Enzyme-linked immunosorbent assay

Enzyme-linked immunosorbent assay

.

# enzyme conjugated to an antibody reacts with colorless substrate to generate a colored

reaction product.
# enzymes; alkaline phosphatase, peroxidase and p- nitrophenyl phosphatase ;mixed with substrate cocolored reaction products
# fluorescence to give light under the UV light.
#Safer & less costly
#qualitatively detect the presence of Ab or Ag
#it used substrate & stop solution
#in 5 seconds it able to read 96 sample
ELISA

ELISA

fluoroELISA
Vidas or minividas those give fluorescence not need substrate or stop solution

Types

1-Direct ELISA
Microwell plates are coated with target Ag.
Add enzyme labeled Ab
Wash
Add substrate color
Add stop solution
Measure the color which is directly proportional with the concentration of Ag


Cont…………/Types
2-indirect ELISA
Microwell plates are coated with target Ag
Add detection Ab(primary Ab)
Add enzyme linked Ab (secondary Ab)
Wash
add substrate color

Cont…………/Types

3-Sandwich ELISA
Microwell plates are coated with target Ab( capture Ab)
Add Ag
wash
Add enzyme linked Ab (secondary Ab)
add substrate color
We use 2-3 standard Ag with known concentration in order to draw the standard curve which represent the relation between the color and the concentration.

Cont…………/Types

3-competitive ELISA
Incubate Ab with Ag
Add(Ag-Ab) to antigen coated well
wash
Add enzyme linked Ab (secondary Ab)
wash
add substrate no color which is an inhibition type assy the concentration of antigen is inversely proportional to the color produced.,the higher concentration of Ag in the original sample ,the lower the absorbance


Applications;
1-detection & quantitation of immunoglobulins
their classes
2-detection of viral Ag & their Ab
3-detection of bacterial Ag
4-detection of malaria Ag
5- detection of antitoxoplasm Ab
6-estimation of hormone level in the blood
7-estimation of drug level in the blood
8-detection of autoantibodies in the blood .