CN107561289B - The double crush syndrome detection kit of human cardiac troponin I - Google Patents
The double crush syndrome detection kit of human cardiac troponin I Download PDFInfo
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Abstract
The invention discloses a kind of double crush syndrome detection kits of human cardiac troponin I, it is capture antibody with monoclonal antibody 7D2, monoclonal antibody 7H4 is detection antibody, wherein, the hybridoma cell strain 7D2 of secrete monoclonal antibody 7D2 was stored in China typical culture collection center (CCTCC) on August 23rd, 2017, and deposit number is CCTCC NO:C2017120；The hybridoma cell strain 7H4 of secrete monoclonal antibody 7H4 was stored in China typical culture collection center (CCTCC) on August 23rd, 2017, and deposit number is CCTCC NO:C2017121.Detection kit high sensitivity, it is at low cost, analysis means rapidly and efficiently are provided for the detection of human cardiac troponin I.
Description
Technical field
The present invention relates to field of immunodetection, more particularly, to the ELISA detection kit of human cardiac troponin I.
Background technique
Cardiac troponin is the systaltic key protein of regulation.By cardiac muscle troponin I (cTnI), myocardium myo calcium egg
White C(cTnC) and serum cardiac troponin T (cTnT) three subunits composition trimer compositions.CTnI is the inhibition of actin
Subunit, by inhibit actin atriphos (ATPase) activity come the mutual of modulate actin and myosin
Effect.The molecular weight of cTnI is about 24kDa, is made of 209 amino acid, is a kind of albumen rich in α spiral, theoretical isoelectric point
It is 9.87.TnI(Troponin I) there are three kinds of hypotypes: there are fast skeletal muscle types and slow bone in skeletal troponin I(sTnI)
Bone flesh type, they have similar molecular weight (20KD), but amino acid sequence between the two about has 40% difference；Third
Kind is myocardium type.There is also 40% difference, the ends cTnI for the amino acid sequence of cardiac muscle troponin I (cTnI) and skeletal muscle type
31 amino acid more than the end sTnI, and cTnI only exists a kind of hypotype in cardiac muscle cell, determines cTnI myocardium thin
Specificity in born of the same parents.The 22nd of cTnI molecule and the 23rd is serine.The two amino acid can be in vivo by albumen
Kinases A phosphorylation, thus there are four kinds of different phosphate acidification types by cTnI in vivo.
Acute myocardial infarction (Acute myocardial infarction, AMI) be the most common cardiovascular disease it
One, it is on the basis of coronary artery pathological changes, blood flow coronarius is sharply reduced or interrupted, and corresponding cardiac muscle occurs serious
And enduringly coronary artery pathological changes caused by acute ischemia, finally cause the ischemic necrosis of cardiac muscle.When being in a bad way, heart
Its function of pumping blood can not be exercised again, will lead to patient's die by visitation of God.Therefore, the early diagnosis of acute myocardial infarction is pre- for its
Anti- and treatment is very important.CTnI is considered that AMI diagnoses best one of Biological indicators by researcher, since it is in cardiac muscle
Just occur in blood when impaired, and time of occurrence is early, the duration is long, high specificity, therefore international clinical chemistry alliance
(IFCC), the biochemical meeting of U.S. clinical (NACB) and Chinese medical examine the authoritative institutions such as branch to examine cTnI as AMI
Disconnected " goldstandard ".
In myocardial infarction marker, cTnI be considered as it is sensitiveer than other markers, specificity is higher " gold mark ".
