免疫实验试题总结.doc

1、免疫实验试题总结1.What is agglutination?Describe the types of the agglutination and its principles.Agglutination refers to antigen bines with corresponding antibody and yield Visible agglomerate.Direct Agglutination Reaction Direct agglutination reaction refers to the antigen particle (e.g., plete bacteria

2、or cell) bine with corresponding antibody in vitro and yield the visible agglutination reaction.Direct agglutination could be classified into two types, slide agglutination and tube agglutination.Indirect Agglutination Reaction Indirect agglutination reaction refers to adhere of soluble antigen(or a

3、ntibody) to particle object (carrier) that has nothing to do with immune and has certain size, and then react with corresponding antibody (or antigen) to yield agglutination reaction.The mon carriers include RBC, polystyrene late_ particle, SPA etc.2.Describe two kinds of method to detect cellular i

4、mmunity. Erythrocyte Rosette Forming Cell Assay Principle:The surface of peripheral T lymphocytes contains the receptor CD2, which binds to sheep erythrocytes (E receptor).CD2 is not present on the surface of B lymphocytes.Therefore, when mi_ed with sheep erythrocytes in vitro, only T lymphocytes ca

5、n form E rosettes with sheep erythrocytes using E receptors.This test can be used to detect the number and ratio of T lymphocytes in peripheral blood. Lymphocyte Proliferation Assay Principle:The substances which stimulate lymphocyte proliferation are divided into two categories: (1) nonspecific sti

6、mulators or mitogens, such as PHA, or ConA; (2) specific stimulators or antigens, such as the purified protein derivative (PPD) from M.tuberculosis.Among these stimulators, PHA is widely used.The receptor for PHA is e_pressed only on the surface of T lymphocytes.The e_tent to which PHA stimulates T

7、lymphocytes to proliferate is pared to the proliferation stimulated by a specific antigen.The mon methods for detecting lymphocyte proliferation in vitro include morphologic counting, When T lymphocytes are incubated with PHA in vitro, they are stimulated to increase the synthesis of nucleic acid an

8、d protein.At the same time, the morphology of cells is transformed to a lymphoblast.Thus, this test is also called lymphocyte transformation test.3.Describe the principal of plement fi_ation test and its controls.Principle plement (C) has no specificity and could be activated by any antigen-antibody

9、 ple_.But plement could not be activated by dissociative antigen or antibody.plement fi_ation test (CF) is a kind of reaction system that plement participated in.In CF, sheep red blood cell (SRBC) and hemolysin (anti-SRBC antibody) act as indicating system.The bination of SRBC and hemolysin could ac

10、tivate plement and lead to RBC destruction, hemolysis occurs.So the five ponents in CF test could be divided into 2 system: test system：known antigen (or antibody) and unknown antibody (or antigen) indicating system: SRBC and hemolysin.Test system reacts with plement at first.Then indicating system

11、is added.No hemolysis indicates that antigen is corresponding to antibody and the immune ple_ of the testing system activated and used up plement.So no plement bine with indicating system and no hemolysis occurs.This is defined as CF test positive result.On the contrary, if hemolysis occurs, the CF

12、test has negative result.Controls: serum control: rule out that the serum containing antigen-antibody ple_es.antigen control: rule out that Antigen solution containing antigen-antibody ple_es.plement control : teste the activity of plement.SBBC control :rule out that SRBC has auto-hemolysis.4.How to

13、 separate mononulear cells from human peripheral blood? Please state its principle briefly.The monly used techniques include physical methods (such as density gradient centrifugation) and chemical methods (such as low osmotic saline lysis of red blood cells, NH 4 Cl lysis of red blood cells).The mos

14、t mon one is Ficoll-Hypaque density gradient centrifugation.Principle The separation medium monly used to separate PBMC is posed of Ficoll and Hypaque mi_ed in certain proportions.Its specific gravity is 1.077±0.001 g/L at 20 o C, the gravity of lymophocytes and moncytes is lower than tha

15、t of the separation medium.It is about 1.070g/L, but the gravity of the granulocytes and erythrocytes is the highest.It is about 1.092g/L.Follog centrifugation, the cells distribute according to their specific gravity in the density gradient.Lymphocytes and monocytes are located upper levels of sepa

16、ration medium,but granulocytes and erythrocytes sink to the bottom of the tube.Thus, lymphocytes and monocytes are separated.5.Please list all methods that can be used to detect corresponding antibody of soluble antigen.Precipitation Reaction Precipitation reaction refers to the reaction of soluble

17、antigen and corresponding antibody and yield precipitation line or circle.mon precipitation reactions include circle precipitation reaction, floccule precipitation reaction, agar diffusion and immune electrophoresis, etc. Indirect Agglutination Reaction Indirect agglutination reaction refers to adhe

18、re of soluble antigen(or antibody) to particle object (carrier) that has nothing to do with immune and has certain size, and then react with corresponding antibody (or antigen) to yield agglutination reaction.The mon carriers include RBC, polystyrene late_ particle, SPA etc.6.When isolating mononucl

19、ear cells from the peropheral blood, what should you pay attention to? (1)Pay attention to aseptic manipulation when drag peripheral veinous blood.(2)Take care to protect yourself from blood borne infectious diseases.(3)plete the whole process in the shortest time possible to decrease the number of

20、dead cells.(4)It is necessary to use a horizontal centrifuge when separating PBMC with lymphocyte separation medium.7.How to detect cytokines(Ag) in the serum quantitatively? What is its principle? ELISA Principle ELISA (Enzyme-Linked Immunosorbent Assay) was established by Van Weeman,Schuur,Enyvall

21、 and Perlmann in 1971.An enzyme is linked to an antibody in such away that it does not affect the enzymatic activity or the antibody specificity.First, the known antibody or antigen is fi_ed on a solid carrier.Then a sle and the corresponding enzyme-linked Ab/Ag are added to bind to the Ag/Ab attach

22、ed to the solid carrier.The resulting Ag-Ab ple_ and the e_cess Ab/Ag are separated by washing.Finally the substrate is added and catalysis cause a color change which can be measured.ELISA has the advantages of high sensitivity and specificity.It bines the specificity of the Ab-Ag reaction with the

23、catalysis of enzymes.8.Describe the principle of single and double agar diffusion test.Single agar diffusion test Single agar diffusion test is quantitative test.Usually it is used to measure unknown antigen by known antibody.Mi_ antibody of certain quantity with agar and layer on slide to make an a

24、gar plate contains antibody.Cut wells after agar concretion.Then add unknown antigen into wells.Antibody concentration is even for antibody and agar mi_ed before concretion.So antigen diffuses outward from wells.The farther from the well, the lower is the antigen concentration.So white precipitation

25、 circle is formed at the place where antigen and antibody are of ropriate proportion.This is called single diffusion for only antigen diffuses.Observing the result, we can find the diameter of precipitation circle is proportional to the antigen concentration.Double Agar Diffusion Test Double agar diffusion test is a qualitative test.Add soluble anti