Optimizing diagnostics for galactofuranose containing antigens

Abstract

Disclosed herein are methods of detecting microbial infection in mammalian subjects comprising treatment of a sample and detection of galactofuranose (galF)-containing antigenic components utilizing monoclonal antibodies. The methods disclosed provide for pretreatment of biological samples, such as urine samples, to maximize detection of galF antigens and improvement of sensitivity of galF antigen detection assays. The methods include minimizing intelectin-1 binding to galF antigens and improvement of monoclonal antibody binding. The detection methods are useful for identifying the presence of microbial antigens related to bacterial, fungal, and parasitic pathogens, including Streptococcus pneumoniae, Aspergillus species, Fusarium species, Coccidioides species, Cryptococcus species, Histoplasma species, and Leishmania species.

1 - 11 . (canceled) 
     
     
         12 . A method for diagnosing a microbial infection in a human subject suspected of having, having, or susceptible to having a fungal infection by detecting the presence of at least one polysaccharide comprising a galactofuranose residue in a urine sample of the human subject comprising:
 a) contacting the urine sample with an antibody which recognizes at least one epitope of a galactofuranose residue of a polysaccharide which is attached to a first agent which renders the antibody detectable on a solid surface, wherein the antibody specific for at least one epitope of the galactofuranose residue of the polysaccharide or glycan is selected from the group consisting of monoclonal antibody 205 (MAb 205) comprising a variable heavy (V.sub.H) domain encoded by SEQ ID NO:1 and a variable light (V.sub.L) domain encoded by SEQ ID NO:2; monoclonal antibody 24 (MAb 24) comprising a V.sub.H domain encoded by SEQ ID NO:3 and a V.sub.L domain encoded by SEQ ID NO:4; monoclonal antibody 686 (MAb686) comprising a V.sub.H domain encoded by SEQ ID NO:5 and a V.sub.L domain encoded by SEQ ID NO:6; monoclonal antibody 838 (MAb 838) comprising a V.sub.H domain encoded by SEQ ID NO:7 and a V.sub.L domain encoded by SEQ ID NO:8; and monoclonal antibody 476 (MAb 476) comprising a V.sub.H domain encoded by SEQ ID NO:9 and a V.sub.L domain encoded by SEQ ID NO:10;   b) contacting the urine sample with a lateral flow device on which is immobilized a second agent capable of capturing the antibody specific for at least one epitope of the galactofuranose residue of the polysaccharide or glycan when it has reacted with said epitope, wherein the lateral flow device consists of an enzyme-linked immunosorbent assay (ELISA); and   c) detecting a fungal infection in accordance with instructions provided with a kit also containing the antibody and the lateral flow device.   
     
     
         13 . The method of claim  1 , wherein the antibody specific for at least one epitope of the galactofuranose residue of the polysaccharide or glycan is monoclonal antibody 476 (MAb 476) comprising a V.sub.H domain encoded by SEQ ID NO:9 and a V.sub.L domain encoded by SEQ ID NO:10. 
     
     
         14 . The method of  claim 12 , wherein the first agent is an optically detectable label. 
     
     
         15 . The method of  claim 14 , wherein the optically detectable label is a latex bead impregnated with a fluorescent tag. 
     
     
         16 . The method of  claim 12 , wherein the lateral flow device comprises a solid surface along which the antibody specific for at least one epitope of the galactofuranose residue of the polysaccharide or glycan attached to the first agent can migrate by capillary action. 
     
     
         17 . The method of  claim 16 , wherein also immobilized on the lateral flow device is a third agent capable of capturing the antibody specific for at least one epitope of the galactofuranose residue of the polysaccharide or glycan when it has not reacted with said epitope. 
     
     
         18 . The method of  claim 12 , wherein the ELISA comprises a direct ELISA or a sandwich ELISA. 
     
     
         19 . The method of  claim 12 , wherein a mechanism for removing an inhibitor found in urine samples which interferes with the binding of the antibody specific for at least one epitope of the galactofuranose residue of the polysaccharide or glycan to such epitopes is utilized. 
     
     
         20 . The method of  claim 12 , wherein the diagnosis involves screening the human subject for the risk of developing a fungal infection, or for detecting the presence of a fungal infection. 
     
     
         21 . The method of  claim 20 , wherein the fungal infection is caused by  Aspergillus  species. 
     
