CN103012595B - Stable NT-probBNP calibrator and application thereof - Google Patents
Stable NT-probBNP calibrator and application thereof Download PDFInfo
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Abstract
The invention provides a stable NT-probBNP calibrator and application thereof, and belongs to a biological detection reagent. The stable NT-probBNP calibrator is characterized in that the stable NT-probBNP calibrator is polypeptide, the linear structure of which is NT-proBNP captured antibody epitope peptide fragment-connecting peptide-NT-probBNP detecting antibody epitope peptide fragment. Based on the structure, the calibrator has better stability and dissolvability proved by a series of experiments. The stable NT-probBNP calibrator can be used for a calibrator in a reagent kit for detecting NT-proBNP and proBNP protein.
Description
Technical field
The present invention relates to medical science detection reagent, particularly a kind of NT-proBNP calibration object and preparation method thereof and application.
Technical background
1988, Japanese scholars Sudoh was first from separating and obtain a kind of polypeptide with powerful sharp sodium, diuresis, expansion blood vessel and hypotensive effect in pig brain, called after brain natriuretic peptide or claim natriuretic peptide (Brain Natriuretic Peptide, BNP).After this research shows, in the process of organic evolution, development produces one group of polypeptide A NP, BNP including BNP, CNP, DNP, VNP etc. gradually, is called natriuretic peptide family, and its function is the stable state of the capacity, osmotic pressure and the pressure that maintain the recycle system.BNP is mainly present in ventricle barrier film particle, and volume expansion and pressure load that its secretion depends on ventricle increase.As the most responsive and the most special index of cardiac disorder, BNP has important clinical meaning., as far back as ESC " chronic heart failure guide (2001) ", then in U.S. ACC/AHA " chronic heart failure guide (2005) ", recommend using blood BNP level determination as diagnosis and prognosis index in heart failure.ESC in 2008 " acute and chronic heart failure guide " and 2009 AHA " guide in heart failure " this has been done to further recommendation.
When after myocardial cell's irriate, can produce containing 134 amino acid whose PROBNPs (pre-proBNP), under the effect of relevant enzyme, cut away subsequently the signal peptide sequence of N end, form containing 108 amino acid whose proBNPs (proBNP), the latter is cracked into the N end product NT-proBNP (NT-proBNP) that contains 76 amino acid, lifeless matter activity and contains 32 activated C end product B type natriuretic peptides of amino acid (BNP) under the effect of restriction endonuclease.The removing of BNP is mainly by removing receptors bind with BNP, and NT-proBNP is mainly by glomerular filtration, and therefore, its blood concentration is subject to renal function to be greater than BNP.The BNP transformation period short (22min), vitro stability is poor, and the NT-proBNP transformation period is grown (120min), and vitro stability is strong, and the concentration in heart failure patient is high compared with BNP, is more being conducive in some cases diagnosis in heart failure.The U.S. has delivered respectively BNP clinical application Consensus of experts and international NT-proBNP Consensus of experts at 2004 and 2008, systematically discussed biology and the clinical application of BNP and NT-proBNP.
BNP and NT-proBNP detect at the beginning of 21 century and successively enter China, over 10 years, are widely used in clinical practice by situation of all-level hospitals and doctor, become especially diagnosing patients with heart failure and the very useful biomarker of assessment of cardiovascular diseases.China's 2007 " diagnosis of chronic congestive heart failure treatment guide " and 2010 " acute heart failure diagnoses and treatment guide " also recommends NT-proBNP and BNP to judge for diagnosis and prognosis in heart failure.But in kit developing and use procedure, the less stable of calibration solution, especially for the diagnostic reagent of exempting from platform based on board-like enzyme, because its each mensuration all needs to calibrate simultaneously, so higher to the stability requirement of test kit calibration object.
The albumen that NT-proBNP is made up of 76 amino acid, manufacturer, in test kit and performance history, adopts recombinant protein as calibration object more.But a large amount of research discovery, due to the hypoproteinosis glycosylation that RT-PCR is expressed, its stability is poorer compared with native protein.Do not see at present the NT-proBNP calibration object of excellent in stability.
Summary of the invention
Demand and blank that the present invention exists according to above-mentioned field, provide a kind of stable NT-proBNP calibration object, and technical scheme is as follows:
A kind of NT-proBNP calibration object, is characterized in that: described NT-proBNP calibration object is polypeptide, and its linear structure is: NT-proBNP capture antibody epitope peptide section-connection peptides-NT-proBNP detects antibody epitope peptide section.
