本發(fā)明涉及一種血氨（AMM）檢測技術領域，特別涉及一種穩(wěn)定性強的單試劑的液態(tài)血氨（AMM）檢測試劑。

背景技術：

體內(nèi)各組織各種氨基酸分解代謝產(chǎn)生的氨以及由腸管吸收進來的氨進入血液，形成血氨。血氨正常值18-72μmol/L。血氨的產(chǎn)生分為內(nèi)源性產(chǎn)生和外源性獲取，內(nèi)源性產(chǎn)生主要是體內(nèi)代謝產(chǎn)生的氨稱為內(nèi)源性氨，主要來自氨基酸的脫氨基作用，部分來自腎小管上皮細胞中谷氨酰胺分解產(chǎn)生的氨。胺類的分解也可產(chǎn)生氨。外源性獲取則由消化道吸收人體內(nèi)的氨稱為外源性氨。它包括腸道內(nèi)未被消化的蛋白質(zhì)和未被吸收的氨基酸，經(jīng)腸道細菌作用產(chǎn)生的氨。血中尿素擴散到腸道，經(jīng)細菌尿素酶作用水解生成的氨。

血氨增高是肝性腦病臨床特征之一，他的檢測結果的準確性對于肝性腦病、兒科Reye綜合征等的準確診斷有非常重要的意義。血氨在人體中主要以NH4+和NH3兩種形式存在，血液中血氨的主要來源主要是腸道產(chǎn)生的氨和腎臟分泌的氨。醫(yī)學中檢測血液中氨的方法非常多，比如去蛋白波氏比色法、氨電極法、擴散法、離子交換層析法、酶法和干化學法等方法。其中擴散法和波氏比色法靈敏度較低，準確性和精密度滿足不了臨床的要求，離子交換層析法則需要非常長的時間的處理，氨電極法準確性和精密度非常好，但是檢測結果的穩(wěn)定性受到多種因素的影響，并且電極需要經(jīng)常的保養(yǎng)和更換，檢測的成本和操作要求較高，無法在普通醫(yī)院中普及。目前使用的較多的為干化學法和酶法，這兩種方法可以直接應用在半自動和全自動生化分析儀器上，成本較低，操作相對較低，但是干化學法需要將干粉在使用前進行手工處理，這無疑增加了操作的繁瑣性，也增加操作的誤差，并且干粉一旦被溶解后保存時間較短，試劑中關鍵組分容易衰減，無法保證檢測結果的穩(wěn)定性和準確性，而液體的酶兩點動力學法則不需要做前期的工作，直接可以配合儀器使用，但是目前市場出現(xiàn)的酶兩點法試劑為了保證試劑的穩(wěn)定性，多為四試劑或者為三試劑，因此客戶在使用的時候必須將試劑按照比例調(diào)配混勻，試劑一旦在混合之后多不穩(wěn)定，也必須要盡快使用，不然的話試劑會發(fā)生衰減，影響結果的準確性，因此發(fā)明一種準確性強又穩(wěn)定的酶兩點法單試劑，能夠大大簡化操作的流程，方便臨床的應用，有很大的應用前景。

技術實現(xiàn)要素：

本發(fā)明的目的是提供一種穩(wěn)定性強的單試劑的液態(tài)血氨（AMM）檢測試劑以及檢測方法。

基本原理：

谷氨酸脫氫酶兩點法檢測血氨的基本原理為：在乳酸脫氫酶（LDH）的作用下，血清中干擾氨測定的丙酮酸在預反應中被除去。氨在谷氨酸脫氫酶的作用下與α-KG 和 NADH 反應，與同樣處理的校準液比較，可計算出血清中氨的含量。

第一步： 乳酸脫氫酶

丙酮酸 + NADH + H+ L-乳酸+NAD+

第二步： GLDH

α-酮戊二酸+氨+NADH 谷氨酸+NAD+ +H2O。

本發(fā)明是通過以下步驟得到的：

一種穩(wěn)定性強的單試劑液態(tài)血氨試劑試劑組成如下：

緩沖液_{···························································································································}50mmol/L，

LDH(乳酸脫氫酶)_{································································································}5KU/L-10KU/L

重金屬離子螯合劑_{·······································································································}3g/L-10g/L

離子平衡劑_{·····················································································································}2g/L-5g/L

