L10 Monoclonal Antibody Production Flashcards
Learning outcomes: Explain how murine hybridomas are produced and understand the problems that may arise. Understand the differences between conventional serum and monoclonal antibody derived from a hybridoma. Learn about industrial production of mAbs
How does harvesting of B cells from animal differ for monoclonal antibodies?
Instead of harvesting of antiserum (blood) from live animals, the animals are sacrificed, their spleens removed and B cells are isolated from this.
Once the desired B cell is identified, what must occur before antibodies can be produced?
Must be immortalised. Done via hybridoma process with an immortal cancer cell.
How are B cells obtained from spleen?
Mechanically broken apart and put through density gradient centrifugation, separates cells.
B cell fraction is taken.
Which two genes are removed from myeloma cells during hybridoma formation?
HGRPT
Ig
What solute is added during fusion to enhance the process?
Polyethylene glycol (PEG)
Aids fusion through association of water between two cell membranes or through creation of fusion pore.
How is the issue of spontaneous cell fusions being low frequency addressed?
Use of PEG.
Or electrofusion - aligns cells and joins membranes together using pusled electric field, promotes fusion.
After fusion, what cells will be seen?
How do the two gene knockouts + aminopterin treatment address this to isolate successful hybridoma cells?
Fused and unfused myeloma cells - these die because they are HGPRT- and aminopterin blocks de novo pathway.
Fused and unfused B cells - die because they have not been given immortality (short lifespan).
Hybridoma cells - Surive as B cell has confered HGPRT+, and they can divide indefninitely.
How does HAT media kill undesired cells?
Contains three toxins. Aminopterin blocks a nucleotide snythesis pathway only cells capable of nucleotide synthesis by salvage pathway (HGPRT key enzyme in this) survive in HAT medium. Only B cells and hybridoma cells can survive. And B cells die shortly after as they are not immortalised.
Once hybridomas are selected, what issue is now faced?
They must be selected to become monoclonal.
Diluted in such a way that one cell is added to each well in a multi-well plate.
After hybridoma propagation in multi-well plates, what must be done to ensure best antibody is used?
Must be checked that antibody reacts to antigen.
Tissue culture media is tested from each of the well, ELISA performed to identify the well with the strongest reaction to antigen.
As well as strongest Ig response, what must also be selected for when finding best hybridoma line?
Class and subclass of antibody produced!
IgG1 is the best.
This is detected using antibodies for IgG1 hinge region.
What is the rapid ELISA isotyping kit and how does it aid moAb production?
ELISA kit that simplifies process of isotyping an antibody. Only one probing-incubating step.
Substrate added after incubation and reveals antibody isotype based on whic well strips produce colour.
How are antibodies purified from tissue culture media?
Purification is done using Protein A-coated beads.
IgG binds to protein A on beads, every other serum component is eluted away in wash.
Then IgG is eluted in seperate solution with acidic solution. It is then dialysed (to neutrialise pH) and avaible for therapy.
Murine antibodies are problematic in therapy - can be immunogenic themselves in patient.
How is this overcome?
Humanised antibodies.
Variable region of Ab fused to backbome of human antibody. Can also be more specific in using only CDR region.
But best way is transgenic mice that express human Ig gene, therefore synthesise fully human Ab.
What are the three characeristics of moAbs?
highly specific - directed against one antigen epitope only.
Invariant in nature - all molecules the same.
Supply is unlimited - produced by immortal cells or by recombinant methods.
