LOS诱导的特异性抗体分泌细胞的ELISPOT法检测

目的 :动态测定卡他性莫拉氏菌 (Moraxellacatarrhalis,M .cat)脱毒脂寡糖 (dLOS)蛋白质结合疫苗诱导的抗体分泌细胞的应答状态。方法 :以M .catdLOS蛋白质结合疫苗滴鼻免疫BALB/c小鼠。应用酶联免疫斑点试验 (ELISPOT)检测免疫小鼠不同免疫诱导部位和免疫效应部位 ,包括 :鼻相关淋巴组织 (NALT)、脾脏、颈部淋巴结、鼻内容物、肺脏和派伊尔氏结的特异性抗体分泌细胞 ,并同时测定血清、鼻冲洗液、肺泡灌洗液、唾液及粪便提取液中特异性IgA、IgG和IgM的水平。结果 :M .catdLOS蛋白质结合疫苗免疫小鼠的NALT、脾脏、颈部淋巴结、鼻内容物、肺脏和派伊尔氏结中 ,均测出分泌LOS特异抗体的抗体分泌细胞 ,以鼻内容物中IgA分泌细胞的数目最多 ,其次是在NALT和肺脏中 ,这与特异性抗体测定的结果相一致。结论 :M .catdLOS蛋白质结合疫苗经滴鼻免疫 ,能刺激产生LOS特异的黏膜和全身抗体分泌细胞的应答。ELISPOT试验具有快速、灵敏、特异的优点 ,为动态分析单个抗体分泌细胞应答规律提供了新方法。

Detection of LOS-specific antibody-secreting cells by ELISPOT assay

AIM: To detect dynamically the response of specific antibody-secreting cells elicited by a detoxified-lipooligosaccharide-cross-reactive mutant (dLOS-CRM) of diphtheria toxin conjugate vaccine for Moraxella catarrhalis (M.cat). METHODS: BALB/c mice were intranasally immunized with dLOS-CRM conjugate vaccine. The specific antibody-secreting cells responding to LOS of M. cat in different inductive and effective sites, including nasally associated lymphoid tissues (NALT), spleen, cervical lymph nodes (CLN), nasal passages (NP), lungs and Peyer's patches (PP) were detected by an enzyme-linked immunospot assay (ELISPOT). The levels of LOS-specific antibodies IgA, IgG and IgM in serum, nasal flush fluid, alveolar douche fluid, saliva and fecal extract were also detected by ELISA. RESULTS: Intranasal immunization with dLOS-CRM plus cholera toxin induced a significantly dose-dependent enhancement of immune response. LOS-specific antibody (IgA,IgG or IgM)-secreting cells were found in NALT, spleens, CLN, NP, lungs and PP with most LOS-specific IgA antibody-secreting cells located in nasal passages, and next, NALT and lungs. It was correlated well with the level of LOS-specific IgA, IgG or IgM antibody titers in nasal flush fluid, alveolar douche fluid, saliva, serum and fecal extract. CONCLUSION: dLOS-CRM can induce specific mucosal and systemic humoral immune response through intranasal immunization. ELISPOT assay is quick, sensitive, specific, and would be a very useful tool to analyze dynamically the mechanism of single antibody-secreting cell response.