Antibody Production

Polyclonal Antibody Production [back to top]

BioServ UK offers custom polyclonal anti-sera production in a variety of species to include the following:

design and preparation of protein antigen or advice with peptide synthesis and conjugation, typically peptide-KLH as antigen and peptide-BSA as screening reagent;
immunisation schedule using BioServ UK SOPs;
determination and quantification of serum titer in end use assay e. g. , ELISA, Western Blot, IP;
affinity chromatography purification.

Monoclonal Antibody Production [back to top]

Although BioServ UK treats each contract as a custom project, most of the following elements are applicable and help to ensure a quality product is produced:

consultation with client to discuss epitope selection;
critical assessment of suitable screening methods and relevance to intended end use assay;
ongoing liaison between client and BioServ UK during hybridoma development process;
full confidentiality assured;
project segmentation into different phases to provide researcher flexibility for terminating project at the end of each phase.

Phase I: Antigen Design and Immunisation (6–10 wks)

Design and preparation of protein or cellular antigens or advice with peptide synthesis and conjugation.
Immunisation schedule using BioServ UK SOPs.
Determination and quantification of serum titer in end use assay e. g. , ELISA, Western Blot, IP.

Phase II: Cell Fusion and Screening of Candidate Colonies (3–6 wks)

Hybridoma fusion using BioServ UK SOP into 10x96 well plates.
Initial screen by ELISA to select strongest positive candidate 'binders'. There is opportunity at this stage to select isotype specific antibodies using defined secondary reagents.
Expansion into 24-well plates for harvest of tissue culture supernatant for further antibody screening (ELISA, Western Blot or FACS).
Option for client to assess antibody performance: dispatch of 5ml of supernantant from best 15–20 colonies.

Phase III: Limiting Dilution Cloning and Cryogenic Preservation (3–5 wks)

Limiting dilution cloning performed with strongest parental colonies.
Subclones with high specificity expanded and cryopreserved (5 vials each).
Shipment of hybridoma cells and up to 5ml of supernatant from each subcloned cell line.

Phase IV: Large Scale Antibody Production (3–6 wks)

Milligramme to gramme quantities produced.
Production performed in vitro using a variety of bioreactor systems with option for protein free adaptation.
Purification using protein A or G affinity chromatography.

Antibody Purification and Related Services [back to top]

Serum from our polyclonal schedules can be processed further:

Protein A/G purification;
ELISA titering of peptide-antibody;
SDS-PAGE analysis of antibody;
Antibody fragmentation e. g. Fab F(ab')₂;
Antibody labelling e. g. HRP, FITC, Biotin;
Preparation of affinity matrices using customer-specific immobilised peptide and protein ligands;
Metal chelate affinity chromatography.