1、基于量子點(diǎn)技術(shù)同步檢測(cè)輸血傳染病病原體抗原的方法建立        【中文摘要】目的與意義在獻(xiàn)血及輸血治療的過程中, HBV、HCV、HIV-1/2以及TP的檢測(cè)是國家強(qiáng)制性檢測(cè)項(xiàng)目,HBV與HCV是造成輸血后肝炎的主要病原體。目前對(duì)上述多種病原微生物的實(shí)驗(yàn)室診斷方法較多,但各有不足。如ELISA無法同步檢測(cè)多項(xiàng)指標(biāo)、時(shí)間較長,金標(biāo)法靈敏度較低,PCR和化學(xué)發(fā)光法需借助特殊設(shè)備,對(duì)實(shí)驗(yàn)室要求較高等。為解決輸血性傳染病的多種病原體的抗原同步檢測(cè),本研究擬以乙肝表面抗原和丙肝核心抗原的同步檢測(cè)為例,建立一種高靈敏度和特

2、異性、穩(wěn)定性好的抗原同步快速檢測(cè)體系。將量子點(diǎn)技術(shù)與磁微粒技術(shù)相結(jié)合,以磁微粒富集以進(jìn)步靈敏度,利用不同量子點(diǎn)在相同波長激發(fā)光下發(fā)射不同熒光的特性達(dá)到同步檢測(cè)的目的。為建立針對(duì)多種輸血傳染病病原體抗原的同步檢測(cè)液相芯片奠定基礎(chǔ)。材料與方法1.磁微粒的活化及其與抗體的連接:選擇粒徑3m的羧基化磁微粒,采用碳二亞胺共價(jià)交聯(lián)法,在活化劑EDC與NHS的不同濃度組合條件下活化磁微粒,選擇最適的活化劑濃度組合。以不同濃度的鼠抗HBsAg-IgG、鼠抗HCcAg-IgG連接磁微粒,在不同反應(yīng)時(shí)間、pH值的條件組合下考察抗體與磁微粒的連接效果,以Folin-酚法評(píng)價(jià)抗體連接效率。2.量子點(diǎn)的活化及其與抗體的

3、連接:采用碳二亞胺共價(jià)交聯(lián)法以EDC及NHS活化發(fā)射波長為581nm、618nm的羧基化CdSe量子點(diǎn),以不同濃度的兔抗HBsAg-IgG、兔抗HCcAg-IgG連接量子點(diǎn)并在不同反應(yīng)時(shí)間、pH值的條件組合下考察抗體與磁微粒的連接效果,以Folin-酚法評(píng)價(jià)抗體連接效率。3.單指標(biāo)檢測(cè)的體系建立:以連接抗體的量子點(diǎn)作為熒光標(biāo)記物,以連接抗體的磁微粒為載體作固-液分離,確定最適標(biāo)本檢測(cè)時(shí)間。作陰性血清檢測(cè)以檢測(cè)熒光強(qiáng)度并以2倍陰性對(duì)照均值+空缺對(duì)照均值作為單指標(biāo)檢測(cè)Cut off值判讀結(jié)果。對(duì)不同濃度的HBsAg、HCcAg抗原標(biāo)準(zhǔn)品作單指標(biāo)檢測(cè)以確定檢測(cè)范圍。并作重復(fù)性實(shí)驗(yàn)和正確度評(píng)價(jià)。4.同

4、步檢測(cè)HBsAg和HCcAg體系的建立:在單指標(biāo)檢測(cè)的基礎(chǔ)上整合反應(yīng)條件及最適標(biāo)本檢測(cè)時(shí)間,作陰性血清檢測(cè)以檢測(cè)熒光強(qiáng)度并以2倍陰性對(duì)照均值+空缺對(duì)照均值作為單指標(biāo)檢測(cè)Cut off值判讀結(jié)果。混合激發(fā)光的記錄以濾光片濾往一種顏色光線后記錄一項(xiàng)結(jié)果,更換濾光片后記錄另一結(jié)果。5.對(duì)不同濃度HBsAg、HCcAg抗原標(biāo)準(zhǔn)品混合物作同步檢測(cè)以確定檢測(cè)范圍。6.對(duì)188例臨床標(biāo)本進(jìn)行檢測(cè),并與ELISA試劑盒檢測(cè)結(jié)果相比較。7.選取10ng/mL、100ng/mL、1g/mL三個(gè)濃度抗原標(biāo)準(zhǔn)品作同步檢測(cè)的批內(nèi)、天間重復(fù)性實(shí)驗(yàn)。8.以脂血、溶血、黃疸標(biāo)本作干擾實(shí)驗(yàn)。以HAV、TP抗體陽性標(biāo)本作特異性實(shí)

