全自動化學(xué)發(fā)光免疫分析儀運行原理.ppt

1、美國雅培i2000化學(xué)發(fā)光免疫分析儀,吉林市中心醫(yī)院 滕彥玲,化學(xué)發(fā)光基本原理,反應(yīng)體系中的某種物質(zhì)的分子(反應(yīng)物、產(chǎn)物、中間體或熒光物質(zhì)) 吸收了反應(yīng)所釋放的能量而由基態(tài)躍遷至激發(fā)態(tài)，然后再從激發(fā)態(tài)返回基態(tài)，同時將能量以光輻射的形式釋放出來，產(chǎn)生化學(xué)發(fā)光. 將化學(xué)發(fā)光反應(yīng)應(yīng)用于分析化學(xué)，根據(jù)某一時刻的發(fā)光強度或反應(yīng)的發(fā)光總量來確定體系的相應(yīng)組分含量的分析方法叫化學(xué)發(fā)光分析法.,化學(xué)發(fā)光的反應(yīng)必須具備兩個條件,一、是該反應(yīng)能釋放出一定的能量，且釋放出的能量可以被某種反應(yīng)產(chǎn)物或中間體所吸收，使之處于激發(fā)態(tài)； 二、是這種激發(fā)態(tài)產(chǎn)物應(yīng)具有一定的化學(xué)發(fā)光量子產(chǎn)率，或者可以將其能量有效地轉(zhuǎn)移給某種熒光物

2、質(zhì)，產(chǎn)生光輻射. 基于此，并不是任何化學(xué)反應(yīng)都能產(chǎn)生化學(xué)發(fā)光反應(yīng)，因此，如果測量條件適當(dāng)，化學(xué)發(fā)光分析會有足夠的選擇性.,化學(xué)發(fā)光現(xiàn)象的發(fā)現(xiàn),最早發(fā)現(xiàn)的化學(xué)發(fā)光現(xiàn)象發(fā)生在生物體內(nèi)，即熒火蟲，現(xiàn)在稱之為生物發(fā)光(Bioluminescence). 到了十九世紀(jì)后期人們發(fā)現(xiàn)簡單的非生物有機化合物也能產(chǎn)生化學(xué)發(fā)光. 1877年，發(fā)現(xiàn)洛汾堿(2，4，5-三苯基咪唑)在堿性介質(zhì)中被過氧化氫等試劑氧化時發(fā)出綠色的光. 1928年，觀察到魯米諾(3-氨基苯二甲酰肼)在堿性介質(zhì)中的 化學(xué)發(fā)光行為. 1935年，第一個報告了光澤精(N,N-二甲基二吖啶硝酸鹽)與過氧化氫反應(yīng)產(chǎn)生化學(xué)發(fā)光. 到現(xiàn)在的吖啶酯、三聯(lián)吡

3、啶釕等發(fā)光標(biāo)記物應(yīng)用技術(shù)的成熟。,化學(xué)發(fā)光分析法的特點,由于化學(xué)發(fā)光分析不使用任何光源，避免了背景光和雜散光的干擾，降低了噪聲，大大提高了信噪比，因而，化學(xué)發(fā)光分析法一般都有很高的靈敏度，通?？蓽y定納克級或皮克級的化學(xué)成分. 測量化學(xué)發(fā)光強度，多采用光電轉(zhuǎn)換裝置，用函數(shù)記錄儀或數(shù)顯裝置，記錄化學(xué)發(fā)光的相對強度. 以最大發(fā)光強度(曲線的峰高)定量被測組分的濃度. 由于流動注射分析(FIA)技術(shù)的發(fā)展，流動注射化學(xué)發(fā)光儀也廣泛使用. 配備微機系統(tǒng)，自動進樣，儲存記錄，打印結(jié)果，使化學(xué)發(fā)光分析的速度更快.,化學(xué)發(fā)光的分類,化學(xué)發(fā)光反應(yīng)參與的免疫測定分為二種類型。 第一種是以發(fā)光劑作為酶免疫測定的底物

4、，通過發(fā)光反應(yīng)增強測定的敏感性。 如：化學(xué)發(fā)光酶免疫測定（CLEIA，與酶標(biāo)ELISA法類似，即最后一步酶反應(yīng)所用底物為發(fā)光劑 ） 第二種是以發(fā)光劑作為抗體或抗原的標(biāo)記物，直接通過發(fā)光反應(yīng)檢測標(biāo)本中抗原或抗體的含量。 如：化學(xué)發(fā)光標(biāo)記免疫測定亦稱化學(xué)發(fā)光免疫測定（CLIA）、電化學(xué)發(fā)光免疫測定（ECLI）,ABBOTT i2000SR 結(jié)構(gòu),1. i2000SR processing module 2. RSH (retest sample handler) 3. SCC (system control center),ABBOTT i2000SR 結(jié)構(gòu),1. Front processing

5、 center cover 2. Processing module keypad 3. Supply and waste center door 4. Card cage door,1. Rear processing center cover 2. Rear processing center access panel 3. Power supply panel 4. Pump bay panel,ABBOTT i2000SR 結(jié)構(gòu),Sample hardware components: Provide sample aspiration and dispense. 2. Reagent

6、hardware components: Provide reagent aspiration and dispense. 3. Process path hardware components: Position the RVs for sample and reagent aspiration, mixing, washing, and CMIA processing,1. Sample pipettor,STAT pipettor,Sample and STAT pipettors (S and ST): 2. Sample and STAT syringes (SS and STS):

