Method to detect dirofilaria immitis infection

Abstract

The present invention includes a method to detect D. immitis infection in a host animal using a D. immitis Di33 protein to detect anti- D. immitis Di33 antibodies in a bodily fluid of the animal. Also included is a method to detect D. immitis infection in a host animal using a D. immitis anti-Di33 protein to detect Di33 proteins in a bodily fluid of the animal. The present invention also relates to D. immitis detection kits that include either a Di33 protein or an anti-Di33 antibody; such kits also include a composition to detect an immunocomplex between the anti-Di33 antibody and D. immitis Di33 protein. The present invention also includes Di33 proteins, nucleic acid molecules encoding such proteins, as well as recombinant molecules and recombinant cells comprising such nucleic acid molecules, and anti-Di33 antibodies. Also included are methods to produce such proteins, nucleic acid molecules and antibodies.

What is claimed is:  
     
         1 . A method to detect  D. immitis  in a non-adapted host comprising: 
 (a) contacting a bodily fluid collected from said host with a formulation comprising an isolated  D. immitis  Di33 protein under conditions sufficient to form an immunocomplex between Di33 protein and anti-Di33 antibodies; and    (b) measuring immunocomplex formation between said Di33 protein and anti-Di33 antibodies, if any, in said fluid, wherein the presence of said immunocomplex indicates that said host is or has recently been infected with  D. immitis.      
     
     
         2 . The method of  claim 1 , wherein said method detects  D. immitis  in said host prior to maturation of said  D. immitis  into an adult heartworm.  
     
     
         3 . The method of  claim 1 , wherein said method detects  D. immitis  in said host within at least about ten weeks post infection of said host with  D. immitis.    
     
     
         4 . The method of  claim 1 , wherein said method detects  D. immitis  infection in a non-adapted host harboring adult heartworms.  
     
     
         5 . The method of  claim 1 , wherein said method detects  D. immitis  infection resulting in the maturation of a single adult heartworm.  
     
     
         6 . The method of  claim 1 , wherein said method detects  D. immitis  infection resulting in the maturation of a single adult male heartworm.  
     
     
         7 . The method of  claim 1 , wherein said method detects  D. immitis  infection resulting in the maturation of a single adult female heartworm.  
     
     
         8 . The method of  claim 1 , wherein said antibody in said immunocomplex is selected from the group consisting of IgG, IgM and IgA antibodies.  
     
     
         9 . The method of  claim 1 , wherein said antibody in said immunocomplex is an IgE antibody.  
     
     
         10 . The method of  claim 1 , wherein said host is selected from the group consisting of a cat and a ferret.  
     
     
         11 . The method of  claim 10 , wherein said cat is a domestic cat.  
     
     
         12 . The method of  claim 1 , wherein said immunocomplex is measured by contacting said Di33 protein-contacted bodily fluid with a composition that selectively binds to an antibody selected from the group consisting of an IgG antibody, an IgE antibody, an IgM antibody, and an IgA antibody.  
     
     
         13 . The method of  claim 12 , wherein said composition is selected from the group consisting of an anti-IgG antibody, an anti-IgE antibody, an anti-IgM antibody, an anti-IgA antibody, an Fc γ  receptor molecule, an Fc ε  receptor molecule, an Fc μ  receptor molecule, and an Fc α  receptor molecule.  
     
     
         14 . The method of  claim 12 , wherein said composition is conjugated to a detectable marker.  
     
     
         15 . The method of  claim 14 , wherein said detectable marker is selected from the group consisting of an enzyme label, a radioactive label, a fluorescent label, a chemiluminescent label, a chromophoric label, and a ligand.  
     
     
         16 . The method of  claim 12 , wherein said composition is immobilized on a substrate.  
     
     
         17 . The method of  claim 16 , wherein said substrate comprises a material selected from the group consisting of plastic, glass, gel, celluloid, paper, and particulate materials.  
     
     
         18 . The method of  claim 16 , wherein said substrate is selected from the group consisting of a well, a plate, a dipstick, a bead, a lateral flow apparatus, a membrane, a filter, a tube, a dish, a celluloid-type matrix, and a magnetic particle.  
     
     
         19 . The method of  claim 1 , wherein said Di33 protein is immobilized on a substrate.  
     
     
         20 . The method of  claim 19 , wherein said substrate comprises a material selected from the group consisting of plastic, glass, gel, celluloid, paper, and particulate materials.  
     
     
         21 . The method of  claim 19 , wherein said substrate is selected from the group consisting of a well, a plate, a dipstick, a bead, a lateral flow apparatus, a membrane, a filter, a tube, a dish, a celluloid-type matrix, and a magnetic particle.  
     
