WO2014121715A1 - 25羟维生素d检测试剂盒及其制备方法 - Google Patents
25羟维生素d检测试剂盒及其制备方法 Download PDFInfo
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- WO2014121715A1 WO2014121715A1 PCT/CN2014/071605 CN2014071605W WO2014121715A1 WO 2014121715 A1 WO2014121715 A1 WO 2014121715A1 CN 2014071605 W CN2014071605 W CN 2014071605W WO 2014121715 A1 WO2014121715 A1 WO 2014121715A1
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- Prior art keywords
- hydroxyvitamin
- antigen
- buffer
- microspheres
- vitamin
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/82—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving vitamins or their receptors
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
Definitions
- the present invention relates to the field of clinical in vitro diagnosis and medical immunology; in particular, to an immunoassay reagent; and further, the present invention relates to a 25-hydroxyvitamin D detection kit and a preparation method thereof. Background technique
- Vitamin D is also called anti-caries vitamins, and can be divided into vitamin D2 and vitamin D3.
- Vitamin D2 is mostly contained in plant foods. It is synthesized from the ergosterol of plants by sunlight. Vitamin D3 can be synthesized from human skin and adipose tissue by 7-dehydrocholesterol.
- Vitamin D2 is a fat-soluble vitamin. Vitamin D from food is absorbed through the small intestine together with fat. It forms chylomicrons with the help of bile. It enters the bloodstream from the lymphatic vessels and is transported into the liver with its own synthetic vitamin D3. In the liver, 25-hydroxyvitamin 0 is formed by the action of a monooxygenase system (25-hydroxylase) in hepatocyte microsomes.
- 25-hydroxyvitamin D is converted to l,25(OH) 2 D3 in the mitochondrial ⁇ -hydroxylase system of renal proximal tubular epithelial cells, which is the largest biological action form of vitamin D and can promote intestinal calcium-binding protein. synthesis. And 25-hydroxyvitamin D is the main form of circulation in metabolism, which can reflect the body's vitamin D level.
- Vitamin D deficiency is also known as vitamin D deficiency rickets. Common in infants and young children, more common in the lack of vitamin D in the diet, or the lack of sunlight in the human body. Calcium and phosphorus metabolism disorders caused by insufficient vitamin D in the body, calcium salts can not normally deposit on the growth part of the bone, so that a chronic nutritional disease characterized by growth of bone formation lesions is called rickets. If the disease occurs in an adult, calcification of the mature bone occurs, called osteomalacia. Studies have also shown that vitamin D deficiency may increase the risk of certain cancers, cardiovascular diseases, autoimmune diseases, and infectious diseases.
- the determination methods of 25-hydroxyvitamin D mainly include enzyme-linked immunosorbent assay (such as the method and kit disclosed in CN102628872 A, and the 25-OH vitamin D (total) detection kit EIA5396 provided by DRG), and radioimmunoassay (such as Hollis).
- enzyme-linked immunosorbent assay such as the method and kit disclosed in CN102628872 A, and the 25-OH vitamin D (total) detection kit EIA5396 provided by DRG
- radioimmunoassay such as Hollis
- BW et al., 1985 liquid chromatography-mass spectrometry
- chemiluminescence eg, ⁇ et al., 2011; 25-hydroxy total vitamin D quantitative assay kit produced by Sorin (Chemiluminescence method) ).
- the degree of automation of enzyme-linked immunosorbent assay is not high, and it is greatly affected by human factors.
- the radioimmunoassay has environmental pollution problems; liquid chromatography mass spectrometry is complicated and takes a long time; while the chemiluminescence method has high sensitivity, but the detection cost is high, and a specific chemiluminometer is required, which results in a small application range.
- the invention adopts latex enhanced immunoturbidimetric method to determine the concentration of 25-hydroxyvitamin D in human samples.
- the method has low requirements on instruments and equipment, and has no problems such as environmental protection and protection of operators themselves.
- the method is simple, rapid, sensitive and reliable.
- Ordinary automatic or semi-automatic biochemical analyzers can be used. , has a large application range and practical value. Summary of the invention
- a 25 hydroxyvitamin D detecting kit comprising a first reagent, and a second reagent.
- the first reagent comprises a 25 hydroxyvitamin D antibody, a vitamin D binding protein antibody and a buffer
- the second reagent comprises microspheres and a buffer bound to the 25 hydroxyvitamin D antigen.
