Sample Preparation and Specific Capture for Multiplex Detection of Target Analytes (i.e., Bacteria, Viruses, Etc.)

Abstract

A method and apparatus for the rapid and reliable preparation of a sample for use in testing for target analytes such as bacteria, viruses, toxins and pathogenic agents in various products. A sample for testing the target analyte is collected from various sources. The method for sample preparation provides for an express process for preparing collected samples for testing. The collected sample may be concentrated by centrifugation, filtration, or other means suitable for sample concentration, homogenized with the addition of a broth and enriched for specified period of time. Immunomagnetic separation of the sample occurs with receptor-coated magnetic microspheres in different test-specific formulations (singleplex or multiplex). An automatic testing system is disclosed which includes a biological testing cassette, in which testing of the sample occurs using liquid crystal diagnostic methodologies.

What is claimed is: 
     
         1 . A method for preparing a sample containing multiple target analytes of antigenic materials for detection of the analytes comprising:
 homogenizing and incubating the sample with an enrichment media;   adding magnetic microspheres having different preselected formulations and a solution to the sample to form a mixture, the magnetic microspheres being structured and arranged to bind to specific preselected antigenic materials contained within the mixture;   precipitating the antigenic materials out of the mixture when the antigenic materials becomes specifically bound to the magnetic microspheres to form a precipitate and a supernatant;   discarding the supernatant; and   isolating the precipitate to produce a prepared sample.   
     
     
         2 . The method of  claim 1 , wherein the precipitating step further comprises the step of applying of a magnetic field to the precipitate. 
     
     
         3 . The method of  claim 1 , further comprising the steps of:
 washing the prepared sample in a PBS or PBST solution to form a mixture with the magnetic microspheres;   applying a magnetic field to the mixture to stabilize the magnetic microspheres; and   collecting the antigen-containing materials to form an isolated prepared sample.   
     
     
         4 . The method of  claim 1 , wherein the solution is a buffer solution. 
     
     
         5 . The method of  claim 1 , wherein the mixture further comprises a surfactant. 
     
     
         6 . The method of  claim 5 , wherein the surfactant is a nonionic surfactant. 
     
     
         7 . The method of  claim 1 , wherein the magnetic microspheres are comprised of a material selected from the group consisting of iron, iron oxide, iron nitride, iron carbide, nickel and cobalt, and mixtures and alloys thereof. 
     
     
         8 . The method of  claim 1 , wherein the magnetic microspheres are paramagnetic microspheres. 
     
     
         9 . The method of  claim 1 , wherein the magnetic microspheres are non-functionalized microspheres. 
     
     
         10 . The method of  claim 9 , wherein the magnetic microspheres are plain polystyrene beads 
     
     
         11 . The method of  claim 1 , wherein the magnetic microspheres have an average diameter within the range of approximately 0.1 μm to approximately 100 μm. 
     
     
         12 . The method of  claim 1 , wherein the magnetic microspheres have an average diameter within the range of approximately 1 μm to approximately 50 μm. 
     
     
         13 . The method of  claim 12 , wherein the magnetic microspheres have an average diameter within the range of approximately 1 μm to approximately 5 μm. 
     
     
         14 . The method of  claim 1 , wherein the antigenic materials are selected from the group consisting of immunoglobulins, proteins, (lipo)polysaccharides, or enzymes. 
     
     
         15 . The method of  claim 1 , wherein the prepared sample is structured and arranged for use in liquid crystal diagnostics. 
     
     
         16 . The method of  claim 1 , wherein the magnetic microspheres are functionalized microspheres. 
     
     
         17 . The method of  claim 16 , wherein the functionalized microspheres are coated with an affinity ligand. 
     
     
         18 . The method of  claim 1 , wherein the sample is a meat, produce, pet food, a swab/surface, rinse or fecal sample. 
     
     
         19 . An apparatus for preparing samples for multiplex detection of analytes, comprising:
 a plurality of magnetic microspheres that are structured and arranged to bind to antigenic materials contained within the samples;   one or more microsphere holding devices;   a magnetic holder having a magnet for holding one or more of the microsphere holding devices; and   a cassette containing a diagnostic media, the cassette being configured to hold the prepared sample.   
     
     
         20 . The apparatus of  claim 19 , further including a buffer solution. 
     
     
         21 . The apparatus of  claim 19 , further including a surfactant. 
     
     
         22 . The apparatus of  claim 21 , wherein the surfactant is a non-ionic surfactant. 
     
     
         23 . The apparatus of  claim 19 , wherein the magnetic microspheres are comprised of a material selected from the group consisting of iron, iron oxide, iron nitride, iron carbide, nickel and cobalt, and mixtures and alloys thereof. 
     
     
         24 . The apparatus of  claim 19 , wherein the magnetic microspheres are paramagnetic microspheres. 
     
     
         25 . The apparatus of  claim 19 , wherein the magnetic microspheres are magnetic latex microspheres. 
     
     
         26 . The apparatus of  claim 19 , wherein the magnetic microspheres have an average diameter within the range of approximately 0.1 μm to approximately 100 μm.