Abbott Laboratories Corporation

Houston F Voss

Jacob J Plattner

Thomas R Herrin

Susan E Magic

Stephen D Stroupe

The present invention encompasses a method for determining ligands in test samples comprising intermixing with the test sample a ligand analog-irreversible enzyme inhibitor conjugate and a binding protein bindable to the ligand and the ligand analog-irreversible enzyme inhibitor conjugate and wherein the amount of ligand analog-irreversible enzyme inhibitor conjugate bound by the binding protein is related to the amount of ligand in the test sample, said binding protein inactivating the irreversible enzyme inhibitor when bound to the ligand analog portion of the conjugate; intermixing an enzyme which is irreversibly inhibited by the ligand analog-irreversible enzyme inhibitor conjugate unbound by the binding protein; and intermixing substrate to the enzyme and monitoring the enzyme substrate reaction. The invention also includes ligand analog-irreversible enzyme inhibitor conjugates useful as reagents in practicing the method. Methods and reagents of the present are particularly useful in determining drugs, hormones, and the like in biological fluids.

Ligand analog-irreversible enzyme inhibitor conjugates

This invention encompasses a method for measuring unsaturated thyroxine binding globulin in serum comprising intermixing with the serum:

Determination of thyroxine binding globulin

Ligand analog-irreversible enzyme inhibitor conjugates and methods for

The present invention comprises compounds of the formula ##STR1## and the biologically acceptable acid addition salts thereof wherein R.sub.1 represents hydrogen, or lower alkyl having 1-4 carbon atoms; R.sub.2 represents p-hydroxybenzyl or benzyl; R.sub.3 represents an alkyl having 1-6 carbon atoms; R.sub.4 represents amino or guanidino; R.sub.5 represents p-nitrophenyl, methylnitrophenyl, dinitrophenyl, naphthyl, or nitronaphthyl; and n is 3 or 4.

Chromogenic substrates

The present invention encompasses acid addition salts of a compound of the formula ##STR1## wherein: R.sub.1 represents phenylsulfonyl, benzoyl, carbobenzoxy, and the halo, loweralkyl having 1-4 carbon atoms, loweralkoxy having 1-3 carbon atoms, phenyl, or hydroxy substituted derivatives thereof; or alkanoyl having 2-12 carbon atoms; R.sub.2 represents alkyl having 1-10 carbon atoms, alkoxyalkyl having 2-6 carbon atoms, cycloalkyl having 5-7 carbon atoms, benzyl and the halo, loweralkyl having 1-4 carbon atoms, loweralkoxy having 1-3 carbon atoms, carboxy, hydroxy, or substituted derivatives thereof; R.sub.3 represents hydrogen or guanyl, and m is 3 or 4. Compounds of the present invention are useful as analytical reagents. Enzymatic hydrolysis provides --S--R.sub.2 which can be further reacted with 5,5'-dithiobis (2-nitrobenzoic acid) to provide a colored product by which the enzyme concentration can be determined spectrophotometrically.

The present invention encompasses the acid addition salts of a compound of the formula ##STR1## wherein R.sub.1 represents phenylsulfonyl, benzoyl, carbobenzoxy, and the halo, loweralkyl having 1-4 carbon atoms, loweralkoxy having 1-3 carbon atoms, phenyl, or hydroxy substituted derivatives thereof; or alkanoyl having 2-12 carbon atoms; and R.sub.2 represents alkyl having 1-10 carbon atoms, or alkoxyalkyl having 2-6 carbon atoms, cycloalkyl having 5-7 carbon atoms, or benzyl and the halo, loweralkyl having 1-4 carbon atoms, loweralkoxy having 1-3 carbon atoms, hydroxy, carboxy, or phenyl substituted derivatives thereof. Compounds of the present invention are useful as analytical reagents. Enzymatic hydrolysis provides --S--R.sub.2 which can be further reacted with 5,5'-dithiobis (2-nitrobenzoic acid) to provide a colored product by which the enzyme concentration can be determined spectrophotometrically.

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Libertyville, IL, United States of America

Houston F Voss