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

Biochem Biophys Res Commun. 2003 Aug 22;308(2):240-50. doi: 10.1016/s0006-291x(03)01364-0.

Abstract

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.

MeSH terms

  • Animals
  • Antineoplastic Agents, Phytogenic / analysis
  • Enzyme-Linked Immunosorbent Assay / methods*
  • Enzyme-Linked Immunosorbent Assay / statistics & numerical data
  • Humans
  • 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
  • Recombinant Proteins / analysis
  • Ribosome Inactivating Proteins, Type 2
  • Sensitivity and Specificity
  • Toxins, Biological / analysis

Substances

  • Antineoplastic Agents, Phytogenic
  • Immunoglobulin G
  • Indicators and Reagents
  • Plant Preparations
  • Plant Proteins
  • Proteins
  • Recombinant Proteins
  • Ribosome Inactivating Proteins, Type 2
  • Toxins, Biological
  • ribosome inactivating protein, Viscum