Enzyme-Linked Immunosorbent Assay to Verify Chemically-Coupled Antibody-Antigen Conjugates

Abstract

Source: Volckmar, J. et al., Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo. J. Vis. Exp. (2021)

This video demonstrates the detection of chemically conjugated antibody-antigen complexes via the sandwich enzyme-linked immunosorbent assay, or ELISA, technique. The captured antibody binds to the specific epitope of the antigen of the antibody-antigen conjugate, which can be later visualized using an enzyme-linked secondary antibody.

Protocol

1. Production of αDEC-205 from the hybridoma cell line NLDC-145 For antibody production, thaw cryopreserved NLDC-145 cells producing αDEC-205 at 37 °C in a water bath. Expand the cells at 37 °C and 5% CO2. Two 75 cm2 bottles will be needed to proceed to antibody production. Cell culture procedures should be performed in a safety cabinet to ensure safe working conditions and prevent contamination of the cultures. Resuspend 1 mL of thawed cells (1 x 106…

Materials

Blocking buffer (ELISA)			10 % FBS in PBS
Cell culture flask T25	Greiner Bio-One	690175	we use standard CELLSTAR filter cap cell culture flasks; alternatively use suspension culture flask (690195 )
Cell culture flask T75	Greiner Bio-One	658175	we use standard CELLSTAR  filter cap cell culture flasks; alternatively use suspension culture flask (658195)
Cell culture flask T175	Greiner Bio-One	661175	we use standard CELLSTAR filter cap cell culture flasks; alternatively use suspension culture flask (661195)
Centrifugal concentrator MWCO 10 kDa	Sartorius	VS2001	Vivaspin 20 centrifugal concentrator
Centrifugal protein concentrator MWCO 100 kDa, 5 – 20 ml	Thermo Fisher Scientific	88532	Pierce Protein Concentrator, PES 5 -20 ml; we use the Pierce Concentrator 150K MWCO 20mL (catalog number 89921), which is however no longer available
Centrifuge bottles	Nalgene	525-2314	PPCO (polypropylene copolymer) with PP (polypropylene) screw closure, 500 ml; obtained from VWR, Germany
Coating buffer (ELISA)			0.1 M sodium bicarbonate (NaHCO3) in H2O (pH 9.6)
Desalting columns MWCO 7 kDa	Thermo Fisher Scientific	89891	Thermo Scientific Zeba Spin Desalting Columns, 7K MWCO, 5 mL
Detection reagent ELISA (HRPO substrate)	Sigma-Aldrich/Merck	T8665-100ML	3,3′,5,5′-Tetramethylbenzidine (TMB) liquid substrate system
ELISA 96-well plate	Thermo Fisher Scientific	442404	MaxiSorp Nunc-Immuno Plate
Fetal calf serum	PAN-BIOtech	P40-47500	FBS Good forte
ISF-1 medium	Biochrom/bioswisstec	F 9061-01
NLDC-145 hybridoma	ATCC	HB-290	if not already at hand, the hybridoma cells can be acquired from ATCC
Ovalbumin	Hyglos (via BioVendor)	321000	EndoGrade OVA ultrapure with <0.1 EU/mg
Penicillin/Streptomycin	Thermo Fisher Scientific	15140122	Gibco Penicillin/Streptomycin 10.000 U/ml; alternatively Gibco Penicillin/Streptomycin 5.000 U/ml (15070-063) can be used
PETG polyethylene terephthalate glycol cell culture roller bottles	Nunc In Vitro	734-2394	standard PDL-coated, vented (1.2X), 1050 cm², 100 – 500 ml volume; obtained from VWR, Germany
pH indicator strips	Merck	109535	pH indicator strips 0-14
Polyclonal goat αrat-IgG+IgM-HRPO antibody  (ELISA)	Jackson ImmunoResearch	112-035-068	obtained from Dianova, Germany; used at 1:2000 for ELISA
Polyclonal rabbit αOVA (ELISA)	Abcam	ab181688	used at 3 ng/µl
Protein G Sepharose column	Merck/Millipore	P3296	5 ml Protein G Sepharose, Fast Flow are packed onto an empty column PD-10 (Merck, GE 17-0435-01)
Rubber plug	Omnilab	5230217	DEUTSCH & NEUMANN rubber stoppers (lower Φ 17 mm; upper Φ 22 mm)
Silicone tube	Omnilab	5430925	DEUTSCH & NEUMANN (inside Φ 1 mm; outer Φ 3 mm)
Slim-Fast			we have used regular Slim-Fast Chocolate freely available at the pharmacy as in this western blot approach it yielded better results than milk powder
Stopping solution (ELISA)			1M H2SO4
Sulfo-SMCC	Thermo Fisher Scientific	22322	Pierce Sulfo-SMCC Cross-Linker; alternatively use catalog number A39268 (10 x 2 mg)
Syringe filter unit 0.22 µm	Merck/Millipore	SLGV033RS	Millex-GV Syringe Filter Unit, 0.22 µm, PVDF, 33 mm, gamma sterilized
Syringe 10 ml	Omnilab		Disposable syringes Injekt® Solo B.Braun
Sterican® cannulas	B. Braun		Sterican® G 20 x 1 1/2""; 0.90 x 40 mm; yellow
TCEP-HCl	Thermo Fisher Scientific	A35349
Tubing connector	Omnilab		Kleinfeld miniature tubing connectors for silicone tube