Lecture 12 Flashcards
What do antibodies do
They act as very specific labels for infectious material.
Labelled material is “eliminated”
What are antibiotics
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
Useful properties of antibodies
- 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)
What does MAB efficacy depend on
The quality of the the interaction between hypervariable region and target regions. Downstream effects are key to efficacy and safety.
What are antibodies generally used for
To label and identify molecules in complex mixtures.
Examples of type of label and the technique used
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
How do antibodies recognise their targets
Antibodies recognise epitopes on an antigen.
Antibodies can bind monovalently to single epitopes on an antigen or multivalently to repeated epitopes
What are epitopes and the types
They’re shapes antibodies bind with
Linear: adjacent in sequence (non-conformational)
Discontinuous: non-adjacent (conformational)
Immunogenecity
The ability of an antigen to induce an effective immune response
Properties to consider for generating antibodies
- 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
Advantages of polyclonal antibodies
- Relatively cheap, robust (may recognise partially denatured/ unfolded antigen
Disadvantages of polyclonal antibodies
-Specific for multiple epitopes, need pure antigen to immunise, can be difficult to standardise- different animals respond differently
What is cross reactivity and why is it a problem. How do we overcome this?
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
How do you create monoclonal antibodies
- Immortalise b cells - fusing immortal cell line (from myeloma) with b cells
What do unfused myeloma cells lack
The enzyme HGPRT
What are the advantages of monoclonal antibodies
-Highly specific, they bind to a single epitope, can be standardised, pure antigen is not needed for immunisation
What are the disadvantages of monoclonal antibodies
-Often conformation sensitive, expensive- requires special equipment and knowledge
What do rodent antibodies cause
“Serum sickness”- severe reaction to monoclonal antibodies.
How can antibodies be engineered
- 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
How are humanised antibodies created
- Splicing human framework region genes and mouse CDR region genes aka. CDR grafting
Whats the downside of humanised antibodies
May lose affinity/ specificity, framework regions are also important. Time consuming to carry out this genetic engineering
What are some strategies for generating fully human antibodies:
- Transgenic mice expressing human immunoglobulin genes. Mouse antibody genes are replaced by human antibody genes
- Antibody gene libraries: antibody v genes are cloned from naive/ immune b cells using a suitable vector and used to make a large library.
How to generate antibodies from gene library
- Isolate mRNA from antibody producing cells e.g. blood, lymphoid tissue, bone marrow
- Reverse transcribe mRNA. Amplify Fab or Fv cDNA or paired variable regions by PCR- can increase from small number of b cells
- Clone and express Fab or FV cDNA in bacteria/phage (phage display)
- Screen antibody phage display library vs solid phage antigen “panning”
What do phagemid vectors allow
Phage particles to express soluble proteins in bacteria or on the surface of filamentous phage particles
