Amplification method for solid phase immunoassays

Abstract

The present invention is directed to immunoassays for detecting one or more target analytes in a fluid sample wherein the detection reaction occurs on a solid support and involves an amplification system. In particular, the invention is directed to making and using a test device having at least one site for detecting the presence of at least one target analyte, wherein a conjugate comprising dextran-polystreptavidin is immobilized at the test site(s) as a capture reagent for a complex containing the target analyte.

1 . An immunoassay method comprising use of a dextran-polystreptavidin conjugate immobilized at a discrete site on a porous carrier material, the dextran-polystreptavidin conjugate deposited at the discrete site and then incubated under conditions sufficient to immobilize the dextran-polystreptavidin conjugate at the discrete site.  
   
   
       2 . The method of  claim 1  wherein immobilization occurs in the absence of a vacuum.  
   
   
       3 . The method of  claim 1  wherein immobilization occurs in the absence of latex particles.  
   
   
       4 . The method of  claim 1  wherein incubation is for about 10 h to about 15 h at about 60° C., or for about 7 days to about 14 days at about 20° C.  
   
   
       5 . The method of  claim 1  used in a lateral flow immunoassay.  
   
   
       6 . An immunoassay amplification system comprising 
 a porous carrier containing a dextran-polystreptavidin conjugate immobilized at a test site for a target analyte, the target analyte comprising a target antigen or a target antibody,    at least one biotinylated antibody or antigen, or fragment thereof, for a respective target antigen or antibody,    at least one labeled antibody or antigen, or fragment thereof, for the respective target antigen or antibody,    the polystreptavidin-dextran conjugate capturing the biotinylated antibody, antigen, or fragment thereof at the test site, the biotinylated antibody, antigen, or fragment thereof forming a sandwich at the test site with target analyte, if present, and the labeled antibody, antigen, or fragment thereof.    
   
   
       7 . The system of  claim 6  wherein the target analyte is at least one of influenza A antigen or influenza B antigen.  
   
   
       8 . The system of  claim 6  wherein the target analyte is at least one of an infectious disease antigen, an antibody, a hormone, or a receptor.  
   
   
       9 . A solid phase immunoassay amplification method comprising dextran-polystreptavidin deposited at a test site on a porous carrier under conditions sufficient to immobilize the dextran-polystreptavidin at the test site and thus amplify the amount of a target analyte bound to a labeled antibody at the test site in a sandwich immunoassay.  
   
   
       10 . The method of  claim 9  wherein the method sandwiches the target antigen at the test site with a biotinylated antibody bound to the polystreptavidin at the test site and a labeled antibody to the target antigen.  
   
   
       11 . The method of  claim 9  wherein the biotinylated and labeled antibodies are the same or different.  
   
   
       12 . The method of  claim 9  wherein the label is at least one of an enzyme, an enzyme fragment, a metal sol, a latex particle, a fluorescent molecule, or a dye.  
   
   
       13 . The method of  claim 9  wherein the biotinylated antibody is provided at a discrete unoccupied site on the porous carrier.  
   
   
       14 . The method of  claim 9  wherein the biotinylated antibody is provided on a discrete porous carrier.  
   
   
       15 . A solid phase immunoassay comprising 
 a first porous carrier comprising a dextran-polystreptavidin conjugate immobilized at a test site,    a second porous carrier upstream of and in wicking flow with the first porous carrier, the second porous carrier comprising a sample receiving area and a conjugate site, the conjugate site comprising a mobilizable biotinylated antibody, and    a labeled antibody, the antibodies directed against a target antigen.    
   
   
       16 . The immunoassay of  claim 15  further comprising a backing layer.  
   
   
       17 . The immunoassay of  claim 15  further comprising an absorbent pad downstream of said test site and optional control site.  
   
   
       18 . The immunoassay of  claim 15  in a single housing.