5、驗(yàn)。9.將預(yù)備好的試劑及反應(yīng)后的復(fù)合物于4保存一定時(shí)間后觀察保存時(shí)間對(duì)檢測(cè)效果的影響。主要結(jié)果1.磁微粒的選擇及連接抗體的條件選擇:鼠抗HBsAg-IgG連接磁微粒的活化劑濃度為EDC 6mg/L、NHS 4mg/L,連接時(shí)間120min,最適pH值6.6,最適濃度20mg/mL,連接效率41%,并制備試劑1(R1HBV)。鼠抗HCcAg-IgG連接磁微粒的活化劑濃度為EDC 4mg/L、NHS4mg/L,連接時(shí)間120min,最適pH值6.6,最適濃度20mg/mL,連接效率44%,并制備試劑2(R2HBV)。上述R1HBV、R1HCV清洗后加進(jìn)1%BSA封閉磁微粒表面剩余位點(diǎn)過夜。2.量子

6、點(diǎn)連接抗體的條件選擇:兔抗HBsAg-IgG連接581nm量子點(diǎn),連接時(shí)間120min,最適pH值6.2,最適濃度20mg/mL,連接效率45%,并制備試劑1 (R1HCV)。兔抗HBsAg-IgG連接581nm量子點(diǎn),連接時(shí)間120min,最適pH值6.2,最適濃度10mg/mL,連接效率45%,并制備試劑2 (R2HCV)。3.量子點(diǎn)磁微粒單指標(biāo)檢測(cè)體系:于EP管內(nèi)同時(shí)加進(jìn)標(biāo)本50L,R1各100L,R2各5L,反應(yīng)體系pH值7.4,計(jì)算Cut off值為122和136(HBsAb與HCcAg),檢測(cè)范圍為2ng/mL-10g/mL、5ng/mL-5g/mL。檢測(cè)時(shí)間分別縮短到30min、

7、40min。4.量子點(diǎn)磁微粒體系單項(xiàng)檢測(cè)HBsAg和HCcAg標(biāo)準(zhǔn)品,批內(nèi)重復(fù)性實(shí)驗(yàn)均勻CV值為9.7%、9.8%;天間重復(fù)性實(shí)驗(yàn)均勻CV值為10.3%、10.1%。5.量子點(diǎn)磁微粒同步檢測(cè)體系:于EP管內(nèi)同時(shí)加進(jìn)標(biāo)本5100L,R1各100L,R2各5L,反應(yīng)體系pH值7.4,計(jì)算Cut off值為133/140,檢測(cè)范圍為2ng/mL-10g/mL,5ng/mL-5g/mL。檢測(cè)時(shí)間從1h/3h同步縮短到40min。6.對(duì)188份臨床標(biāo)本的同步檢測(cè)結(jié)果,HBsAg陽性:PCR方法36例,量子點(diǎn)-磁微粒法35例,ELISA方法34例。HCcAg陽性:PCR方法11例,量子點(diǎn)-磁微粒法11例,

8、 ELISA方法8例。對(duì)2例HBV與HCV混合感染的臨床標(biāo)本及5例標(biāo)準(zhǔn)品混合樣本成功檢出。HBsAg檢測(cè)結(jié)果與PCR方法比較,符合率98.4%,靈敏度94.4%,特異度99.3%;HCcAg檢測(cè)結(jié)果與ELISA方法比較符合率97.9%,靈敏度81.8%,特異度98.9%。7.同步檢測(cè)HBsAg和HCcAg標(biāo)準(zhǔn)品,批內(nèi)重復(fù)性實(shí)驗(yàn)均勻CV值為10.0%、10.1%;天間重復(fù)性實(shí)驗(yàn)均勻CV值為10.7%、10.6%。8.檢測(cè)肉眼可見的溶血、黃疸、脂血標(biāo)本,對(duì)結(jié)果無干擾。與其它常見致輸血后感染的病原體(TP、HAV)無交叉反應(yīng)。9.試劑R1、R2于4保存4周后使用,與新配置試劑檢測(cè)標(biāo)準(zhǔn)品結(jié)果無明顯差異

