免疫学常用实验方法共63页
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待测样品的合理稀释
•不同类型的检测方法
2. Immunohistochemistry (免疫组化)
原理：抗原抗体反应，抗体标记技术（荧光、酶） 用途：组织或细胞内抗原的定性和定位
流程：
Copyright ©2019 American Association for Cancer Research
（1）固相的抗原或抗体，
即"免疫吸附剂"（immunosorbent）;
（2）酶标记的抗原或抗体，
称为"结合物"（conjugate）；
（3）酶反应的底物。
•ELISA基本的实验过程
a.包被 b.抗原抗体反应 c.酶促反应,显色 d.终止显色，读取数据。 •对照和标准曲线
阳性对照 阴性对照 定量测定：标准品制作标准曲线
Specificity 特异性
Reversibility可逆性
Ag+Ab→Ag·Ab
解离后的抗原或抗体均能保持原有的结构和活性。 在一定条件下(如低pH、高浓度盐等)可以解离。
Ratio最适比例
Sensitivity 敏感性
化学比色法： mg/ml
酶反应测定法： 5-10μg/ml (免疫测定中凝胶扩散法和浊度法的敏感度与酶反应法相仿)
Figure 3. mTNF induces infiltration of innate immune cells and angiostasis in tumors from TNFR1–/– but not TNFR1–/–/R2–/– mice. A to F, TNFR1–/– mice or TNFR1–/–/R2–/– mice were injected s.c. with 5 x 106 of J-mTNF10 cells. Ten days after tumor cell challenge, tissue sections of the injection site were stained for Mac-1+ (A and B), Gr-1+ (C and D), and CD31+ (E and F) cells as indicated. G and H, for confocal microscopy analysis, tissue sections were stained with Cy3-labeled anti-CD31 for blood vessel endothelial cells (green) and the VasoTACS in situ kit to detect apoptosis (red). Arrows, apoptotic endothelial cells in the tumor.
•Ag-Ab reation:
ELISA, Immunohistochemistry, FACS, Western blot, Hybridoma & monoclonal antibody
•Cell function: 3H－TdR proliferation MTT CFSE Cytotoxicity 51Cr
•Animal experiment
I. Antigen-Antibody Reaction
非非共共价价键键结合结合 抗体的互补决定区 (CDRs)
抗原决定簇(antigenic determinant) 又称表位 (epitope)
Complementarity-determining regions (CDRs):
Lequin, R. M. Clin Chem 2019;51:2415-2418 Copyright ©2019 American Association for Clinical Chemistry
ELISA
•基础: 抗原或抗体的固相化 抗原或抗体的酶标记
•用途：目标蛋白的定性或定量分析
•三个必要试剂：
Estimates of the number of articles published per 5-year period from 1960 to 2019
Figure. Estimates of the number of articles published per 5-year period from 1960 to 2019. The search was done in February 2019 in PubMed/National Library of Medicine, NIH, with the search terms: enzymeimmunoassay, enzyme-linked immunosorbent assay (EIA/ELISA combined), and RIA. (Ordinate), number of articles in which the keywords are quoted. (Abscissa), 5-year periods from 1960 to 2019. , combined EIA/ELISA; , RIA. [Note: I do not pretend that the numbers in this figure are precise; the trends, however, are evident.]
