L8: Antibodies and Immunoassays Flashcards
Define immunity and list immune cell types.
• Composed of effector cells and molecules – Recognition – Effector functions – Regulation – Memory Immune cells • Leukocytes (or White Blood Cells) – Myeloid (bone marrow) or Lymphoid – Granulocytes or Agranulocytes
See slide 6 for hematopoesis
List immune molecules integral to cellular response.
• Cytokine: secreted proteins that affect the behaviour of
neighbouring cells
• Chemokine: secreted proteins that act as
chemoattractants (i.e. attract neutrophils out of the
blood and into infected tissue)
• Complement: a group of over 30 proteins involved in
the killing of pathogens
Describe the basics of innate and adaptive immune response.
Innate
• Immediately available
• Non-specific
• Lacks ‘memory’
• Innate defences control pathogens that carry certain
molecular patterns or that induce other non-specific
defences
‒ Pathogen-associated molecular patterns [PAMPs])
• Toll-like receptors (TLRs)
• Pattern recognition receptors (PRRs)
Adaptive
• Has to be developed
• Response to a specific infection
• Has ‘immunologic memory’; can confer lifetime
protection
• Adaptive immunity is required to recognize and battle the
wide array of pathogens present
Each consists of a humoral and cellular component (humor = cell-free bodily fluid) (see slide 9 for time response)
Provide examples of PAMPs.
Bacterial Lipopolysaccharides (LPS) (a.k.a. Endotoxins) ‒ Prototypical PAMPs ‒ Outer membranes of Gram-negative bacteria • Other PAMPs include: ‒ Bacterial flagellin ‒ Lipoteichoic acid (Gram+) ‒ Petidoglycan (Gram+) ‒ Chitin (fungi) ‒ dsRNA (viruses)
Define antigen and antibodies.
Antigen
• A molecule, or part thereof, that is specifically recognized
by the recognition proteins of lymphocytes
‒ B-cells have membrane bound B-cell receptor (BCR)
Antibody
• Produced by Plasma cells (terminally differentiated B-cells)
• Bind specifically to a pathogen, or pathogen product, that
produced the immune response
• Recruit other cells/molecules to destroy the pathogen
Describe three roles of antibodies.
- Neutralization
- Opsonization
- Compliment Activation
(14-16)
Describe the structure of an antibody.
An antibody, also known as an immunoglobulin, is a Y-shaped structure which consists of four polypeptides — two heavy chains and two light chains. This structure allows antibody molecules to carry out their dual functions: antigen binding and biological activity mediation.
Variable region
• Defines antigen specificity and binds to the antigen
Constant region
• Determines the isotype/allotype
• Recruits effector molecules
Define antibody isotype and subclasses.
Isotypes • Five heavy chain (g, a, μ, d, e) and two light chain (k, l) • Heavy chain isotype determines the class of Ig
Subclasses
• Encoded by separate genes
• Four IgG (1-4)
‒ IgG1 is most abundant and makes up ~70% of IgG
• Two IgA (1-2)
‒ IgA2 is minor component in serum but major in
secretions
(see slide 22)
Define allotype and idiotype.
Allotype
• Allelic variations found in the constant region
‒ Can be antigenic determinants
Idiotype
• Differences due to rearrangement of the variable regions
‒ Specific to each Ig molecule
Anti-idiotypic antibodies
Epitope is part of the variable region of other antibodies
Two types:
• Alpha
‒ Recognizes epitopes in variable region
‒ Insensitive to Ab-Ag interaction of target antibody
• Beta
‒ Recognizes the paratope
‒ Sensitive to Ag-Ab interaction of target antibody
(does not bind if Ag is bound)
Describe antibody fragments.
cleaved into functional fragments
• Fragment antigen binding (Fab)
• Fragment crystallizable (Fc)
Define antigen and describe the difference between immunogen and hapten.
Any substance that can bind to an antibody
Immunogen
• Can induce an adaptive immune response
Hapten
• A small molecule that is unable to elicit an immune
response on its own
‒ When coupled to a carrier, it is able to do so
‒ The carrier does not have to be immunogenic
Describe immunogenic factors of antigen.
see slide 27
Define epitope.
