controlling line for obtaining step (1).
As a kind of preferred embodiment of the invention, the preparation method of the kit includes the following steps:
(1) preparation of sample pad: it is dry after glass fibre membrane is handled with Tween-20 solution, obtain sample pad；
(2) preparation of reaction solution:
1. the preparation of the capture antibody of fluorescent microsphere label: fluorescent microsphere is diluted using MES (2-morpholine ethane sulfonic acid) solution,
EDC (1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride) solution is added and activates fluorescent microsphere 20min, is lived
The fluorescent microsphere of change；
Take cardiac muscle troponin I monoclonal antibody 41-49 and 83-93 mixing as capture antibody, the monoclonal antibody
41-49 and 83-93 mass ratio is 1:1, and it is small that mixing stirring 2 is added into activated fluorescent microsphere solution in capture antibody
When, BSA processing is added in the capture antibody of obtained fluorescent microsphere label, end mark reaction, the precipitating being centrifuged is i.e.
For the capture antibody of fluorescent microsphere label.
2. the preparation of the fluorescein isothiocynate antibody of fluorescent microsphere label: fluorescein isothiocynate antibody being taken to be added to
Stirring 2 hours is mixed in activated fluorescent microsphere solution, BSA processing is added, end mark reaction, the precipitating being centrifuged is i.e.
For the fluorescein isothiocynate antibody of fluorescent microsphere label.
By the fluorescein isothiocynate of the capture antibody of the above-mentioned fluorescent microsphere label being prepared and fluorescent microsphere label
Antibody mixing is added the appropriate dilution and is resuspended up to reaction solution.
Those skilled in the art can send out this according to the actual conditions of sample and the concrete condition of detection when for detecting
Reaction solution in the bright kit uses inspection of the diluted of the present invention for different working concentrations for sample
It surveys.
As the preferred embodiment of the present invention, the extension rate of the reaction solution is 5~20 times.
(3) preparation of nitrocellulose filter: the polyclonal antibody of cardiac muscle troponin I is crossed on nitrocellulose filter
Obtain detection line；The BSA of marked by fluorescein isothiocyanate is crossed on nitrocellulose filter and obtains nature controlling line；Inspection will be coated with
The nitrocellulose filter drying for standby of survey line and nature controlling line.
(4) preparation of test card: sample pad, nitrocellulose filter and the water absorption pad that step (1), (3) are prepared from
From left to right is successively pasted on plastic bottom board, through cutting out to obtain test card.
The present invention also provides the kits that the kit or the preparation method are prepared to detect myocardium myo
Application in calcium protein I.
The beneficial effects of the present invention are: the present invention is by screening suitable cardiac muscle troponin I immune chromatography method inspection
The capture antibody of survey detects antibody and combinations thereof mode, and discovery selects cardiac muscle troponin I monoclonal antibody when capture antibody
The combination of 41-49 and 83-93 when detection antibody selects cardiac muscle troponin I polyclonal antibody, while being aided with containing polyethylene pyrrole
The dilution of pyrrolidone significantly improves the sensitivity of detection and the range of linearity of detection, it is dense that 0.05~50ng/mL may be implemented
The detection of the cardiac muscle troponin I of range is spent, while ensure that the accuracy and precision of detection, can be used for cardiac troponin
The efficient detection of I.
Detailed description of the invention
Fig. 1 is the canonical plotting detected in embodiment 2.
Specific embodiment
The preferred embodiment of the present invention is described in detail below in conjunction with embodiment.It will be appreciated that following real
Providing merely to play the purpose of explanation for example is applied, is not used to limit the scope of the present invention.The skill of this field
Art personnel without departing from the spirit and purpose of the present invention, can carry out various modifications and replace to the present invention.
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified, wherein the heart
All monoclonal antibodies and polyclonal antibody of flesh Troponin I are purchased from Hai Tai biotech firm.
