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MLT immunology > Unit 3 > Flashcards

Unit 3 Flashcards

1
Q

What is precipitation?

A

Combine soluble antigen and stable antibody to produce insoluble complexes

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2
Q

What is agglutination

A

Antigen -antibody complexes cross-link, causing clumping

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3
Q

Why are precipitation and agglutination
Considered unlabeled immunoassays?

A

Because a marker label is not needed to detect the reaction

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4
Q

What is affinity?

A

Initial force of attraction that exists between a single Fab site on antibody and a single epitope or determinant site on corresponding antigen

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5
Q

What does the strength of attraction depend on?

A
  • Specificity of antibody for a particular antigen
  • the epitopes shape and way it fits with binding siteson antibody determine whether band is stable or not
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6
Q

What is cross-reactivity?

A

-The more the cross-reacting antigen resembles the original antigen, the stronger the bond will be between the antigen and binding sites
- cross reacting antigens have lower affinity

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7
Q

What is serology?

A

Study of fluid components in the blood, especially antibodies

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8
Q

Describe serum

A

-liquid portion of blood minus coagulation factors
-most frequently encounter specimen in immunologic testing
- can be separated from other components of blood via centrifuge

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9
Q

What Color tube tops are serum collected in

A

Red top or tiger top

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10
Q

What is the additive for the red tube top?

A

None

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11
Q

How should serum be stored if testing is delayed?

A

-2-8°C for up to 72 hours
-frozen at -20°C or below

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12
Q

What is the additive of the red and grey tiger top?

A

Gel barrier

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13
Q

Why would a speciemen be rejected?

A
  • hemolyzed
  • clotted speciemen
  • wrong tube or container
  • tube not labeled
  • inadequate quantity
  • tube mislabeled
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14
Q

What are the 3 phases of laboratory testing?

A
  • preanalytical
    -analytical
    -postanalytical
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15
Q

What are examples of preanalytical testing?

A
  • Speciemen collection
  • Transport
  • processing
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16
Q

What are some examples of analytical testing?

A

Testing

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17
Q

What are some examples of postanalytical phase

A
  • Testing results
  • transmission
    -interpretation
    -follow up
  • retesting
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18
Q

What phases of the laboratory testing constitutes for 90% of the errors that occur?

A
  • Pre-analytical
  • post-analytical
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19
Q

What are the three phases of antigen/antibody complexes?

A

① primary phenomenon
② secondary phenomenon
③tertiary phenomenon

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20
Q

Describe the primary phenomenon phase of antigen/antibody complexes

A
  • Combination of binding site on antibody with a single epitope on antigen
    -reversible: occurs in mili-seconds
  • not easily detectable-hard to measure
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21
Q

Describe the secondary phenomenon phase of antigen/antibody complexes

A
  • Precipitation, agglutination, and complement fixation
  • easily detectable
  • most serological tests based
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22
Q

Describe the tertiary phenomenon phase of antigen/antibody complexes

A

-inflammation, phagocytosis, depression of immune complexes,immune adherence, chemotaxis (all in vivo)

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23
Q

What is complement fixation?

A

Triggers the classical complement pathway to detect antibody or antigen in the patients serum

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24
Q

What is avidity?

A

-Is the sum oo the attractive forces between an antigen and an antibody
- strength with which a multivalent antibody binds a multivalent antigen

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25
Q

What does avidity measure?

A

Overall stability of an antigen- antibody complex

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26
Q

The more bonds that form between antigen and antibody, the higher the ________

A

Avidity

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27
Q

What is the law of mass action?

A

Free reactants are in equilibrium with bound
Reactants

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28
Q

What is the law of mass action equation?

A

K=[Ag/Ab]/[Ab][Ag]

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29
Q

In law of mass action, what does the Kvalue depend on?

A

The strength of binding between antibody and antigen

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30
Q

The higher the K value……

A

-the larger the amount of antigen-antibody complexes
- the more visible or easily detectable the reaction

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31
Q

In the law of mass action equation, what does [Ag/Ab] represent?

