一種穩(wěn)定、抗干擾能力強的5’-核糖核苷酸水解酶檢測試劑及檢測方法技術(shù)

本發(fā)明專利技術(shù)涉及5’-NT檢測技術(shù)領(lǐng)域，特別涉及一種5’-NT檢測試劑，試劑R1中含有緩沖液，β-甘油磷酸鈉，MgCl2，Toos，防腐劑；試劑R2中含有緩沖液，5’-IMP，EHSPT，PNP，XOD，POD，抗壞血酸氧化酶，4-AA，防腐劑。采用磷酸鹽緩沖液，添加穩(wěn)定劑β-甘油磷酸鈉、抗壞血酸氧化酶，大大增強了試劑的穩(wěn)定性；不僅顯著改善測定的性能，而且增強了試劑的穩(wěn)定性和抗干擾能力。

Stable, strong anti-interference 5 '- ribose nucleotide hydrolase detection reagent and detection method

The present invention relates to the technical field of 5 -NT detection, in particular to a 5 '-NT kit, containing buffer reagent R1, beta glycerophosphate, MgCl2, Toos, preservative; buffer reagent containing R2, -IMP, EHSPT, 5' PNP, XOD, POD, 4-AA, ascorbic acid oxidase, preservatives. Using phosphate buffer, adding stabilizer sodium glycerophosphate and ascorbic acid oxidase, greatly enhanced the stability of the reagent; not only significantly improve the determination of performance, but also enhance the stability and anti-interference ability of reagent.

