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Clinical Chemistry > Week 8 > Flashcards

Week 8 Flashcards

1
Q

What is an ag

A

-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

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2
Q

what is an AB

A

-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

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3
Q

specificity, affinity, avidity for an AB

A

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

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4
Q

Ab structure

A

-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

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5
Q

what happens in an immune response normally

A

-AG stimulates B lymph production
- B lymphs differentiate into plasma cells and proliferate
-production of Ab = primary and secondary response

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6
Q

PRIMARY AND SECONDARY HUMORAL RESPONSE

A

Primary Response:
-low
-low levels
-short lived
-IgM followed later by IgG

Secondary Response:
-rapid
-stronger response
-long lived
-IgG > IgM

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7
Q

What is an immunoassay

A

-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

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8
Q

Precipitin reaction principle

A

-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

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9
Q

The Precipitin Curve - parts

A

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

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10
Q

What is a simple immunoassay

A

-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

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11
Q

What type of label is used in a simple immunoassay

A

-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

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12
Q

what is in an immunoassay

A

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

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13
Q

how are immunoassays classified

A

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

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14
Q

what are two major types of reactions in immunoassays

A

Competitive
Limited reagent assays
RIA, FIA, EIA

Noncompetitive
Excess reagent, two-site, or sandwich assays
Immunoradiometric assay (IRMA), microparticle enzyme immunoassay (MEIA)

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15
Q

What is a Competitive Immunoassay

A

-“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

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16
Q

how are reagents mixed in a competitive immunoassay

A

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.

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17
Q

What happens in the SIMULTANEOUS APPROACH of reagent mixing

A
  • 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

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18
Q

What is the Reaction Relationships in SIMULTANEOUS APPROACH

A

-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

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18
Q

what is a Noncompetitive Immunoassay(or “How to make a sandwich”)

A

-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

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18
Q

Typical Competitive Dose-response Curve

A

-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

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19
Q

what occurs in the adsorption phase of Heterogeneous Assays

A

-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

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19
Q

What is the dose response curve

A

-determines signal of AG of interest based on standards with known concentrations

-dose= concentration = on X axis independent

-response - signal = Y axis and dependent

Plot curve: signal (y-axis) from a series of standards vs. concentration (x-axis, antigen of interest)

-As concen of AG increases the concen of labeled AG that binds to AB decreases
-if there is 0 unlabeled AG then all the labeled AG will bind
-when there is equal amount of both then they will both have equal affinity to AB

In a competitive immunoassay, less Ag* measures in the assay means more of the unlabeled (test sample) Ag is present

19
Q

what happens in the reaction of a
Noncompetitive Immunoassay

A

-Capture Ab is bound to the surface of a solid phase
-Ag from patient sample is allowed to react with solid-phase capture Ab
-Wash
-Ab* is added that reacts with bound Ag through a second and distinct epitope
-Wash
-Measure signal from bound label

The concentration of the Ag is directly proportional to the bound labelled Ab - linear on graph

20
Q

how can NONCOMPETITIVE immunoassay be changed to detect AB

A

-immobilized AG captures the AB and then a labeled AB is added that binds to the captured AB and detected

Amount of bound labeled Ab is directly proportional to the amount of specific Ab present

20
Q

What is a heterogeneous immunoassay

A

-a competitive binding assay where you have to mechanically separate the protein-bound labeled antigen from the unbound

-if the signals from both the bound and free forms of the labeled Ag are the same, we need to separate them from each other
Enzyme-Linked Immunosorbent Assay (ELISA)

Physical separation achieved by: adsorption, precipitation and interaction with a solid phase

20
Q

What is a Homogeneous immunoassay

A

-a competitive binding assay in which it is not necessary to separate the bound from the free fraction

-if the signals from the bound and free forms of the labeled Ag are different (because the signal is modulated by antibody binding), we do not need to separate them from each other
Enzyme Multiplied Immunoassay Technique (EMIT)

21
Q

What occurs in the precipitation phase of Heterogeneous Assays

A
  • ammonium sulfate, polyethylene glycol precipitate proteins nonspecifically
    -free Ab and Ag-Ab complexes will precipitate

-Soluble Ag-Ab complexes can be precipitated by a second Ab that recognized the primary Ab in the complex = result in an insoluble precipitate
-Separation method is specific because only the primary Ab is precipitated

22
Q

What occurs in the solid phase of Heterogeneous Assays

A

-Separate free from bound labeled reactant after washing
-Solid-phase support (magnetic beads, polystyrene surfaces, etc) is an inert surface to which reagent Ag or Ab is attached
-Immobilized Ag or Ab may be adsorbed to the solid-phase support; their spontaneous release is prevented by covalent linkages
-Easier to perform, automate and require less manipulation and time to perform

23
Q

What are examples of these labels:
Radioisotope
Enzyme
Fluorophor
Chemiluminescent

