Antibody Applications Flashcards
Why do antibodies have a vast range?
Somatic rearrangement
Affinity maturation
Class switching
They have a high specificity and affinity, stability and their effector Fc region provides an ideal mechanism for binding and eliminating antigens
What are the general applications of antibodies?
Physiological role- fighting disease
Research- detecting “invisible substances”, blocking function, etc
Diagnosis- detecting disease markers, blood types, pregnancy hormones
Therapeutics- anti-venom, passive vaccination, immunotherapy
Industrial uses- abzymes, detectors
What are the types of antibodies?
Polyclonal and monoclonal antibodies
Polyclonal antibodies are produced by many different B cells responding to the same antigen
They contain a mixture of antibodies targeting different parts of the protein
Monoclonal antibodies are produced by a population of identical clonal - B cells
They are homogeneous and all recognise the same epitope
How are polyclonal antibodies produced?
Production:
1. An animal is immunised with a purified protein and an adjuvant (a chemical that helps increase local immune response)
Adjuvant needed to elicit local immune response
If using a small hapten requires a carrier protein to elicit an immune response
2. Whole sera (liquid fraction of clotted blood) is collected and purified
Multiple boosts generates higher titre, higher affinity antibodies
Class switching and hypermutation generates high affinity IgG immunoglobulin
What are some uses of polyclonal antibodies?
Physiological response
Research (ELISA, FACS, Biopsy staining, functional blocking, western blots)
Diagnosis (ELISA, FACS, Biopsy staining)
Anti-venom (other complex mixture of poisons), passive immunity (rabies), prevention of Hemolytic Disease of the Newborn (anti-D)
What are some advantages of polyclonal antibodies?
Technically easy to obtain
Antibodies are against numerous epitopes - allows more effective crossing-linking/neutralisation also higher chance of cross-reactivity
Examples- passive protect against rabies, neutralisation of snake venom, Anti-D
What are some disadvantages of polyclonal antibodies?
Due to large complex mixture of different antibodies its hard to manipulate via recombinant means
Every antisera preparation differs in specificity, average affinity, cross-reactive specificities, etc.
Not optimised for application
Supply is limited
Disadvantages can be circumvented via monoclonal antibodies
What is a disease linked to polyclonal antibodies?
Hemolytic Disease of the Newborn - Rhesus disease
Rh- mother is sensitised to Rh+ foetus during first pregnancy/birth
Production of IgG against Rh by memory B cells during second pregnancy destroys foetus (IgG is transferred across placenta)
Therapeutic anti-Rh Ab before birth prevents memory response
How are monoclonal antibodies produced?
- A mouse is injected with the epitopes of the antigen
- Isolate the spleen cells that are immune cells producing antibodies
- Fuse these cells with myeloma cells (tumour cells) to form hybridomas
To fuse the myeloma cells with the antibody producing cells we add polyethylene glycol and HGPRT (hypoxanthine guanine phosphoribosyl transferase - The hybridomas’ are screened for production of the desired antibody
- The antibody producing hybridomas are cloned in 96 well plates
- Through clonal expansion have monoclonal antibodies
What are some uses of monoclonal antibodies?
Research - tissue staining, western blot
Diagnostic/prognostic indicators - pregnancy test
(hCG - Human chorionic gonadotropin is detected/bound)
Therapeutic antibodies - Rheumatoid arthritis, Crohn’s disease, Infliximab, Efalizumab, Rituximab and Herceptin
Abzymes
What are some advantages of monoclonal antibodies?
Consistent Limitless supply of specific reagent More easily tested for cross-reactivity Can optimise for application Can be manipulated via recombinant technologies
What are some disadvantages of monoclonal antibodies?
Technically more difficult and time consuming, less likely to neutralise/cross react
Repeat doses are often immunogenic, resulting in neutralising anti-antibody antibodies
Side-effects mediated via Fc region, eg complement activation, FcR activation
Using mice leads to us not being able to generate antibodies against very homologous (similar) proteins
Solution - In vitro generation, phage display technology
What is phage display?
- Rearranged variable-region gene segments are randomly combined using recombinant molecular biology techniques to yield a large phage ‘library’ of antibodies
- Bacteriophage library is used to infected E. Coli which display the “antibody” fragment on the surfaces of the generated phage
- Panning selects binding phage
- Binding phage are then isolated and used to re-infect E. Coli
- Totally human-derived antibody fragment with known sequence, ready for manipulation
What are some antibody alternatives?
Variable lymphocyte receptor (VLR) genes from vertebrates consist of leucine rich repeats (LRRs)
