Isolating a target analyte from a body fluid

Abstract

The invention generally relates to using magnetic particles and magnets to isolate a target analyte from a body fluid sample. In certain embodiments, methods of the invention involve introducing magnetic particles including a target-specific binding moiety to a body fluid sample in order to create a mixture, incubating the mixture to allow the particles to bind to a target, applying a magnetic field to capture target/magnetic particle complexes on a surface, and washing with a wash solution that reduces particle aggregation, thereby isolating target/magnetic particle complexes.

1 . A method for isolating a target analyte from a body fluid sample, the method comprising the steps of:
 introducing magnetic particles comprising a target-specific binding moiety to a body fluid sample in order to create a mixture;   incubating said mixture to allow said particles to bind to a target;   applying a magnetic field to capture target/magnetic particle complexes on a surface; and   washing with a wash solution that reduces particle aggregation, thereby isolating target/magnetic particle complexes.   
     
     
         2 . The method of  claim 1 , wherein said target-specific binding moiety is an antibody. 
     
     
         3 . The method of  claim 2 , wherein said antibody is specific for bacteria. 
     
     
         4 . The method of  claim 1 , wherein said particles are superparamagnetic beads. 
     
     
         5 . The method of  claim 1 , wherein said incubating step comprises incubating said mixture in a buffer that inhibits cell lysis. 
     
     
         6 . The method of  claim 1 , wherein said body fluid is selected from blood, sputum, urine, saliva, sweat, and cerebral spinal fluid. 
     
     
         7 . The method of  claim 5 , wherein said buffer comprises Tris(hydroximethyl)-aminomethane hydrochloride at a concentration of about 75 mM. 
     
     
         8 . The method of  claim 4 , wherein said magnetic particles comprise at least 70% superparamagnetic beads by weight. 
     
     
         9 . The method of  claim 4 , wherein said superparamagnetic beads are from about 100 nm to about 250 nm in diameter. 
     
     
         10 . The method of  claim 1 , further comprising retaining said magnetic particles in a magnetic field during said washing step. 
     
     
         11 . The method of  claim 1 , wherein washing comprises:
 removing the magnetic field, thereby re-suspending the target/magnetic particle complexes;   introducing the wash solution;   flowing the re-suspended target/magnetic particle complexes over the surface in the presence of a re-applied magnetic field, thereby re-capturing the target/magnetic particle complexes on the surface.   
     
     
         12 . The method of  claim 2 , wherein said antibody is specific for fungi. 
     
     
         13 . The method of  claim 3 , wherein said bacteria is pathogenic. 
     
     
         14 . The method of  claim 3 , wherein the bacteria is gram positive bacteria. 
     
     
         15 . The method of  claim 3 , wherein the bacteria is gram negative bacteria 
     
     
         16 . The method of  claim 3 , wherein said bacteria is selected  from E. coli, Listeria, Clostridium, Mycobacterium, Shigella, Borrelia, Campylobacter, Bacillus, Salmonella, Staphylococcus, Enterococcus, Pneumococcus, Streptococcus , and a combination thereof. 
     
     
         17 . The method of  claim 1 , wherein the magnetic particle is an iron containing magnetic particle. 
     
     
         18 . The method of  claim 17 , wherein the magnetic particle comprises iron oxide or iron platinum. 
     
     
         19 . A method for isolating a target microorganism from a body fluid sample, the method comprising the steps of:
 introducing magnetic particles comprising a target-specific binding moiety to a body fluid sample in order to create mixture;   incubating said mixture to allow said particles to bind to target;   applying a magnetic field to isolate on a surface magnetic particles to which target is bound;   washing with a wash solution that reduces particle aggregation; and   lysing the captured bacteria and extracting DNA for further analysis by PCR, microarray hybridization or sequencing.   
     
     
         20 . A method for isolating a target analyte from a body fluid sample, the method comprising the steps of:
 introducing magnetic beads comprising 70% by weight superparamagnetic particles and having a target-specific binding moiety to a body fluid sample in order to create mixture;   incubating said mixture to allow said particles to bind to target;   applying a magnetic field to isolate on a surface magnetic particles to which target is bound; and   washing with a wash solution that reduces particle aggregation.   
     
     
         21 . A method for isolating a target analyte from a body fluid sample, the method comprising the steps of:
 introducing superparamagnetic particles having a diameter from about 100 nm to about 250 nm and comprising a target-specific binding moiety to a body fluid sample in order to create a mixture;   incubating said mixture to allow said particles to bind to a target;   applying a magnetic field to isolate target/magnetic particle complexes on a surface; and   washing with a wash solution that reduces particle aggregation.   
     
     
         22 . A method for isolating as low as 1 CFU/ml of bacteria in a blood sample, the method comprising the steps of:
 introducing superparamagnetic particles having a diameter from about 100 nm to about 250 nm and comprising a bacteria-specific binding moiety to a body fluid sample in order to create a mixture;   incubating said mixture to allow said particles to bind to bacteria;   applying a magnetic field to isolate bacteria/magnetic particle complexes on a surface; and   washing with a wash solution that reduces particle aggregation, thereby isolating as low as 1 CFU/ml of bacteria in the blood sample.