详细介绍

人稳定型骨钙素（1-43/49）特异性酶免试剂盒

美国*

【型号】 KT-809

【预期用途】

这个ELISA试剂盒是用来对人体骨钙素（1-43）和骨钙素（1-49）（也被称为N端和中部骨钙素或稳定型骨钙素）的浓度进行定量检测。该检测项目可以用来检测人的骨形成活性或者成骨细胞活性，他们与代谢性骨疾病中骨更新率的变化相关，例如骨质疏松，原发性甲状旁腺功能亢进症，甲状腺功能亢进，畸形性骨炎以及肾性骨营养不良。

【简介】

骨钙素[也叫做骨钙蛋白质（BGP）]是骨骼和牙本质中的一种主要的非胶原蛋白质。骨钙素的合成物包括维生素K和维生素D3。新合成的骨钙素部分释放到血流中，部分渗入到骨基质中。骨钙素（1-49）及其片段以及骨钙素（1-43）都会释放到血流中去。在血液循环、肝、肾中，骨钙素（1-49）降解生成稳定型血清骨钙素（1-43）。当抽取人体样本进行体外降解时，同样能生成稳定型血清骨钙素（1-43），因为骨钙素（1-49） C端的六个氨基酸不稳定，在体外时，这六个氨基酸极易在室温下脱落。一些研究证明，临床上对稳定型骨钙素[骨钙素（1-43/49）] 进行测定，其效果更好，它能对骨更新率提供更加精确的评估。

骨钙素由成骨细胞产生，在骨骼形成过程中，它通常作为一个生物化学指标或者生物指标。在用骨形成药物（如Forteo）治疗骨质疏松时，通常能观察到稳定型血清骨钙素水平的增高与骨质密度的增加呈现出很强的正相关。在许多药物治疗研究中，骨钙素是骨形成的特定疗效跟踪和评定的*生化指标。

【检验原理】

这个ELISA的设计，研发和生产的目的是对血清或者血浆样品中稳定型人骨钙素（1-43）和骨钙素（1-49）进行定量检验。该试剂盒是基于运用两种特异性抗体与人骨钙素的不同抗原表位结合的“双位点夹心”技术。

将标准品、质控品、和待测样品加入链霉亲和素包被的微孔板中。随后，加入生物素偶联的人骨钙素N端特异性多克隆抗体和过氧化物酶标记的人骨钙素20-43区域特异性单克隆抗体混合物。经过*个保温孵育期后，形成了“生物素抗体-人骨钙素-HRP单克隆抗体”的“双位点夹心”结构，该免疫复合体在特异性“链霉亲和素-生物素”亲和结合的作用下被吸附到微孔板的内壁。游离的单抗和缓冲基质则被洗涤剂冲走。微孔板加入过氧化物酶基质（3,3',5,5'-四甲基联苯胺，TMB）并经过一段时间的反应后，测量吸光度。微孔板内壁的免疫复合体的酶活性与样本中人骨钙素的浓度成正比。根据标准品中不同的人骨钙素浓度的吸光度，按照“点到点”或者“4 - 参数法”绘制标准曲线，待测样品中骨钙素浓度可以直接从标准曲线按测量到的吸光度得到。

【组成成分】

1. 链霉亲和素包被的微孔板：1块×98个

2. HRP标记骨钙素抗体：1瓶×1.2mL

3. 生物素标记骨钙素抗体：2瓶×12mL

4. ELISA 浓缩洗涤液，30X ：1瓶×30mL

5. ELISA HRP基质：1瓶×22mL

6. ELISA终止液：1瓶×12mL

7. 人骨钙素标准品：6瓶

8. 人骨钙素质控品：2瓶

【简要实验步骤】

1. 添加25µL标准品、质控品和病人样本到孔中；

2. 在各孔中添加200µL抗体混合物；

3. 在室温下，以350 rpm振动培养60分钟；

4. 将各孔清洗5次；

5. 添加200 µLELISA HRP基质到各个孔中；

6. 将微孔板密封，室温下静置20分钟；

7. 添加50 µLELISA终止液到各个孔中；

8. 读取450 nm的吸光度。

Osteocalcin （1-43/49） Specific ELISA Kit

Catalog Number: KT-809

Storage：This test kit must be stored at 2 – 8°C upon receipt.

