Methods and materials for capture antibody targeted fluorescent in-situ hybridization (cat-fish)

Abstract

The subject invention concerns materials and methods for detecting a target cell in a population. Methods of the invention comprise internally labeling cells via fluorescence in situ hybridization (FISH) using probes that target rRNA, followed by binding of capture antibodies targeted (CAT) for specific cell surface epitopes on the target cells. In one embodiment, the target cells are bacterial cells.

We claim: 
     
         1 . A method for detecting a target cell in a population of cells, wherein the method comprises:
 a) obtaining a sample of cells from the population of cells;   b) fixing the cells in a solution that will allow for antibody binding to one or more antigens on the surface of the cells after hybridization processing steps;   c) dehydrating the fixed cells;   d) contacting the fixed cells with a detectably labeled oligonucleotide probe that targets and hybridizes with rRNA of the target cell under suitable hybridization conditions;   e) contacting the cells after step d) with antibody immobilized on a surface platform, wherein the antibody binds to one or more antigens on the surface of the target cell; and   f) detecting the detectably labeled oligonucleotide probe hybridized to rRNA of the target cell.   
     
     
         2 . The method according to  claim 1 , wherein cells are isolated from the sample following step a). 
     
     
         3 . The method according to  claim 2 , wherein the cells are isolated by centrifugation of the sample. 
     
     
         4 . The method according to  claim 1 , wherein the solution in step b) is a non-cross linking fixative. 
     
     
         5 . The method according to  claim 4 , wherein the fixative is a methacarn solution. 
     
     
         6 . The method according to  claim 1 , wherein the cells are dehydrated in step c) using one or more ethanol washes and/or air drying. 
     
     
         7 . The method according to  claim 1 , wherein the detectably labeled oligonucleotide probe is labeled with a fluorescent label, a chemiluminescent reagent, a bioluminescent reagent, an enzyme, or a radioisotope. 
     
     
         8 . The method according to  claim 1 , wherein after step d) the cells are centrifuged and resuspended in a suitable buffer one or more times. 
     
     
         9 . The method according to  claim 1 , wherein the surface platform is a bead; microtiter plate; microarray; fiber optic waveguide; planar array biosensor; or microfluidic chip. 
     
     
         10 . The method according to  claim 9 , wherein the bead can be attracted by a magnetic field and the bead having target cells bound thereto is isolated by applying a magnetic field. 
     
     
         11 . The method according to  claim 10 , wherein the bead is a magnetic or super paramagnetic bead. 
     
     
         12 . The method according to  claim 1 , wherein the bound target cells are washed one or more times prior to detecting the detectably labeled oligonucleotide probe. 
     
     
         13 . The method according to  claim 1 , wherein the antibody is a monoclonal antibody, or an antigen binding fragment thereof. 
     
     
         14 . The method according to  claim 1 , wherein the antibody is a polyclonal antibody, or an antigen binding fragment thereof. 
     
     
         15 . The method according to  claim 1 , wherein the rRNA is 16S and/or 23S RNA. 
     
     
         16 . The method according to  claim 1 , wherein the target cell is a bacterial cell. 
     
     
         17 . The method according to  claim 16 , wherein the bacterial cell is  Nitrospira  spp.,  Nitrosospira  spp.,  Nitrobacter  spp.,  Nitrosomonas  spp.,  Clostridium  spp.,  Bacillus  spp., methanogenic archaea, coliforms,  Salmonella  spp.,  Bacteroides  spp.,  Staphylococcus  spp.,  Streptococcus  spp.,  Neisseria  spp.,  Haemophilus  spp.,  Bordetella  spp.,  Listeria  spp.,  Mycobacterium  spp.,  Shigella  spp.,  Pseudomonas  spp.,  Brucella  spp.,  Treponema  spp.,  Mycoplasma  spp.,  Yersinia  spp.,  Vibrionaceae  spp.,  Chlamydia  spp.,  Legionella  spp.,  Escherichia  spp.,  Acinetobacter  spp.,  Burkholderia  spp.,  Thiobacillus  spp.,  Rickettsia  spp.,  Sphinomonas  spp.,  Francisella  spp.,  Campylobacter  spp., or  Helicobacter  spp. 
     
     
         18 . The method according to  claim 1 , wherein the hybridization conditions in step d) are stringent hybridization conditions. 
     
     
         19 . The method according to  claim 9 , wherein the bead is a microbead or wherein the bead contains a detectable label or fluorophore. 
     
     
         20 . A kit for detecting a target cell, wherein the kit comprises in one or more containers:
 a) a detectably labeled probe that can bind to or hybridize with rRNA of the target cell;   b) a surface platform that can have an antibody attached thereto.