Method for detecting BCMACAR affinity
An affinity, positive cell technology, applied in the field of cell therapy, can solve the problems of low expression and no expression of B cell marker proteins
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Embodiment 1
[0020] Example 1: Binding affinity of BCMA IgG to BCMA positive cell lines
[0021] The specific steps are: use the expressed and purified Anti-human BCMA IgG (clone number: C11D5.3) prepared by our company, set the initial concentration to 100ug / ml, and then dilute 7 points according to 1:3 gradient, respectively with 3× 10 5 The human BCMA-positive cell line MM.1S and H929 were co-incubated (see Table 1 for specific groups), and then the flow cytometry antibody PE-anti human IgG-Fc was used to detect the BCMA IgG bound to the target cells, and its fluorescence was detected by flow cytometry strength and judge binding affinity.
[0022] Table 1 Grouping of BCMA IgG binding affinity test to BCMA-positive cell lines
[0023]
[0024] Example results see figure 1 , figure 2 . Among them, the EC50 of MM.1S binding affinity to BCMA IgG is 37nM, and that of H929 reaches 57nM.
Embodiment 2
[0025] Embodiment 2: ELISA detection of binding affinity between BCMA IgG and BCMA
[0026] The specific steps are as follows: the test first uses 2 μg / ml Human BCMA protein to coat the ELISA plate, washes off the free protein after 1 hour, and then dilutes 8 concentration gradients with the purified Anti-human BCMA IgG 2ug / ml 1:3 made by our company. Incubate at 37°C for 1 hour, wash off excess antibody, and then use secondary antibody Biotin Goat anti mouse F(ab) in sequence 2 Incubate with the third antibody Streptavidin-HRP at 37°C for 1 hour, and finally develop the color, and read the OD450 / 620 value on a microplate reader.
[0027] The results of this example are shown in figure 1 , figure 2 . Coat the plate with excess Human BCMA-Fc protein, then incubate with 8 concentrations of Anti-human BCMA IgG with an initial concentration of 2ug / ml and 1:3 gradient dilution, and detect its binding affinity by ELISA, with an EC50 value of 1.2 nM.
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