Becton Dickinson and Company Limited

James P O'Connell

Daniel B Wagner

Patrick D Mize

Robert L Campbell

Joel L Williams

Burton H Sage

Randal A Hoke

David B Montgomery

Terry S Dunn

Uri Piran

Beverly M Fleming

Daniel A McLaurin

A method for enzyme immunoassay for a ligand suspected to be present in a liquid sample includes signal amplification by use of at least two enzymes and a blocked modulator for one of the enzymes. Ligand present in the liquid binds to an antiligand and an enzyme-labeled tracer. The resulting bound fraction is separated and the enzyme in the tracer removes the blocking group from the blocked modulator. The modulator activates or inhibits a second enzyme which catalyzes the conversion of a substrate to a product. The presence or absence of the ligand in the liquid is indicated by a signal, such as a color change or a rate of color change, associated with the product. The invention includes a new class of enzyme inhibitors and blocked inhibitors and a kit of materials useful for performing the method of the invention.

Fluoroketone enzyme inhibitors

A method for enzyme immunoassay for a ligand suspected to be present in a liquid sample includes signal amplification by use of at least two hydrolases and a blocked fluoroketone for one of the hydrolases. Ligand present in the liquid binds to an antiligand and a hydrolase-labeled tracer. The resulting bound fraction is separated and the hydrolase in the tracer removes the blocking group from the blocked fluoroketone. The fluoroketone activates or inhibits a second hydrolase which catalyzes the conversion of a substrate to a product. The presence or absence of the ligand in the liquid is indicated by a signal, such as a color change or a rate of color change, associated with the product. The invention includes a new class of hydrolase inhibitors and blocked fluoroketones and a kit of materials useful for performing the method of the invention.

Signal enhancement in immunoassay by modulation of enzymatic catalysis

A method for assay of an analyte and assay device therefor. The assay device includes an assay component affixed to a permeable membrane. The membrane preferably rests on a permeable membrane support and covers an opening through a receptacle for holding a liquid. The support is adjacent the open upper end of a double ended needle, the lower end of which is adapted to puncture a plug inserted in an open end of a preevacuated container. Puncturing of the plug establishes fluid communication between the prevacuated interior of the container and the opening in the receptacle whereby liquid in the receptacle is passed through the membrane. The membrane has a bubble point sufficient to prevent passage of air through the membrane after all of the liquid has been pulled through as long as the membrane remains wet so that vacuum in the tube is not lost. The device may be part of an assay workstation which comprises a canister and various utensils useful in performing the assay.

Vacuum actuated assay device and method of using same

Solid phase assay for an analyte wherein binder is supported on a solid support, such as nitrocellulose, and the tracer is comprised of ligand labeled with a particulate label, such as a liposome, including a detectable marker which is not visible.

Solid phase assay

Solid phase assay for an analyte wherein binder is supported on a solid support, such as nitrocellulose, and the tracer is comprised of ligand labeled with a colored particulate label, such as a liposome including a dye. The assay has a high sensitivity, and the tracer is visible on the support under assay conditions, whereby tracer can be determined, without instrumentation, and without further treatment thereof.

Solid phase assay with visual readout

A sac, in particular a vesicle, including as a detectable marker an absorbing dye having an extinction coefficient of at least 100,000 and a solubility in water of at least 0.1M; in particular, sulforhodamine B or salt thereof. The sacs have improved stability to leakage and are useful in a variety of assays; in particular, when sensitized with a ligand.

Sac or liposome containing dye (sulforhodamine) for immunoassay

A method for immunoassay of an analyte in a fluid includes contacting the analyte with an antianalyte to give a bound fraction. The bound fraction activates the first component of complement whereby a substrate present in the fluid is modulated to provide a detectable signal. The signal may be detected to establish the presence or absence of the analyte in the fluid, or it may be quantitatively measured to determine the concentration of the analyte in the fluid. The invention includes a kit of materials useful in performing the method of the invention.

Immunoassay using complement

The disclosure is of a method to improve the antithrombogenicity which results when heparin is attached to a polymeric surface. The polymeric surface is activated by treatment with a plasma. The surface thus activated is rendered receptive to the binding of a surface active agent. The surface active agent in turn reacts with heparin to provide a polymeric surface of exceptional antithrombogenicity when contacted with blood for an extended period of time.

Heparinization of plasma treated surfaces

Reticulocytes are stained with thioflavin T for detection in a flow cytometer.

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Chapel Hill, NC, United States of America

James P O'Connell