The preparation of 1 detection kit of embodiment
1, the preparation of sample pad: sample pad selects glass fibre membrane, glass fibre element film is placed in the PVC board of white,
0.1% Tween-20 solution is prepared as sample pad treatment fluid, according to 45 μ L/cm2Treating capacity sample pad treatment fluid is uniformly spread
It falls on glass fibre element film, then the obverse and reverse for uniformly smearing nitrocellulose filter is put until no any white point
It is placed in equilibrium at room temperature 60min ± 15min on screen window., be placed in 37 DEG C it is 30-72 hours dry；
2, the preparation of reaction solution:
(1) (using Eu as fluorescent material, the partial size of fluorescent microsphere is 200nm to polystyrene fluorescent microsphere, and fluorescent microsphere contains
Carboxyl modified) use 20mM MES (2-morpholine ethane sulfonic acid) (pH 6.0) to dilute 10 times, ultrasonic 1min；
(2) with EDC (1- (3- dimethylamino-propyl) -3- ethyl carbon of 20mM MES solution (pH 6.0) configuration 5mg/mL
Diimmonium salt hydrochlorate) solution；
(3) EDC solution is added into the fluorescent microsphere of ultrasonic treatment to (dosage is that 1mg fluorescent microsphere uses 0.02mg
EDC), it is uniformly mixed, reaction 10min-30min carries out the activation of fluorescent microsphere；
(4) activated fluorescent microsphere is ultrasonically treated, supernatant is abandoned in centrifugation；
(5) fluorescent microsphere precipitating dilutes 10 times with 20mM MES (pH 6.0), and ultrasound is uniformly；
(6) take two plants of cTnI monoclonal antibody 41-49 and 83-93 mixing as capture antibody, two plants of monoclonal antibodies
Mass ratio is 1:1, and mixed antibody rapidly joins in the fluorescent microsphere of step (5) preparation that (1mg fluorescent microsphere uses 0.04mg
CTnI monoclonal antibody 41-49 and 0.04mg cTnI monoclonal antibody 83-93), it is uniformly mixed, room temperature marks 2-3 hours.
(7) obtained fluorescent microsphere labelled antibody is ultrasonically treated, 2%BSA is added and handles 1 hour, end mark reaction,
Supernatant is abandoned in ultrasonic treatment, centrifugation.
The mark fluorescent micro-sphere method of fluorescein isothiocynate monoclonal antibody is as described above.
(8) prepared by reaction solution: the capture antibody of the above-mentioned fluorescent microsphere label being prepared and fluorescent microsphere are marked
The mixing of fluorescein isothiocynate monoclonal antibody is added a small amount of dilution and is resuspended, reaction solution is prepared；
3, the processing of nitrocellulose filter
(1) preparation of detection line: by polyclonal antibody (detection antibody) the coating buffer of cardiac muscle troponin I
It is 2.0mg/ml that (10mM PBS, 2% trehalose, 0.1%PC300), which is diluted to concentration, by the antibody after dilution with 1.0 μ l/cm
Content cross on nitrocellulose filter and obtain detection line.
(2) preparation of nature controlling line: weighing 40mg BSA, with 0.025M K2CO3Solution allocation concentration is the BSA of 10mg/mL
Solution, be fitted into bag filter (D6mm 12-14KD) clip it is spare.8mg fluorescein isothiocynate is weighed, with 0.01M PBS (pH
7.4) being configured to concentration is that 0.1mg/mL fluorescein isothiocynate solution is fitted into 100mL beaker, is put into rotor.BSA will be housed
The bag filter of solution is put into equipped in fluorescein isothiocynate solution beaker, after 2-8 DEG C of dialysis is stirred 24 hours, takes out bag filter
In solution be fitted into 7mL vial, be added 0.004mLPC300, the final concentration of the BSA solution of marked by fluorescein isothiocyanate
For 10.0mg/mL.
Nature controlling line coating buffer (10mM PBS, 2% trehalose, 0.1%PC300) is moved on station, it is first accurate
The coating buffer of nature controlling line needed for measuring is added into corresponding clean container, then measures required fluorescein isothiocynate BSA label
Solution is added into corresponding nature controlling line coating buffer, mixes, makes its final concentration 1.0mg/mL, existed with the content of 0.8 μ l/cm
Scribing line obtains nature controlling line on nitrocellulose filter.Nature controlling line distance PVC bottom plate lower end 47mm, detection line distance PVC bottom plate lower end
42mm, nature controlling line are arranged in parallel with detection line, and distance is 5mm or so between the two.
(3) nitrocellulose filter handled well is placed in 55 DEG C of dryings 3 days, dried nitrocellulose filter envelope is standby
With.
4, the assembling of test card: 8cm plastic bottom board and water absorption pad are that this field test card prepares general component.It will be above-mentioned
The sample pad that is prepared, the nitrocellulose filter for being coated with detection line and nature controlling line and water absorption pad are successively pasted from left to right
In plastic bottom lining, the intermediary posted is fitted into card with the reagent strip that cutting machine is cut into 4.0mm width, the examination detected
Paper card.
5, the preparation of dilution: the formula of dilution is as follows: 0.12mol/L glycine, 0.85% sodium chloride, 1%BSA,
0.15% casein, 2% trehalose, 0.5% Tween-20,0.5%PVP-40,0.5%PC300, pH7.5.
