Chapter 10. 免疫学检测方法与免疫技术 一、概述 免疫学检验方法和免疫化学技术主要包括：

1. Chapter 10. 免疫学检测方法与免疫技术 一、概述 免疫学检验方法和免疫化学技术主要包括： 抗原抗体的制备、纯化和鉴定，免疫扩散、免疫电泳、免疫凝集试验、补体结合试验，免疫细胞分离、纯化和鉴定，免疫功能检测，细胞因子检测，放射免疫检测，免疫酶标检测，荧光和发光免疫技术，免疫组化实验方法，原位杂交免疫组化，免疫PCR技术，免疫微球的应用，免疫电镜技术，细胞凋亡的检测方法，膜受体分析，胞内钙镁浓度的测定和细胞间通讯，流氏细胞仪技术及应用等。 从上述内容可以看出，免疫技术是免疫学和物理、化学及电子信息和分子生物学理论和技术的结合产物。其应用涉及生命科学的各个领域，已成为现代医学和生物学研究工作不可缺少的有效工具。

2. 免疫技术的原理和特点： • 基于免疫应答的理论，即抗原抗体的特异性反应。基于免疫细胞的结构、应答特性和分子基础。 具有特异性 高度灵敏性； 可重复性；广泛适用性；快速反应性；可观察性；可定性、定量，即可控性；组化定位特性等特点。 概括起来可分为： • 沉淀反应，即可溶性抗原和抗体间的反应。 • 凝集反应，即颗粒性抗原和抗体间的反应。 • 免疫标记，即用酶、同位素、荧光素或电子致密物质标记。 • 免疫印渍，即用标记抗体与待测蛋白质印迹结合。 • 单抗及工程抗体技术，即分子生物技术。 • 流氏细胞术，即荧光标记，流体喷射，激光和能谱检测，电 • 脑分析。

3. Ag-Ab reactionsTests for Ag-Ab reactions

4. http://www.med.sc.edu:85/chime2/lyso-abfr.htm Source: Li, Y., Li, H., Smith-Gill, S. J., Mariuzza, R. A., Biochemistry 39, 6296, 2000 Nature of Ag/Ab Reactions • Lock and Key Concept • Non-covalent Bonds • Hydrogen bonds • Electrostatic bonds • Van der Waal forces • Hydrophobic bonds • Multiple Bonds • Reversible

5. Low Affinity High Affinity Ab Ab Ag Ag Affinity • Strength of the reaction between a single antigenic determinant and a single Ab combining site Affinity =  attractive and repulsive forces

6. [Ag-Ab] Keq = [Ag] x [Ab] Calculation of Affinity Ag + Ab  Ag-Ab Applying the Law of Mass Action:

7. Y Y Y Y Y Y Y 104 106 1010 Keq = Avidity Affinity Avidity Avidity • The overall strength of binding between an Ag with many determinants and multivalent Abs

8. Specificity • The ability of an individual antibody combining site to react with only one antigenic determinant. • The ability of a population of antibody molecules to react with only one antigen.

9. Cross reactions Anti-A Ab Anti-A Ab Anti-A Ab Ag B Ag C Shared epitope Similar epitope Ag A Cross Reactivity • The ability of an individual Ab combining site to react with more than one antigenic determinant. • The ability of a population of Ab molecules to react with more than one Ag

10. Ab excess Ag excess Equivalence – Lattice formation Factors Affecting Measurement of Ag/Ab Reactions • Affinity • Avidity • Ag:Ab ratio • Physical form of Ag

11. Tests Based on Ag/Ab Reactions • All tests based on Ag/Ab reactions will have to depend on lattice formation or they will have to utilize ways to detect small immune complexes • All tests based on Ag/Ab reactions can be used to detect either Ag or Ab

12. Agglutination Tests Lattice Formation

13. Qualitative agglutination test • Ag or Ab Y +  Y Y Agglutination/Hemagglutination • Definition - tests that have as their endpoint the agglutination of a particulate antigen • Agglutinin/hemagglutinin

14. 1/1024 1/256 1/512 1/128 1/16 1/64 1/32 Pos. 1/8 Neg. 1/4 1/2 Titer Patient 64 1 8 2 512 3 <2 4 32 5 128 6 32 7 4 8 Agglutination/Hemagglutination • Quantitative agglutination test • Titer • Prozone

15. 1/256 1/512 1/128 1/16 1/64 1/32 1/8 1/4 1/2 Agglutination/Hemagglutination • Definition • Qualitative test • Quantitative test • Applications • Blood typing • Bacterial infections • Fourfold rise in titer • Practical considerations • Easy • Semi-quantitative

16. Y Y +  Y Passive Agglutination/Hemagglutination • Definition - agglutination test done with a soluble antigen coated onto a particle • Applications • Measurement of antibodies to soluble antigens