- the buffer in the first reagent and the second reagent is selected from the group consisting of a phosphate buffer, a glycine buffer, a MES buffer, a boric acid buffer, an acetate buffer, a 2-morpholine ethanesulfonic acid buffer. Or one or more of ammonium chloride buffer; buffer concentration of 10-500 mM.
- the buffer types in the first reagent and the second reagent may be the same or different; the buffer concentrations in the first reagent and the second reagent may be the same or different. In some preferred embodiments, the buffers in the first reagent and the second reagent are of the same type; the buffer concentrations in the first reagent and the second reagent are different.
- the buffer of the first reagent is 50 mM MES buffer; the buffer of the second reagent is 20 mM MES buffer.
- the pH of the buffer may vary as factors such as buffer type, concentration, and the like change.
- the 25 hydroxyvitamin D antibody is a polyclonal antibody or a monoclonal antibody;
- the vitamin D binding protein antibody is a polyclonal antibody or a monoclonal antibody; wherein the polyclonal antibody is derived from rabbit, sheep or chicken, monoclonal antibody source From rats or rabbits.
- the 25 hydroxyvitamin D antigen is a natural antigen or a synthetic antigen.
- the microspheres are latex particles polymerized from one or more of polystyrene, acrylic acid, or acrylic acid related esters; the microspheres are between 150 and 500 nm in diameter. In a specific embodiment, the microspheres are polystyrene latex particles having a diameter of 223 nm.
- the 25-hydroxyvitamin D test kit further includes a calibrator which is mainly used for calibrating a measurement system, evaluating a measurement program, or assigning a value to a sample to be tested, as needed. Therefore, the calibrator contains a known concentration of 25 hydroxyvitamin D, and the value of the calibrator can even be traced back to the reference material or traced back to the reference method (IS017511: 2003).
- a calibrator which is mainly used for calibrating a measurement system, evaluating a measurement program, or assigning a value to a sample to be tested, as needed. Therefore, the calibrator contains a known concentration of 25 hydroxyvitamin D, and the value of the calibrator can even be traced back to the reference material or traced back to the reference method (IS017511: 2003).
- Those skilled in the art can prepare a suitable concentration of the calibration product according to the concentration range of the substance to be tested, or use a commercially available calibrator, or a work calibration product provided by the manufacturer, by using a method commonly
- the 25 hydroxyvitamin D detection kit according to the present invention further comprises a plurality of different concentrations of calibrators, such as 2, 3, 4, 5 or even more concentrations of calibrators.
- the 25 hydroxyvitamin D test kit of the present invention comprises five different concentrations of calibrators.
- the calibrator contains 25 hydroxyvitamin D (respectively, but not limited to, 20 ng/ml, 45 ng/ml, 65 ng/ml, 100 ng/ml, and 150 ng/ml), buffer, and, if appropriate, stabilizers, preservatives, etc. .
- the calibrator can be prepared in liquid form, dry powder or lyophilized form.
- a method for preparing a microsphere incorporating a 25-hydroxyvitamin D antigen comprising the steps of: Activating 25 hydroxyvitamin D antigen;
- the activated 25-hydroxyvitamin D antigen is attached to the activated microspheres to obtain microspheres incorporating the 25-hydroxyvitamin D antigen.
- the procedure for activating the 25 hydroxyvitamin D antigen is interchangeable with the sequence of steps for activating the microspheres.
- the reagent for the step of activating the 25 hydroxyvitamin D antigen is selected from the group consisting of carbonyl diimidazole, anhydrous acetone, diaminodipropylimine, ethyl dimethylaminopropyl carbodiimide, and 2- One or more of morpholine ethanesulfonic acid.
- the microspheres are carboxyl modified microspheres.
- the first reagent comprises sufficient vitamin D binding protein antibody to separate vitamin D from the vitamin binding protein; further comprising sufficient 25 hydroxy vitamin D antibody, a sample of the subject (eg whole blood) 25-hydroxyvitamin D in the blood paddle, serum) can specifically bind to the antibody; the second reagent contains microspheres bound to the 25-hydroxyvitamin D antigen, 25-hydroxyvitamin D antigen on the microspheres and unbound sample The remaining antibody of 25-hydroxyvitamin D can react specifically; the principle of using the latex to enhance the immunoturbidimetry, the absorbance generated by the reaction of the microspheres with the remaining antibody can be used to calculate the content of 25-hydroxyvitamin D in the sample.
