Gene cloning, expression and serological evaluation of the 12-kDa antigen-B subunit from Echinococcus granulosus

Abstract A 12-kDa subunit of antigen B from Echinococcus granulosus has recently been cloned, expressed and used in diagnostic ELISA to test human sera for evidence of cystic echinococcosis. The performance of the ELISA based on the recombinant antigen (rAgB) was compared with that of similar assays based on native antigen B (nAgB) or hydatid-cyst fluid. For the preparation of the rAgB, total RNA was extracted from Ec. granulosus protoscoleces so that antigen-B complementary DNA could be synthesised, amplified by PCR, and then cloned into the pQE30 expression vector. The recombinant plasmid was transformed in Escherichia coli and induced using isopropyl-β-D-thiogalactopyrano-side. Bacterial samples were collected, lysed and then analysed by SDS–PAGE and western blotting. The recombinant protein was purified by affinity chromatography. Although the performance of the ELISA based on cyst fluid appeared identical to that of the assay based on the recombinant antigen (with a sensitivity, specificity, positive predictive value and negative predictive value of 96.0%, 97.0%, 97.2% and 95.5%, respectively), the corresponding results for the ELISA based on nAgB (98.6%, 100%, 100% and 98.5%) were slightly better. Despite this difference (which was not statistically significant), the comparative ease with which large quantities of the recombinant antigen could be produced make the antigen a potentially useful tool in the diagnosis of cystic echinococcosis.

[1]  M. Bandehpour,et al.  Application of recombinant Echinococcus granulosus antigen B to ELISA kits for diagnosing hydatidosis , 2010, Parasitology Research.

[2]  P. Torgerson,et al.  Echinococcosis: diagnosis and diagnostic interpretation in population studies. , 2009, Trends in parasitology.

[3]  Mb Rokni.  Echinococcosis /hydatidosis in Iran , 2009 .

[4]  H. Ertabaklar,et al.  [The comparative evaluation of Enzyme Linked Immunosorbent Assay (ELISA), Indirect Hemagglutination Test (IHA) and Indirect Fluorescent Antibody Test (IFAT) in the diagnosis of cystic echinococcosis]. , 2009, Turkiye parazitolojii dergisi.

[5]  A. Khamesipour,et al.  Gene Cloning of Iranian Leishmania major Mannose-1- Phosphate Guanyltransferase , 2009 .

[6]  K. Monteiro,et al.  Recombinant subunits as tools for the structural and functional characterization of Echinococcus granulosus antigen B. , 2008, Experimental parasitology.

[7]  J. Bart,et al.  Molecular characterization of a novel gene encoding an 8-kDa-subunit of antigen B from Echinococcus granulosus genotypes 1 and 6. , 2007, Parasitology international.

[8]  A. Muro,et al.  Usefulness of Four Different Echinococcus granulosus Recombinant Antigens for Serodiagnosis of Unilocular Hydatid Disease (UHD) and Postsurgical Follow-Up of Patients Treated for UHD , 2007, Clinical and Vaccine Immunology.

[9]  F. Ayala,et al.  Redundancy and recombination in the Echinococcus AgB multigene family: is there any similarity with protozoan contingency genes? , 2006, Parasitology.

[10]  D. Carmena,et al.  Antigens for the immunodiagnosis of Echinococcus granulosus infection: An update. , 2006, Acta tropica.

[11]  B. Kazemi,et al.  Cloning and Expression of Two Genes Encoding Subunits of Echinococcus Granulosus Antigen B. , 2006 .

[12]  C. Budke,et al.  Global Socioeconomic Impact of Cystic Echinococcosis , 2006, Emerging infectious diseases.

[13]  S. M. Sadjjadi.  Present situation of echinococcosis in the Middle East and Arabic North Africa. , 2006, Parasitology international.

[14]  M. Rokni,et al.  EVALUATION OF THE ENZYME-LINKED IMMUNOELECTRO TRANSFER BLOT (EITB) TECHNIQUE USING HYDATID CYST ANTIGENS B/5 AND TOTAL IGG ANTIBODIES IN LABORATORY DIAGNOSIS OF HUMAN HYDATIDOSIS , 2006 .

[15]  M. Rokni,et al.  Comparative Evaluation of Fast Enzyme Linked Immunosorbent Assay (Fast-ELISA) and Standard-ELISA For The Diagnosis Of Human Hydatidosis , 2006 .

[16]  N. Xiao,et al.  Recent advances in characterization of Echinococcus antigen B. , 2006, Parasitology international.

