Afuco™ Anti-Human TNF ADCC Recombinant Antibody (AVX-470), ADCC Enhanced (CAT#: AFC-601CL)

• Host Species
• Cow

• Derivation
• Cow

• Type
• ADCC enhanced antibody

• Species Reactivity
• Human

• Applications
• Neut, FuncS

• Related Disease
• Ulcerative Colitis

• Purity
• >95% as determined by analysis by SDS-PAGE

• Storage
• ≤1 year at -20°C.

• Application Notes
• AVX-470 and AVX-470m TNF-Binding and Neutralization Activities
  The ability of AVX-470 and AVX-470m to bind to TNF was measured in a direct enzyme-linked immunosorbent assay (ELISA). Microtiter plates were coated with human or mouse TNF at 1 µg/mL and binding was detected using horseradish peroxidase-conjugated sheep anti-bovine antibody using standard techniques. Titers, expressed as µg/mL, were defined as the concentration of test article resulting in an absorbance value of 0.2. The ability of antibody to neutralize TNF was determined using the murine fibroblast cell line L929. These cells are killed by TNF, especially in the presence of actinomycin D, and a neutralizing anti-TNF antibody will prevent this cytotoxicity15. IC50 values were calculated as the concentration of AVX-470/AVX-470m resulting in 50% inhibition of TNF-mediated killing of L929 cells. Affinity of AVX-470A and AVX-470mA for TNF by Competition ELISA Serial dilutions of human or mouse TNF were incubated with defined levels of anti-TNF antibodies or control bovine Ig. The levels of TNF-specific antibodies used in these assays were previously determined to be in the linear portions of titration curves in direct ELISAs: AVX-470A (35 ng/mL), AVX-470mA (50 ng/mL), infliximab (2 ng/mL) and TN3 (TN3-19.12, 10 ng/mL). The antibody/TNF dilution mixtures were then added to the TNF-coated plates which were incubated, washed, incubated with horseradish peroxidase-labeled anti-bovine, human or hamster IgG secondary antibodies, washed and read on a plate reader. Data were analyzed using Gen5 Data Analysis Software and affinities were estimated as dissociation constants (KD) calculated from the amount of soluble TNF required to inhibit 50% of the binding of the antibodies to plate-bound TNF. Mouse models of colitis Animal experiments were conducted in the AAALAC-approved facility of Biomodels, LLC with ethical review approval from Biomodels' Institutional Animal Care and Use Committee. Colitis was induced in male C57BL/6 mice by exposure to 3% DSS in drinking water from Day 0 to Day 512 or by intrarectal administration of 4 mg TNBS in 50% ethanol on Day 0. Treatments with vehicle (saline), AVX-470m (1-10 mg/day), control Ig (3 or 10 mg/day), or prednisolone (3 mg/kg/day) were administered in volumes of 0.2 mL/mouse twice a day by oral gavage. Treatments with etanercept (10 mg/kg) were administered by intraperitoneal injection every other day. In prevention studies, treatment began one day before the induction of colitis and continued through day 3 for TNBS colitis or through day 12 for DSS colitis. In treatment studies, treatment began on day 6 and continued through day 19. Colitis severity was assessed using video endoscopy (Karl Storz Endoskope, Tuttlingen, Germany) a method that provides a robust clinical readout of disease severity14,16. Images were scored by a blinded observer: 0 = normal, 1 = loss of vascularity, 2 = loss of vascularity and friability, 3 = friability and mucosal erosions, and 4 = ulcerations and bleeding.

• Alternative Names
• TNF; DIF; TNFA; TNFSF2; TNF-alpha; tumor necrosis factor

• Gene ID
• 7124

• UniProt ID
• P01375

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