CN103012595B - Stable NT-probBNP calibrator and application thereof - Google Patents

Stable NT-probBNP calibrator and application thereof Download PDF

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CN103012595B
CN103012595B CN 201210548527 CN201210548527A CN103012595B CN 103012595 B CN103012595 B CN 103012595B CN 201210548527 CN201210548527 CN 201210548527 CN 201210548527 A CN201210548527 A CN 201210548527A CN 103012595 B CN103012595 B CN 103012595B
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probnp
nt
calibrator
peptide
amino acid
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CN103012595A (en )
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肖智
焦守恕
李全
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同昕生物技术(北京)有限公司
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Abstract

本发明提供一种稳定的NT-proBNP校准品及应用,属于生物检测试剂。 The present invention provides a stable calibrator and NT-proBNP application belongs biological detection reagent. 其特征在于:所述NT-proBNP校准品为多肽,其线性结构为:NT-proBNP捕获抗体表位肽段-连接肽-NT-proBNP检测抗体表位肽段。 Wherein: said NT-proBNP calibrator is a polypeptide which is a linear structure: NT-proBNP capture antibody epitope peptide - connecting peptide -NT-proBNP epitope peptide segment detection antibody. 基于该结构,发明人提供了一序列经试验验证具有较好稳定性、溶解性的校准品。 Based on this structure, the inventors provide a test verification sequence by having better stability, solubility calibrator. 可作为检测NT-proBNP和proBNP蛋白的试剂盒中的校准品。 As NT-proBNP and proBNP detection kit protein calibrator.

Description

稳定的NT-proBNP校准品及应用 Stable NT-proBNP calibrators and Applications

技术领域 FIELD

[0001] 本发明涉及医学检测试剂,特别是一种ΝΤ-ρι.οΒΝΡ校准品及其制备方法与应用。 [0001] The present invention relates to medical testing reagent, particularly a ΝΤ-ρι.οΒΝΡ calibrator preparation method and application.

技术背景 technical background

[0002] 1988年,日本学者Sudoh首次从猪脑内分离得到一种具有强力的利钠、利尿、扩血管和降压作用的多肽,命名为脑钠肽或称钠尿肽(Brain Natriuretic Peptide, BNP)。 [0002] In 1988, Japanese scholar Sudoh first isolated polypeptide potent natriuretic, diuretic, vasodilator and antihypertensive effects having from pig brain, brain natriuretic peptide or named, said natriuretic peptide (Brain Natriuretic Peptide, BNP). 此后的研究表明,在生物进化的过程中逐渐发展产生包括BNP在内的一组多肽ANP、BNP、CNP、DNP、VNP等,称为利钠肽家族,其功能是维持循环系统的容量、渗透压和压力的稳态。 After studies show progressive development to generate a set including BNP polypeptide comprising ANP, BNP, CNP, DNP, VNP or the like in the process of biological evolution, it referred natriuretic peptide family, whose function is to maintain the capacity of the circulatory system, the permeate pressure and pressure homeostasis. BNP主要存在于心室隔膜颗粒中,其分泌依赖于心室的容积扩张和压力负荷增加。 BNP membrane particles primarily in the ventricle, which is dependent on the secretion capacity ventricular dilation and pressure overload increases. 作为心功能紊乱最敏感和最特异的指标,BNP具有重要的临床意义。 Cardiac dysfunction as the most sensitive and the most specific indicators, BNP has important clinical significance. 最早在ESC《慢性心力衰竭指南(2001)》,继而在美国ACC/AHA《慢性心力衰竭指南(2005)》中推荐将血液BNP水平测定作为心力衰竭的诊断和预后指标。 In the first ESC "chronic heart failure guidelines (2001)," and then recommended the assay as a diagnostic and prognostic indicator of heart failure in the "Guide for chronic heart failure (2005)" American ACC / AHA blood levels of BNP. 2008年ESC《急性和慢性心力衰竭指南》和2009年AHA《心力衰竭指南》对此作了进一步的推荐。 In 2008 ESC "acute and chronic heart failure guidelines" AHA ​​"Heart Failure Guidelines" and 2009 This is further recommended.

