To generate hyper-immune Ig for passive transfer, the immune serum samples from each NHP were diluted 1:50 with PBS, added to protein A columns, and incubated overnight at 4°C. After washing the columns briefly, captured antibodies were eluted with low-pH IgG elution buffer (ThermoFisher Scientific) and the eluates were immediately neutralized by adding 1 M Tris-HCl (pH 8.0) to a final concentration of 100 mM. Purified polyclonal antibodies were dialyzed two times against PBS, concentrated to ~20 mg ml−1 and stored at −80°C until use. BALB/cAnNHsd mice (Envigo) were given intraperitoneally 0.2 mg of FI6v3 (approximately 10 mg kg−1) or 10 mg of purified polyclonal Ig from individual NHPs. Twenty-four hours later, the mice were infected intranasally with 25× or 10× LD50 of H5N1 or H7N9 viruses (Supplementary Table 4 ) at Bioqual. The animals were monitored twice daily for development of clinical signs of infection and weighed daily for 14 days. Any animals that lost 20% or more of their initial body weight were euthanized.
Low ph igg elution buffer
Manufactured by Thermo Fisher Scientific
The Low-pH IgG elution buffer is a laboratory reagent designed to facilitate the elution of immunoglobulin G (IgG) antibodies from affinity chromatography columns. The buffer is formulated to provide a low pH environment, which can disrupt the binding interactions between the IgG and the column matrix, allowing the IgG to be efficiently recovered.
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5 protocols using low ph igg elution buffer
1
Passive Transfer of Hyper-immune Ig Against Influenza
2
Hyperimmune Ig Generation Protocol
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To generate hyperimmune Ig, 100 female BALB/cJ mice (6–8 weeks old; Jackson Laboratory) were immunized three times with H1ssF gN38 protein (2 μg per immunization with SAS) at weeks 0, 4, and 8 and sera were collected at 1 and 2 weeks after the last immunization prior to terminal bleed at 3 weeks after the last immunization. Briefly, 10 mg H7 AN13 HA protein was immobilized on NHS-Activated Agarose Dry Resin (Thermo Scientific) for 2 h at RT. After coupling, the resin was washed three times with PBS, then pooled immune sera was added. The column was incubated overnight at 4 °C. After washing the column briefly, captured antibodies were eluted with low-pH IgG elution buffer (Thermo Scientific) and the eluates containing H7-specific antibodies were immediately neutralized by adding 1 M Tris-HCl (pH 8.0) at a final concentration of 100 mM. Purified H7-specific antibodies were dialyzed two times against PBS, concentrated to ~1.0 mg ml−1 and stored in −80 °C until use.
3
Recombinant Antibody Production in HEK293FS
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All antibodies were expressed in HEK293FS cells by co-transfecting equal amount of heavy chain and light chain plasmids. FreeStyle™ MAX Reagent (Thermo Fisher Scientific; 16447500) was used for transfection according to manufacturer’s protocol. Cell-free supernatants were collected after 5-day incubation at 37 °C and filtered through a 0.22-μm filter. Antibody was purified by passing the supernatants through protein A column. After washing with PBS, protein was eluted with low-pH IgG elution buffer (Thermo Fisher Scientific; 21009), followed by neutralization with 100 mM Tris-HCl pH 9.0. The eluted protein was dialyzed to PBS and stored at 1 mg/ml at −80 °C.
4
Monoclonal Antibody CR3022 Production
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A plasmid construct that encodes heavy and light chain genes of a monoclonal antibody (mAb) CR3022 in backbone of pDR12, a two-promoter plasmid, was kindly provided by Dr. Tianlei Ying (Fudan University, Shanghai, China). CR3022 was expressed by transfecting the plasmid into FreeStyle 293F cells with 293Fectin transfection reagent. Cell culture medium was harvested 5 days after transfection and clarified by centrifugation. Clarified medium was diluted 1:1 with protein A IgG Binding Buffer (Thermo Scientific) and filtered through 0.22 μm filter to remove any precipitate before incubation with Protein A Plus Agarose (Thermo Scientific). After binding, antibody was eluted from the column using low pH IgG Elution Buffer (Thermo Scientific) and neutralized immediately in collection tubes. The purified antibody was buffer-exchanged with PBS and concentrated using Amicon Ultra concentrator with a 30 kDa cut-off filter.
5
Passive Transfer of Hyper-immune Ig Against Influenza
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