定量ELISA检测小鼠血清中抗LDH-C4IgG2b抗体的水平

用定量EL ISA 的方法检测背部携带分泌抗LDH - C4 IgG2b 抗体杂交瘤小鼠的血清中IgG2b 抗体浓 度, 结果表明: 本法稳定, 批间变异系数为7104 %～13130 % , 批内变异系数为3161 %～10120 %; 标准曲线 相关性良好, 相关系数从01962 884～01996 795 ; 灵敏度较高, 最低检出浓度为0101 mg/ L ; 特异性亦较好.

Circulating immunoglobulin A- and immunoglobulin G-secreting hybridoma cells in peripheral blood preferably migrate to female genital tracts. The role of sex hormones

Antigen-specific circulating immunoglobulin-secreting cells (ISC) migrate to various secondary and tertiary lymphoid tissues. To understand the migration of the cells into the genital tract and its regulation by sex hormones, spleen-derived SG2 hybridoma cells secreting immunoglobulin G2b (IgG2b) and Peyer's patch-derived PA4 hybridoma cells secreting polymer IgA were labelled with (3) H-TdR, and intravenously injected into syngeneic mice of both sexes. Using flow cytometry, surface molecular markers of plasma cells, CD38 and CD138, and adhesion molecules, CD49d, CD162, and CD11a were found to be positive in SG2 and PA4 cells, but CD62L, alpha4beta7 and CD44 were not expressed on these cells. The relative distribution indexes (RDIs) of the cells in genital tract and other tissues were measured. The means of RDIs of SG2 and PA4 cells in female genital tissues were 6.5 and 4.5 times as many as the means in male genital tissues, respectively. The treatment of ovariectomized mice with beta-oestradiol significantly increased the RDIs of PA4 cells in cervix and vagina, but decreased the RDIs of SG2 cells in vagina, horn of uterus, uterus and rectum (P <0.05). Progesterone treatment increased the RDIs of PA4 cells in vagina and rectum (P <0.05). The treatment with testosterone significantly increased the RDIs of SG2 and PA4 cells in epididymis and accessory sex glands (P <0.05). These results demonstrate that the female genital tract is the preferable site for the migration of circulating hybridoma cells to the male genital tract, and sex hormones play an important role in regulation of the migration of circulating ISC to genital tracts.

Migration of mouse antibody-secreting hybridoma cells from blood to genital tract and its regulation by sex hormones are associated with the differential expression patterns of adhesion molecules and chemokines in the tract rather than in the antibody-secreting cells

To understand better the molecular mechanisms of differential migration of antibody-secreting cells (ASCs) into mouse genital tracts, and regulation by sex hormones, surface markers, hormone receptors and adhesion molecules in mouse SG2 and PA4 hybridoma cells, respectively, secreting IgG2b and polymeric IgA antibody were detected by flow cytometry or RT-PCR. Semiquantitative RT-PCR was also used for measuring mRNA expression of adhesion molecules and chemokines (VCAM-1, ICAM-1, P-selectin, JAM-1 and CXCL12) in genital tracts of various adult mouse groups. The mRNAs of androgen receptor, estrogen receptor beta and CXCR4 were expressed in the ASCs. Sex hormones had no effect on expression of these molecules in ASCs. Except for VCAM-1, mRNA of all examined genes was expressed in normal mouse genital tracts. The mean of relative amounts of ICAM-1 and CXCL12 mRNA in all examined organs of females were higher (2.1- and 1.9-fold) than those in males. After orchiectomy or ovariectomy, the expression of ICAM-1, CXCL12 and P-selectin mRNA in the examined organs increased, except JAM-1 in male and CXCL12 in female. Sex hormone treatment recovered the changes to normal levels of mRNA expression in many examined genital tissues. In combination with our previous work, preferential migration of ASCs into female genital tract and regulation of migration by sex hormones are associated with expression patterns of adhesion molecules and chemokines in genital tract rather than in ASCs. (C) 2006 Elsevier Ireland Ltd. All rights reserved.