[0003] 当心肌细胞受刺激后,会产生含134个氨基酸的B型利钠肽原前体(pre-proBNP),随后在相关酶的作用下切掉N端的信号肽序列,形成含108个氨基酸的BNP前体(proBNP),后者在内切酶的作用下裂解为含有76个氨基酸、无生物活性的N端产物N末端B型利钠肽原(NT-proBNP)和含有32个氨基酸有活性的C端产物B型利钠肽(BNP)。 [0003] When cardiomyocytes stimulated, will produce B-type natriuretic peptide contains 134 amino acids of the original precursor (pre-proBNP), and then cut off the signal peptide sequence of the N-terminus by the action of enzymes form containing 108 amino acids the BNP precursor (proBNP), the latter including the action of Dicer cleavage containing 76 amino acids, N-terminal end of the B product N-type natriuretic peptide biologically inactive original (NT-proBNP) has 32 amino acids and comprising activity of the C-terminal B-type natriuretic peptide product (BNP). BNP的清除主要通过与BNP清除受体结合,而NT-proBNP则主要由肾小球滤过,因此,其血浓度受肾功能影响大于BNP。 BNP is cleared by clearing the major receptor binding BNP, NT-proBNP but mainly by the glomerular filtration, and therefore, the blood concentration of BNP greater than by renal impact. BNP半衰期短(22min),体外稳定性差,而NT-proBNP半衰期较长(120min),体外稳定性强,在心力衰竭患者中的浓度较BNP高,在有些情况下更有利于心力衰竭的诊断。 BNP short half-life (22min), a difference in vitro stability, longer half-life and NT-proBNP (120min), strong in vitro stability, in heart failure patients the concentration of BNP higher than, in some cases, more favorable diagnosis of heart failure. 美国在2004年和2008年分别发表了BNP临床应用专家共识和国际NT-proBNP专家共识,系统阐述了BNP和NT-proBNP的生物学和临床应用。 The United States in 2004 and 2008 were published expert consensus and clinical application of BNP NT-proBNP international expert consensus, the system describes the biology and clinical applications of BNP and NT-proBNP.

[0004] BNP和NT-proBNP检测在21世纪初先后进入我国,10年来已经被各级医院和医师广泛用于临床实践,成为心血管病尤其是心`力衰竭诊断和评估十分有用的生物标志物。 [0004] BNP and NT-proBNP is detected in the early 21st century has entered the country, 10 years, all levels of hospitals and physicians has been widely used in clinical practice, cardiovascular disease, especially heart become `force failure diagnosis and evaluation of useful biomarkers thereof. 我国2007年《慢性心力衰竭诊断治疗指南》和2010年《急性心力衰竭诊断治疗指南》也推荐将NT-proBNP和BNP用于心力衰竭的诊断和预后判断。 Our 2007 "Guidelines for Patients with chronic heart failure," and the 2010 "Guidelines for Patients with acute heart failure" is also recommended to NT-proBNP and BNP for the diagnosis and prognosis of heart failure. 但是在试剂盒开发和使用过程中,定标液的稳定性较差,尤其对于基于板式酶免平台的诊断试剂,由于其每次测定均需同时定标,所以对试剂盒校准品的稳定性要求较高。 However, the development of kits and in use, poor stability of calibrators, in particular for diagnostic agents based on ELISA plate platform that measures are required at the same time since each calibration, the calibration stability of the product kit demanding.

[0005] NT-proBNP是由76个氨基酸组成的蛋白,生产商在试剂盒和开发过程中,多采用重组蛋白作为校准品。 [0005] NT-proBNP is a protein of 76 amino acids, the kit manufacturer and the development process, the use of recombinant protein as calibrators. 但是大量的研究发现,由于原核重组表达的蛋白缺乏糖基化,其稳定性较天然蛋白更差。 However, a large number of studies found that, due to the expression of a prokaryotic lacks glycosylation, worse stability than the native protein. 目前没有见到稳定性优良的ΝΤ-ρι.οΒΝΡ校准品。 There is no superior stability seen ΝΤ-ρι.οΒΝΡ calibrator.

发明内容 SUMMARY

[0006] 本发明根据上述领域存在的需求和空白,提供一种稳定的ΝΤ-ρι.οΒΝΡ校准品,技术方案如下: [0006] According to the present invention, the presence of gaps and areas of need, to provide a stable calibrator ΝΤ-ρι.οΒΝΡ, technical solutions are as follows:

[0007] 一种NT-proBNP校准品,其特征在于:所述NT-proBNP校准品为多肽,其线性结构为:ΝΤ-ρι.οΒΝΡ捕获抗体表位肽段一连接肽一NT-proBNP检测抗体表位肽段。 [0007] An NT-proBNP calibrators, wherein: said NT-proBNP as a calibrator polypeptide, its linear structure: ΝΤ-ρι.οΒΝΡ capture antibody epitope peptide segments a connecting peptide NT-proBNP a detection antibody epitope peptide.

[0008] 所述线性结构为从氨基端到羧基端为:ΝΤ-ρι.οΒΝΡ捕获抗体表位肽段一连接肽一NT-proBNP检测抗体表位肽段。 [0008] The linear structures from amino terminus to carboxy terminus is to: ΝΤ-ρι.οΒΝΡ capture antibody epitope peptide segments a connecting peptide NT-proBNP a detection antibody epitope peptide.