Described linear structure for to be from aminoterminal to carboxyl terminal: NT-proBNP capture antibody epitope peptide section-connection peptides-NT-proBNP detects antibody epitope peptide section.
Described connection peptides is the small peptide that 4～10 hydrophilic amino acid residues form.
In described connection peptides, do not contain amino acid or the peptide sequence of easy formation corner and alpha-helix.
The aminoacid sequence of described joining peptide as Seq ID No.2,3 and 8 ~ 13 arbitrary as shown in.
As shown in the NT-proBNP capture antibody epitope peptide section aminoacid sequence of described NT-proBNP calibration object is as arbitrary in Seq ID No.15, No.17.
The NT-proBNP of described NT-proBNP calibration object detect antibody epitope peptide section aminoacid sequence as arbitrary in Seq ID No.14, No.16 as shown in.
Described NT-proBNP calibration object, as shown in its aminoacid sequence is as arbitrary in Seq ID No.18 ~ 25.
The purposes as calibration object of above-mentioned arbitrary NT-proBNP calibration object in the test kit that detects NT-proBNP and proBNP albumen.
The invention provides a kind of NT-proBNP calibration object of artificial design, it comprises the stable NT-proBNP epitope that a pair of connection peptides by artificial design connects.
In order to improve the stability of NT-proBNP calibration object of the present invention, the connection peptides designing in the present invention is 4～10 amino acid compositions.
In order to prevent polypeptide dimer formation, the space folding of linear order and the solvability of synthetic polypeptide, size, aminoacid sequence composition and the spatial character thereof of linker further optimized in test, design obtains the connection peptides as shown in Seq ID No.2 in table 1, experimental results show that the calibration object that adopts the present invention of these connection peptides to obtain has better solvability.
For preventing dimeric formation, in the design process of linker, reject the selection of halfcystine; In order to keep the linearity of polypeptide, linker selects to have rejected proline(Pro) etc. and easily forms the amino acid of corner, according to this design as.Connection peptides as shown in Seq ID No.2 in table 1, experimental results show that the calibration object that adopts the present invention of these connection peptides to obtain has better solvability and stability.
According to the literature, epi-position EP1(5-12 in NT-proBNP) and epi-position EP2(67-76) comparatively stable and sugar based site, so not only can prevent epi-position degraded, and can keep the epi-position of preparation consistent with natural epi-position, therefore in one embodiment of the present of invention, select epi-position EP1 and the epi-position EP2 antibody identification meter position as quantitative analysis, as SeqID No.14, No.15 in table 2.
In a preferred embodiment of the present invention, in order to reduce the impact of linker and other factors antagonist-epi-position identification, assay optimization select epi-position EP1 ' (4-13), epi-position EP2 ' is (66-76) as quantitative analysis antibody identification meter position, as SeqID No.16, No.17 in table 2.
To sum up, calibration object provided by the invention, owing to having the epi-position of NT-proBNP, so can its corresponding antibodies.Meanwhile, owing to having rejected the unsettled aminoacid sequence of NT-proBNP, select comparatively stable epi-position, and two epi-positions are connected with stable polypeptide linker, so the calibration object of preparation is more stable thus.
Calibration object provided by the invention can be used for the calibration object of NT-proBNP and proBNP labile protein test kit.
Calibration object provided by the invention, owing to being all small peptide, preferably adopts the method preparation of synthetic.
Terminological interpretation:
NT-proBNP capture antibody epi-position: refer in NT-proBNP that the region that anti-NT-proBNP antibody works to it effect of catching is to be made up of one section of aminoacid sequence.
NT-proBNP detects antibody epitope: refer in NT-proBNP that the region that anti-NT-proBNP antibody plays detection effect to it is to be made up of one section of aminoacid sequence.
Connection peptides, refer to the small peptide of artificial design, in the present invention, mainly play the stability that improves the calibration object being made up of NT-proBNP capture antibody epi-position and NT-proBNP detection antibody epitope, the space structure of realizing the epi-position of two synthetic makes catching and measuring ability of its performance antagonist.
P/N: i.e. male/female, refer to the ratio of the positive (P, the positive) value of reading and negative (N, negative) value of reading, this ratio is higher represents that the ability of test kit differentiation yin and yang attribute is stronger.