     
         22 . A method for diagnosing a microbial infection related to bacterial, fungal, and parasitic pathogens, including  Aspergillus  species,  Fusarium  species,  Coccidioides  species,  Cryptococcus  species,  Histoplasma  species, and  Leishmania  species in an immunocompromised human subject suspected of having, having, or at risk for having a microbial infection by detecting the presence of at least one polysaccharide or glycan comprising a galactofuranose residue in a urine sample of the immunocompromised human subject comprising:
 a) obtaining a kit containing a binding molecule which carries the amino acid sequences encoded by both SEQ ID NOS. 1 and 2, or SEQ ID NOS. 3 and 4, or SEQ ID NOS. 5 and 6, or SEQ ID NOS. 7 and 8, or SEQ ID NOS. 9 and 10, recognizes at least one epitope of a galactofuranose residue of a polysaccharide or glycan secreted by a microbe and either carries a detectable label or is accompanied by an additional reagent that carries a detectable label and also carries one epitope of a galactofuranose residue of a polysaccharide secreted by the microbe, a lateral flow device on which is immobilized either the binding molecule or an agent capable of capturing the binding molecule either before or after it has reacted with the epitope, wherein the lateral flow device consists of an enzyme-linked immunosorbent assay (ELISA); and instructions for using the binding molecule and the lateral flow device to diagnose the microbial infection;   b) contacting the urine sample with the binding molecule;   c) contacting the urine sample with the lateral flow device; and   d) detecting the microbial infection.   
     
     
         23 . The method of  claim 22 , wherein the diagnosis involves screening the human subject for the presence of pathogens related to  Aspergillus  species,  Fusarium  species,  Coccidioides  species,  Cryptococcus  species,  Histoplasma  species, and  Leishmania  species. 
     
     
         24 . The method of  claim 22 , wherein the ELISA comprises a direct ELISA or a sandwich ELISA. 
     
     
         25 . The method of  claim 22 , wherein the kit further comprises a desalting column for removing the effects of an inhibitor found in urine samples which interferes with the binding of the binding molecule which recognizes at least one epitope of the galactofuranose residue of the polysaccharide or glycan to such epitopes. 
     
     
         26 . A method for diagnosing a microbial infection using a biological sample from a mammalian subject suspected of having, having, or at risk for having a microbial infection by detecting the presence of at least one polysaccharide comprising a galactofuranose residue in a biological sample of the mammalian subject, the method comprising:
 (a) treating the biological sample to inhibit human intelectin (hIntL) binding of galactofuranose residues present in the sample, wherein the treatment of the biological sample comprises contacting the biological sample with at least one ligand which binds directly to intelectin, or substrates which bind divalent cations with high affinity;   (b) contacting the treated sample of (a) with at least one antibody specific for at least one polysaccharide comprising a galactofuranose residue in an effective amount to produce a detectable amount of antibody-polysaccharide complex, wherein the at least one antibody specific for at least polysaccharide comprising a galactofuranose residue is selected from the group consisting of monoclonal antibody 205 (MAb 205) comprising a variable heavy (V.sub.H) domain encoded by SEQ ID NO:1 and a variable light (V.sub.L) domain encoded by SEQ ID NO:2; monoclonal antibody 24 (MAb 24) comprising a V.sub.H domain encoded by SEQ ID NO:3 and a V.sub.L domain encoded by SEQ ID NO:4; monoclonal antibody 686 (MAb686) comprising a V.sub.H domain encoded by SEQ ID NO:5 and a V.sub.L domain encoded by SEQ ID NO:6; monoclonal antibody 838 (MAb 838) comprising a V.sub.H domain encoded by SEQ ID NO:7 and a V.sub.L domain encoded by SEQ ID NO:8; and monoclonal antibody 476 (MAb 476) comprising a V.sub.H domain encoded by SEQ ID NO:9 and a V.sub.L domain encoded by SEQ ID NO:10;   (c) contacting the biological sample with a lateral flow device on which is immobilized a second agent capable of capturing the antibody specific for at least one epitope of the galactofuranose residue of the polysaccharide or glycan when it has reacted with said epitope, wherein the lateral flow device consists of an enzyme-linked immunosorbent assay (ELISA); and   (d) detecting the presence of at least one antibody-polysaccharide complex, wherein the detection of the presence of at least one antibody-antigen complex is diagnostic of a microbial infection in a mammalian subject.   
     
     
         27 . The method of  claim 26 , wherein the ELISA comprises a direct ELISA or a sandwich ELISA. 
     
     
         28 . The method of  claim 26 , wherein in step (a) treating the sample comprises contacting the sample with a substrate. 
     
     
         29 . The method of  claim 28 , wherein the substrate comprises a polyacrylamide resin and/or a desalting column. 
     
     
         30 . The method of  claim 29 , wherein the desalting column has a molecular weight cut off of less than 10 kDa, or a molecular weight cut off of 7 kDa. 
     
     
         31 . The method of  claim 26 , wherein the microbial infection comprises a fungal infection. 
     
     
         32 . The method of  claim 31 , wherein the fungal infection comprises an infection caused by an organism related to  Aspergillus  species. 
     
     
         33 . The method of  claim 26 , wherein the mammal is a human. 
     
     
         34 . The method of  claim 33 , wherein the human is immunocompromised. 
     
     
         35 . The method of  claim 26 , wherein the biological sample is urine.