When the cell membrane of cardiac muscle cells is complete, cTnI can not be entered in blood through cell membrane.Only cardiac muscle cells
When impaired, cTnI is discharged into blood from cardiac muscle cell rapidly, starts to increase in 2-8 hours, and peak is reached in 1-2 days, and
And the amount that cTnI still can be measured after 3-8 days is increasing, the time that theoretically cTnI is maintained in serum is more than 10 days, more long
The diagnostic window phase diagnosis of myocardial infarction is very important.Research has shown that, normal person and Patients With Myocardial Infarction serum
The critical value of middle cTnI is 50pg/mL, and blood level is up to 100~300 μ g/L when morbidity.Most cTnI is in blood
Exist in the form of cTnI-T-C cTnI-C compound.Discovery cTnI is initially to be discharged into the form of monomer under study for action
In blood, but as Myocardial injury degree increases, cTnI is degraded in order to prevent, and cTnI can exist in the form of compound,
Then due to the influence of various factors, compound resolve into monomer, so the antibody for detecting cTnI should be able to be with same again
Efficiency identification blood in various complex forms cTnI, and there is a serious shortage of such monoclonal antibody resources in the prior art.
Summary of the invention
The present invention in order to overcome at least one of the drawbacks of the prior art described above, provides a kind of cardiac muscle troponin I
ELISA detection kit.
It is an object of the present invention to provide a kind of double crush syndrome detection kits of human cardiac troponin I.
A kind of double crush syndrome detection kit of human cardiac troponin I is that capture is anti-with monoclonal antibody 7D2
Body, monoclonal antibody 7H4 are detection antibody, wherein the hybridoma cell strain 7D2 of secrete monoclonal antibody 7D2 is in August, 2017
It is stored within 23rd China typical culture collection center (CCTCC), deposit number is CCTCC NO:C2017120；Secrete Dan Ke
The hybridoma cell strain 7H4 of grand antibody 7H4 was stored in China typical culture collection center (CCTCC) on August 23rd, 2017,
Deposit number is CCTCC NO:C2017121.
Preferably, which further includes negative control, positive control, terminate liquid, developing solution, elisa plate, coating buffer,
PBS-T buffer, PBS.
Preferably, detection antibody 7H4 is by horseradish peroxidase-labeled.
Preferably, developing solution TMB.
Preferably, terminate liquid is 10% H2SO4。
Preferably, coating buffer is 0.05 M, and the carbonate of pH 9.6 is coated with buffer.
Preferably, positive control is cTnI albumen.
Monoclonal antibody 7H4 is to utilize monoclonal antibody prepared by immune holoprotein cTnI-T-C, specific preparation method
It is as follows: Balb/c mouse is immunized with holoprotein cTnI-T-C, takes the spleen cell of mouse to merge with myeloma cell, by indirect
ELISA filters out positive hybridoma cell strain, can secrete the miscellaneous of stabilization of antibodies by the acquisition of limited dilution cloning technology three times
Tumor cell strain is handed over, ascitic type monoclonal antibody is prepared, obtains monoclonal antibody after purification.
Monoclonal antibody 7D2 be using the 13-24 amino acids epitope on cTnI, coupling carrier protein B SA, formation it is complete
Monoclonal antibody prepared by mouse is immunized in holoantigen, specific the preparation method is as follows: synthesizing the 13-24 amino acids table on cTnI
Position, coupling carrier protein B SA form comlete antigen, and Balb/c mouse is immunized, the spleen cell of mouse is taken to melt with myeloma cell
It closes, filters out positive hybridoma cell strain by indirect ELISA, can be secreted by the acquisition of limited dilution cloning technology three times steady
The hybridoma cell strain for determining antibody prepares ascitic type monoclonal antibody, obtains monoclonal antibody after purification.
With coating buffer dilution 7D2 to 2 μ g/mL, every hole adds 100 μ L, and 4 DEG C overnight.The closing one of 5% skimmed milk power is small
When.50 μ L samples to be tested are added in every hole, and 37 DEG C are incubated for 1 hour.100 μ L are separately added into according to the diluted HRP-7H4 of 1:2000,
It is incubated for one hour.100 μ L developing solution TMB are added, develop the color 10 minutes, 50 μ L terminate liquids are added.Microplate reader reads 450nm extinction
Degree value OD450, analyze data.
Double crush syndrome method has many advantages, such as that intuitive is strong, detection sensitivity is high, is usually used in detection with multiple antigens
The macromolecular antigen of epitope, such as macro-molecular protein, virus, pathogenic bacteria.
