Hat supplement

For the immunization of F2-hybrid mice, 0.5 mg of C3a peptide in 1 mL of PBS was mixed with equal volume of Freund’s complete adjuvant (or Freund’s incomplete adjuvant for reimmunization). The reimmunization was carried out after 30 days; 12 days after that, the lymphocytes from the inguinal, peritoneal and axillary lymph nodes were collected for hybridization with Sp2/0 myeloma cells in the ratio 2:1. Cell hybridization was performed in PEG/DMSO (Sigma), using serial dilution with culture media [31 (link)]. The hybrids were transferred to 96-well culture plates, and cultivated for 12–16 days in RPMI-1640 medium (Sigma) containing 20% fetal calf serum. The HAT supplement (Sigma) was added to the culture medium solution for selection of hybridomas.
Clone screening was performed using ELISA in a 96-well microplate. Briefly, 50 µL of culture media was added to wells precoated with C3a antigen, and after incubation for 1 h, peroxidase-conjugated goat anti-mouse IgGs were added. Detection was performed with 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution.
For C3a ELISA development, two monoclonal murine antibodies to C3a, with different epitope specificities designated as CC3a-5 and CC3a-1, were selected. CC3a-5 was immobilized on a solid phase as a capture antibody, while CC3a-1 was conjugated with horseradish peroxidase and was used as a detection antibody.

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.

A dose of 100 μg of prokaryotically expressed pB602L mixed with an equal volume of complete Freund’s adjuvant or incomplete Freund’s adjuvant (Sigma, USA) was used to immunize mice. Four six-week-old female BALB/c mice were immunized four times at 14-day intervals. Three days after the fourth immunization, spleen cells were harvested and fused with SP2/0 cells. The fused cells were cultured in DMEM medium containing HAT Supplement (Sigma, USA) and 20% FBS. After 10 days, the supernatant of the fused cells was collected, and antibodies against pB602L were detected using an indirect ELISA (iELISA). Positive clones were selected and subcloned three times by limiting dilution. Monoclonal cells were cultured in DMEM medium containing 20% FBS and 1% penicillin-streptomycin. Ten-week-old female BALB/c mice were used for the preparation of ascites containing antibodies against the pB602L protein. The antibody titers in the ascites were determined using iELISA. An SBA Clonotyping System-HRP kit (Southern Biotech, USA) was used to identify the subtypes of the monoclonal antibodies. The specificity of the monoclonal antibodies against the recombinant protein and virions was evaluated using Western blot and IFA.