Immunoassays

Immunoassays
By:Diane C. Jette, Ph.D.
BioMedica Diagnostics Inc.

Test Menu
* Infectious Diseases
* Cardiac Markers
* Cancer Screening
* Fertility / Hormones
* Drugs of Abuse
* Other Tests
o IgE
o Rh Factor
o Occult Blood
o TSH, T3, T4
o Blood Grouping

Test Formats Available
* Latex Agglutination
* DIPSTICK
* Card Format
* Cartridge Format
* ELISA Format with Plate Reader

Structure of IgG
(150,000 Daltons)
Antigen
Binding Site
Fab
Fc

Structure of IgM
(900,000 Daltons)
Binding Sites
Sandwich ELISA Procedure
* Antibody coated on plate.
* Incubate with sample containing antigen.
* Wash away unbound material.
* Incubate with antibody-enzyme conjugate.
* Wash away unbound conjugate.
* Add substrate.
* Enzyme acts on substrate and produces color change.
* Measure amount of color produced.
* Amount of antigen present is proportional to the amount of color produced.

ELISA: Sandwich Format
Sandwich Assay Results
Color Intensity (OD)
Antigen Concentration
Linear Range
ELISA: Inhibition Assay Procedure
* Antigen coated on plate.
* Incubate with sample containing antigen and enzyme-antibody conjugate.
* Antigen in solution binds to enzyme-antibody conjugate preventing binding to antigen on plate.
* Wash away all unbound material.
* Add substrate.
* Enzyme acts on substrate to produce a color change.
* Measure the amount of color.
* The amount of antigen present is inversely proportional to the amount of color produced.
ELISA: Inhibition Format
Inhibition Assay Results
Color Intensity (OD)
Antigen Concentration
Linear Range
Agglutination Assay Procedure
Antibody Detection
* Antigen coated on latex particles.
* Incubate with sample containing antibody.
* Antibody binding to antigen causes cross-linking of latex beads.
* Agglutination is observed.
Antigen Detection
* Antibody coated on latex particles.
* Incubate with sample containing antigen.
* Antigen binding to antibody causes cross-linking of latex beads.
* Agglutination is observed.
Latex Agglutination Assay
Latex Agglutination
Negative Result
Smooth gray appearance
Positive Result
Large aggregates in the center or periphery of the test circle
DIPSTICK Format
Absorbing Pad
Test Area
Maximum Level
Sample Pad
Negative Result
Positive Result
CARD Format
CONTROL
TEST
SAMPLE
Negative Result
CONTROL
TEST
SAMPLE
Positive Result
Cartridge Format
Negative Result
S T C
Positive Result

Immunoassays.ppt