Epitope (Antigenic Determinant)
• The part of the antigen that the antibody binds to
Epitopes on proteins can be:
• Continuous/linear
‒ Made up of a single segment of a polypeptide chain
• Discontinuous/conformational
‒ Composed of amino acids from different parts of the polypeptide chain that are brought together by protein folding
Define paratope
Paratope
• The part of the antibody that binds the epitope
Each variable region has hyper-variable regions which are responsible for antigen recognition and binding
Describe binding of antibody to antigen reaction and the factors impacting the strength of the reaction. What is the Scatchard model?
Binding of Ab to Ag is reversible and obeys the law of mass action:
• The rate of the reaction is proportional to the concentration of the reactants
The strength of the interaction is based on the ‘fit’ of the epitope and paratope
Non-covalent forces:
• Electrostatic (1/d2)
• Hydrogen bonds (stronger at colder temp)
• Van der Waals forces (1/d6)
• Hydrophobic forces (provide up to 50% strength in interaction)
Real Ab-Ag interactions are complicated
The Scatchard model is used for the quantitative description of the multiple
equilibria occurring
• Assumes independent and noninteracting binding sites
Describe affinity.
The strength of binding between a single epitope - paratope complex
‒ Determined by the equilibrium constant of the association
reaction between antibody (Ab) and antigen (Ag)
(see equation on Slide 34)
Describe avidity.
Avidity (a.k.a. functional affinity)
• The strength of interaction between an antigen with multiple epitopes to an antibody with multiple paratopes
Multiple interactions are involved making accurate modeling of the interaction difficult
• Avidity can be many times greater than the sum of affinities
‒ When multiple interactions are present (i.e. IgM with a bacterium) the probability that every interaction will dissociate simultaneously is exceedingly small)
(see slide 39 for equation)
What factors include Ag-Ab interactions in vitro?
• Synergistic effects (Avidity)
• pH, Temperature and Ionic strength of the solution
‒ Cationic salts inhibit binding with cationic haptens and anionic
salts inhibit binding with anionic haptens
• Cs+ > Rb+ >NH4+ > K+ > Na+ > Li+
• SCN- > NO3- > I- > Br- > Cl- > F-
• Polymer effect
‒ Soluble linear high MW polymers increase the effective
concentration of Ag and Ab and increase the rate of complex
formation (i.e. PEG 6000)
• Relative [Ab] and [Ag]
• Higher concentrations lead to increase rate of reaction
Define cross vs shared reactivity.
Cross reactivity
• Binding of structurally different epitopes by the same antibody (different affinities)
Shared reactivity
• Common epitopes on different antigens
List the antibody requirements for immunoassays.
- Reproducible
- Specific
- High Affinity
- Stable
- Large-scale production
- Readily purified
Describe antibody clonality.
- Antibodies are produced plasma cells (terminally differentiated B-cells)
- Each B-cell encodes a single unique immunoglobulin
- Plasma cells originating from the same B-cell produce identical antibodies and are ‘clones’ of each other
Monoclonal antibodies
‒ Generated from a single plasma cell clone
Polyclonal antibodies
‒ Generated from multiple plasma cell clones
Describe polyclonal antibodies and their characteristics.
‒ Generated from multiple plasma cell clones
• Classical immune response to an antigen
• An animal is immunized and bled and the resulting antiserum is used as the reagent
‒ Rabbits, sheep, goats, donkeys
• The larger the animal, the more serum that can be collected but the harder they are to maintain
Polyclonal antisera:
• Reacts with several epitopes on the antigen
• Includes antibodies of varying specificities and affinities
• Includes antibodies of varying classes and subclasses
• Will include a wide variety of irrelevant antibodies (unless purified and/or animal is kept in sterile conditions)
• Will vary in antibody composition each time it is collected from an animal
• Have a finite supply (depends on lifespan of the animal)
• Are relatively inexpensive to produce
Describe monoclonal antibodies and their characteristics.
• Artificial way to produce antibodies from a single clone of plasma cells
• B-cells are cultured from the spleen of an immunized animal, immortalized, isolated and used to produce antibodies
‒ Mice, rabbits, humans
‒ Size of the animal does not matter (cells culture)
- React with a single epitope on an antigen
- Include a single antibody of a defined class and subclass
- Include a single antibody of a defined specificity and affinity
- Does not include any irrelevant antibodies
- Are completely reproducible
- Have an infinite supply available
- Are expensive to produce
In antibody production, how do you choose choice of animal?