[17]  K. Monteiro,et al.  Comparative Analysis of the Diagnostic Performance of Six Major Echinococcus granulosus Antigens Assessed in a Double-Blind, Randomized Multicenter Study , 2005, Journal of Clinical Microbiology.

[18]  M. Rokni,et al.  EVALUATION AND COMPARISON OF ANTIGEN B-ELISA AND ANTIGEN B-IMMUNOBLOTTING IN IMMUNODIAGNOSIS OF CYSTIC HYDATID DISEASE , 2005 .

[19]  Haniloo.  EVALUATION AND COMPARISON OF ANTIGEN B-ELISA AND ANTIGEN B-IMMUNOBLOTTING IN IMMUNODIAGNOSIS OF CYSTIC HYDATID DISEASE , 2005 .

[20]  D. McManus,et al.  A novel recombinant antigen for immunodiagnosis of human cystic echinococcosis. , 2003, The Journal of infectious diseases.

[21]  K. Monteiro,et al.  A set of recombinant antigens from Echinococcus granulosus with potential for use in the immunodiagnosis of human cystic hydatid disease , 2003, Clinical and experimental immunology.

[22]  U. Hellman,et al.  Echinococcus granulosus antigen 5 is closely related to proteases of the trypsin family. , 2003, The Biochemical journal.

[23]  P. Nahar,et al.  Covalent immobilization of proteins onto photoactivated polystyrene microtiter plates for enzyme-linked immunosorbent assay procedures. , 2002, Journal of immunological methods.

[24]  M.C.U.-P.H. Marie-France Biava,et al.  Laboratory Diagnosis of Cystic Hydatic Disease , 2001, World Journal of Surgery.

[25]  A. Osuna,et al.  Comparative sensitivity of six serological tests and diagnostic value of ELISA using purified antigen in hydatidosis , 2001, Journal of clinical laboratory analysis.

[26]  R. Riganò,et al.  Native and recombinant antigens in the immunodiagnosis of human cystic echinococcosis , 2000, Parasite immunology.

[27]  G. González-Sapienza,et al.  Improved Immunodiagnosis of Cystic Hydatid Disease by Using a Synthetic Peptide with Higher Diagnostic Value Than That of Its Parent Protein, Echinococcus granulosus Antigen B , 2000, Journal of Clinical Microbiology.

[28]  A. Nieto,et al.  Characterization and optimization of bovine Echinococcus granulosus cyst fluid to be used in immunodiagnosis of hydatid disease by ELISA. , 2000, Revista do Instituto de Medicina Tropical de Sao Paulo.

[29]  A. Zaha,et al.  Comparative analysis of two different subunits of antigen B from Echinococcus granulosus: gene sequences, expression in Escherichia coli and serological evaluation. , 2000, Acta tropica.

[30]  D. McManus,et al.  Indication of the presence of two distinct strains of Echinococcus granulosus in Iran by mitochondrial DNA markers. , 1998, The American journal of tropical medicine and hygiene.

[31]  C. Fernández,et al.  Two different 8 kDa monomers are involved in the oligomeric organization of the native Echinococcus granulosus antigen B , 1996, Parasite immunology.

[32]  C. Fernández,et al.  Molecular characterisation of a novel 8-kDa subunit of Echinococcus granulosus antigen B. , 1996, Molecular and biochemical parasitology.

[33]  M. Frosch,et al.  Sequence heterogeneity of the echinococcal antigen B. , 1994, Molecular and biochemical parasitology.

[34]  D. McManus,et al.  Identification and diagnostic value of a major antibody epitope on the 12 kDa antigen from Echinococcus granulosus (hydatid disease) cyst fluid , 1994, Parasite immunology.

[35]  D. McManus,et al.  A protein secreted in vivo by Echinococcus granulosus inhibits elastase activity and neutrophil chemotaxis. , 1991, Molecular and biochemical parasitology.

[36]  M. Lightowlers,et al.  Subunit composition and specificity of the major cyst fluid antigens of Echinococcus granulosus. , 1989, Molecular and biochemical parasitology.

[37]  V. Tsang,et al.  A specific diagnostic antigen of Echinococcus granulosus with an apparent molecular weight of 8 kDA. , 1989, The American journal of tropical medicine and hygiene.

[38]  D. McManus,et al.  Specific and cross-reactive antigens of Echinococcus granulosus hydatid cyst fluid. , 1987, Molecular and biochemical parasitology.

[39]  R. Galen,et al.  Predictive value and efficiency of laboratory testing. , 1980, Pediatric clinics of North America.

[40]  J. F. Williams,et al.  Purification of lipoprotein antigens of Echinococcus granulosus from sheep hydatid fluid. , 1971, The American journal of tropical medicine and hygiene.