Chimeric target molecules having a regulatable activity
The present invention relates to a chimeric target molecule having an activity which can be regulated or modulated by a binding molecule. The invention also relates to methods of using the chimeric target molecule to detect the presence and/or amount of a desired analyte in a sample. The analyte is a binding molecule, or a competitor of a binding molecule, which binding molecule, upon binding to the target molecule, alters the activity of the target molecule in a detectable way. In one aspect of the invention, a binding molecule binds to the chimeric molecule, inactivating it. An analyte in a test sample competes and/or displaces the binding molecule from the chimera, reactivating it. The reappearance of activity in the presence of the analyte indicates its existence in the test sample existence and amount. Another aspect of the invention relates to a binding molecule which regulates a chimeric target molecule and methods of producing it.
1. A method for determining the presence or amount of an analyte in a test sample, comprising:
forming a mixture of (1) a chimeric enzyme comprising β-lactamase and a binding site moiety, said binding site moiety including at least one amino acid, said chimeric enzyme having a sequence of said binding site moiety inserted in said enzyme or replacing at least one amino acid of said enzyme with the proviso that the activity of the chimeric enzyme is modulated upon binding of a binding molecule to the binding site moiety, (2) a test sample, (3) a binding molecule which binds to a binding site moiety of the chimeric enzyme and modulates the activity of the enzyme, and (4) a substrate upon which the chimeric enzyme catalytically acts; and
detecting the amount of catalysis of the substrate and thereby determining the presence or absence of said analyte of interest.
2. The method of claim 1 , wherein the analyte competes with the chimeric enzyme for binding to the binding molecule.
3. The method of claim 1 , wherein the binding molecule is said analyte.
4. The method of claim 1 , wherein the binding molecule is an antibody.
5. The method for determining the presence or amount of an analyte in a test sample, comprising:
forming a mixture of (1) a chimeric enzyme comprising β-lactamase and a binding site moiety, said binding site moiety including at least one amino acid, wherein said chimeric enzyme having a sequence of said binding site moiety inserted in said enzyme or replacing at least one amino acid of said enzyme with the proviso that the activity of the chimeric enzyme is modulated upon binding of a binding molecule to the binding site moiety, (2) test sample, and (3) a substrate upon which the chimeric enzyme catalytically acts; and
detecting the amount of catalysis of the substrate and thereby determining the presence or absence of said analyte of interest.
6. The method of claim 5 , wherein the analyte is an antibody.
7. The method of claim 1 , wherein the enzymatic activity of the chimeric enzyme in the unbound state is equivalent to that of the β-lactamase.
8. The method of claim 5 , wherein the enzymatic activity of the chimeric enzyme in the unbound state is equivalent to that of the β-lactamase.