免疫學技術在科研中的應用培訓講學

1、免疫學技術在科研中的應用抗原或抗體的檢測w抗原抗體反應的特點w抗原抗體反應的影響因素w常用的抗原抗體反應凝集反應w直接凝集反應 血型鑒定 肥大反應w間接凝集反應w間接凝集抑制試驗w微粒捕獲酶免疫分析技術間接凝集反應w將可溶性抗原或抗體吸附在與免疫無關的顆粒載體上，形成致敏顆粒，再與相應抗體或抗原進行反應產(chǎn)生的凝集反應，稱為間接凝集反應。w顆粒載體有紅細胞、聚苯乙烯乳膠顆粒、活性炭顆粒，而相應的凝集現(xiàn)象分別稱為間接血球凝集、間接乳膠凝集、間接炭粒凝集反應。微粒捕獲酶免疫分析技術w將已知特異性抗體致敏的免疫微粒與生物素親和素酶放大系統(tǒng)相結合，最后酶作用于熒光底物，使之發(fā)熒光，通過檢測熒光強度判斷待

2、測抗原的含量。w檢測腫瘤標記物CAl99、CEA、CAl25、AFP、性激素、甲狀腺素T-T+、葉酸、HIV抗體、HCG等微量可溶性抗原。沉淀反應w速率散射比濁法免疫比濁法w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 免疫印跡技術速率散射比濁法免疫比濁法w將已知抗體與相應抗原在液相中按一定比例混合形成可溶性免疫復合物，這些復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)。w抗體含量固定并處于抗體過剩時，免疫復合物的多少直接取決于抗原的濃度，抗原的終濃度通過標準品繪出的標準曲線查出。w提高了靈敏度，通過自動化，可同時檢測多個樣品并進行精確的定量

3、分析。w檢測前白蛋白、酸性蛋白酶、巨球蛋白、轉鐵蛋白、尿微量蛋白及IgG、IgM、IgA及補體，藥物含量分析。免疫印跡技術w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記蛋白轉移到固相載體膜上，再用標記的特異性的抗血清或單克隆抗體對蛋白質進行定性及定量分析的技術，其鑒定蛋白質的敏感性為15ng。w檢測可溶性抗原、細胞成分的鑒定與分析，檢測與自身變性細胞核成分結合的抗體(抗核抗體)，HIV的明確診斷。免疫印跡法的基本步驟w電泳分離蛋白抗原，SDS是一種陰離子去污劑，與蛋白質牢固結合，十二烷基磺酸根帶負電荷，使樣品中各種蛋白質與SDS形成SDS-蛋白質復合物，由于蛋白質表面

4、均帶有相同密度的負電荷，使分子構型幾乎相同(線性)。因此SDS-PAGE中，SDS多肽復合物的電泳遷移率只與其分子質量有關，而不受所帶電荷及分子形態(tài)的影響。w將SDS-PAGE分離到的蛋白條帶轉移至固相的硝酸纖維素膜上。w蛋白條帶可用酶、同位素標記的一抗或二抗進行特異性反應，加入顯色底物或放射自顯影以顯示結果。免疫標記技術w免疫酶測定法w免疫熒光技術w放射免疫測定法w免疫膠體金技術免疫酶測定法w是一種用酶標記一抗或二抗檢測特異性抗原或抗體的方法。w將抗原抗體反應的高度特異性與酶對底物的高效催化作用有效地結合起來，通過酶分解底物產(chǎn)生有色物質(也可作用于熒光底物，產(chǎn)生熒光)，肉眼觀察顏色深淺或酶標

5、儀測定光密度值(OD)，以反映抗原或抗體的含量。w本法靈敏度高，檢測可溶性抗原或抗體、組織或細胞表面特異性抗原。ELISAw雙抗體夾心法(sandwich assay) 檢測血清、腦脊液、胸、腹水等各種液相中的可溶性抗原w間接法 測定細胞及組織表面抗原酶聯(lián)免疫斑點試驗(enzymelinked immunospot assay，ELISPOT)w用已知細胞因子的抗體包被固相載體，加入待檢效應細胞，溫育一定時間后洗去細胞，如待檢效應細胞產(chǎn)生相應細胞因子，則與已包被的抗體結合，再加入酶標記抗該細胞因子抗體，加底物顯色。w一般選擇硝酸纖維素膜(NC)或聚偏二氟乙烯(PVDF)膜覆蓋微量反應板作為固相