A

Concentration of the antigen- antibody complex (mol/L)

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32
Q

In the law of mass action equation, what does [Ab] represent?

A

Concentration of free antibody (mol/L)

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33
Q

In the law of mass action equation, what does [Ag] represent?

A

Concentration of free antigen (mol/L)

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34
Q

What does precipitation require antigens and antibodies to have?

A
  • Multiple binding sitesfor one another
    -equivalent concentrations
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35
Q

What are the 3 groups of the precipitation curve?

A
  • zone of equivalence
  • prozone
  • postpone
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36
Q

For optimal precipitation, reactions must be run in what phase of the precipitation curve?

A

Zone of equivalent

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37
Q

What is the zone of equivalence of the precipitation curve?

A
  • Number of multi-valent sites of antigen and antibody are approximately equal
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38
Q

Describe the prozone of precipitation curve

A
  • Antibody excess
  • antigen combine with one or two antibodies
  • ## No cross linking
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39
Q

What could cause a false negative result in the prozone phase of the precipitation curve?

A
  • High antibody concentration
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40
Q

What should be done if a false negative result is suspected in the prozone phase of precipitation curve?

A
  • Diluting out the antibody and performing the test again may produce a positive result
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41
Q

Describe the postzone phase of the precipitation curve

A

-antigen excess
- small aggregates are surrounded by excess antigen
- No lattice network formed

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42
Q

What could cause a false negative result in the postzone phase of the precipitation curve?

A

-presence of a small amount of antibody

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43
Q

What should be done is false negative results occur in the postzone phase of the precipitation curve?

A

-test may be repeated a week later with another speciemen to give more time for antibody production

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44
Q

What are immunoturbidimetry and Nephleometry used to test for?

A
  • Complement
  • C-reactive protein
  • immunoglobulins
  • other serum protein
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45
Q

What is the sensitivity of immunoturbidity and nephelometry?

A

1-10 ug AB/ML

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46
Q

What is the principle of immunoturbidity and Nephelometry?

A

Light that is scattered at an angle is measured, indicating the amount of antigen or antibody present

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47
Q

What is radial immunodiffusion used to test for?

A
  • Complement
  • immunoglobulins
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48
Q

What is the sensitivity of radial immunodiffusion?

A

10-50 ug AB/ML

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49
Q

What is the principle of radial immunodiffusion?

A

Antigen diffuses out into gel that is infused with antibody. Measurement of the radius indicates concentration of antigen

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50
Q

What is Ouchterlony double diffusion used to test for?

A
  • Complex antigens, such as fungal antigens
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51
Q

What is the sensitivity of the Ouchterlony double diffusion?

A

20-200 ug AB/ML

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52
Q

What is the principle of Ouchterlony double diffusion?

A

Both antigen and antibody diffuse out from wells in a gel. The lines of precipitate formed indicate the relationship of antigens

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53
Q

What does immunoelectrophoresis test for?

A

Differentiation of serum proteins

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54
Q

What is the sensitivity of immunoelectrophoresis?

A

20-200 ug AB/ML

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55
Q

What is the principle of immunoelectrophoresis?

A

Electrophoresis of serum is followed by diffusion of antibody from the wells

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56
Q

What is immunofixation electrophoresisuse to test for?

A

Over or under production of antibody

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57
Q

What is the sensitivity of immunofixation electrophoresis?

A

Varies

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58
Q

What is the principle of immunofixation electrophoresis

A

Electrophoresis of serum is followed by direct application of antibody to the gel

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59
Q

What machines could be used for immunoturbidity?

A
  • Spectrophotometer
    -automated clinical chemistry analyzer
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60
Q

What machine is used for nephelometry?

A

Nephelotometer

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61
Q

Describe a nephelotometer

A
  • Contains photodetector located between 30-90 degrees from light source
    -amount of light scatter increases as the number of immune complexes increases and is an index of the concentration of antigen or antibody in solution
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62
Q

What does agarose gel do?