防腐劑_{··························································································································}0.1g/L-1g/L

α-酮戊二酸（α-KG）_{················································································}7mmol/L-12 mmol/L

NADH.NA2(還原性輔酶I二鈉鹽)_{····················································}0.18mmol/L-0.36 mmol/L

表面活性劑_{···············································································································}1ml/L-5ml/L

保護劑1_{·······················································································································}3g/L-5 g/L

保護劑2_{························································································································}2g/L-5g/L

保護劑3_{························································································································}2g/L-5g/L

所述的一種穩(wěn)定性強的單試劑的血氨檢測試劑，試劑R中緩沖液為25℃，PH=8.1的，濃度為50mmol/L的TES（N-三(羥甲基)甲基-2-氨基乙磺酸）緩沖液；

所述的一種穩(wěn)定性強的單試劑的血氨檢測試劑，所述的重金屬離子螯合劑為環(huán)已烷二胺四醋酸(CDTA)；

所述的一種穩(wěn)定性強的單試劑的血氨檢測試劑，所述離子平衡劑為氯化鉀；

所述的一種穩(wěn)定性強的單試劑的血氨檢測試劑，所述防腐劑為氯霉素；

所述的一種穩(wěn)定性強的單試劑的血氨檢測試劑，所述表面活性劑為Emulgen A90；

所述的一種穩(wěn)定性強的單試劑的血氨檢測試劑，所述的保護劑1為ADP.NA2；

所述的一種穩(wěn)定性強的單試劑的血氨檢測試劑，所述的保護劑2為聚乙烯醇AH-26；

所述的一種穩(wěn)定性強的單試劑的血氨檢測試劑，所述的保護劑3為黃原膠；

所述的一種穩(wěn)定性強的單試劑的血氨檢測試劑，使用全自動生化分析儀利用終點法進行測定，檢測主波長為340nm；

所述的試劑為液態(tài)單試劑。

本發(fā)明的創(chuàng)新處：

1）本試劑建立的是谷氨酸脫氫酶兩點法動力學法的單試劑，有效的簡化了使用的操作，且保證了產(chǎn)品有效成分的穩(wěn)定性；

2）本發(fā)明緩沖體系的建立優(yōu)先選擇了TES（N-三(羥甲基)甲基-2-氨基乙磺酸）作為緩沖液，在起到基本的緩沖能力之外，能夠對NADH起到較好的保護作用；

3）在試劑中選擇了環(huán)已烷二胺四醋酸(CDTA)作為重金屬里的螯合劑，能夠較好的螯合重金屬對GLDH和LDH兩種酶的影響，同時還不會對GLDH的催化活性產(chǎn)生抑制，并且能夠較好的去除重金屬離子的對NADH穩(wěn)定性的影響；

4）在試劑中優(yōu)先選擇了氯化鉀作為例子平衡劑，能夠對體系產(chǎn)生良好的平衡作用；

5）在試劑R1中加入了環(huán)己二胺四乙酸（CDTA），能夠有效的螯合重金屬離子，并且能夠較好的提高試劑的準確性；

6）本發(fā)明在試劑中添加了Emulgen A90表面活性劑作為乳化劑，能夠非常好的提高檢測結果的重復性并且對酶的催化作用有增效作用；

7）本發(fā)明優(yōu)選ADP.NA2、聚乙烯醇AH-26、黃原膠三種物質(zhì)作為添加保護劑，對GLDH、LDH和NADH三種物質(zhì)具有非常好的保護作用。

附圖說明

圖1 為15天開瓶穩(wěn)定性實驗驗證結果曲線變化圖，

圖2 為37℃ 7天熱穩(wěn)定性實驗驗證結果曲線變化圖。

具體實施方式

下面結合具體實施例對本發(fā)明進行進一步說明：

實施例1

一種現(xiàn)有的三試劑的血氨檢測試劑：

三試劑：

試劑1a（R1a）：

Tirs緩沖液 PH8.0 50mmol/L

LDH 2KU/L

EDTA 5.3mmol/L

試劑1b（R1b）：

α-KG 18.0mmol/L

NADH 0.18mmol/L

試劑2（R2）：

GLDH 25.0KU/L

實施例2

所述的一種穩(wěn)定性強的單試劑的血氨檢測試劑組成如下：

TES（N-三(羥甲基)甲基-2-氨基乙磺酸）緩沖液_{·····················································}50mmol/L，