The monoclonal antibody that the present invention is uniform with physicochemical property height, specificity is good, can largely prepare, the sandwich method of foundation
High sensitivity, it is at low cost, analysis means rapidly and efficiently are provided for the detection of human cardiac troponin I.
Compared with prior art, the invention has the following beneficial effects: human cardiac troponin I Dan Ke provided by the invention
Grand antibody 7D2,7H4 are the cTnI mouse monoclonal antibody for being high-affinity, high specific, can be in conjunction with free
CTnI, dimer complex cTnI-C, tri- kinds of forms of trimer compositions cTnI-T-C cTnI, therefore can be used for detecting blood
CTnI content in clear lays the foundation to develop quickly detection myocardial infarction kit.
Detailed description of the invention
Fig. 1 is 3 CDR(complementary determining regions of antibody 7D2 light and weight chain variable region) area.
Fig. 2 is 3 CDR(complementary determining regions of antibody 7H4 light and weight chain variable region) area.
Fig. 3 is that the SDS-PAGE of antibody 7D2 and 7H4 after purification detects figure, wherein 1 is albumen Marker, and 2 be 7D2, and 3 are
7H4。
Fig. 4 is the bioactivity ELISA result figure of antibody 7D2 and 7H4, wherein figure A is 7D2, and figure B is 7H4.
Fig. 5 is that the affinity constant of antibody 7D2 and 7H4 detect ELISA figure, wherein figure A is 7D2, and figure B is 7H4.
Fig. 6 be antibody 7D2 and 7H4 with cTnI and cTnI-C response diagram, wherein figure A be cTnI monomer, scheme B be
CTnI-C compound.
Fig. 7 is the detection curve of 7D2/HRP-7H4 and 7H4/HRP-7D2.
Fig. 8 is 7D2/HRP-7H4 examination criteria curve graph.
Specific embodiment
The present invention is made with specific embodiment with reference to the accompanying drawings of the specification and further being elaborated, the embodiment
It is served only for explaining the present invention, be not intended to limit the scope of the present invention.Test method as used in the following examples is such as without spy
Different explanation, is conventional method；Used material, reagent etc., unless otherwise specified, for the reagent commercially obtained
And material.
Embodiment 1
The preparation of hybridoma cell strain and its monoclonal antibody of secretion, includes the following steps:
One, antigen
Monoclonal antibody 7D2 is small by the way that Balb/c is immunized with the Antigenic Peptide that BSA is coupled with 13-24 amino acids on cTnI
The monoclonal antibody that mouse prepares, the Antigenic Peptide the preparation method comprises the following steps: with Discovery Studio4.0 software simulate cTnI-T-C
Structure analyzes epitope on cTnI subunit, picks out 13-24 amino acids as epitope antigen.Synthesize 13- on cTnI
24 amino acids simultaneously are coupled to form comlete antigen with BSA, this step is synthesized by Hangzhou Zhong Tai company.
Monoclonal antibody 7H4 antibody is prepared by the way that Balb/c mouse is immunized with whole protein complex cTnI-T-C
Monoclonal antibody, wherein cTnI-T-C is bought from Hytest company, Finland.
Two, the preparation of hybridoma cell strain: hybridoma cell strain 7D2 is identical with the preparation method of hybridoma cell strain 7H4,
The two only uses different antigen, and preparation method is specific as follows:
1, animal immune
(1) initial immunity: 50 μ g antigens are emulsified with isometric complete Freund's adjuvant volume, subcutaneous multi-point injection；
(2) booster immunization: interval 2 weeks, amount of antigen same as initial immunity and isometric incomplete Freund's adjuvant emulsify,
Subcutaneous multi-point injection；Its potency is surveyed in blood sampling after being immunized 10 days in the 3-5 times；
(3) 72 hours before fusion, the antigen 25 μ g of adjuvant is not added in intraperitoneal injection.
