An In Vitro Method to Determine the Anti-Apoptotic Activity of Antibodies against TNFα

Published: January 31, 2024

Abstract

Source: Tierrablanca-Sánchez, L., et al. Determination of the Relative Potency of an Anti-TNF Monoclonal Antibody (mAb) by Neutralizing TNF Using an In Vitro Bioanalytical Method. J. Vis. Exp. (2017)

This video demonstrates a technique for assessing the efficacy of antibodies against tumor necrosis factor-alpha (TNFα), an inflammatory cytokine, through an in vitro bioanalytical approach. TNFα, when introduced to stressed cells, triggers apoptosis. However, binding to the antibodies confines TNFα, averting apoptosis. The degree of apoptosis is quantified using a bioluminescence assay.

Protocol

1. Preparation of the Media and Solutions Prepare the culture medium: RPMI-1640 with 10% fetal bovine serum (FBS), pH 7.4. Prepare assay culture medium: RPMI-1640 without phenol red but with 1% FBS, pH 7.4. Prepare cell wash solution: Dulbecco's phosphate-buffered saline (DPBS) Mg- and Ca-free solution with 0.02% EDTA, pH 7.4. Prepare cell detachment solution: 0.125% trypsin with 1 mM ethylenediaminetetraacetic acid (EDTA). Thaw 100 mL of a 0.25% …

Representative Results

Figure 1: Disposition of samples in the assay plates. B1 to G11 are well coordinates in the microplates and describe the positions where the sample dilutions are placed. Missing coordinates are wells filled with controls and assay culture medium (A1-A12 and H1-H12). This random distribution of samples (forward and reverse dilutions in the microplates) helps to eliminate bias in the results due to…

Divulgazioni

The authors have nothing to disclose.

Materials

WEHI 164 ATCC CRL-1751 Fibrosarcoma cells from Mus musculus
RPMI-1640 Medium ATCC 30-2001 Store medium at 2 °C to 8 °C
RPMI 1640 Medium, no phenol red GIBCO 11835-030 Store medium at 2 °C to 8 °C
Trypsin-EDTA(0.25%),phenol red GIBCO 25200-056 Store medium at -10 °C to -20 °C
DPBS, no calcium, no magnesium GIBCO 14190-136 Store medium at 2 °C to 8 °C
Recombinant Human TNF-alpha Protein R&D Systems 210-TA-020 Store at -20 °C to -70 °C
Fetal Bovine Serum (U.S), Super Low IgG HyClone SH3089803 Store at -10 °C to -20 °C
Fetal Bovine Serum (U.S.), Characterized HyClone SH3007103 Store at -10 °C to -20 °C
Caspase-Glo 3/7 Assay kit Promega G8093 Store the Caspase-Glo. 3/7 Substrate and Caspase-Glo. 3/7 Buffer at –20 ºC protected fromLight
EDTA, Disodium Salt, Dihydrate, Crystal, A.C.S. Reagent J.T.Baker 8993-01
Sample mAb Adalimumab Probiomed NA Final concentrations in the microplate are: 0.666, 0.333, 0.167, 0.111, 0.083, 0.056, 0.042, 0.028, 0.014 and 0.004 μg/mL
Reference and Control mAb Adalimumab Abbvie NA Final concentrations in the microplate are: 0.666, 0.333, 0.167, 0.111, 0.083, 0.056, 0.042, 0.028, 0.014 and 0.004 μg/mL
Microplate Reader Molecular Devices 89429-536 SpectraMax M3 Multi-Mode
Microplate reader Software Molecular Devices SoftMax Pro 6.3 GxP
Incubator Revco 30482 Revco RNW3000TABB Forced-Air CO2
Laminar Flow Hood The Baker Company 200256 Baker SG603A-HE | High Efficiency, Class II Type A2

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Citazione di questo articolo
An In Vitro Method to Determine the Anti-Apoptotic Activity of Antibodies against TNFα. J. Vis. Exp. (Pending Publication), e21918, doi: (2024).

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