L11 - Recombinant antibody technology Flashcards
Describe the features of the antigen binding site
Structural plasticity;
- hypervariability in the loop region:complementarity-determining regions = CDRs
- Variability of peptide length and side chains
- backbone conformational variability
Coformational flexibility:
- disordered loops in the absence of antigen
- induced fit upon complex formation
What is the difference between classical and modern isolation of antibodies?
Classical: mouse model was immunised and b cells isolated, then screen for specificity
Modern
- blood from individuals, isolate lymphocytes
- extract the genes which encode antibodies
- clone these genes
provides a linking of genotype and phenotype
- can also reconstitute in various sequences, can take out constant region, AB/FB fragments
Cloning the antibody genes into e.coli for single chain Fv fragment allows what?
Stitching together the variable light and variable heavy chains
In Bacteriophage Display, the antibody _____ is expressed, shunted out to surface of bacteriophage produced in single ___ cell. You therefore get the ______ coming along in the phage, expressed as one ______ antibody
In Bacteriophage Display, the antibody vector is expressed, shunted out to surface of bacteriophage produced in single e.`coli cell. You therefore get the genotype coming along in the phage, expressed as one individual antibody
How can you then select for your antibodies in bacteriophage display?
There is a huge population of phages with antibodies displayed on surface - need to isolate the right ones
- immobilise the phage displaying the antibody of interest on solid surface with antigens
- then wash, amplify and enrich
- then extract the genes and sequence to see what it is
What are some other Display technologies which link genotype and phenotype?
Phage Display* Ribosome display DNA display cell display mRNA display In vitro compartmentalization
Ribosome Display utilises ___ _ _______ and selection of ______affinity complexes which emulates the _____ antibody system.
Once the RNA is recovered it is cloned, _______ , _____ and _____ . This process is repeats to produce an optimised product
Ribosome Display utilises in-vitro translation and selection of highest affinity complexes which emulates the human antibody system.
Once the RNA is recovered it is cloned, sequenced, expressed and screened. This process is repeats to produce an optimised product
What are the different types of approved therapeutic antibodies?
in order of increase human DNA
Mouse
Chimeric
Humanized
Human
true or false
it is difficult to get individual human domains
true
What are the two examples in nature of single variable domains
in sharks and camels
What is the differences in shark vs. human antibodies?
similar antibodies but sharks have a few single chain antibodies called IgNARs
have to get their diversity from the 3CDR (complementarity-determining regions) loops - they have extra long CDR regions that can recognise antigens
How do you generate a shark immune library?
blood sample from shark
isolate the lymphocytes
mRNA preparation made from lymphocytes
cDNA copies corresponding to mRNA from antibody producing cells
true or false
Sharks have the CDR2, same as mouse and human as well as the long CDR3
false
shark is missing CDR2
What is the advantages of the IgNAR with the long CDR3 region?
the long cleft antibody can penetrate areas otherwise conserved epitopes
Though IgNAR is single chain, with a long CDR3 it makes up for less variable region by being able to penetrate with a long CDR3
true or false
CDr3 can also do induced fit binding where there is a conformational change which stabilises and fits better with the antigen
true
