Gold nanoparticle enhanced immuno-PCR for ultrasensitive detection of Hantaan virus nucleocapsid protein.
A functionalized gold nanoparticle (GNP) enhanced ultrasensitive immuno-PCR assay (GNP-IPCR on ELISA plate), which was modified from the recent developed bio-barcode assay (BCA) technique, was developed to detect Hantaan virus nucleocapsid protein (HNP). During the assay, the target antigen HNP was captured by a polyclonal antibody coated on ELISA microplate wells, followed by adding GNP dually modified with oligonucleotides and a HNP specific monoclonal antibody L13 (mAb L13) to form a sandwich immuno-complex. The oligonucleotides on the GNP contained two strands: one as capture DNA immobilized on the surface of the GNP through Au-S bond and the other as signal amplification DNA, which was partially complementary with the capture DNA. After the immuno-complex was formed, the signal DNA was released by heating, and consequently characterized by PCR/gel electrophoresis and SYBR-Green real time PCR. The detection limit of this method could reach down to 10 fg/mL for detecting purified HNP in buffers as well as in human serum, which was approximately 7 orders of magnitude more sensitive than that of conventional ELISA. The current assay format might be adopted for other proteins that need ultra-high sensitive detection.
Chen
Longyan
L
State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Science, Wuhan, PR China.
Wei
Hongping
H
Guo
Yongchao
Y
Cui
Zongqiang
Z
Zhang
Zhiping
Z
Zhang
Xian-En
XE
D000956:Antigens, Viral / Q000097:blood*; D036022:Capsid Proteins / Q000097:blood*; D004797:Enzyme-Linked Immunosorbent Assay; D006046:Gold; D018063:Hantaan virus / Q000378:metabolism*; D018778:Hantavirus Infections / Q000175:diagnosis* / Q000821:virology; D006801:Humans; D053768:Metal Nanoparticles; D015336:Molecular Probe Techniques; D016133:Polymerase Chain Reaction; D011237:Predictive Value of Tests; D012680:Sensitivity and Specificity; D014758:Viral Core Proteins / Q000097:blood*
D000956:Antigens, Viral; D036022:Capsid Proteins; D014758:Viral Core Proteins; C111782:nucleocapsid protein, Hantaan virus; D006046:Gold