Week 8 Flashcards
What is an ag
-when introduced induces an immune response - AB formation
-binds AB with different specificities
-binding sites on an AG is referred to as AGenic determinant or epitope
what is an AB
-able to bind specifically to AG
-Five classes: IgG, IgA, IgM, IgD, and IgE
-has a functional domain known as F (ab) which binds the Ag site
-proteins made by plasma
-specificity, affinity, avidity, cross-linking
specificity, affinity, avidity for an AB
specificity- binding only to AG of interest
affinity-strength between epitope and SINGLE combining site on AB
avidity - OVERALL strength of binding of AG with many multivalent AB
Ab structure
-2 light chains and 2 heavy chains
-immunoglobulins named according to their heavy chains
-constant and variable regions
Fc = region of constant structure within an antibody class
Fab = contains the Ag binding site that varies between different Ab
IgM is a pentamer
IgA - secretory - dimer
the rest are monomers
what happens in an immune response normally
-AG stimulates B lymph production
- B lymphs differentiate into plasma cells and proliferate
-production of Ab = primary and secondary response
PRIMARY AND SECONDARY HUMORAL RESPONSE
Primary Response:
-low
-low levels
-short lived
-IgM followed later by IgG
Secondary Response:
-rapid
-stronger response
-long lived
-IgG > IgM
What is an immunoassay
-measures AB-AG complex (immuno complex)
-different from other types of assays because it measures AB-AG complexes to generate a signal that can be measured
-very sensitive and specific
-used to measure substances that are hard to measure by classical methods
Precipitin reaction principle
-crosslinking of AG by AB is more likely when there is an optimal combining ratio of both AG and AB
-dont need exact molar equivalence of reactants
-slight excess of AB
-can be seen by precipitate formation
The Precipitin Curve - parts
Prozone - AB excess as crosslinking and lattice formation cannot occur as AG is covered by AB
Equivalence zone - Crosslinking occurs. Antigenic sites are available for AB and precipitates are formed
Postzone - too much AG, not enough AB so AG-AB complexes stay soluble
there are triplets 2 AG: 1 AB . no precipitate formed
What is a simple immunoassay
-using AG:AB binding without the immune complex precipitation but with a label
Gel diffusion
Immunoelectrophoresis
Agglutination assays
direct testing of erythrocytes for blood group: ABO, Rh, other antigens
What type of label is used in a simple immunoassay
-tag that helps with detection but doesnt interfere with the reaction
-labeled and unlabeled AG behave the same
-bind with equal affinity so the label doesnt interfere with AG activity and both forms bind with EQUAL AFFINITY to AB
-improves sensitivity
Can label antigen OR antibody
Types of labels:
Radioisotopes
Enzymes
Fluorescent compounds
Chemiluminescent compounds
Bioluminescent compounds
Competitive: usually antigen labeled
Non-competitive: usually the 2nd antibody labeled
what is in an immunoassay
AG- what we want to measure and what binds to AB
AB- binding agent (any compound with capacity to bind)
in a cell receptor assay = a cell receptor
in a protein binding assay = an endogenous protein
label - on AG or AB
Separation method if needed
Data reduction method - needed for methodology
how are immunoassays classified
Identified by type of binding agent
-Antibody: immunoassay
-Receptor: cell receptor assay
-Protein: protein binding assay
Identified by type of label
-Radioactive isotope: radioimmunoassay (RIA)
-Fluorescent compound: fluorescent immunoassay (FIA)
-Enzyme: enzyme immunoassay (EIA)
Identified by type of reaction format; two major types
-competitive vs non competitive
what are two major types of reactions in immunoassays
Competitive
Limited reagent assays
RIA, FIA, EIA
Noncompetitive
Excess reagent, two-site, or sandwich assays
Immunoradiometric assay (IRMA), microparticle enzyme immunoassay (MEIA)
What is a Competitive Immunoassay
-“competition” between labeled and unlabeled antigen for antibody binding sites
-antibody and labeled antigen come from the reagent we provide (fixed supply
-unlabeled antigen comes from the patient’s sample (variable supply)
-if calibrating, then unlabeled antigen comes from our standards
how are reagents mixed in a competitive immunoassay
together simultaneously OR sequentially:
Simultaneous approach
Ab + Ag + Ag* Ab:Ag + Ab:Ag* + Ag + Ag*
(free) (bound)
Sequential approach
(1) Ab + Ag - > Ab:Ag + Ab
(2) Ab:Ag + Ab + Ag* -> Ab:Ag + Ab:Ag* + Ag*
Separate bound from free and measure the bound label.
The sequential approach has a better detection limit since it allows for improved patient Ag binding.
What happens in the SIMULTANEOUS APPROACH of reagent mixing
- L-AG* is constant, unL-AG for pt, R-AG is constant. They are incubated together yield bound labeled Ag (AgAb), bound unlabeled Ag (AgAb) and free labeled Ag (Ag)
-both types of AG have same affinity to AB and will compete for binding sites
-after incubation is completed the free labeled and unbound AG is removed and bound labeled AG is measured in principate
-if there is more unlabeled free AG then more will bind to AB which is why we remove so we can have maximum binding by labeled AG
What is the Reaction Relationships in SIMULTANEOUS APPROACH
-Probability of Ab binding Ag* is inversely proportional to concentration of Ag in patient’s sample
-due to competition; higher concentrations of patient Ag will mean lower concentrations of Ag* that are bound
-Signal (from bound Ag*) is inversely proportional to [Ag] in the patient sample
what is a Noncompetitive Immunoassay(or “How to make a sandwich”)
-two site assay that uses two antibodies and sandwiches the Ag (pt sample) between them
-capture antibody: immobilized on a solid phase (can be mag beads, wall of test tube)
-detection antibody: conjugated to a label
-the AB is already labelled and the reagents are in excess so there is no competition for binding sites
Disadvantages :false positive and false negative interferences due to heterophile antibodies (Ab formed from patients with autoimmune disease and other disorders)
AG detection
Typical Competitive Dose-response Curve
-signal is from the label as: radioactive counts, light from a chemiluminescent compound, a coloured product from an enzyme reaction,
-seen as a curved line on the analyzer as its calibration curb but typically seen as linear to show inverse relationship between signal and concentration in competitive immunoassay
what occurs in the adsorption phase of Heterogeneous Assays
-uses particles to trap small labeled or unlabeled Ags
-charcoal is used because it is porous and can combine with small molecules to remove them from solution and dextran gives a non specific protein binding to charcoal
-after absorption and centrifugation free labeled AG is found in the precipitate
-Binding of the capture Ab to paramagnetic particles is used in ACCESS2; separation takes place by applying a powerful magnet adhering bound analytes to each reaction chamber
