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Serology / Immunology > Immunologic Techniques > Flashcards

Immunologic Techniques Flashcards

1
Q

substance or molecule that elicits immune response or triggers the production of an antibody

A

antigen

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2
Q

one that is produced in response to an antigen

A

antibody

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3
Q

basic reaction of antigen-antibody binding

A

sensitization

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4
Q

factors that affect ag-ab reaction

A

concentration of reactants.
temperature.
length of incubation.
pH of the system.

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5
Q

distinct phases of ag-ab combination

A

primary phenomenon.
secondary phenomenon.
tertiary phenomenon.

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6
Q

describe primary phenomenon

A

sensitization.
represents the initial ag-ab binding.
single ab to single ag site.
tests to detect these reactions are technically difficult, complex, expensive, and may require special equipment and time consuming.

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7
Q

examples of primary phenomenon

A

immunofluorescence, radioimmunoassay (RIA), enzyme immunoassay (EIA)

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8
Q

describe secondary phenomenon

A

aggregation of complexes.
lattice formation to create large molecules that are easily detectable.
methods used are quick and easy to perform, less expensive, less time consuming, and do not require special equipment.
less specific, less sensitive, and more interferences.

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9
Q

combination of ab and multivalent ag to reduce a stable network and results in a visible reaction

A

lattice formation

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10
Q

examples of secondary phenomenon

A

precipitation, agglutination, complement fixation

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11
Q

describe tertiary phenomenon

A

ag-ab reaction is not visible but is detected by the effect of the reaction on tissues or on cells.

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12
Q

examples of tertiary phenomenon

A 
inflammation, 
phagocytosis, 
deposition of immune complexes,
chemotaxis,
immune adherence.
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13
Q

method of choice in immunology

A

secondary phenomenon = in-vitro presence of ag or ab

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14
Q

soluble ag and soluble ab produce insoluble complexes

A

precipitation

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15
Q

process by which particulate ag such as cells are aggregated to form large visible aggregates if the specific ab is present

A

agglutination

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16
Q

triggering of the classical complement pathway due to combination of antigen with specific antibody.

A

complement fixation

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17
Q

primary union of ag and ab depends on which two characteristics

A

affinity and avidity

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18
Q

initial force of attraction that an ab has for a specific epitope or determinant

A

affinity

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19
Q

sum of all attractive forces between an ag and an ab

A

avidity

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20
Q

describe epitope

A

part of ag.
determinant site.
portion of the immunogen in which the immune response is directed.

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21
Q

examples of force / chemical bonds (weak)

A

ionic,
hydrogen,
hydrophobic,
van der waals.

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22
Q

occurs between oppositely charged particles.

A

ionic

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23
Q

involved on attraction between polar molecules that have a slight charge separation and which a positive charge resides on a H atom

A

hydrogen

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24
Q

occurs between nonpolar molecules that associate with one another and exclude molecules of water as they do so

A

hydrophobic

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25
Q

due to the interaction between electron clouds of oscillating dipoles

A

van der waals

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26
Q

all of these bonds are _________ and dissociation can easily occur

A

rather weak bonds

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27
Q

the strength of the reaction depends on the _____________________

A

specificity of ab to ag

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28
Q

describe avidity

A

the sum of all attractive forces between an ab and ag.
the force that stabilizes the ag-ab reaction keeping the molecules together.
the stronger the chemical bonds which form between ag and ab, the less likely the reaction will reverse.

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29
Q

describe affinity

A

initial force of attraction that an ab has for a specific epitope or determinant.

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30
Q

governs the reversibility of the ag-ab reaction

A

law of mass action

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31
Q

describe law of mass action.

A

governs the reversibility of the ag-ab reaction.
free reactants are in equilibrium with bound reactants.
the equilibrium constant represents the rate of the ag-ab binding and dissociation.
measures the goodness of fit.
as the avidity increases, the rate of dissociation decreases.

32
Q

simplest method of detecting ag-ab reaction

A

precipitation.

when ag and ab mix, initial turbidity is followed by precipitation.

33
Q

excess ag, falsely negative

A

postzone

34
Q

excess ab, falsely negative

A

prozone

35
Q

optimum precipitation occurs where the number of multivalent sites of ag and ab are approx. equal

A

zone of equivalence

36
Q

describe zone of quivalence

A

optimum precipitation occurs where the number of multivalent sites of ag and ab are approx. equal

37
Q

denote turbidity or cloudiness of solution

A

turbidimetry.
a detection device is placed on the direct line with an incident collecting light after it has passed through the solution.
measures the reduction of light intensity due to reflection, absorption of scatter.
scattering of light occurs in proportion to the size, shape, and concentration of molecules to the amount of solution.
amount of substance is calculated based on the results obtained standard and control.
very simple method but not very sensitive.

38
Q

measures the light that scattered at a particular angle from an incident beam as it passes through the suspension.

A

nephelometry.
the amount of light scattered is depencent on the number and size of particles scattered in the light beam..
measures light scatter from 10o to about 7o.

39
Q

kinds of nephelometry

A

endpoint nephelometry and kinetic nephelometry.

40
Q

reaction is allowed to run to completion but particles tend to fall out of solution and decrease light scatter

A

endpoint nephelometry

41
Q

describe endpoint nephelometry

A

reaction is allowed to run to completion but particles tend to fall out of solution and decrease light scatter.

42
Q

measures the rate of increase in scattering immediately after the reagent is added. this change of rate is directly proportional to ag-ab concentration.

A

kinetic nephelometry.

43
Q

kinetic nephelometry is used to

A

quantitate Ig, complement components, CRP, and other acute phase proteins.

