Preparation of recombinant serpins B3 and B4 and investigation of their specific interactions with antibodies using hydrogel-based microarrays

Differential quantification of SCCA1 and SCCA2 cancer antigens using a hydrogel biochip

“…The preparation of recombinant SCCA1 and SCCA2 was described previously. 26 Additionally, recombinant SCCA1 and SCCA2 were affinity puried on Sepharose 4B (GE Healthcare Bio-Sciences, # 17-0430-01, Piscataway, NJ, USA) with immobilized antibody C5 or A11. The concentrations of SCCA1 and SCCA2 in solutions were determined using an SCC EIA test kit (CanAg-Fujirebio, # 800-10-96, Sweden).…”

“…Monoclonal antibodies H30, C5, H5, H81 and G9 have been described before (clone H30 was originally described as H3 and subsequently renamed). 26 Antibodies F8, A11, H31 and D10 were generated by immunizing BALB/c mice with recombinant SCCA1 and SCCA2. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals (NRC 2011), the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientic Purposes, Council of Europe (ETS 123), and "The Guidelines for Manipulations with Experimental Animals" (the decree of the Presidium of the Russian Academy of Sciences of April 02, 1980, no.…”

“…The antibody activity was monitored by solidphase enzyme immunoassay as described elsewhere. 26 Antibodies C5, A11 and H31 were additionally puried by PEG-8000 (Sigma-Aldrich # P-5413, USA) precipitation in 0.01 M sodium phosphate, pH 8.0; then, the precipitates were collected, dissolved in the same buffer, and pelleted for 15 min at 10 000 rpm at +7 C. The pellets were puried by ion-exchange chromatography on DEAE-cellulose (DE-52, Whatman # 4057-050, UK). The puried antibodies were eluted using a gradient of 0-0.4 M sodium chloride in 0.01 M sodium phosphate, pH 8.0.…”

“…Monoclonal antibodies H30, C5, H5, H81 and G9 have been described before (clone H30 was originally described as H3 and subsequently renamed). 26 The procedure was performed twice with an interval of two weeks with a dosage of 50 mg of the protein per immunization. The rst immunization was performed using Freund's complete adjuvant and the second through Freund's incomplete adjuvant.…”

“…Previously, we have demonstrated the possibility of determining SCCA1 in the presence of SCCA2. 26 The purpose of this study was to develop a method for the simultaneous differentiated measurement of two highly homologous proteins (SCCA1 and SCCA2) within one analysis on a biochip in clinically relevant concentrations. Our aim was also to determine the analytical characteristics of the method and to test it on actual biological samples in comparison to a reference method, used in clinical practice.…”