医学免疫学教学资料：Exp 4 ELIS

1、Exp IV. 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:Ra

2、dioimmunoassay(RIA): 131I，32PImmunofluorescence technique: FITC，PEEnzyme Immunoassay: HRP，APImmunological labeling techniques3. Labeling techniques：Direct labeling techniques: Each Ag or AbIndirect labeling techniques: Secondary AbIsotype of antibody:Immunize RabbitMouse IgG1Rabbit anti-mouse IgG1Se

3、condary AbImmunofluorescence technique：Enzyme ImmunoAssay (EIA)Ag-Ab reactions with enzyme-labeled Ag or Ab.horseradish peroxidase (HRP)，alkaline phosphatase (AP)Characteristics:High specificity: Ag-Ab reactionHigh sensitivity: enzyme catalytic reaction (pg level)Qulitative or quantitative assay: Co

4、lor change or OD valueClassification:ELISA: Soluble Ag or AbImmunochemistry: Ag in tissues or in the surface of cellsImmunohistochemistryEnzyme-Linked ImmunoSorbent Assay (ELISA)Unknown Ab or Ag in blood or culture medium are detected by enzyme-labeled Ag or Ab.1.Definition:2.Classification:Indirect

5、 ELISA:Known Ag, enzyme-labeled secondary AbUnknown AbSandwich ELISA:Known Ab, enzyme-labeled AbUnknown, soluble AgCompetitive ELISA:Known Ab or Ag, enzyme-labeled Ab or AgUnknown Ag or AbEnzyme-Linked ImmunoSorbent Assay (ELISA)3. Principles (Indirect ELISA for example):Experiment: Assay of hemolys

6、in by Indirect ELISA Qualitative assayMaterials:Defribinated SRBC AgRabbit anti-SRBC Ab(hemolysin) Primary AbHRP-labeled goat anti-rabbit IgG Secondary AbCoating buffer：0.05M pH9.6 bicarbonate bufferWashing buffer：0.01M PBS(pH7.4) containing 0.05% Tween 20Substrate buffer： pH5.0 phosphate-citrate bu

7、ffer solutionSubstrate solution：OPD 10mg,Substrate buffer 25ml,30% H2O2 40LMicrowell plateMethods1. Preparation of Ag:Defibrated SRBCAdd 3mL N.S and mix2000rpm，5minTake 2mL packed SRBCAdd 2mL DDW and shatter RBCDilute with coating buffer in a ratio of 1：4002. Coating Ag:Add 100L of Ag to each well o

8、f ELISA plate4，in a humidified box, 18hAdd 3mL N.S and mix2000rpm，5minSRBCMouseAnti-SRBC Ab(hemolysin) Immunization3 times / 3wsIgGSerumYYYYMethods3. Prepare the serumRemove the eyeball of the SRBC-immunized miceCollect the blood into Ep tubeCentrifuge at 3000 rpm for 10 minTake the supernatantMetho

9、dsDiscard the Ag solution in ELISA plate(4 wells/group)Wash the plate 3 times(1min each time)4. Add test serumSign and add 100L of solution to each well1234NegativeBlankPositiveTest5. Add secondary Ab37 for 30min in a humidified boxDiscard the solution in ELISA plateAdd 100L of HRP-labeled secondary

10、 Ab to each well6. Showing colorAdd 100L of substrate solution to each wellShow color in dark for 10min7. Observe the resultMethodsWash the plate 3 times(1min each time)37 for 30min in a humidified boxDiscard the solution in ELISA plateWash the plate 3 times(1min each time)Anticipated resultsPositive：yellowNegative：blank1 2 3 4NegNegPosPosAttentionsWash t