[0009] 所述连接肽为4~10个亲水氨基酸残基构成的短肽。 [0009] The linker peptide of 4 to 10 hydrophilic amino acid residues of the peptide.

[0010] 所述连接肽中不含有易形成转角和α -螺旋的氨基酸或多肽序列。 [0010] The connecting peptide does not contain easy to form and angle α - amino acid or polypeptide sequence helix.

[0011] 所述连接肽段的氨基酸序列如Seq ID N0.2、3和8~13任一所示。 [0011] The connector and amino acid sequence as Seq ID N0.2,3 Claims 8 to 13 and a FIG.

[0012] 所述NT-proBNP校准品的NT-proBNP捕获抗体表位肽段氨基酸序列如Seq IDN0.15、N0.17 任一所示。 [0012] The calibrator of NT-proBNP NT-proBNP amino acid sequence of the capture antibody epitope peptide as Seq IDN0.15, any of a N0.17 FIG.

[0013] 所述NT-proBNP校准品的NT-proBNP检测抗体表位肽段氨基酸序列如Seq IDN0.14、N0.16 任一所不。 [0013] The amino acid sequences of NT-proBNP detection antibody epitope peptide NT-proBNP as a calibrator Seq IDN0.14, not any one N0.16.

[0014] 所述NT-proBNP校准品,其氨基酸序列如Seq ID N0.18~25任一所示。 [0014] The NT-proBNP calibrator, such as the amino acid sequence of any of Seq ID N0.18 ~ 25 a in FIG.

[0015] 上述任一NT-proBNP校准品在检测NT-proBNP和proBNP蛋白的试剂盒中的作为校准品的用途。 [0015] The use of any preceding calibrators NT-proBNP NT-proBNP and proBNP detection kit protein as calibrator.

[0016] 本发明提供一种人工设计的ΝΤ-ρroΒΝΡ校准品,其包含一对通过人工设计的连接肽连接的稳定的NT-proBNP抗原表位。 [0016] The present invention provides an artificially designed ΝΤ-ρroΒΝΡ calibrator which comprises a pair of stabilizing the NT-proBNP epitope peptides linked by a linker artificially designed.

[0017] 为了提高本发明的ΝΤ-ρroΒΝΡ校准品的稳定性,本发明中设计的连接肽为4~10个氨基酸组成。 [0017] In order to improve the stability of the calibrators ΝΤ-ρroΒΝΡ the present invention, a connecting peptide of the present invention is designed for 4 to 10 amino acids.

[0018] 为了防止多肽二聚体形成、线性序列的空间折叠及合成多肽的溶解性,试验进一步优化了linker的大小、氨基酸序列组成及其空间特性,设计获得如表1中Seq ID N0.2所示的连接肽,实验证明采用这些连接肽的本发明获得的校准品具有更好的溶解性。 [0018] In order to prevent the polypeptide-dimer formation, folding and space linear sequence of synthetic polypeptide solubility, the size of the test further optimize the linker, and the amino acid sequence of the spatial characteristics, the design is obtained as shown in Table 1 Seq ID N0.2 connecting peptide shown, these experiments show that use of the calibrator of the present invention, a connecting peptide obtained has better solubility.

[0019] 为防止二聚体的形成,linker的设计过程中剔除了半胱氨酸的选择;为了保持多肽的线性,linker选择剔除了脯氨酸等易形成转角的氨基酸,根据此设计出如。 [0019] To prevent the formation of dimers, Linker design process removed the selected cysteine; linear polypeptide in order to maintain, eliminate selected Linker amino acid proline is easy to form the corner, according to this design as . 如表1中Seq ID N0.2所示的连接肽,实验证明采用这些连接肽的本发明获得的校准品具有更好的溶解性和稳定性。 The connecting peptide shown in Table 1 Seq ID N0.2, these experiments show that use of the present invention, a connecting peptide obtained calibrator has better solubility and stability.

[0020] 据文献报道,NT-proBNP中表位EPl (5-12)和表位EP2 (67-76)较为稳定且无糖基化位点,这样不仅可以防止表位降解,而且可以保持制备的表位与天然的表位相一致,因此本发明的一个实施例中,选择表位EPl和表位EP2作为定量分析的抗体识别表位,如表2中SeqID N0.14、N0.15。 [0020] According to the literature, NT-proBNP epitope EPl (5-12) epitope and EP2 (67-76) is more stable and aglycosylated sites, which can not only prevent degradation of the epitope, but may remain prepared epitopes coincides with the natural epitope, therefore an embodiment of the present invention, the selected epitope EPl and EP2 epitope as the antibody recognizes an epitope of quantitative analysis, as shown in table 2 SeqID N0.14, N0.15.

