本發(fā)明涉及5’-核糖核苷酸水解酶檢測技術(shù)領(lǐng)域，特別涉及一種抗干擾能力強(qiáng)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。

背景技術(shù)：

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(xì)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(jī)磷酸鹽的酶，最適pH為6.6-7.0，受Mg^2＋或Mn^2＋激活，為Ni^2＋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(jià)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(shí)，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(shí)，病人血5’-NT升高較在慢性活動(dòng)型肝炎時(shí)敏感。肝細(xì)胞損傷發(fā)生肝昏迷時(shí)，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(shí)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(xiàn)報(bào)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(xué)發(fā)光法、NADPH測定法、尿素生成法等。這些方法準(zhǔn)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，本發(fā)明在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(gè)酶促反應(yīng)，建立了一種既能用于手工操作，又能用于自動(dòng)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(shí)現(xiàn)要素：

本發(fā)明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(qiáng)，穩(wěn)定性好等優(yōu)點(diǎn)。

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)

POD

2H2O2 + 4-AA + EHSPT 4 H2O+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。

本發(fā)明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/L

β-甘油磷酸鈉_{····························································}6g/L

MgCl_{2··········································································}10g/L

Toos_{·············································································}0.5g/L

防腐劑_{·········································································}0.5g/L；

2）試劑R2的組分為：

緩沖液_{········································································}100mmol/L

5’-IMP_{·········································································}11mmol/L

PNP_{·············································································}0.3U/mL

XOD_{···········································································}0.4U/mL

POD_{············································································}0.1U/mL

抗壞血酸氧化酶_{························································}2.7U/mL

EHSPT _{····································································}4mmol/L

4-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(dòng)生化分析儀利用速率法進(jìn)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。

本發(fā)明的有益效果：

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(qiáng)了抗干擾能力，適宜批量試樣全自動(dòng)分析；

3）試劑的準(zhǔn)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(shí)施方式

下面結(jié)合具體實(shí)施例對本發(fā)明進(jìn)行進(jìn)一步說明：

實(shí)施例1

5’-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/L

β-甘油磷酸鈉_{····························································}12g/L

MgCl_{2··········································································}20g/L

Toos_{·············································································}0.5g/L

防腐劑_{·········································································}0.5g/L；

2）試劑R2的組分為：

緩沖液_{········································································}100mmol/L

5’-IMP_{·········································································}11mmol/L

PNP_{·············································································}0.3U/mL

XOD_{···········································································}0.4U/mL

POD_{············································································}0.1U/mL

抗壞血酸氧化酶_{························································}2.7U/mL

EHSPT _{·······································································}4mmol/L

4-AA_{·············································································}4mmol/L；

3）本實(shí)施例試劑的使用方法：

本實(shí)施例描述的5’-NT檢測試劑，在使用時(shí)采用具有雙試劑功能的全自動(dòng)生化分析儀，如日立7180全自動(dòng)分析儀等，利用速率法進(jìn)行測定。將R1和R2按照2:1的比例放置到對應(yīng)的試劑位上，在樣品盤的對應(yīng)位置放置好蒸餾水、標(biāo)準(zhǔn)品和樣本，操作如表1：

表1 實(shí)施例1試劑檢測方法

計(jì)算：5’-NT含量（U/L）＝（?A測定÷?A標(biāo)準(zhǔn)）×C標(biāo)準(zhǔn)。

實(shí)施例2

干擾性試驗(yàn)：取新鮮混合血清，分成2等份，然后將每等份再分成5等份，加入不同的干擾物質(zhì)，使其在血清中的濃度達(dá)到表2的要求。然后分別用實(shí)施例1所得試劑，與市場常見并認(rèn)可的5’-NT試劑同時(shí)對比測定血清中5’-NT的含量，對照組測定結(jié)果與加入不同干擾物質(zhì)后各組的測定結(jié)果見表2。相對偏差（%）=（干擾樣本的測定均值-對照樣本的測定均值）/對照樣本的測定均值×100%。

由表2可以看出，實(shí)施例1試劑在抗壞血酸≤1704μmol/L、膽紅素≤684μmol/L、血紅蛋白≤10 g/L、甘油三酯≤22.6 mmol/L對測試結(jié)果沒有明顯干擾。而對照組試劑在上述濃度干擾物質(zhì)存在時(shí)，受到明顯干擾，這說明實(shí)施例1試劑的抗干擾性能遠(yuǎn)遠(yuǎn)優(yōu)于對比試劑。

表2實(shí)施例1試劑抗干擾性能比較

實(shí)施例3

相關(guān)性實(shí)驗(yàn)：利用實(shí)施例1配方配制試劑，與市場常見的國家食品藥品監(jiān)督管理局認(rèn)可的某公司的5’-NT試劑盒進(jìn)行對照檢測，同時(shí)檢測了20個(gè)臨床血清樣本，檢測結(jié)果如表3所示。并獲得了兩種試劑的相關(guān)性曲線（如圖1所示），通過檢測結(jié)果顯示，兩個(gè)試劑盒的相關(guān)系數(shù)為0.9929，說明了兩者有極大的相關(guān)性。

表3實(shí)施例1試劑與市場常見并得到認(rèn)可的5’-NT測定試劑盒對比檢測結(jié)果

實(shí)施例4

試劑的穩(wěn)定性對比試驗(yàn)：對實(shí)施例1中的試劑，均勻分裝13組，每組的試劑量為R1為20mL，R2為10mL；并且取13組市場常見的國家食品藥品監(jiān)督管理局認(rèn)可的某公司的5’-NT試劑盒作對照。放置到2-8℃冰箱中，每月的同一天取出一組試劑檢測5’-NT質(zhì)控品，檢測結(jié)果如圖2所示，實(shí)施例1試劑在2-8℃儲(chǔ)存條件下比市場常見的5’-NT測定試劑盒更加穩(wěn)定。

通過驗(yàn)證，本試劑與同類檢測試劑對比相關(guān)性好，臨床檢測樣本結(jié)果一致，能夠達(dá)到市場對產(chǎn)品的應(yīng)用要求，并且抗干擾性能好，是一種更加穩(wěn)定、良好的5’-NT檢測試劑。