- Figure 1 Standard curve of the 25-hydroxyvitamin D kit.
- Figure 2 Comparison of standard curves between reagents prepared by the method of the invention and the control method. ⁇ method of the invention; ⁇ control method.
- Figure 3 Correlation of the kit of the invention with serum measurements by enzyme-linked immunoassay. detailed description
- % means mass/volume.
- the specific materials used in the embodiments of the present invention and their sources are provided below. However, it should be understood that these are merely exemplary and are not intended to limit the invention, and materials that are the same or similar to the type, model, quality, nature or function of the reagents and instruments described below can be used in the practice of the invention. The following examples were tested with serum as an example, but those skilled in the art will understand that blood plasma and whole blood are the same.
- the sample can be used as a sample according to the method and the kit of the present invention, because the test object is 25 hydroxyvitamin D in the sample, and the operations of collecting, storing, pretreating, etc. of serum, blood paddle and whole blood can be followed. Conventional methods in the field of clinical testing are performed.
- the first reagent consists of:
- Vitamin D binding protein monoclonal antibody 0.2%
- the vitamin D-binding protein antibody and the 25-hydroxyvitamin D antibody were diluted at room temperature with a 50 mM MES solution containing 0.15 M NaCl, 0.1% NaN 3 , 0.1% BSA to a final antibody concentration of 0.2%.
- the second reagent preparation process is as follows:
- the second reagent preparation process is as follows:
- c) weigh 1.5 mg of diaminodipropylimine into b) and stir for 10 to 60 minutes, for example 30min; d) adding the polystyrene latex particles obtained in c) to a), mixing, and reacting at 30 ° C for 150 hours in a shaker for 1-4 hours, for example 3 hours;
- f) dilute the latex particles obtained by e) with 20 mM MES solution (pH 5.0) to 1.25%, 0.1% preservative can be added as needed;
- the assay steps of the kit of the invention are as follows:
- the kit of the invention has a linear range of up to 0-150 ng/ml.
- the blank (deionized water) and the same low-concentration sample (serum) diluted with physiological saline were repeatedly measured 15 times, and the amount of change in absorbance was read by a method known to those skilled in the art. Then, the absorbance values of each sample after subtracting the blank absorbance were calculated, and the mean value and standard deviation were calculated. A minimum detection limit of 99.7% was used to calculate the minimum detection limit. The mean value of each sample is subtracted by 3 times the respective standard deviation, and then compared with the standard deviation of 3 times of the blank liquid. If the former is higher than the latter, we assume that the minimum absorbance of 99.7% is necessarily greater than Blank absorbance, can report results quantitatively. The measurement results are shown in Table 2 and Table 3.
- Example 5 Comparison of the preparation method (Example 1) of the present invention and the control method (Example 2)
- the reagent prepared by the preparation method of the present invention and the reagent prepared by the control method were subjected to the operation as described in Example 3, and a standard curve was drawn as shown in Fig. 2.
- the calibration absorbance is increased by more than one time, that is, in the case where the vitamin D antigen mass is fixed.
- the method of application can crosslink more vitamin D to the microspheres, greatly improving the utilization of vitamin D antigen; that is, reducing the input of vitamin D antigen at the same absorbance level, and reducing the cost of reagents.
