Fully human TNFalpha-Fab antibody and its PEG antibody
An antibody and whole-human technology, applied in the direction of antibodies, anti-inflammatory agents, chemical instruments and methods, etc., can solve the problems of accelerated metabolism, increased antigenicity of Fab fragments, shortened half-life, etc., to achieve good affinity, avoid short half-life, and prolong the effect of time
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Embodiment 1
[0030] Example 1 Obtaining of B cells secreting anti-human TNFa antibody
[0031] Take 5 milliliters of peripheral blood from patients with active rheumatoid arthritis, separate leukocytes with lymphocyte separation medium, culture them, and identify positive clones according to the results of ELISA.
[0032] 1. Select blood sample
[0033]In order to obtain human B cells that secrete anti-human TNFα, human TNFα (purchased from Shenzhen Jingmei Company) was used to routinely coat a 96-well plate, and each well was coated with 250 ng of the protein overnight. Then, it was blocked with 5% skimmed milk powder at room temperature for 2 hours, and the milk powder was prepared with pH7.2 PBS. After washing, 100 microliters of sera from different patients were added and incubated at room temperature for 1 hour. Then add peroxidase-labeled goat anti-human IgG and let stand at room temperature for 1 hour. After washing at least 5 times, add TMD or other chromogenic reagents and trea...
Embodiment 2
[0038] Example 2 Obtaining of the Variable Region of the Whole Human TNFa-Fab Antibody Gene
[0039] Take 5,000-10,000 positive clone cells, use Trizol (GIBCO) to separate total RNA, and use Superscript III First-strand reverse transcriptase (invitrogen company product) to obtain cDNA. The above operations were carried out in accordance with the manufacturer's instructions. PCR was carried out with the following primers and conditions, and the amplification enzyme used was KOD-plus of TOYOBO to ensure that possible mutations were reduced during the amplification process.
[0040] Light chain upstream primer: GCTAGCGCCGCCACCATGGACATGCGGG (SEQ ID NO: 1)
[0041] Light chain downstream primer: TTTGATCTCAAGCTTGGTCC (SEQ ID NO: 2)
[0042] Heavy chain upstream primer: CCGGAATTCGCCGCCACCATGGAGTT (SEQ ID NO: 3)
[0043] Heavy chain downstream primer: ACTCGAGACGGTGACCAGTGTA (SEQ ID NO: 4)
[0044] PCR conditions:
[0045] a) The composition of the reaction system:
[0046]
...
Embodiment 3
[0060] Example 3 Preparation of fully human TNFa-Fab antibody
[0061] 1. Acquisition of human IgG1 light chain constant region and heavy chain Fd fragment coding sequence
[0062] We synthesized the light chain constant region of human IgG1 and the heavy chain CH1 gene sequence in order to constitute the complete human TNFa-Fab antibody gene. According to the literature, the DNA coding sequence of the human IgG1 light chain constant region is as follows: (5'→3') (SEQ ID NO: 9)
[0063] CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG
[0064] The human IgG1 heavy chain CH1 gene coding sequence is as follows:
[0065] (5'→3') (SEQ ID NO: 10)
[0066] GCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCAC...
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