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 nonmalignant 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 mu g/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 mu g/lane and serum dilution decreased to 1:10. Conclusion: 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.