2, cell fusion
(1) raising of myeloma cell SP2/0.The fusion preceding ten days SP2/0 cells frozen of recovering, and carry out expansion training
It supports.A part of SP2/0 HAT culture medium culture 24 hours is taken, whether observation cell can apoptosis.
(2) feeder cells prepare: fusion the previous day takes the feeder cells of the Balb/c mouse of health to carry out bed board, and cell is dense
Degree is cell to 1 × 105A/mL, every 100 μ L of hole.
(3) preparation of SP2/0.Cell is gently blown down, is collected into centrifuge tube, overall solution volume is write down, is mixed simultaneously
It is counted after taking a small amount of cell suitably to dilute.The preparation of splenocyte.The spleen for taking out fusion mouse, with 1640 basal mediums
Spleen is rinsed, and is in spleen in the environment moistened, grinds spleen, is remained on needle core and strainer with basal medium flushing
Spleen cell, collect lapping liquid, record total volume.Partial mill liquid is taken to be counted.
(4) SP2/0 cell and splenocyte mix in centrifuge tube according to the ratio of 1:5~1:10,1000rpm/min centrifugation
7 min abandon supernatant.1mL PEG preheated in 37 DEG C of water is added, then instills the 1640 of the 15mL having had been warmed up
Basal medium terminates reaction, and 1000 rpm/min are centrifuged 7min, abandon supernatant, and HAT complete medium is added and is resuspended, every hole is added
100 μ L place and cultivate in cell incubator, observe fusion results after five days.
3, positive cell screening and cloning
(1) the 5th day observation cell after fusion, carries out the biggish hole of cell mass to change liquid, third day takes cell supernatant
To being surrounded by the plate of the hole 50ng/ antigen, ELISA testing inspection, positive hole needs to change a not good liquor again, detects again every other day, to two
The hole of secondary all test positive carries out cloning.
(2) cloning is carried out to positive cell with limiting dilution assay, generally carries out cloning three times.
After positive cell screening is with cloning, hybridoma cell strain 7D2 and hybridoma cell strain 7H4 is screened, two
Person was stored in China typical culture collection center (CCTCC), the guarantor of hybridoma cell strain 7D2 respectively on August 23rd, 2017
Hiding number is CCTCC NO:C2017120, and the deposit number of hybridoma cell strain 7H4 is CCTCC NO:C2017121.In preservation
The address of the heart are as follows: Wuhan City, Hubei Province Wuchang District Wuhan University collection.
Three, the preparation of monoclonal antibody:
Monoclonal is prepared by the method for hybridoma cell strain 7D2 preparation monoclonal antibody 7D2 and by hybridoma cell strain 7H4
The method of antibody 7H4 is identical, specific as follows:
1, the preparation of ascitic type antibody
(1) 7~14 days BaLb/c mouse peritoneals to inoculation inject the incomplete of 500 μ L before injecting hybridoma cell strain
Freund's adjuvant.
(2) every mouse peritoneal injection 2 × 105~1 × 106A hybridoma cell strain, volume injected 500L.General 7~
Mouse web portion obviously swells after 14 days, and ascites can be extracted when slow in action.
(3) 4 DEG C of the ascites being collected into, 12000 rpm/min are centrifuged 30min, take supernatant, -20 degree of packing saves.
2, the purifying of antibody
(1) ammonium sulfate precipitation method: 1. taking out ascites from -20 DEG C, 4 DEG C of defrostings.2. 4 DEG C of 12000rpm/min centrifugations
30min takes supernatant, and records the volume of supernatant.It is stirred 3. ascites is placed under 4 DEG C of ice water hybird environment, side stirring
While isometric saturated ammonium sulfate solution is slowly added dropwise, it can be observed that there is white precipitate generation, continue to stir 15min, 4 DEG C quiet
It sets overnight.4. the ascites after saturated ammonium sulphate is centrifuged 30min in 4 DEG C, 7500 rpm, supernatant is abandoned, with the PBS of 1.5mL
(being filtered with 0.015M filter membrane) is resuspended.