- Example 6 Correlation between 25 hydroxyvitamin D reagent and enzyme-linked immunosorbent assay (Elisa) of the present invention
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Abstract
本发明涉及25羟维生素D检测试剂盒及其制备方法。该试剂包括第一试剂、第二试剂,可选地还可以包括校准品。第一试剂包含25羟维生素D抗体、维生素D结合蛋白抗体和缓冲液;第二试剂含有结合有25羟维生素D抗原的微球和缓冲液。该试剂盒利用样本中的25羟维生素D与结合在微球表面的抗原竞争结合25羟维生素D抗体,使得结合有25羟维生素D的微球与25羟维生素D抗体反应形成的浊度下降,通过该浊度的下降程度推算样本中25羟维生素D的含量。
Description
25羟维生素 D检测试剂盒及其制备方法 技术领域
本发明涉及临床体外诊断及医学免疫学领域; 具体地涉及一种免 疫检测试剂; 更进一步地, 本发明涉及一种 25羟维生素 D检测试剂盒 及其制备方法。 背景技术
维生素 D也称抗佝偻病维生素,又可分为维生素 D2和维生素 D3。 维生素 D2多含于植物性食物中,它是由植物的麦角固醇经阳光照射而 合成的,维生素 D3可由人体皮肤和脂肪组织在 7-脱氢胆固醇经过阳光 照射合成。 维生素 D2属脂溶性维生素, 来自食物中的维生素 D与脂 肪一起经小肠吸收, 在胆汁协助下形成乳糜微粒, 由淋巴管进入血液, 与自身合成的维生素 D3—起转运入肝脏。在肝脏中经肝细胞微粒体中 的单氧酶系统 (25-羟化酶) 的作用, 形成 25羟维生素0。 25羟维生 素 D在肾近端小管上皮细胞线粒体内 α羟化酶系统的作用下转变为 l,25(OH)2D3 , 它是维生素 D最大的生物作用形式, 可以促进肠道钙结 合蛋白的合成。而 25羟维生素 D是代谢中的主要循环形式, 能够反映 机体的维生素 D水平。
维生素 D缺乏又称维生素 D缺乏性佝偻病。 常见于婴幼儿, 多见 于膳食中缺乏维生素 D, 或者是人体缺乏阳光的照射。 由于体内维生 素 D不足而引起钙、 磷代谢障碍, 钙盐不能正常地沉积于骨骼的生长 部分, 以致出现生长骨发生病变为特征的一种慢性营养性疾病, 称为 佝偻病。 如果该病发生于成人, 则出现发育成熟骨骼的钙化不全, 称 为骨软化症。 研究还表明, 维生素 D缺乏可能使某些癌症、 心血管疾 病、 自身免疫性疾病和感染性疾病的发病风险升高。
根据当前糖尿病报告杂志上发表的结果, 3,262例年龄 50-70岁之 间的中国人参加的一项研究表明 94%的人维生素 D缺乏或不足, 这些 人中有 42 %还有代谢综合征。 其它一些研究表明, 维生素 D缺乏和不 足在中国的孕妇以及 6-16岁的儿童中也很常见。 而长期摄入过多的维 生素 D (5000IU), 将引起高血钙和高尿钙。 特征为食欲减退, 过度口
渴、 恶心、 呕吐、 烦躁、 体弱、 便泌腹泻交替出现, 严重者将因肾钙 化、 心脏和大动脉钙化而死亡。 随着医疗专业人员和患者意识到维生 素 D缺乏的潜在健康风险, 实验室检验量激增, 需要我们提供更快、 更准、 更有效的检测方法, 帮助医生和患者更早的得到检测结果。
目前 25 羟维生素 D 的测定方法主要有酶联免疫法 (如 CN102628872 A中公开的方法和试剂盒, 以及 DRG提供的 25-OH维 他命 D (总)检测试剂盒 EIA5396)、 放射免疫法(如 Hollis BW等人, 1985 )、 液相色谱 -质谱检测法(如郑晓艳等人, 2012 ) 以及化学发光法 (如单咏梅等人, 2011;索灵生产的 25-羟基总维生素 D定量测定试剂 盒 (化学发光法) )。 酶联免疫法自动化程度不高, 且受人为因素影响 较大。 放射免疫法存在着环境污染问题; 液相色谱质谱操作复杂, 用 时较长; 而化学发光法灵敏度虽高, 但检测成本较高, 需要特定化学 发光仪, 这些原因导致其应用范围较小。
