A real-time immuno-PCR assay for routine ultrasensitive quantification of proteins

A fast and robust assay, based on the combination of the highly sensitive immuno-PCR (IPCR), employing standardized self-assembled DNA–protein conjugates as reagents, and the well-established, reliable, and fast real-time PCR detection by means of the TaqMan principle is introduced in this work. The...

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Bibliographic Details
Published in:Biochemical and biophysical research communications 2003-08, Vol.308 (2), p.240-250
Main Authors: Adler, Michael, Wacker, Ron, Niemeyer, Christof M.
Format: Article
Language:English
Subjects:
Animals
Antibodies
Antineoplastic Agents, Phytogenic - analysis
Assay development
ELISA
Enzyme-Linked Immunosorbent Assay - methods
Enzyme-Linked Immunosorbent Assay - statistics & numerical data
Humans
Immuno-PCR
Immunoglobulin G - analysis
Indicators and Reagents
Mice
Plant Preparations - analysis
Plant Proteins
Polymerase Chain Reaction - methods
Polymerase Chain Reaction - statistics & numerical data
Proteins - analysis
Rabbits
Real-time detection
Recombinant Proteins - analysis
Ribosome Inactivating Proteins, Type 2
Secondary reagents
Sensitivity and Specificity
Toxins, Biological - analysis
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Summary:A fast and robust assay, based on the combination of the highly sensitive immuno-PCR (IPCR), employing standardized self-assembled DNA–protein conjugates as reagents, and the well-established, reliable, and fast real-time PCR detection by means of the TaqMan principle is introduced in this work. The use of anti-species immunoglobulin reagents allows one for easy adaptation of this assay to basically any existing ELISA application. The use of an internal competitor in the real-time IPCR (rtIPCR) further increases the sensitivity and significance of this assay; 0.1–0.01 amol (500–50 fg/mL) IgG from several species (mouse, rabbit, goat, and human) were detectable using direct, indirect, and sandwich model rtIPCR assays, thereby increasing the detection limit of the analogous ELISA tests about 100- to 1000-fold. The robustness of this method was demonstrated in two typical applications by detecting 40 pg/mL of the novel anti-cancer drug rViscumin in human plasma samples as well as 100 pg/mL of a research antibody in cell culture media. In both cases, a comparable ELISA was 1000-fold less sensitive.
ISSN:0006-291X
1090-2104
DOI:10.1016/S0006-291X(03)01364-0