6、，在分泌相應細胞因子的細胞所在局部呈現(xiàn)有色斑點，一個斑點表示一個分泌相應細胞因子的細胞，通過計數(shù)可推算出分泌某種細胞因子細胞的頻率。免疫熒光技術w用熒光素標記一抗或二抗，檢測特異性抗原或抗體的方法。w常用的熒光素有異硫氰酸熒光素(nuorescein isothiocyanate，F(xiàn)ITC)、藻紅蛋白(phycoerythrin，PE)等。在激發(fā)光的作用下，可直接發(fā)射熒光，前者發(fā)黃綠色熒光，后者發(fā)紅色熒光。Immunofluorescent stain of immunoglobulin G (IgG) showing linear pattern in Goodpastures syndro

7、me 天皰瘡免疫熒光染色天皰瘡免疫熒光染色放射免疫測定法w用放射性同位素標記抗原或抗體進行的免疫測定。w既有同位素的敏感性又有抗原抗體結合的特異性，同時具有重復性好、準確性高、標本用量少等優(yōu)點。w廣泛應用于激素、藥物等微量物質的檢測。免疫膠體金技術w氯金酸(HAuCl+)在還原劑作用下，產(chǎn)生分散狀態(tài)的膠體金顆粒。堿性條件下，金顆粒表面帶負電荷，與蛋白質正電荷基團結合。w膠體金可標記白蛋白、免疫球蛋白、糖蛋白、激素、脂蛋白、植物血凝素、卵白素等。大分子以單層形式吸附在金顆粒表面。w不同還原劑作用于氯金酸，產(chǎn)生的膠體金粒徑大小不相同(550nm)，小粒徑的膠體金由于穿透性好，電子密度高，常被用于免

8、疫電鏡技術。這些小粒徑的金顆粒，經(jīng)銀顯影液處理后，金粒子還原銀離子生成銀顆粒而吸附在金顆粒周圍呈黑褐色，從而放大了金顆粒的顯色效果，又稱免疫金銀法。w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，進一步提高靈敏度。w當膠體金的粒徑較大、濃度密集時肉眼水平即可觀察，即膠體金斑點滲濾試驗和膠體金斑點免疫層析試驗。免疫細胞的檢測w免疫細胞的分離 磁珠分離法 fluorescence-activated cell sorter，F(xiàn)ACSw免疫細胞功能的測定 T細胞 B細胞磁珠分離法w特異性分離所需淋巴細胞的方法。w將特異性抗體(如抗C

9、D3、抗CD4、抗CD8等)吸附在鐵顆粒(磁珠)上，加至細胞懸液中，具有相應抗原的細胞與磁珠上的特異性抗體結合。w反應管置于磁場中，鐵顆粒受磁場的吸引，攜帶有相應細胞的磁球吸附于靠近磁鐵的管壁上。w棄細胞懸液，重新解離細胞與磁珠。fluorescence-activated cell sorter，F(xiàn)ACST細胞鑒定及功能測定w使用酶、免疫熒光標記單抗進行鑒定w淋巴細胞轉化試驗wE花環(huán)形成試驗w混合淋巴細胞培養(yǎng)wCTL介導的細胞毒試驗抗原肽MHC分子四聚體技術tetramerw用生物素化的抗原肽MHC分子復合物與熒光標記的親合素結合，由于1個熒光素標記的親合素可結合4個生物素分子，能使4個MH

10、C抗原肽復合物形成一個復合體，將該復合體標記熒光素后，即成抗原特異性四聚體。w抗原特異性四聚體能與樣品中的特異性T細胞的TCR結合，由于四聚體能同時結合一個T細胞表面的4個TCR，親和力大大提高。用流式細胞術即可確定待檢標本中抗原特異性CTL細胞的頻率。wMHC分子可為工類或類分子，與抗原肽形成的四聚體復合物，可分別鑒定表達特異性TCR的CD8+T細胞及CD4+T細胞的頻率。B細胞的鑒定及功能測定w檢測B細胞分化抗原w測定B細胞產(chǎn)生抗體的能力w溶血空斑試驗wELISPOT細胞因子的檢測w生物活性檢測 細胞增生或增生抑制法 細胞病變抑制法 趨化作用測定法w免疫學檢測法w分子生物學技術基因敲除技術

