人穩(wěn)定型骨鈣素（1-43/49）特異性酶免試劑盒
美國(guó)*
【型號(hào)】 KT-809
【預(yù)期用途】
這個(gè)ELISA試劑盒是用來對(duì)人體骨鈣素（1-43）和骨鈣素（1-49）（也被稱為N端和中部骨鈣素或穩(wěn)定型骨鈣素）的濃度進(jìn)行定量檢測(cè)。該檢測(cè)項(xiàng)目可以用來檢測(cè)人的骨形成活性或者成骨細(xì)胞活性，他們與代謝性骨疾病中骨更新率的變化相關(guān)，例如骨質(zhì)疏松，原發(fā)性甲狀旁腺功能亢進(jìn)癥，甲狀腺功能亢進(jìn)，畸形性骨炎以及腎性骨營(yíng)養(yǎng)不良。
【簡(jiǎn)介】
骨鈣素[也叫做骨鈣蛋白質(zhì)（BGP）]是骨骼和牙本質(zhì)中的一種主要的非膠原蛋白質(zhì)。骨鈣素的合成物包括維生素K和維生素D3。新合成的骨鈣素部分釋放到血流中，部分滲入到骨基質(zhì)中。骨鈣素（1-49）及其片段以及骨鈣素（1-43）都會(huì)釋放到血流中去。在血液循環(huán)、肝、腎中，骨鈣素（1-49）降解生成穩(wěn)定型血清骨鈣素（1-43）。當(dāng)抽取人體樣本進(jìn)行體外降解時(shí)，同樣能生成穩(wěn)定型血清骨鈣素（1-43），因?yàn)楣氢}素（1-49） C端的六個(gè)氨基酸不穩(wěn)定，在體外時(shí)，這六個(gè)氨基酸極易在室溫下脫落。一些研究證明，臨床上對(duì)穩(wěn)定型骨鈣素[骨鈣素（1-43/49）] 進(jìn)行測(cè)定，其效果更好，它能對(duì)骨更新率提供更加精確的評(píng)估。
骨鈣素由成骨細(xì)胞產(chǎn)生，在骨骼形成過程中，它通常作為一個(gè)生物化學(xué)指標(biāo)或者生物指標(biāo)。在用骨形成藥物 （如Forteo） 治療骨質(zhì)疏松時(shí)，通常能觀察到穩(wěn)定型血清骨鈣素水平的增高與骨質(zhì)密度的增加呈現(xiàn)出很強(qiáng)的正相關(guān)。在許多藥物治療研究中，骨鈣素是骨形成的特定療效跟蹤和評(píng)定的*生化指標(biāo)。
【檢驗(yàn)原理】
這個(gè)ELISA的設(shè)計(jì)，研發(fā)和生產(chǎn)的目的是對(duì)血清或者血漿樣品中穩(wěn)定型人骨鈣素（1-43）和骨鈣素（1-49）進(jìn)行定量檢驗(yàn)。該試劑盒是基于運(yùn)用兩種特異性抗體與人骨鈣素的不同抗原表位結(jié)合的“雙位點(diǎn)夾心”技術(shù)。
將標(biāo)準(zhǔn)品、質(zhì)控品、和待測(cè)樣品加入鏈霉親和素包被的微孔板中。隨后，加入生物素偶聯(lián)的人骨鈣素N端特異性多克隆抗體和過氧化物酶標(biāo)記的人骨鈣素20-43區(qū)域特異性單克隆抗體混合物。經(jīng)過*個(gè)保溫孵育期后，形成了“生物素抗體-人骨鈣素-HRP單克隆抗體”的“雙位點(diǎn)夾心”結(jié)構(gòu)，該免疫復(fù)合體在特異性“鏈霉親和素-生物素”親和結(jié)合的作用下被吸附到微孔板的內(nèi)壁。游離的單抗和緩沖基質(zhì)則被洗滌劑沖走。微孔板加入過氧化物酶基質(zhì)（3,3',5,5'-四甲基聯(lián)苯胺，TMB）并經(jīng)過一段時(shí)間的反應(yīng)后，測(cè)量吸光度。微孔板內(nèi)壁的免疫復(fù)合體的酶活性與樣本中人骨鈣素的濃度成正比。根據(jù)標(biāo)準(zhǔn)品中不同的人骨鈣素濃度的吸光度，按照“點(diǎn)到點(diǎn)”或者“4 - 參數(shù)法”繪制標(biāo)準(zhǔn)曲線，待測(cè)樣品中骨鈣素濃度可以直接從標(biāo)準(zhǔn)曲線按測(cè)量到的吸光度得到。
【組成成分】
1. 鏈霉親和素包被的微孔板：1塊×98個(gè)
2. HRP標(biāo)記骨鈣素抗體：1瓶×1.2mL
3. 生物素標(biāo)記骨鈣素抗體：2瓶×12mL
4. ELISA 濃縮洗滌液，30X ：1瓶×30mL
5. ELISA HRP基質(zhì)：1瓶×22mL
6. ELISA終止液：1瓶×12mL
7. 人骨鈣素標(biāo)準(zhǔn)品：6瓶
8. 人骨鈣素質(zhì)控品：2瓶
【 簡(jiǎn)要實(shí)驗(yàn)步驟】
1. 添加25µL標(biāo)準(zhǔn)品、質(zhì)控品和病人樣本到孔中；
2. 在各孔中添加200µL抗體混合物；
3. 在室溫下，以350 rpm振動(dòng)培養(yǎng)60分鐘；
4. 將各孔清洗5次；
5. 添加200 µLELISA HRP基質(zhì)到各個(gè)孔中；
6. 將微孔板密封，室溫下靜置20分鐘；
7. 添加50 µLELISA終止液到各個(gè)孔中；
8. 讀取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.
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