A

Radioisotope
-first labels used-3H, 14C, 125I, 32P

Enzyme
-alkaline phosphatase, horseradish peroxidase
-convert compound from colourless to coloured

Fluorophor
fluorescein, rhodamine - light excitation and emission

Chemiluminescent
-acridinium esters, ruthenium complex
-light emission initiated chemically or electrogenerated

24
Q

What do you have to consider when choosing a label

A

Is there a radiation hazard?
What is the sensitivity?
What is the specific activity (affects detection limit)?
Potential for automation?
Do we need specialized equipment?
e.g. fluorometers, photomultipliers

25
Q

What are the dis/advantages for using Radioisotopes as Labels

A

Advantages
-high detectability (can detect low quantities)
-produces a high measurable signal
-results in increased sensitivity (so a big signal change for small change in concentration)

Disadvantages
Expensive
Licence required
Strict paperwork
High cost of waste disposal
Exposure risk
Loss of reagent by decay (unstable) = limited shelf life
Need a radiation detector
Not amenable to automation

Radiation detector: gamma, alpha- or beta-particle counter.

26
Q

Radioisotope must be evaluated for

A

Specific activity
-radioactivity present to amount of AG present
-low specific activity

Reactivity with receptors or binding AG
-radiolabelling of antigen may damage antigen, change reactivity

To improve detectability/sensitivity of assay
-need higher specific activity to produce higher signal
-high radiation destroys analyte faster
-reduce radioisotope to match the lowest AG concentration that the assay measures

27
Q

What occurs in a competitive, sequential immunoassay (radioimmunoassay).

A

Unlabeled Ag (hormone) competes with labeled Ag* from the reagent for binding sites on the antibody (top and middle rows).

A high [Ag] means a low signal (positive sample column), and a low [Ag] means a high signal (negative sample column).

28
Q

What occurs when you use Enzymes as labels

A

-using catalytic properties of enzymes to detect and quantitate immunological reactions
-After reaction, add enzyme substrate and monitor resultant decrease in [S] or increase in [P]

Examples of enzyme labels:
alkaline phosphatase
horseradish peroxidase
glucose-6-phosphate dehydrogenase (G6PD)

29
Q

What are the dis/advantages to using enzymes as labels

A

Advantages
-high specific activity - reacts with alot of substrates
-provides amplification - many products per enzyme
-long stable shelf life
-cheap
-can be changed for automation

Disadvantages
-less detectable and sensitive than RIA
can be solved by replacing photometric detection reaction by chemi- or bioluminescent reaction ***

Environmental conditions
-pH, temperature, substrate concentration, time must be optimized for a) enzyme activity, b) Ag-Ab reaction

30
Q

Fluorophors as Labels

A

-Fluorophors are compounds that when exposed to a light of a shorter wavelength emit light of LONGER wavelength
-can be used an indicators of progress
-e.g. fluorescein, umbelliferone, europium chelates, rhodamine (umbelliferone are stronger fluorophors than fluorescein

31
Q

What is Stokes shift
why should it be wide

A

-difference between the excitation & emission wavelengths: the wider the better

-if the excitation and emission wavelengths are too close they are too tough to differentiate making it hard to measure the emitted fluorescent light intensity

32
Q

What are the advantages and disadvantages of using Fluorophors as Labels

A

Advantages
safe
easy to automate
long shelf-life

disadvantages
-less sensitivity & detectability than RIA
-for a larger signal use more intense excitation light (e.g. xenon arc, mercury)
-background fluorescence: overcome using time-resolved and lanthanide chelates fluorescence
-quenching occurs if there is a decrease in fluorescence because of an interaction with another solvent or solute

33
Q

Difference between Direct & Indirect Immunofluorescence

which is better

A

Direct
-Antibody specific to Ag is directly tagged with fluorophore

Indirect
Antibody specific for Ag is unlabeled & a second fluorophor-labeled antibody is directed toward the first

Better?
Indirect because the labeled Ab has no direct interaction with the Ag.
- immunohistochemistry tissue sections stained using AG-AB reactions in order to highlight specific features of the tissue

34
Q

What are Chemiluminescent Compounds as Labels

A

-Chemiluminescence: light emission produced during a chemical reaction
-enzyme-catalyzed oxidation of isoluminol by hydrogen peroxide produces long-lived light emission
-oxidation of an acridinium ester by hydrogen peroxide produces a rapid flash of light

Emitted light measured by a luminometer
Examples: luminol, luciferin, acridinium

35
Q

What are the dis/advantages of using Chemiluminescent Compounds as Labels

A

Advantages
Long shelf-life
Safe to handle; no waste disposal
Sensitivity equivalent to or better than RIA
Many automated instruments
Wide applications
Little background interference because other substances in serum do not exhibit chemiluminescence

36
Q

What receptor properties do AB have that determine how sensitive and specific our assay is

Diagnostic sensitivity:
Diagnostic specificity
Positive predictive value:
Negative predictive value:

A

Diagnostic sensitivity: ability of test to detect a given disease

Diagnostic specificity: ability of test to correctly identify the absence of a given disease.

