Detection of rViscumin in plasma samples by immuno-PCR.
To allow for pharmacokinetic studies in adjunction with the current clinical developments of the potent cytostatic anti-cancer drug rViscumin, a sandwich immuno-PCR (IPCR) assay was developed for the detection of rViscumin in blood plasma. The IPCR was carried out with a commercially available reagent kit, consisting of pre-assembled rViscumin-specific antibody-DNA conjugates as well as a specific competitor DNA fragment to be amplified by PCR. Various combinations of capture- and detection-antibodies were compared for performance in IPCR. Using the optimized assay, as few as 50 zeptomol (approx. 100 fg/ml) rViscumin (MW 57 kDa) was detectable in standardized human serum samples. The IPCR assay was very selective for rViscumin and in spiking experiments in proband plasma samples, signal recovery rates between 70% and 120% were obtained. The linear sensitivity range of the assay covered more than five orders of magnitude. Repeated measurements of rViscumin resulted in a mean standard deviation value of 14.2%.
Adler
Michael
M
CHIMERA BIOTEC GmbH, Emil-Figge-Str. 76 A, D-44227 Dortmund, Germany. adler@chimera-biotec.de
Langer
Martin
M
Witthohn
Klaus
K
Eck
Jürgen
J
Blohm
Dietmar
D
Niemeyer
Christof M
CM
D000818:Animals; D000918:Antibody Specificity; D003429:Cross Reactions / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D037121:Plant Lectins / Q000097:blood* / Q000235:genetics / Q000276:immunology; D028321:Plant Preparations / Q000097:blood* / Q000276:immunology; D010940:Plant Proteins; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D015203:Reproducibility of Results; D054790:Ribosome Inactivating Proteins, Type 2; D012680:Sensitivity and Specificity; D014118:Toxins, Biological / Q000097:blood* / Q000235:genetics / Q000276:immunology
D037121:Plant Lectins; D028321:Plant Preparations; D010940:Plant Proteins; D054790:Ribosome Inactivating Proteins, Type 2; D014118:Toxins, Biological; C009674:ribosome inactivating protein, Viscum