Microgenics Corporation

Boehringer Mannheim Gmbh

Glenda L Choate

Pyare L Khanna

Wayne B Manning

Galactopyranoside derivative compounds for use as substrates for hydrolysis by enzymes with .beta.-galactosidase activity are provided. The concentration of the reaction products can be measured by spectroscopy. The compounds find particular use in diagnostic assays for detection of analyte.

Substrates for .beta.galactosidase

Substrates for .beta.-galactosidase

Diagnostic assays are provided comprising complementary enzyme fragments of .beta.-galactosidase, where one of the fragments is bound to a support and the other fragment is conjugated to an immunologically cross-reactive epitope of the analyte or complementary to an analyte receptor. Binding to the receptor allows for discrimination between complexed enzyme fragment conjugate and uncomplexed enzyme fragment conjugate. The assay medium is then allowed to wick onto a support to which the complementary fragment is substantially uniformly bound in the detection region. The support is then developed, where color formation from the substrate in the detection region is used as a measure of the presence of analyte in the sample.

Enzyme quantitation wicking assay

A .beta.-galactosidase complementation assay for determining the presence of an analyte which is a member of a specific binding pair (sbp) in a sample is provided, which assay permits visual discrimination between those samples wherein the analyte is present above a predetermined, threshold concentration. The method comprises combining the sample with a hydrophobic enzyme donor- (ED-) analyte conjugate and the complementary member of the sbp to form an sbp complex-containing assay medium. A small volume of the assay medium is spotted onto an enzyme acceptor (EA) affixed to a bibulous, solid support, followed by development with enzyme substrate solution. A substantially larger area is detectable when analyte in the sample is below as compared to above a threshold concentration. The assay is particularly useful in field testing applications such as determining the presence of antibiotics in milk, toxins in water, or drugs in serum or urine. Kits facilitating the method are also provided.

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Concord, CA, United States of America

Glenda L Choate