Immunoassay and antibody selection methods using cell surface expressed libraries

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.

What is claimed is:  
     
         1 . A method for binding an analyte, comprising the steps of: 
 (a) obtaining a host cell that expresses an anti-analyte antibody or analyte-combining antibody fragment on the surface of the 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 . The method of  claim 1 , further defined as a method for quantifying the amount of an analyte in a sample, the method further comprising the step of detecting the amount of antibody/analyte complex so formed.  
     
     
         4 . The method of  claim 3 , wherein the amount of said antibody/analyte complex is detected by means of a detectable label linked to said analyte.  
     
     
         5 . The method of  claim 3 , wherein: 
 (a) the host cell is contacted with a test sample that includes an unknown amount of analyte and a known concentration of the analyte linked to a detectable label;    (b) analyte present in the test sample competes for binding with the analyte-combining antibody on the surface of the cell; and    (c) where a decrease in the amount of detectable label in solution as determined in step (b) in comparison to step (a) is indicative of the presence of said analyte in said test sample.    
     
     
         6 . The method of  claim 5 , wherein the amount of the analyte is determined by collecting the detectably labeled analyte free of said analyte in solution and quantifying the amount of the label.  
     
     
         7 . The method of  claim 5 , wherein the amount of the labeled analyte is determined by collecting the detectably labeled analyte free from said analyte and quantifying the amount of the label.  
     
     
         8 . The method of  claim 7 , wherein the amount of said antibody/analyte complex is determined by means of a second antibody linked to a detectable label, the second antibody having binding affinity for the analyte.  
     
     
         9 . The method of  claim 7 , wherein the amount of said antibody/analyte complex is determined by means of a second antibody having binding affinity for said analyte and a third antibody linked to a detectable label, the third antibody having binding affinity for said second antibody.  
     
     
         10 . The method of  claim 3 , wherein the amount of said antibody/analyte complex is determined by means of detecting a radioactive label, biotin, avidin, a fluorescent label, an electrochemiluminescent label or an enzyme that generates a colored product upon contact with a chromogenic substrate.  
     
     
         11 . A method for conducting a competitive binding assay, comprising the steps of: 
 (a) obtaining a first and a second host cell, each host cell expressing the same antibody or antigen-combining antibody fragment on the surface of the cell;    (b) contacting said first host cell with a standard antigen sample that contains a known amount of an antigen linked to a detectable label, under conditions effective to form a first antibody/antigen admixture;    (c) contacting said second host cell with said standard antigen sample admixed with a test sample suspected of containing said antigen, under conditions effective to form a second antibody/antigen admixture; and    (d) determining a decrease in the amount of labeled antigen in the second antibody/antigen admixture in comparison to said first antibody\antigen admixture.    
     
     
         12 . The method of  claim 11 , wherein determining the amount of the first and second antibody/antigen admixtures is achieved by collecting the detectably labeled antigen free from said first and second antibody/antigen admixtures and determining the amount of the label associated with the free antigen.  
     
     
         13 . The method of  claim 11 , wherein determining the amount of the first and second antibody/antigen admixtures is achieved by collecting the detectably labeled first and second antibody/antigen admixtures free from said antigen and determining the amount of the label associated with the antibody/antigen admixtures.  
     
     
         14 . A method for selecting an antibody or antibody fragment from a plurality of candidate antibodies or antibody fragments, the method comprising the steps of: 
 (a) obtaining an expression vector library that encodes a plurality of distinct candidate antibodies or antibody fragments;    (b) expressing said library in Gram negative host cells that express an antibody fragment on the surface of the cell; and    (c) selecting a host cell that expresses a desired antibody.    
     
     
         15 . The method of  claim 14 , wherein selecting a host cell that expresses a desired antibody comprises the steps of: 
 (a) contacting said antibody- or antibody fragment-expressing cells with a selected antigen; and    (b) identifying cells that bind to said selected antigen.    
     
     
         16 . The method of  claim 14 , wherein said expression vector library is obtained by a method comprising the steps of: 
 (a) administering to an animal an immunologically effective amount of a composition comprising a selected antigen;    (b) obtaining from the animal a plurality of distinct DNA segments that encode distinct antibodies or antibody fragments; and    (c) incorporating said plurality of DNA segments into a plurality of expression vectors, the vectors expressing antibodies or antibody fragments on the outer membrane surface of a Gram negative host cell.    
     
     
         17 . The method of  claim 16 , wherein said plurality of DNA segments are obtained by a method comprising the steps of: 
 (a) isolating mRNA from antibody-producing cells of said animal;    (b) amplifying a plurality of distinct RNA segments using a set of nucleic acid primers having sequences complementary to antibody constant region or antibody framework region nucleic acid sequences; and    (c) preparing a plurality of distinct DNA segments having sequences complementary to said amplified RNA segments.    
     
