Muteins of .beta.-galactosidase fragments having increased activity

An immunoassay method for determining an analyte in a sample suspected of containing said analyte comprising:

• (a) contacting said sample with
• (i) an enzyme donor polypeptide conjugate,
• (ii) an analyte-binding protein specific for said analyte,
• (iii) an enzyme acceptor polypeptide of .beta.-galactosidase wherein said enzyme acceptor polypeptide is characterized by forming with said enzyme donor polypeptide an active enzyme complex having .beta.-galactosidase activity in the absence of analyte-binding protein binding to said conjugate, said enzyme acceptor polypeptide being further characterized by having an amino acid other than cysteine substituted at position 500, and
• (iv) a substrate capable of reacting with said active enzyme complex, such that the rate of conversion of said substrate by said active enzyme complex can be monitored, and wherein said enzyme donor conjugate is capable of competing with said analyte to bind to said analyte-binding protein, thereby inhibiting the formation of active enzyme complex;
• (b) measuring the rate of conversion of substrate;
• 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.

An immunoassay method for determining an analyte in a sample suspected of containing said analyte comprising:

• (a) contacting said sample with
• (i) an enzyme donor polypeptide conjugate,
• (ii) an analyte-binding protein specific for said analyte,
• (iii) an enzyme acceptor polypeptide of .beta.-galactosidase wherein said enzyme acceptor polypeptide is characterized by forming with said enzyme donor polypeptide an active enzyme complex having .beta.-galactosidase activity in the absence of analyte-binding protein binding to said conjugate, said enzyme acceptor polypeptide being further characterized by having an amino acid other than methionine substituted at position 443 and
• (iv) a substrate capable of reacting with said active enzyme complex, such that the rate of conversion of said substrate by said active enzyme complex can be monitored, and wherein said enzyme donor conjugate is capable of competing with said analyte to bind to said analyte-binding protein, thereby inhibiting the formation of active enzyme complex;
• (b) measuring the rate of conversion of substrate;
• 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.

An immunoassay method for determining an analyte in a sample suspected of containing said analyte comprising:

• (a) contacting said sample with
• (i) an enzyme donor polypeptide conjugate,
• (ii) an analyte-binding protein specific for said analyte,
• (iii) an enzyme acceptor polypeptide of .beta.-galactosidase wherein said enzyme acceptor polypeptide is characterized by forming with said enzyme donor polypeptide an active enzyme complex having .beta.-galactosidase activity in the absence of analyte-binding protein binding to said conjugate, said enzyme acceptor polypeptide being further characterized by having an amino acid other than cysteine substituted at position 76, and
• (iv) a substrate capable of reacting with said active enzyme complex, such that the rate of conversion of said substrate by said active enzyme complex can be monitored, and wherein said enzyme donor conjugate is capable of competing with said analyte to bind to said analyte-binding protein, thereby inhibiting the formation of active enzyme complex;
• (b) measuring the rate of conversion of substrate;
• 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.