ExerciseⅧ Enzyme-Linked lmmunosorbent Assay（ELlSA）
Principle
ELISA（Enzyme-Linked Immunosorbent Assay）was established by Van Weeman，Schuur，Enyvall and Perlmann in 1971.An enzyme is linked to an antibody in such away that it does not affect the enzymatic activity or the antibody specificity.First，the known antibody or antigen is fixed on a solid carrier.Then a sample and the corresponding enzyme-linked Ab/Ag are added to bind to the Ag/Ab attached to the solid carrier.The resulting Ag/Ab complex and the excess Ab/Ag are separated by washing.Finally the substrate is added and catalysis cause a color change which can be measured.
ELISA has the advantages of high sensitivity and specificity.It combines the specificity of the Ab/Ag reaction with the catalysis of enzymes.
Methods
There are three different kinds of ELISAs，which can detect the presence of protein in serum or supernatent.
1.Indirect ELISA—detecting antibodies（Figure 1）
（1）coat the plate with the appropriate antigen，wash after incubating at 4℃overnight
（2）Add the samples（primary Ab）to the appropriate wells.Wash after incubating for 1 hour at RT.
（3）Add the secondary Ab conjugated to an enzyme.Wash after incubating for 1hr at RT.
（4）Add the substrate and incubate at RT until the color change is evident.Read plates on an ELISA plate reader.
2.Double antibody sandwich ELISA—detecting antigens（Figure 2）
（1）Coat the plate with an appropriate antibody.Wash after incubating overnight at 4℃.
（2）Add the test samples to the wells.Wash after incubating for 1h at RT.(https://www.daowen.com)
（3）Add the appropriate Ab conjugated to an enzyme.Wash after incubating for 1h at RT.
（4）Add the substrate to each well.Incubate and assess results.
3.competitive method—detecting antigens（Figure 3）
1）Coat the plate with the appropriate antibody.Wash after incubating.
2）Add a antigen conjugated to an enzyme and the test sample containing the antigen.Wash after incubating.
3）Add the substrate and incubate at RT until the color change is evident.
Figure 1 Indirect ELISA
Figure 2 Double antibody sandwich ELISA
Figure 3 competitive ELISA