Accompanying drawing explanation
Fig. 1. typical curve,
Fig. 2. methodology comparative result.
Embodiment
By embodiment, technical scheme of the present invention and effect thereof are described below.
Test required solution and reagent:
1．10mM?PB：
Take 0.27g potassium primary phosphate (KH2PO4) (traditional Chinese medicines, 10017608), 1.42g Sodium phosphate dibasic (Na2HPO4) (traditional Chinese medicines, 100203008), 1mL Proclin300(Sigma, 48914-U) in 900mL ultrapure water, after dissolving completely, regulate pH to 7.4, be then settled to 1L with volumetric flask.
2．10mM?PBS：
Take 0.27g potassium primary phosphate (KH2PO4) (traditional Chinese medicines, 10017608), 1.42g Sodium phosphate dibasic (Na2HPO4) (traditional Chinese medicines, 100203008), 8g sodium-chlor (NaCl) (traditional Chinese medicines, 10019308), 0.2g Repone K (KCl) (traditional Chinese medicines, 10016308), 1mL Proclin300(Sigma, 48914-U) in 900mL ultrapure water, after dissolving completely, regulate pH to 7.4, be then settled to 1L with volumetric flask.
3．0.5%BSA?10mM?PBS：
Contain 0.5%(W/V) the 10mM PBS solution of BSA bovine serum albumin (amresco, 0332).
4. washings:
Contain 0.05%(V/V) Tween-20(sigma, 44112) 10mM PBS solution.
5. synthetic polypeptide: in the present invention, the listed polypeptide of table 3 is given birth to work biosynthesizing by Shanghai.
6.NT-proBNP antibody 4NT1-29D12,4NT1-24E11 are all purchased from Hytest.
7. restructuring NT-proBNP(1-76 amino acid, 8NT2) albumen is purchased from Hytest.
8. substrate A and substrate B (Thermo, 34080)
The connection peptides designing in table 1 the present invention
| Sequence number | Connection peptides aminoacid sequence |
| Seq?ID?No.1 | STQNG |
| Seq?ID?No.2 | QNQQASNQNQ |
| Seq?ID?No.3 | PVPFLPMFVP |
| Seq?ID?No.4 | VDIRFPIYMF |
| Seq?ID?No.5 | Q |
| Seq?ID?No.6 | QN |
| Seq?ID?No.7 | QNQ |
| Seq?ID?No.8 | QNQQ |
| Seq?ID?No.9 | QNQQA |
| Seq?ID?No.10 | QNQQAS |
| Seq?ID?No.11 | QNQQASN |
| Seq?ID?No.12 | QNQQASNQ |
| Seq?IDNo.13 | QNQQASNQN |
The polypeptide epitope of selecting in table 2 the present invention
| Sequence number | Aminoacid sequence | Sequence number | ? |
| EP1 sequence: Seq ID No.14 | SPGSASDL | EP ' 1 sequence Seq ID No.16 | GSPGSASDLE |
| EP2 sequence: Seq ID No.15 | MVLYTLRAPR | EP ' 2 sequences: Seq ID No.17 | KMVLYTLRAPR |
The aminoacid sequence of the NT-proBNP calibration object that table 3 the present invention optimizes
| Sequence number | The aminoacid sequence of synthetic NT-proBNP calibration object |
| Seq?ID?No.18 | GSPGSASDLEQNQQKMVLYTLRAPR |
| Seq?ID?No.19 | GSPGSASDLEQNQQAKMVLYTLRAPR |
| Seq?ID?No.20 | GSPGSASDLEQNQQASKMVLYTLRAPR |
| Seq?ID?No.21 | GSPGSASDLEQNQQASNKMVLYTLRAPR |
| Seq?ID?No.22 | GSPGSASDLEQNQQASNQKMVLYTLRAPR |
| Seq?ID?No.23 | GSPGSASDLEQNQQASNQNKMVLYTLRAPR |
| Seq?ID?No.24 | GSPGSASDLEQNQQASNQNQKMVLYTLRAPR |
| Seq?ID?No.25 | GSPGSASDLE?PVPFLPMFVP?KMVLYTLRAPR |
The selection of embodiment 1, NT-ProBNP antibody
According to the literature, the sequence of NT-p roBNP antigen N end and C end is comparatively stable, therefore, the 4NT1-29D12(aminoacid sequence 5-12 that selection Hytest company provides), 4NT1-24E11(aminoacid sequence 67-76) as the antibody pair of double antibodies sandwich method.
