Phage display mediated immuno-PCR

Abstract

Immuno-PCR (IPCR) is a powerful detection technology in immunological study and clinical diagnosis due to its ultrasensitivity. Here we introduce a new strategy termed phage display mediated immuno-PCR (PD-IPCR). Instead of utilization of monoclonal antibody (mAb) and chemically bond DNA that required in the conventional IPCR, a recombinant phage particle is applied as a ready reagent for IPCR experiment. The surface displayed single chain variable fragment (scFv) and phage DNA themselves can directly serve as detection antibody and PCR template, respectively. The aim of the design is to overcome shortcoming of low detection sensitivity of scFv so as to largely facilitate the real application of scFv in immunoassay. The idea has been demonstrated by applying hantaan virus nucleocapsid protein (NP) and prion protein (PrP) as detection targets in three experimental protocols (indirect, sandwich and real-time PD-IPCR assays). The detection sensitivity was increased 1000- to 10,000-folds compared with conventional enzyme-linked immunosorbent assays (ELISAs). This proof-of-concept study may serve as a new model to develop an easy to operate, low cost and ultrasensitive immunoassay method for broad applications.

Figure 1

Schematic diagram of phage display mediated IPCR.

Figure 2

Detection sensitivity of indirect PD-IPCR and ELISA for NP. (a) Indirect PD-IPCR experiment. The polystyrene microtitre plate was coated with 100 µl of 10-fold serial dilutions of NP. Bound phages were used for PCR and amplification products were run on a 1.5% agarose gel. Lane 1 to 7 represent amplification products from 10⁶ to 1 pg/ml. Lane 8 was negative control without NP coating. Lane M is DNA marker (DL2000) with 2000, 1000, 750, 500, 250 and 100 bp. (b) Sensitivity comparison between PD-IPCR and ELISA. The amplification band of PD-IPCR was quantified by a computer assisted image analyzer. The color development in ELISA was achieved with HRP-labeled anti-M13 monoclonal conjugate secondary antibody and TMB substrate. Average values of three independent experiments are given.

Figure 3

Real-time quantitative PD-IPCR assay of NP. (a) Quantitative curve of indirect PD-IPCR for purified NP. Curves A–F represent serial 10-fold dilutions of NP from 10⁶ pg/ml to 10 pg/ml. G stands for the negative control I that had no NP antigen coating. No fluorescent signal was observed for negative control II that had no any template. Note the high similarity of the duplicate determinations, the regular intervals between the signals of the 10-fold dilutions and the difference between the control I and the control II. (b) Calibration plot of log purified NP concentration versus threshold cycles (Cts) in indirect PD-IPCR. NP coating was 100 µl of 4-fold serial dilutions from 200 to 0.8 ng/ml. The results from three individual experiments were averaged, and the error bars give the SD. (c) Quantitative curve of sandwich PD-IPCR for purified NP. The plate was coated with 100 µl of L13 mAb. Curves A–F represent serial 10-fold dilutions of NP from 10⁶ pg/ml to 10 pg/ml, each had two duplicates. G stands for the negative control I that had no NP coating. No fluorescent signal was observed for negative control II that had no any template. (d) Calibration plot of log purified NP concentration versus Ct in sandwich PD-IPCR. The plate was coated with 100 µl of L13 mAb, followed by the addition of 4-fold serial dilutions of NP from 2000 to 2 ng/ml. Again, the results from three individual experiments were averaged, and the error bars give the SD.

Figure 4

Detection sensitivity of sandwich PD-IPCR and sandwich ELISA for NP. The amplification band of sandwich PD-IPCR was quantified by a computer assisted image analyzer. The color development in sandwich ELISA was achieved with HRP-labeled anti-M13 monoclonal conjugate secondary antibody and TMB substrate. Average values of three independent experiments are given.

Figure 5

Detection sensitivity of sandwich PD-IPCR and sandwich ELISA for PrP. The amplification band of PD-IPCR was quantified by a computer assisted image analyzer. The color development in ELISA was achieved with HRP-labeled anti-M13 monoclonal conjugate secondary antibody and TMB substrate. Average values of three independent experiments are given.