[0021 ] 本发明的一个优选实施例中,为了降低I inker及其他因素对抗体-表位识别的影响,试验优化选择表位ΕΡt (4-13)、表位EP2' (66-76)作为定量分析抗体识别表位,如表 [0021] In a preferred embodiment of the present invention, in order to reduce I inker antibodies and other factors - Effect of epitope recognition, epitope Optimization of test ΕΡt (4-13), an epitope EP2 '(66-76) as quantitative analysis of epitope recognized by an antibody, as shown in table

2 中SeqID N0.16、N0.17。 2 SeqID N0.16, N0.17.

[0022] 综上,本发明提供的校准品,由于具有ΝΤ-ρroΒΝΡ的表位,所以可以与其相应的抗体结合。 [0022] In summary, the present invention provides a calibrator, since ΝΤ-ρroΒΝΡ epitope can be bound to its corresponding antibody. 同时,由于剔除了ΝΤ-ρroΒΝΡ不稳定的氨基酸序列,选择较为稳定的表位,并用稳定的多肽linker将两个表位连接,所以由此制备的校准品更加稳定。 Meanwhile, since the amino acid sequence removed ΝΤ-ρroΒΝΡ unstable, stable epitope selection, and with a stable polypeptide linker connecting the two epitopes, whereby it is more stable calibrators prepared.

[0023] 本发明提供的校准品可用于NT-proBNP和proBNP不稳定蛋白试剂盒的校准品。 [0023] The present invention may be used to provide calibrator calibrator NT-proBNP and proBNP labile protein kit.

[0024] 本发明提供的校准品由于都是短肽,优选采用人工合成的方法制备。 [0024] The present invention provides a calibrator because short peptides are preferably prepared using synthetic methods.

[0025] 术语解释: [0025] Explanation of terms:

[0026] NT-proBNP捕获抗体表位:指NT-proBNP中,抗NT-proBNP抗体对其起捕获作用的区域,是由一段氨基酸序列构成。 [0026] NT-proBNP capture antibody epitope: refers to the NT-proBNP, the anti-NT-proBNP capture antibody for its role play area is constituted by an amino acid sequence. [0027] NT-proBNP检测抗体表位:指NT-proBNP中,抗NT-proBNP抗体对其起检测作用的区域,是由一段氨基酸序列构成。 [0027] NT-proBNP epitope detected: means NT-proBNP, the anti-NT-proBNP antibody for its action is detected, a region is constituted by an amino acid sequence.

[0028] 连接肽,指人工设计的短肽,本发明中主要起到提高由ΝΤ-ρι.οΒΝΡ捕获抗体表位和ΝΤ-ρι.οΒΝΡ检测抗体表位构成的校准品的稳定性,实现两个人工合成的表位的空间结构使其发挥对抗体的捕获及检测功能。 [0028] connecting peptide, refers to a short peptide artificially designed, in the present invention mainly plays stability calibrator antibody epitopes and ΝΤ-ρι.οΒΝΡ detection antibody epitope constituted improve captured by ΝΤ-ρι.οΒΝΡ, achieve two artificial synthetic epitope spatial structure so as to play the antibody capture and detection.

[0029] P / N:即阳性/阴性,指阳性(P, positive)读值与阴性(N, negative)读值的比值,此比值越高代表试剂盒区分阴阳性的能力越强。 [0029] P / N: i.e. the stronger the positive / negative, positive refers to the ratio (P, positive) reading the negative (N, negative) the value read, the higher this ratio representative of a kit of the ability to distinguish between Yin and Yang.

附图说明 BRIEF DESCRIPTION

[0030] 图1.标准曲线, [0030] FIG 1. Standard curve

[0031] 图2.方法学比较结果。 [0031] Figure 2. Comparison of the results of the method.

具体实施方式 detailed description

[0032] 以下通过具体实施方式说明本发明的技术方案及其效果。 [0032] The following description of the invention and the technical effect of the specific embodiments.

[0033] 试验所需溶液和试剂: [0033] The test solutions and reagents required:

[0034] 1.1OmM PB: [0034] 1.1OmM PB:

[0035] 称取称取0.27g磷酸二氢钾(KH2P04)(国药,10017608)、1.42g磷酸氢二钠(Na2HP04)(国药,100203008)、ImL Proclin300 (Sigma,48914-U)于900mL 超纯水中,完全溶解后调节PH至7.4,然后用容量瓶定容至1L。 [0035] Weigh weighed 0.27g potassium dihydrogen phosphate (KH2P04) (Sinopharm, 10017608), 1.42g disodium hydrogen phosphate (Na2HP04) (Sinopharm, 100203008), ImL Proclin300 (Sigma, 48914-U) in 900mL ultrapure water and completely dissolved to adjust the PH to 7.4, and then the flask volume to 1L.

