Amplified flow-cytometric separation-free fluorescence immunoassays

Journal Article · Sun Dec 01 04:00:00 EST 1985 · Clin. Chem. (Winston-Salem, N.C.); (United States)

OSTI ID:5636811

Saunders, G C; Jett, J H; Martin, J C

An equilibrium-type competitive-binding fluorescence immunoassay with high sensitivity and excellent precision is described that obviates separation of free from bound label. In the assay relatively large (10 microns diameter) antibody-coated non-fluorescent particles and very small (0.10 micron diameter) antigen-coated fluorescent latex particles are used. Soluble nonlabeled antigen competes with antigen on the microspheres for antibody binding sites on the larger spheres. After equilibrium is attained, the fluorescence distribution of 5000 of the large spheres is measured in a flow cytometer. The mean values for the fluorescence distribution obtained from samples containing known concentrations of soluble antigen are used to construct a standard displacement curve. In a prototype assay for the antigen horseradish peroxidase, a sensitivity of 10(-12) mol/L has been achieved. Undiluted serum can be assayed without loss of sensitivity. Preliminary experiments also indicate that double-antibody sandwich-type assays of very high sensitivity (10(-14) mol/L) are also possible when this dual-sphere concept is exploited.

• OSTI ID: 5636811
• Journal Information: Clin. Chem. (Winston-Salem, N.C.); (United States), Journal Name: Clin. Chem. (Winston-Salem, N.C.); (United States) Vol. 12; ISSN CLCHA
• Country of Publication: United States
• Language: English

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59 BASIC BIOLOGICAL SCIENCES
ANTIBODIES
ANTIGEN-ANTIBODY REACTIONS
ANTIGENS
BIOASSAY
CELL FLOW SYSTEMS
ENZYMES
FLUORESCENCE
IMMUNITY
LABELLING
LUMINESCENCE
MICROSPHERES
OXIDOREDUCTASES
PEROXIDASES