Monoclonal antibodies and methods for using same
A monoclonal antibody, antibody technology, applied in the field of biotechnology and biomedicine, can solve problems such as decline
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Embodiment 1
[0203] Example 1. Production and purification of antibodies
[0204] Nucleic acids encoding the variable domains of antibody heavy and light chains (SEQ ID NO: 10, 12, 14, 16, 18) were obtained by amplifying the DNA fragment using overlapping primers and high-fidelity Q5 polymerase (NEB, USA). ). The resulting nucleic acid was purified on a column from Quagen (Germany) using a kit (# 28104) and targeted cloned into a commercially available pFuse vector containing human heavy (IgG1) and light (κ ) chain gene constant region (Invivogen, USA). The sequence of the cloned fragment was confirmed by sequencing using the Sanger method.
[0205] As a result, plasmids were obtained comprising the coding sequences for four antibody heavy chain variants:
[0206] HV anti-TRBV9-1, its nucleotide and amino acid sequences are shown in SEQ ID NO: 20 and 21;
[0207] HV anti-TRBV9-2, its nucleotide and amino acid sequences are shown in SEQ ID NO: 22 and 23;
[0208] HV anti-TRBV9-3, its...
Embodiment 2
[0221] Example 2. Use of anti-TRBV9 monoclonal antibodies for labeling T lymphocytes expressing TCR β-chains belonging to the TRBV9 family The purpose of the cells.
[0222] Monoclonal antibodies (anti-TRBV9-1, anti-TRBV9-2, anti-TRBV9-3, anti-TRBV9-4, characterized by heavy chain CDR3 sequences) were produced as described in Example 1. For visualization of antibodies, they were labeled with fluorescein using fluorescein isothiocyanate reagent (Sigma, USA) according to the manufacturer's protocol. The amount of fluorophore reactive with the antibody molecule is controlled by the ratio of the absorption spectrum at 495 / 280 nm wavelength. The labeled antibody was used to detect T lymphocytes expressing the TCR β-chain of the TRBV9 family in the mononuclear fraction of human blood.
[0223] Peripheral blood from 5 healthy donors was used to obtain the fractions. Blood was collected in EDTA Vacuette tubes (2x9ml each) and mononuclear fractions were isolated according to stan...
Embodiment 3
[0230] Example 3. Functional activity of antibodies
[0231] Monoclonal antibodies (anti-TRBV9-1, anti-TRBV9-2, anti-TRBV9-3, anti-TRBV9-4, characterized by heavy chain CDR3 sequences) were obtained as described in Example 1. Mononuclear fractions of human blood were obtained as described in Example 2.
[0232] Further, natural killer cells were isolated from a part of the mononuclear fraction using Human NK Cell Isolation Kit # 130-092-657 (Miltenyi biotec, USA). Use manufacturer's protocol. The quality of NK cell isolation was assessed by cytofluorometry (BD FACS ARIA III, USA) using labeled antibodies CD16-FITC, CD56-PE, CD3-VioBlue. The enrichment of NK cells was 85-95%.
[0233] To assess cytotoxicity, antibodies (anti-TRBV9-1, anti-TRBV9-2, anti-TRBV9-3, or anti-TRBV9-4) were added to aliquots of the mononuclear fraction containing 1 x 106 cells to 5 Final concentration in μg / ml. Antibody at the same concentration as anti-TRBV9 antibody was used as a negative cont...
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