Novel anti-human igβ antibody
A human, antibody technology, applied in the direction of antibodies, anti-animal/human immunoglobulins, fungi, etc., can solve the problem that humoral immunity cannot be properly controlled
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Embodiment 1
[0180] (Example 1: acquisition of human and monkey Igβ-Flag proteins)
[0181] A protein in which a Flag tag is bound to human Igβ (human Igβ-Flag protein) and a protein in which a Flag tag is bound to monkey Igβ (monkey Igβ-Flag protein) were obtained. Human Igβ-Flag gene (SEQ ID NO: 13) was introduced into GS vector pEE6.4 (Lonza Biologics, Inc.). The monkey Igβ-Flag gene (SEQ ID NO: 14) was introduced into GS vector pEE6.4 (Lonza Biologics, Inc.). The correspondingly prepared vector genes were transferred to FreeStyle 293 cells (Life Technologies, Inc.) using FreeStyle MAX reagent (life Technologies, Inc.). The corresponding cells were cultured in a serum-free culture system for 1 week using FreeStyle 293 expression medium (Life Technologies, Inc.), and culture supernatants containing human Igβ-Flag protein and monkey Igβ-Flag protein were obtained, respectively. The protein was purified from the obtained culture supernatant using an anti-Flag M2 antibody affinity gel (SI...
Embodiment 2
[0182] (Example 2: Acquisition of anti-human Igβ antibody)
[0183] In order to obtain an anti-human Igβ antibody, the human Igβ-Flag protein and monkey Igβ-Flag protein obtained in Example 1 were injected into C3H / HeJJmsSlc-lpr / lpr mice (Japan SLC, Inc.) together with an adjuvant, using To elicit an immune response for immunization. The mice were immunized several times and a final immunization was performed. The spleen and lymph nodes of the immunized mice were extracted according to conventional methods, and lymphocytes were collected and subjected to cell fusion with mouse myeloma cells SP2 / 0 (ATCC CRL-1581), thereby preparing hybridomas. Limiting dilution samples of the hybridomas were prepared, and the hybridomas were monocloned. The corresponding clones were expanded and cultured, the medium was replaced with a serum-free medium called Hybridoma SFM (Life Technologies, Inc.), and then the clones were cultured for 3 to 5 days. The antibody was purified from the obtain...
Embodiment 3
[0185] (Example 3: Preparation of humanized antibody)
[0186] The CDRs of the heavy and light chains of CL6_40 were grafted to other human antibodies, and the genes of the heavy and light chains of multiple humanized antibodies were prepared. Expression vectors containing both heavy and light chain genes of the corresponding humanized antibodies were constructed using GS vectors (Lonza Biologics, Inc.). Specifically, the gene encoding the signal sequence (N.Whittle et al., ProteinEng., Vol.1, p.499-505, 1987) and the constant region gene of human Igγ1 with amino acid mutations S239D, H268D and L328W (identified by SEQ ID NO: 1 base sequence from 358 to 1350 bases) were respectively connected to the 5' side and 3' side of the heavy chain variable region gene of the corresponding humanized antibody, and then the heavy chain gene was inserted into GS vector pEE6.4. In addition, the gene encoding the signal sequence (N. Whittle et al., as described above) and the constant regio...
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