Serological diagnosis of protostrongylidae infections and identification of unique antigens

Abstract

Novel Protostrongylidea antigens and early and accurate diagnostic methods for Protostrongylidae infection are disclosed. Novel P. tenuis -specific antigens and methods of discriminating between P. tenuis infection and infection with other closely-related members of the Protostrongylidae family are provided. Novel E. cervi -specific antigens and methods of discriminating between E. cervi infection and infection with other closely-related members of the Protostrongylidae family are provided.

What is claimed is:  
     
         1 . An isolated immunogenic Protostrongylidae antigen selected from the group consisting of a  P. tenuis -specific 20 kDa antigen, a  P. tenuis -specific 37 kDa antigen, an  E. cervi -specific 37 kDa antigen, a 52 kDa antigen and a  P. tenuis -specific 75 kDa antigen, a common 105 kDa antigen or a common 158 kDa antigen, as determined by SDS-PAGE gel electrophoresis and immunoblotting.  
     
     
         2 . An isolated antibody that recognizes an epitope on an antigen of  claim 1 .  
     
     
         3 . A polynucleotide encoding an antigen according to  claim 1 .  
     
     
         4 . An isolated immunogenic  P. tenuis -specific antigen.  
     
     
         5 . A  P. tenuis  antigen according to  claim 4  selected from the group consisting of a  P. tenuis -specific 20 kDa antigen, a  P. tenuis -specific 37 kDa antigen and a  P. tenuis -specific 75 kDa antigen, as determined by SDS-PAGE gel electrophoresis.  
     
     
         6 . A  P. tenuis -specific antigen according to  claim 4  wherein the  P. tenuis -specific antigen is a 37 kDa antigen, as determined by SDS-PAGE gel electrophoresis.  
     
     
         7 . An isolated antibody that recognizes an epitope on the antigen of  claim 4 .  
     
     
         8 . A polynucleotide encoding an antigen according to  claim 4 .  
     
     
         9 . A polynucleotide encoding an antigen according to  claim 6 .  
     
     
         10 . An isolated immunogenic  E. cervi -specific antigen.  
     
     
         11 . An  E. cervi -specific antigen according to  claim 10  selected from the group consisting of a  E. cervi -specific 37 kDa antigen and a  E. cervi -specific 52 kDa antigen, as determined by SDS-PAGE gel electrophoresis.  
     
     
         12 . An  E. cervi -specific antigen according to  claim 10  wherein the  E. cervi -specific antigen is a 37 kDa antigen, as determined by SDS-PAGE gel electrophoresis.  
     
     
         13 . An isolated antibody that recognizes an epitope on the antigen of  claim 10 .  
     
     
         14 . A polynucleotide encoding an antigen according to  claim 10 .  
     
     
         15 . A polynucleotide encoding an antigen according to  claim 12 .  
     
     
         16 . A method of diagnosing Protostrongylidae infection in a vertebrate subject, comprising detecting the presence of at least one common Protostrongylidae antigen in a biological sample obtained from the subject, wherein the presence of the at least one common antigen is indicative of Protostrongylidae infection.  
     
     
         17 . The method of  claim 16  wherein the method comprises detecting the presence of a common 105 kDa antigen or a common 158 kDa antigen.  
     
     
         18 . The method of  claim 16 , wherein the method comprises detecting the presence of more than one common antigen.  
     
     
         19 . The method of  claim 18 , wherein the method comprises detecting the presence of a common 105 kDa antigen and a common 158 kDa antigen.  
     
     
         20 . The method of  claim 16 , wherein the at least one common antigen is detected using an antibody.  
     
     
         21 . The method of  claim 16 , wherein the at least one common antigen is detected using a nucleic acid probe.  
     
     
         22 . The method of  claim 16 , wherein the at least one common antigen is detected using PCR.  
     
     
         23 . The method of  claim 16 , wherein the Protostrongylidae infection is caused by  P. tenuis  or  E. cervi.    
     
     
         24 . The method of  claim 20 , wherein the method comprises: 
 (a) reacting the biological sample with one or more isolated common Protostrongylidae antigens under conditions which allow anti-Protostrongylidae antibodies, when present in the sample, to specifically bind with said common antigens;    (b) removing unbound antibodies;    (c) providing one or more moieties capable of associating with the bound antibodies; and    (d) detecting the presence or absence of the one or more moieties, thereby detecting the presence or absence of Protostrongylidae infection.    
     
     
         25 . The method of  claim 24 , wherein the one or more moieties comprises a detectably labeled immunoglobulin antibody.  
     
     
         26 . The method of  claim 24 , wherein the one or more Protostrongylidae common antigens are a common 105 kDa antigen or a common 158 kDa antigen.  
     
     
         27 . The method of  claim 16 , wherein the biological sample is a serum sample.  
     
     
         28 . A method for specifically diagnosing  P. tenuis  infection in a subject, the method comprising detecting the presence of one or more  P. tenuis -specific antigens in a biological sample obtained from the subject.  
     
     
         29 . The method of  claim 28 , wherein the one or more  P. tenuis -specific antigens are detected using an antibody.  
     
     
         30 . The method of  claim 28 , wherein the one or more  P. tenuis -specific antigens are detected using nucleic acid hybridization.  
     
     
         31 . The method of  claim 28 , wherein the one or more  P. tenuis -specific antigens are detected using PCR.  
     
     
         32 . The method of  claim 28 , wherein the method comprises, 
 (a) reacting the biological sample with one or more isolated  P. tenuis -specific antigens under conditions which allow antibodies, when present in the sample, to specifically bind with the specific antigens;    (b) removing unbound antibodies;    (c) providing one or more moieties capable of associating with the bound antibodies; and    (d) detecting the presence or absence of the one or more moieties, thereby detecting the presence or absence of  P. tenuis  infection.    
     