The sensitivity analysis of 2 kit of embodiment detection
1, the preparation of cTnI examination criteria product
Using normal human serum as dilution cTnI antigen standard, compound concentration is 0ng/mL and 50ng/mL respectively
Standard items；The cTnI standard solution of two kinds of concentration is successively mixed according to a certain concentration, prepares 0~50ng/mL respectively
Concentration gradient cTnI examination criteria product.
2, detection reaction
By the reaction solution in kit prepared by embodiment 1 be added after 10 times of diluted step 1 obtain it is each dense
The 60 μ L of examination criteria product of degree, mixes well 5s~10s.
The examination criteria product of above-mentioned each concentration and the 90 μ L of mixed liquor of reaction solution are drawn respectively, are added drop-wise to Test paper card sample
In well on product pad, when sampling, pays attention to avoiding sucking bubble, is placed at room temperature for 15 minutes.
3, the detection of fluorescence signal: test paper is placed in 01 dry type fluorescence immunity analyzer of maccura R and obtains fluorescence
Signal strength, and draw corresponding standard curve.
The fluorescence signal intensity of examination criteria product is as shown in table 1, and the standard curve according to this production is as shown in Figure 1, result
Show using cTnI monoclonal antibody 41-49 and 83-93 as capture antibody, using cTnI polyclonal antibody as detection antibody, examination
The range of linearity of the detection of paper card is larger, when cTnI concentration is 0.05ng/mL~50ng/mL, the fluorescence signal and mark of detection
The concentration of quasi- product has good linear relationship (R2=0.9899).The sensitivity that kit detects cTnI is 0.05ng/mL, inspection
The upper limit of survey is 50ng/mL.
The fluorescence signal intensity of 1 various concentration cTnI standard items of table
| CTnI concentration (ng/mL) | Fluorescence signal (mean value) |
| 0 | 11 |
| 0.05 | 40 |
| 0.125 | 83 |
| 0.25 | 135 |
| 0.5 | 236 |
| 1 | 442 |
| 2 | 867 |
| 4 | 1708 |
| 8 | 3337 |
| 16 | 5980 |
| 32 | 10075 |
| 40 | 12090 |
| 50 | 13892 |
Fluorescence signal mean value is the average value of the fluorescence signal of 3 detections in table 1
The accuracy of 3 kit of embodiment detection and Precision Analyze
The detection of the cTnI of 3 parts of clinical serum samples is carried out using the kit that embodiment 1 is prepared.According to three parts of samples
The content height of cTnI is divided into low value, intermediate value and three groups of high level in this, and wherein low value definite value sample clinical measures are 0.15ng/
ML, intermediate value definite value sample clinical measures are 0.57ng/mL, l.89ng/mL high level definite value sample clinical measures are.To 3 parts
Definite value sample respectively carries out replication 10 times, and testing result is as shown in table 2.The results show that the kit prepared using embodiment 1
The detection of 3 parts of definite value samples, testing result and clinical measures are carried out very close to (average value and clinic of 10 detections are surveyed
Magnitude differs only in 0.01ng/mL or so), show that cTnI detection kit of the invention has the accuracy of height；10 weights
The CV value surveyed is rechecked between 4.42%~8.47%, shows that cTnI detection kit has detection accuracy well.
The repetition detection of 23 parts of table clinical definite value samples
| Number | Low value | Intermediate value | High level |
| 1 | 0.158 | 0.545 | 1.929 |
| 2 | 0.187 | 0.556 | 1.821 |
| 3 | 0.142 | 0.605 | 1.872 |
| 4 | 0.158 | 0.616 | 1.986 |
| 5 | 0.147 | 0.528 | 1.775 |
| 6 | 0.174 | 0.540 | 1.931 |
| 7 | 0.158 | 0.594 | 1.933 |
| 8 | 0.150 | 0.608 | 1.926 |
| 9 | 0.171 | 0.498 | 2.057 |
| 10 | 0.158 | 0.548 | 1.824 |
| Average value | 0.160 | 0.564 | 1.905 |
| Standard deviation | 0.014 | 0.040 | 0.084 |
| CV | 8.47% | 7.04% | 4.42% |
Detection kit of the comparative example 1 using 2 kinds of monoclonal antibodies as capture antibody
Preparation method preparation of the kit of this comparative example using kit described in embodiment 1, the area with embodiment 1
It is not only that, the capture antibody used is two kinds of Monoclonal Antibody Mixture as shown in Table 3 and Table 4.Testing result such as 3 He of table
Shown in table 4, the results showed that, use other two different monoclonal antibodies mixing as the sensitivity for the detection for capturing antibody compared with
41-49 and 83-93 mixing is substantially reduced as the sensitivity of capture antibody.