Characterization and application of monoclonal antibodies against turbot (Scophthalmus maximus) rhabdovirus

Five monoclonal antibodies (mAbs), 1G8, 1H9, 2D2, 2D3, and 2F5, against Scophthalmus maximus rhabdovirus (SMRV) were prepared. Characterization of the mAbs included indirect enzyme-linked immunosorbent assay, isotyping, viral inhibition assay, immunofluorescence staining of virus-infected cell cultures, and Western blot analysis. Isotyping revealed that 1G8 and 1H9 were of the IgG2b subclass and that the other three were IgM. 2D2, 2D3, and 2F5 partially inhibited SMRV infection in epithelioma. papulosum cyprinid (EPC) cell culture. Western blotting showed that all five mAbs could react with two SMRV proteins with molecular masses of approximately 30 kDa (P) and 26 kDa (M). These two proteins were localized within the cytoplasm of SMRV-infected EPC cells by immunofluorescence assay. Also, progressive foci of viral replication in cell cultures were monitored from 6 to 24 h, using mAb 2D3 as the primary antibody. A flow cytometry procedure was used to detect and quantify SMRV-infected (0.01 PFU/cell) EPC cells with mAb 2D3, and 10.8% of cells could be distinguished as infected 36 h postinfection. Moreover, mAb 2D3 was successfully applied for the detection of viral antigen in cryosections from flounder tissues by immunohistochemistry tests.

抗 LDH－C4 IgA 和IgG 抗体在粘膜免疫系统的分布及其抗生育作用的研究

已有的研究表明，小鼠背部携带能分泌抗原特异的IgA 单克隆抗体的杂交细 胞瘤，可以保护小鼠抵抗微生物和病毒等多种病原体经粘膜途径感染机体。我们 利用背部携带能分泌抗精子特异抗原（LDH-C4）的IgA 和IgG 杂交细胞瘤、以 及抗DNP 的IgA 骨髓细胞瘤的小鼠为动物模型，采用定量ELISA 法研究了抗 LDH－C4 IgA 与抗DNP IgA 单克隆抗体在呼吸道、肠道及生殖道内转运和分布， 抗LDH-C4 IgG2b 在肠道内转运与分布，以及抗LDH-C4 IgA 和IgG 单克隆抗体 在体内抗生育作用。 研究结果表明，带瘤小鼠血液中含有较高水平抗原特异的 IgA 和IgG 单克 隆抗体。PA4 和MOPC IgA 单克隆抗体在呼吸道、肠道以及雌性生殖道分泌物 内有较高的分布水平。在肠道，PA4 和MOPC IgA 单克隆抗体的分布水平显著 高于IgG（p<0.01 和p<0.05）。在肠道和生殖道的不同部位，IgA 抗体的分布水 平不同。在肠道，结肠分泌物中的IgA 单克隆抗体显著高于其它肠道部位 （p<0.01）。在生殖道，IgA 单克隆抗体分布水平以子宫角分泌物中最高。雄性 的前列腺也有较高的IgA 抗体水平。在呼吸道、肠道以及雌性生殖道相应部位的 分泌物内，PA4 IgA 单克隆抗体的水平显著高于MOPC IgA 单克隆抗体的分布水 平（p<0.05）。PA4 和MOPC IgA 单克隆抗体在粘膜分泌物内的分布水平差异可 能与其IgA 聚合形式的不同有关。另外，除气管外，在两时间点间分泌物中的 IgA 抗体水平没有显著差异。 检测背部带瘤小鼠交配后的两细胞胚胎期，发现携带PA4 和G2b 杂交细瘤 的雌性小鼠的受精率与对照组并没有显著的差异，这表明抗LDH-C4 IgA 和IgG 单克隆抗体在体内不能明显抑制小鼠的精子和卵子的结合或受精过程。注射细 胞后的27 天，检测鼠着床胚胎时，发现带瘤两性小鼠均携带PA4 时或者只有 雌性携带PA4 杂交瘤时，以及雌雄两性小鼠均携带G2b 杂交瘤细胞时，交配后的怀孕率与能分泌抗DNP 抗体的MOPC 的骨髓瘤细胞瘤的相应组别相比，显 著降低（p<0.01）。但PA4 各组与G2b 各组之间无显著差异(p>0.05）。然而，雌 雄的小鼠均带瘤时，最高怀孕减少率未能达100%。这些结果提示，抗LDHC4 IgA 和IgG 单克隆抗体在小鼠体内不能有效地抑制小鼠的精子与卵子的结 合，但能显著地抑制小鼠受精后胚胎的发育。抗LDH-C4 的IgA 和IgG 单克隆 抗体单独存在时，在体内均具有抗生育作用，但不能完全抑制生育。

