title: Detection of antibodies against 60-, 65- and 70-kDa heat shock proteins in paediatric patients with various disorders using Western blotting and ELISA.
abstract: BACKGROUND
  We examined antibodies against 60-, 65- and 70-kDa heat shock proteins (HSPs) in paediatric healthy individuals, patients with juvenile idiopathic arthritis (JIA) and those undergoing allogeneic stem-cell transplantation for various malignant and non-malignant diseases.
  
  
  METHODS
  Western blotting and ELISA were used to examine HSP-directed humoral immune responses.
  
  
  RESULTS
  Using ELISA we detected anti-Hsp60, -Hsp65 and -Hsp70 IgG antibodies in patient sera before, during and after conditioning and at all post-transplant times, as well as in JIA patients and controls. Western blotting showed positivity for anti-Hsp60 and anti-Hsp65 antibodies in all samples with a HSP concentration of 0.5 microg/lane. However, anti-Hsp70 antibodies were not detected at all when both sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and native PAGE were used, except for one JIA patient, for whom a positive signal was only achieved in native PAGE when Hsp70 was increased to 2 microg/lane and serum dilution decreased to 1:10.
  
  
  CONCLUSIONS
  Western blotting is convenient for the detection of anti-Hsp60 and anti-Hsp65 antibodies, but it is not sensitive enough for the detection of anti-Hsp70 antibodies. ELISA, which is more sensitive, might be preferentially used to screen anti-Hsp60, -Hsp65 and -Hsp70 antibodies in sera of children with various disorders.
authors.affiliation: Cell Biology Laboratory, Department of Paediatrics, 2nd Medical Faculty and University Hospital Motol, Charles University in Prague, Prague, Czech Republic. thuhien71@yahoo.com
authors.forename: Thi Thu Hien
authors.initials: TT
authors.lastname: Nguyen
authors.forename: Denisa
authors.initials: D
authors.lastname: Zlacka
authors.forename: Pavla
authors.initials: P
authors.lastname: Vavrincova
authors.forename: Petr
authors.initials: P
authors.lastname: Sedlacek
authors.forename: Ilona
authors.initials: I
authors.lastname: Hromadnikova
mesh_terms: D000293:Adolescent; D000328:Adult; D000906:Antibodies / Q000097:blood*; D001171:Arthritis, Juvenile / Q000276:immunology; D001426:Bacterial Proteins / Q000276:immunology; D015153:Blotting, Western / Q000379:methods; D018834:Chaperonin 60 / Q000276:immunology; D018833:Chaperonins / Q000276:immunology; D002648:Child; D002675:Child, Preschool; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005260:Female; D018840:HSP70 Heat-Shock Proteins / Q000276:immunology; D006360:Heat-Shock Proteins / Q000276:immunology*; D018380:Hematopoietic Stem Cell Transplantation; D006801:Humans; D007223:Infant; D008297:Male; D012680:Sensitivity and Specificity; D014181:Transplantation Immunology
chemical_list: D000906:Antibodies; D001426:Bacterial Proteins; D018834:Chaperonin 60; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; C074119:heat-shock protein 65, Mycobacterium; D018833:Chaperonins