antibody technology 2 Flashcards
(16 cards)
nanobodies vs antibodies
Nanobodies are much smaller
not produced in humans
produced naturally in camelids
singular domain so small
extraction of nanobodies from camelids
Inject the llama or alpaca with something (antigen)
→ This makes its immune system create special antibodies.
Take a blood sample
→ The blood contains white blood cells making nanobody-type antibodies.
Take out the useful genetic material (mRNA) from the white blood cells
→ This holds the instructions to make nanobodies.
Make DNA copies and find the nanobody genes
→ Scientists use a lab method called PCR to copy just the nanobody parts.
Put the nanobody genes into bacteria
→ Bacteria are used like tiny factories to make nanobodies.
Grow the bacteria and collect the nanobodies
→ The nanobodies are purified and ready to be used in research or medicine.
what are the 2 strategies to obtain IgG fragments whilst retaining antigen binding
- Enzymatic digestion - cleavage with pepsin or papain
- Recombinant technology -
what does enzymatic digestion of an antibocy with pepsin result in
cleavage in Fc domain = retain disulphide linked antigen binding domains Fab2
what does enzymatic digestion of an antibocy with papain result in
results in monomeric Fab domain. ie not two linked together
how are nanobodies produced via recombinant technology
Recombinant technology
- Single chain variable fragment (scFv)
- Consist of variable region only - resopinsible fot binding to antigen
- Linked with short peptide linker = single polypeptide chain
Genecoding for scFv fragment is inderted into host such as bacteria to produce scFv
encoded by one gene
how is multivalency achived from a monovalent fragment
stringing them together
benefit of multivalency
Higher affinity of nanobody to therapeutic target
altered pharmacological properties
How is dual specificity therapeutics achieved
stringing different nanobodies together to improve efficacy
why are camelid and shark antibodies used as a source of nanobodies
heavy chain only
encoded as one chain so variable heavy chain can be extracted
required humanisation
what problem with IgGs do nanobodies solve
can access recessed binding sites in drug targets through hypervariable regions with longer CDR loops
produces convex binding surface capable of recognising internal binding sites
What is multivalency
replication of multivalent binding of IgG in nanobodies
what is conjgation in respect to nanobodies
addition og peptides, lipids, proteins, toxins and radionuclotides to nanobodies or domains
how does conjugation in nanobodies achive effect as delivery vehicles
- recruitment of immune system
- anticancer therapeutics - delivery of radionuclodtides and cytotoxins
- medical imaging - nucleotide labelling for PET scanning
how can CNS penetration be achieved by conjugation of nanobodies
Hijacking of receptors that mediate transport across the BBB can be achieved by conjugation with particular ligands