CDR1 CDR2
CDR1
CDR2 CDR3
CDR3
H
FR1
CDR1 FR2 CDR2 FR3
CDR3
FR4
L
CDR: Complementarity Determining Region FR: Framework Region
Antigenic Determinant
Features of Ag-Ab Reaction
标记的免疫测定法： ng/ml 水平 (例如，用放射免疫测定法或酶免疫测定法测定HBsAg，其 敏感度可达0.1ng /ml)
Often used methods
ELISA------酶联免疫吸附试验 Immunohistochemistry --------免疫组织化学 FACS------流式细胞术 Western Blotting-------免疫印迹技术
1. ELISA(酶联免疫吸附试验 Enzyme Linked ImmunoSorbent Assay)
RIA (radioimmunoassay, Berson&Yalow， 1960)
IRMA(immunoradiovelric assayபைடு நூலகம் Miles&Hiles, 1968)
E(L)I(S)A (Engvall&Perlmann, van Weemen and Schuurs , 1971)
•不同类型的检测方法
2. Immunohistochemistry (免疫组化)
原理：抗原抗体反应，抗体标记技术（荧光、酶） 用途：组织或细胞内抗原的定性和定位
流程：
Copyright ©2019 American Association for Cancer Research
（1）固相的抗原或抗体，
即"免疫吸附剂"（immunosorbent）;
（2）酶标记的抗原或抗体，
称为"结合物"（conjugate）；
（3）酶反应的底物。
•ELISA基本的实验过程
a.包被 b.抗原抗体反应 c.酶促反应,显色 d.终止显色，读取数据。 •对照和标准曲线
阳性对照 阴性对照 定量测定：标准品制作标准曲线
Specificity 特异性
Reversibility可逆性
Ag+Ab→Ag·Ab
解离后的抗原或抗体均能保持原有的结构和活性。 在一定条件下(如低pH、高浓度盐等)可以解离。
Ratio最适比例
Sensitivity 敏感性
化学比色法： mg/ml
酶反应测定法： 5-10μg/ml (免疫测定中凝胶扩散法和浊度法的敏感度与酶反应法相仿)
Figure 3. mTNF induces infiltration of innate immune cells and angiostasis in tumors from TNFR1–/– but not TNFR1–/–/R2–/– mice. A to F, TNFR1–/– mice or TNFR1–/–/R2–/– mice were injected s.c. with 5 x 106 of J-mTNF10 cells. Ten days after tumor cell challenge, tissue sections of the injection site were stained for Mac-1+ (A and B), Gr-1+ (C and D), and CD31+ (E and F) cells as indicated. G and H, for confocal microscopy analysis, tissue sections were stained with Cy3-labeled anti-CD31 for blood vessel endothelial cells (green) and the VasoTACS in situ kit to detect apoptosis (red). Arrows, apoptotic endothelial cells in the tumor.
•Ag-Ab reation:
ELISA, Immunohistochemistry, FACS, Western blot, Hybridoma & monoclonal antibody
•Cell function: 3H－TdR proliferation MTT CFSE Cytotoxicity 51Cr
•Animal experiment
I. Antigen-Antibody Reaction
非非共共价价键键结合结合 抗体的互补决定区 (CDRs)
抗原决定簇(antigenic determinant) 又称表位 (epitope)
Complementarity-determining regions (CDRs):
Lequin, R. M. Clin Chem 2019;51:2415-2418 Copyright ©2019 American Association for Clinical Chemistry
ELISA
•基础: 抗原或抗体的固相化 抗原或抗体的酶标记
•用途：目标蛋白的定性或定量分析
•三个必要试剂：
Estimates of the number of articles published per 5-year period from 1960 to 2019
Figure. Estimates of the number of articles published per 5-year period from 1960 to 2019. The search was done in February 2019 in PubMed/National Library of Medicine, NIH, with the search terms: enzymeimmunoassay, enzyme-linked immunosorbent assay (EIA/ELISA combined), and RIA. (Ordinate), number of articles in which the keywords are quoted. (Abscissa), 5-year periods from 1960 to 2019. , combined EIA/ELISA; , RIA. [Note: I do not pretend that the numbers in this figure are precise; the trends, however, are evident.]
CDR1 CDR2
CDR1
CDR2 CDR3
CDR3
H
FR1
CDR1 FR2 CDR2 FR3
CDR3
FR4
L
CDR: Complementarity Determining Region FR: Framework Region
Antigenic Determinant
Features of Ag-Ab Reaction
标记的免疫测定法： ng/ml 水平 (例如，用放射免疫测定法或酶免疫测定法测定HBsAg，其 敏感度可达0.1ng /ml)
Often used methods
ELISA------酶联免疫吸附试验 Immunohistochemistry --------免疫组织化学 FACS------流式细胞术 Western Blotting-------免疫印迹技术
1. ELISA(酶联免疫吸附试验 Enzyme Linked ImmunoSorbent Assay)
RIA (radioimmunoassay, Berson&Yalow， 1960)
IRMA(immunoradiovelric assayபைடு நூலகம் Miles&Hiles, 1968)
E(L)I(S)A (Engvall&Perlmann, van Weemen and Schuurs , 1971)