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LMP1203H > L8: Antibodies and Immunoassays > Flashcards

L8: Antibodies and Immunoassays Flashcards

1
Q

Define immunity and list immune cell types.

A 
• 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

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2
Q

List immune molecules integral to cellular response.

A

• 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

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3
Q

Describe the basics of innate and adaptive immune response.

A

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)

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4
Q

Provide examples of PAMPs.

A 
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)
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5
Q

Define antigen and antibodies.

A

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

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6
Q

Describe three roles of antibodies.

A
  • Neutralization
  • Opsonization
  • Compliment Activation

(14-16)

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7
Q

Describe the structure of an antibody.

A

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

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8
Q

Define antibody isotype and subclasses.

A 
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)

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9
Q

Define allotype and idiotype.

A

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)

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10
Q

Describe antibody fragments.

A

cleaved into functional fragments
• Fragment antigen binding (Fab)
• Fragment crystallizable (Fc)

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11
Q

Define antigen and describe the difference between immunogen and hapten.

A

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

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12
Q

Describe immunogenic factors of antigen.

A

see slide 27

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13
Q

Define epitope.

A

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

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14
Q

Define paratope

A

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

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15
Q

Describe binding of antibody to antigen reaction and the factors impacting the strength of the reaction. What is the Scatchard model?

A

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

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16
Q

Describe affinity.

A

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)

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17
Q

Describe avidity.

A

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)

18
Q

What factors include Ag-Ab interactions in vitro?

A

• 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

19
Q

Define cross vs shared reactivity.

A

Cross reactivity
• Binding of structurally different epitopes by the same antibody (different affinities)

Shared reactivity
• Common epitopes on different antigens

20
Q

List the antibody requirements for immunoassays.

A
  • Reproducible
  • Specific
  • High Affinity
  • Stable
  • Large-scale production
  • Readily purified
21
Q

Describe antibody clonality.

A
  • 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

22
Q

Describe polyclonal antibodies and their characteristics.

A

‒ 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

23
Q

Describe monoclonal antibodies and their characteristics.

A

• 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
24
Q

In antibody production, how do you choose choice of animal?

A

• 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)

25
Q

In antibody production, what are common considerations in immunization procedures?

A

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

26
Q

Describe important considerations for antibody purification.

A

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)

27
Q

Describe types of antibody purification.

A

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

28
Q

Describe considerations for storage of antibodies.

A

• 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

29
Q

Describe different types of isotopic antibody labelling.

A

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

30
Q

Describe direct iodination and conjugation labelling.

A

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

31
Q

What are the benefits and limitations of conjugation labelling?

A 
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

32
Q

What are some types of non-isotopic labels?

A

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
33
Q

Describe using primary amines for conjugation labelling.

A
  • 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
34
Q

Describe using sulfhydryl groups for conjugation labelling.

A
  • 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
35
Q

Describe using carbonyl groups for conjugation labelling.

A
  • 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)
36
Q

Describe using carboxyl groups for conjugation labelling.

A

• 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

37
Q

Provide an overview of antibody conjugation pros and cons.

A

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

38
Q

Describe classes of cross-linkers.

A

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

39
Q

Provide important considerations for conjugation.

A

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

40
Q

Describe the Biotin-Streptavidin system

A

• 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
41
Q

List important considerations for choosing a label.

A 
Need to consider several factors:
• Health/environmental hazards (i.e. radioactivity)
• Sensitivity
• Stability/shelf life
• Ability to automate
• Need for special equipment

LMP1203H (6 decks)

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