抗LDH-C4 IgA和IgG抗体在带瘤小鼠生殖道和其它粘膜分泌物内分布特性的研究

中文摘要 　 已有的研究表明，小鼠背部携带能分泌抗原特异的IgA单克隆抗体的杂交细胞瘤，可以保护小鼠抵抗微生物和病毒等多种病原体经粘膜途径感染机体。我们利用背部携带能分泌抗精子特异抗原（LDH-C4）的IgA和IgG杂交细胞瘤、以及抗DNP的IgA骨髓细胞瘤的小鼠为动物模型，采用定量ELISA法研究了抗LDH-C4 IgA与抗DNP IgA单克隆抗体在呼吸道、肠道及生殖道内转运和分布，抗LDH-C4 IgG2b在肠道内转运和分布，以及抗LDH-C4 IgA和IgG单克隆抗体与体内抗生育作用的关系。研究结果表明，带瘤小鼠血液中含有较高水平抗原特异的IgA和IgG单克隆抗体。PA4和MOPC IgA单克隆抗体在呼吸道、肠道以及雌性生殖道分泌物内有较高的分布水平。在肠道，PA4和MOPC IgA 单克隆抗体的分布水平显著高于IgG（p < 0.01和p < 0.05）。在肠道和生殖道的不同部位，IgA抗体的分布水平不同。在肠道，结肠分泌物中的IgA单克隆抗体显著高于其它肠道部位（p < 0.01）。在生殖道，IgA单克隆抗体分布水平以子宫角分泌物中最高。雄性的前列腺也有较高的IgA抗体水平。在呼吸道、肠道以及雌性生殖道相应部位的分泌物内，PA4 IgA单克隆抗体的水平显著高于MOPC IgA单克隆抗体的分布水平（<0.05）。PA4和MOPC IgA单克隆抗体在粘膜分泌物内的分布水平差异可能与其IgA聚合形式的不同有关。另外，除气管外，在两时间点间分泌物中的IgA抗体水平没有显著差异。检测背部带瘤小鼠交配后的两细胞胚胎期，发现携带PA4或G2b杂交细胞瘤的雌雄小鼠的受精率与对照组并没有显著性差异，这表明抗LDH-C4 IgA和IgG单克隆抗体在体内不能显著抑制小鼠的精子和卵子的结合或受精过程。注射细胞后的27天，检测着床胚胎时，发现带瘤两性小鼠均携带PA4时或者只有雌性携带PA4杂交瘤时，以及雌雄性小鼠均携带G2b杂交瘤时，交配后的怀孕率与带能分泌抗DNP抗体的MOPC骨髓瘤细胞瘤的相应组别相比，显著降低（p < 0.01）。但PA4各组与G2b各组之间无显著差异（p < 0.05）。然而，雌雄小鼠均带瘤时，最高怀孕减少率也未达到100%。这些结果提示，抗LDH-C4 IgA和IgG单克隆抗体在小鼠体内不能有效地抑制小鼠的精子和卵子的结合，但能显著地抑制小鼠受精后胚胎的发育。抗LDH-C4 的IgA或者IgG单克隆抗体单独存在时，在小鼠体内均具有抗生育作用，但不能完全抑制生育。