     
         18 . The method of  claim 14 , wherein said expression vector library is obtained by a method comprising the steps of: 
 (a) obtaining a DNA segment that encodes a selected antibody or antibody fragment;    (b) mutagenizing said DNA segment to provide a plurality of DNA segments that encode antibodies or antibody fragments with distinct binding specificities; and    (c) incorporating said plurality of DNA segments into a plurality of expression vectors, the vectors expressing antibodies or antibody fragments on the surface of a Gram negative host cell.    
     
     
         19 . The method of  claim 14 , wherein said selected cells that express a desired antibody are subjected to cleavage to release the selected antibody or antibody fragment from the surface of the outer membrane.  
     
     
         20 . The method of  claim 15 , wherein said selected antigen is linked to a detectable label.  
     
     
         21 . The method of  claim 20 , wherein said selected antigen is linked to a radioactive label, biotin, avidin or an enzyme that generates a colored product upon contact with a chromogenic substrate.  
     
     
         22 . The method of  claim 21 , wherein said cells that bind to said selected antigen are identified by a method comprising the steps of: 
 (a) contacting said plurality of cells with said detectably labeled antigen under conditions effective to allow specific antigen-antibody binding;    (b) removing non-specifically bound antigen from said cells; and    (c) identifying the antibody- or antibody fragment-expressing cells by detecting the presence of the bound detectable label.    
     
     
         23 . The method of  claim 20 , wherein said selected antigen is linked to a fluorescent label.  
     
     
         24 . The method of  claim 23 , wherein said cells that bind to said selected antigen are identified by a method comprising the steps of: 
 (a) contacting said plurality of cells with said fluorescently labeled antigen under conditions effective to allow specific antigen-antibody binding;    (b) subjecting said cells to automated cell sorting; and    (c) identifying the antibody- or antibody fragment-expressing cells by detecting the fluorescently labeled sorted cells.    
     
     
         25 . The method of  claim 24 , wherein said cells are subjected to sorting by flow cytometry.  
     
     
         26 . The method of  claim 24 , wherein said cells are subjected to a first and a second round of automated cell sorting.  
     
     
         27 . The method of  claim 26 , wherein regrowth of sorted cells is conducted between said first and said second rounds of cell sorting.  
     
     
         28 . The method of  claim 20 , wherein said selected antigen is linked to an electrochemiluminescent label.  
     
     
         29 . The method of  claim 28 , wherein said selected antigen is linked to the electrochemiluminescent label Ru(bipy) 3   2+.    
     
     
         30 . The method of  claim 28 , wherein said cells that bind to said selected antigen are identified by a method comprising the steps of: 
 (a) contacting said plurality of cells with said electrochemiluminescently labeled antigen under conditions effective to allow specific antigen-antibody binding; and    (b) identifying the antibody- or antibody fragment-expressing cells by detecting chemiluminescence.    
     
     
         31 . An expression vector library that encodes a plurality of antibodies or antibody fragments having distinct antigen-combining regions or metal binding sites or catalytic antibodies or antibody conjugates, the library expressing said antibodies or antibody fragments on the outer membrane surface upon incorporation into a Gram negative host cell.  
     
     
         32 . A kit comprising, in suitable container means, an aliquoted expression vector library that encodes a plurality of distinct antibodies or antigen-combining antibody fragments expressible on the outer membrane surface of a Gram negative host cell.  
     
     
         33 . The kit of  claim 32 , further comprising, in suitable container means, a sample of viable Gram negative host cells.  
     
     
         34 . A heterogeneous population of Gram negative host cells that express, on the surface of their outer membranes, a plurality of distinct antibodies or a plurality of antibody fragments having distinct antigen-combining regions.  
     
     
         35 . The heterogeneous population of cells of  claim 34 , prepared by the process of  claim 14 .  
     
     
         36 . A kit comprising, in suitable container means, a population of viable Gram negative host cells, each cell expressing a distinct antibody or a distinct antigen-combining antibody fragment on the surface of the outer membrane.  
     
     
         37 . The kit of  claim 36 , further comprising, in suitable container means, an antigen linked to a detectable label or an antigen immobilized on a solid support.  
     
     
         38 . A method for catalyzing a chemical reaction, comprising the steps of: 
 (a) obtaining a Gram negative host cell that expresses a catalytic antibody or catalytic antibody fragment on the surface of the outer membrane; and    (b) contacting said host cell with a sample containing the necessary substrates for said chemical reaction.    
     
     
         39 . A method for stimulating an immune response, comprising administering to an animal a pharmaceutical composition comprising an immunologically effective amount of a Gram negative host cell that expresses an antibody or antigen-combining antibody fragment on the surface of the outer membrane.  
     
     
         40 . The method of  claim 39 , wherein a sample is obtained from said animal to provide a polyclonal or monoclonal antibody.  
     
     
         41 . A method of providing an antibody to an animal, comprising administering to said animal a pharmaceutical composition comprising a Gram negative host cell that expresses an antibody or antigen-combining antibody fragment on the surface of the outer membrane, the composition provided in an amount effective to bind to a detrimental antigen that may be present within said animal.