Antigens and Antibody Technologies
Chapter 4 pp 73 - 75
Chapter 5 pp 83 - 86
(however distinction between Td and
Ti antigens will be covered later)
Appendix F for descriptions
of antibody technologies
Self-Test Questions:
Chap 4: C all
Chap 5: D all
Appendix F: in Question Bank
Antibody Fab region bound to
a sequential antigen
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Some pertinent terminology
Affinity vs Avidity
Affinity – Strength of molecular forces
Avidity – Affinity x binding valence
(# of binding sites)
Antigenicity vs Immunogenicity
Antigenicity –> Characteristic of a molecule
-- ability to trigger an immune response
Immunogenicity –> Characteristic of the host
-- ability to respond to an antigen
Influenced by:
•
•
•
•
genetics
route of administration
dosage
tolerance
“In a natural infection, invading
bacteria and viruses appear as
collections of proteins,
polysaccharides and other
macromolecules to the host’s
immune system. Within this
collection, there are many
immunogens, each inducing its own
adaptive immune response.”
P 83
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What factors influence antigenicity?
-- ability to bind to TCR and antibody
Key characteristics
Chemical complexity
Size
Foreignness
Peptides, glycoproteins, complex carbohydrates, lipids
-- not sugars, amino acids, acids, etc
What forces hold Antigen to receptor?
various non-covalent bonds
Image from Goldsby et al Immunolgy 5th ed. Fig 6.1
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Receptors do not recognize
entire antigenic molecules
Epitopes (Binding site of Ab is called the “paratope”)
Sequential (linear) (B-cells and T-cells)
Conformational (B-cells only)
-- destroyed by denaturing
Sperm whale myoglobin sequential epitopes
CHIPS protein of S. aureus: inhibits leukocyte chemotaxis by blocking C5a receptor
(Gustafsson, et al. 2009. BMC Immunol. 10: 1-13.)
Antibody models
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“Haptens” are a special case
BSA
Important in the study of AB/AG binding
Mice injected with
Antibodies formed
DNP
(Not to scale)
Hapten (DNP)
only
None
Protein (BSA)
only
Anti-BSA
BSA-DNP
Anti-DNP (major)
Anti BSA (minor)
“Adjuvants” stimulate immunogenicity
Provide danger signals
Freund’s -- /mycobacterium/oil immersion
various TLR ligands
Create aggregates/deposits/slow release
Alum (aluminium hydroxide gel)
– in many vaccines
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How to prepare antibodies to a single epitope
Monoclonal antibodies
Monoclonals have same AG-binding specificity
Myelomas
Hybridomas
Uses:
Research
Cancer detection
Drug testing
HLA testing
on & on & on…
McGraw-Hill
Monoclonal antibodies
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Antibodies have many uses:
analytical, preparative, diagnostic and therapeutic
Some analytical methodologies…
Elisa (Enzyme-linked Immunosorbent Assay)
Indirect: quantifying antibodies
e.g.,: titer of anti-HIV antibodies in serum
1O antibody (in serum)
2O antibody (enzyme-linked)
chromogen
Sandwich: quantifying antigens
(skip ‘Competitive’)
-- e.g., amount of GP120
(HIV protein) in serum
Rows: different patients
Columns: different amounts of
serum (2X dilution series)
Quantifying results
-- use “plate reader”
-- “titer”
Sumanas
Elisa
Which patient has the highest
titer?
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Western Blotting
To detect proteins in
electrophoresed samples
1. Extract proteins
2. Electrophorese
3. Electroblot to nitrocellulose
4. Probe with 1O & 2O Abs
why use 2O ab?
why use enzyme?
5. Stain
Mouse melanoma tissue
electrophoresed and probed
with anti-actin antibodies
A: molecular Wt standards
B: stained gel
C: probed nitrocellulose
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Immunohistology
Immunofluorescence detection of
Herpes Simplex Virus I
To look for antigens in histological sections
Enzyme-linked antibodies (Immunohistochemistry)
or Immunofluorescence
indirect is more sensitive… why?
Hodgkin’s disease: immunohistochemical
staining for Epstein-Barr virus
© 2002 Novocastra Laboratories Ltd.
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Some preparative techniques
Immunoprecipitation
Affinity Chromatography
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