Immunometric assay of low molecular weight haptens containing primary amino groups

Abstract

A new enzyme immunometric assay of small haptens containing primary amino groups (thyroxine, MW 777; substance P, MW 1347; endothelin, MW 2492) is described. The procedure involves different sequential steps: (1) immunocapture of the haptens (standard or sample) by monoclonal anti-hapten antibodies coated on 96-well microtiter plates; (2) cross-linking of haptens via their amino groups to the wells using homobifunctional reagents (glutaraldehyde or disuccinimidyl suberate); (3) denaturing treatments (HCl or methanol); (4) measurement of linked epitope using the same monoclonal anti-hapten antibodies labeled with acetylcholinesterase. A minimal detectable concentration in the 4-10 fmoL/mL range was observed. Each assay appeared to be 70-200 times more sensitive than conventional competitive enzyme immunoassay using the same monoclonal antibody-coated plate technology and acetylcholinesterase-hapten conjugates as enzymatic tracers. Precision and specificity were very satisfying. Good correlation was noted between this assay and the competitive assays performed for different biological samples (plasma, tissues, or supernatant cell culture).

Selective and Efficient Immunoprecipitation of the Disease-associated Form of the Prion Protein Can Be Mediated by Nonspecific Interactions between Monoclonal Antibodies and Scrapie-associated Fibrils

“…The Solid-phase Immobilized Epitope Immunoassay (SPIE-IA) Technique-The SPIE-IA technique was developed several years ago by some of us (9,10). This new format was originally developed to allow the determination of low molecular weight haptens in excess reagent conditions (immunometric assays) but is also suitable for measuring proteins in a sandwich format using a single epitope (11).…”

Surfactant Protein A Suppresses Lipopolysaccharide-Induced IL-10 Production by Murine Macrophages

“…Briefly, the solid phase immunoenzyme assay (35,36) is an immunometric assay for murine IL-10 that uses the same monoclonal anti-murine (JES-2A5) Ab for both capture and revelation steps. The assay relies on the reaction of the thiol groups of mAb FabЈ with maleimido groups previously introduced into acetylcholinesterase (AchE) as previously described (37).…”

Highly Sensitive and Rapid Assay of Substance P and Streptolysin O in Human Serum Using Immuno-liposomes and Gramicidin Channels

“…The lower detection limit for SP was 0.32 pg mL -1 , as calculated from the fluorescence intensity at three-times the standard deviation of the fluorescence intensity in the absence of SP. The lower detection limit was comparable to that (0.25 pg mL -1 ) of the competitive fluorometric immunoassay, 34 but it was much superior to those of the liposome array method (sub-pg mL -1 ), 31 as well as the radioimmunoassay (10 -20 pg mL -1 ), 35,36 the solid-phase immobilized epitope immunoassay (6 pg mL -1 ) 37 and the amperometric micro-immunosensor (10 pg mL -1 ). 18 …”