Positive predictive value: chance of an individual having a given disease if the test is abnormal.

Negative predictive value: chance of an individual having a given disease if the test is within the reference interval.

37
Q

Questions

A

slide 66 - when it mentions receptor what does it refer to? is the receptor the AB or is it the Fab region ON the AB

  • slide 57 says less detec and sensi than RIA but then it says in combination with ampli the EIA becomes one of the most sensitive but if amplification is already part of the reaction why is it under a disadvat if we state its one of the most sensitive

-slide 42- how is only the primary AB precipitated if both freeAB and AG-AB complexes are precipitated. Doesnt the 2nd AB bind to the complex and precipitates with it?

slide 65 can HRP act as both an enzyme label and Chemiluminescent Labels? What is meant as the substrate

38
Q

What receptor properties do AB have that determine how sensitive and specific our assay is
Assay sensitivity
Assay detection limit
Ab specificity
Ab cross-reactivity (%)

A

Assay sensitivity
-size of change in signal relative to the change in AG concentration
-given by the slope of the dose-response curve
-depends on the affinity of the specific receptor and the specific activity of the labeled AG

Assay detection limit
-smallest amount of AG that can be reliably distinguished from zero

Ab specificity
-Ability to bind only the antigen of interest
-Ability of the receptor to measure one specific AB

Ab cross-reactivity (%)
-Degree to which our Ab reacts with Ag analogues (look-alikes)
-Cross-reactivity must be as low as possible

39
Q

What are immune receptors

A

-if purified AG is injected in an animal , B lymph will be stimulated and produce plasma cell lines that secrete AB with different specificities
-Antibodies derived in this manner are termed polyclonal

40
Q

how are monoclonal AB derived (directed against only one epitope on an antigen)

A

-Ag injected into a mouse, then lymphocytes taken from spleen or lymph node fused with myeloma cell line (immortal B-cell line; tumour cells have lost ability to die/ do not respond to death signals) from tissue culture

-Fused hybrid cell line is grown in selective (HAT) medium that kills unfused cells

Colonies screened for Ab production

Cell lines secreting Ab of desired specificity are cloned in subcultures

Isolation of a single clonal line producing an Ab with specificity for a single epitope

41
Q

What are the advantages of poly and monoclonal AB

A

improved specificity and reaction efficiency over polyclonal Abs

unlimited supply of one particular kind of Ab

42
Q

after we make our antisera (poly/mono) how do we check how useful it is

A
  1. determine titre or optimum concentration of Ab
  2. determine strength of binding
    related to equilibrium constant (K)
    related to detection limit
  3. determine cross-reactivity of Ab
    related to specificity
43
Q

how do we determine the titre

A

-Incubate progressive dilutions of Ab with a fixed amount of Ag*
-Dilution that binds 50% of Ag* is considered optimum concentration (i.e. titre)
-If titre selected is too high (too concentrated) there will be little or no competition between Ag and Ag* for binding sites
-If titre selected is too low there will be insufficient Ab to bind Ag and Ag*

44
Q

What is the binding between AG and AB related to

A

-Concentration of each reactant,
-Specificity of the Ab for the Ag
-Affinity and avidity for the pair
-Environmental conditions

Closeness of fit (high affinity) is the most important factor in affecting strength of bond - the closer they can get the better the bond

-receptor assays and competitive binding assays use receptor proteins or transport proteins as binding agents

affinity/avidity will determine K (kinetic constant that can be calculated)

45
Q

What is cross reactivity

A

-An Ab can react with an Ag that is structurally similar to the homologous Ag
-The greater the similarity between cross-reacting Ag and the homologous Ag, the stronger is the bond with the Ab
-the stronger the affinity and avidity, the greater is the possibility of cross-reactivity
-When cross-reactivity is low, specificity is high
-presence of analogue implies increased cross-reactivity and therefore a potential for falsely elevated results.

-Increased cross-reactivity is also a consequence of antibodies with low affinity or low avidity for their antigen

46
Q

Receptor type: Transport protein

A

-protein acting as binding agent instead of AB

e.g. thyroxine binding globulin (TBG) in plasma is used to bind thyroxine (the ligand AG)
-drawback: patient’s serum also contains TBG & interferes, resulting in decreased specificity

47
Q

Receptor type: CELLULAR RECEPTORS

A

-a cellular (membrane-bound) receptor can act as the binding agent instead of an antibody
-used in receptor assays
-e.g. thyroid cell membrane is used to measure thyroid stimulating hormone (TSH)
-specificity directed toward biologically active site (vs. the immunological site for antibodies)

-measures only biologically active antigen
have limited shelf life
-more common in research applications
-(biologically active site is the catalytic site or antigen-binding site.)

Clinical Chemistry (8 decks)

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