Cell culture and total cell extract preparation
The human colon adenocarcinoma cell lines HT29 and SW620 were kindly supplied by Oryzon Genomics (Barcelona, Spain), originally acquired from the American Tissue Culture Collection (ATCC)(Manassas, VA, USA).
The HT29 and SW620 cell lines were cultured with F12 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco, Grand Island, NY). The cells were grown to confluency at 37°C and 5% CO2. To obtain total cell extracts, cell monolayers were washed twice with cold phosphate buffered saline (PBS), and then were scraped and centrifuged at 1500 × rpm for 5 min at 4°C. The cellular pellets were resuspended in lysis buffer (10 mM Tris-HCl, pH 7.5, 140 mM NaCl, 5 mM EDTA, and 1.5% v/v Triton X-100) supplemented with protease inhibitor cocktail complete EDTA free (Roche, Basilea) and sonicated.
Preparation of bovine leukocytes suspensions
A blood sample from cow was collected for leukocytes isolation. The cow received humane care in accordance with the Guidelines for the accommodation and care of animals used for experimental and other scientific purpose according Animal Wellfare Laws (National Laws RD 1201/2005). The animal experiment was approved by Ethical Committee "Nucleo Zoosanitario" (No ES-80790000095). The sample was collected into heparin (Sigma, Inc., St.Louis, MO) and centrifuged at 1000 × g for 15 min at room temperature (RT). The supernatant was removed and the pellet was treated with lysis buffer (310 mM NH4Cl, 24 mM NaHCO3, 0.5 mM Na2 EDTA, pH 7.4) for 15 min at room temperature. The sample was centrifuged again at 1000 × g during 15 min and the supernatant was removed. The pellet was resuspended in 400 μl 25 mM bicarbonate buffer.
Human lymphocytes purification
Fresh human lymphocytes were isolated from 50 ml blood from a healthy volunteer blood donor by using Histoplaque density gradients purchased from Sigma (St.Louis, MO), following the protocol of the manufacturer. The human blood sample was obtained in accordance with the declaration of Helsinki from 1997 and its revision from 2004 and Spanish Biomedical Law 121/000104. Written informed consent was obtained from the participants after oral and written information was provided. The local ethics committee and the ethic committee of the consortium Oncnosis Pharma AIE approved all procedures. The lymphocytes were lysed by lysis buffer (10 mM Tris-HCl, pH 7.5, 140 mM NaCl, 5 mM EDTA, and 1.5% v/v Triton X-100) supplemented with protease inhibitor cocktail complete EDTA free (Roche, Basilea) and sonicated.
Cloning, expression and purification of recombinant CTHRC1 and NFE2L3
A full-length of the human CTHRC1 gene was cloned into the Gateway System expression vector pDEST17 (Invitrogen, Carlsbad, CA) from pDONR233 (Open Biosystem BC014245), by recombination based on the lambda recombination system. The sequence was confirmed with an automatic DNA sequencer. The new vector was called pDEST17-CTHRC1.
A truncated form of NFE2L3 gene (844nt-2345nt)(corresponding to residues 30-694) was amplified by PCR using cDNA obtained by reverse transcription of total RNA from tumour cell line SW620 as described previously . Two oligonucleotides NFE2L3 REVERSE (5'-CTCACTTTCTCTTTCCCTTTTGGG-3') and NFE2L3 FORWARD (5'-AGAGAAAAGCACGAAGCTGTG-3') were designed and the amplification reaction was carried out in a total volume of 50 μl with 1 μl cDNA, 1 U DNA Taq polymerase (Roche), 200 μmol of each deoxyribonucleotide triphosphate, and 100 ng of each primer. Amplification involved 35 cycles at 94°C during 30 seconds, 55°C 30 seconds, 72°C 2 minutes 35 cycles, and a final extension step at 72°C during 10 minutes. The PCR product was purified and cloned into the TOPO Gateway vector (Invitrogen, San Diego, CA). The cloned fragment was subsequently transferred to the expression vector pDEST17 by using the Gateway system (Invitrogen). The new vector was called pDEST17-NFE2L3. All sequences were confirmed by sequencing.
In both cases the genes concerned were expressed as a fusion protein that included His-Tag. The recombinant CTHRC1 and NFE2L3 proteins were expressed by transformation of E.coli BL21 strain (Invitrogen) cells. Overnight cultures of transformed bacteria in LB supplemented with 100 μg/ml of ampicillin and 50 μg/ml carbenicillin were inoculated in fresh medium. Cultures were grown at 37°C until they reached mid-log phase (OD600 = 0.5). At that moment, expression of the CTHRC1 and NFE2L3 recombinant proteins were induced with the addition of L-arabinose to a final concentration of 0.2% w/v and incubated during three hours at 37°C. Then, cultures were centrifuged, resuspended in lysis buffer containing 400 mM NaCl, 100 mM KCl, 10% glycerol, 0.5% Triton X-100, and 10 mM Imidazol. Due to the insolubility of the proteins, an additional step was done consisting in a ClGn-6 M extraction and finally an additional purification step in a column prepacked with Ni Sepharose (His GraviTrap™ from GE Healthcare, Buckinghamshire, UK) was carried out.
Production of anti-NFE2L3 polyclonal antibodies
Purified recombinant NFE2L3 protein was used to immunize rabbits. All rabbits received humane care in accordance with the Guidelines for the accommodation and care of animals used for experimental and other scientific purpose according Animal Wellfare Laws (National Laws RD 1201/2005). The animal experiment was approved by Ethical Committee "Nucleo Zoosanitario" (No ES-80790000095). Aliquots containing 200 μg of protein in 500 μl of PBS were mixed with an equal volume of Freund's complete adjuvant for the first injection and Freund's incomplete adjuvant for boosters. All injections in rabbits were administered subcutaneously in the leg of the rabbit. Injections were repeated 3 times at 2-week intervals. Immunoglobulins presented in serum were purified by a protein G column. The protocol suggested by the manufacturer was followed (GE Healthcare Live Science, Uppsala, Sweden). The purified immunoglobulins were dialysed against PBS, diluted with glycerol (1:1) and stored at -20°C.
Production of anti-CTHRC1 and NFE2L3 monoclonal antibodies
After immunization of mice with CTHRC1 or NFE2L3, several MAbs specific for CTHRC1 or NFE2L3 were obtained. Immunisation protocols, production and purification of MAbs were performed as previously described [24, 25]. All mice received humane care in accordance with the Guidelines for the accommodation and care of animals used for experimental and other scientific purpose according Animal Wellfare Laws (National Laws RD 1201/2005). The animal experiment was approved by Ethical Committee "Nucleo Zoosanitario" (No ES-80790000095).
Production of anti-NFE2L3 Fab fragments
The antibody library FAB-310 (Dyax Corp, Cambridge, MA) was used to select recombinant monoclonal antibodies in the form of Fab fragments using the phage display technology. FAB-310 is a human Fab library that has an unique combination of immunoglobulin sequences captured from human donors and synthetic diversity in key antigen contact sites in heavy-chain complementarity-determining regions 1 and 2 . The Fab fragments in the FAB-310 library are expressed on the surfaces of M13 fused with pIII protein. Fab-310 has a diversity of 3.5 × 1010 (1.5 × 1010 of kappa and 2 × 1010 of lambda). The selection procedure was done as it was recommended by the manufacturer in the experimental handbook of the product (Dyax Corp, Cambridge, MA).
Positive Fab clones against recombinant CTHRC1 and NFE2L3 were reformated to soluble Fab fragments (sFab) by removing the pIII gene. The plasmids pMID21 carrying the Fab fragments were digested with MluI enzyme, re-ligated and transformed into E. coli TG-1 starin. 94 randomly chosen individual colonies from each selection round were analysed as recommended by the manufacturer (Dyax Corp, Cambridge, MA). Supernatant material was analysed in indirect ELISA using 0.1 μg CTHRC1, NFE2L3 or a non related biomarker as negative control protein per well and detecting bonded sFabs with anti C-myc HRP (Pierce, Waltham, MA) or anti Fab peroxidase-conjugated antibodies (Sigma). Specific fragments were defined as sFabs clones that showed a sign of ELISA at least 3 times higher in the plate covered with CTHRC1 or NFE2L3 than in the plate covered with negative control.
sFab producing clones were grown until they reached a cellular density of 0.9 (OD600) on a stirrer at 37°C. sFab production was induced with a final Isopropyl-beta-d- thiogalactopyranoside (IPTG) concentration of 1 mM and the cultures were grown on a stirrer at 30°C during the night. sFabs were separated from bacterial sediment through centrifugation and purified by affinity with protein A-Sepharose (GE Healthcare, Buckinghamshire, UK).
To determine the binding specificity of PAbs, MAbs, and sFab, indirect ELISA trials were carried out using non conjugated or biotin-conjugated antibodies. CTHRC1 or NFE2L3 (30-694) were used to cover 96-well Maxisorp plates (Nunc) in a 1-5 μg/ml concentration. The bindings with increasing biotin-conjugated antibodies concentrations were measured with streptavidin-peroxidase (Sigma, Inc., St.Louis, MO). PAb, MAbs, or sFabs binding with other non-related proteins, such as lysozyme, fibrinogen, albumin, ovalbumin, human IgG purchased from Sigma (St. Louis, MO) or with other non-related cancer biomarkers were also determined.
Immunologic detection of proteins was performed by Western blot. 50-100 μg cellular extract was loaded in a sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and then transferred to a nitrocellulose membrane. The MAb CH21D7 against CTHRC1 was used in a concentration of 25 μg/ml. The peroxidase-conjugated PAb against NFE2L3 was used in a dilution of 1:2500. The monoclonal antibody 41HF8 and the sFab E5 against NFE2L3 were used in a concentration of 10 μg/ml. All secondary antibodies coupled to horseradish peroxidase directed against mouse IgG (Amersham) and rabbit IgG were diluted 1:5000 and anti human Fab fragment 1:2000 (Sigma, Inc., St.Louis, MO).
Peroxidase and biotin conjugation of antibodies
The PAb were conjugated with peroxidase using the periodate coupling method described by Nakane and co-workers . The PAbs, MAbs and sFabs were biotinylated using (+)-Biotin N-hydroxysuccinimide ester following the protocol developed by Bayer and Wilchek .
Three assay formats were developed: one format to detect CTHRC1 and two formats to detect NFE2L3.
In the CTHRC1 DAS-ELISA, the MAb CH21D7 was used as captured antibody with a concentration of 0.5 μg/ml and biotin-conjugated CH24G2 as detection antibody with a concentration 0.5 ng/ml.
In the NFE2L3 DAS-ELISA, the sFab E5 was used as captured antibody with a concentration of 1.2 μg/ml and the PAb or biotin-conjugated 41HF8 as detection antibody with a concentration of 5 ng/ml. In an alternative DAS-ELISA was used PAb as captured antibody with a concentration of 5 ug/ml and biotin-conjugated 41HF8 as detection antibody with a concentration of 5 ng/ml.
The plates were blocked with 3% BSA in PBS 0.1% Tween. Cellular extracts, or control antigens were added to the wells and incubated 1 h 24°C or overnight at 4°C. The control antigens were recombinant proteins (CTHRC1 or NFE2L3). The negative control was NFE2L3 in the CTHRC1 DAS-ELISA and CTHRC1 in the NFE2L3 DAS ELISA. All further incubations were performed at room temperature. The biotin-conjugated antibodies were detected with the streptavidin-peroxidase (Sigma, Inc., St.Louis, MO).
Dissociation-enhanced lanthanide fluorescent immunoassay
The colorimetric DAS-ELISAs against CTHRC1 and NFE2L3 were converted to a single Dissociation-Enhanced Lanthanide Fluorescent Immunoassay (DELFIA). Chessboard reagent titration experiments were carried out to define optimal assay parameters. In the DELFIA assay, sFab E5 and MAb CH21D7 were used as captured antibodies with a concentration of 0.5 and 0.06 μg/well respectively in yellow plates. The recommendations of the manufacturer were followed (Perkin Elmer, Turku, Finland). The plates were blocked with blocking solution (50 mM NaH2PO4, 6% trehalose, 0.1% BSA-TSA, 0.1% Germal II)(Sigma, Inc., St.Louis, MO). Recombinant CTHRC1, NFE2L3 or a non-related cancer biomarker as negative control protein were added to the wells and incubated at room temperature during 1 hour. The captured antigens were detected with a mixture of PAb against NFE2L3 and biotin-conjugated CH24D7, followed by a mixture of Europium-labelled anti rabbit antibody and Samarium-labelled streptavidin. The signal was enhanced adding enhancement solution (Perkin Elmer, Turku, Finland). The fluorescence was measured with Envision Multilabel Plate reader (Perkin Elmer, Turku, Finland). Antibody levels were shown as fluorescence units.