本发明采用胶乳增强免疫透射比浊法测定人样本中 25羟维生素 D 浓度, 该方法对仪器设备要求不高, 没有环保和操作人员自身防护等 问题。 而目前市场上还未出现胶乳增强免疫透射比浊法测定人样本中 25羟维生素 D浓度的试剂盒,与其它测定方法比较,本方法简便快速、 灵敏可靠, 普通自动或半自动生化分析仪就可, 有较大应用范围和实 用价值。 发明内容
根据本发明的第一方面, 提供了一种 25羟维生素 D检测试剂盒, 其包含第一试剂, 和第二试剂。第一试剂包含 25羟维生素 D抗体、 维 生素 D结合蛋白抗体和缓冲液, 第二试剂包含结合有 25羟维生素 D 抗原的微球和缓冲液。 在一些实施方式中, 第一试剂和第二试剂中的 缓冲液选自磷酸缓冲液、 甘氨酸缓冲液、 MES缓冲液、 硼酸缓冲液、 醋酸盐缓冲液、 2-吗啉乙磺酸缓冲液或氯化铵缓冲液中的一种或几种; 缓冲液浓度为 10-500 mM。
在一些实施方式中, 第一试剂和第二试剂中的缓冲液类型可以相 同也可以不同; 第一试剂和第二试剂中的缓冲液浓度可以相同也可以 不同。
在一些优选实施方式中, 第一试剂和第二试剂中的缓冲液类型相 同; 第一试剂和第二试剂中的缓冲液浓度不同。
在一个具体实施方式中,第一试剂的缓冲液是 50mM MES缓冲液; 第二试剂的缓冲液是 20mM MES缓冲液。 本领域技术人员可以理解, 随着缓冲液类型、 浓度等因素的改变, 缓冲液的 pH值可以有所不同。
在一些实施方式中, 25羟维生素 D抗体为多克隆抗体或单克隆抗 体; 维生素 D结合蛋白抗体为多克隆抗体或单克隆抗体; 其中多克隆 抗体源自兔、 羊或鸡, 单克隆抗体源自鼠或兔。
在一些实施方式中,所述 25羟维生素 D抗原为自然抗原或人工合 成抗原。
在一些实施方式中, 所述微球是由聚苯乙烯、 丙烯酸、 丙烯酸相 关酯类中的一种或几种聚合而成的胶乳颗粒; 微球直径在 150-500 nm 之间。 在一个具体实施方式中, 所述微球是聚苯乙烯胶乳颗粒, 微球 直径为 223 nm。
根据需要,根据本发明的 25羟维生素 D检测试剂盒还包括校准品, 校准品主要用于校准测量系统、 评价测量程序或为待测样本赋值。 因 此, 所述校准品包含已知浓度的 25羟维生素 D, 校准品的值甚至可以 追溯至参考物质或者追溯至参考方法 (IS017511:2003 ) 。 本领域技术 人员可以根据待测物质的浓度范围, 采用本领域常用的方法自行制备 适当浓度的校准品, 也可以使用市售校准品、 或制造商提供的工作校 准品等。
在一些具体实施方式中,根据本发明的 25羟维生素 D检测试剂盒 还包括若干不同浓度的校准品, 例如 2个、 3个、 4个、 5个甚至更多 浓度的校准品。
在一个实施方式中,本发明的 25羟维生素 D检测试剂盒包括 5个 不同浓度的校准品。 校准品含有 25羟维生素 D (分别为, 但不限于, 20ng/ml、 45ng/ml、 65ng/ml、 lOOng/ml和 150ng/ml) 、 缓冲液, 适当 时还含有稳定剂、 或防腐剂等。 校准品可制备成液体形式、 干粉或冻 干粉形式。
根据本发明的另一方面,提供了一种结合有 25羟维生素 D抗原的 微球的制备方法, 包括步骤:
活化 25羟维生素 D抗原;
活化微球;
将活化的 25羟维生素 D抗原与活化的微球连接,从而获得结合有 25羟维生素 D抗原的微球。 其中活化 25羟维生素 D抗原的步骤与活 化微球的步骤的操作顺序是可互换的。
在一些实施方式中,活化 25羟维生素 D抗原的步骤所用的试剂选 自羰基二咪唑、 无水丙酮、 二氨基二丙基亚胺、 乙基二甲基胺丙基碳 化二亚胺和 2-吗啉乙磺酸中的一种或多种。
在一些实施方式中, 所述微球为羧基修饰的微球。
采用本发明的试剂盒, 第一试剂包含足够多的维生素 D结合蛋白 抗体, 其能够将维生素 D与维生素结合蛋白分开; 还包含足够多的 25 羟维生素 D抗体, 受试者样本(如全血、 血桨、 血清) 中的 25羟维生 素 D可以和该抗体发生特异性结合;第二试剂含有结合有 25羟维生素 D抗原的微球, 微球上的 25羟维生素 D抗原和未结合样本中 25羟维 生素 D的剩余抗体可以发生特异性反应; 利用胶乳增强免疫比浊的方 法原理, 检测微球与剩余抗体反应产生的吸光度, 可以推算出样本中 25羟维生素 D的含量。 附图说明