Table 3 captures the testing result that antibody is 2 kinds of different monoclonal antibodies
Table 4 captures the testing result that antibody is 2 kinds of different monoclonal antibodies
Comparative example 2 is using monoclonal antibody as detection antibody
Preparation method preparation of the kit of this comparative example using kit described in embodiment 1, the area with embodiment 1
It is not only that, the detection antibody used is the mixture of two kinds of monoclonal antibodies as shown in table 5.Testing result is as shown in table 5,
The result shows that using two different monoclonal antibody mixing as the sensitivity of the detection of detection antibody relatively uses Anti-TNF-α
Body is substantially reduced as the sensitivity of detection antibody.
Table 5 detects the testing result that antibody is 2 kinds of different monoclonal antibodies
The fluorescent microsphere mark mode that comparative example 3 captures antibody is different
Preparation method preparation of the kit of this comparative example using kit described in embodiment 1, the area with embodiment 1
It is not only that, in reaction solution preparation, the capture antibody of fluorescent microsphere label is prepared as using two kinds of lists of 41-49 and 83-93
After clonal antibody distinguishes mark fluorescent microballoon, then the fluorescent microsphere that two kinds are marked mixes (the quality that two kinds of fluorescent microspheres mix
Than for 1:1).The results are shown in Table 6, the results showed that, it mixes, examines again after distinguishing mark fluorescent microballoon using two kinds of monoclonal antibodies
The sensitivity of survey is substantially reduced.
Table 6 captures the testing result of the different fluorescent microsphere mark modes of antibody
The dilution that comparative example 4 captures antibody mark fluorescent microballoon is different
Preparation method preparation of the kit of this comparative example using kit described in embodiment 1, the area with embodiment 1
It is not only that, dilution is different.Wherein, experimental group is the dilution in embodiment 1, the dilution component of use are as follows:
0.12mol/L glycine, 0.85%NaCl, 1%BSA, 0.15% casein, 2% trehalose, 0.5% Tween-20,0.5%
PVP-40,0.5%PC300, pH7.5.
The dilution component that control group uses are as follows: 0.12mol/L glycine, 0.85%NaCl, 1%BSA, 0.15% junket egg
It is white, 2% trehalose, 0.5% Tween-20,0.5%PC300, pH7.5.
The above mass percentage is mass/volume percentage composition.
Testing result is as shown in table 7, the results showed that, by adding PVP-40 in dilution as thickener, on surface
Under the action of activating agent, when sample progress antibody capture reaction is added, antigen and the coated myocardium myo of fluorescent microsphere can be increased
The reaction time of calcium protein I antibody, the detection sensitivity of kit significantly improve.
Table 7 uses the testing result of different dilutions
Although above the present invention is described in detail with a general description of the specific embodiments, at this
On the basis of invention, it can be modified or is improved, this will be apparent to those skilled in the art.Therefore,
These modifications or improvements without departing from theon the basis of the spirit of the present invention are fallen within the scope of the claimed invention.
Sequence table
<110>mikey Biological Co., Ltd.
<120>a kind of detection kit of cardiac muscle troponin I and preparation method thereof
<130> KHP181113354.7
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 209
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 1
Ala Asp Gly Ser Ser Asp Ala Ala Arg Glu Pro Arg Pro Ala Pro Ala
1 5 10 15
Pro Ile Arg Arg Arg Ser Ser Asn Tyr Arg Ala Tyr Ala Thr Glu Pro
20 25 30
His Ala Lys Lys Lys Ser Lys Ile Ser Ala Ser Arg Lys Leu Gln Leu
35 40 45
Lys Thr Leu Leu Leu Gln Ile Ala Lys Gln Glu Leu Glu Arg Glu Ala
50 55 60
Glu Glu Arg Arg Gly Glu Lys Gly Arg Ala Leu Ser Thr Arg Cys Gln
65 70 75 80
Pro Leu Glu Leu Ala Gly Leu Gly Phe Ala Glu Leu Gln Asp Leu Cys
85 90 95
Arg Gln Leu His Ala Arg Val Asp Lys Val Asp Glu Glu Arg Tyr Asp
100 105 110
Ile Glu Ala Lys Val Thr Lys Asn Ile Thr Glu Ile Ala Asp Leu Thr
115 120 125
Gln Lys Ile Phe Asp Leu Arg Gly Lys Phe Lys Arg Pro Thr Leu Arg
130 135 140
Arg Val Arg Ile Ser Ala Asp Ala Met Met Gln Ala Leu Leu Gly Ala
145 150 155 160
Arg Ala Lys Glu Ser Leu Asp Leu Arg Ala His Leu Lys Gln Val Lys
165 170 175
Lys Glu Asp Thr Glu Lys Glu Asn Arg Glu Val Gly Asp Trp Arg Lys
180 185 190
Asn Ile Asp Ala Leu Ser Gly Met Glu Gly Arg Lys Lys Lys Phe Glu
195 200 205
Ser
Claims (10)
1. a kind of detection kit of cardiac muscle troponin I, which is characterized in that the kit includes detection antibody and tracer
The capture antibody of substance markers, the capture antibody are two kinds of monoclonal antibodies respectively in connection with cardiac muscle troponin I different loci,
The detection antibody is the polyclonal antibody of cardiac muscle troponin I.
