新生儿苯丙氨酸荧光分析法的建立及其试剂盒的临床评价

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新生儿苯丙氨酸荧光分析法的建立及其试剂盒的临床评价

谭玉华

季涛

李贵情

华丽

赵德华

欧明才

沈健

冯建明

何海荣

吴道贫

摘要目的建立新生儿苯丙氨酸荧光分析法并评价其试剂盒性能。方法以滤纸为载体制备标准品和质控品干

血片,琥珀酸盐缓冲液、L -亮氨酸-L -丙氨酸二肽和茚三酮溶液作为反应液,硫酸铜为主要成分的溶液作为增强剂和稳定剂,应用荧光定量法检测新生儿滤纸干血片中的苯丙氨酸,并对方法进行性能评价。比对试验采用线性回归分析,配对计数资料结果的一致性采用Kappa 检验分析。结果自研试剂的曲线线性相关系数r 大于099,检测限不高于05m g/d,l 标准品的效价

比在09667~10060,批内、批间的变异系数均不高于10%,20m g/d l 以内的常见的氨基酸干扰物对检测方法无明显影响;2

~8

保存的试剂可以稳定13个月;与国外试剂比对,回归方程为Y (自研试剂盒)=08565X (An i L a b systems K I T)+01448,r =09853;不

同包装规格的试剂比对检测,线性回归方程Y (96人份/盒)=10101X (960人份/盒)-00098,r =09854。国内外的检测仪器比对检测,回归方程为Y (TAK E E N )=1011X (1420)-00058,r =09948;Y (TAK E E N )=1027X (FL UO ROS K AN)-0011,r =09937;参考值定为20m g/d;l 临床研究评价一致程度百分比可达9981%。结论

自研试剂盒灵敏度高,特异性强,准确度好,精密度好,稳定性

良好,与Ani Labsyste m s 的试剂盒比对试验检测结果的相关性好,符合率高,不同包装规格的试剂盒分析性能一致,并且适合于国内外的检测仪器,自建方法和自研试剂盒能满足临床新生儿苯丙酮尿症筛查的需要。

关键词

婴儿,新生;

苯丙酮尿症;

苯丙氨酸;

荧光分析

E stab lishm en t of fluor escence ana lysis of n eona ta l phenyla l a n i ne and eva l ua t i on of it s test r ea gent

TA N Y u -hua,JI Ta o ,LI

Gui -qi ng,HU A Li ,ZHA O De -hua,OU M ing -ca i ,S HEN J ian ,FENG J ian-m ing ,HE H a i -ron g,WU Da o -pin .Gua ngz hou F enghua B io -engineering Co .,L TD.,Guangzhou 510730,Ch i na

Corres ponding author:TA N Y u-hua,Ema il :tany why @yahoo .co

Ab stra ct

Ob jective

To estab lish a m ethod of fluorescence ana l ysis for the quantitative deter m i nati on of neonata l

phenylalani ne i n blood spec i m ens dr i ed on filter paper as an a i d in screening newborns for neonata l Phenylke to nuria ,and to prepare it s test reagent .M e thods The filter paper was used f or carr i e rs of standa rds and control s .Succ i nate buff e r ,L -l eucine dipepti de -L -a l anine sol uti on and n i nhydri n sol uti on was used f or reacti on sol uti on .The copper su lfate sol u tion was used for enhancer and stab ilizer .The fl uorescence ana l ysi s i ntened for quan ti ta ti ve dete r m ina tion of neona tal phenyla l an i ne fro m dried blo od spots .The assaym ethodol ogywas eva l uated .Co mparati ve experi m ents were ana l yzed by li near regressi on ,and the Kappa test was used f or m atched count da ta ana l ysi s .R esu lts The correlati on coe fficient of se l-f m ade kit s curve wasm ore than 0.99.The li m it of detecti on was better than 05m g/d.l The re lati ve potency ofAn i Labsyste m s ca li brators t o t he k it cali brators was 09667-1006.The i ntra -assay and the i n ter -assay coefficients of va riati on(CV)of control s were not m ore than 10%.There was no sig n ificant effect o n the detecti on m etho d by testi ng the co mm o n a m i no ac i d i nte rferences wh i ch concertrati on we re less than 20m g/d.l The stab ility of se l-f m ade k itwas 13m o nths when stored at 2-8.Co mpared with i m port k it ,t he regressi on equa ti on was Y (ho m emade kit)=08565X (i m portk it)+01448,r =09853.Co mpared with the resu lt of i m port deter m i nati on equ i p m en t ,the regressi on equatio n was Y (TAK E EN)=1011X (1420)-00058,r =09948;Y (T AKEEN)=1027X (FL UO ROS K AN)-0011,r=09937,respecti ve l y .The reference val ue of k itwas 20m g/d.l The co m pli ance rate of cli n i ca l research was up to 9981%.C onc l u si on s The sel-f m ade k it wh i ch has several advan tages inc l udi ng hi gh sensiti vity ,stro ng specificity ,n ice

precisio n ,good accuracy ,and good stab ility .Its pe rf or m ance i nd ica tors are as we ll as i m port kit .The develo ped k it is va l uable for

cli n i ca l app licati on .

DO I :10.3877/c m a ..j is sn .1674-0785.2011.06.011

基金项目:粤港关键领域重点突破项目(2008A030600003);省粤港关键领域重点突破项目开发区资金配套类(2009S -P076);科技难题求解招贤计划项目(6I );省国际合作项目(B 5)

作者单位53广州市丰华生物工程有限公司(谭玉华、季涛、李贵情、沈健、冯建明、何海荣、吴道贫);广州医学院免疫教研室(谭玉华);沈阳市妇女儿童保健中心(华丽);郑州大学第三附属医院新生儿筛查中心(赵德华);四川省妇幼保健院新生儿筛查中心(欧明才)

通讯作者谭玉华,y y @y 2009C -111200900700047:1070:Em ai:l tan wh ahoo .co m .cn