Antibodies Flashcards
1
Q
What are antibodies
A
- immunoglobulin protein produced as immune defense against foreign agents (antigens)
- each antibody has a region binding specifically to a particular antigen
- made of light and heavy chains
2
Q
Humoral Immune System
A
control of circulating pathogens
- B cell binds to antigen. T dependent B cell requires T helper cell.
- B cell differentiation + proliferation
- plasma cells proliferate and produce antibodies against antibody
3
Q
Cellular Immune System
A
control of intracellular pathogens
- T cell binds MHC-antigen complex activating the T cell
- activation of macrophage
- CD8 T cell becomes cytotoxic T lymphocyte able to induce apoptosis of target cell
- cytokines transform B cells into antibody providing plasma cells
4
Q
IgG structure
A
- S-S disulfide bonds connect hinge region and the light/heavy chains
- constant and variable sequences
- light chain: one variable and one constant
- heavy chain: one variable and three constant
5
Q
Light Chain seuqneces
A
- 2 domains
- 214 amino acids in two domains
- variable sequences have differences in the N terminal domain
- constant regions are identical in C terminal domain
6
Q
Heavy chain sequences
A
- heavy chain is 445 amino acids in 4 domains
- hinge gives flexibility to bind to antigen
- 1 variable and 3 constant domains
7
Q
Ig Fold
A
- B strand arrangement of B sandwich motif of sheets linked by loops
- loops are the CDRs with variation
- gives binding specificity
- L and H chain fold to yield 3 CDR in each chain to form walls of Ab binding groove
- therefore there are 3 CDR per subunit and 6 total forming the antigen binding site
8
Q
Benefits of catalytic antibodies
A
- high affinity and specific binding of an antibody
- non-toxic
- stabilise the TS
- but enzymes alone cannot be used in treatment : immune response from body
9
Q
Abzymes
A
- catalytic Abs having structural complementarity for the TS of an enzyme catalysed reaction
- strong binding of the TS with high association constant enhancing reaction rate
- reduce rotational entropy to reduce the free Gibbs energy of the reaction
- idea is antibody with high substrate affinity yet are able to have catalytic activity-
Abs bind TS to lower activation energy and enhance reaction rate - you need it to target TS and NOT the substrate
10
Q
Hapten
A
- small molecule reacting specifically with an antibody but is incapable of stimulating antibody production except in combination with an associated protein molecule
- hapten in combination with a larger protein becomes antigenic and can elicit and immune response
- will bind to antibody but if inject won’t provoke them
11
Q
Cocaine
A
- alkaloid from coca plant
- serotonin-norepinephrine-dopamine reuptake inhibitor
- nervous system stimulator increasing concentrations of the neurotransmitters (not reuptaken)
- TS hapten approach used to treat overdoses
12
Q
Cocaine Degradation
A
- degraded into inactive molecules prior to crossing blood brain barrier
- hydrolysis at benzoyl ester or the methyl ester
- non toxic secreted product
13
Q
Butyrylcholinesterase
A
- hydrolyses butyrylcholine
- hydrolyzes cocaine into methyl ester and benzoic acid
- can hydrolyze toxic compounds containing an ester
- human esterases are slow to degrade cocaine
14
Q
BChE Mechanism
A
- glutamate H bonds His to form base
- His deprotonates Ser-OH group
- Ser nucleophilically attacks the ester bond
- oxyanion hole stabilised by enzyme backbone
- His acts as conjugate acid to donate a proton
- release of choline
- His deprotonates water
- water attacks acetylated Ser
- charge delocalisation and oxyanion hole
15
Q
Cocaine Degradation by Cholinesterase
A
- Ser deprotonated by His to make acetylated intermediate
- oxyanion hole
- negative charge collapse
- pick up His proton
- release of product intermediate
- water deprotonated by His base
- nucleophilic attack on Ser
- 2nd intermediate and collapse
- His proton regenerates Ser active site
- similar to how the native substrate (butyrylcholine) operates
- promiscuity in esterase cleavage of ester bond in cocaine
16
Q
Anzyme Design
A
