L11 - Recombinant antibody technology Flashcards Preview

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Flashcards in L11 - Recombinant antibody technology Deck (18):
1

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

2

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

3

Cloning the antibody genes into e.coli for single chain Fv fragment allows what?

Stitching together the variable light and variable heavy chains

4

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

5

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

6

What are some other Display technologies which link genotype and phenotype?

Phage Display*
Ribosome display
DNA display
cell display
mRNA display
In vitro compartmentalization

7

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

8

What are the different types of approved therapeutic antibodies?

in order of increase human DNA

Mouse
Chimeric
Humanized
Human

9

true or false

it is difficult to get individual human domains

true

10

What are the two examples in nature of single variable domains

in sharks and camels

11

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

12

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

13

true or false

Sharks have the CDR2, same as mouse and human as well as the long CDR3

false

shark is missing CDR2

14

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

15

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

16

True or false

camels found the same solution as sharks with the IgNARs, but arrived at it independently proving the biological excellence of it

true

17

IgNAR variable regions have an interesting architecture with ____molecules able to interact with ________or________ regions

IgNAR variable regions have an interesting architecture with charged molecules able to interact with hydrophilic/hydrophobic regions

18

What can we take from these snark antibodies?

The principles of shark antibodies are trying to be combined to make a more human like synthetic antibody - company from La trobe doing this