[0036] 2.1OmM PBS:`[0037]称取 0.27g 磷酸二氢钾(KH2P04)(国药,10017608)、1.42g 磷酸氢二钠(Na2HP04)(国药,100203008),8g 氯化钠(NaCl)(国药,10019308)、0.2g 氯化钾(KCl)(国药,10016308)、lmL Proclin300 (Sigma,48914-U)于900mL 超纯水中,完全溶解后调节pH 至 [0036] 2.1OmM PBS: `[0037] Weigh 0.27g potassium dihydrogen phosphate (KH2P04) (Sinopharm, 10017608), 1.42g disodium hydrogen phosphate (Na2HP04) (Sinopharm, 100203008), 8g of sodium chloride (NaCl) (Medicines, 10019308), 0.2g potassium chloride (KCI) (Sinopharm, 10016308), lmL Proclin300 (Sigma, 48914-U) in 900mL ultrapure water. after complete dissolution pH was adjusted to

7.4,然后用容量瓶定容至1L。 7.4, then the flask volume to 1L.

[0038] 3.0.5%BSA IOmM PBS: [0038] 3.0.5% BSA IOmM PBS:

[0039]含有 0.5% (ff/V) BSA 牛血清白蛋白(amresco,0332)的IOmM PBS 溶液。 [0039] containing 0.5% (ff / V) BSA Bovine Serum Albumin (amresco, 0332) of IOmM PBS solution.

[0040] 4.洗涤液: [0040] 4. The washing solution:

[0041]含有 0.05% (V/V) Tween-20 (sigma,44112)的IOmM PBS 溶液。 [0041] containing 0.05% (V / V) Tween-20 (sigma, 44112) of IOmM PBS solution.

[0042] 5.合成多肽:本发明中表3所列的多肽均由上海生工生物合成。 [0042] The synthetic polypeptide: a polypeptide listed in Table 3 of the present invention is synthesized by Shanghai Sangon.

[0043] 6.NT-proBNP 抗体4NT1-29D12、4NT1-24E11 均购于Hytest。 [0043] 6.NT-proBNP antibodies were purchased from 4NT1-29D12,4NT1-24E11 Hytest.

[0044] 7.重组NT-proBNP (1-76 氨基酸,8NT2)蛋白购于Hytest。 [0044] 7. The recombinant NT-proBNP (1-76 amino acids, 8NT2) was purchased in Hytest.

[0045] 8.底物A 和底物B (Thermo, 34080) [0045] 8. The substrate A and the substrate B (Thermo, 34080)

[0046] 表1本发明中设计的连接肽 [0046] Table 1, the present invention is designed linker peptide

[0047] [0047]

序列号 I连接肽氨基酸序列 I linker peptide SEQ ID NO amino acid sequence

Seq ID N0.1 STQNG Seq ID N0.1 STQNG

Seq ID N0.2 QNQQASNQNQ Seq ID N0.2 QNQQASNQNQ

Figure CN103012595BD00061

[0048] 表2本发明中选用的多肽表位 [0048] Table 2, the present invention is selected polypeptide epitopes

[0049] [0049]

Figure CN103012595BD00062

[0050] 表3本发明优选出的NT-proBNP校准品的氨基酸序列 [0050] Table 3. The amino acid sequences of the present invention preferably NT-proBNP calibrator

[0051] [0051]

Figure CN103012595BD00063
Figure CN103012595BD00071

[0052] 实施例1、NT-ProBNP抗体的选择 1, NT-ProBNP selection of antibodies according to [0052] Embodiment

[0053] 据文献报道,NT-p roBNP抗原N端和C端的序列较为稳定,因此,选择Hytest公司提供的4NT1-29D12 (氨基酸序列5_12)、4NT1_24E11 (氨基酸序列67-76)作为双抗夹心法的抗体对。 [0053] According to the literature, NT-p roBNP antigen sequence N-terminal and C-terminal is more stable, and therefore, choose Hytest provided by 4NT1-29D12 (amino acid sequence 5_12), 4NT1_24E11 (amino acid sequence 67-76) was used as the sandwich method the antibody pairs.

[0054] 实施例2、合成多肽表位的选择 [0054] Example 2, a synthetic polypeptide selected epitope

[0055]将合成 EP1-STQNG-EP2、EP1' -STQNG-EP2'、重组NT-proBNP 抗原(Hytest,8NT2)配制高值(H,15000pg/ml )、中值(M,5000pg/ml )、低值(L,100pg/ml)梯度样本,测定方法同实施例6,并计算P/N。 [0055] The synthetic EP1-STQNG-EP2, EP1 '-STQNG-EP2', recombinant NT-proBNP antigen (Hytest, 8NT2) formulating a high value (H, 15000pg / ml), the value (M, 5000pg / ml), low (L, 100pg / ml) gradient sample measurement method described in Example 6, and calculates the P / N.

