Methods for protein binding enzyme complementation assays

Claim 1

An enzyme assay method for determining the amount of a suspected analyte in a sample which comprises the steps of: (a) Forming a reaction mixture by combining in a medium (1) sample; (2) an enzyme-donor polypeptide conjugate; (3) an analyte-binding protein specific for said analyte; and (4) an enzyme-acceptor polypeptide consisting essentially of a fragment of β-galactosidase, wherein said enzyme-donor polypeptide conjugate and enzyme acceptor polypeptide are prepared by recombinant DNA techniques or chemical polypeptide synthesis techniques, wherein said enzyme-donor polypeptide is characterized by forming with said enzyme-acceptor polypeptide, an active enzyme complex having β-galactosidase activity in the absence of analyte-binding protein binding to said conjugate, said enzyme donor polypeptide conjugate being further characterized by having substantially as the internal amino acid sequence, amino acids 6-51 of β-galactosidase, having one cysteine, with analyte joined to said one cysteine, and having an N-terminal sequence and a C-terminal sequence or having substantially as the amino acid sequence amino acids 7-44 of β-galactosidase, having one cysteine with analyte joined to said one cysteine and having at least an N-terminal sequence and (5) a substrate capable of reacting with said active enzyme complex, such that the rate of conversion by said active enzyme complex can be monitored; and wherein said enzyme-donor conjugate is capable of competitively binding to the analyte-binding protein inhibiting the formation of active enzyme complex; (b) measuring the rate of conversion of substrate in the reaction mixture; and (c) determining the amount of analyte in the sample by comparing the rate of conversion of substrate to a rate of conversion of substrate obtained using a known concentration of the analyte.

Claim 5

A method according to any of claims 1, 2, or 3, wherein said N-terminal sequence has the sequence: ______________________________________ M D P S G N P Y G I D P T E S S P G N I D P R A S S N, M D P S G D P R A S S N, M D P R A S S N, or C I T D; ______________________________________ and said C-terminal sequence has the sequence: ______________________________________ A Q P E W G L E S R S A G M P L E, R S L N G L E S R S A G M P L E, or R S L N G E L C G V K Y R T D A, ______________________________________ with the proviso that where said N-terminal or C-terminal sequence has said one cysteine, said internal amino acid sequence lacks said cysteine.