Immunoassay and Fluorescence Microscopy

Radioimmunoassay (RIA) labels

Radioactive labels with known emission properties

• competitive RIA - Ag labeled aka tracer

• non-competitive - Ab labeled

Enzyme labels

enzyme catalyzes substrate molecules and amplifies signal

enzyme activity monitored directly or couple with co-enzyme action, producing a colored product, measure photometrically

Fluorescent labels

compounds that absorb radiant energy at one wavelength and emit radiant energy at longer wavelength

Chemiluminescent labels

organic compounds can emit photon of light in response to chemical reaction as they revert to ground state

Competitive assays

competition between labeled Ag and unlabeled Ag for limited number of Ab binding sites

• amount of Ag indirectly related to amount of label signal

• more target Ag is present in sample = more Ab bound to target Ag and less Ab are left to bind Ag coating the wells = lower signal

Non-competitive assays

analyte sandwiched between two specific antibody reagents

concentration of labeled Ag directly proportional to bound Ab

Homogeneous immunoassay methods

do not require separation, limited washings

Heterozygous immunoassay methods

require separation or washing of bound Ag/Ab complexes on solid phase

Fluorescence

excitation light occurs at smaller wavelength and higher energy

emission light is at higher wavelength and lower energy

Conventional microscope

uses light to illuminate sample and produce magnified image of sample

Fluorescence microscope

uses higher intensity light to illuminate sample

Photoluminescence

ability of specimens to absorb and subsequently re-radiate light

Epifluorescence

combination of excitation and emission wavelengths travel through specimen emitting fluorescence

Fluorescent techniques

Anti-nuclear antibodies (ANA test)

fluorescent treponemal antibody (FTA-ABS)

fluorescence polarization (FPIA)

Nephelometry

quantitation of immunoglobulins

light scattering application used to detect Ag/Ab complexes at 90 degrees