Generating Recombinant Monoclonal Antibodies From Mammalian Cell Cultures

Abstract

Source: Nogales-Gadea, G. et al., Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells. J. Vis. Exp. (2015)

This video demonstrates a method of antibody generation from mammalian cells by introducing plasmid vectors with heavy and light chain genes. These vectors interact with polyethylenimine and enter the cells by forming polyplexes; later, they are translated into individual proteins and assembled into complete antibodies, which are secreted into the extracellular medium.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Isolation of Peripheral Blood Mononuclear Cells (PBMCs)

1. Centrifuge 25 ml of the participants' heparinized blood at 900 x g for 15 min, as soon as possible after the blood extraction. If there is less blood, scale down the reagents accordingly. Perform all the next steps in a hood.
2. Transfer the serum to a clean tube. It can be used in later steps for freezing PBMCs or in case of autoimmune patients, to test auto-antibodies.
3. Dilute the blood with 10 ml of Roswell Park M....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Histopaque-1077	Sigma-Aldrich	10771	solution containing polysucrose and sodium diatrizoate
FACSAria II cell sorter	BD Biosciences
96 U-bottom micro well plates	Costar	3799
Advanced Roswell Park Memorial Institute (RPMI) 1640 medium	Gibco, Life Technologies	12633-020
30% v/v EBV-containing supernatant of the B95-8 cell line	ATCC	CRL-1612	3.4 x 108 copies/ml
CpG2006	Invivogen	ODN 7909
Wi38 cells	Sigma-Aldrich	90020107
Interleukin-2	Roche	10799068001
ELISA plates	Greiner Bio-One, Microlon	655092
AffiniPure F(ab')2 Fragment Goat Anti-Human IgG, Fcγ Fragment Specific (unconjugated)	Jackson ImmunoResearch	109-006-008
4% non-fat dry milk (Blotting Grade Blocker)	Biorad	170-6404
Human IgG	Sigma	I 2511	HUMAN IgG purified Immunoglobulin, 5.6 mg/ml
Goat F(ab)2 antihuman IgG Fcγ (conjugated with peroxidase (PO))	Jackson ImmunoResearch	109-036-008
ELISA reader (Perkin Elmer 2030)	Perkin Elmer	2030-0050
Peroxidase-conjugated AffiniPure Rabbit Anti-Human IgM, Fc5µ	Jackson ImmunoResearch	309-035-095
SuperScript III Cells Direct cDNA Synthesis System	Invitrogen	18080-200
Applied Biosystems (ABI) GeneAm PCR System 2700	Applied Biosystems
High Pure RNA Isolation Kit	Roche	11828665001
Reverse transcription system kit	Promega	A3500
Recombinant Taq DNA Polymerase	TAKARA	R001A
Primers (2μl)	Sigma
Ultrapure Agarose	Invitrogen	16500-500
100 bp ladder	Invitrogen	15628-019
Quantity One 4.5.2 (Gel Doc 2000)	Biorad	170-8100
QIAquick PCR purification kit	QIAGEN	28106
BigDye Terminator v3.1 cycle sequencing kit	Applied Biosystems	4337455
0.1 ml reaction plate (MicroAMP Optical 96-well)	Applied Biosystems	4346906
Genetic analyser ABI300	Applied Biosystems	4346906
DH5α competent cells (E. coli)	Invitrogen	18263-012
pFUSEss-CHIg-hG1 (4493 bp)	Invivogen	pfusess-hchg1
pFUSEss-CHIg-hG4 (4484 bp)	Invivogen	pfusess-hchg4
pFUSE2ss-CLIg-hk (3875 bp)	Invivogen	pfuse2ss-hclk
pFUSE2ss-CLIg-hl2 (3883 bp)	Invivogen	pfuse2ss-hcll2
SOC medium	Invitrogen	15544-034
LB-based agar medium supplemented with Zeocin (Fast-Media Zeo Agar)	Invivogen	fas-zn-s
Terrific Broth (TB)-based liquid medium supplemented with Zeocin (Fast-Media Zeo TB)	Invivogen	fas-zn-l
DNA Miniprep kit	Omega Bio Technology	D6942-02
Nanodrop (ND1000 Spectrophotometer)	Nanodrop
LB-based agar medium supplemented with Blasticidin (Fast-Media Blast Agar)	Invivogen	fas-bl-s
Terrific Broth (TB)-based liquid medium supplemented with Blasticidin (Fast-Media Blast TB)	Invivogen	fas-bl-l
EcoRI	New England Biolabs	R0101S	20,000 U/ml, in 10x NEBuffer EcoRI
NheI	New England Biolabs	R0131S	10,000 U/ml, in 10x NEBuffer 2.1
2-Log DNA ladder	New England Biolabs	N3200S	0.1-10.0 kb, 1,000 μg/ml
XmaI	New England Biolabs	R0180S	10,000 U/ml, in 10x CutSmart Buffer
BsiWI	New England Biolabs	R0553S	10,000 U/ml, in 10x NEBuffer 3.1
AvrII	New England Biolabs	R0174S	5,000 U/ml, in 10x CutSmart Buffer
FastAP Thermosensitive Alkaline Phosphatase	Thermo Scientific	EF0651	1 U/µL, in 10x FastAP Buffer
DH5α competent cells	Invitrogen	18263-012
PE Mouse Anti-Human IgG	BD Pharmingen	555787
anti-CD22, PerCP-Cy5.5, Clone: HIB22	Fisher scientific	BDB563942
QIAprep Spin Miniprep Kit	QIAGEN	27106
BigDye Terminator v3.1	Applied Biosystems	4337455