Development of an Immuno-PCR Protocol for Detection of a Small Amount of Antigen.
- Author:
Chang Ho JEON
1
;
Eun Ha KOH
Author Information
1. Department of Laboratory Medicine, School of Medicine, Catholic University of Daegu, Daegu, Korea. chjeon@cu.ac.kr
- Publication Type:Original Article
- Keywords:
Immuno-PCR;
Non specific binding;
Neutravidin;
Casein block;
Detection limit
- MeSH:
Antibodies;
Caseins;
Employment;
Enzyme-Linked Immunosorbent Assay;
False Positive Reactions;
Indicators and Reagents;
Limit of Detection;
Streptavidin
- From:The Korean Journal of Laboratory Medicine
2005;25(1):66-70
- CountryRepublic of Korea
- Language:Korean
-
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.
