antibody engineering

Question 1

where are igs present

Answer 1

plasma, secretion and lymphatics

Question 2

what do igs recognise

Answer 2

antigen and trigger effector fucntion

Question 3

where do antigens bind igs

Answer 3

tip of the fab region

Question 4

what is an n linked ogliosaccharide

Answer 4

ogliosaccaride attached to asparagine - helps stabilise structure

Question 5

what are the 2 functions of antibodies

Answer 5

recognition - mediated by fab

effector - mediated by fc with effector molecules

Question 6

what are hypervariable loops

Answer 6

variable domains in light and ehavy chains which vary between diff igs

Question 7

what does cdr stand for

Answer 7

complementary determining region

Question 8

what is the cdr

Answer 8

antigen binding site - close lying loops at one end of v domain

Question 9

how are antigen binding sites formed

Answer 9

Vh and Vl domains are paired and 6 CDRs create the binding site

Question 10

what is the structure of the antigen binding site

Answer 10

3d complementary structure, antigenic epitope provides high specificity and high affinity

Question 11

how many sub classes are there of IgG and where do they vary

Answer 11

4 - heavy chains differ they are encoded by separate gene

difference particularly seen in the hinge region

Question 12

describe the classical pathway of complement activation

Answer 12

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

Question 13

what is the diff types of human FcyR

Answer 13

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

Question 14

where does FcyR interact with igG

Answer 14

amino acid sequences within the igG subclasses in the binding site

Question 15

rank diff igG in order of ability to bind and trigger FcyR

Answer 15

1 and 3 most effective
2 is ineffective
4 is + and -

Question 16

4 function of FcyR receptor

Answer 16

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

Question 17

monoclonal abs

Answer 17

homogenous prep of antibody
consistent quality and characteristic
single ig class
defined affinity of antigen target
all are identicle

Question 18

polyclonal abs

Answer 18

in vivo produced in response to foreign materials
mix of many abs of varying specificity
diff ig classes
diff affinities for antigen target

Question 19

how does hybridoma technology work and what does it produce

Answer 19

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

Question 20

3 problems that are caused by using mAbs in humans

Answer 20

1. immunogenic response - HAMA - human anti mouse antibody
   - repetitive dose may result in serum sickness
2. relatively quick clearance of mouse mabs in humans
3. unconjugated mouse mabs are poor at recruiting human effector systems

Question 21

what does cdr grafting create

Answer 21

a humanised antibody where only hypervariable loops are mouse

Question 22

how to avoid using animals to generate mAbs

Answer 22

immortalise memory b cells
phage display of ab libraries
use transgenic mice carrying human ig genes

Question 23

how does human b cell immortalisation work

Answer 23

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

Question 24

how to gain human mAbs from early antibody secreting cells

Answer 24

1. individual is given booster immunisation
2. seven days later peripheral lymphocytes are sorted
3. igV regions amplified by single cell RT-PCR
4. subclone and screen mAbs

Question 25

how are human ig variable region repertoire libraries made

Answer 25

1. primer design
2. 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
3. generate Vl in the same manner
4. randomly combine vh and vl frags to give a large library of different combos
5. diff vh vl combos displayed on surfaces of phage [articles which = phage display

Question 26

hoe can transgenic mice produce human abs

Answer 26

1 inactivate mouse ab genes

2. replace with unrearranged human abs genes for both heavy and light chains
3. immunise with antibody of interest
4. generate select hybridomas in a conventional way
5. hybridomas produce fully human abs