• Animals used need to be healthy
• Gender does not matter
‒ Except when sharing cages (i.e. male mice are more aggressive; injuries may lead to infections)
• Older animals generally have poorer immunologic response to injected antigens
• The species used will depend on the type and amount of antibody you need
Polyclonal
• Rabbits are most common (low maintenance costs)
• Larger animals are generally used for secondary Ab
production with broad uses (e.g. goat anti-mouse IgG)
• Animals are genetically outbred with respect to MHC antigens; each reacts differently to an antigen
Monoclonal
• Usually Mouse (most popular), Rat or Human
• Require inbred animals in order to produce hybridomas
• Human hybridomas may be chimeric (rodent-human)
In antibody production, what are common considerations in immunization procedures?
Size of the Antigen
• All natural B cell antigens are large and so antigens must be presented in this context
• Small molecules that cannot elicit an immune response (haptens) need to be coupled to a carrier
‒ BSA, ovalbumin, or keyhole limpet hemocyanin (KLH)
Chemical nature of the Antigen
• Carbohydrates elicit a large B cell response
• Proteins: lower homology à larger response
• Nucleic acids and lipids are poor antigens on their own
Adjuvants
• Used as an immunopotentiator
• Slows the release of antigens (mimicking natural
antigens) thus increases immune response
• Addition of bacteria to adjuvants were thought to increase the immune response, but may actually divert the response away from your antigen
‒ Most common is complete Freund’s adjuvant (CFA) which is suspension of killed mycobacteria in mineral oil. Incomplete Freund’s adjuvant (IFA) is the mineral oil without bacteria
Route of immunization • Subcutaneous ‒ Most commonly used ‒ Used for soluble antigens emulsified with CFA or IFA • Intraperitoneal ‒ Used for cellular antigens in mice • Intravenous ‒ May be used for final boost
Amount of Antigen
• Immune response does not directly correlate with the amount of antigen and large amounts will not provide a better response
Describe important considerations for antibody purification.
Degree of purification depends on the intended use
• Unlabeled immunoassay: limited purification
• Labeled: ≥80% purity
• In vivo: completely pure
Contaminants
• Serum: Albumin (50%); Other Ig (12-25%)
• Cell culture: FBS; other proteins (secreted, shed)
Describe types of antibody purification.
Typical rough purification techniques include:
1. Ammonium Sulfate Precipitation
• Nonselective precipitation
• Used more for concentration of Ab
2. DEAE and QAE Columns
• Positively charged columns used primarily to remove albumin (anion exchange)
More selective purification can be performed using Protein A, G or L
• Bacterial proteins that bind to mammalian immunoglobulins
• A and G bind to the Fc region of IgG (A binds weakly to IgG3, G binds strongly to all subclasses and is better at binding mouse IgG)
• L binds to variable region of kappa
• Provides rough purification for polyclonal Ab
Selective purification can be achieved by affinity
chromatography
‒ Negative selection: use carrier bound to a solid support and keep the flow through
‒ Positive selection: use your antigen bound to a solid support, wash off all non-specific proteins and elute your Ab of interest
Describe considerations for storage of antibodies.
• IgG are very stable; can maintain activity for weeks at RT
• Antibodies collected as serum (polyclonal) or culture media (monoclonal) contain proteases and may be nonsterile and need to be processed
‒ Heat to 60 oC for 5’-10’ to inactivate most proteases
‒ Store frozen (-70 oC) to minimize proteolytic activity
‒ Add chemicals to inhibit bacterial growth or protease activity
Describe different types of isotopic antibody labelling.
Internal Labels
• Existing atom in a molecule is replaced by a radioactive
isotope of the same atom
External Labels
• More common than internal labels
• Radioactive isotope is covalently linked to the molecule
• Most common is 125I; others include 3H,131I, 57Co, 75Se, 32P
• More being replaced by non-isotopic (external) labels
Describe direct iodination and conjugation labelling.