11、和轉基因動物w應用基因同源重組，將外源有功能基因(基因組中原先不存在、或已失活的基因)，轉入細胞與基因組中的同源序列進行同源重組，插人到基因組中，在細胞內獲得表達。w通過同源重組產(chǎn)生目標基因缺失或失活的轉基因動物可以確定被敲除的基因在體內代謝過程中的作用，還可確定被敲除基因在分化、發(fā)育、生存等過程中的作用和必要性。w轉基因動物可以作為疾病模型。w可以用于藥物篩選的動物模型。w轉基因動物可作為“生物反應器”生產(chǎn)藥物。Th細胞在細胞在mCTL介導的腫介導的腫瘤保護中作用的研究瘤保護中作用的研究相關說明wOT-1 mice : 表達SIINFEKL特異性TCRwOVA：雞卵白蛋白wOVA CTL e

12、pitope: SIINFEKLwOVA Th epitope:ISQAVHAAHAEI-NEAGROVA （OVT）w RAG-1 KO mice:wC57BL/6 mice:wIFA/CFA/KLHwEG7 cell linewEL4 cell line相關步驟wmCTL and eCTL generation OT-1細胞轉輸RAG-1-基因敲除小鼠，2天后以 OT-1 TCR 特異性表位多肽 SIINFEKL 免疫；wspecific Th generation OVA 特異性和非特異性Th表位多肽免疫C57BL/6小鼠以產(chǎn)生特異性和非特異性Th細胞；wmCTL transfer 記憶

13、性CTL轉輸已產(chǎn)生特異性和非特異性Th的 C57BL/6 小鼠；wtumor challenge 接種表達OVA抗原的腫瘤細胞EG7；Generation of Ovalbumin-specific Memory CD8 T Cells.OT-1 lymph nodes cells were transferred to RAG-/- mice through tail vein injection, and mice were immunized with 50ug of SIINFEKL peptide in CFA 1 day later. At day 7 , 14 , 21, mic

14、e were sacrificed, and splenocytes or lymph nodes cells were isolated and analyzed. The percentage of CD44high, SIINFEKL-specific CD8 T cells was assessed by three-color FACS staining with SIINFEKL MHC class I tetramer and antibodies to CD8 and CD44. Plots shown are gated on the SIINFEKL tetramer po

15、sitive lymphocytes; values are mean percentages of CD44 cells within the SIINFEKL tetramer-positive population.CD8+ Memory T Cells Need Antigen-specific CD4+T-helper Cells to Achieve Tumor ProtectionOvalbumin CTL epitope (SIINFEKL)-specific T cells were parked in Rag-/- mice for 42 days to generate

16、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 incomplete Freunds adjuvant or 20 ug keyhole limpet hemocyanin (KLH) protein as control in incomplete Freunds adjuvant . Mice were challenged with ovalbumin-e

17、xpressing 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+KLHCTLThKLHControl0.00.20.40.60.81.0CTL+ThCTL+KLHCTLThKLHControlTumour weight (g)Absence of tumor protection in mice without anti

18、gen-specific T-helper 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

19、. 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 T-helper peptide to determine the presence of ovalbumin-specific CD8+ CTLs and CD4+ T-helper cells. In contrast to mCTLs, eCTLs do not n e e d

20、T h e l p t o k i l l t u m o r. 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). Ovalbumi

21、n-expressing 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 long-lived memor

22、y CD8+ T cells generated in C57BL/6J mice.Ovalbumin CTL epitope (SIINFEKL)-specific OT-1 cells were adoptively transferred to syngeneic C57BL/6 mice ; mice were immunized with SIINFEKL/CFA to activate ovalbumin-specific CD8+ T cells, and the mice were kept for 100 days to ensure that all transferred

23、 OT-1 are of long-lived memory phenotype before challenging with tumor. Eight days before tumor challenge, two groups of mice were immunized with 50 ug of OVT in incomplete Freunds adjuvant. Untreated C57BL/6 mice were also challenged with tumor as negative control. Mice were challenged under scruff

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