Specific Probe Selection from Landscape Phage Display Library and Its Application in Enzyme-Linked Immunosorbent Assay of Free Prostate-Specific Antigen

Abstract

Probes against targets can be selected from the landscape phage library f8/8, displaying random octapeptides on the pVIII coat protein of the phage fd-tet and demonstrating many excellent features including multivalency, stability, and high structural homogeneity. Prostate-specific antigen (PSA) is usually determined by immunoassay, by which antibodies are frequently used as the specific probes. Herein we found that more advanced probes against free prostate-specific antigen (f-PSA) can be screened from the landscape phage library. Four phage monoclones were selected and identified by the specificity array. One phage clone displaying the fusion peptide ERNSVSPS showed good specificity and affinity to f-PSA and was used as a PSA capture probe in a sandwich enzyme-linked immunosorbent assay (ELISA) array. An anti-human PSA monoclonal antibody (anti-PSA mAb) was used to recognize the captured antigen, followed by horseradish peroxidase-conjugated antibody (HRP-IgG) and o-phenylenediamine, which were successively added to develop plate color. The ELISA conditions such as effect of blocking agent, coating buffer pH, phage concentration, antigen incubation time, and anti-PSA mAb dilution for phage ELISA were optimized. On the basis of the optimal phage ELISA conditions, the absorbance taken at 492 nm on a microplate reader was linear with f-PSA concentration within 0.825-165 ng/mL with a low limit of detection of 0.16 ng/mL. Thus, the landscape phage is an attractive biomolecular probe in bioanalysis.

Selectively assaying CEA based on a creative strategy of gold nanoparticles enhancing silver nanoclusters' fluorescence

“…Accordingly, investigating the amounts of CEA plays an important role towards clinical purpose. Currently, immunoassays and related techniques have been considered as major analytical methods towards detections of CEA mainly including radioimmunoassay, enzyme immunoassay, chemiluminescence immunoassay and fluorescence immunoassay (Lang et al, 2014;Li et al, 2010;Yu et al, 2013;Yuan et al, 2001).…”

Continuous microfluidic assortment of interactive ligands (CMAIL)

“…Biopanning involves the immobilization of target molecules or antigens onto a solid support or magnetic beads followed by the incubation with the phage library. After extensive washing to remove nonadherent or weakly binding phages, bound phages are eluted and recovered using elution buffer, and may be subsequently amplified in a bacteria host for further enrichment101112. Three to five repeated rounds of incubation, washing, elution, and amplification steps are usually carried out in order to obtain high-affinity phages, which can be quite laborious, lengthy and experimentally challenging for a number of reasons.…”

Peptide-based biosensor for the prostate-specific antigen using magnetic particle-bound invertase and a personal glucose meter for readout

“…This method and some other techniques, which can greatly improve the sensitivity of the PSA detection, are listed in Table 1. This is more than 300, 200, 160 -fold lower, respectively, than the previously reported detection limits of most sensitive assay for PSA [30][31][32] and equal to the most sensitive assay [33].…”