LDH(乳酸脫氫酶)_{··············································································································}5KU/L

CDTA_{······································································································································}3g/L

氯化鉀_{·····································································································································}2g/L

氯霉素_{··································································································································}0.1g/L

α-酮戊二酸（α-KG）_{····································································································}7mmol/L

NADH.NA2(還原性輔酶I二鈉鹽)_{···········································································}0.18mmol/L

Emulgen A90_{·························································································································}1ml/L

ADP.NA2_{··························································································································}3mmol/L

聚乙烯醇AH-26_{·····················································································································}2g/L

黃原膠_{·····································································································································}2g/L

實施例3

1)本實施例描述的是一種關鍵原材料增加的穩(wěn)定性強的單試劑的血氨檢測試劑組成如下:

TES（N-三(羥甲基)甲基-2-氨基乙磺酸）緩沖液_{···················································}50mmol/L，

LDH(乳酸脫氫酶)_{··········································································································}10KU/L

CDTA_{··································································································································}10g/L

氯化鉀_{···································································································································}5g/L

氯霉素_{···································································································································}1g/L

α-酮戊二酸（α-KG）_{·······························································································}12mmol/L

NADH.NA2(還原性輔酶I二鈉鹽)_{········································································}0.36mmol/L

Emulgen A90_{·····················································································································}5ml/L

ADP.NA2_{······················································································································}5mmol/L

聚乙烯醇AH-26_{·················································································································}5g/L

黃原膠_{···································································································································}5g/L

2）校準品和質(zhì)控品:

校準品：自制標準，其中AMM的含量為30μmol/L

質(zhì)控品：自制高值質(zhì)控：靶值：73.6μmol/L，靶值范圍：58.88-88.32μmol/L

自制低值質(zhì)控：靶值：18.0μmol/L，靶值范圍：14.4-21.6μmol/L

3)試劑穩(wěn)定性驗證具體操作方法：

（1）關于試劑的穩(wěn)定性驗證，將分為試劑的15天開平穩(wěn)定性和7天37℃熱穩(wěn)定性驗證。首先將本實施例中的檢測試劑和實施例1中的檢測試劑，按照配方配置好，分別取相同的兩組，一組做15天開瓶穩(wěn)定性，將試劑開瓶放置在儀器的2-8℃冷藏箱中（15天不取出），作為15天開瓶穩(wěn)定性檢測，另一組做37℃熱穩(wěn)定性，封閉放置在37℃恒溫水浴鍋中（每天僅在檢測的時候取出，檢測完畢后，依然封口放回37℃水浴鍋中，連續(xù)7天），作為7天37℃熱穩(wěn)定性驗證。將試劑同時在日立7180全自動生化分析儀器上，按照如下表1方法進行檢測，并在儀器上建立標準曲線。分別取高、低質(zhì)控，各自平均分成15份，4℃儲存，每天高、低值質(zhì)控各取一個，并且跟蹤檢測結果，其跟蹤監(jiān)測趨勢如附圖1和附圖2

（2）試劑檢測使用方法：

本實施例描述的所述的一種穩(wěn)定性強的單試劑的血氨檢測試劑，采用全自動生化分析儀，如日立7180全自動分析儀等，將實施例2和實施例3利用兩點動力學法，按照表1中的要求進行測定；實施例1使用雙試劑終點法進行操作，首先將試劑1(a)和試劑1（b）按照要求比例混勻后形成使用的R1，然后按照表2中的檢測方法進行操作。

表1 實施例2和3試劑檢測方法

計算：一種穩(wěn)定性強的單試劑的血氨檢測試劑（μmol/L）＝（?A測定÷?A標準）×C標準。

表2 實施例1試劑檢測方法

計算：現(xiàn)有的三試劑的血氨檢測試劑（μmol/L）＝（?A測定÷?A標準）×C標準。

(3）通過圖1和圖2中的曲線變化明顯能夠發(fā)現(xiàn)，實施例2和實施3中的單試劑明顯比實施例1中的多試劑混合后要穩(wěn)定很多，從而證明了本發(fā)明的對于產(chǎn)品的穩(wěn)定性的效果。