Mutty C E, Timm E A, Stewart C C
Laboratory of Flow Cytometry, Roswell Park Cancer Institute, Buffalo, New York 14263, USA.
Cytometry. 1999 Aug 1;36(4):303-11. doi: 10.1002/(sici)1097-0320(19990801)36:4<303::aid-cyto4>3.0.co;2-#.
The combination of in situ PCR and cell phenotyping by antibody labeling (ISPCR/Flow) allows for the identification of cell subsets carrying a particular genetic sequence. ISPCR utilizes thermal cycling for genetic amplification, which can reduce the effectiveness of surface antibody labeling. This study explored and characterized the effects of thermal exposure on antibody labeling using CD4 and CD45.
Single temperature incubations and thermal cycling exposures were performed on leukocytes labeled with either direct antibody conjugates or with biotinylated antibodies and PE-streptavidin.
Fluorescence emission decreased above 70 degrees ( )C when cells were stained with directly conjugated antibodies or a biotinylated antibody and PE-streptavidin prior to high heat exposure. If counter stained with PE-streptavidin after heat, fluorochrome fluorescence was detectable. We tested a second CD4 clone, that provided poor results under similar labeling conditions, suggesting the combination of fixation and heat may have an epitope specific effect for the same cellular antigen.
Immunophenotyping can be combined with ISPCR, but each antibody must be tested to determine its efficacy. The denaturation of protein above 70 degrees C appears to be the main reason for loss of fluorescence. The best procedure is to first stain cells with a biotinylated antibody to an epitope that survives fixation and thermocycling. The cells are then subjected to the desired PCR procedure. Finally they are stained with a fluorochrome conjugated streptavidin.
原位聚合酶链反应（ISPCR）与抗体标记细胞表型分析（ISPCR/Flow）相结合，可用于鉴定携带特定遗传序列的细胞亚群。ISPCR利用热循环进行基因扩增，这可能会降低表面抗体标记的有效性。本研究探讨并表征了热暴露对使用CD4和CD45进行抗体标记的影响。
对用直接抗体偶联物或生物素化抗体及PE-链霉亲和素标记的白细胞进行单温度孵育和热循环暴露。
当细胞在高温暴露前用直接偶联抗体或生物素化抗体及PE-链霉亲和素染色时，荧光发射在70℃以上会降低。如果在加热后用PE-链霉亲和素复染，则可检测到荧光染料荧光。我们测试了第二个CD4克隆，在类似的标记条件下结果不佳，这表明固定和加热的组合可能对相同细胞抗原具有表位特异性作用。
免疫表型分析可与ISPCR相结合，但每种抗体都必须进行测试以确定其有效性。70℃以上蛋白质的变性似乎是荧光丧失的主要原因。最佳方法是先用生物素化抗体对在固定和热循环后仍能存活的表位对细胞进行染色。然后对细胞进行所需的PCR程序。最后用荧光染料偶联的链霉亲和素对其进行染色。