- Ab recognises, binds, and catalyses cocaine
- stablises the TS for better catalysis
- products released so the Ab is recycled and free : therefore cannot bind tightly
- to generate Abs with catalytic activity we generate an immune response against molecule mimicking the TS of cocaine degradation
- hapten must be stable and mimic the TS tetrahedral intermediate structural/electronic properties
- Hapten elicits Abs stabilising the TS of the reaction
17
Q
TS in Cocaine Hydrolysis
A
- 1st step of hapten is the generate a cocaine like molecule with TS behaviour
- intermediate between cocaine and ecogonine methyl ester product
- can make TS analogs (tetrahedral) that are modified to cause no further catalysis
- these tetrahedral hapten types elicit hydrolytic antibodies
18
Q
Linker
A
- 2nd step is after making the TS analogue to conjugate to a larger protein
- allows exposure of epitope to the Ab
- allow to induce immune response
- must determine the linker type, tether site, length
- linked to a carrier protein : BSA or ovalbumin
19
Q
Link Length
A
- too short of a linker makes the carrier + phosphonate mono-ester TS analog seen as one identity and the Abs made against BSA not the analog
- too long of a link means blood proteases cleave the linker before the immune response
- cleaves the TS/hapten so the Abs are also against the carrier
20
Q
Linker Site
A
- site is important as you don’t want the linker to interfere with molecular catalysis
- 3 sites for linker attachment on the phosphonate mono-ester TS analog
21
Q
Linker Attachment
A
- modifying the lysine amine group to get many hapten molecule attached to increase chances of Ab recognition
- want more Ab binding a large protein carrier relative to analog
- this increases hapten antibodies by having a large ratio
- more accessible sites on the hapten and less on the carrier
22
Q
Catalytic Ab production
A
Conventional:
- antigen inoculation of mouse
- remove spleen cells
- obtain antiserum: mixture of Abs specific for each antigenic determinant
Monoclonal:
- fuse spleen cells with myeloma cells into hybrid myeloma cells
- grown on medium selecting the nonfused cells (myeloma cells lack HGPRT and fusion allows alternative nucleotide pathway)
- obtain individual populations of cloned antibodies
- screen for catalytic activity
23
Q
Degradation Kinetics
A
- want a lower Km so you need less substrate to achieve 1/2 Vmax
- kcat/k0 higher means higher efficiency at low substrate concentrations
- review*
24
Q
Cocaine Toxicity
A
- rat survival after infusion of lethal dose of cocaine significantly improved after selected Abzyme administered
- can it work in humans in vivo?
25
Q
Antibody Catalytic Mechanism
A
- mechanistically different to esterase (doesn’t bind substrate tightly like normal enzymes)
- Tyr aligns the binding site for cocaine coordination
- water molecule activated to attack the ester
- active site of antibody stabilised tetrahedral intermediate of the oxyanion hole (Tyr-OH)
- collapse of negative charge
- product release and Abzyme regeneration
- products are benzoate and methyl ester
26
Q
Prodrug Activation
A
- prodrug has no activity and is activated inside the body by the metabolism
- Abzyme delivering prodrug to a site and activating it or having catalytic antibodies in a specific location activating drugs on arrival
27
Q
Chloramphenicol Abzyme
A
- raise Abs against hapten
- Ab can catalyse prodrug ester hydrolysis to release chloramphenicol
28
Q
Abzyme Limitations
A
- haptens fail to generate antibodies catalysing the desired reaction
- if haptens too closely resemble the final product there is slow product release or inhibition
- hapten may be difficult to synthesize
- catalytic efficiency depends on solvent exposed binding site : if too hydrophobic it gets stuck in membrane
- Hammond Postulate : TS position relative to product/substrate affects the affinity
29
Q
Therapeutic Abs
A
- murine, chimeric, humanized, human
- humanised are best as only the CDR loops are grafted onto a human Ab
- chimeric consists of variable regions derived from a mouse and constant regions derived from human
