antibody engineering Flashcards
where are igs present
plasma, secretion and lymphatics
what do igs recognise
antigen and trigger effector fucntion
where do antigens bind igs
tip of the fab region
what is an n linked ogliosaccharide
ogliosaccaride attached to asparagine - helps stabilise structure
what are the 2 functions of antibodies
recognition - mediated by fab
effector - mediated by fc with effector molecules
what are hypervariable loops
variable domains in light and ehavy chains which vary between diff igs
what does cdr stand for
complementary determining region
what is the cdr
antigen binding site - close lying loops at one end of v domain
how are antigen binding sites formed
Vh and Vl domains are paired and 6 CDRs create the binding site
what is the structure of the antigen binding site
3d complementary structure, antigenic epitope provides high specificity and high affinity
how many sub classes are there of IgG and where do they vary
4 - heavy chains differ they are encoded by separate gene
difference particularly seen in the hinge region
describe the classical pathway of complement activation
activated by 2 or more igG
C1q is the 1st component - binding of globular heads to the CH2 domain
motif for igG is on the upper part of C1q
what is the diff types of human FcyR
CD64/ FcyR1 - highest binding affinity, possess gamma chain
CD32/ FcyR2A/B - activation and inhibition effect
CD16/ FcyR3 A/B - one has y chain that is transmembrane and the other doesnt and binds to plasma membrane
where does FcyR interact with igG
amino acid sequences within the igG subclasses in the binding site
rank diff igG in order of ability to bind and trigger FcyR
1 and 3 most effective
2 is ineffective
4 is + and -
4 function of FcyR receptor
phagocytosis - more effective when target is coated/opsonised with igG
antibody dependent cell-mediated cytotoxicity (ADCC)
respiratory burst - release of activated oxygen species
release of inflam mediators and enzymes
monoclonal abs
homogenous prep of antibody consistent quality and characteristic single ig class defined affinity of antigen target all are identicle
polyclonal abs
in vivo produced in response to foreign materials
mix of many abs of varying specificity
diff ig classes
diff affinities for antigen target
how does hybridoma technology work and what does it produce
produces monoclonal abs
spleen from a mouse immunised with antigen of interest is fused with a myeloma cells using polyethylene glycol fusion to produce a hybridoma cell
after fusion - cells are plated in selection medium
culture kills off all unfused lymphocytes
selected hybridoma cells secrete antigen-specific mouse mono-clonal antibodies
3 problems that are caused by using mAbs in humans
- immunogenic response - HAMA - human anti mouse antibody
- repetitive dose may result in serum sickness - relatively quick clearance of mouse mabs in humans
- unconjugated mouse mabs are poor at recruiting human effector systems
what does cdr grafting create
a humanised antibody where only hypervariable loops are mouse
how to avoid using animals to generate mAbs
immortalise memory b cells
phage display of ab libraries
use transgenic mice carrying human ig genes
how does human b cell immortalisation work
isolation of memory b cell from peripheral blood of patients who have recovered from sars-cov and H5N1 avian flu infections
add in epstein barr virus
increase efficiency of EDV B cell immortalisation by adding CpG oglionucleotides and tlr9 agonist
leads to immortalisation then screening
how to gain human mAbs from early antibody secreting cells
- individual is given booster immunisation
- seven days later peripheral lymphocytes are sorted
- igV regions amplified by single cell RT-PCR
- subclone and screen mAbs
how are human ig variable region repertoire libraries made
- primer design
- take mRNA rom human b lymphocytes
- reverse transcribe to give cDNA
- use cDNA as a template for pcr using primers to generate millions of Vh fragments - generate Vl in the same manner
- randomly combine vh and vl frags to give a large library of different combos
- diff vh vl combos displayed on surfaces of phage [articles which = phage display
hoe can transgenic mice produce human abs
1 inactivate mouse ab genes
- replace with unrearranged human abs genes for both heavy and light chains
- immunise with antibody of interest
- generate select hybridomas in a conventional way
- hybridomas produce fully human abs