     
         33 . The method of  claim 32 , wherein the one or more moieties comprises a detectably labeled immunoglobulin antibody.  
     
     
         34 . The method of  claim 28 , wherein the one or more  P. tenuis -specific antigens are selected from the group consisting of a  P. tenuis -specific 20 kDa antigen, a  P. tenuis -specific 37 kDa antigen and a  P. tenuis -specific 75 kDa antigen.  
     
     
         35 . The method of  claim 28 , wherein the  P. tenuis  specific antigen is a  P. tenuis -specific 37 kDa antigen.  
     
     
         36 . The method of  claim 28 , wherein the biological sample is a serum sample.  
     
     
         37 . A method for specifically diagnosing  E. cervi  infection in a subject, the method comprising detecting the presence of one or more  E. cervi -specific antigens in a biological sample obtained from the subject.  
     
     
         38 . The method of  claim 37 , wherein the one or more  E. cervi -specific antigens are detected using an antibody.  
     
     
         39 . The method of  claim 37 , wherein the one or more  E. cervi -specific antigens are detected using nucleic acid hybridization.  
     
     
         40 . The method of  claim 37 , wherein the one or more  E. cervi -specific antigens are detected using PCR.  
     
     
         41 . The method of  claim 37 , wherein the method comprises, 
 (a) reacting the biological sample with one or more isolated  E. cervi -specific antigens under conditions which allow antibodies, when present in the sample, to specifically bind with the specific antigens;    (b) removing unbound antibodies;    (c) providing one or more moieties capable of associating with the bound antibodies; and    (d) detecting the presence or absence of the one or more moieties, thereby detecting the presence or absence of  E. cervi  infection.    
     
     
         42 . The method of  claim 41 , wherein the one or more moieties comprises a detectably labeled immunoglobulin antibody.  
     
     
         43 . The method of  claim 37 , wherein the one or more  E. cervi -specific antigens are 37 kDa or 52 kDa antigens.  
     
     
         44 . The method of  claim 37 , wherein the  E. cervi -specific antigen is a 37 kDa antigen.  
     
     
         45 . The method of  claim 37 , wherein the biological sample is a serum sample.  
     
     
         46 . A method of detecting antibodies to parasites in a biological sample, comprising 
 (a) reacting the biological sample with an antigen preparation selected from the group consisting of an ES-L3 antigen preparation and an sL3 antigen preparation, under conditions which allow parasitic antibodies to bind to an antigen in the antigen preparations and form an antigen:antibody complex; and    (b) detecting the presence or absence of said complex, thereby detecting the presence or absence of parasitic antibodies in said biological sample.    
     
     
         47 . The method of  claim 46  wherein the parasites are Protostrongylidae.  
     
     
         48 . A kit for use in the diagnostic method according to  claim 28 , comprising in a suitable packaging: 
 one or more common or  P. tenuis - or  E. cervi -specific antigens immobilized on a solid support; and    a reagent suitable for detecting, in a biological sample, the presence of antibodies to the one or more common or  P. tenuis - or  E. cervi -specific antigens.    
     
     
         49 . A kit for use in the diagnostic method according to  claim 37 , comprising in a suitable packaging: 
 one or more common or  P. tenuis - or  E. cervi -specific antigens immobilized on a solid support; and    a reagent suitable for detecting, in a biological sample, the presence of antibodies to the one or more common or  P. tenuis - or  E. cervi -specific antigens.    
     
     
         50 . An antigen according to  claim 1  obtained by: 
 (a) providing a cDNA library which expresses protostrongylidae genes;  
 (b) screening the expressed genes of the cDNA library with a source of anti-protostrongylidae antibodies to identify cDNA clones which express common antigens; and  
 (c) transforming a host cell with the cDNA clones which express the common antigen, thereby producing the antigen.  
 
     
     
         51 . An antigen according to  claim 4  obtained by: 
 (a) providing a cDNA library which expresses  P. tenuis  genes;  
 (b) screening the expressed genes of the cDNA library with a source of anti- P. tenuis  antibodies to identify cDNA clones which express  P. tenuis -specific antigens; and  
 (c) transforming a host cell with the cDNA clones which express the  P. tenuis -specific antigen, thereby producing the antigen.  
 
     
     
         52 . An antigen according to claims  10  obtained by: 
 (a) providing a cDNA library which expresses  E. cervi  genes;  
 (b) screening the expressed genes of the cDNA library with a source of anti- E. cervi  antibodies to identify cDNA clones which express  E. cervi -specific antigens; and  
 (c) transforming a host cell with the cDNA clones which express the  E. cervi -specific antigen, thereby producing the antigen.  
 
     
     
         53 . A method of isolating one or more common Protostrongylidae antigens, comprising: 
 (a) purifying proteins from the excretory-secretory (ES) Protostrongylidae; and    (b) determining ES purified proteins that react with anti-Protostrongylidae antibodies, thereby isolating one or more common Protostrongylidae antigens.    
     
     
         54 . A method of isolating one or more  P. tenuis -specific antigens, comprising: 
 (a) purifying proteins from the excretory-secretory products (ES) of  P. tenuis ; and    (b) determining ES purified proteins that react with anti- P. tenuis  antibodies, thereby isolating one or more  P. tenuis -specific antigens.    
     
     
         55 . A method of isolating one or more  E. cervi -specific antigens, comprising: 
 (a) purifying proteins from the excretory-secretory products (ES) of  E. cervi ; and    (b) determining ES purified proteins that react with anti- E. cervi  antibodies, thereby isolating one or more  E. cervi -specific antigens.