Abstract
A liposome immunosorbent assay enhanced by immuno-PCR (iPCR-LISA) using antigen-coupled liposomes encapsulating a signal plasmid (pCR-script Amp KS(+)), was developed for the competitive measurement of antigens. The number of signal plasmid DNA molecules per liposome was found to be 2115. Signal plasmid concentration was determined by SYBR-Green real-time PCR, in which the multi-cloning site (MCS) region of the plasmid was amplified using M13 primer sets. The threshold cycle value (CT) was correlated to antigen concentration. A competitive LISA using antigen-coupled liposomes encapsulating a fluorescent marker, carboxyfluorescein (CF), was also performed (CF-LISA). The effects of the measuring conditions and the amount of antigen coupled to liposomes on assay characteristics were determined.The detection limit of the competitive iPCR-LISA was almost the same as that of the competitive CF-LISA. Both the competitive iPCR LISA and CF-LISA could measure a concentration range of 10−7–10−2 μmol/ml, which is much wider than that of 2.7 × 10−5–3.3 × 10−4 μmol/ml using the conventional indirect ELISA method.
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