Antibody Applications

Question 1

Why do antibodies have a vast range?

Answer 1

Somatic rearrangement
Affinity maturation
Class switching

They have a high specificity and affinity

Question 2

What are the general applications of antibodies?

Answer 2

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

Question 3

What are the types of antibodies?

Answer 3

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

Question 4

How are polyclonal antibodies produced?

Answer 4

1. An animal is immunised with a purified protein and an adjuvant (a chemical that helps increase 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

Question 5

What are some uses of polyclonal antibodies?

Answer 5

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)

Question 6

What are some advantages of polyclonal antibodies?

Answer 6

Technically easy to obtain
Antibodies are against numerous epitopes - allows more effective crossing-linking/neutralisation also higher chance of cross-reactivity

Question 7

What are some disadvantages of polyclonal antibodies?

Answer 7

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

Question 8

What is a disease linked to polyclonal antibodies?

Answer 8

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

Question 9

How are monoclonal antibodies produced?

Answer 9

1. A mouse is injected with the epitopes of the antigen
2. Isolate the spleen cells that are immune cells producing antibodies
3. 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
4. The hybridomas’ are screened for production of the desired antibody
5. The antibody producing hybridomas are cloned in 96 well plates
6. Through clonal expansion have monoclonal antibodies

Question 10

What are some uses of monoclonal antibodies?

Answer 10

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

Question 11

What are some advantages of monoclonal antibodies?

Answer 11

Consistent
Limitless supply of specific reagent
More easily tested for cross-reactivity
Can optimise for application
Can be manipulated via recombinant technologies

Question 12

What are some disadvantages of monoclonal antibodies?

Answer 12

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

Question 13

What is phage display?

Answer 13

1. Rearranged variable-region gene segments are randomly combined using recombinant molecular biology techniques to yield a large phage ‘library’ of antibodies
2. Bacteriophage library is used to infected E. Coli which display the “antibody” fragment on the surfaces of the generated phage
3. Panning selects binding phage
4. Binding phage are then isolated and used to re-infect E. Coli
5. Totally human-derived antibody fragment with known sequence, ready for manipulation

Question 14

What are some antibody alternatives?

Answer 14

Variable lymphocyte receptor (VLR) genes from vertebrates consist of leucine rich repeats (LRRs)