图 1 : 25羟维生素 D试剂盒标准曲线。
图 2: 本发明方法和对照方法所制备的试剂之间标准曲线的比较。 ♦本发明方法; ■ 对照方法。
图 3 : 本发明的试剂盒与酶联免疫分析法的血清测值的相关性。 具体实施方式
为了使本发明易于理解, 下面结合具体实施例进一步阐述本发明。 除非另有指明, "% "表示质量 /体积。 以下提供了本发明实施方式中 所使用的具体材料及其来源。 但是, 应当理解的是, 这些仅仅是示例 性的, 并不意图限制本发明, 与如下试剂和仪器的类型、 型号、 品质、 性质或功能相同或相似的材料均可以用于实施本发明。 以下实施例以 血清为例进行了测试, 但是本领域技术人员能够理解, 血桨和全血同
样可用作根据本发明的方法和试剂盒的受试样本, 因为其检测对象均 为样本中的 25羟维生素 D, 血清、 血桨和全血的采集、 保存、 预处理 等操作可以按照临床检验领域中的常规方法进行。 实施例
实施例 1: 25羟维生素 D检测试剂盒的制备
1. 第一试剂制备过程如下:
第一试剂组成:
pH 7.3
MES 50 mM
NaCl 0.15M
NaN3 0.1%
BSA 0.1%
维生素 D结合蛋白单克隆抗体 (鼠源) 0.2%
抗 25羟维生素 D抗体多克隆抗体 (兔源) 0.2%
用含有 0.15M NaCl、 0.1% NaN3、 0.1 %BSA的 50 mM MES溶液, 在室温下稀释维生素 D结合蛋白抗体和 25羟维生素 D抗体, 至抗体 终浓度为 0.2%。
2. 第二试剂制备过程如下:
a)用无水丙酮在室温下稀释 4ml人 25羟维生素 D抗原( 10mg/ml), 至抗原终浓度为 2mg/ml;
b )称取 20mg羰基二咪唑加入到上述溶液中, 混匀, 在 30°C 150 rpm摇床中反应 0.5-2小时, 例如 1小时;
c ) 用 20mM MES溶液 (pH 5.0 ) 在室温下稀释 1ml 223 nm聚苯 乙烯胶乳颗粒, 至胶乳浓度为 4%;
d)称取 10mg二氨基二丙基亚胺溶于 7.5ml无水丙酮中, 加入 1.5 mg乙基二甲基胺丙基碳化二亚胺搅拌 10至 60 min, 例如 30min; e )将 c )获得的聚苯乙烯胶乳颗粒滴加进 d),混匀, 30°C 150 rpm 摇床中反应 1-3小时, 例如 2小时;
f)将 e )获得的活化的聚苯乙烯胶乳颗粒滴加进 b ) 中, 混匀, 30 V 150 rpm摇床中反应 1-4小时, 例如 3小时;
g) 将 0 获得的液体离心, 并用去离子水清洗 2-3遍, 去上清, 获得胶乳颗粒沉淀;
h) 用 20 mM MES溶液 (pH 5.0)稀释 g)获得的胶乳颗粒至 1.25%, 根据需要可以加入 0.1%防腐剂;
i) 超声分散后, 获得第二试剂。
3. 校准品的制备过程:
校准品组成:
MES 50mM
NaCl 0.15M
NaN3 0.1%
BSA 0.5%
无水乙醇 10%
25羟维生素 D纯品 20ng/ml; 45ng/ml;
65ng/ml; lOOng/ml; 150ng/ml 其余为去离子水
按参考校准品所需要浓度,将 25羟维生素 D纯品加入含有 0.15M NaCl、 0.1% NaN3、 0.5%BSA、 10%无水乙醇、 5%蔗糖的 50 mM MES 溶液中, 制得 20ng/ml、 45ng/ml、 65ng/ml、 lOOng/mL 150ng/ml浓度 的 25羟维生素 D参考校准品。
尽管上述方法中提供了具体的实验条件, 但是本领域技术人员应 当理解, 其可以在本发明的精神范围内, 针对实验规模等因素进行适 当的调整。 例如, 当制备规模不同时, 可以改变上述试剂的用量, 并 选择适当的反应设备以匹配规模的需求; 再比如, 搅拌速度不同时, 搅拌时间可以适当地延长或缩短。 实施例 2: 对照制备方法