[0056] 试验结果显示,EP1'-STQNG-EP2'合成多肽表位与重组NT-proBNP抗原的P/N (H/N、M/N、L/N)均高于EP1-STQNG-EP2合成多肽表位,表明为获得与重组抗原一致的反应特性,合成多肽抗原表位应较抗体的识别表位的N端和C端至少各多含一个氨基酸。 [0056] The test results show, EP1'-STQNG-EP2 'P synthetic polypeptide epitope recombinant NT-proBNP antigen / N (H / N, M / N, L / N) were higher than EP1-STQNG-EP2 Synthesis of polypeptide epitope shown to be consistent with the response characteristics recombinant antigens, synthetic peptides should each epitope containing more than one amino acid N-terminal epitope recognized by the antibody and the C-terminus at least.

[0057] 表4不同校准品P/N差异 [0057] Table 4 calibrators different P / N difference

[0058] [0058]

Figure CN103012595BD00072

[0059] 实施例3、linker长度选择 [0059] Example 3, linker length selection

[0060] 用合成的多肽抗原(ΕΡ1' -linker-EP2')配制高值(H,15000pg/ml)、中值(M,5000pg/ml)、低值(L,100pg/ml)梯度样本,测定方法同实施例6,并计算P/N。 [0060] The preparation of high value (H, 15000pg / ml) with a synthetic peptide antigen (ΕΡ1 '-linker-EP2'), the median (M, 5000pg / ml), low (L, 100pg / ml) gradient sample, measurement method in Example 6, and calculates the P / N.

[0061] 试验结果显示,Linker长度大于4个氨基酸序列的合成多肽表位的P/N (H/N、M/N、L/N)明显高于其它Linker的多肽,表明Linker的长度对影响表位与抗体的结合,其可能的原因是空间位阻效应,因此试验建议选择含4-10氨基酸的linker的多肽序列作为试剂盒的校准品。 [0061] The test results show, Linker length greater than four synthetic polypeptide epitope amino acid sequence P / N (H / N, M / N, L / N) was significantly higher than that of other polypeptide Linker, indicating the influence of the length of the Linker binding epitope to the antibody, which may cause a steric effect, therefore we recommended to choose the test polypeptide linker containing 4-10 amino acid sequence as a calibrator kit.

[0062] 表5 linker氨基酸序列 [0062] Table 5 linker amino acid sequence

[0063] [0063]

Figure CN103012595BD00073

[0064] 注:non-无linker,即将EPl'与EP2'直接连接。 [0064] Note: non- no linker, coming EPl 'and EP2' direct connection.

[0065] 表6不同linker制备的校准品P/N差异 Calibrator P [0065] 6 prepared different tables linker / N difference

[0066] [0066]

Figure CN103012595BD00081

[0067] 注:non_无linker,即将EPl'与EP2'直接连接。 [0067] Note: non_ no linker, EPl coming directly connected 'to the EP2'.

[0068] 实施例4、linker氨基酸组成 [0068] 4, linker amino acid composition Example

[0069] 用合成的多肽抗原(ΕΡl' -linker_EP2')配制高值(H,15000pg/ml)、中值(M,5000pg/ml)、低值(L,100pg/ml)梯度样本,测定方法同实施例6,并计算P/N。 [0069] The preparation of high value (H, 15000pg / ml) with a synthetic peptide antigen (ΕΡl '-linker_EP2'), (L, 100pg / ml) Sample gradient method of measuring low value (M, 5000pg / ml), in Example 6, and calculates the P / N.

[0070] 试验结果显示,由亲水氨基酸组成Linker 10-1 (SEQ ID N0.2)的合成多肽表位的P/N (H/N、M/N、L/N)高于由疏水氨基酸组成Linker 10-2 (SEQ ID N0.3)的多肽,表明Linker的氨基酸组成影响表位与抗体的结合,其可能的原因是疏水氨基酸含量过高影响合成多肽的溶解性。 [0070] The test results showed that a synthetic polypeptide composed of hydrophilic amino acid epitope Linker 10-1 (SEQ ID N0.2) of the P / N (H / N, M / N, L / N) is higher than the hydrophobic amino acids composition Linker 10-2 (SEQ ID N0.3) polypeptide Linker amino acid composition indicated that the epitope binding of the antibody, which may be the reason for the solubility of a hydrophobic amino acid content is too high for effective synthesis of polypeptides. 此外,由于Linker 10-3 (SEQ ID N0.4)的合成多肽中含有大量的折叠因素,因此其P/N明显低于含Linkerl0-l、10-2的合成多肽表位。 Further, since the Linker 10-3 (SEQ ID N0.4) of the synthetic polypeptide contains a large number of folding elements, so the P / N was significantly lower than that containing Linkerl0-l, 10-2 epitope synthetic polypeptide. 因此试验选择含亲水性氨基酸较高的、空间的linker的多肽序列作为试剂盒的校准品。 Thus hydrophilic amino acid containing test selected higher, polypeptide sequence as a linker space calibrator kit.