(2) it the salt ion in desalting column protein isolate matter: 1. balances: pillar (preventing air from entering pillar) is connected, with 5 times
The PBS of column volume filtered washes away 20% ethyl alcohol in pillar for preservation.2. loading: speed is slow when loading.3. receiving
Sample: since protein molecule is bigger than the volume of salt ion, protein molecule cannot by gap narrow in filler in pillar,
It can only be come out in big gap first from the side, therefore only receive the sample of first absorption peak.4. balance: with greater than 5 times of cylinders
The filtered PBS of product rinses pillar.5. saving: column is sealed with 20% filtered ethyl alcohol, prevents from polluting, 4 DEG C of preservations.
(3) affinity column Protein G separates foreign protein: 1. balancing: connecting pillar (preventing air from entering pillar), uses
The PBS of 5 times of column volumes filtered washes away 20% ethyl alcohol in pillar for preservation.2. loading: speed is slow when loading.
3. receiving sample: what first absorption peak came out is foreign protein, is not collected.After PBS develops foreign protein, eluent is used
(pH2.7 glycine solution) elutes destination protein, collects albumen, and the elution of every 1mL at once when peak value rises
Albumen adds the neutralizer (pH9.0Tris-HCL) of 100 μ L.4. balance: rinsing pillar with the filtered PBS of 5 times of column volumes is greater than.
5. saving: column is sealed with 20% filtered ethyl alcohol, prevents from polluting, 4 DEG C of preservations.
(4) ultrafiltration: taking the super filter tube of 50KD, fills it up with filtered PBS, and 5000 rpm/min are centrifuged 10min, repeats three
It is secondary.So that film internal and external equilibrium.Protein sample is added, 5000 rpm/min are centrifuged 10min.The concentrate being collected on collection membrane.
PBS is filled it up with, 5000 rpm/min centrifugation 10min fills it up with 20% ethyl alcohol, 4 DEG C of preservation super filter tubes in triplicate.
Four, monoclonal antibody performance detection
The total serum IgE of hybridoma cell strain 7D2 and 7H4 are extracted respectively, is cDNA by RNA reverse transcription, to antibody variable region base
Because carrying out PCR amplification, pMD-18T carrier is connected, massive amplification in the bacillus coli DH 5 alpha of competence is finally imported, extracts plasmid
Send to sequencing.The light and heavy chain variable region amino acid sequence of monoclonal antibody 7D2 is known as shown in Figure 1, monoclonal antibody 7H4
Light and heavy chain variable region amino acid sequence such as Fig. 2.
Using the purity of monoclonal antibody 7D2 and 7H4 that SDS-PAGE test for identification purifies, as a result as shown in figure 3,
Using the potency of monoclonal antibody 7D2 and 7H4 that ELISA method detection purifying obtains, as a result as shown in figure 4, utilizing the side ELISA
The affinity constant of monoclonal antibody 7D2 and 7H4 that method detection purifying obtains, as a result as shown in Figure 5.
With the combination situation of the other two kinds of forms of 2 strain antibody of indirect ELISA test for identification and cTnI: cTnI monomer (Finland
The purchase of Hytest company) and cTnI-C compound (expressing certainly in laboratory), 2 strain antibodies energy and cTnI, cTnI-C can be obtained by Fig. 6
In conjunction with illustrating that 2 strain antibodies in the present invention can identify various forms of cTnI.According to existing research (cTnI-linker-
The prokaryotic expression of TnC fusion protein and identification, Chinese biological engineering magazine, 2015,35 (4): 48-53) and the present embodiment it is found that
7D2,7H4 can with cTnI, cTnI-C or cTnI-T-C occur specific binding react, show its can with it is various forms of
CTnI is combined.
