Lecture 12

Question 1

What do antibodies do

Answer 1

They act as very specific labels for infectious material.

Labelled material is “eliminated”

Question 2

What are antibiotics

Answer 2

They are compounds produced by one species of microbe that can kill or inhibit the growth of other microbes.

Most are polyclonal are are produced by many distinct B lymphocytes- different specificity to target antigens

Question 3

Useful properties of antibodies

Answer 3

• They’re diverse (108 specificities)
• They’re specific and have high affinity (they exhibit selective toxicity)
• Stable domain structure that facilitates engineering (retains overall stable folding pattern)
• Multivalent (improved binding, cross linking can be useful)
• Effector properties (useful in some techniques)

Question 4

What does MAB efficacy depend on

Answer 4

The quality of the the interaction between hypervariable region and target regions. Downstream effects are key to efficacy and safety.

Question 5

What are antibodies generally used for

Answer 5

To label and identify molecules in complex mixtures.

Question 6

Examples of type of label and the technique used

Answer 6

Fluorescent e.g. fluorescein: immunofluorescence microscopy, FACS (fluoresence activated cell sorting)

Enzyme -> coloured product: ELISA (Enzyme - linked immunosorbent assay), immunoblotting, immunohistology

Radioisotope: Radioimmunoassay, imaging e.g. of tumours

Gold particles: immuno-electron microscopy

Sephraose: affinity purification, immunoprecipitation

Question 7

How do antibodies recognise their targets

Answer 7

Antibodies recognise epitopes on an antigen.

Antibodies can bind monovalently to single epitopes on an antigen or multivalently to repeated epitopes

Question 8

What are epitopes and the types

Answer 8

They’re shapes antibodies bind with

Linear: adjacent in sequence (non-conformational)

Discontinuous: non-adjacent (conformational)

Question 9

Immunogenecity

Answer 9

The ability of an antigen to induce an effective immune response

Question 10

Properties to consider for generating antibodies

Answer 10

• Foreigness: sequence homology between antigen and equivalent protein in recipient

-Molecular size (can link larger protein with increasing molecular weight so molecule stays in the body for longer)

• Chemical composition: aromatic groups, charged residues. Some non-covalent interactions are stronger than others

-Use of adjuvants: induce inflammation, “danger signals”- used in vaccines and producing experimental antibodies

Question 11

Advantages of polyclonal antibodies

Answer 11

• Relatively cheap, robust (may recognise partially denatured/ unfolded antigen

Question 12

Disadvantages of polyclonal antibodies

Answer 12

-Specific for multiple epitopes, need pure antigen to immunise, can be difficult to standardise- different animals respond differently

Question 13

What is cross reactivity and why is it a problem. How do we overcome this?

Answer 13

When different antigens share the same epitopes. This causes a problem when you want to have an antibody for a single epitope

We overcome this by using monoclonal antibodies

Question 14

How do you create monoclonal antibodies

Answer 14

• Immortalise b cells - fusing immortal cell line (from myeloma) with b cells

Question 15

What do unfused myeloma cells lack

Answer 15

The enzyme HGPRT

Question 16

What are the advantages of monoclonal antibodies

Answer 16

-Highly specific, they bind to a single epitope, can be standardised, pure antigen is not needed for immunisation

Question 17

What are the disadvantages of monoclonal antibodies

Answer 17

-Often conformation sensitive, expensive- requires special equipment and knowledge

Question 18

What do rodent antibodies cause

Answer 18

“Serum sickness”- severe reaction to monoclonal antibodies.

Question 19

How can antibodies be engineered

Answer 19

• Antibody chimeras: splice mouse V region genes to human c region genes. Constant regions are the most immunogenic

Hypervariable loops are mainly responsible for antigen binding

Question 20

How are humanised antibodies created

Answer 20

• Splicing human framework region genes and mouse CDR region genes aka. CDR grafting

Question 21

Whats the downside of humanised antibodies

Answer 21

May lose affinity/ specificity, framework regions are also important. Time consuming to carry out this genetic engineering

Question 22

What are some strategies for generating fully human antibodies:

Answer 22

1. Transgenic mice expressing human immunoglobulin genes. Mouse antibody genes are replaced by human antibody genes
2. Antibody gene libraries: antibody v genes are cloned from naive/ immune b cells using a suitable vector and used to make a large library.

Question 23

How to generate antibodies from gene library

Answer 23

1. Isolate mRNA from antibody producing cells e.g. blood, lymphoid tissue, bone marrow
2. Reverse transcribe mRNA. Amplify Fab or Fv cDNA or paired variable regions by PCR- can increase from small number of b cells
3. Clone and express Fab or FV cDNA in bacteria/phage (phage display)
4. Screen antibody phage display library vs solid phage antigen “panning”

Question 24

What do phagemid vectors allow

Answer 24

Phage particles to express soluble proteins in bacteria or on the surface of filamentous phage particles

Question 25

What are nanobodies

Answer 25

Single domain antibodies. The heavy chains bind antigen very well. They’re chemically very stable, easily expressed in bacteria or yeast.