AIM： We designed two synthetic-core-specific peptides core 1 （C1） and core 2 （C2）, and an E1-specific peptide （El）. We produced specific polyclonal antibodies against these peptides and used the antibodies for ...AIM： We designed two synthetic-core-specific peptides core 1 （C1） and core 2 （C2）, and an E1-specific peptide （El）. We produced specific polyclonal antibodies against these peptides and used the antibodies for detection of HCV antigens on surface and within infected peripheral blood leukocytes. METHODS： Peripheral blood from a healthy individual who tested negative for HCV RNA was incubated with HCV type 4 infected serum for i h and 24 h at 37 ℃. Cells were stained by direct and indirect immunofluorescence and measured by flow cytometry. RESULTS： After 1 h of incubation, antibodies against C1, C2, and El detected HCV antigens on the surface of 27%, 26% and 73% of monocytes respectively, while 10%, 5% and 9% of lymphocytes were positive with anti-C1, anti-C2 and anti-E1 respectively. Only 1-3% of granulocytes showed positive staining with anti-C1, anti-C2 and anti E1 antibodies. After 24 h of incubation, we found no surface staining with anti-C1, anti-C2 or anti-E1. Direct immunostaining using anti-C2 could not detect intracellular HCV antigens, after 1 h of incubation with the virus, while after 24 h of incubation, 28% of infected cells showed positive staining. Only plus strand RNA was detectable intracellularly as early as 1 h after incubation, and remained detectable throughout 48 h post-infection. Interestingly, minus RNA strand could not be detected after 1 h, but became strongly detectable intracellularly after 24 h post-infection. CONCLUSION： Monocytes and lymphocytes are the preferred target cells for HCV infection in peripheral blood leukocytes. Our specific anti-core and anti-E1 antibodies are valuable reagents for demonstration of HCV cell cycle. Also, HCV is capable of infecting and replicating in peripheral blood mononuclear cells as confirmed by detection of minus strand HCV RNA as well as intracellular staining of core HCV antigen.展开更多
目的建立流式细胞术(flow cytom etry,FCM)检测抗核抗体(an tinuclear an tibody,ANA)的方法。方法收集经间接免疫荧光法(ind irect imm unofluorescen t assay,IIF)测定的55例血清标本作为检测对象,36例健康体检者血清作为阴性对照,采...目的建立流式细胞术(flow cytom etry,FCM)检测抗核抗体(an tinuclear an tibody,ANA)的方法。方法收集经间接免疫荧光法(ind irect imm unofluorescen t assay,IIF)测定的55例血清标本作为检测对象,36例健康体检者血清作为阴性对照,采用提取的H e la细胞核作为靶抗原,对所有血清标本进行FCM分析。结果将阳性细胞百分率大于12%的标本定为ANA阳性。IFA和FCM两种方法阴性和阳性的总符合率为92.7%(51/55),FCM测定值随着ANA滴度的增高而增高,并大部分集中于某一个范围。结论FCM分析法具有省时、客观、可定量分析的特点,是ANA检测的一种新方法。展开更多
文摘AIM： We designed two synthetic-core-specific peptides core 1 （C1） and core 2 （C2）, and an E1-specific peptide （El）. We produced specific polyclonal antibodies against these peptides and used the antibodies for detection of HCV antigens on surface and within infected peripheral blood leukocytes. METHODS： Peripheral blood from a healthy individual who tested negative for HCV RNA was incubated with HCV type 4 infected serum for i h and 24 h at 37 ℃. Cells were stained by direct and indirect immunofluorescence and measured by flow cytometry. RESULTS： After 1 h of incubation, antibodies against C1, C2, and El detected HCV antigens on the surface of 27%, 26% and 73% of monocytes respectively, while 10%, 5% and 9% of lymphocytes were positive with anti-C1, anti-C2 and anti-E1 respectively. Only 1-3% of granulocytes showed positive staining with anti-C1, anti-C2 and anti E1 antibodies. After 24 h of incubation, we found no surface staining with anti-C1, anti-C2 or anti-E1. Direct immunostaining using anti-C2 could not detect intracellular HCV antigens, after 1 h of incubation with the virus, while after 24 h of incubation, 28% of infected cells showed positive staining. Only plus strand RNA was detectable intracellularly as early as 1 h after incubation, and remained detectable throughout 48 h post-infection. Interestingly, minus RNA strand could not be detected after 1 h, but became strongly detectable intracellularly after 24 h post-infection. CONCLUSION： Monocytes and lymphocytes are the preferred target cells for HCV infection in peripheral blood leukocytes. Our specific anti-core and anti-E1 antibodies are valuable reagents for demonstration of HCV cell cycle. Also, HCV is capable of infecting and replicating in peripheral blood mononuclear cells as confirmed by detection of minus strand HCV RNA as well as intracellular staining of core HCV antigen.
文摘目的建立流式细胞术(flow cytom etry,FCM)检测抗核抗体(an tinuclear an tibody,ANA)的方法。方法收集经间接免疫荧光法(ind irect imm unofluorescen t assay,IIF)测定的55例血清标本作为检测对象,36例健康体检者血清作为阴性对照,采用提取的H e la细胞核作为靶抗原,对所有血清标本进行FCM分析。结果将阳性细胞百分率大于12%的标本定为ANA阳性。IFA和FCM两种方法阴性和阳性的总符合率为92.7%(51/55),FCM测定值随着ANA滴度的增高而增高,并大部分集中于某一个范围。结论FCM分析法具有省时、客观、可定量分析的特点,是ANA检测的一种新方法。