7、 3. Sample and STAT wash stations (SW and STW):,Reagent carousel 2. Reagent bar code reader 3. Reagent pipettors 4. Reagent syringes 5. Reagent wash stations,1. Load diverter 2. RV access door 3. RV loader and hopper assembly 4. STAT diverter5. Vortexers (Mix)6. Wash zone diverter 7. Wash zone manif

8、olds (WZ1, WZ2):8. Process path drive motor 9. Pre-trigger/trigger manifold 10. CMIA reader (CMIA)11. Liquid waste arm 12. RV unloader,ABBOTT i2000SR檢測原理,Architect I系統(tǒng)采用化學(xué)發(fā)光微粒子免疫分析技術(shù)（Chemiluminesent Microparticle ImmunoAssay, CMIA）檢測樣品中的抗原，抗體和分析物。 即：磁性微粒子包被的捕捉分子（抗原，抗體或病毒顆粒）特異的與被分析物中抗原、抗體結(jié)合形成抗原抗體復(fù)合物，

9、再與吖啶酯標(biāo)記的連接物反應(yīng)形成雙抗體夾心抗原抗體復(fù)合物。吖啶酯在過氧化氫的稀堿溶液中發(fā)生氧化還原反應(yīng)生成10-甲基吖啶酮，當(dāng)它恢復(fù)到基態(tài)時發(fā)光，根據(jù)發(fā)光強度可計算出分析物的濃度 。,ABBOTT i2000SR試劑組成,試劑組成 中圈（RI）：包被有抗體的順磁性微粒子 內(nèi)圈（R2）：吖啶酯包被的抗體 外圈：樣本稀釋液 預(yù)激發(fā)液：1.32%的過氧化氫 激發(fā)液：0.35mol./L的氫氧化鈉 清洗緩沖液：磷酸鹽緩沖液,R1試劑,R2試劑,ABBOTT i2000SR反應(yīng)類型,1.雙抗原（抗體）夾心一步法 2.雙抗原（抗體）夾心兩步法 Assay processing for One step 25

10、 Assay processing for Two step 18-4 STAT assay processing for One step 11 STAT assay processing for Two step 4-4,Assay processing for Two step 18-4 (iSystem),1. At position 1 the sample pipettor dispenses the sample into the RV (reaction vessel). 2. At position 2 the R1 pipettor dispenses the microp

11、articles. 3. At position 3 the vortexer mixes the sample and microparticles. 4. At positions 4 - 63 the reaction mixture incubates for 18 minutes.,共112個RV杯空位，每18秒轉(zhuǎn)一個空位，200T/h。常規(guī)28Min/TEST,Assay processing for Two step 18-4 (iSystem),5. At positions 64 - 67 the wash zone 1 manifold washes the reactio

12、n mixture in the RV, and then removes unbound materials,Assay processing for Two step 18-4 (iSystem),6. At position 71 the R2 pipettor dispenses acridinium-labeled conjugate. 7. At position 72 the vortexer mixes the reaction mixture. 8. At positions 73 - 86 the reaction mixture incubates for 4 minut

13、es.,Assay processing for Two step 18-4 (iSystem),9. At positions 87 - 90 the wash zone 2 manifold washes the reaction mixture in the RV, and then removes unbound materials.,Assay processing for Two step 18-4 (iSystem),10. At position 94 the pre-trigger nozzle dispenses Pre-Trigger Solution to the re

14、action mixture, and then mixes using the vortexer. 11. At position 98 the CMIA optical system takes a background read, the trigger nozzle dispenses Trigger Solution to the reaction mixture, and then the CMIA optical system takes an activated read. 12. At position 100 the liquid waste arm aspirates t

15、he liquid waste from the RV. 13. At position 109 the RV unloader removes the RV,Assay processing for One step 25 (iSystem),1. At position 1 the sample pipettor dispenses the sample into the RV (reaction vessel). 2. At position 2 the R1 pipettor dispenses the microparticles and acridinium-labeled con

16、jugate. NOTE: For a delayed one-step assay the R2 pipettor adds the acridinium-labeled conjugate at position 71 and the vortexer mixes the reaction mixture at position 72. 3. At position 3 the vortexer mixes the sample, microparticles, and conjugate. 4. At positions 4 - 86 the reaction mixture incub

17、ates for 25 minutes.,Assay processing for One step 25 (iSystem),5. At positions 87 - 90 the wash zone 2 manifold washes the reaction mixture in the RV, and then removes unbound materials. Next position is same to the two step18-4.,STAT assay processing for One step 11 (iSystem),1. At position 47 the

18、 STAT pipettor dispenses the sample into the RV (reaction vessel). 2. At position 48 the R2 pipettor dispenses the microparticles and acridinium-labeled conjugate. 3. At position 49 the vortexer mixes the sample, microparticles, and conjugate. 4. At positions 50 - 86 the reaction mixture incubates f

19、or 11 minutes.,STAT assay processing for One step 11 (iSystem),5. At positions 87 - 90 the wash zone 2 manifold washes the reaction mixture in the RV, and then removes unbound materials. Next position is same to the two step18-4.,STAT assay processing for Two step 4-4 (iSystem),1. At position 47 the

20、 STAT pipettor dispenses the sample into the RV (reaction vessel). 2. At position 48 the R2 pipettor dispenses the microparticles. 3. At position 49 the vortexer mixes the sample and microparticles. 4. At positions 50 - 63 the reaction mixture incubates for 4 minutes.,STAT assay processing for Two step 4-4 (iSystem),5. At positions 64 - 67 the wash