【技術(shù)實現(xiàn)步驟摘要】
一種穩(wěn)定、抗干擾能力強的5’-核糖核苷酸水解酶檢測試劑及檢測方法
本專利技術(shù)涉及5’-核糖核苷酸水解酶檢測
，特別涉及一種抗干擾能力強的5’-核糖核苷酸水解酶檢測試劑，還涉及使用此檢測試劑的檢測方法。
技術(shù)介紹
5’-核苷酸酶全稱5’-核糖核苷酸磷酸水解酶（5’-Nucleoticlase或5’-NT），廣泛分布在肝、膽、胰、腸、心、腦、肺、腎、垂體、甲狀腺、前列腺、睪丸等臟器和組織中，定位在細胞膜上，在肝內(nèi)主要存在于膽小管和竇狀間隙內(nèi)，是一種催化核苷5’-單磷酸水解生成核苷和無機磷酸鹽的酶，最適pH為6.6-7.0，受Mg2＋或Mn2＋激活，為Ni2＋所抑制。眾多研究資料表明，血清5’-NT活性升高主要見于肝膽胰系統(tǒng)疾病及某些惡性腫瘤，故有較特異的診斷價值。膽管結(jié)石或腫瘤所致之肝外膽管阻塞，以及氯丙嗪、肝癌或肝硬變引起肝內(nèi)膽汁郁積時，病人血清5’-NT活性可升高2-6倍。原發(fā)性或繼發(fā)性肝硬變時，病人血5’-NT升高較在慢性活動型肝炎時敏感。肝細胞損傷發(fā)生肝昏迷時，病人血清5’-NT活性多呈正常。原發(fā)性或繼發(fā)性肝癌患者此酶活性升高的陽性率為88.4％-97.2％。急性胰腺炎病人血清5’-NT略有升高，慢性胰腺炎時血清酶活性正常。發(fā)生肝繼發(fā)癌灶的胰體或胰尾癌病人血清酶活性明顯升高，以肝內(nèi)有繼發(fā)病灶的胰頭癌病人，血清5’-NT活性升高最為顯著。原發(fā)性乳腺癌患者約有65％血清5’-NT活性升高，在發(fā)生全血性廣泛轉(zhuǎn)移的病人血清5’-NT活性全部升高。測定血液、體液中的5’-NT及其同工酶水平對某些疾病的診斷、鑒別診斷、治療及免疫功能的研究越來越受到臨床的重視。目前，文獻報道的5’-NT活性的測定方法有鉬酸銨反應(yīng)法、氨量測定法、化學發(fā)光法、NADPH測定法、尿素生成法等。這些方法準確性差、靈敏度低、儀器設(shè)備要求高、試劑成本高，難于全面推廣應(yīng)用。鑒于此，本專利技術(shù)在5’-NT催化次黃嘌呤核苷酸水解生成氨和次黃嘌呤核苷反應(yīng)的基礎(chǔ)上，再依次偶聯(lián)三個酶促反應(yīng)，建立了一種既能用于手工操作，又能用于自動生化分析儀的四酶偶聯(lián)測定5’-NT活性的新方法。
技術(shù)實現(xiàn)思路
本專利技術(shù)的目的是提供一種用于檢測5’-NT的試劑及使用該試劑檢測5’-NT含量的方法。該試劑盒采用四酶偶聯(lián)法，可以有效檢測5’-NT的含量，抗干擾能力強，穩(wěn)定性好等優(yōu)點。基本原理：次黃嘌呤核苷酸在5’-NT作用下，生成次黃嘌呤核苷和磷酸，5’-NT次黃嘌呤核苷酸+H2O次黃嘌呤核苷+磷酸次黃嘌呤核苷在嘌呤核苷磷酸化酶（PNP）催化下，與磷反應(yīng)生成次黃嘌呤和核糖1-磷酸PNP次黃嘌呤核苷+Pi次黃嘌呤+核糖1-磷酸次黃嘌呤在黃嘌呤氧化酶（XOD）催化氧化生成尿酸和H2O2：XOD次黃嘌呤+2H2O+O2尿酸+2H2O2再偶聯(lián)由過氧化物酶催化的反應(yīng)POD2H2O2+4-AA+EHSPT4H2O+醌染料通過測定紅色醌化合物在550nm處吸光度的變化速率（ΔA/min）可測得5’-NT活性。本專利技術(shù)是通過以下步驟得到的：一種5’-NT檢酸測試劑，包括試劑R1和試劑R2，所述試劑R1和試劑R2的組成如下：1）試劑R1的組分為：緩沖液··········································································100mmol/Lβ-甘油磷酸鈉····························································6g/LMgCl2··········································································10g/LToos·············································································0.5g/L防腐劑·········································································0.5g/L；2）試劑R2的組分為：緩沖液········································································100mmol/L5’-IMP·········································································11mmol/LPNP·············································································0.3U/mLXOD···········································································0.4U/mLPOD············································································0.1U/mL抗壞血酸氧化酶························································2.7U/mLEHSPT····································································4mmol/L4-AA·············································································4mmol/L；以上試劑均用pH7.4的磷酸鹽緩沖溶液。以上5’-NT檢測試劑，所述防腐劑為NaN3。注：EHSPT：N-乙基-N-(2-羥基-3硫代丙基)-3-甲基苯胺4-AA：4-氨基氨替吡啉POD：過氧化物酶。所述的5’-NT檢測試劑來檢測5’-NT的檢測方法，使用全自動生化分析儀利用速率法進行測定，檢測主波長為550nm。所述的檢測方法，R1試劑和R2試劑的比例為2:1。本專利技術(shù)的有益效果：1）采用新的緩沖體系和穩(wěn)定劑，顯著改善了試劑的穩(wěn)定性；2）采用四酶偶聯(lián)法，不僅顯著改善測定的性能，而且增強了抗干擾能力，適宜批量試樣全自動分析；3）試劑的準確度和穩(wěn)定性良好，使用方便，完全可以滿足臨床需要。附圖說明圖1為兩種試劑的相關(guān)性曲線圖，圖2為兩種試劑效期穩(wěn)定性曲線圖。具體實施方式下面結(jié)合具體實施例對本專利技術(shù)進行進一步說明：實施例15’-NT的檢測試劑，包試劑R1和試劑R2：1）試劑R1的組分為：緩沖液··········································································100mmol/Lβ-甘油磷酸鈉····························································12g/LMgCl2·························································本文檔來自技高網(wǎng)...

【技術(shù)保護點】
一種5’?NT檢測試劑，其特征在于包括試劑R1和試劑R2，所述試劑R1和試劑R2的組成如下：1）試劑R1的組分為：緩沖液

【技術(shù)特征摘要】
1.一種5’-NT檢測試劑，其特征在于包括試劑R1和試劑R2，所述試劑R1和試劑R2的組成如下：1）試劑R1的組分為：緩沖液··········································································100mmol/Lβ-甘油磷酸鈉····························································12g/LMgCl2··········································································20g/LToos·············································································0.5g/L防腐劑·········································································0.5g/L；2）試劑R2的組分為：緩沖液········································································100mmol/L5’-IMP·········································································11mmol/LPNP··················································...

【專利技術(shù)屬性】
技術(shù)研發(fā)人員：甘宜梧，史秀明，謝清華，譚柏清，
申請(專利權(quán))人：山東博科生物產(chǎn)業(yè)有限公司，
類型：發(fā)明
國別省市：山東,37