Development of bispecific anti-proPSA × cotinine antibody for multiplex detection of proPSA using raman signal

Abstract

Introduction: A truncated precursor form of prostate-specific antigen (PSA), proPSA, is a well-known biomarker for prostate cancer. Since the amount of proPSA in serum or semen is quite low (1), it is impractical to purify it from biological fluid. Thereby, recombinant proPSA proteins must highly pure to use as a standard reference. For detecting [-2]proPSA and [-5/-7]proPSA in human serum, generation of selective and specific antibodies are essential. Based on reference materials and antibodies, an assay detecting [-2]proPSA and [-5/-7]proPSA in human serum simultaneously would offer a usefulness for detecting the prostate cancer.

Methods: In this study, proPSAs were expressed in HEK 293F cell and purified recombinant proteins from cell culture supernatant. Expressed proteins were identified by protein Edman sequencing and LC-MS/MS. The protein properties such as aggregation, monomeric or oligomeric forms and stability were analyzed by SEC-HPLC. After chicken immunization with identified proteins and propeptide-carrier protein conjugates, generation of polyclonal antibodies in chicken serum was tested by immunoblot analysis. Bio-panning and phage ELISA using phage display technique, antibodies binding [-2]proPSA and [-5/-7]proPSA were individually developed. The bispecific antibodies composed of 2 scFvs linked with human Fc domain were expressed in HEK 293F cells and purified by protein A beads. The characteristics of single walled carbon nanotube with bispecific antibody (SNA) were tested by Raman specrometry and BIAcore assay. The sensitivity and selectivity of SNA were analyzed by Raman imaging.

Results: In the developed expression and purification system, [-2]proPSA and [-5/-7]proPSA were generated stably, and production yields of recombinant proteins were approximately 585.6 µg/L, 522.2 µg/L, respectively. The antibodies targeting [-2]proPSA, [-5/-7]proPSA were developed form phage libraries constructed form chickens immunized with peptide conjugates or recombinant proteins. The screened out antibodies specifically bound to target proteins without cross-reactivity to other proteins and peptides in ELISA and immunoblot analysis. An interaction between cotinine on synthesized SNAs and anti-cotinine antibody in a bispecific antibody was confirmed in Raman specrometry and BIAcore assay. In Raman imaging, it was proved that SNAs complexed with anti-HER 2 × cotinine antibody attached selectively on HER2 molecules on SK-BR-3 cell surface.

Conclusions: Taken together, [-2]proPSA, [-5/-7]proPSA were generated purely in developed system, and antibodies for [-2]proPSA and [-5/-7]proPSA were discovered from immunized chicken libraries. SNAs with bispecific antibodies were synthsized well and its characteristics were confirmed by Raman spectroscopy and imaging. Based on developed materials such as recombinant proPSAs as the reference material, anti-proPSA antibodies and SNA with bispecific antibody, the assay detecting [-2]proPSA and [-5/-7]proPSA simultanousely in a single tubes contaning human serum is under developing. If the assay is developed in near future, it could offer a usefulness for detecting the prostate cancer

* These works are published in Biotechnology and applied Biochemistry(2), and Nanoscale(3).