Lateral flow chromatographic binding assay device

Abstract

The present invention relates to improved specific binding assay devices comprising a chromatographic medium including a reaction site at which a specific binding reagent is immobilized, a sample application well located adjacent to the chromatographic medium and offset upstream from the reaction site, and liquid absorption blotter offset downstream from the reaction site.

1. A test device for determining the presence or amount of an analyte substance in a sample by means of one or more specific binding reactions comprising:
 a bibulous chromatographic medium which extends throughout said device and is free of chromatographic transport- facilitating agent , defining a flowpath having capillarity and the  capacity for chromatographic solvent transport of one or more reactive sample components and non-immobilized reagents; said chromatographic medium comprising (1) a prefiltering zone; (2) a reaction site  zone disposed downstream from said prefiltering zone at which is present  containing an immobilized reagent capable of binding  which specifically binds a member selected from the group consisting of said analyte substance and a labeled specific binding material, and at which the presence or amount of immobilized specific binding material, may be  is capable of being detected; and (3) a downstream zone free of said immobilized reagent disposed downstream from said reaction site  zone,  
 a sample application means selected from the group consisting ofa single undivided well and an absorbent paddefined by a housing element comprising an upper and lower portion; wherein said upper portion defines the spatial boundaries of said well and said lower portion comprises a portion of said chromatographic medium which is upstream of said prefiltering zone, said well having sufficient capacity to hold a volume of sample for chromatographic movement of said volume of sample from said well to a liquid absorption means, said well being located adjacent to and disposed in fluid contact with said prefiltering zone of said chromatographic medium and offset upstream from said reaction sitezone, and  
 said liquid absorption means consisting of a quantity of blotter material disposed in fluid contact with said downstream zone of said chromatographic medium and offset downstream from said reaction sitezone.  
 
     
     
       2. The test device according to  claim 1  wherein said sample application means consists of a well. 
     
     
       3. The test device according to  claim 1  wherein said sample application means consists of an absorbent pad. 
     
     
       4. The test device according to  claim 1  wherein said well comprises a non-removable filter. 
     
     
       5. The test device according to  claim 1  wherein a member of a signal generating substrate/cofactor group is immobilized at said reaction zone. 
     
     
       6. The test device according to  claim 5  wherein nitroblue tetrazolium is immobilized at said reaction zone. 
     
     
       7. A method for determining the presence or amount of an analyte substance in a sample which method utilizes a device comprising:
 a bibulous chromatographic medium which extends throughout said device and is free of chromatographic transport- facilitating agent , defining a flowpath having capillarity and the  capacity for chromatographic solvent transport of one or more non-immobilized reagents and reactive sample components; said chromatographic medium comprising (1) a prefiltering zone; (2) a reaction site  zone disposed downward from said prefiltering zone at which is present  containing an immobilized reagent capable of binding  which specifically binds a member selected form the group consisting of said anlayte substance and labeled specific binding material, and at which the presence or amount of immobilized labeled specific binding material may be  is capable of being detected and (3) a downstream zone free of said immobilized reagent disposed downstream from said reaction site  zone,  
 a sample application means selected from the group consisting ofa single, undivided well and an absorbent paddefined by a housing element having an upper and lower portion; wherein said upper portion defines the spatial boundaries of said well and said lower portion comprises a portion of said chromatographic medium which is upstream of said prefiltering zone, said well being of sufficient capacity to hold a volume of sample for chromatographic movement from said well to a liquid absorption means, said well being located adjacent to and disposed in fluid contact with said prefiltering zone of said chromatographic materialmedium and offset upstream from said reaction sitezone, and  
 asaid liquid absorption means consisting of a quantity of blotter material disposed in fluid contact with said downstream zone of said chromatographicchromatographic medium and offset downstream from said reaction sitezone, said method comprising: 
 (a) applying a  said volume of said sample sample application means  to said well whereby said sample is free of chromatographic transport-facilitating agent and is transported  absorbed onto said chromatographic medium at  and is chromatographically transported through said prefiltering zone and along said chromatographic medium through said reaction site  zone, and said downstream zone to said sample  liquid absorption means,    (b) contacting said labeled specific binding material to said reaction site  zone, and    (c) determining the presence or amount of said labeled specific binding material immobilized at said reaction sites  zones as an indication of the presence or amount of the analyte substance in the sample.    
 
     
     
       8. The method according to  claim 7  wherein unbound sample materials and unbound specific binding materials are removed from the reaction zone by means of a wash step. 
     
     
       9. The method according to  claim 7  wherein said unlabeled specific binding material participates in a specific binding reaction with said analyte substance. 
     
     
       10. The method according to  claim 7  wherein said reaction site  zone includes an immobilized member of a signal generating substrate/cofactor group, said method further comprising the step (c′) of contacting one or more additional members of said signal generating substrate/cofactor group to said reaction site to initiate a signal generating reaction. 
     
     
       11. The method according to  claim 10  wherein nitroblue tetrazolium is immobilized at said reaction site  zone and bromochlorindolylphosphate is added to initiate a signal generating reaction. 
     
     
       12. A test device for determining the presence or amount of an analyte substance in a sample by means of one or more specific binding reactions comprising:
   a bibulous chromatographic medium - free of chromatographic transport - facilitating agent having capillarity and the capacity for lateral chromatographic transport of one or more reactive sample components and non - immobilized reagents, said medium including a reaction zone comprising an immobilized reagent which binds a member selected from the group consisting of said analyte substance and a labeled specific binding material, said reaction zone occupying less than the entire area of the medium such that there remains an upstream portion and a downstream portion;        a single undivided well defined by a housing element comprising an upper and lower portion; wherein said upper portion defines the spatial boundaries of said well and said lower portion comprises a part of said upstream portions, said well being in fluid contact with the upstream portion of said chromatographic medium and being of sufficient capacity to retain a volume of fluid sample for chromatographic movement of said volume of sample along said chromatographic medium from said well through said reaction zone and into a liquid absorption means;        said liquid absorption means consisting of a quantity of blotter material disposed in - fluid contact with said downstream zone of said chromatographic medium.      
     
     
       13. The test device according to  claim 1  wherein said chromatographic medium is selected from the group consisting of nitrocellulose or mixed nitrocellulose ester.  
     
     
       14. The method according to  claim 7  wherein said chromatographic medium is selected from the group consisting of nitrocellulose or mixed nitrocellular ester.  
     
     
       15. The test device according to  claim 14  wherein said chromatographic medium is selected from the group consisting of nitrocellulose or mixed nitrocellulose ester.  
     
     
       16. The test device according to  claim 12  wherein said well includes a non- removable filter.