Siitari H, Hemmilä I, Soini E, Lövgren T, Koistinen V
Nature. 1983 Jan 20;301(5897):258-60. doi: 10.1038/301258a0.
Conventional fluoroimmunoassay (FIA) methods based on various fluorescence principles have not achieved the sensitivity of radioimmunoassay (RIA) mainly because of problems of background fluorescence arising, for example, from the biological specimen. We now describe an immunoassay of hepatitis B surface antigen (HBsAg) based on time-resolved (TR) fluorescence using a lanthanide as label. The assay initiates the development of a new generation of immunoassays. The fluorescence intensity is measured after a selected delay time which almost completely eliminates background fluorescence, which has a fast decay time. The excitation is performed with a flashing light source. The molecules with a long fluorescent lifetime consist of chelates of rare earth metals (Eu, Tb, Sm, Dy). They absorb strongly the excitation radiation and transfer the energy to the chelated central atom which in turn produces an emission spectrum characteristic of the lanthanide used. A long Stokes' shift (greater than 270 nm) helps to reduce the background in the emission region of the chelate and thus optimizes measurement of the relevant fluorescence. The present TR-FIA uses 2-naphthoyltrifluoroacetone as chelating agent because it creates an intense fluorescence with the rare earth metals. Synergistic agents such as trioctylphosphineoxid further enhance the fluorescence of the chelate. Depending on the instrumentation used for measuring time-resolved fluorescence and the conditions used for chelate formation, lanthanides can be detected at 10(-12)-10(-14)M concentrations.
基于各种荧光原理的传统荧光免疫分析（FIA）方法尚未达到放射免疫分析（RIA）的灵敏度，主要是因为存在背景荧光问题，例如由生物样本产生的背景荧光。我们现在描述一种基于时间分辨（TR）荧光的乙型肝炎表面抗原（HBsAg）免疫分析方法，使用镧系元素作为标记。该分析方法开启了新一代免疫分析的发展。在选定的延迟时间后测量荧光强度，这几乎完全消除了具有快速衰减时间的背景荧光。激发由闪光灯源进行。具有长荧光寿命的分子由稀土金属（铕、铽、钐、镝）的螯合物组成。它们强烈吸收激发辐射并将能量转移到螯合的中心原子，该中心原子进而产生所用镧系元素特有的发射光谱。大的斯托克斯位移（大于270nm）有助于减少螯合物发射区域的背景，从而优化相关荧光的测量。目前的时间分辨荧光免疫分析（TR-FIA）使用2-萘甲酰三氟丙酮作为螯合剂，因为它与稀土金属产生强烈荧光。三辛基氧化膦等协同剂进一步增强了螯合物的荧光。根据用于测量时间分辨荧光的仪器和用于形成螯合物的条件，镧系元素可以在10^(-12)-10^(-14)M的浓度下被检测到。
