Elution of the CD19-IgG1Fc fusion was performed under denaturing or non-denaturing (native) conditions according to the manufacturers instructions. samples by flow cytometry, but may need development for each construct and present significant background in samples from xenograft models. Methods A specific reagent for the detection of anti-CD19 CAR expression was developed, a fusion protein consisting of human CD19 extracellular domains and the Fc region of human IgG1 (CD19sIg). Genes encoding CD19sIg fusion proteins were constructed by fusing either exons 1 to 3 (CD19sIg1-3) or exons 1 to 4 (CD19sIg1-4) of the human CD19 cDNA to a human IgG1Fc fragment. These fusion proteins are intended to work in similar fashion as the MHC Tetramers used for identification of antigen-specific T-cells, and may also have other applications in studies of activation of anti-CD19 CAR bearing cells. The CD19sIg proteins Mevastatin were produced from 293?T cells by stable lentiviral vector transduction and purification from culture medium. Results ELISA assays using several different monoclonal antibodies to CD19 exhibited dose-related specific binding by the fusion molecule CD19sIg1-4, but no binding by CD19sIg1-3. Conjugation of the CD19sIg1-4 fusion protein to Alexa Fluor 488 allowed specific and sensitive staining of anti-CD19 CAR-bearing cells for flow cytometry assays, detecting as low as 0.5% of CAR-modified primary cells with minimal background staining. Conclusions This fusion molecule is usually a sensitive reagent for detection of anti-CD19 CAR derived from any monoclonal antibody present in CAR-modified T-cells. Keywords: CD19, Chimeric antigen receptor, Fusion protein, Flow cytometry Background Chimeric Antigen Receptors (CARs) have been used over the last twenty years to redirect specificity of T-cells for use in immunotherapy research approaches. Among multiple targeted antigens, CD19 has been increasingly studied for its expression in most of the B lineage hematological malignancies, with significant responses in animal models using adoptive transfer of T-cells armed with CD19-specific CAR. The same approach is currently being evaluated in clinical trials with initial success reported [1]. Detection of CAR-bearing cells has usually been performed by flow cytometry, with the use of antibodies against the extracellular structure of the molecule, such as the hinge (using an anti-IgG Fc antibody or F(ab)2 fragment) or the antigen-binding domains (as in the case of the use of an anti-idiotypic antibody). More recently, protein L isolated from Peptostreptococcus was proposed for detection of CAR expression by flow cytometry [2]. Using the antigen specificity of CAR as the determinant of a more specific reagent for the detection of the CD19-specific CAR, we developed a CD19/Fc molecule that can be labeled for its use primarily as a reagent in flow cytometry studies. Our approach consisted of fusing the extracellular domains of the human CD19 protein [3-5] to the human immunoglobulin Fc domain name. Fusion to the Fc domain name Mevastatin has been used to Mevastatin allow secretion of peptide sequences, with enhanced solubility and stability, and a fusion protein of murine extracellular CD19 and Fc domain name has been previously described [6]. We describe the studies for the development and evaluation of this fusion protein. The properties of this reagent make possible sensitive detection by flow cytometry of cells modified with CD19-specific CAR. Material and methods Construction of CD19-IgG1Fc expressing plasmids The CD19-IgG1Fc fusion proteins, CD19sIg1-3 and CD19sIg1-4, were constructed by fusing either exons 1 to 3 (E13) or exons 1 to 4 (E14) of the human CD19 cDNA (Origene, Rockville, MD) to a human IgG1Fc (Fc) fragment [7] by PCR-based cloning. Exons 1 through 3 of hCD19 were amplified using primers UNF (5-CTGGCTAGCGTTTAAACGGG-3) and X3R (5-CTGGCTGAGGCTCTGGTTC-3). Exons 1 through 4 of hCD19 of hCD19 were amplified using primers UNF and X4R (5- TGGCCGAGCAGTGATCTC-3). The IgG1Fc region was amplified using a 5phosphorylated primer FCF (5- TCTGCAGAGCCCAAATCTTG-3) and the reverse primer ERFC (5- GTCCAGTGTGGTGGAATTCG FHF3 -3). The hCD19 PCR products were digested with EcoR1 and the IgG1Fc product was digested with EcoR1. The digested products Mevastatin were ligated into the Fc fragment-containing expression plasmid (pCMV-Fc), previously digested with NheI and EcoR1 to create either pCMV-CD19sIg1-3 or pCMV-CD19sIg1-4. The CD19sIg1-3 and CD19sIg1-4 fragments were then removed from the pCMV-CD19sIg1-3 and pCMV- CD19sIg1-4 expression vectors by restriction enzyme digestion with EcoRI and XhoI. The lentiviral plasmid pCCLc-EFS-hADA-WPRE (from Adrian Thrasher, University College London, London, UK) contains the short human EF1a promoter [8] and the ADA (adenosine deaminase) transgene. ADA was removed by BamH1 digestion and the backbone.