Optimizing diagnostics for galactofuranose containing antigens
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. A method for diagnosing a microbial infection in a biological sample from a mammalian subject suspected of having, having, or susceptible to 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 (VH) domain of SEQ ID NO:1 and a variable light (VL) domain of SEQ ID NO:2; monoclonal antibody 24 (MAb 24) comprising a VH domain of SEQ ID NO:3 and a VL domain of SEQ ID NO:4; monoclonal antibody 686 (MAb 686) comprising a VH domain of SEQ ID NO:5 and a VL domain of SEQ ID NO:6; monoclonal antibody 838 (MAb 838) comprising a VH domain of SEQ ID NO:7 and a VL domain of SEQ ID NO:8; and monoclonal antibody 476 (MAb 476) comprising a VH domain of SEQ ID NO:9 and a VL domain of SEQ ID NO:10; and
(c) detecting the presence of at least one antibody-polysaccharide complex, wherein the detection of the presence of at least one antibody-polysaccharide complex is diagnostic of a microbial infection in a mammalian subject.
2. The method of claim 1 , wherein in step (a) treating the sample comprises contacting the sample with a substrate.
3. The method of claim 2 , wherein the substrate comprises a polyacrylamide resin.
4. The method of claim 2 , wherein the substrate comprises a desalting column.
5. The method of claim 4 , wherein the desalting column has a molecular weight cut off of less than 10 kDa.
6. The method of claim 4 , wherein the desalting column has a molecular weight cut off of 7 kDa.
7. A method for optimizing an assay for detecting the presence of at least one polysaccharide comprising a galF residue in a biological sample of the mammalian subject, the method comprising:
(a) treating the biological sample comprising contacting the sample with a substrate which binds salts with high affinity, thereby inhibiting human intelectin (hIntL) binding of galF residues present in the sample;
(b) contacting the treated sample of (a) with at least one antibody specific for at least one polysaccharide comprising a galF 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 (VH) domain of SEQ ID NO:1 and a variable light (VL) domain of SEQ ID NO:2; monoclonal antibody 24 (MAb 24) comprising a VH domain of SEQ ID NO:3 and a VL domain of SEQ ID NO:4; monoclonal antibody 686 (MAb 686) comprising a VH domain of SEQ ID NO:5 and a VL domain of SEQ ID NO:6; monoclonal antibody 838 (MAb 838) comprising a VH domain of SEQ ID NO:7 and a VL domain of SEQ ID NO:8; and monoclonal antibody 476 (MAb 476) comprising a VH domain of SEQ ID NO:9 and a VL domain of SEQ ID NO:10; and
(c) detecting the presence of at least one antibody-polysaccharide complex.
8. The method of claim 7 , wherein the substrate comprises a polyacrylamide resin.
9. The method of claim 7 , wherein the substrate comprises a desalting column.
10. The method of claim 9 , wherein the desalting column has a molecular weight cut off of less than 10 kDa.
11. The method of claim 9 , wherein the desalting column has a molecular weight cut off of 7 kDa.