The selection of embodiment 2, synthetic polypeptide epitope
To synthesize EP1-STQNG-EP2, EP1 '-STQNG-EP2 ', restructuring NT-proBNP antigen (Hytest, 8NT2) preparation high value (H, 15000pg/ml), intermediate value (M, 5000pg/ml), low value (L, 100pg/ml) gradient sample, measuring method is with embodiment 6, and calculates P/N.
Test-results shows, P/N(H/N, M/N, the L/N of the synthetic polypeptide epitope of EP1 '-STQNG-EP2 ' and restructuring NT-proBNP antigen) all higher than the synthetic polypeptide epitope of EP1-STQNG-EP2, be indicated as and obtain the response characteristic consistent with recombinant antigen, synthetic polypeptide antigen epi-position should be at least each many containing an amino acid compared with the N end of the identification epi-position of antibody and C end.
The different calibration object P/N of table 4 difference
Embodiment 3, linker length are selected
With the high value of synthetic polypeptide antigen (EP1 '-linker-EP2 ') preparation (H, 15000pg/ml), intermediate value (M, 5000pg/ml), low value (L, 100pg/ml) gradient sample, measuring method is with embodiment 6, and calculates P/N.
Test-results shows, Linker length is greater than P/N(H/N, M/N, the L/N of the synthetic polypeptide epitope of 4 aminoacid sequences) apparently higher than the polypeptide of other Linker, show that the length of Linker is on affecting the combination of epi-position and antibody, its possible reason is space steric effect, and therefore test suggestion selection contains the peptide sequence of the amino acid whose linker of 4-10 as the calibration object of test kit.
Table 5 linker aminoacid sequence
Note: non-, without linker, is directly connected with EP2 ' by EP1 '.
Calibration object P/N difference prepared by the different linker of table 6
Note: non-, without linker, is directly connected with EP2 ' by EP1 '.
Embodiment 4, linker amino acid composition
With the high value of synthetic polypeptide antigen (EP1 '-linker-EP2 ') preparation (H, 15000pg/ml), intermediate value (M, 5000pg/ml), low value (L, 100pg/ml) gradient sample, measuring method is with embodiment 6, and calculates P/N.
Test-results shows, formed P/N(H/N, M/N, the L/N of the synthetic polypeptide epitope of Linker 10-1 (SEQ ID No.2) by hydrophilic amino acid) higher than the polypeptide that is formed Linker 10-2 (SEQ ID No.3) by hydrophobic amino acid, show that the amino acid composition of Linker affects the combination of epi-position and antibody, its possible reason is the solvability of the synthetic polypeptide of hydrophobic amino acid too high levels impact.In addition, in the synthetic polypeptide due to Linker 10-3 (SEQ ID No.4), contain a large amount of folding factors, therefore its P/N is starkly lower than the synthetic polypeptide epitope containing Linker10-1,10-2.Therefore test and Selection containing hydrophilic amino acid higher, the peptide sequence of the linker in space is as the calibration object of test kit.
Table 7 linker aminoacid sequence
Note: a.DNAman software secondary structure prediction.
Table 8 linker aminoacid sequence
Embodiment 5, calibration object Stability Determination
With synthetic polypeptide antigen (EP1 '-QNQQASNQNQ-EP2 ') and restructuring NT-proBNP(Hytest, 8NT2) the high value of antigen preparation (H, 15000pg/ml), intermediate value (M, 5000pg/ml), low value (L, 100pg/ml) gradient sample, is then placed in respectively 4 ℃ and 37 ℃ and places 6d.
Measuring method, with embodiment 6, calculates P/N.
Result shows, synthetic polypeptide antigen is significantly higher than recombinant antigen (in table 9) 37 ℃ of stability (37 ℃/4 ℃) of placing 3d, 6d.
Table 9 calibration object stability-1
Table 10 calibration object stability-2
Embodiment 6, NT-proBNP detection kit (chemoluminescence method) preparation:
1) coated: preparation 1 μ g/mL 24E11 antibody (hytest, 4NT1) 10mM PB solution, add in micropore luminescent screen, 100 μ L/ holes, hatch 2h for 37 ℃;
2) sealing: coating buffer in microwell plate is dried, add 5%BSA 10mM PB solution, 200 μ L/ holes,, hatch 1h by 37 ℃;
3) antigen: confining liquid in microwell plate is dried, add 100 μ L/ hole antigens, 37 ℃, hatch 30min;
4) enzyme labelled antibody: by washings washing 3 times drying for microwell plate, add the 29D12 antibody (Hytest, 4NT1) of 100 μ L/ hole HRP marks (HRP-29D12), 37 ℃, hatch 30min;
5) substrate: by washings washing 5 times drying for microwell plate, add respectively 50 μ L/ hole substrate A and substrate B (Thermo, 34080), lucifuge reaction 5min, measures luminous value.
