临床免疫学检验实验：ELISA Detect Hepatitis B Virus

1、ELISA Detect Hepatitis B VirusConception of ELISA enzyme-linked immunosorbent assay:Enzyme-linked :enzyme labeled antibody or antigenImmuno: The antigen and antibody react with each other Sorbent: the reaction happens on the surface of solid phase carrier. The antigen antibody reaction on the surfac

2、e of solid phase carrier, under the catalysis of enzyme, substance color will change though hydrolysis, oxidation or reduction reaction. According to the color, we can detect the concentration of antigen or antibody.Application:Antigen detection: double antibody sandwich method: suitable for protein

3、 or macromolecular antigen. competitive inhibition method: suitable for small molecules Antibody detection: double antigen sandwich method indirect method, competitive inhibition method capture method.What can we detect about HBV? HBsAg has immunogenicity which is a symbol of HBV infection. HBsAb is

4、 a kind of protective antibody. Its appearance means body is in the recovery period or already has immunity. HBeAg is not only a symbol of HBV duplicate, but also an Infectious index. HBeAb and HBcAb are both indexes of infection that patient got in the past. If HBcAb-IgG appears which means body is

5、 infected now and virus begin to duplicate. method HBsAg: Double antibody sandwich method HBsAb: Double antigen sandwich method HBeAg: Double antibody sandwich method HBeAb: Neutral Competitive method HBcAb: Competitive inhibition method 1、Double antibody sandwich method detected antigen (two-steps

6、method)coatingantibodySample IncubatewashIncubatewashEEEenzyme labelled Ab substanceIncubateStop bufferSandwich formingSuitable forprotein and otherlarge molecules, such as HBsAg、HBeAg. EEEEEE1、Double antibody sandwich method detected antigen (one-step method)coatingantibodySample IncubatewashEEEenz

7、yme labelled Ab substanceIncubateStop bufferSandwich formingToday, we only use one-step method to detect HBsAg、 HBeAg. EEEEEETwo-steps method and one-step methodTwo-steps methodone-step methoddifferencesSample and enzyme labelled Ag/Ab is added at the different time. The whole process has two times

8、of incubation and washing.Sample and enzyme labelled Ag/Ab is added at the same time. The whole process has just one incubation and washing.advantagesResult is more accurate.Shorten the reaction time.Operation process is easily.disadvantagesReaction time is long.Operation process is complicated.Hook

9、 effect: double Ab sandwich method may have false negative result.reaction curveMethod content Antigen concentrationAntigen concentrationabsorbance absorbance 2、Double antigen sandwich method detected antibody (one-step method) coatingantigenSample IncubatewashEEEenzyme labelled Ag substanceIncubate

10、Stop bufferSandwich formingEEEEEESuitable for HBsAb3、competitive inhibition method detected antibodycoatingantigenSample IncubatewashsubstanceIncubateStop bufferSandwich formingSuitable for HBcAb EEEenzyme labelled Ab EE4、neutral competitive method detected antibodycoatingantibodyNeutral AntigenIncu

11、batewashEEEenzyme labelled Ab substanceIncubateStop bufferSandwich formingEEEESample Suitable for HBeAb. HBeAg is not stable. If coated HBeAg directly, HBeAg will be easily transform into HBcAg which lead to an inaccurate result. 1、 immunosorbent: every hole of the plate is already coated with speci

12、fic antibody or antigen. We can use it directly. Material preparation For ELISA96 holes plate2、 enzyme： HRP is commonly used to label antigen or antibody. 3、substance：（chromogenic agent ） Ingredient: liquid A: contain 0.5 H2O2 liquid B：contain 0.26 TMB Mix two liquid together and use. 4、cleaning sol

13、ution ： Before using it needs dilute first. cleaning solution 1 cleaning solution+ water 205、 stop buffer: concentration of H2SO4 is less than 2mol/l.6、 Controlset：positive control and negative control.7、 Neutral reagent: only prepared in HBeAb detecting reagent set. =Detection Steps (one-step metho

14、d) : 1、 quantitative analysis ：standard sample: Establishan appropriate concentration gradient: 1:2，1:22，1:23，1:24.2n1:21:41:81:161:321 ml diluentsample232323242424252525Standard sampleAdding standard sample and sample: Negative controlpositive controlsample50L /holeStandard curve1:11:21:41:81:161:3

15、21:64Negative controlpositive controlsample50L /holequalitative analysis: no standard curveDetection of HBcAb, the sample should be diluted 30 times with cleaning solution .2、Adding neutral antigen: Only needed to detect HBeAb, 50L /hole3、 Adding enzyme-labelled antibody: 50L /hole4、Incubating: mix

16、together, cover the plate with sticky paper, 37 for 30min. 5、Washing: abandon the liquid. Inject cleaning solution to each hole then shake plate gently for 1 minute. Abandon the liquid. Repeat 5 times. (Note: the last time pat the plate and make sure the plate is dry )6、Adding substance: Add 50L liq

17、uid A and 50L liquid B. Mix together. 7、Incubating: cover the plate with sticky paper, 37 for 10min. 8、Terminal reaction: Add stop buffer 50L/ hole to terminate reaction. The color will change to yellow. Test the result in 10min. 9、Result judging: Observing by eyes:holescolorresultNegative controlNo

18、 colornegativePositive controlyellowpositivesampleyellow？sampleNo color？HBsAg， HBsAb， HBeAg HBeAb ， HBcAbholescolorresultNegative controlyellownegativePositive controlNo colorpositivesampleyellow？sampleNo color？ Colorimetry : The detection wavelength is 450nm. Before testing the result, we have to a

19、djust the blank hole to “0”. HBsAg， HBsAb， HBeAg sample OD value negative control OD value HBeAb ， HBcAb negative control OD value sample OD value 2.1 positive 2.1 positive HBsAgHBeAgHBsAbHBcAbHBeAb methodDouble Ab methodDouble Ab methodDouble Ag methodCompetitioninhibition methodNeutral competi-tit

20、ive Method coatinganti-HBs anti-HBeHBsAgHBcAganti-HBe1、sample50L/ hole50L/ hole50L/ holeDiluted first 50L/ hole50L/ hole2、 neutral Ag-HBeAg3、 HRP-Ab/AgHRP-AbHRP-AbHRP-AgHRP-AbHRP-Ab4、incubationMix together. Cover the plate with sticky paper, 37 for 30min.5、washingAbandon the solution. wash 5 times.

21、Dry it.6、substance50L liquid A and 50L liquid B mix together. 7、incubation Cover the plate with sticky paper, 37 for 10min.8、terminationAdd stop buffer and observe the result. Test it under 450 nm. One-Step method:stepsitemsHBsAganti-HBsHBeAganti-HBeanti-HBcClinic significance+-Latency period of acu

22、te HBV infection+-+-early period of acute HBV infection and it can contagious to other people. +-+-+Acute or chronic HBV infection, viral replication is active. Infectivity is strong.（大三阳）+-+Acute or chronic HBV infection. Virus replication reduces and infectious is weak. （小三阳） +-+Acute or chronic HBV infection. HBeAg converse to anti HBe which has certain infectivity+-+Acute or chronic HBV infection. +-+In the recovery period, body produces immunity.Resul