44
Q

describe kinetic nephelometry

A

measures the rate of increase in scattering immediately after the reagent is added. this change of rate is directly proportional to ag-ab concentration.

45
Q

precipitation of ag-ab complexes can also be determined in support medium

A

passive immunodiffusion

46
Q

tyes of support medium - gel

A

agar - high MW complex polysaccharide derived from seaweed.
agarose - purified / modified agar.

agarose is often performed since agar has a stron neg charge; interaction between gel and reagent are minor agarose?

47
Q

describe passive imunodiffusion

A

precipitation of ag-ab complexes can also be determined in support medium.
the reactant is added.
ag and ab combination occurs by diffusion.
when no electric current is used to speed up the process, it is called passive immunodiffusion.

48
Q

rate of diffusion is affected by:

A 
gel viscosity,
size of particles,
temperature,
amount of hydration,
interactions between matrix and reactants.
49
Q

types of diffusion

A

single diffusion, single dimension - one reactant is moving (either up or down);

single diffusion, double dimension - one reactant is moving (moving out radially from a well);

double diffusion, single dimension - both reactants moving (either up or down);

double diffusion, double dimension - both reactants moving (moving out radially from a well).

50
Q

describe oudin single diffusion

A

ab is added to agarose and placed in test tube.
ag is then layered over gelled ab and diffuses down to agarose
ab present. reacts with the ag and precipitin bond will form in the gel.

51
Q

describe radial immunodiffusion

A

single diffusion, double dimension.
ab is incorporated in a gel (plate or petri dish).
the amount of precipitate formed is directly related to the amount of ag present.

52
Q

example of radial immunodifusion.

A

C3 - measure by a ruler (or a standard)

53
Q

methods of radial immunodiffusion

A

endpoint - allows reaction to go to completion.

kinetic - employs measurement taken before the zone of equivalence is reached.

54
Q

technical sources of error in radial immunodiffusion

A 
overfilling or underfilling of the well.
spilling sample outside well.
nicking the well.
improper incubation time or temperature.
plate unleveled.
bumping the plate.
plate dries out.
55
Q

describe ouchterlony

A

double diffusion, double dimension.
both ag and ab diffuse from wells and travel toward each other.
method for comparison of antigens.

56
Q

patterns of ouchterlony

A

non-identity - cross lines

partial identity - fusion of 2 lines with a spur.

  • 2 ag share a common determinant but some ab molecules are not captured by ag and travel to the initial precipitin line combined by the additional determinant found in mor comlex ag.
  • spur slways point to the simpler ab.

identity

57
Q

factors affecting accuracy of ouchterlony

A

drying out of gel.
inadequate time for diffusion.
bacterial or fungal contamination of gel.

58
Q

immunodiffusion can be combined with

A

electric current

59
Q

a technique which separates molecules accdg to differences in electrical charge when exposed to electric current

A

electrophoresis.

60
Q

reads the patterns

A

densitometer.

for protein and Hb electrophoresis

61
Q

explain electrophoresis

A 
direct current is applied to gel.
ag-ab migrate.
diffusion.
precipitin bonds are formed.
may be single or double diffusion.
62
Q

explain rocket immunoelectrophoresis (RIEP)

A

as the concentration of ag changes, there is this dissolution on the formation of ppt at increasing intervals.
result: precipitin line in conical shape.
height is measured and is directly proportional to concentration of ag standard deviation.

63
Q

advantage of RIEP over RID

A

results obtained in few hours.
primarily used for Ig and
-too low for nephelometry but too high for RID

64
Q

explain immunofxation electrophoresis (IFE)

A

excellent screening test to differentiate serum proteins and detect abnormalities such as MM and other proliferative disorders.

similar to except that after electrophoresis, ab are applie in a gel rather than placed in a trough?

ppt occurs in a shorter time with higher resolution

65
Q

sources of error in IFE

A

current applied in wrong direction.
incorrect buffer pH.
incorrect timing.
amount of current applied.

66
Q

explain agglutination

A

process by which a particulate ag such as cells are coated by ab in such a way large complexes/aggregates are formed.

67
Q

steps in agglultination

A

sensitization.
antibody production.
lattice formation - combination of ab and multivalent ag to produce.
enhancement of agglutination.

68
Q

factors that govern lattice formation

A

pH.
ionic strength.
temperature.

69
Q

things about ionic strength

A

LISS, albumin (conc.5-30%), enzyme

70
Q

explain LISS

A

lowers ionic strength of buffer.

71
Q

explain albumin

A

5-30%;

to neutralize surface charges and facilitate agglutination.

72
Q

explain use of enzyme

A

increases viscosity, agitation, or centrifugation by adding reagents: dextran, polyvinyl pyrrolidone, serum albumin.

73
Q

most frequently used enzyme:

A

papain, bromylene, ficin.

-reduces surface charges of RBCs through cleaving chemical groups, decreasing migration.

74
Q

types of agglutination

A

direct - when ag are found naturally on a particle (identification of bacterial types).
-if agg involves reaction of RBCs: ABO blood types (hemagglutination)

indirect/passive - ag are artificially attached to such a particle (polystyrene latex, bentonite, charcoal)

reverse-passive - ab rather than ag is attached to a carrier particle

agglutination-inhibition - based on competition between particulate and soluble ag for limited ab binding site and lack of agglutination is an indicator of positive rxn.
hemagglutination-inhibition use the same principle except that RBCs are the indicator particles.

coagglutination - utilizes bacteria. the carrier particle to which the ab is attached.
-S. aureus is most freq used bec it has protein A or the outer surface called proteases.

75
Q

antiglobulin-mediated

A

coomb’s test.

a technique that detects non-agglutinating ab by means of coupling with 2nd ab

Serology / Immunology (2 decks)

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