Direct Iodination
• Iodine can be incorporated into the aromatic ring of
tyrosine residues
• May also be incorporated into other aromatic side chains (H, F and W) but reaction is 30-80x less efficient
Conjugation labeling
• Carrier that incorporates a phenol or imidazole group that can be iodinated, and an amine group that can be coupled directly to carboxyl groups on the ligand
What are the benefits and limitations of conjugation labelling?
Pros • Does not require tyrosine • Can be used on nonpeptides • Simple to perform • Not very damaging to the ligand
Cons
• Larger than an individual iodine atom; may alter the properties of the tracer
What are some types of non-isotopic labels?
Label - Example
Chemiluminescent - Acridium ester, isoluminol
Electrochemiluminescent - Ruthenium complexes
Cofactor - ATP, NAD
Enzyme - ALP, b-gal, G6PD, HRP
Fluorophore - Europium chelate, fluorescein, phycoerythrin
- Unlike radioisotopic labels, they are not single atoms
- Covalently linked (conjugated) to the Ab/Tracer
Describe using primary amines for conjugation labelling.
- Most common target
- Found on lysine and the N-terminus of each polypeptide chain.
- Usually outward facing on proteins and available for conjugation
- Abundant, widely distributed and easily modified
- Several chemistries available
- N-hydroxysuccinimidyl ester (NHS)
- Glutaraldehyde
Describe using sulfhydryl groups for conjugation labelling.
- In native proteins, -SH are commonly found in the oxidized form (disulfide bonds) which participate in tertiary and quaternary protein structure
- Conjugation requires thiols be present in sulfhydryl form, so reducing agents necessary
- Several chemistries exist
- Maleimide
- Iodoacetyl
Describe using carbonyl groups for conjugation labelling.
- Carbohydrates must first be oxidized to create reactive aldehydes
- Aldehyde-activated carbohydrates can be reacted directly to primary amines (-NH2) or to labels activated with hydrazide groups (N-N)
Describe using carboxyl groups for conjugation labelling.
• Found on Gln, Glu, and at the C-terminus of each polypeptide chain
• Usually outward facing on proteins and available for conjugation
• Abundant, widely distributed and easily modified
• Couple to primary amines after carboxyl activation -
most commonly achieved with carbodiimides
• EDC: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
Provide an overview of antibody conjugation pros and cons.
Primary amines (–NH2)
• Numerous and distributed over the entire antibody
• K residues in the paratope region may affect binding (especially if adding a high density of labels)
Sulfhydryl groups (–SH) • Disulfides in the hinge region are most susceptible to reduction and can be selectively reduced • Provides consistent labeling at a defined location
Carbonyls (-CO)
• Glycosylation sites are predominantly found in the Fc region of IgG
• Results in labeled Ab with high activity
Aldehydes (-CHO)
Carboxyls (-COOH)
• Not generally used to conjugate to Ab
Describe classes of cross-linkers.
Homobifunctional
• Two identical functional groups
• Can cause self-coupling of Ab
• i.e. Glutaraldehyde
Heterobifunctional
• Two different functional groups, allowing 2-step reaction
• Limits self-coupling, and thus polymer formation
• Numerous types are available
Provide important considerations for conjugation.
Detectability
• Can limit the sensitivity of an assay
• Conjugation reaction (e.g. temp, pH) does not affect label
Reactivity
• The binding ability of reactants (i.e. Ab, Ag, biotin, avidin)
need to be preserved after conjugation
Nonspecific binding
• Minimal binding of labeled reactants to solid phase
Stability
• Stable cross-linker under storage and reaction conditions
Describe the Biotin-Streptavidin system
• Based on the highly specific interaction of avidin with
Biotin (Vitamin H/B7)
• One of the strongest known non-covalent interaction between a ligand and protein (Ka=1015 M-1)
• Biotin is small, so many molecules can be conjugated onto a single protein without affecting its function
- Streptavidin (60 kDa protein purified from the Streptomyces avidinii)
- Avidin (found in egg whites of birds, reptiles and amphibians)
- Four biotin binding sites per molecule of avidin allowing for multiple assay configurations as well as signal amplification
List important considerations for choosing a label.
Need to consider several factors: • Health/environmental hazards (i.e. radioactivity) • Sensitivity • Stability/shelf life • Ability to automate • Need for special equipment
