title: Determination of intracellular heat shock protein 70 using a newly developed cell lysate immunometric assay.
abstract: 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.
authors.affiliation: Geriatric Unit, Academic Hospital, Free University of Brussels (VUB), Laarbeeklaan 101, B-1090, Brussels, Belgium.
authors.forename: Rose
authors.initials: R
authors.lastname: Njemini
authors.forename: Christian
authors.initials: C
authors.lastname: Demanet
authors.forename: Tony
authors.initials: T
authors.lastname: Mets
mesh_terms: 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
chemical_list: D000911:Antibodies, Monoclonal; D002457:Cell Extracts; D018840:HSP70 Heat-Shock Proteins