Generation of Murine Monoclonal Antibodies by Hybridoma Technology

Summary

An optimized protocol is presented for the generation of monoclonal antibodies based on the hybridoma technology. Mice were immunized with an immunoconjugate. Spleen cells were fused by PEG and an electric impulse with immortal myeloma cells. Antibody-producing hybridoma cells were selected by HAT and antigen-specific ELISA screening.

Abstract

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding molecules is time-consuming and laborious due to the complicated handling and lack of alternatives. The aim of this protocol is to provide one standard method for the generation of monoclonal antibodies using hybridoma technology. This technology combines two steps. Step 1 is an appropriate immunization of the animal and step 2 is the fusion of B lymphocytes with immortal myeloma cells in order to generate hybrids possessing both parental functions, such as the production of antibody molecules and immortality. The generated hybridoma cells were then recloned and diluted to obtain stable monoclonal cell cultures secreting the desired monoclonal antibody in the culture supernatant. The supernatants were tested in enzyme-linked immunosorbent assays (ELISA) for antigen specificity. After the selection of appropriate cell clones, the cells were transferred to mass cultivation in order to produce the desired antibody molecule in large amounts. The purification of the antibodies is routinely performed by affinity chromatography. After purification, the antibody molecule can be characterized and validated for the final test application. The whole process takes 8 to 12 months of development, and there is a high risk that the antibody will not work in the desired test system.

Introduction

The hybridoma technology presented in this protocol was first described by Köhler and Milstein¹ in 1975 and, except for some technical improvements, the main procedure has not changed dramatically during the last 40 years². The aim of this protocol is to explain a more appropriate immunization strategy, a standard method for the generation of monoclonal antibodies, and an example for a validation method (ELISA).

Antibodies are incredible tools and contribute to a wide range of technological approaches, such as flow cytometry, magnetic cell sorting, or immunofluorescence, as well as to diagnostic and therapeut....

Protocol

Balb/c or NMRI mice (Mus musculus) from our breeding colony at the University of Potsdam (Potsdam, Germany) were used for the production of monoclonal antibodies. The animal work was conducted according to relevant national and international guidelines. The study was approved by the Brandenburg Ministry of Environment, Health, and Consumer Protection (reference number V3-2347-A16-4-2012).

1. Preparation of Immunoconjugates

1. For coupling the antigen to a carrier, use standard coupling protocols via amino-, carboxy, or sulfo-groups, such as by glutaraldehyde, N-(3-dimethylaminopropyl)-N-ethylcarbodiimide ....

Results

Figure 1 shows an example of the antigen-specific serum titer for one immunization performed in the lab. In this figure, the immune sera A and B were titrated from 1:50 to 1:5,000,000 in comparison to a serum of a naïve mouse. The antigen was coated on the solid phase, and the specific antibodies were detected with a POD-conjugated goat anti-mouse Ig antibody. Both sera of the immunized mice showed a significantly higher antigen-specific titer compared to the naï.......

Discussion

The generation of monoclonal antibodies by hybridoma technology requires an intense and detailed epitope analysis, especially with regard to the final application, when the antibody should recognize the target. This is often underestimated by users and leads to antibodies with weak performances. The fusion process is always random, which means that the outcome of specific hybridomas is highly dependent upon the cell ratio and the vitality at this point. After limited dilution, the cells are very unstable and require stri.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
glutaraldehyde	Sigma Aldrich	G5882
N-(3-dimethylaminopropyl)-N-ethylcarbodiimide (EDC)	Sigma Aldrich	39391-10ML
Sulfo-GMBS	Perbio Science Germany	22324
ovalbumin	Sigma Aldrich	A5503
bovine serum albumin	Sigma Aldrich	A2153
keyhole limpet hemocyanin	Sigma Aldrich	H8283
Falcon tubes 15 mL	Biochrom GmbH	P91015
reaction vials, 1.5 mL	Carl Roth GmbH & C0.KG	CNT2.1
hollow needle	Carl Roth GmbH & C0.KG	C724.1
glass wool	Carl Roth GmbH & C0.KG	6574.1
Sephadex G25 coarse	Sigma Aldrich	GE-17-0034-02
Freund´s adjuvant, complete	Sigma Aldrich	F5881-10ML
ELISA plates, 96 well	Greiner bio-one	655101
neonatal calf serum	Biochrom GmbH	S1025
TipOne Tips 1,000 µL	Starlab	S1111-2021
Pipette tips 200 µL	Greiner bio-one	739291
HRP-conjugated goat-anti-mouse IgG antibody	Dianova	115-035-003
tetramethylbenzidine	Carl Roth GmbH & C0.KG	6350.2
Natriumdihydrogenphosphat	Carl Roth GmbH & C0.KG	K300.2
peroxide/urea
sulphuric acid	Carl Roth GmbH & C0.KG	4623.3
RPMI 1640	Life technologies GmbH	31870074
L-glutamine	Carl Roth GmbH & C0.KG	HN08.2
beta-mercaptoethanol	Sigma Aldrich	M6250
fetal calf serum	Invitrogen	10270106
TC-flask 25 cm2	Peske GmbH	86-V025
TC-flask 75 cm2	Peske GmbH	86-V075
ethanol, 96%	Carl Roth GmbH & C0.KG	P075.1
cell strainer	VWR international	734-0002
Falcon tubes 50 mL	Biochrom GmbH	P91050
PEG 8000	Sigma Aldrich	1546605
electroporation cuvette, 2 mm	Biodeal Handelsvertretung Edelmann e.K.	EKL2,25
hypoxanthine	Sigma Aldrich	H9636-25G
azaserine	Sigma Aldrich	A4142
thymidine	USB Europa GmbH	22305 1 GM
TC-plates 96 well	Biochrom GmbH	P92696
TC-plates 24 well	Biochrom GmbH	P92424
cryotubes, 1 mL	Sigma Aldrich	V7384-1CS
dimethylsulfoxide	Carl Roth GmbH & C0.KG	4720.1
protein A sepharose	Sigma Aldrich	P3391-1G
SDS sample loading buffer, Roti-Load 1	Carl Roth GmbH & C0.KG	K929.1
unstained protein ladder	BioRad Laboratories	161-0363
comassie brilliant blue R-250	BioRad Laboratories	161-0406