Antibody selection methods using cell surface expressed libraries

• US 5,866,344 A
• Filed: 05/23/1995
• Issued: 02/02/1999
• Est. Priority Date: 11/15/1991
• Status: Expired due to Term

• Current Assignee: Board of Regents of the University of Texas System (University of Texas System)
• Original Assignee: Board of Regents of the University of Texas System (University of Texas System)
• Inventors: Georgiou, George
• Primary Examiner(s): Green, Lora M.
• Assistant Examiner(s): MUSTO, NEAL
• Application Number:
  US08/447,402
• Time in Patent Office: 1,351 Days
• Field of Search: 436/518, 436/519, 435/7.1
• US Class Current: 435/7.21
• CPC Class Codes: C07K 14/245 Escherichia (G)
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/16 against material from plants
• CPC Class Codes: C07K 16/44 against material not provided for elsewhere , e.g. haptens, metals, DNA, RNA, amino acids
• CPC Class Codes: C07K 2317/565 Complementarity determining region [CDR]
• CPC Class Codes: C07K 2317/622 Single chain antibody (scFv)
• CPC Class Codes: C07K 2317/92 Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/035 containing a signal for targeting to the external surface of a cell, e.g. to the outer membrane of Gram negative bacteria, GPI- anchored eukaryote proteins
• CPC Class Codes: C12N 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• CPC Class Codes: C12N 15/625 containing a sequence coding for a signal sequence
• CPC Class Codes: C12N 9/16 acting on ester bonds (3.1)
• CPC Class Codes: C12N 9/86 acting on amide bonds in cyclic amides, e.g. penicillinase (3.5.2)
• CPC Class Codes: C40B 40/02 Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors

Abstract

The invention relates to novel competitive immunoassays that are useful in detecting and quantitatively measuring analytes down to the nanomolar range. The invention also includes methods of selecting antibodies from libraries of polypeptides expressed on a cell surface. In conducting immunoassays, anti-analyte antibody molecules are expressed on the surface of a bacterial cell and then used to bind with labeled analyte. Quantitation is performed by competitively displacing the bound labeled analyte with a known amount of analyte and measuring the label. The method is rapid and inexpensive and may be performed with readily available safe labeling reagents such as fluorescent compounds.

Claims

1. A method for binding an analyte, comprising the steps of:

(a) obtaining a host cell that expresses from a vector within said host cell an anti-analyte antibody or analyte-combining antibody fragment on the surface of the host cell; and

(b) contacting said host cell with a sample suspected of containing an analyte that binds to said anti-analyte antibody or analyte-combining antibody fragment under conditions effective to allow the formation of an antibody/analyte complex.

2. The method of claim 1, further defined as a method for removing an analyte from a sample, the method comprising contacting said host cell with a sample containing an unwanted analyte and obtaining the sample free from said complexed analyte.

3. A method for binding an analyte, comprising the steps of:

(a) obtaining a gram negative bacterial host cell that expresses from a vector within said host cell, an anti-analyte antibody or analyte-combining antibody fragment on the surface of the host cell; and

(b) contacting said host cell with a sample suspected of containing an analyte that binds to said anti-analyte antibody or analyte-combining antibody fragment under conditions effective to allow the formation of an antibody/analyte complex.

4. The method of claim 3, further defined as a method for removing an analyte from a sample, the method comprising contacting said host cell with a sample containing an unwanted analyte and obtaining the sample free from said complexed analyte.