9、。結(jié)合目標(biāo)抗原后的復(fù)合物標(biāo)本,經(jīng)4放置4周后熒光強(qiáng)度無明顯衰減。最長放置8周后仍可檢出熒光。結(jié)論1.本研究利用不同量子點(diǎn)在相同激發(fā)光照射下可發(fā)射不同顏色熒光的特性,選用輸血傳染病中造成輸血后肝炎的兩種主要病原體HBV和HCV的抗原檢測(cè)為例,成功達(dá)到了同步檢測(cè)不同抗原的目的,為建立針對(duì)多種輸血傳染病病原體抗原的檢測(cè)體系建立奠定了基礎(chǔ)。同時(shí)由于針對(duì)抗原作檢測(cè),相對(duì)抗體檢測(cè)較大幅度提前了陽性檢出時(shí)間,避免了因抗體形成的窗口期造成的漏檢。2.以碳二亞胺法連接了不同源的抗體于羧基化磁微粒和量子點(diǎn)表面,以雙抗體夾心法檢測(cè)抗原,由于采用磁微粒作為固相載體,在不借助大型特殊設(shè)備的條件下獲得了較高的靈敏度,對(duì)H

10、BsAg與HCcAg檢測(cè)靈敏度分別為2ng/mL、5ng/mL。3.對(duì)臨床標(biāo)本的檢測(cè)結(jié)果與ELISA試劑比較無明顯性差異,但檢測(cè)時(shí)間明顯縮短,對(duì)HBsAg與HCcAg的檢測(cè)時(shí)間分別從ELISA所需的1h、3h左右同步縮短到40min-50min。研究結(jié)果顯示,抗原濃度與熒光強(qiáng)度呈正相關(guān),通過圖像軟件計(jì)算可做到半定量分析。4.試劑制備所需較短,標(biāo)本檢測(cè)后的復(fù)合物易保存,熒光漂白作用小,利于結(jié)果的復(fù)查和動(dòng)態(tài)監(jiān)測(cè)。');【Abstract】 Objective and significanceDuring blood donation and transfusing, HBV, HCV, H

11、IV-1, HIV-2 and syphilis testing are compulsorily required in China nowadays. Hepatitis B virus and Hepatitis C virus mostly caused the PTH (post transfusion hepatitis). And the clinical laboratories usually use ELISA (Enzyme linked immunosorbent assay) to analyze those pathogenic microorganisms. Mo

12、st of these assays are for antibody test and the delay of antibody production (window phase) is inevitably can not be checked out. Morever, the different of testing kits use caused the different testing time.As the Quantum dots (QDs) with different size can be inspired different fluorescence by mono

13、chromatic light and the Magnetic Microspheres (MMS) can be enriched in a magnetic field, we may establish and apply testing system with high sensitivity, specificity, efficiency and speed for the simultaneously detection of antigens in liquid. Materials and methods1. The conjugation of MMS and Ab (r

14、eady for Reagent 1, R1): Chose the 3m diameter Carboxyl MMS and use the carbodiimide cross-link technique activate the MMS with EDC and NHS in different concentration, then linked mice anti-HBsAg-IgG and mice anti-HCcAg-IgG on them.To view the effect with different concentration of Ab, bounding time

15、 and pH value which act on the conjugation of MMS and Ab. Then examine the ratio of conjugation by folin-phenol method.2. The conjugation of QDs and Ab (ready for Reagent 2, R2): Activate QDs with emission wavelengths at 581nm and 618nm by EDC/NHS at the volume proportion of 1:50. To view the effect

16、 with different concentration of Ab, bounding time and pH value which act on the conjugation of QDs and Ab. Then examine the ratio of conjugation by folin-phenol method.3. The single test systems: Use the QD-Ab as the fluorescence labels and the MMS-Ab as carriers to solid-liquid separate. After obt

17、ained the optimal value of react time we analyzed the negative serums to get the average of double negative control adds average of blank as cut off values. Then we used R1, R2 to analyze mixed standard antigen at different concentrations of HBsAg and HCcAg to determine the detection limits.4. To es

