1. Antibody Fab region bound to a sequential antigen
Antigens and Antibody Technologies
Chapter 4 pp
Chapter 5 pp
(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
Antigens and Technologies

2. Antigens and Technologies
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
Antigens and Technologies

3. Antigens and Technologies
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
What factors influence antigenicity of molecules?
Complexicity
glycogen vs protein vs glycoprotein
Size
-- Macromolecules
-- haptens a special case
Foreigness
-- most cells that might potentially bind to self-antigens are eliminated
during cell maturation
-- creates a condition known as “tolerance”
-- acquired immune system recognizes non-self antigens
-- failure of tolerance leads to autoimmune reactions
MHC only binds short peptide fragments
-- 8 – 15 AA in length
Image from Goldsby et al Immunolgy 5th ed. Fig 6.1
Antigens and Technologies

4. Antigens and Technologies
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 BMC Immunol. 10: 1-13.)
Antibody models
Antigens and Technologies

5. Antigens and Technologies
BSA
“Haptens” are a special case
Important in the study of AB/AG binding
Mice injected with
Antibodies formed
Hapten (DNP) only
None
Protein (BSA) only
Anti-BSA
BSA-DNP
Anti-DNP (major)
Anti BSA (minor)
DNP
(Not to scale)
“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
Antigens and Technologies

6. Monoclonal antibodies
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
Antigens and Technologies

7. Antigens and Technologies
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
Quantifying results
-- use “plate reader”
-- “titer”
Rows: different patients
Columns: different amounts of serum (2X dilution series)
Which patient has the highest titer?
Sumanas
Elisa
Antigens and Technologies

8. Antigens and Technologies
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
Antigens and Technologies

9. Immunohistology Immunofluorescence detection of
To look for antigens in histological sections
Enzyme-linked antibodies (Immunohistochemistry)
or Immunofluorescence
indirect is more sensitive… why?
Immunofluorescence detection of
Herpes Simplex Virus I
Using antibodies in disease diagnosis.
Billion dollar industry [ bring Vector / Oncogene catalogs ]
Can be used to detect the presence of pathogens, of cells with specific characteristics.
Primary antibodies are Abs that bind to specific antigenes
-- monoclonals & polyclonals
-- against antigens of pathogens, oncogenes, tumor antigens, etc
-- typically raised in animals
Secondary Abs are antibodies against other antibodies
-- used to detect binding of a primary antibody
-- detection system may be fluorescence or enzymatic
Immunohistochemistry
Hodgkin’s disease: immunohistochemical
staining for Epstein-Barr virus
© 2002 Novocastra Laboratories Ltd.
Antigens and Technologies

10. Antigens and Technologies
Some preparative techniques
Immunoprecipitation
Affinity Chromatography
Antigens and Technologies