     
         22 . The method of  claim 19 , wherein said substrate further comprises a detectable marker.  
     
     
         23 . The method of  claim 19 , wherein said substrate is conjugated to a detectable marker.  
     
     
         24 . The method of  claim 1 , wherein said immunocomplex forms in solution.  
     
     
         25 . The method of  claim 1 , wherein said step of measuring comprises an assay selected from the group consisting of an enzyme-linked immunoassay, a radioimmunoassay, a fluorescence immunoassay, a lateral flow assay, an agglutination assay, a particulate-based assay, an immunoprecipitation assay, and an immunoblotting assay.  
     
     
         26 . The method of  claim 1 , wherein said Di33 protein is conjugated to a detectable marker.  
     
     
         27 . The method of  claim 1 , wherein said bodily fluid is selected from the group consisting of blood, serum, plasma, urine, tears, saliva, lymph, nasal secretions and feces.  
     
     
         28 . The method of  claim 1 , wherein said formulation further comprises a carrier.  
     
     
         29 . The method of  claim 1 , wherein said formulation further comprises a  D. immitis  antigen other than said Di33 protein.  
     
     
         30 . The method of  claim 1 , wherein said formulation further comprises an antibody raised against a  D. immitis  antigen.  
     
     
         31 . The method of  claim 1 , wherein said  D. immitis  antigen comprises a circulating parasite antigen.  
     
     
         32 . A method to detect  D. immitis  in a host animal, said method comprising: 
 (a) contacting a bodily fluid collected from said animal with a formulation comprising an isolated  D. immitis  Di33 protein under conditions sufficient to form an immunocomplex between Di33 protein and anti-Di33 IgE antibodies; and    (b) measuring immunocomplex formation between said Di33 protein and anti-Di33 IgE antibodies, if any, in said fluid, wherein the presence of said immunocomplex indicates that said animal is or has recently been infected with D. immitis.    
     
     
         33 . The method of  claim 32 , wherein said animal is selected from the group consisting of cats, dogs and ferrets.  
     
     
         34 . The method of  claim 32 , wherein said method detects  D. immitis  in said animal within at least about ten weeks post infection of said animal with  D. immitis.    
     
     
         35 . The method of  claim 32 , wherein said method detects  D. immitis  infection resulting in the maturation of a single adult heartworm.  
     
     
         36 . The method of  claim 35 , wherein said adult heartworm is male.  
     
     
         37 . The method of  claim 35 , wherein said adult heartworm is female.  
     
     
         38 . A method to detect  D. immitis  infection in a non-adapted host within 10 weeks of infection, said method comprising detecting anti-Di33 antibodies in a bodily fluid collected from said host.  
     
     
         39 . The method of  claim 38 , wherein said method detects  D. immitis  infection resulting in the maturation of a single adult heartworm.  
     
     
         40 . The method of  claim 39 , wherein said adult heartworm is male.  
     
     
         41 . The method of  claim 39 , wherein said adult heartworm is female.  
     
     
         42 . A kit to detect  D. immitis  infection comprising an isolated  D. immitis  Di33 protein and a composition to detect antibodies capable of forming an immunocomplex with said Di33 protein.  
     
     
         43 . The kit of  claim 42 , wherein said antibodies are selected from the group consisting of IgG, IgE, IgM, and IgA antibodies.  
     
     
         44 . The kit of  claim 42  further comprising a substrate on which said Di33 protein can be immobilized.  
     
     
         45 . The kit of  claim 42 , wherein said Di33 protein is immobilized on a substrate.  
     
     
         46 . The kit of  claim 42  further comprising a substrate on which said composition can be immobilized.  
     
     
         47 . The kit of  claim 42 , wherein said composition is immobilized on a substrate.  
     
     
         48 . The kit of  claim 42 , wherein said composition is selected from the group consisting of an anti-IgG antibody, an anti-IgE antibody, an anti-IgM antibody, an anti-IgA antibody, an Fc γ  receptor molecule, an Fc ε  receptor molecule, an Fc μ  receptor molecule, and an Fc α  receptor molecule.  
     
     
         49 . The kit of  claim 42  further comprising an apparatus comprising: 
 (a) a support structure defining a flow path;  
 (b) a labeling reagent comprising a bead conjugated to said Di33 protein, wherein said labeling reagent is impregnated within the support structure in a labeling zone; and  
 (c) a capture reagent comprising said composition, wherein said capture reagent is located downstream of said labeling reagent within a capture zone fluidly connected to said labeling zone in such a manner that said labeling reagent can flow from said labeling zone into said capture zone.  
 
