Genentech, Inc.

Wai Lee Wong

Paul J Godowski

Melanie Rose Mark

Michael D Sadick

David L Shelton

Ross G Clark

Lena M Carlsson

P Mickey Williams

Yu-Ju G Meng

Anthony H Dodge

Paul W Sims

Dominick V Sinicropi

An oligonucleotide assay is useful for the detection of target compounds in samples which may contain the target compound.

PCR assay

Kinase receptor activation assay

An assay for measuring activation (i.e., autophosphorylation) of a tyrosine kinase receptor of interest is disclosed.

An assay for measuring activation (i.e., autophosphorylation) of a tyrosine kinase receptor of interest is disclosed. (a) A first solid phase is coated with a substantially homogeneous population of cells so that the cells adhere to the first solid phase. The cells have either an endogenous tyrosine kinase receptor or have been transformed with DNA encoding a receptor or 'receptor construct' and the DNA has been expressed so that the receptor or receptor construct is presented in the cell membranes of the cells. (b) A ligand is then added to the solid phase having the adhering cells, such that the tyrosine kinase receptor is exposed to the ligand. (c) Following exposure to the ligand, the adherent cells are solubilized, thereby releasing cell lysate. (d) A second solid phase is coated with a capture agent which binds specifically to the tyrosine kinase receptor, or, in the case of a receptor construct, to the flag polypeptide. (e) The cell lysate obtained in step (c) is added to the wells containing the adhering capture agent so as to capture the receptor or receptor construct to the wells. (f) A washing step is then carried out, so as to remove unbound cell lysate, leaving the captured receptor or receptor construct. (g) The captured receptor or receptor construct is exposed to a labelled antiphosphotyrosine antibody which identifies phosphorylated residues in the tyrosine kinase receptor. (h) Binding of the anti-phosphotyrosine antibody to the captured receptor or receptor construct is measured.

A ligand-mediated immunofunctional assay (LIFA) method for detecting the presence and the concentration of polypeptide hormone binding proteins which comprises capturing the binding protein with a solid phase bound first antibody, saturating the bound hormone binding protein with the ligand polypeptide hormone, and detecting the bound ligand polypeptide hormone with a detectably labeled second antibody specific for the ligand polypeptide hormone. In the absence of added saturating polypeptide hormone, the LIFA measures the amount of hormone binding protein bound to the endogenous ligand polypeptide hormone. A growth hormone binding protein assay illustrates the method of the present invention. LIFA assay results indicate that increased binding protein substantially increases growth hormone activity. Methods of use and formulations of growth hormone binding protein, growth hormone, insulin-like growth factor-I and insulin-like growth factor binding protein are disclosed.

Ligand-mediated immunofunctional hormone binding protein assay method

A ligand-mediated immunofunctional assay (LIFA) method for detecting the presence and the concentration of polypeptide hormone binding proteins which comprises capturing the binding protein with a solid phase bound first antibody, saturating the bound hormone binding protein with the ligand polypeptide hormone, and detecting the bound ligand polypeptide hormone with a detectably labeled second antibody specific for the ligand polypeptide hormone. In the absence of added saturating polypeptide hormone, the LIFA measures the amount of hormone binding protein bound to the endogenous ligand polypeptide hormone. A growth hormone binding protein assay illustrates the method of the present invention.

Growing community of inventors

Los Altos Hills, CA, United States of America

Wai Lee Wong