L8: Antibodies and Immunoassays Flashcards

Question

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

Answer 1

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

Answer 2

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)

Answer 3

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

Answer 4

• 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

Answer 5

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

Answer 6

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

Answer 7

``` 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

Answer 8

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

Answer 9

* 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

Answer 10

* 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

Answer 11

* 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)

Answer 12

• 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

Answer 13

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

Answer 14

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

Answer 15

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

Answer 16

• 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

Answer 17

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