FIGURE 1.

Development of Abs specific for the sCTLA-4 isoform. (A) Human (NP_001032720.1), rabbit, and mouse (ENSMUSP00000095327) amino acid sequences at the C-terminal region of sCTLA-4 are aligned with the synthetic peptides used for immunization of rabbits (P1_rabbit) and mice (P2_mouse). The cytoplasmic tail of TM-CTLA-4 is shown for comparison at the bottom of the panel (P16410, UniProt). Mouse and human nucleotide sequences encoding sCTLA-4 have been confirmed in our laboratory by sequencing. Rabbit amino acid sequence was determined from the deposited Tm-CTLA-4 nucleotide sequence (CTLA4_rabbit ENSOCUG00000017274) after alignment with human sCTLA-4 nucleotide sequence (NM_001037631.2). The 9-aa peptide used to immunize rabbits differs at a single residue from the rabbit sequence, a methionine for valine substitution at position 174 of rabbit sCTLA-4. The 23-aa synthetic peptide having the human sequence used to immunize mice differs at position 158 from the endogenous mouse sequence; a proline replaces serine. A serine for cysteine substitution in the 23-aa peptide was made at the residue corresponding to position 165 of the mature sCTLA-4 protein to prevent the formation of disulfide bonds in the peptide preparation and the peptide-KLH conjugate used for immunization. (B) The 4B8 mAb, 10D1 mAb, and 4017 polyclonal Ab validation by Western blot under reducing conditions on detergent lysates of HeLa cells expressing human recombinant Tm-CTLA-4, sCTLA-4, and vector only. Specific detection of Tm-CTLA-4 is performed with CTLA-4 (C19) polyclonal Ab. (C) sCTLA-4 Ab specificity was confirmed by flow cytometry using HeLa cells transfected with sCTLA-4 (top row) or Tm-CTLA-4 (middle row). As a control, HeLa cells transfected with Tm-CTLA-4 were stained with isotype-matched control Abs (bottom row); similar results were obtained using HeLa cells transfected with sCTLA-4 (data not shown). All experiments are representative of at least three independent observations.