免疫学技术 1

1、Chapter Immunotechnology,Section A Antibodies as research and diagnostic tools,抗体作为研究和诊断的工具,A variety of assays have been developed which provide specific qualitative and quantitative measurement of Ag or Ab, both of which are often of considerable research and clinical relevance. Ab to an organism

2、in the serum of a patient demonstrates infe- ction by the organism. Ab with defined specificity is used to determine the presence of disease associated antigens in a patient. As tools in molecular and cellular research, Abs permit localization and characterization of Ags.,Key notes,Methods for measu

3、ring antigen-antibody reactions have been well established and include those that have direct biologic relevance (Table 1). The combination of Ab with biologically active Ag (virus, toxin, enzyme and hormone) can be detected by neutralization of the virus infection, toxicity, enzymatic and hormone a

4、ctivity, respectively. Precipitation and agglutination have also been adapted fo development of several useful assays.,Antibody and assays,Table 1. Effects of combination of antigen and antibody Agglutination Antigenic particle + specific Ab results in aggregation of particles Precipitation Soluble

5、Ag + specific Ab results in lattice formation and precipitation C Activation Ag in solution or on particle + specific Ab results in activation of C Cytolysis Cell + anti-cell Ab + C may result in lysis of the cell Opsonization Antigenic particle + Ab + C enhances phagocytosis by Mo, M0, PMNs Neutral

6、ization Toxins, viruses, enzymes, etc. + specific Abs may result in their inactivation * C, Complement; Mo, monocytes; M0, macrophages; Ab, antibody; PMNs, polymorpho-nuclear cells,A variety of other assays have been developed which provide specific qualitative and quantitative measurement of Ag or

7、Ab for both research and diagnostic purposes.,Since the immune system recognizes and remembers virtually all Ag that are introduced into an individual, assays which demonstrate the presence of Ab to an organism in the serum of a patient have become a standard way of determining that the patient has

8、had contact with, was infected by, the organism,Diagnostic purpose,e.g the presence of Ab to HIV in the serum of a patient usually means that the patient has been infected with HIV.,Diagnose for AIDs,Alternatively, Abs with defined specificity (e.g. (to Ags associated with cancer cells) can be used

9、to determine the presence of disease associated Ags in a patient.,Abs are also extremely important tools in molecular and cellular research as they permit the localization and characterization of Ags.,Research,Section B precipitation and agglutination,沉淀和凝集,Combination of Ab with Ag results in latti

10、ce formation and precipitation if there is sufficient Ag and Ab (equivalence). These reactions are the basis for qualitative and quantitative assays for Ag or Ab, including radial immunodiffusion（辐射免疫扩散） and immunoelectrophoresis（免疫电泳）.,Key notes,Precipitation assays,The interaction of surface Ags o

11、n insoluble particles (e.g. cells) with specific Ab to these Ags results in agglutination of the particles. Agglutination can be used to determine blood types; the presence of Ab to bacteria in serum is an indication of previous or current infection; and in the Coombs test autoantibodies to erythroc

12、ytes can be assayed.,Key notes,Agglutination assays,As previously described , when there is both sufficient Ag and sufficient Ab, the combination of Ag and Ab proceeds until large aggregates are formed which are insoluble in water and precipitate (equivalence). The extent to which a lattice forms de

13、pends on the relative amounts of Ag and Ab present.,1. Precipitation assays,Lattice formation, and precipitation are the basis for several qualitative and quantitative assays for Ag or Ab. These assays are done in semisolid gels into which holes are cut for Ag and/or for Ab and diffusion occurs unti

14、l Ag and Ab are at equivalence and precipitate.,In radial immunodiffusion, Ab (e.g. horse anti-human IgG) is incorporated into the gel and Ag (e.g. human serum) is placed in a hole cut in the gel. Ag diffuses radially out of the well into the gel and interacts with the Ab forming a ring of precipita

15、tion, the diameter of which is related to the concentration of the Ag (Fig. 1).,Radial immunodiffusion,Fig. 1. Measurement ofAg by precipitation In gels. Ab-containing gel is placed on a glass or plastic surface. Holes are cut in the gel and filled with Ag which diffuses radially out of the well and

16、 interacts with the Ab in the gel. Soluble complexes are initially formed but as more Ag diffuses equivalence is reached resulting in a lattice and precipitation. The diameter of the precipitation ring Is related to the concentration of the Ag and, using known standards, can be quantitated and compa

17、red with the levels of Ag in other samples.,Similar assays have been developed in which a voltage gradient (electrophoresis) is used to speed up movement of Ag into the Ab containing gel (rocket immunoelectrophoresis火箭免疫电泳).,In immunoelectrophoresis, Ags (e.g. serum) are placed in a well cut in a ge

18、l (without Ab) and electrophoresed, after which a trough is cut in the gel into which Abs (e.g. horse anti-human) are placed. The Abs diffuse laterally（横向扩散） to meet diffusing Ag, and lattice formation and precipitation occur permitting determination of the nature of the Ags (Fig. 2).,immunoelectrophoresis,Fig.2 identification of antigens using gel electrophoresis. Ag(Ee.g. serum)is placed in a well cut in a gel and subjected to a voltage gradient which causes the various antigens to migrate different distances through the gel dependent on their charge. After electrophoresis, a tro