1. 第一试剂的制备同实施例 1。
2. 第二试剂制备过程如下:
a) 用 20mM MES溶液 (pH 5.0) 在室温下稀释 4ml人 25羟维生 素 D抗原 (10mg/ml) , 至抗原终浓度为 2mg/ml;
b) 用 20mM MES溶液 (pH 5.0) 在室温下稀释 1ml 223 nm聚苯 乙烯胶乳颗粒, 至胶乳浓度为 4%;
c) 称取 1.5 mg 二氨基二丙基亚胺加入 b) 中搅拌 10至 60 min, 例如 30min;
d)将 c)获得的聚苯乙烯胶乳颗粒滴加进 a),混匀, 30°C 150 rpm 摇床中反应 1-4小时, 例如 3小时;
e) 将 d) 获得的液体离心, 并用去离子水清洗 2-3遍, 去上清, 获得胶乳颗粒沉淀;
f) 用 20 mM MES溶液(pH 5.0)稀释 e)获得的胶乳颗粒至 1.25%, 根据需要可以加入 0.1%防腐剂;
g) 超声分散后, 获得第二试剂。
3. 校准品的制备过程同实施例 1。 实施例 3 : 25羟维生素 D校准曲线的绘制
本发明试剂盒的测定步骤如下:
以校准品浓度为横轴, 相应的 AOD57。为纵轴, 采用非线性拟合,
实施例 4: 25羟维生素 D试剂的线性和最低检测限
1. 线性实验:
采用本领域技术人员已知的方法,将某一高 25羟维生素 D浓度样 本 (血清) 做倍比稀释, 测定稀释后的浓度, 测定三次求平均值, 与 表 1. 线性结果 (ng/ml)
线性 读取 1 读取 2 读取 3 均值 理论值 偏差%
1 156.70 158.11 159.56 158.12 157.73 0.25
0.5 76.73 77.97 78.53 77.74 78.58 -1.06
0.25 39.38 39.56 38.76 39.23 39.00 0.60
0.125 18.69 19.53 19.28 19.17 19.21 -0.24
0.0625 9.37 9.48 9.58 9.48 9.32 1.69
0.03125 4.54 4.57 4.49 4.53 4.37 .69 从表 1可以看出本发明试剂盒线性范围可达 0-150ng/ml。
2. 最低检测限:
采用本领域技术人员已知的方法, 将空白液 (去离子水) 和同一 份用生理盐水进行稀释的几个低浓度样品 (血清) , 重复测定 15次, 读取吸光度变化量。 然后计算出扣除空白吸光度后的各样品的吸光度 值, 计算均值, 标准偏差。采用 99.7%的置信度来计算最低检测限。将 每个样本的均值减去 3倍的各自的标准偏差, 然后与空白液的 3倍的 标准偏差比较,如果前者高于后者,我们就认定有 99.7%的可能性出现 的最小吸光度一定大于空白吸光度, 能定量的报告结果。 测定结果如 表 2和表 3。
表 2. 样本的吸光度测量结果
本测值 ng/ml
测值序号 1 2 3 4 水空白
1 7.99 4.43 2.52 1.05 -0.0016
2 7.35 4.00 2.52 0.43 -0.0071
3 6.73 4.00 1.90 1.26 0.0008
4 5.47 4.21 1.48 0.43 -0.0024
5 6.52 3.35 2.09 0.64 -0.0008
6 8.21 3.35 2.31 0.00 0.0000
7 6.95 4.00 2.31 0.21 -0.0016
8 7.56 4.00 2.52 -0.43 -0.0016
9 7.78 4.21 2.31 0.00 0.0000
10 6.52 4.43 2.74 0.83 -0.0031
11 8.21 4.00 2.31 0.21 -0.0008
12 7.13 3.78 2.95 0.43 0.0024
13 7.56 4.00 1.69 1.05 -0.0024
14 8.61 3.78 1.90 0.21 -0.0016
15 7.99 4.21 2.74 0.21 0.0016
均值 7.37 3.98 2.29 0.44 -0.0012