[0071] 表7 linker氨基酸序列 [0071] Table 7 linker amino acid sequence

[0072] [0072]

Figure CN103012595BD00091
Figure CN103012595BD00101

[0074] 注:a.DNAman软件二级结构预测。 [0074] Note: a.DNAman secondary structure prediction software.

[0075] 表8 linker氨基酸序列 [0075] Table 8 linker amino acid sequence

Figure CN103012595BD00102

[0077] 实施例5、校准品稳定性测定 5, the stability of calibrators measured Example [0077] Embodiment

[0078]用合成的多肽抗原(ΕΡ1'-QNQQASNQNQ-EP2' )和重组NT-proBNP (Hytest, 8NT2)抗原配制高值(H, 15000pg/ml)、中值(M,5000pg/ml)、低值(L, 100pg/ml)梯度样本,然后分别置于4°C和37°C放置6d。 [0078] with a synthetic peptide antigen (ΕΡ1'-QNQQASNQNQ-EP2 ') and recombinant NT-proBNP (Hytest, 8NT2) antigen preparation of high value (H, 15000pg / ml), the value (M, 5000pg / ml), low value (L, 100pg / ml) gradient samples were then placed in 4 ° C and 37 ° C is placed 6d.

[0079] 测定方法同实施例6,计算P/N。 [0079] The measurement method described in Example 6, calculated P / N.

[0080] 结果表明,合成的多肽抗原在37°C放置3d、6d的稳定性(37°C /4°C)显著高于重组抗原(见表9)。 [0080] The results showed that the synthesized polypeptide antigen shelf stability 3d, 6d of (37 ° C / 4 ° C) was significantly higher than the recombinant antigen (see Table 9) at 37 ° C.

[0081 ] 表9校准品稳定性-1 [0081] Table 9 the stability of calibrators -1

Figure CN103012595BD00111

[0083] 表10校准品稳定性-2 [0083] Table 10 Stability of calibrators -2

Figure CN103012595BD00112

[0085] 实施例6、NT-proBNP检测试剂盒(化学发光法)制备: [0085] Example 6, NT-proBNP assay kit (chemiluminescent) was prepared:

[0086] I)包被:配制I μ g/mL 24E11抗体(hytest,4NT1) IOmM PB溶液,加入微孔发光板中,100 μ L/ 孔,37°C孵育2h ; [0086] I) coating: formulation I μ g / mL 24E11 antibody (hytest, 4NT1) IOmM PB was added to the micro-well luminometer plate, 100 μ L / hole, 37 ° C incubation 2H;

[0087] 2)封闭:将微孔板中包被液甩干,加入5%BSA IOmM PB溶液,200 μ L/孔,37°C,孵育Ih; [0087] 2) Blocking: the coating liquid drying microtiter plates, was added 5% BSA IOmM PB solution, 200 μ L / hole, 37 ° C, incubated Ih is;

[0088] 3)抗原:将微孔板中封闭液甩干,加入100 μ L/孔抗原,37°C,孵育30min ; [0088] 3) Antigen: The blocking solution spinning microplate was added 100 μ L / well of the antigen, 37 ° C, incubated for 30 min;

[0089] 4)酶标抗体:将微孔板用洗涤液洗涤3次并甩干,加入100 μ I/孔HRP标记的29D12 抗体(Hytest,4ΝΤ1) (HRP-29D12),37°C,孵育30min ; [0089] 4) enzyme-labeled antibody: The microplate was washed 3 times with washing solution and drying, 29D12 antibody (Hytest, 4ΝΤ1) (HRP-29D12) was added 100 μ I / hole HRP-labeled, 37 ° C, incubation 30min;

[0090] 5)底物:将微孔板用洗涤液洗涤5次并甩干,分别加入50 μ L/孔底物A和底物B(Thermo, 34080),避光反应5min,测定发光值。 [0090] 5) Substrate: The microplate was washed five times with a washing solution and drying, were added 50 μ L / well bottom substrate A and B (Thermo, 34080), the dark reaction 5min, luminescence value is measured .

[0091] 6)发光强度与样本中抗原的浓度成线性关系(四参数拟合)。 [0091] 6) the concentration of the antigen in the sample emission intensity is a linear relationship (four parameter fit).