Embodiment 2
It is capture antibody using monoclonal antibody 7D2,7H4 is that detection antibody establishes double-antibody sandwich elisa detection method,
Exploitation quickly detects myocardial infarction kit.The establishment process of double-antibodies sandwich ELISA is as follows:
1, the selection of antibody is matched
(1) it is coated with: antibody 7D2 being diluted to 2 μ g/mL with coating buffer, adds 100 μ L to 96 hole elisa plates.4 DEG C overnight.With
PBS-T buffer washs three times, each 3min.
(2) it closes: being added in elisa plate with 5% skimmed milk power, 200 μ L are added in every hole, and 37 DEG C are closed one hour.Use PBS-T
Buffer washs three times, each 3min.
(3) add antigen: antigen (cTnI) being diluted to 1000ng/mL, 500ng/mL, 250ng/ respectively with PBS buffer solution
ML, 125ng/mL, 62.5ng/mL, 31.25ng/mL, 15.625ng/mL.Every hole adds 100 μ L.37 DEG C are incubated for one hour.With
PBS-T buffer washs three times, each 3min.Only plus PBS is as negative control.
(4) marking HRP antibody 7H4: HRP is diluted to suitable multiple (OD in Salmonella with PBS-T buffer450
The corresponding extension rate near 2.0) 100 μ L of every hole, 37 DEG C are incubated for one hour.Five times are washed with PBS-T buffer, every time
3min。
(5) it develops the color: 100 μ L TMB developing solutions is added, room temperature is protected from light 10min.
(6) every hole adds 50 μ L terminate liquids.
(7) microplate reader reads 450nm absorption values OD450, analyze data.
2, the 7D2 established is capture antibody, and 7H4 is the double-antibody sandwich elisa detection method detection curve for detecting antibody
Foundation and detection range establishment: with 0.05 M, the carbonate coating buffer of pH 9.6 dilutes antibody 7D2 to 2 μ g/mL,
Every hole adds 100 μ L, and 4 DEG C overnight.5% skimmed milk power is closed one hour.Be separately added into various concentration uses PBS-T buffer soln
The cTnI albumen of diluted purchase as antigen (1000ng/mL, 500ng/mL, 250ng/mL, 125ng/mL, 62.5ng/mL,
31.25ng/mL, 15.625ng/mL), every 50 μ L of hole, 37 DEG C are incubated for 1 hour.It is diluted according to 1:2000 to be separately added into 100 μ L
HRP-7H4 is incubated for one hour.100 μ L developing solution TMB are added, develop the color 10 minutes, 50 μ L terminate liquid (10% H are added2SO4).Enzyme
It marks instrument and reads 450nm absorption values OD450, data are analyzed, make standard curve according to data.In cTnI concentration in 30ng/mL
Within the scope of~500ng/mL, with the raising of cTnI concentration, ELISA reacts OD450Value increases, cTnI concentration and OD450Value is in line
Property related, therefore the range of the detection of this method are as follows: 30ng/mL~500ng/mL(Fig. 7).And it is anti-for capture to depict 7D2
Body, 7H4 are the standard curve (Fig. 8) for detecting the double-antibody sandwich elisa detection method of antibody.
3, the detection of clinical sample: double crush syndrome detection kit of the invention to 30 parts of clinical serum samples into
Row detection, wherein it is 22 parts positive, it is 8 parts negative.Statistic analysis result, positive rate 77.3%, negative recall rate are 100%.
Comparative example 1
It is capture antibody using monoclonal antibody 7H4,7D2 is that detection antibody establishes double-antibody sandwich elisa detection method,
It can be used for developing quickly detection myocardial infarction kit.The establishment process of double-antibodies sandwich ELISA is as follows:
1, the selection of antibody is matched
(1) it is coated with: antibody 7H4 being diluted to 2 μ g/mL with coating buffer, adds 100 μ L to 96 hole elisa plates.4 DEG C overnight.With
PBS-T buffer washs three times, each 3min.
(2) it closes: being added in elisa plate with 5% skimmed milk power, 200 μ L are added in every hole, and 37 DEG C are closed one hour.Use PBS-T
Buffer washs three times, each 3min.
