Antibody:antigen interactions

Question 0

Hapten

Answer 0

Small molecule
Binds to antibody but too small to create a response (vs immunogen)
Carrier proteins bind -> endocytosed -> induce T cell response

Ex:
Penicillin = hapten -> binds host protein -> antipenicillin Ig’s
Vaccines - design to recruit carrier proteins -> T cell -> follicular response (bypasses immature marginal zone B cells)

Question 1

Antibody:antigen binding

Answer 1

All non-covalent interactions
Distance is key to strength
Number of interactions (sum determines strength)
Poor “fit” can repel (usu steric)

Affinity maturation is through alterations in residues or shape to maximize binding affinity/time

Question 2

Physical antigen:antibody formations

Answer 2

Agglutination - insoluble antigen crosslinked by antibody ->
- used for diagnostics - ex Coombs test
- only small amounts of antibody needed (vs precipitation)
Precipitation - soluble antigen and antibody form “lattice”
- occurs most at matched concentrations = equivalence zone (everything bound to each other)
- can get false test results - ex antigen overwhelms dye available
- most common with carbs (repeated epitopes) or protein + multiclonal Ab’s

Question 3

Cross-reactivity

Answer 3

Epitopes on different structures are identical or similar enough to bind same antibody

• > innate immunity from widely reactive CD5/marginal zone antibodies
• > blood type reactions - A, B are similar to many pathogens
• > autoimmune
• > rheumatic fever - Strep pyogenes “M” protein is similar to heart, brain, joints

Question 4

Coombs test

Answer 4

Agglutination of RBC’s if Ig’s are present in serum

Direct - Ig’s bound to RBC’s -> add anti-Ig Ig (from goat) -> complex agglutinizes
- ex patient has hemolytic anemia
Indirect - Ig’s in serum -> add to exogenous RBC’s -> add anti-Ig Ig -> agglutinization of complex
- ex test whether patient’s Ig’s bind to transfusion cells

Question 5

Principles of antibody testing

Answer 5

Highly sensitive due to affinity
Highly specific
Reproducible - via monoclonal antibodies

Use to confirm infection, immunity, or allergy (IgE)
Can track changes over time (ex AIDS patient)
Can measure either antigen or antibody

Question 6

Monoclonal antibodies

Answer 6

Fuse immortal B-cell with antibody producing cell (ie from vaccinated animal)
Select antibody products for specificity
-> immortal production of monoclonal Ab

• > reproducible diagnostics
• > treatments (ex anti-CD20 or anti-TNF-a for autoimmune)
• > passive immunity (anti-RSV for premature infants)

Question 7

ELISA

Answer 7

Enzyme-linked immunoabsorbant assay

Insoluble antibodies or antigens on plate
Incubate sample -> binding if correct ligand
Secondary antibody (with label - radio, fluorescent or enzyme)

Can get concentrations if you have standardized curve

Question 8

FACS

Answer 8

Fluorescence activated cell sorting

Use fluorescent antibodies vs membrane components (or cytoplasmic with diffusible antibody)
Can track multiple characteristics - ex CD3 vs CD8 to identify cell types and charracteristics

Question 9

Immunohistochemistry

Answer 9

Histology specimens (vs FACS for blood cells)

Label samples with enzyme-linked mAb’s
Detect color change with enzyme substrate