title: An ELISA serum assay for autoantibodies to HSP70 in immune-mediated hearing loss.
abstract: A Western blot to detect anti-HSP70 autoantibodies has been reported to be of diagnostic value for immune-mediated hearing loss patients. While setting up this Western blot in our lab, we detected two main problems. First, some patients were positive for antibodies to a 70-kDa protein when tested against a whole cell lysate, but negative if the antigen used was purified HSP70. Second, if high amounts of purified HSP70 were loaded on the gel, both patients and healthy controls were positive. We have developed and optimized an ELISA as an alternative to the Western blot. This assay is more appropriate to identify positive and negative individuals because it is semi-quantitative. The ELISA is also more sensitive, requiring very low concentrations of the antigen and thus minimizing false positives. Finally, we demonstrated that immune-mediated hearing loss patients recognize mainly the native form of HSP70, a fact that potentially leads to false negatives when a denaturing Western blot assay is used for diagnosis. To test the diagnostic value of the ELISA, we performed a blind test with 70 hearing loss patients, as well as 30 healthy controls. A sensitivity of 84% and a specificity of 93% were obtained, superior to what has been reported so far for the Western blot.
authors.affiliation: Faculdade de Biociências and Instituto de Pesquisas Biomédicas, Hospital São Lucas, Pontifícia Universidade Católica do Rio Grande do Sul, Av. Ipiranga, 6690 2o andar 90610-000 RS Porto Alegre, Brazil.
authors.forename: Leonardo
authors.initials: L
authors.lastname: Munari
authors.forename: Sheila
authors.initials: S
authors.lastname: Charchat
authors.forename: Luiz
authors.initials: L
authors.lastname: Rodrigues
authors.forename: Carlos Alberto
authors.initials: CA
authors.lastname: von Muhlen
authors.forename: Anne Rose
authors.initials: AR
authors.lastname: Baú
authors.forename: Luiz
authors.initials: L
authors.lastname: Lavinsky
authors.forename: Cristina
authors.initials: C
authors.lastname: Bonorino
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001323:Autoantibodies / Q000097:blood*; D015153:Blotting, Western; D002648:Child; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D018840:HSP70 Heat-Shock Proteins / Q000276:immunology*; D034381:Hearing Loss / Q000276:immunology*; D006801:Humans; D008297:Male; D008875:Middle Aged
chemical_list: D001323:Autoantibodies; D018840:HSP70 Heat-Shock Proteins