Development and evaluation of a sandwich ELISA for quantification of the 20S proteasome in human plasma.
Because quantification of the 20S proteasome by functional activity measurements is difficult and inaccurate, we have developed an indirect sandwich enzyme-linked immunosorbent assays (ELISA) for quantification of the 20S proteasome in human plasma. This sandwich ELISA uses a combination of a monoclonal antibody (mcp 20) recognizing the C2-beta subunit of human 20S proteasome (Mr approximately 30,000) and a polyclonal rabbit anti-20S antibody which labels different subunits of the complex. The detection limit of the assay was established as 10 ng/ml (n=10, mean of zero standard+2 S.D.) and the recovery rate ranged from 96% to 104%. The within-run and between-run coefficients of variation (CV) ranges were 2.8-3.3 and 3.0-3.4, respectively. Using serial dilutions of plasma to which various amounts of purified 20S proteasome were added, a linear dose-response was observed between 102 and 2050 ng/ml with a slope of 1.004 and a coefficient of determination r(2) of 0.99. In a preliminary experiment performed on a limited number of patients, the present assay was used to quantify the 20S proteasome in plasma from healthy subjects (n=11) and from a limited number of patients with various diseases (two patients with each of the following diagnoses: acute myeloid leukaemia, chronic myeloproliferative syndromes, Hodgkin's disease and solid tumors). The average concentration of 20S proteasome in plasma from normal subjects was found to be 2319+/-237 ng/ml (n=11). With reference to this normal range, the plasma proteasome concentration was found to be increased in most of these pathological state and as high as 1200% when solid tumors had been detected. For patients with Hodgkin's disease, the changes were more variable whereas in patients with chronic lymphocytic leukaemia, the proteasome concentration was raised during the acute phase of disease and decreased during therapy. We suggest that this robust, accurate and highly reproducible assay could be used to quantify proteasome in human plasma and investigate its value as a biological marker for various malignant and nonmalignant diseases.
Dutaud
Dominique
D
Biochemistry Team, SRV, INRA Theix, 63122 Saint Genès Champanelle, France.
Aubry
Laurent
L
Henry
Laurent
L
Levieux
Didier
D
Hendil
Klavs B
KB
Kuehn
Lothar
L
Bureau
Jean Paul
JP
Ouali
Ahmed
A
D000818:Animals; D015415:Biomarkers; D003546:Cysteine Endopeptidases / Q000097:blood* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006689:Hodgkin Disease / Q000097:blood; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000097:blood; D009097:Multienzyme Complexes / Q000097:blood* / Q000276:immunology; D046988:Proteasome Endopeptidase Complex; D011817:Rabbits; D012680:Sensitivity and Specificity
D015415:Biomarkers; D009097:Multienzyme Complexes; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex