Sp2 o ag14

A synthetic peptide (CSRSR(pThr)P(pSer)LP(pThr)PPTREPKK) corresponding to amino acids 208–225 in the 2N/4R human tau isoform with residues Thr212, Ser214 and Thr217 phosphorylated was used for immunization. This peptide was chosen as it contains all 3 phosphorylated amino acid residues with additional adjacent residues allowing for appropriate size for the immunization peptide. The peptide included an added Cys residue at the amino-terminus that allowed for conjugation to inject maleimide-activated mariculture keyhole limpet hemocyanin (mcKLH) (Thermo Scientific, Waltham, MA, USA). All procedures were performed according to the NIH Guide for the Care and Use of Experimental Animals and were approved by the University of Florida Institutional Animal Care and Use Committee. The peptide–KLH conjugate was used to immunize female BALB/c mice (Jackson Laboratory, Bar Harbor, ME, USA) as previously described [75 (link)]. Spleens from the mice were harvested, and the white blood cells were fused with mouse myeloma cells (Sp2/O-Ag14; ATCC, Manassas, VA, USA). Hybridoma clones were selected using HAT supplement (Sigma Aldrich, St. Louis, MO, USA) and the surviving clones were initially screened for reactivity by enzyme-linked immunosorbent assay (ELISA) using the immunization peptide, but further assessed using a series of peptides and recombinant tau proteins, as described below.

Antibodies 1A1 and 1A2 are new mouse monoclonal antibodies generated by immunizing female BALB/c mice using a synthetic peptide (CEEGAPQEGILE) corresponding to amino acid residues 104-114 within the C-terminal region of human αS which was synthesized and purified by GenScript Biotech Corp. The peptide included an added cysteine residue at the amino terminus that allowed for conjugation to Imject maleimide-activated mcKLH (Thermo Scientific). The peptide-KLH conjugate was used to immunize female BALB/c mice (Jackson Laboratory) as previously described (48 (link)). All procedures were performed according to the NIH Guide for the Care and Use of Experimental Animals and were approved by the University of Florida Institutional Animal Care and Use Committee. Spleens from the mice were harvested, and the white bloods cells were fused with mouse myeloma cells (Sp2/O-Ag14; ATCC) as previously described (48 (link)). Hybridoma clones were selected using HAT supplement (Sigma Aldrich) and the surviving clones were initially screened for reactivity by ELISA using the respective peptide used for immunization, and further characterized using FL αS and a series of C-truncated αS proteins as detailed above. Antibody isotypes were determined using a mouse monoclonal isotyping kit (Millipore Sigma).

For MAbs production, 6/8 week-old Balb/c mice were inoculated with heat-inactivated and sonicated E. coli O104:H4 preparations. Animal experimentation was carried out in compliance with Italian national law (Decreto legislativo 27 Gennaio, 1992 , n. 116) implementing Directive 86/609/EEC of the Council of the European Communities on the protection of animals used for experimental and other scientific purposes (European Commission [EC], 1986 ). The protocol was approved by the Italian Ministry of Health with number 5146 of 26.04.2012. The whole antigen, diluted to a protein concentration of 100 μg/ml, was emulsified with complete Freund adjuvant (Sigma, St. Louis, MO, USA) and administered intraperitoneally; 14 days later a second immunization was performed using the same concentration of antigen emulsified with incomplete Freund adjuvant (Sigma). Subsequently, on days 28 and 56, 100 μg/ml of antigen diluted in PBS was given (intravenous booster). Three days later, the mice were sacrificed, the spleen collected and splenocytes subjected to cell fusion with murine myeloma cells Sp2/O-Ag-14 (ATCC CRL-1581^TM).