세포소기관내 단백질의 표지화를 위한 면역전자현미경법 및 Biochip platform에 Tyramide signal amplification (TSA)법의 적용

Application of Tyramide Signal Amplification (TSA) Both to Biochip Platform and to the Immunoelectron Microscopy to Label Proteins within the Organelle

항원∙항체 복합체가 결합된 horse raddish peroxidase (HRP) 효소가 외부에서 넣어 준 biotinylated tyramide를 HRP의 주변에 있는 단백질에 침착 시킨다는 사실을 이용한 Tyramide Signal Amplification (TSA) 방법 (일종의 신호증폭법)을 바이오칩과 면역전자현미경법에 적용하여 보았다. 먼저, 바이오칩에서는 Dako EnvisionTM (goat anti-mouse immunoglobulins IgG conjugated to peroxidase labelled-dextran polymer)과 tyramide-Cy3로 신호(signal)를 키웠으며 이것을 goat antimouse IgG-Cy3 conjugate만을 반응시킨 샘플과 비교하였을 때 결과는 tyramide를 사용한 방법이 충분히 민감한 반응을 보임을 알 수 있었다. 다음으로, 면역세포화학의 모델로서는 세포안에 있는 세포소기관의 막 안쪽 구조물에도 효과적으로 TSA 방법을 적용할 수 있는지를 보고자 하였다. 즉, 멜라닌 세포의 멜라닌소체 내 단백질인 gp100을 검출하기 위하여 glutaraldehyde로 고정하고 1% sodium borohydride (PBS 용액)로 처리한 후에 일차 항체, biotinylated 이차 항체, streptavidin-HRP, biotinyltyramide, 그리고 streptavidin-nanogold를 이용하여 신호를 증폭시켰으며 이 방법을 streptavidin-HRP와 biotinyltyramide의 과정을 생략한 비증폭법(non-amplified protocol)과 비교하였다. 결과는 tyramide를 사용한 방법이 뛰어난 민감성 (sensitivity)을 보여주었다. 더불어 일차 항체를 제외한 실험으로 비특이적 반응 (non-specific binding)이 없음을 확인하였고, 멜라닌 소체에 국한되어 나타나는 금 입자(gold particle) 신호를 확인함으로서 높은 정밀성(high resolution)도 확인할 수 있었다. 이것은 TSA 방법이 적당한 막투과성이 요구되는 고정조건에서만이 표지화(labeling)에 성공할 수 있는 세포소기관(intracellular organelle) 속의 단백질의 표지화에도 적용할 수 있음을 보여주었다.

The tyramide signal amplification (TSA) technique, based on the ability of HRP to catalyze the deposition of tyramide onto the surrounding proteins, has been proved to detect scarce tissue antigens. In this study we applied this technique to a biochip platform and an immunocytochemistry at the electron microscopic level. First, in the optical fluorescence sensing, the signal was amplified by Dako EnvisionTM (goat anti-mouse immunoglobulins IgG conjugated to peroxidase labelled-dextran polymer) and tyramide-Cy3, which was then compared to the non-amplified control using goat antimouse IgG-Cy3 conjugate instead. The result showed that the tyramide method produced a more sensitive signal than the control method. Secondly, in the pre-embedding immunocytochemistry, we investigated to see whether it is possible to label proteins within a organelle in the cell using the TSA method. The signal was amplified by a primary antibody, a biotinylated secondary antibody, streptavidin-HRP, biotinyl-tyramide, and streptavidinnanogold followed by silver enhancement and gold toning. Then, this protocol was compared to the non-amplified or simple protocol that does not include the steps of streptavidin-HRP and biotinyl-tyramide. With the TSA protocol, the labeling for a membrane bound antigen (gp100) that is known to be exclusively localized to melanosomes in melanocyte, was tested in a melanoma cell line (G361) and found to be highly sensitive and more enhanced than with the simple protocol. Moreover, the gold particles were well localized to the subcellular structures or melanosomes both in the TSA and simple protocols, which indicates that resolution of the signals remains high. Control experiment with omission of the primary antibody demonstrated that background levels or nonspecific bindings are negligible. This result showed that the TSA method can be successfully applied to label the intra-organelle protein that is known to be labeled only in the specific fixation condition with the optimal permeability.