Determination of intracellular heat shock protein 70 using a newly developed cell lysate immunometric assay.
Heat shock proteins (Hsp) have been associated to several clinical relevant conditions. Currently used methods to determine Hsp 70 possess certain drawbacks. Therefore, we developed a cell lysate immunometric assay (CLIA) for the quantification of intracellular Hsp 70. This CLIA uses a combination of two distinct monoclonal antibodies that recognize different epitopes on the Hsp 70 molecule. A recombinant human Hsp 70 was used as the standard material. The detection range of the CLIA was 4-4000 ng/ml. The intra- and interassay coefficients of variation were, on average, 5% and 12%, respectively. The recovery varied between 81% and 116%. The Hsp 70 levels assayed after serial dilution of cell lysates varied linearly with dilution (between 97% and 120%). The reliability of the CLIA was assessed by comparison with the values determined by flow cytometric procedure; these two sets of values showed a highly significant correlation (r=0.896, p<0.0001), indicating that the two methods are comparable. We conclude that this assay represents a low-cost alternative of the flow cytometric technique.
Njemini
Rose
R
Geriatric Unit, Academic Hospital, Free University of Brussels (VUB), Laarbeeklaan 101, B-1090, Brussels, Belgium.
Demanet
Christian
C
Mets
Tony
T
D000911:Antibodies, Monoclonal / Q000276:immunology; D002457:Cell Extracts / Q000737:chemistry; D004306:Dose-Response Relationship, Immunologic; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005434:Flow Cytometry; D018840:HSP70 Heat-Shock Proteins / Q000032:analysis* / Q000276:immunology; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000737:chemistry; D015203:Reproducibility of Results; D013997:Time Factors
D000911:Antibodies, Monoclonal; D002457:Cell Extracts; D018840:HSP70 Heat-Shock Proteins