2. kit according to claim 1, which is characterized in that the capture antibody be and cardiac muscle troponin I
Appointing in the binding site respectively position 41-49 of the amino acid sequence as shown in SEQ ID NO.1,83-93 and 190-196
It anticipates two kinds of monoclonal antibodies.
3. kit according to claim 1 or 2, which is characterized in that it is described capture antibody two kinds of monoclonal antibodies with
The binding site of cardiac muscle troponin I be respectively the amino acid sequence such as SEQ ID NO.1 shown in the position 41-49 with 83-93.
4. described in any item kits according to claim 1~3, which is characterized in that the tracer is fluorescent microsphere；
Preferably, one of rare earth doped lanthanide series of the fluorescent microsphere or a variety of, the partial size of the fluorescent microsphere are 100
~300nm.
5. kit according to claim 4, which is characterized in that the fluorescent microsphere carries the chemical base of amino or carboxyl
Group's modification；
Preferably, the fluorescent microsphere is that polystyrene fluorescent microsphere, polyphosphazene fluorescent microsphere, melamino-formaldehyde fluorescence are micro-
Ball, Lauxite fluorescent microsphere, any one in silica fluorescent microballoon.
6. described in any item kits according to claim 1~5, which is characterized in that the kit further includes dilution, institute
Stating dilution includes polyvinylpyrrolidone；
Preferably, the dilution includes polyvinylpyrrolidone and Tween-20, further includes glycine, sodium chloride, BSA, junket egg
One of white, trehalose is a variety of.
7. described in any item kits according to claim 1~6, which is characterized in that the kit includes test card, reaction
Liquid and dilution；
The test card includes following component: bottom plate, sample pad, nitrocellulose filter and water absorption pad, the nitrocellulose filter
The nature controlling line that the BSA of the upper detection line and marked by fluorescein isothiocyanate crossed containing the detection antibody crosses；
The fluorescein isothiocynate antibody of capture antibody and fluorescent microsphere label containing fluorescent microsphere label in the reaction solution；
The dilution includes polyvinylpyrrolidone and Tween-20.
8. the preparation method of the described in any item kits of claim 1~7, which is characterized in that preparation including reaction solution and
The preparation of test card；
The preparation method for the capture antibody that fluorescent microsphere marks in the reaction solution includes the following steps:
(1) activation of fluorescent microsphere；
(2) take cardiac muscle troponin I monoclonal antibody 41-49 and 83-93 mixing as capture antibody, the monoclonal antibody
The mass ratio of 41-49 and 83-93 is 1:1, capture antibody is added, reaction is marked into the fluorescent microsphere solution of activation；
(3) BSA processing is added in the capture antibody for the fluorescent microsphere label that step (2) obtains, end mark reaction obtains glimmering
The capture antibody of light microballoon label.
9. preparation method according to claim 8, which is characterized in that the test card is by the sample pad, nitric acid
Cellulose membrane and water absorption pad, which are fixed on bottom plate, to be prepared；
The preparation method of the nitrocellulose filter includes the following steps:
(1) preparation of detection line and nature controlling line: the detection antibody is crossed on nitrocellulose filter and obtains detection line；It will be different
The fluorescein-labeled BSA of thiocyanic acid crosses on nitrocellulose filter obtains nature controlling line；
(2) the nitrocellulose filter drying for standby for being coated with detection line and nature controlling line for obtaining step (1).
10. the examination that preparation method described in the described in any item kits of claim 1~7 or claim 8 or 9 is prepared
Application of the agent box in detection cardiac muscle troponin I.
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