(3) add antigen: antigen (cTnI) being diluted to 1000ng/mL, 500ng/mL, 250ng/ respectively with PBS buffer solution
ML, 125ng/mL, 62.5ng/mL, 31.25ng/mL, 15.625ng/mL.Every hole adds 100 μ L.37 DEG C are incubated for one hour.With
PBS-T buffer washs three times, each 3min.Only plus PBS is as negative control.
(4) marking HRP antibody 7D2: HRP is diluted to suitable multiple (OD in Salmonella with PBS-T buffer450
The corresponding extension rate near 2.0) 100 μ L of every hole, 37 DEG C are incubated for one hour.Five times are washed with PBS-T buffer, every time
3min。
(5) it develops the color: 100 μ L TMB developing solutions is added, room temperature is protected from light 10min.
(6) every hole adds 50 μ L terminate liquids.
(7) microplate reader reads 450nm absorption values OD450, analyze data.
2, the 7H4 established is capture antibody, and 7D2 is the double-antibody sandwich elisa detection method detection curve for detecting antibody
Foundation and detection range establishment: with 0.05 M, the carbonate coating buffer of pH 9.6 dilutes antibody 7H2 to 2 μ g/mL,
Every hole adds 100 μ L, and 4 DEG C overnight.5% skimmed milk power is closed one hour.It is separately added into being diluted with PBS-T buffer for various concentration
The standard antigen cTnI(0 of purchase, 15.625,31.25,62.5,125,250,500,1000ng/mL), 50 μ L of every hole, 37
DEG C be incubated for 1 hour.100 μ L are separately added into according to the diluted HRP-7D2 of 1:2000, are incubated for 1 hour.100 μ L developing solutions are added
TMB develops the color 10 minutes, and 50 μ L terminate liquid (10% H are added2SO4).Microplate reader reads 450nm absorption values OD450, analyze number
According to, it is known that, in cTnI concentration within the scope of 60ng/mL~500ng/mL, with the raising of cTnI concentration, ELISA reacts OD450
Value increases, cTnI concentration and OD450Value is linear related, but the related slope is small.Therefore, the range of the detection of this method are as follows:
60ng/mL~500ng/mL(Fig. 7).
Comparing embodiment 2 and comparative example 1 are can be found that: according to embodiment 7D2/HRP-7H4 and comparative example 7H4/HRP-
The detection curve of 7D2 determines respective detection range, the detection curve ratio 7H4/HRP-7D2 of root 7D2/HRP-7H4 according to the graph
Detection curve change rate is big, and detection range is wide, high sensitivity.
Claims (7)
1. a kind of double crush syndrome detection kit of human cardiac troponin I, which is characterized in that with monoclonal antibody 7D2
For capture antibody, monoclonal antibody 7H4 be detection antibody, wherein the hybridoma cell strain 7D2 of secrete monoclonal antibody 7D2 in
It is stored on August 23rd, 2017 China typical culture collection center CCTCC, deposit number is CCTCC NO:C2017120；Point
The hybridoma cell strain 7H4 for secreting monoclonal antibody 7H4 was stored in China typical culture collection center on August 23rd, 2017
CCTCC, deposit number are CCTCC NO:C2017121.
2. double crush syndrome detection kit according to claim 1, which is characterized in that the kit further includes yin
Property control, positive control, terminate liquid, developing solution, elisa plate, coating buffer, PBS-T buffer, PBS.
3. double crush syndrome detection kit according to claim 1, which is characterized in that antibody 7H4 is by horseradish for detection
Peroxidase labelling.
4. double crush syndrome detection kit according to claim 2, which is characterized in that developing solution TMB.
5. double crush syndrome detection kit according to claim 2, which is characterized in that terminate liquid is 10% H2SO4。
6. double crush syndrome detection kit according to claim 2, which is characterized in that coating buffer is 0.05 M, pH
9.6 carbonate is coated with buffer.
7. double crush syndrome detection kit according to claim 2, which is characterized in that positive control is cTnI egg
It is white.
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| 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 |
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