미량항원 검출을 위한 면역 중합효소연쇄반응법의 개발 = Development of an Immuno-PCR Protocol for Detection of a Small Amount of Antigen

다국어 초록 (Multilingual 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 oligonucleotide 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.

국문 초록 (Abstract)

배경 : 면역 중합효소연쇄반응법은 우수한 민감도와 특이도를보이는 획기적인 검사법으로 알려져 있으나, 아직 표준화된 검사방법이 제시되지 못하고 있다. 이에 면역 중합효소연쇄반응법의 검사과정을 각 단계별로 분석하여, 표준화된 검사방법을 완성하여 임상검사실에서 용이하게 사용할 수 있는 면역 중합효소연쇄반응법을 개발하고자 하였다.방법 : 면역 중합효소연쇄반응을 기존에 발표된 방법을 조합하여 시행하였으나 위양성이 나타났다. 이에 그 원인을 구명하고자각 시약별로 비특이 결합을 확인하였으며, 올리고핵산 더듬자 및검출용 항체의 희석 완충액을 선정하였다. Angiogenin 항원을 이용하여 완성된 면역 중합효소연쇄반응법의 민감도를 효소면역법과비교하여 관찰하였다.결과 : Streptavidin은 면역중합효소연쇄반응법에 의한 위양성의 주된 원인으로 밝혀져, neutravidin으로 교체하여 사용하였다.올리고핵산 더듬자의 희석완충액으로 CAS 및 Casein Block이,검출용 항체의 희석완충액으로는 10% Casein-T 용액이 각 위양성을 제거할 수 있었다. 완성된 면역 중합효소연쇄반응법의 민감도는 5 fg까지 측정할 수 있었으며, 효소면역법으로 5 pg까지측정되었다.
결론 : 새롭게 제시된 면역 중합효소연쇄반응법은 위양성이 발생하지 않았고, 매우 우수한 민감도를 나타내었다. 향후 효소면역 검사법으로 측정이 어려운 각종 미량항원의 검출에 획기적인 방법으로 활용될 수 있다.

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