     
     
         50 . The kit of  claim 49 , wherein said apparatus further comprises a sample receiving zone located along said flow path.  
     
     
         51 . The kit of  claim 50 , wherein said sample receiving zone is located upstream of said labeling reagent.  
     
     
         52 . The kit of  claim 49 , wherein said apparatus further comprises an absorbent located at the end of said flow path.  
     
     
         53 . The kit of  claim 49 , wherein said support structure comprises a material that does not impede the flow of said bead from said labeling zone to said capture zone.  
     
     
         54 . The kit of  claim 49 , wherein said bead comprises a latex bead.  
     
     
         55 . The kit of  claim 49 , wherein said bead comprises a detectable marker.  
     
     
         56 . The kit of  claim 49 , wherein said Di33 protein is conjugated to a detectable marker.  
     
     
         57 . The kit of  claim 42  further comprising an apparatus comprising: 
 (a) a support structure defining a flow path;  
 (b) a labeling reagent comprising said composition, wherein said labeling reagent is impregnated within the support structure in a labeling zone; and  
 (c) a capture reagent comprising said Di33 protein, wherein said capture reagent is located downstream of said labeling reagent within a capture zone fluidly connected to said labeling zone in such a manner that said labeling reagent can flow from said labeling zone into said capture zone.  
 
     
     
         58 . The kit of  claim 57 , wherein said apparatus further comprises a sample receiving zone located along said flow path.  
     
     
         59 . The kit of  claim 58 , wherein said sample receiving zone is located upstream of said labeling reagent.  
     
     
         60 . The kit of  claim 42 , wherein said kit further comprises a  D. immitis  antigen other than said Di33 protein.  
     
     
         61 . A method to produce an isolated  D. immitis  Di33 protein comprising: 
 (a) culturing a bacterium transformed with a  D. immitis  Di33 nucleic acid molecule to produce a  D. immitis  Di33 protein-containing culture;    (b) recovering insoluble material comprising said Di33 protein from said culture; and    (c) purifying said Di33 protein from said insoluble material.    
     
     
         62 . The method of  claim 61 , wherein said step of purifying comprises the steps of: 
 (a) disrupting the insoluble material of step (b) to form a solution comprising Di33 protein;    (b) submitting said solution to cation exchange chromatography; and    (c) recovering said Di33 protein.    
     
     
         63 . The method of  claim 61 , wherein said step of purifying comprises the steps of: 
 (a) disrupting the unsoluble material of step (b) to form a solution comprising Di33 protein;    (b) submitting said solution to cation exchange chromatography to obtain a Di33 protein-containing fraction;    (c) submitting said fraction to hydrophobic interaction chromatography; and    (d) recovering said Di33 protein.    
     
     
         64 . An isolated  D. immitis  Di33 protein produced by the method of  claim 61 .  
     
     
         65 . A method for purifying a  D. immitis  Di33 protein comprising recovering Di33 protein from cation exchange chromatography of disrupted insoluble material obtained from a culture of  D. immitis  Di33 protein-producing recombinant cells.  
     
     
         66 . An isolated  D. immitis  Di33 protein produced by the method of  claim 65 .  
     
     
         67 . A protein selected from the group consisting of PHIS-PDi33 234  and PDi33 217 .  
     
     
         68 . A nucleic acid molecule selected from the group consisting of nDi33 346 , nDi33 750 , nDi33 702 , nDi33 708 , and nDi33 651 .  
     
     
         69 . A recombinant molecule comprising a nucleic acid molecule of  claim 68 .  
     
     
         70 . The recombinant molecule of  claim 69 , wherein said recombinant molecule is selected from the group consisting of pλPR-nD-i33 708  and pλPR-nDi33 651 .  
     
     
         71 . A recombinant cell comprising a nucleic acid molecule of  claim 68 .  
     
     
         72 . A method to produce an isolated  D. immitis  Di33 protein comprising culturing a recombinant cell comprising a nucleic acid molecule of  claim 68  and recovering said protein.  
     
     
         73 . A method to detect  D. immitis  in a non-adapted host comprising: 
 (a) contacting a bodily fluid collected from said host with a formulation comprising an isolated anti-Di33 antibody under conditions sufficient to form an immunocomplex between said anti-Di33 antibody and  D. immitis  Di33 protein; and    (b) measuring immunocomplex formation between said anti-Di33 antibody and  D. immitis  Di33 protein, if any, in said fluid, wherein the presence of said immunocomplex indicates said host is or recently has been infected with  D. immitis.      
     
     
         74 . A kit to detect  D. immitis  infection comprising an isolated anti-Di33 antibody and a composition to detect an immunocomplex between said anti-Di33 antibody and  D. immitis  Di33 protein.