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

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

1. Prepare the culture medium: RPMI-1640 with 10% fetal bovine serum (FBS), pH 7.4.
2. Prepare assay culture medium: RPMI-1640 without phenol red but with 1% FBS, pH 7.4.
3. Prepare cell wash solution: Dulbecco's phosphate-buffered saline (DPBS) Mg- and Ca-free solution with 0.02% EDTA, pH 7.4.
4. Prepare cell detachment solution: 0.125% trypsin with 1 mM ethylenediaminetetraacetic acid (EDTA).
   1. Thaw 100 mL of a 0.25% solution of trypsin-EDTA and transfer to a sterile 500-mL flask.
   2. Mix with 100 mL of cell wash solution and dispense 15 mL aliquots into 15 mL s....

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-H1.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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