18、tablish the simultaneously detection system: On the basis of single test systems we combined the conditions and choose the best react time.Then analyzed the negative serums to get the average of double negative adds average of blank as cut off values. Because of the overlapping of MMS, we use the fi

19、lters to obtain the different colors of fluorescence partly. Then we used R1, R2 to analyze mixed standard antigen at different concentrations of HBsAg and HCcAg to determine the detection limits.5. 188 unknown samples were analyzed by the simultaneously detection system and compared the results wit

20、h ELISA.6. We analyzed the individual and mixed standards at concentration of 10ng/mL, 100ng/mL, 1g /mL as the repetitive experiments.7. The samples with hemolysis, jaundice and lipemia were analyzed as the interference factors and the positive HAV, TP serums as the specificity experiments.8. The fl

21、uorescence of prepared reagents and the complexs after reaction were recorded in 8 weeks to investigate the stabilities.Results1. The condition for the conjugation of MMS and Ab: The concentration of activators EDC at 6mg/L and NHS at 4mg/L for mice anti-HBsAg-IgG, bonding time t=120min, pH value 6.

22、6 and the concentration of Ab was 20mg/mL. EDC at 4mg /L and NHS at 4mg /L for rabbit anti-HCcAg-IgG, bounding time at 120min, pH value 6.6 and the concentration of Ab was 20mg/mL. The efficiency ratios of conjugation were 41% and 44%. Add 1% BSA in R1 and store at room temperature for 4 hours and w

23、ashed with pH7.4 PBS for use.2. The condition for the conjugation of QDs and Ab: Bonding time t=120min, pH value 6.2 and the concentration of rabbit-anti-HBsAg-IgG was 20mg/mL to QD581. Bonding time t=120min, pH value 6.2 and the concentration of rabbit anti-HCcAg-IgG was 10mg/mL to QD618. The effic

24、iency ratios of conjugation were 45% and 45%.3. The single test systems: Serum 50L; R1, 100L; R2, 5L (after enrichment), add in EP tube totally then react under the condition of pH7.4, 37for 20min and 30min (HBV and HCV). The cut off value were 122,136. The detecting ranges were 2ng/mL10g/mL for HBs

25、Ab and 5ng/mL5g/mL for HCcAg. The time of detection were shortened from 1h and 3h to 30min and 40min.4. The parallel test system: Serum 100L; Each R1, 100L and each R2, 5L (after enrichment), add in EP tube totally then react at the condition of pH7.4, 37for 40min. The cut off value were 133,140 of

26、HBsAg and HCcAg. The detecting ranges were 2ng/mL10g/mL for HBsAb and 5ng/mL5g/mL for HCcAg.5. The results of 188 unknown samples analysis: Positive HBsAg serums were 36, 35, 34 and HCcAg were 11, 11, 8 by PCR, parallel test system and ELISA. The mixed 5 standards and 2 multiple infection serums wer

27、e detected correctly. The accordance rates, sensitivity, specificity of parallel test system were 98.4%, 94.4%, 99.3% for HBsAg and 97.9%, 81.8%, 98.9% for HCcAg between PCR. The accordance rates of parallel test system were 95.8%, 98.4% between ELISA for HBsAb and HCcAg.6. Standards analyze results

28、 of repetitive experiments show that the average coefficient variation (CV) of within-run and the inter-day-run of single test systems were 9.7%, 9.8% and 10.3%, 10.1% for HBsAb and HCcAg. The CV of within-run were 10.0%, 10.1% and of the inter-day-run were 10.7%, 10.6% for HBsAb, HCcAg of parallel

29、test system7. The analysis of samples with hemolysis, jaundice and lipemia show theses factors do not interfere the results and the test of positive HAV, TP serums show a good specificity.8. It show that the using effect of reagents and the intensity of fluorescence of the complexes after reacted wh

30、ich stored at 4did not change obviously after 4weeks.Conclusions1. In this study, by using the speciality of the different QDs can be inspired different fluorescence by monochromatic light, we took HBV and HCV which mostly caused PTH as examples and established the method that can detect different antigens in parallel.This research may helpful to the foundation of the antigens detect system in parallel and ensure the transfusion security. Because of the aiming at antigen detect