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.
The authors have nothing to disclose.
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 |