17. Y Y + Y Y Y Y  Y Y Y Y Y Y Y Y Y Y Y Y Y Patient’s RBCs Coombs Reagent (Antiglobulin) Coombs (Antiglobulin)Tests • Incomplete Ab • Direct Coombs Test • Detects antibodies on erythrocytes

18. Step 1 Y +  Y Y Y Y Target RBCs Patient’s Serum Y Y Y Step 2 Y Y Y Y Y Y Y Y Y Y Y Y +  Y Y Y Y Coombs Reagent (Antiglobulin) Coombs (Antiglobulin)Tests • Indirect Coombs Test • Detects anti-erythrocyte antibodies in serum

19. Coombs (Antiglobulin)Tests • Applications • Detection of anti-Rh Ab • Autoimmune hemolytic anemia

20. Prior to Test Y Y +  Y Y Test Y + +  Y Patient’s sample Agglutination/Hemagglutination Inhibition • Definition - test based on the inhibition of agglutination due to competition with a soluble Ag

21. Agglutination/Hemagglutination Inhibition • Definition • Applications • Measurement of soluble Ag • Practical considerations • Same as agglutination test

22. Precipitation Tests Lattice Formation

23. Ab in gel Ag Ag Ag Ag Diameter2 Ag Concentration Radial Immunodiffusion (Mancini) • Method • Ab in gel • Ag in a well • Interpretation • Diameter of ring is proportional to the concentration • Quantitative • Ig levels

24. - + Ag Ag Ab Ag Ab Immunoelectrophoresis • Ab is placed in trough cut in the agar • Method • Ags are separated by electrophoresis • Interpretation • Precipitin arc represent individual antigens

25. Immunoelectrophoresis • Method • Interpretation • Qualitative • Relative concentration

26. - + Ab Ag Countercurrent electrophoresis • Method • Ag and Ab migrate toward each other by electrophoresis • Used only when Ag and Ab have opposite charges • Qualitative • Rapid

27. Radioimmuoassays (RIA)Enzyme-Linked Immunosorbent Assays (ELISA) Lattice formation not required

28. Prior to Test Y Y +  Labeled Ag Test Y Y + + +  Labeled Ag Patient’s sample Competitive RIA/ELISA for Ag • Method • Determine amount of Ab needed to bind to a known amount of labeled Ag • Use predetermined amounts of labeled Ag and Ab and add a sample containing unlabeled Ag as a competitor

29. Solid Phase Solid Phase Test Y Y + + +  Labeled Ag Patient’s sample Competitive RIA/ELISA for Ag • Method cont. • Determine amount of labeled Ag bound to Ab • NH4SO4 •  anti-Ig • Immobilize the Ab • Concentration determined from a standard curve using known amounts of unlabeled Ag • Quantitative • Most sensitive test

30. Labeled Anti-Ig Ab in Patient’s sample Y Y Ag Immobilized Solid Phase Solid Phase Non-Competitive RIA/ELISA • Ab detection • Immobilize Ag • Incubate with sample • Add labeled anti-Ig • Amount of labeled Ab bound is proportional to amount of Ab in the sample • Quantitative

31. Labeled Ab Ag in Patient’s sample Y Ag Y Immobilized Solid Phase Solid Phase Non-Competitive RIA/ELISA • Ag detection • Immobilize Ab • Incubate with sample • Add labeled antibody • Amount of labeled Ab bound is proportional to the amount of Ag in the sample • Quantitative

32. Tests for Cell Associated Antigens Lattice formation not required

33. Fluorochrome Labeled Ab Y Ag Tissue Section Immunofluorescence • Direct • Ab to tissue Ag is labeled with fluorochrome

34. Fluorochrome Labeled Anti-Ig Unlabeled Ab Y Y Ag Tissue Section Immunofluorescence • Indirect • Ab to tissue Ag is unlabeled • Fluorochrome-labeled anti-Ig is used to detect binding of the first Ab. • Qualitative to Semi-Quantitative

35. Flow Tip FL Detector Light Scatter Detector Laser Immunofluorescence • Flow Cytometry • Cells in suspension are labeld with fluorescent tag • Direct or Indirect Fluorescence • Cells analyzed on a flow cytometer

36. Two Parameter Histogram Green Fluorescence Intensity Red Fluorescence Intensity Immunofluorescence • Flow Cytometry cont. • Data displayed One Parameter Histogram Unstained cells FITC-labeled cells Number of Cells Green Fluorescence Intensity

37. Assays Based on Complement Lattice formation not required

38. No Ag Ag Patient’s serum Y Y Y Y Y Y Y Y Y Y Complement Fixation • Methodology • Standard amount of complement is added • Ag mixed with test serum to be assayed for Ab • Erythrocytes coated with Abs is added • Amount of erythrocyte lysis is determined Ag Ag