Korean J Lab Med.  2005 Feb;25(1):66-70.

Development of an Immuno-PCR Protocol for Detection of a Small Amount of Antigen

• Jeon CH
• Koh EH

Abstract

BACKGROUND: Immuno-PCR has been known as a highly sensitive and specific method, yet no standardized protocol is available. We analyzed each step of immuno-PCR to develop a reliable standardized method.
METHODS
We made a protocol modified from several methods reported previously, and performed immuno-PCR, but false positive reactions were noted. To reduce the false positivity, we investigated the buffer reagents and biotin-labelled oligo-nucleotide probe. Using a finally determined protocol, we compared the detection-limits of the immuno-PCR and ELISA methods.
RESULTS
Streptavidin was identified as a main reagent causing a non-specific binding, thus it was replaced by neutravidin. The employment of CAS block as a dilution buffer for the biotin-labelled oligo-nucleotide probe and Casein block as a buffer for the detection antibodies resulted in a dramatic reduction in the false positive reactions. The standardized immuno-PCR detected angiogenin antigen at a concentration as low as 5 fg/mL, while an ELISA method detected 5 pg/mL.
CONCLUSIONS
The immuno-PCR procedure newly described in this study was ultra-sensitive with no false positivity. This method can be utilized as an epochal tool for detection of a small amount of antigen which would not be discovered by ELISA method.

Keyword

Immuno-PCR; Non specific binding; Neutravidin; Casein block; Detection limit

MeSH Terms

Antibodies
Caseins
Employment
Enzyme-Linked Immunosorbent Assay
False Positive Reactions
Indicators and Reagents
Limit of Detection
Streptavidin
Antibodies
Caseins
Indicators and Reagents
Streptavidin