Experiment ten To detect the HBsAg with ELISA. ELISA Enzyme linked immunosorbent assay is a normal and simple means to detect the Ag and Ab of HBV and.

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Presentation transcript:

Experiment ten To detect the HBsAg with ELISA

ELISA Enzyme linked immunosorbent assay is a normal and simple means to detect the Ag and Ab of HBV and other pathogens

ELISA (Enzyme-Linked Immunosorbent Assay)

ELISA for Antibody Detection (Enzyme-Linked Immunosorbent Assay) Capture of antigens in microplate Capture of antigens in microplate Washes Washes Block unbound sites in wells Block unbound sites in wells Washes Washes Add antibody serum to wells Add antibody serum to wells Washs Washs Add 2 nd antibody (attach enzyme) Add 2 nd antibody (attach enzyme) Washes Washes Color development Color development Ex: HIV test Ex: HIV test

Sandwich ELISA for Antigen Detection (Enzyme-Linked Immunosorbent Assay) Capture 1 st antibody in microplate Capture 1 st antibody in microplate Washes Washes Block unbound sites in wells Block unbound sites in wells Washes Washes Add antigen serum to wells Add antigen serum to wells Washes Washes Add labeled 2 nd antibody specific to antigen of interest (with substrate) Add labeled 2 nd antibody specific to antigen of interest (with substrate) Washes Washes Color development Color development Ex: Pregnancy test Ex: Pregnancy test

Enzyme-Mediated Detection Enzyme Horseradish Peroxidase SubstrateAbbrev.Color DiaminobenzidineDABBrown Enzyme Alkaline Phosphatase (AP) SubstrateAbbrev.Color BromochloroindolylphosphateNitro Blue Tetrazolium BCIP (AP substrate) NBT (Enhance color) Purple

Horseradish Peroxidase Enzyme DAB Substrate DAB + H HRP > DAB ppt + H 2 0

AP Enzyme BCIP &NBT Substrate

Process to detect HBsAg with ELISA Preparation of washing solution Add specimens (blank control, positive control, negative control)50ul and serum from patients 50ul Add enzyme: Enzyme labeled Ab 50ul(1 drop), except blank control Mixed Incubator--37 ℃,30mins Washing 4times Coloration: substrate A ＆ B.50ul--37 ℃,15mins Incubator

Result