醫(yī)學(xué)免疫學(xué)實(shí)驗(yàn)：4 B淋巴細(xì)胞膜表面免疫球蛋白的檢測(cè)—直接熒光法

1、BackgroundMaterials and MethodsResultsDiscussion2SmIg 是B細(xì)胞的抗原識(shí)別受體，也是B細(xì)胞特異的表面標(biāo)志，用直接免疫熒光法可檢查出SmIg。用熒光素標(biāo)記的抗Ig 抗體，在一定條件下與淋巴細(xì)胞混合，熒光素標(biāo)記的抗Ig 抗體可以與B 細(xì)胞表面的Ig 結(jié)合，在熒光顯微鏡下觀察可見(jiàn)B 細(xì)胞膜上出現(xiàn)熒光。此方法可用來(lái)鑒定B 淋巴細(xì)胞。3實(shí)驗(yàn)材料實(shí)驗(yàn)材料:ICR 小鼠FITC標(biāo)記兔抗小鼠Ig抗體Hanks 液 (pH 7.4, 0.1% NaN3)離心管，移液器離心機(jī)4實(shí)驗(yàn)方法實(shí)驗(yàn)方法準(zhǔn)備細(xì)胞6取脾于6ml Hanks液中研磨輕柔混勻，吸取1ml細(xì)胞到離

2、心管1000rpm 離心，5min6Methods準(zhǔn)備細(xì)胞免疫熒光標(biāo)記熒光顯微鏡觀察加入 FITC標(biāo)記抗體 100l4 避光，孵育30min 洗2遍取脾于6ml Hanks液中研磨輕柔混勻，吸取1ml細(xì)胞到離心管1000rpm 離心，5min光鏡熒光顯微鏡B cells percentage in splenic lymphocyte = N(Fluo)N(Light)118256= 44.5%7B cells percentage in splenic lymphocyte44.5%Reported value:40%Fell A H, Preston P M. Int. J. Parasi

3、tol., 1992, 22(4): 491-501.55% (BALB/c mouse)Pellegrini A, Guiaz N, Aoki M P, et al. Int. immunol., 2007, 19(12): 1395-1402.8Experimental mistakes analysisPipetting too many times in washing step Weak signalExposure to light Weak signalSmear too thick in specimen preparation View cannot matchNo cont

4、rol group False positive移液多次洗滌步驟微弱信號(hào)；曝光微弱信號(hào)；涂抹太厚不易觀察；沒(méi)有對(duì)照組假陽(yáng)性Direct ImmunofluorescenceIndirect ImmunofluorescenceAdvantage Shorter sample staining time Simpler dual and triple labeling procedureAdvantage Greater sensitivity Commercially produced secondary antibodies are Inexpensive, availableDisadva

5、ntage Lower signal Less flexibility Higher costDisadvantage Potential for cross-reactivity9Application of Immunofluorescence12Application of immunofluorescenceDirect immunofluorescence (DIF)Skin/mucosaBullous pemphigoidPemphigus vulgarisLupusRenalPulmonary transplantationIndirect immunofluorescence

6、(IIF)Pemphigus vulgarisParaneoplastic pemphigusBullous and/or cicatricialpemphigoidOther fields of applicationsBone marrowCell surface markersInfective disordersViral (Influenza virus, EBV, adeno virus, herpes virus, varicella zoster, CMV and HIV)Bacterial (M. Pneumoniae, M. Tuberculosis, T. Pallidi

7、um)Parasitic (Entamoeba, Giardia, Cyclospora)Amyloid, Mucin, LipidArvind Babu R S, Chandrasekar P, Chandra K L, et al. J. Orofac. Sci., 2013, 5(1): 2.Limitations of immunofluorescenceFactors affect the quality of a fluorescence image:Nonspecific antibody binding / dilute antibodyNot proper handling of the biopsy specimenSignal is weakened due to exposure to light.10Dermatologic disease diagnosis:Immunofluore