Detection of antibodies against 60-, 65- and 70-kDa heat shock proteins in paediatric patients with various disorders using Western blotting and ELISA.
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.
Nguyen
Thi Thu Hien
TT
Cell Biology Laboratory, Department of Paediatrics, 2nd Medical Faculty and University Hospital Motol, Charles University in Prague, Prague, Czech Republic. thuhien71@yahoo.com
Zlacka
Denisa
D
Vavrincova
Pavla
P
Sedlacek
Petr
P
Hromadnikova
Ilona
I
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
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