Anti-apoc3 antibodies and methods of use thereof
A technology of antibodies and amino acids, applied in the direction of antibodies, antibody medical components, chemical instruments and methods, etc., can solve the problem of small and effective plasma triglycerides
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Embodiment 1
[0204]Example 1: Construction of anti-ApoC3 antibody phage display library from immunized animals
[0205] This example describes the initial construction of an anti-ApoC3 antibody library. Human ApoC3 (huApoC3) protein purified from human serum was obtained from Athens Research and Technology. This huApoC3 protein was used in this example as well as in some of the following examples. HuApoC3 is complexed with dimyristoylphosphatidylcholine (DMPC) liposomes to closely mimic a physiologically relevant conformation. Following the procedure described by Klarenbeek and colleagues (Klarenbeek et al., Protein Eng Des Sel (2016) 29(4):123-133), two llamas were bred by intramuscular injection with 6 doses of huApoC3 with incomplete Freund's adjuvant. Immunization, which is hereby incorporated by reference in its entirety. The immunization schedule consisted of weekly injections of the first two doses of 100 μg huApoC3 each and four additional doses of 50 μg huApoC3 each, followed b...
Embodiment 2
[0207] Embodiment 2: screening anti-ApoC3 monoclonal antibody
[0208] This example describes the screening of antibodies that bind to ApoC3 in the scFv phage display library prepared in Example 1. Two ELISA-based screening methods were employed, one with purified ApoC3 coated directly on the plate and the other with biotinylated ApoC3 captured by streptavidin coated on the plate. Each method revealed many antibody clones with affinity for ApoC3. There was significant overlap between clones identified from these two assays, whereas differences likely reflect differences in ApoC3 protein conformation and epitope exposure under the two conditions.
[0209] 2.1 Analysis of affinity for directly coated ApoC3
[0210] Antibody clones selected from the scFv phage display library constructed in Example 1 were analyzed by ELISA assay using purified huApoC3 (Athens Research and Technology) coated directly on plates. Human native ApoC3 at a concentration of 1-10 μg / ml (100 μl / well) d...
Embodiment 3
[0221] Example 3: Lipid Competition Assay
[0222] This example describes the characterization of anti-ApoC3 antibodies as to whether they compete with lipids for ApoC3 binding. An ELISA-based assay was first performed to identify antibodies that abolished or did not abolish lipid binding. To further validate the results, surface plasmon resonance (SPR) based assays were then performed with certain clones.
[0223] 3.1 ELISA-based assay
[0224] An ELISA-based lipid competition assay was performed to determine whether the anti-ApoC3 antibodies identified from Example 2 interfered with the interaction between huApoC3 protein and lipids immobilized on ELISA plates. Briefly, 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (Avanti Polar Lipids DMPC; 14:0PC) was diluted in chloroform in a mixture of methanol:chloroform:water (2.0:1.0:0.8). stock solution in . 5 μg of DMPC was dispensed into each well of a Greiner U-bottom high binding plate (#850345) and the chloroform, methanol and ...
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