Methods and apparatus for assays of bacterial spores

Abstract

A sample of unknown bacterial spores is added to a test strip. The sample of unknown bacterial spores is drawn to a first sample region on the test strip by capillary action. Species-specific antibodies are bound to the sample when the unknown bacterial spores match the species-specific antibodies, otherwise the sample is left unbound. DPA is released from the bacterial spores in the bound sample. The terbium ions are combined with the DPA to form a Tb-DPA complex. The combined terbium ions and DPA are excited to generate a luminescence characteristic of the combined terbium ions and DPA to detect the bacterial spores. A live/dead assay is performed by a release of the DPA for live spores and a release of DPA for all spores. The detection concentrations are compared to determine the fraction of live spores. Lifetime-gated measurements of bacterial spores to eliminate any fluorescence background from organic chromophores comprise labeling the bacterial spore contents with a long-lifetime lumophore and detecting the luminescence after a waiting period. Unattended monitoring of bacterial spores in the air comprises the steps of collecting bacterial spores carried in the air and repeatedly performing the Tb-DPA detection steps above. The invention is also apparatus for performing the various methods disclosed above.

We claim:  
     
         1 . A method for lateral flow immunoassay for species-specific bacterial spore detection and quantification comprising: 
 providing a matrix including terbium ions;    releasing DPA from the bacterial spores;    combining the terbium ions with the DPA in solution; and    exciting the combined terbium ions and DPA to generate a luminescence characteristic of the combined terbium ions and DPA to detect the bacterial spores.    
     
     
         2 . The method of  claim 1  where releasing DPA from the bacterial spores comprises microwaving the spores.  
     
     
         3 . The method of  claim 1  where releasing DPA from the bacterial spores comprises germinating the spores with L-alanine.  
     
     
         4 . The method of  claim 1  where releasing DPA from the bacterial spores comprises sonicating the spores with microspheres.  
     
     
         5 . The method of  claim 1  where releasing DPA from the bacterial spores comprises autoclaving the spores.  
     
     
         6 . The method of  claim 1  where exciting the combined terbium ions and DPA to generate a luminescence characteristic of the combined terbium ions and DPA comprises radiating the combined terbium ions and DPA with ultraviolet light.  
     
     
         7 . A method for lateral flow immunoassay for bacterial spore detection and quantification comprising: 
 adding a sample of unknown bacterial spores to a test strip;    drawing the sample of unknown bacterial spores to a first sample region on the test strip;    selectively binding species-specific antibodies to the sample when the unknown bacterial spores match the species-specific antibodies, otherwise leaving the sample unbound;    releasing DPA from the bacterial spores in the bound sample;    combining the terbium ions with the DPA; and    exciting the combined terbium ions and DPA to generate a luminescence characteristic of the combined terbium ions and DPA to detect the bacterial spores.    
     
     
         8 . The method of  claim 7  further comprising: 
 adding a standard of known bacterial spores with known concentration to the test strip;  
 drawing the standard of known bacterial spores to a second sample region on the test strip;  
 selectively binding species-specific antibodies to the standard when the known bacterial spores match the species-specific antibodies, otherwise leaving the standard unbound;  
 releasing DPA from the bacterial spores in the bound standard;  
 combining the terbium ions with the DPA;  
 exciting the combined terbium ions and DPA to generate a luminescence characteristic of the combined terbium ions and DPA to detect the bacterial spores of the standard; and  
 comparing the intensity of the excited luminescence from the sample with the standard to derive a quantification of the spore concentration in the sample.  
 
     
     
         9 . The method of  claim 8  further comprising confirming arrival of the sample and standard in the first and second sample regions respectively by means of a visual indicator.  
     
     
         10 . The method of  claim 8  where releasing DPA from the bacterial spores in the sample and standard comprises microwaving the spores.  
     
     
         11 . The method of  claim 8  where releasing DPA from the bacterial spores in the sample and standard comprises germinating the spores with L-alanine.  
     
     
         12 . The method of  claim 8  where releasing DPA from the bacterial spores in the sample and standard comprises sonicating the spores with microspheres.  
     
     
         13 . The method of  claim 8  where releasing DPA from the bacterial spores in the sample and standard comprises autoclaving the spores.  
     
     
         14 . The method of  claim 8  where exciting the combined terbium ions and DPA in the sample and standard to generate a luminescence characteristic of the combined terbium ions and DPA comprises radiating the combined terbium ions and DPA in the sample and standard with ultraviolet light.  
     