6) concentration of antigen linear (four parameter fittings) in luminous intensity and sample.
Embodiment 7, the application of calibration object clinical detection:
Collect the 178 routine clinical samples serum of (age, 18-75 year and more than 75 years old crowd, comprises normal people and doubtful heart failure patient).Select Roche Natriuretic Peptide diagnostic kit (Electrochemiluminescince) (No. 2402335th, state's food medicine prison tool (entering) word 2011) to carry out methodology comparison.
Determination step:
1) with synthetic calibration object preparation 15000pg/ml, 5000pg/ml, 500pg/ml, 200pg/ml, the 100pg/ml setting point of preparation, make typical curve;
2) measuring method is with embodiment 6.
Test kit typical curve is shown in Fig. 1, correlation coefficient r
2=0.99
Kit measurement result (subordinate list 1) is carried out methodology comparison, correlation coefficient r with Roche Natriuretic Peptide diagnostic kit (Electrochemiluminescince)
2=0.9188.See Fig. 2
Subordinate list 1: clinical sample measurement result
Claims (5)
1. a NT-proBNP calibration object, is characterized in that: described NT-proBNP calibration object is polypeptide, and its linear structure is: NT-proBNP capture antibody epitope peptide section-connection peptides-NT-proBNP detects antibody epitope peptide section,
Described connection peptides is the small peptide that 4～10 hydrophilic amino acid residues form, the aminoacid sequence of described connection peptides as Seq ID No.2,3 and 8～13 arbitrary as shown in.
2. NT-proBNP calibration object according to claim 1, does not contain amino acid or the peptide sequence of easy formation corner and alpha-helix in described connection peptides.
3. NT-proBNP calibration object according to claim 1 and 2, the NT-proBNP capture antibody epitope peptide section aminoacid sequence of described NT-proBNP calibration object is as shown in Seq ID No.17.
4. NT-proBNP calibration object according to claim 3, the NT-proBNP of described NT-proBNP calibration object detects antibody epitope peptide section aminoacid sequence as shown in Seq ID No.16.
5. NT-proBNP calibration object according to claim 4, as shown in its aminoacid sequence is as arbitrary in Seq ID No.18～25.
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| EP2403964B1 (en) | 2009-03-02 | 2021-09-08 | Massachusetts Institute of Technology | Methods and products for in vivo enzyme profiling |
| US10006916B2 (en) | 2011-03-15 | 2018-06-26 | Massachusetts Institute Of Technology | Multiplexed detection with isotope-coded reporters |
| CA2914754A1 (en) | 2013-06-07 | 2014-12-11 | Massachusetts Institute Of Technology | Affinity-based detection of ligand-encoded synthetic biomarkers |
| US11448643B2 (en) | 2016-04-08 | 2022-09-20 | Massachusetts Institute Of Technology | Methods to specifically profile protease activity at lymph nodes |
| US11428689B2 (en) | 2016-05-05 | 2022-08-30 | Massachusetts Institute Of Technology | Methods and uses for remotely triggered protease activity measurements |
| US11519905B2 (en) | 2017-04-07 | 2022-12-06 | Massachusetts Institute Of Technology | Methods to spatially profile protease activity in tissue and sections |
| CN107478848B (en) * | 2017-08-23 | 2019-04-16 | 广州瑞博奥生物科技有限公司 | The kit and preparation method thereof of quantitative detection people NT-proBNP |
| WO2019173332A1 (en) | 2018-03-05 | 2019-09-12 | Massachusetts Institute Of Technology | Inhalable nanosensors with volatile reporters and uses thereof |
| EP3911753A1 (en) | 2019-01-17 | 2021-11-24 | Massachusetts Institute of Technology | Sensors for detecting and imaging of cancer metastasis |
| CN111781385B (en) * | 2020-08-19 | 2023-07-18 | 武汉生之源生物科技股份有限公司 | NT-proBNP detection kit and preparation method thereof |
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