Antigens and Technologies 1

Antigens and Antibody TechnologiesChapter 4 pp 73 - 75Chapter 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 allChap 5: D allAppendix F: in Question Bank

Antibody Fab region bound to a sequential antigen

Antigens and Technologies 2

Some pertinent terminology

Affinity vs AvidityAffinity – Strength of molecular forces

Avidity – Affinity x binding valence (# of binding sites)

Antigenicity vs ImmunogenicityAntigenicity –> Characteristic of a molecule

-- ability to trigger an immune response

Immunogenicity –> Characteristic of the host-- ability to respond to an antigenInfluenced 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

What factors influence antigenicity?-- ability to bind to TCR and antibody

Key characteristicsChemical 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

Antigens and Technologies 4

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

Antibody models

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.)

Antigens and Technologies 5

“Haptens” are a special case

Important in the study of AB/AG binding

“Adjuvants” stimulate immunogenicity

Provide danger signalsFreund’s -- /mycobacterium/oil immersionvarious TLR ligands

Create aggregates/deposits/slow releaseAlum (aluminium hydroxide gel)

– in many vaccines

BSA

DNP(Not to scale)

Mice injected with Antibodies formed

Hapten (DNP) only

None

Protein (BSA) only

Anti-BSA

BSA-DNP Anti-DNP (major)

Anti BSA (minor)

Antigens and Technologies 6

How to prepare antibodies to a single epitope

Monoclonal antibodies

Monoclonals have same AG-binding specificity

Myelomas

Hybridomas

Uses:ResearchCancer detectionDrug testingHLA testingon & on & on…

McGraw-HillMonoclonal antibodies

Antigens and Technologies 7

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?

SumanasElisa

Antibodies have many uses: analytical, preparative, diagnostic and therapeutic

Antigens and Technologies 8

Western Blotting

To detect proteins in electrophoresed samples

1. Extract proteins

2. Electrophorese

3. Electroblot to nitrocellulose

4. Probe with 1O & 2O Abswhy use 2O ab?why use enzyme?

5. Stain

Mouse melanoma tissue electrophoresed and probed with anti-actin antibodiesA: molecular Wt standardsB: stained gelC: probed nitrocellulose

Antigens and Technologies 9

Immunohistology

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

Hodgkin’s disease: immunohistochemical staining for Epstein-Barr virus

© 2002 Novocastra Laboratories Ltd.

Antigens and Technologies 10

Some preparative techniquesImmunoprecipitation

Affinity Chromatography