Patent ID: 7897347

Claim:
A method for preparing an immunoglobulin suitable for determining binding of an immunoglobulin to a target in an intracellular environment, comprising the steps of: i) providing a first fusion polypeptide comprising an immunoglobulin fused to a first molecule, wherein a nucleic acid encoding the immunoglobulin is obtained from a phage library encoding a repertoire of immunoglobulin-encoding nucleic acids, wherein the immunoglobulin is unknown and no prior application of phage display is used to isolate immunoglobulins which bind to a target; ii) providing a second fusion polypeptide comprising said target fused to a second molecule, wherein said first and second molecules are separable domains of a reporter molecule; iii) expressing said first fusion polypeptide together with said second fusion polypeptide in an intracellular environment, wherein binding of said immunoglobulin with said target brings said first molecule and said second molecule into operative association, such that binding of said immunoglobulin and said target leads to stable interaction of said first and second molecules, thus producing a detectable reporter molecule that generates a signal; and iv) detecting said signal from said detectable reporter molecule, wherein said detection of a signal is indicative of stable binding activity between said immunoglobulin and said target in the intracellular environment; and isolating those immunoglobulins that stably bind to the target; the isolating step comprising the steps of: (a) expressing a repertoire of immunoglobulin genes in a selection system and isolating those genes which encode immunoglobulins specific for a desired target; (b) wherein the isolated immunoglobin genes specific for a desired target are cloned in-frame with a nucleic acid encoding the first molecule and expressed, wherein stable interaction of the first molecule with a second molecule generates a signal, in order to produce a fusion polypeptide comprising the immunoglobulin and the first molecule.