Sensitive detection of dengue virus NS1 antigen using affibody-conjugated gold nanoparticle

Abstract

The infection of dengue virus (DENV) is a serious health issue that causes a severe dengue fever and sometimes a lethal complication called dengue hemorrhagic fever. Due to the absence of a licensed vaccine or antiviral drug, rapid and sensitive DENV detection is important to reduce the morbidity and mortality. Here, I developed the highly sensitive enhanced ELISA for dengue NS1 using affibody functionalized gold nanoparticles (AuNPs). First of all, I screened NS1 antigen specific affibody molecules (ZNS112, ZNS116 and ZNS146) from affibody phage library and these affibodies were then expressed and purified from Escherichia coli. Among them, ZNS112 affibody showed the highest equilibrium binding constant (Kd) of 1 μM. This affibody was functionalized on gold nanoparticles with 20 nm. Developed anti-NS1 affibody functionalized gold nanoparticles (ZNS112-AuNPs) were used as carriers in order to achieve an amplification of the signal. ZNS112-AuNPs showed good properties such easy synthesis, high number of affibody conjugation on AuNP, and excellent stability in the harsh condition at high salt and temperature. In addition, this nanoparticle-based enhanced ELISA assay resulted in a 14.2-fold signal amplification performance for dengue NS1 detection in comparison with the conventional ELISA procedures. This method using ZNS112-AuNPs can be detectable in DENV infected patients at the early stage and has potential application for detection of other pathogens in clinical diagnostics.; The infection of dengue virus (DENV) is a serious health issue that causes a severe dengue fever and sometimes a lethal complication called dengue hemorrhagic fever. Due to the absence of a licensed vaccine or antiviral drug, rapid and sensitive DENV detection is important to reduce the morbidity and mortality. Here, I developed the highly sensitive enhanced ELISA for dengue NS1 using affibody functionalized gold nanoparticles (AuNPs). First of all, I screened NS1 antigen specific affibody molecules (ZNS112, ZNS116 and ZNS146) from affibody phage library and these affibodies were then expressed and purified from Escherichia coli. Among them, ZNS112 affibody showed the highest equilibrium binding constant (Kd) of 1 μM. This affibody was functionalized on gold nanoparticles with 20 nm. Developed anti-NS1 affibody functionalized gold nanoparticles (ZNS112-AuNPs) were used as carriers in order to achieve an amplification of the signal. ZNS112-AuNPs showed good properties such easy synthesis, high number of affibody conjugation on AuNP, and excellent stability in the harsh condition at high salt and temperature. In addition, this nanoparticle-based enhanced ELISA assay resulted in a 14.2-fold signal amplification performance for dengue NS1 detection in comparison with the conventional ELISA procedures. This method using ZNS112-AuNPs can be detectable in DENV infected patients at the early stage and has potential application for detection of other pathogens in clinical diagnostics.|뎅기 바이러스는 심각한 뎅기열과 때때로 뎅기 출혈열과 같은 치명적인 합병증을 유발할 수 있는 심각한 감염병이다. 그래서 뎅기 바이러스의 감염률과 사망률을 줄이려면 빠르고 민감하게 뎅기 바이러스를 감지하는 것이 중요하다. 본 연구는 뎅기열 바이러스 비 구조단백질인 NS1 항원에 특이적으로 결합하는 어피바디(인공항체)를 발굴하였고 어피바디에 금 나노 입자를 결합시켜 뎅기 바이러스 NS1 항원에 대한 검출한계를 향상 시켰다. 우선, 어피바디 파지 라이브러리에서 NS1 항원에 특이적인 어피바디 분자(ZNS112, ZNS116 and ZNS146)를 스크리닝 했다. 이어서, 이 어피바디들을 대장균에서 발현시켜 정제하였다. 이들 중 ZNS112 어피바디가 1μM의 친화력(Kd)를 보였으며 고온에서 우수한 안정성을 보였다. 이 어피바디를 20nm 직경의 금 나노입자에 결합시켜 ELISA의 검출한계 향상을 위해 사용하였다. 금 나노입자에 결합 된 어피바디는 NS1 항원에 특이적이고 쉬운 생산과 용이하게 합성할 수 있으며, 금 나노입자 상에 고정화 수도 많았고 뿐만 아니라 고염과 고온의 가혹한 조건 하에서 우수한 안정성을 보이는 장점을 확인하였다. 또한, 금 나노입자 기반 어피바디를 이용한 ELISA가 어피바디만 이용한 ELISA에 비해 NS1 항원 검출에 대한 신호가 14.2배 향상 된 결과를 확인하였다. 따라서 금 나노입자에 결합된 어피바디를 사용하는 ELISA 방법은 초기 단계에 감염된 환자에서 뎅기 바이러스를 검출할 수 있고 이와 같은 방법을 이용한 다면 다른 병원체의 검출에도 적용 할 수 있을 것으로 생각된다.