免疫學(xué)技術(shù)在科研中的應(yīng)用-wenkub.com
2025-08-02 02:35 本頁面
【正文】 values are mean percentages of CD44 cells within the SIINFEKL tetramerpositive population. CD8+ Memory T Cells Need Antigenspecific CD4+Thelper Cells to Achieve Tumor Protection Ovalbumin CTL epitope (SIINFEKL)specific T cells were parked in Rag/ mice for 42 days to generate mCTL and then adoptively transferred to C57BL/6 mice . Two groups of recipient C57BL/6J mice were immunized 8 days prior with 50 ug of OVT in inplete Freund’s adjuvant or 20 ug keyhole limpet hemocyanin (KLH) protein as control in inplete Freund’s adjuvant . Mice were challenged with ovalbuminexpressing tumor cells (EG7) in the scruff of the neck 1 day after adoptive transfer. Normal C57BL/6 control mice were challenged with EG7 without any treatment. CTL+ThCTL+KLHCTLThKLHControl0. 00. 20. 40. 60. 81. 0CT L + T hCT L + K L HC T LThK L HCo n tr o lTumour weight (g)Absence of tumor protection in mice without antigenspecific Thelper cells is not because of lower levels of tumor antigen (SIINFEKL)specific CD8+ T cells. mCTLs were transferred to groups of C57BL/6 mice with or without immunization 8 days prior with 50 ug of OVT or with 20 ug of KLH protein control. C57BL/6 control group was given no treatment. IFN Elispot assays were performed using the splenocytes of these mice, which were challenged in vitro with (A) 1 ug/ml SIINFEKL peptide or (B) 8ug/ml OVA Thelper peptide to determine the presence of ovalbuminspecific CD8+ CTLs and CD4+ Thelper cells. In contrast to mCTLs, eCTLs do not need T help to kill tumor. eCTLs generated from RAG/ mice were transferred at (A) day 7 or (B) day 14 to C57BL/6 mice that had been immunized 8 days earlier with 50 ug of OVT or as control 20 ug of KLH. C57BL/6 mice without any treatment were used as controls (EG7 and EL4 controls). Ovalbuminexpressing tumor cells (EG7 ) were injected under scruff of the neck of some groups of mice at (A) day 7 or (B) day 14. The parent tumor cell line EL4 that does not contain ovalbumin gene was injected into other groups of mice at day7 (A) as nonspecific tumor control. Study of the longlived memory CD8+ T cells generated in C57BL/6J mice. Ovalbumin CTL epitope (SIINFEKL)specific OT1 cells were adoptively transferred to syngeneic C57BL/6 mice 。 ? 轉(zhuǎn)基因動物可以作為疾病模型。用流式細(xì)胞術(shù)即可確定待檢標(biāo)本中抗原特異性 CTL細(xì)胞的頻率。 ? 反應(yīng)管置于磁場中，鐵顆粒受磁場的吸引，攜帶有相應(yīng)細(xì)胞的磁球吸附于靠近磁鐵的管壁上。 ? 膠體金顏色隨顆粒大小而變化，大于 20 nm的金顆粒在光鏡下呈現(xiàn)磚紅色，可在光鏡水平行免疫分析，也可用銀顯影劑增強，進(jìn)一步提高靈敏度。 ? 膠體金可標(biāo)記白蛋白、免疫球蛋白、糖蛋白、激素、脂蛋白、植物血凝素、卵白素等。 ? 既有同位素的敏感性又有抗原抗體結(jié)合的特異性，同時具有重復(fù)性好、準(zhǔn)確性高、標(biāo)本用量少等優(yōu)點。 ? 常用 的熒光素有異硫氰酸熒光素(nuorescein isothiocyanate， FITC)、藻紅蛋白 (phycoerythrin， PE)等。 ? 本法靈敏度高，檢測可溶性抗原或抗體、組織或細(xì)胞表面特異性抗原。 ? 將 SDSPAGE分離到的蛋白條帶轉(zhuǎn)移至固相的硝酸纖維素膜上。 免疫印跡技術(shù) ? 將用 SDS— 聚丙烯酰胺凝膠電泳 PAGE分離得到的按分子量大小排列的非標(biāo)記蛋白轉(zhuǎn)移到固相載體膜上，再用標(biāo)記的特異性的抗血清或單克隆抗體對蛋白質(zhì)進(jìn)行定性及定量分析的技術(shù)，其鑒定蛋白質(zhì)的敏感性為 1— 5ng。 沉淀反應(yīng) ? 速率散射比濁法／免疫比濁法 ? 瓊脂擴散法 ? 單向瓊脂擴散 ? 雙向瓊脂擴散 ? 火箭電泳 ? 對流免疫電泳 ? 免疫印跡技術(shù) 速率散射比濁法／免疫比濁法 ? 將已知抗體與相應(yīng)抗原在液相中按一定比例混合形成可溶性免疫復(fù)合物，這些復(fù)合物微小粒子對一定波長的光照射發(fā)生散射，可通過三個光路系統(tǒng)測定光散射(OD值 )。 常用的抗原抗體反應(yīng) ? 凝集反應(yīng) ? 沉淀反應(yīng) ? 免疫標(biāo)記技術(shù) 凝集反應(yīng) ?
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