Human fvii monoclonal antibodies binding the gla domain and use thereof
A monoclonal antibody, domain technology, applied in the direction of anti-coagulation factor immunoglobulin, anti-animal/human immunoglobulin, peptide, etc., can solve problems such as affecting folding and affecting activity
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Embodiment 1
[0331] Example 1. Recombinant production of antibodies
[0332] In an exemplary embodiment, recombinant mAbs can be produced from the VH and VL sequences of FVII antibodies using the methods described below. Steps 1-3 describe obtaining VH and VL regions from hybridomas or other monoclonal FVII antibody-producing cells. Alternatively, the cDNA encoding the FVII antibody VH and VL sequences for step 4 can be prepared from the sequence information provided in Figure 2 using well-established techniques for synthesizing cDNA fragments. VH and VL fragments of the desired antibody, or mutants or derivatives thereof, can also be cloned into any of a variety of expression vectors described in the scientific literature or commercially available expression vectors, wherein Contains the constant regions of the Ig subclass of interest for expression of full-length antibodies. Additionally, VH and VL fragments of the antibody of interest, or mutants or derivatives thereof, can be cloned ...
Embodiment 2
[0364] Example 2. Using sandwich ELISA to measure the concentration of total factor VIIa in the sample
[0365] Determination of the concentration of total Factor VII in a sample can be determined using a sandwich ELISA using any two monoclonal antibodies directed against two different epitopes, such as anti-EGF domain antibodies:
[0366] Add 100 μL of coating buffer (0.1 M NaHCO) to each well 3 , pH 9.8) 96-well microplates (C96 maxisorp Nunc-Immuno plate from Nalgene Nunc International) were coated with 1 μg of monoclonal F9 anti-FVII (primary antibody) overnight at 4°C. After incubation, wash with 350 μL of wash buffer (20 mM Hepes, 100 mM NaCl, 10 mM CaCl 2 , 0.02% Tween80, pH 7.4) and the plate was washed 4 times. After washing, use 350 μL blocking buffer (20 mM Hepes, pH 7.4, 0.1 M NaCl, 10 mM CaCl 2 , 1% BSA, 0.02% Tween 80) to block the wells for 2.5 hours. Blocking was performed at room temperature using a shaking table.
[0367] 100 μL of peroxidase-conjugated,...
Embodiment 3
[0373] The concentration of FVII in individual wells was determined using the standard curve shown in Figure 3 . Example 3. Control of production parameters for production of active factor VIIa
[0374] Using the standard FVII ELISA described in Example 2, the total FVII production thereof can be monitored in batch or continuous cell cultures. This specific amount and thus the production of FVII comprising the intact GLA domain can be determined using the following sandwich ELISA assay based on antibodies against the GLA domain and antibodies against another epitope of FVII.
[0375] Add 100 μL of coating buffer (50 μg / ml F1A2i, 20 mM Hepes, 100 mM NaCl, 10 mM CaCl to each well) 2 , pH 7.4) 96-well microplate (C96 maxisorp Nunc-Immuno plate from Nalgene Nunc International) was coated with 5 μg of monoclonal Fl anti-FVII (primary antibody) overnight at 4°C. After incubation, wash with 350 μL of wash buffer (20 mM Hepes, pH 7.4, 0.1 M NaCl, 10 mM CaCl 2 , 0.02% Tween 80) was ...
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