Antibody targeting BCMA chimeric antigen receptor, and application of antibody
A technology of chimeric antigen receptors and antibodies, which is applied to coding nucleic acid molecules and its application fields, can solve problems such as inability to distinguish different CAR-T cell numbers, affecting experimental results, and non-expression of genes
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Embodiment 1
[0041] Example 1. Phage antibody library panning to enrich RD103A scFv-specific phage clones
[0042] Negative panning antigens RD103B scFv-rFc, B53scFv-rFc and positive panning target antigen RD103A scFv-rFc were used for alternate panning. At the same time, in the positive panning step, human serum with a final concentration of 5% was added as a competitive negative panning to enrich Collection of specific phage clones targeting RD103B scFv. Table 1 lists the panning process.
[0043] Brief experimental steps:
[0044] 1) Coat the Fc-tagged target antigen RD103A scFv-rFc or the control antigen RD103B scFv-rFc, B53scFv-rFc on a high-binding 96-well ELISA plate (Corning Incorporated 96well EIA / RIA plate, Costar, 3590), and use blocking solution Sealed ELISA plate;
[0045] 2) Incubate the phage antibody library with the coated negative antigen RD103B scFv-rFc or B53 scFv-rFc, and deduct the phage antibody clones that do not specifically bind to the Fc tag or components of...
Embodiment 2
[0069] Example 2. ELISA primary screening of phage monoclonal
[0070] Monoclonals were randomly selected from the phage antibody pool enriched in Example 1, and after being packaged into phages, the binding of the monoclonal phages to RD103A scFv-rFc protein and 103B scFv-rFc protein was detected by phage ELISA to find the specific binding 103A scFv Phage antibody cloning. These clones were sequenced to determine their scFv sequences.
[0071] Brief experimental steps:
[0072] 1) The enriched phage solution was serially diluted, infected with host bacteria XL1-blue, and coated with Amp and Tet plates;
[0073] 2) pick a single clone in a 96-well plate, culture at 37°C, 250 rpm until the logarithmic growth phase, and add helper phage KO7, continue to cultivate, and amplify to obtain a monoclonal phage solution;
[0074] 3) directly or indirectly immobilizing the target antigen and the control antigen in a 96-well plate;
[0075] 4) adding the monoclonal phage supernatan...
Embodiment 3
[0092] Example 3. FACS identification of phage monoclonal
[0093] Through the ELISA preliminary screening in Example 2, we have proved that #1 to #10 clones can specifically bind to 103A scFv-rFc protein, but in actual testing, we need to ensure that they can effectively bind to the CAR molecule Only on the scFv domain can it play the role of detecting CAR-T cells. So we measured the binding of these clones to Jurkat-103A scFv and Jurkat cells by flow cytometry (FACS).
[0094] Brief experimental steps:
[0095] 1. Incubate the monoclonal phage antibody to be tested with Jurkat-103A scFv and Jurkat cells at 4°C for 1 hour, and centrifuge to obtain the first precipitate;
[0096] 2. After washing the first precipitate, incubate with the primary antibody [Anti-M13Bacteriophage Coat Protein g8pantibody, abcam, ab9225] for 30 min, and then incubate with the secondary antibody [FITC horse anti mouse-IgG(H+L), vector, FI2000] after washing 30min, centrifuged to obtain the seco...
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