临床免疫学检验：酶联免疫技术

1、EnzymeImmunoassay1.Pinciple of enzyme immunoassay2. Types of emzyme immunoassay3. Enzyme-linked Immunosorbent assay (ELISA)4. Application Main content一、pinciple of enzyme immunoassayenzyme immunoassay: enzyme: enzyme labelled antibody or antigen - enzyme joins chromogenic reaction of the substrateim

2、muno: The antigen antibody reaction - the specific reaction of antigen and antibody the technology can be used for the positioning analysis、the quantitative or qualitative analysis of antigen or antibody.CharacteristicAntigen-antibody reaction:1、Reversibility: the processof antigen antibody reaction

3、isa dynamic balant reaction: Ag + Ab Ag AbTheequilibrium constant K reflect the antigen-antibody affinity. K = Ag Ab/ AgAb Dissociation degree of Ag Abis associated with Kvalue. After dissociation, Antigenand antibodycan keepthe originalstructure andactivity. 2、specificity： Theantigen determinantcan

4、 combine with antibody binding sites.This reaction has a high degree of specificity because of the complementaryrelationship between chemicalstructure and thespatial structure. when we test a particularsubstance,we dont need separate thesamples first. 3、optimal ratio ： Hook effect: The ratio of anti

5、gen and antibodyis notappropriate andlead to false negativephenomenon prezoneposezone4、sensitivity ：Sensitivity of Colorimetric assaycan reach mg/ml level.Sensitivity of enzymeassayis about 5-10g/ml.Sensitivity of enzyme-labelledimmunoassaycan be up to ng/mllevel. For example,HBsAg,the sensitivityca

6、n reach 0.1ng/ml. characteristics of enzyme: High efficiency: it has good catalytic ability to promote the antigen and antibody reaction in the process. Specificity: the catalytic role to substrate is specific and enzyme is not affected by other reagent in the solution. Stable: easy to react with an

7、tigen or antibody and the immunocompetence of enzyme、antigen or antibody in complex is not affected.Non-toxic, no harm Enzyme Marker Horseradish peroxidase(HRP) HRP is a kind of compound enzyme, which is consists of glycoprotein and ferroheme. PH range: 5-9 Substrate: DH2+H2O2=D+H20 donor receptor H

8、RP Common substrate for HRPDH2characteristicstop bufferwavelenthDBAbrown, insoluble-benzidinebule, insoluble-OPDyellow, soluble 0.5M H2SO4492TMBbule, soluble0.5M H2SO4450TMBSbule, soluble0.5M H2SO44505-ASAbrown, soluble3M NaOH550 liquid A: contain 0.5 H2O2 liquid B：contain 0.26 TMB easy to decompose

9、 and store in the dark bottle Mix two liquid together before using. TMBOther enzymeAlkaline Phospharase(AP)substrate: PNPcharacteristic: yellow production and insolublestop buffle: no needAP substrate cirlulatory system: improve efficiency of APApplication: can be observed by eyes and used for EIBT

10、preparation for enzyme markerSodium periodate oxidation method(过碘酸钠氧化法)： only be used for labeling antigen or antibody by HRPGlutaraldehyde cross-linking method(戊二醛交联法)： one step method: mix antibody、enzyme and glutaraldehyde together and produce the complex. two step method: enzyme react with gluta

11、raldehyde then connect with antibody.二、types of emzyme immunoassayEnzyme ImmunoassayEnzyme Immunohistochemistry(EIH)Enzyme Immunoassay(EIA)Homogenus Enzyme ImmunoassayHeterogenousEnzyme ImmunoassaySolid Phase Enzyme Immunoassayliquid PhaseEnzyme ImmunoassayEnzyme Immunohistochemistry(EIH)Its suatabl

12、e for the analysis of antigen or antibody in tissue or cells.Resultantigenantibodyenzyme labelled antibodyHomogenus Enzyme ImmunoassayIn the process, we dont need to separate the enzyme-labelled complexes from other free enzyme-labelled antigen or antibody in solution.enzyme multiplied immunoassay(E

13、MIT)Unknown antigenEnzyme labelled antigensubstrateenzymatic activity Enzyme labelled antigen antibody complexunknown antigen antibody complexspecific antibodyIts suitable for the detection of small molecule hormones and haptens.Cloned enzyme donor Immunoassay(CEDIA)specific antibodyUnknown haptenha

14、pten enzyme-donorenzyme-receptoradding substrate and read resultHeterogenous Enzyme Immunoassay we have to separate the enzyme-labelled complexes from other free enzyme-labelled antigen or antibody in solution. liquid Phase Enzyme Immunoassay: the reaction of antigen and antibody happen in the solut

15、ion.Heterogenous Enzyme Immunoassay solid Phase Enzyme Immunoassay: the reaction of antigen and antibody happen on the surface of the solid carrier.ELISAEnzyme immunoblotting test (EIBT)Principle:Using nitrocellulose (NC) as solid carrier.Antigen adhered to NC by electrotransfered or absorbed Specif

16、ic antibody or enzyme labelled antibody combine with antigen and produce the complex on the surface of NCAdding the substrate and stop buffer, then adjust the result.Dot Immunoenzyme Filtration Assay (DIEFA)1、adding sample2、adding antibody3、adding enzyme labelled antibody4、adding substrate and read

17、resultEnzyme linked Immunoelectrotransfer blot (EITB)1、adding sample and electrophoresis 2、dyeing2、electrotransferEnzyme linked Immunoelectrotransfer blot (EITB)4、adding specific antibody 3、washing and blocking5、adding enzyme labelled antibody6、substrate and read result三、Enzyme-linked Immunosorbent

18、Assay (ELISA)Antigen detection: double antibody sandwich method: suitable for protein or macromolecular antigen. competitive inhibition method: suitable for small molecules Antibody detection: double antigen sandwich method indirect method, competitive inhibition method capture method to detect anti

19、body.1、Double antibody sandwich method detected antigen (two-steps method)coatingantibodySample IncubatewashIncubatewashEEEenzyme labelled Ab substanceIncubateStop bufferSandwich formingSuitable forprotein and otherlarge molecules, such as HBsAg、HBeAg. EEEEEE1、Double antibody sandwich method detecte

20、d antigen (one-step method)coatingantibodySample IncubatewashEEEenzyme labelled Ab substanceIncubateStop bufferSandwich formingEEEEEETwo-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 tw

21、o times 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 complica

22、ted.Hook 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 substance

23、IncubateStop bufferSandwich formingEEEEEESuitable for HBsAb3、competitive inhibition method detected antibodycoatingantigenSample IncubatewashsubstanceIncubateStop bufferSandwich formingSuitable for HBcAb EEEenzyme labelled Ab EEneutral competitive method detected antibodycoatingantibodyNeutral Antig

24、enIncubatewashEEEenzyme 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. 4、indirect method detected antibodynon-competitive method. its u

25、sually used for antibody detection.Enzyme labelled Ab antigensubstanceUnknow antibodyIncubatewashIncubatewashIncubateStop buffer5、Capture method detectedantibodyantigenenzyme labelled Ab Unknow antibodysubstanceStop bufferSuitable for IgM which is usually show in the early phrase of virus infection.IncubatewashIncubatewashIncubateAnti human antibodyThe 96 holes polystyrene plate is good solid phase carrier. Strongly absorptive ability. Low blank value while testing the result. High transparency of the bottom of the hole. The proper