[0092] 实施例7、校准品临床检测应用: [0092] Example 7, calibrators clinical testing applications:

[0093] 收集178例临床样本(年龄在18-75岁和75岁以上人群,包括正常人和疑似心力衰竭病人)的血清。 [0093] Clinical samples collected 178 cases (aged 18-75 years and over 75 years of age, including normal subjects and patients with suspected heart failure) in serum. 选用Roche脑利钠肽前体诊断试剂盒(电化学发光法)(国食药监械(进)字2011第2402335号)进行方法学比较。 Comparison of brain natriuretic peptide selected Roche diagnostic kits precursor (electrochemiluminescence) (SFDA (forward) word 2011 No. 2402335) method.

[0094] 测定步骤: [0094] Determination of the steps of:

[0095] I)用制备的合成校准品配制15000pg/ml、5000pg/ml、500pg/ml、200pg/ml、IOOpg/ml校准点,作标准曲线;[0096] 2)测定方法同实施例6.[0097] 试剂盒标准曲线见图1,相关系数r2=0.99 [0095] I) formulated with synthetic calibrators prepared 15000pg / ml, 5000pg / ml, 500pg / ml, 200pg / ml, IOOpg / ml calibration point, a standard curve; [0096] 2) Measuring method same as Example 6. [0097] The kit of the standard curve shown in Figure 1, the correlation coefficient r2 = 0.99

[0098] 试剂盒测定结果(附表1)与Roche脑利钠肽前体诊断试剂盒(电化学发光法)进行方法学比较,相关系数r2=0.9188。 [0098] The measurement results reagent cartridge (Table 1) and brain natriuretic peptide precursor Roche diagnostic kits (electrochemiluminescence) methodology before comparison, correlation coefficient r2 = 0.9188. 见图2 Figure 2

[0099] 附表1:临床样本测定结果 [0099] Table 1: Determination results of clinical specimens

[0100] [0100]

Figure CN103012595BD00121

[0101] [0101]

Figure CN103012595BD00131
Figure CN103012595BD00141
Figure CN103012595BD00151
Figure CN103012595BD00161

Claims (5)

  1. 1.一种NT-proBNP校准品,其特征在于:所述NT-proBNP校准品为多肽,其线性结构为:ΝΤ-ρι.οΒΝΡ捕获抗体表位肽段一连接肽一NT-proBNP检测抗体表位肽段, 所述连接肽为4~10个亲水氨基酸残基构成的短肽,所述连接肽的氨基酸序列如SeqID N0.2、3和8~13任一所示。 An NT-proBNP calibrators, wherein: said NT-proBNP as a calibrator polypeptide, its linear structure: ΝΤ-ρι.οΒΝΡ capture antibody epitope peptide segments a connecting peptide NT-proBNP a detection antibody table bit peptide, the linker peptide is a peptide having 4 to 10 hydrophilic amino acid residues of the amino acid sequence of the peptide, such as the connection SeqID N0.2,3 Claims 8 to 13 and a FIG.
  2. 2.根据权利要求1所述的ΝΤ-ρι.οΒΝΡ校准品,所述连接肽中不含有易形成转角和α-螺旋的氨基酸或多肽序列。 According to claim ΝΤ-ρι.οΒΝΡ calibrator of claim 1, said connecting peptide comprising an amino acid or polypeptide sequences is not easy to form a corner and α- helices.
  3. 3.根据权利要求1或2所述的NT-proBNP校准品,所述NT-proBNP校准品的NT-proBNP捕获抗体表位肽段氨基酸序列如Seq ID N0.17所示。 The NT-proBNP or calibrator according to claim 1, said calibrator NT-proBNP NT-proBNP capture antibody epitope peptide segment amino acid sequence table as shown in Seq ID N0.17.
  4. 4.根据权利要求3所述的NT-proBNP校准品,所述NT-proBNP校准品的NT-proBNP检测抗体表位肽段氨基酸序列如Seq ID N0.16所示。 The NT-proBNP calibrator according to claim 3, NT-proBNP epitope detection antibody and amino acid sequence of the NT-proBNP as a calibrator as shown in Seq ID N0.16.
  5. 5.根据权利要求4 所述的NT-proBNP校准品,其氨基酸序列如Seq ID N0.18~25任一所示。 According to claim NT-proBNP calibrator 4, the amino acid sequence as any of Seq ID N0.18 ~ 25 a in FIG.
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US7524635B2 (en) 2003-04-17 2009-04-28 Biosite Incorporated Methods and compositions for measuring natriuretic peptides and uses thereof
CN102608335A (en) 2012-04-19 2012-07-25 协和生物制药(天津)有限公司 Method for preparing NT-proBNP time-resolved fluoroimmunoassay kit

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CN1678635A (en) 2002-08-07 2005-10-05 比奥-拉德巴斯德公司 Specific antibodies proBNP(1-108) for diagnosing heart failure
US7524635B2 (en) 2003-04-17 2009-04-28 Biosite Incorporated Methods and compositions for measuring natriuretic peptides and uses thereof
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