实验三 酶联免疫吸附试验（ELISA).pptVIP

Exp Ⅴ 1. Enzyme-linked Immunosorbent Assay（ELISA)2. Complement fixation test (CFT) 1. Enzyme-linked Immunosorbent Assay （ELISA) Immunological labeling techniques Definition: Ag-Ab reactions are combined with labeling techniques. Known Ag or Ab is labeled with radioisotope, enzyme or fluorescein and unknown Ab or Ag are indirectly detected by labeled molecules. Classification: Radioimmunoassay(RIA): 131I，32P Immunofluorescence technique: FITC，PE Enzyme Immunoassay: HRP，AP Immunological labeling techniques Enzyme ImmunoAssay (EIA) Ag-Ab reactions with enzyme-labeled Ag or Ab. horseradish peroxidase (HRP)，alkaline phosphatase (AP) Characteristics: High specificity: Ag-Ab reaction High sensitivity: enzyme catalytic reaction (pg level) Qulitative or quantitative assay: Color change or OD value Classification: ELISA: Soluble Ag or Ab Immunochemistry: Ag in tissues or in the surface of cells Enzyme-Linked ImmunoSorbent Assay (ELISA) Indirect ELISA: Known Ag, enzyme-labeled secondary Ab Unknown Ab Sandwich ELISA: Known Ab, enzyme-labeled Ab Unknown, soluble Ag Competitive ELISA: Known Ab or Ag, enzyme-labeled Ab or Ag Unknown Ag or Ab Enzyme-Linked ImmunoSorbent Assay (ELISA) Experiment: Assay of hemolysin by Indirect ELISA ——Qualitative assay Materials: Defribinated SRBC —— Ag Rabbit anti-SRBC Ab(hemolysin) —— Primary Ab HRP-labeled goat anti-rabbit IgG —— Secondary Ab Coating buffer：0.05M pH9.6 bicarbonate buffer Washing buffer：0.01M PBS(pH7.4) containing 0.05% Tween 20 Substrate buffer： pH5.0 phosphate-citrate buffer solution Substrate solution：OPD 10mg,Substrate buffer 25ml,30% H2O2 40μL Microwell plate Methods Methods Anticipated results Positive：yellow Negative：blank Attentions Wash thoroughly and avoid cross-contamination Add samples in turn and no bubbles in the bottom of the wells Coating and incubation should be performed in humidified box 2. Complement fixation Test(CFT) Definition Complement fixation reaction: Ag-Ab r