Monoclonal Antibodies

There are two principal routes to generating monoclonal antibodies; either using traditional hybridoma approaches, or by using recombinant antibody technologies such as phage display.

Mouse Hybridoma
Hybridomas, as the name suggests, are a hybrid formed from two different cell lines.  Following immunisation, usually of a mouse or rat, spleen tissue is isolated and mechanically disrupted to release splenocytes.  Some of these are B-cells, each of which produces a single antibody, i.e. monoclonal.  The splenocytes are then fused with a suitable myeloma partner, e.g. murine SP2/0 cells, which imparts immortality to the fusion cell line.  After culturing in selective media, only fusions of a B-cell and a myeloma survive.  These are further cultured in multiple 96 well plates and supernatants screened for antigen reactivity.  Positive wells are cloned out by limiting dilution to yield wells containing single colonies each derived from a single hybridoma cell.  Further screening for antigen positive antibody allows hybridomas to be selected and expanded to yield the final hybridoma clone(s). 

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Custom hybridomas are typically generated against peptides or whole proteins (recombinant or purified native), but can also be raised using crude protein preparations, whole cells, or DNA.  Whole proteins are usually customer supplied; however in-house generation can be discussed.  Scotia Biologics can assist with the design and synthesis of peptide antigens, which need to fulfil a number of specification criteria to give the best chance of success.  These include selecting surface-accessible linear epitopes that are sufficiently hydrophilic to be readily water soluble, and will likely adopt a native structure as a peptide.  These are typically 12 to 20 residues in length, and avoid sequences containing too many hydrophobic residues, glutamines, or internal/multiple cysteines, although a single N- or C-terminal cysteine can aid conjugation to carrier protein.  Non-native cysteine can be included at either terminus for conjugation purposes.

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It is recommended that a monoclonal antibody program using peptide antigens include at least 2 – 3 different peptide sequences to maximise the likelihood of generating an antibody with the desired target specificity.  

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A phased program with multiple checkpoints/invoicing points enables customers to control the costs of projects and balance costs with progress/success.  A Hybridoma project includes the following steps:

i)    Consultation, agreeing project objectives and strategy
ii)   Antigen (peptide) design, synthesis and preparation of conjugates (where not customer supplied immunogen)
iii)  Immunisation of multiple (4-5) mice, provision and screening of serum samples
iv)  Selection of best mouse/mice, cell fusion and initial cloning.  Screening of culture supernatants
v)   Selection of best clones, and further sub-cloning.  Supply of antibody and hybridoma (typically two clones) cryo-stocks.
vi)  Scale-up expression and purification of antibody.

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Scotia will retain some vials of hybridoma for up to 6 months to provide a backup for the customer.

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Although hybridomas are frequently described as immortal, they CAN over extended storage time or after large numbers of passages, show reduced yields or even stop producing antibody.  For this reason, for very important clones Scotia recommends that customers also request our hybridoma sequencing/rescue service. With the sequence of the V-genes determined, an antibody can readily be recovered by synthesising the genes and cloning them into a suitable expression vector.