     
         15 . A method for live/dead assay for bacterial spores comprising: 
 providing a solution including terbium ions in a sample of live and dead bacterial spores;    releasing DPA from viable bacterial spores by germination from a first unit of the sample;    combining the terbium ions with the DPA in solution released from viable bacterial spores; and    exciting the combined terbium ions and DPA released from viable bacterial spores to generate a first luminescence characteristic of the combined terbium ions and DPA to detect the viable bacterial spores;    releasing DPA from dead bacterial spores in a second unit of the sample by autoclaving, sonication or microwaving;    combining the terbium ions with the DPA in solution released from dead bacterial spores; and    exciting the combined terbium ions and DPA released from dead bacterial spores to generate a second luminescence characteristic of the combined terbium ions and DPA to detect the dead bacterial spores; and    generating a ratio of the first to second luminescence to yield a fraction of bacterial spores which are alive.    
     
     
         16 . A method for lifetime-gated measurements of bacterial spores to eliminate any fluorescence background from organic chromophores comprising: 
 providing a solution including terbium ions with a sample of bacterial spores;    labeling the bacterial spore contents with a long-lifetime lumophore;    releasing DPA from the bacterial spores;    combining the terbium ions with the DPA in solution; and    exciting the combined terbium ions and DPA for a first time period;    waiting a second time period before detecting luminescence; and    detecting a luminescence characteristic of the combined terbium ions and DPA after the second time period during a defined temporal window synchronized with luminescence timed from the long lifetime lumophore to detect the bacterial spores.    
     
     
         17 . The method of  claim 16  where the first time period of excitation is of the order of nanoseconds, where the second time period is of the order of microseconds and where the defined temporal window is of the order of milliseconds.  
     
     
         18 . The method of  claim 17  where the first time period of excitation is of the order of 1-10 nanoseconds, where the second time period is of the order of tens of microseconds and where the defined temporal window is of the order of 1-10 milliseconds.  
     
     
         19 . The method of  claim 16  where the first time period of excitation is of the order of nanoseconds, where the second time period is of the order of tenths to tens of milliseconds and where the defined temporal window is of the order of hundreds of microseconds.  
     
     
         20 . A method for unattended monitoring of bacterial spores in the air comprising: 
 collecting bacterial spores carried in the air;    suspending the collected bacterial spores in a solution including terbium ions;    releasing DPA from the bacterial spores;    combining the terbium ions with the DPA in solution; and    exciting the combined terbium ions and DPA to generate a luminescence characteristic of the combined terbium ions and DPA;    detecting the luminescence to determine the presence of the bacterial spores; and    generating an alarm signal when the presence of bacterial spores is detected or the concentration thereof reaches a predetermined magnitude.    
     
     
         21 . The method of  claim 20  where collecting bacterial spores carried in the air comprises capturing the bacterial spores with an aerosol sampler or impactor.  
     
     
         22 . The method of  claim 20  where detecting the luminescence to determine the presence of the bacterial spores comprises monitoring the luminescence with a spectrometer or fluorimeter.  
     
     
         23 . The method of  claim 20  where collecting bacterial spores carried in the air comprising continuously sampling the air and where detecting the luminescence to determine the presence of the bacterial spores comprises continuously monitoring the luminescence.  
     
     
         24 . The method of  claim 23  where releasing DPA from the bacterial spores comprising microwaving the bacterial spores to heat the solution and where combining the terbium ions with the DPA in solution comprises cooling the heated solution to increase the fraction of bound Tb-DPA complex.  
     
     
         25 . An apparatus for unattended monitoring of bacterial spores in the air comprising: 
 a biosampler for capturing the bacterial spores in the air and having a collection vessel containing a solution including terbium ions into which the captured bacterial spores are suspended;    means for releasing DPA from the bacterial spores in the solution to allow the DPA to combine with the terbium ions to form a Tb-DPA complex;    an energy source for exciting the Tb-DPA complex to generate luminescence;    an electro-optical circuit to measure the luminescence; and    an alarm circuit coupled to the electro-optical circuit to detect a bacterial spore concentration above a predetermined threshold.    
     
     
         26 . An apparatus for lateral flow immunoassay for bacterial spore detection and quantification comprising: 
 a strip of material for providing lateral capillary flow of a solution including terbium ions across the strip;    an input region on the strip for receiving a liquid sample containing terbium ions, the first zone being provided with a first antibody for specific binding to a specific specie of bacterial spores;    a sample region of the strip laterally displaced from the input region and communicated thereto by means of capillary flow therebetween, the sample region being provided with a second antibody to capture bacterial spores with the attached first antibody and to immobilize them;    means for releasing DPA from the bacterial spores in the sample region of the strip to then allow the terbium ions to combine with the DPA in solution; and    an energy source to excite the combined terbium ions and DPA in the sample region of the strip to generate a luminescence characteristic of the combined terbium ions and DPA; and    a luminescence detector to identify the presence or measure the concentration of the bacterial spores in the sample region of the strip.    
     
     
         27 . The apparatus of  claim 26  where the means for releasing DPA from the bacterial spores in the sample region of the strip comprises a microwave heater, means for adding L-alanine to the solution, means for sonicating the spores with microspheres, or an autoclave.