DEVELOPMENT OF A 2-SITE IMMUNO-PCR ASSAY FOR HEPATITIS-B SURFACE-ANTIGEN
DEVELOPMENT OF A 2-SITE IMMUNO-PCR ASSAY FOR HEPATITIS-B SURFACE-ANTIGEN
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DOI：
10.1016/0166-0934(94)00145-7
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发表时间：
1995-04-01
影响因子：
3.1
通讯作者：
WANDS, JR
中科院分区：
文献类型：
--
作者：
MAIA, M;TAKAHASHI, H;WANDS, JR
B型肝炎病毒（HBV）基因转录可能发生在非常低的水平，导致血清和肝脏中的HBsAg浓度低于目前可用的免疫测定法的检测限。已经开发了一种检测方法，该方法结合了两种高亲和力抗-HBs单克隆抗体（MAb）的特异性，针对HBsAg“a”结构域中不同和独立的决定簇，并采用高灵敏度聚合酶链反应（PCR）检测方法。在捕获血清样品中存在的HBsAg后，加入生物素化的第二种抗-HBs MAb。第二抗体的结合允许链霉亲和素和衍生自蓝舌病毒（BTV）基因的生物素化线性DNA分子的随后特异性结合。然后使用BTV特异性引物通过PCR检测该DNA的存在。PCR产物通过液相寡核苷酸酶测定进行定量，这进一步增加了该技术的灵敏度。使用双位点单克隆抗体捕获和PCR检测系统的HBsAg被证明大大提高了特异性和灵敏度的测定和检测低至0.5 pg的HBsAg血清样品。提示该技术的原理可应用于血清和生物样本中其他低水平病毒抗原的检测。
Hepatitis B virus (HBV) gene transcription may occur at very low levels resulting in HBsAg concentrations in serum and liver below the limit of detection by currently available immunoassays. An assay has been developed that combines the specificity of two high affinity anti-HBs monoclonal antibodies (MAb) directed against distinct and separate determinants in the 'a' domain of HBsAg with the highly sensitive polymerase chain reaction (PCR) detection method. Following capture of HBsAg present in serum samples, the second anti-HBs MAb, which is biotinylated, is added. Binding of the second antibody allows the subsequent specific binding of streptavidin and a biotinylated linear DNA molecule derived from a bluetongue virus (BTV) gene. Presence of this DNA is then detected by PCR using BTV-specific primers. The PCR product is quantified by a liquid-phase oligonucleotide enzymatic assay, which further increases the sensitivity of the technique. The use of a two-site MAb capture and PCR detection system for HBsAg was shown to greatly enhance the specificity and sensitivity of the assay and detect as little as 0.5 pg of HBsAg in serum samples. It is suggested that the principles of this technique could be applied to measure other low level viral antigens in serum and biological samples.