医学免疫学教学资料：Exp 2 Antibody preparation and agglutination Ⅱ

1、Exp Agglutination reaction and Antibody preparation (2) &Precipitation ReactionTest 1 Agglutination reaction1.Principle:Tube agglutination reactionAg-Ab reactionAgglutination reactionSlide agglutination reactionQualitativeSemi-QuantitativeTest 1 Agglutination reaction1.Principle:Test 1 Agglutination

2、 reaction1) Slide agglutination reaction: Known Ag+Unknown AbAgglutination turbid?2) Tube agglutination reaction: Quantitative Ag+Serially diluted AbDifferent degree of agglutinationYES NODetermine the titer of AbAg: Ab ratio2. ExperimentsTube agglutination testTest 1 Agglutination reaction Material

3、: Inactivated typhiod bacteria (7108/ml), anti-serum2. Method:0.50.50.50.50.50.50.50.50.51:201:401:801:1601:3200.5Discard123450.160.9N.SAnti-H SerumControl0.5ml0.5ml0.5ml0.5ml0.5mlInactivated typhiod bacteria0.5ml0.5ml0.5ml0.5ml0.5ml0.5mlDilution ratio of AbShake56, 1h3. AttentionsSign the tube Use

4、the dropper tube correctlyMix thoroughlyT: 56Record the timeTake the tube carefullyWhat is the titer of Ab? The titer is customarily reported as the reciprocal of the highest dilution of Ab that causes an obvious agglutination.Test 1 Agglutination reaction4. Anticipated results+Very small agglutinat

5、ionturbid-No agglutinationSame as control+Small and depositing partlyObvious turbid+Large and depositing mostlySlightly turbid+Large and depositingClearDegree of agglutinationSedimentSupernantant Tube agglutination testSedimentAgglutination degreeThe highest dilution ratioTiter of the anti-serumTest

6、 2 Precipitation reaction Agar diffusion testA kind of classic Ag-Ab reaction Classification:Agar diffusion testCircle precipitation reactionFoccule precipitation reactionImmune electrophoresisPrecipitation reaction:Precipitation linePrecipitation circleSoluble AgCorresponding Ab+Immune electrophore

7、sisAgar diffusion test:Soluble Ag and Ab diffuse in agar, correspond to each other and yield white precipitation line or circle with appropriate proportion.Classification:Single agar diffustion test: only diffusion of Ag or Ab quantitative testDouble agar diffustion test: diffusion of both Ag and Ab

8、 qualitative test Single agar diffusion test:Ag ConcentrationDiameter2AgAgAgAgAb in gel1:101:201:401:80AgUnknown Double agar diffusion test:Test 2 Double agar diffusion testMethod：1. Melt 1.5% agar in microwave oven2. Cover 5ml Melt agar in a plateConcretion3. Cut 5 wells on agar plate 4. Add sample

9、s in wellsAbAg1Ag1Ag2Ag25. 37, 24h in miost chamberTest 2 Double agar diffusion testAttentions：Agar plate should be made evenly and smoothly (No bubbles).Not too thick agar.Not too much fluid in wells.Test 3 Secondary immunization2. Methods:Uncoagulated SRBCAdd 3ml N.S and mix2000rpm, 5min2000rpm, 5minDiscard the supernantDiscard the supernantMake 25%、40% SRBC solutionTake 0.1ml to immunize the mice subcutan