由表 3得知, 本发明方法所制备的试剂的最低检测限制可达 3.98 ng/ml。 实施例 5 : 本发明的制备方法(实施例 1 )与对照方法(实施例 2 ) 的比较
采用本发明制备方法所制备的试剂与对照方法所制备的试剂, 按 照实施例 3所述的操作, 并绘制出标准曲线, 如图 2所示。
如图 2所示, 采用本发明制备方法所制备的试剂与对照方法所制 备的试剂绘制的标准曲线相比, 定标吸光度增加 1 倍以上, 即在维生 素 D抗原质量固定的情况下, 通过本申请的方法可以将更多的维生素 D交联到微球上, 大大提高了维生素 D抗原的利用率; 亦即在同一吸 光度水平下可以降低维生素 D抗原的投入, 能够降低试剂成本。 实施例 6: 本发明 25羟维生素 D试剂与酶联免疫分析法 (Elisa) 测值相关性
相关性考察所用样本: 肝素钠抗凝血清样本。
本发明的 25羟维生素 D检测试剂盒与现有技术 (酶联免疫分析法) 所得的测定值进行比较 (如图 3 ) , 并进行回归分析, 获知相关性 r2=0.988 , v=1.010 x - 1.856; 显示出本方法与酶联免疫分析法在肝素
钠抗凝血清 25羟维生素 D测定方面具有良好的相关性。 参考文献
Hollis BW, Napoli JL. Improved radioimmunoassay for vitamin D and its use in assessing vitamin D status. Clin Chem. 1985; 31 : 1815—19。
郑晓艳, 马爱国, 张辉珍, 王智, 汪求真, 梁惠。 液相色谱 -串联 质谱法检测人血清 25羟维生素 D3水平及条件控制分析。 卫生研究, 2012, 03期。
单咏梅,杨凡。化学发光法测定 25-羟基维生素 D检测性能的验证 和评估。 《中华医学会第九次全国检验医学学术会议暨中国医院协会 临床检验管理专业委员会第六届全国临床检验实验室管理学术会议论 文汇编》 , 2011年。
Claims
1、 一种 25羟维生素 D检测试剂盒, 其包含:
第一试剂, 和
第二试剂;
其中,
第一试剂包含 25羟维生素 D抗体、 维生素 D结合蛋白抗体和缓 冲液,
第二试剂包含结合有 25羟维生素 D抗原的微球和缓冲液。
2、 根据权利要求 1所述的 25羟维生素 D检测试剂盒, 其中 所述缓冲液选自磷酸缓冲液、 甘氨酸缓冲液、 MES缓冲液、 硼酸 缓冲液、醋酸盐缓冲液、 2-吗啉乙磺酸缓冲液或氯化铵缓冲液中的一种 或几种;
所述缓冲液浓度为 10-500 mM。
3、 根据权利要求 1所述的 25羟维生素 D检测试剂盒, 其中 所述维生素 D结合蛋白抗体为多克隆抗体或单克隆抗体; 所述 25羟维生素 D抗体为多克隆抗体或单克隆抗体;
其中多克隆抗体源自兔、 羊或鸡, 单克隆抗体源自鼠或兔。
4、 根据权利要求 1所述的 25羟维生素 D检测试剂盒, 其中 所述 25羟维生素 D抗原为自然抗原或人工合成抗原。
5、 根据权利要求 1所述的 25羟维生素 D检测试剂盒, 其中 所述微球是由聚苯乙烯、 丙烯酸、 丙烯酸相关酯类中的一种或几 种聚合而成的胶乳颗粒; 微球直径在 150-500 nm之间。
6、 根据权利要求 1所述的 25羟维生素 D检测试剂盒, 其还包括 交准品, 所述校准品包含已知浓度的 25羟维生素0。
7、 一种结合有 25羟维生素 D抗原的微球的制备方法, 所述方法
包括步骤:
活化 25羟维生素 D抗原;
活化微球;
将活化的 25羟维生素 D抗原与活化的微球连接,从而获得结合有 25羟维生素 D抗原的微球。
8、根据权利要求 7所述的制备方法,其中所述活化 25羟维生素 D 抗原的步骤所用的试剂选自羰基二咪唑、 无水丙酮、 二氨基二丙基亚 胺、 乙基二甲基胺丙基碳化二亚胺和 2-吗啉乙磺酸中的一种或多种。
9、 根据权利要求 7所述的制备方法, 其中所述微球为羧基修饰的 微球。
10、 根据权利要求 7所述的制备方法, 其中所述活化 25羟维生素 D抗原的步骤与所述活化微球的步骤的操作顺序是可互换的。
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