INTENDED USE

This ELISA （enzyme-linked immunosorbent assay） kit is intended for the quantitative determination of both human osteocalcin （1-49） and osteocalcin （1-43）（also referred as N-terminal & mid-regional osteocalcin） levels in test samples. This test is useful for assessing the bone formation activity or osteoblast activity in patients associated with changes in the rate of bone turnover in metabolic bone disease, such as osteoporosis, primary hyperparathyroidism, hyperthyroidism, Paget’s disease, and renal osteodystrophy.

INTRODUCTION

Osteocalcin [also as bone Gla protein （BGP）] is a major noncollagenous protein found in bone and dentin. The synthesis of osteocalcin involves vitamin K and vitamin D3. Freshly synthesized osteocalcin is partly released into the blood stream and partly incorporated into the bone matrix. Both osteocalcin （1-49） and its fragments including osteocalcin （1-43） are released into the blood stream. Serum osteocalcin （1-43） also generated by catabolic breakdown of osteocalcin （1-49） in the circulation, liver, kidney, as well as by in vitro degradation during storage of samples, because a labile six-amino acid C-terminal sequence that, in vitro at room temperature, is easily cleaved off. There are several studies that have confirmed the measurement of the much more stable N-terminal and mid-regional osteocalcin [osteocalcin （1-43/49）] as being clinically useful, which may contribute to a more accurate assessment of the bone turnover rate.

As osteocalcin is manufactured by osteoblasts, it is often used as a biochemical marker, or biomarker, for the bone formation process. It has been routinely observed that higher serum-osteocalcin levels are relatively well correlated with increases in bone mineral density （BMD） during treatment with anabolic bone formation drugs for osteoporosis, such as Forteo. In many studies, Osteocalcin is used as a preliminary biomarker on the effectiveness of a given drug on bone formation.

ASSAY PRINCIPLE

This ELISA is designed, developed and produced for the quantitative measurement of human osteocalcin （1-49） and （1-43） in serum or plasma sample. The assay utilizes the two-site “sandwich” technique with two selected antibodies that bind to different epitopes of human osteocalcin.

Assay standards, controls and patient samples are added directly to wells of a microtiter plate that is coated with streptavidin. Subsequently, a mixture of biotinylated human osteocalcin N-terminal region specific polyclonal antibody and a peroxidase labeled human osteocalcin 20 – 43 region specific monoclonal antibody is added to each well. After the first incubation period, a “sandwich” of “biotinylated antibody - human osteocalcin – HRP-monoclonal antibody” is formed and this immunocomplex is also captured to the wall of microtiter plate via a streptavidin-biotin affinity binding. The unbound monoclonal antibodies and buffer matrix are removed in the subsequent washing step. A substrate solution in a timed reaction is then measured in a spectrophotometric microplate reader. The enzymatic activity of the immunocomplex bound to the wall of each microtiter well is directly proportional to the amount of human osteocalcin in a test sample. A standard curve is generated by plotting the absorbance versus the respective human osteocalcin concentration for each standard on point-to-point or 4 parameter curve fit. The concentration of human osteocalcin in test samples is determined directly from this standard curve.

REAGENTS: Preparation

1. Streptavidin Coated Microplate：96 wells

2. HRP Conjugated Osteocalcin Antibody ：1vial× 1.2 mL

3. Biotinylated Osteocalcin Antibody：2 bottles× 12mL

4. ELISA Wash Concentrate，30X：1 bottle× 30mL

5. ELISA HRP Substrate ：1 bottle× 22mL

6. ELISA Stop Solution：1 bottle× 12mL

7. Human Osteocalcin Standards：6 vials

8. Human Osteocalcin Controls：2 vials

Short Assay Procedure:

1. Add 25 µL of standards, controls and patient samples into the designated microwell.

2. Add 200 µL of antibody mixture to each well.

3. Incubate 60 minutes at RT, shaking 350 rpm

4. Wash each well 5 times.

5. Add 200 µL of ELISA HRP Substrate into each of the wells.

6. Cover and incubate plate at room temperature static for 20 minutes.

7. Add 50 µL of ELISA Stop Solution into each of the wells.

8. Read the absorbance at 450 nm.

产品简介

详细介绍

Storage：This test kit must be stored at 2 – 8°C upon receipt.

INTENDED USE

INTRODUCTION

ASSAY PRINCIPLE