A
  • Helps stabilize the diffusion process and allow visualization of the preciptin band
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63
Q

What is passive immunodiffusion?

A

-when no electric currentis used to speed up the migration of the reactants

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64
Q

What can affect the rate of diffusion in passive immunodiffusion?

A
  • Size of particle
  • temperature
  • gel viscosity
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65
Q

What is the end point (Mancini) method?

A

Reaction goes to completion

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66
Q

What is the kinetic (Fahey) method?

A

-faster
-measurements taken before reaction is complete

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67
Q

What is the RID end point method results?

A

The square of the diameter is proportional to the antigen concentration

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68
Q

What are the three possible patterns of Ouchterlony diffusion?

A
  • Identity (arc)
  • partial identity (one line extends)
  • nonidentity (line cross X)
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69
Q

Immunofixation electrophoresis (IFE) is
Performed to visualize what?

A

Increased or decreased productionof antibody classes and to differentiate monoclonal and polyclonal immunoglobulins

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70
Q

What are agglutins?

A
  • Produced by antibodies
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71
Q

What are the two steps of agglutination?

A

① sensitization
② lattice formation

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72
Q

Describe the sensitization step of agglutination

A
  • initial bonding
  • antigen and antibody unite through bonding of antigenic determinant sites
  • fast and reversible
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73
Q

Describe the lattice formation of agglutination

A
  • Creation of large aggregates
    -visible aggregates form as multiple antigen and antibody molecules bind to create a stable lattice
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74
Q

What is sensitization of agglutination affected by?

A

-by nature of the antigens on agglutinating ‘ particles
- RBC and bacterial cells have a slightly negative surface charge
- class of immunoglobulin

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75
Q

What occurs if epitopes are sparse in the sensitization step of agglutination?

A
  • Less likely to interact with antibody
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76
Q

What is Coombs reagent?

A
  • Anti-human immunoglobulin
  • frequently used in blood bank
  • binds to Fc portion of IgG
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77
Q

Briefly describe immunoturbidity

A
  • automated methods
  • measures reduction in light intensity
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78
Q

Briefly describe nephelometry

A
  • Automated methods
    -measures light scatter as immune complexes form
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79
Q

What are the 4 types of agglutination reactions?

A

① direct agglutination
② passive agglutination
③ reverse passive agglutination
④ agglutination inhibition

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80
Q

Describe direct agglutination

A
  • Uses particles with naturally occurring antigens to test for antibodies in patient serum
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81
Q

What does direct agglutination test for?

A
  • Bacteria (Widal test)
  • RBCs
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82
Q

Describe the Widal test

A
  • A rapid screening test used to determine the possibility of typhoid fever
  • uses Salmonella O (somatic) and H (flagellar) antigens
  • direct agglutination
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83
Q

What test are ran for RBCs?

A
  • Hemagglutination
  • ABO typing
    (direct agglutination)
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84
Q

Describe passive agglutination

A

-uses particles that are coated with antigens not normally found on their surfaces: erythrocytes, gelatin, latex, and silicates

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85
Q

What does passive agglutination test for?

A
  • RA
  • antibodies to Group A streptococcus antigens
  • antibodies to viruses such as rubella
  • heterophile antibody in infections mononucleosis
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86
Q

Describe the reverse passive agglutination process

A

① antibody is attached to the carrier particle
② agglutination occurs is antigenis present in the patient sample

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87
Q

What can reverse passive agglutination test for?

A
  • Staphylococcus aureus
  • Streptococcus Groups A and B
  • rotavirus
  • Cryptococcus neoformans
  • detects soluble antigen in urine, spinal fluid and serum
  • rapid ID of antigens from infections
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88
Q

Describe agglutination inhibition

A

-based on competitionbetween particle and soluble antigens for limited antibody- combining sites
- used to detect haptens antigens (illicit drugs)
- lack of agglutination= positive reaction

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89
Q

What is an example of an agglutination inhibition procedure?

A
  • hemagglationation inhibition → uses RBCs
    -used to detect antibodies to certain viruses that can bind to RBCs and agglutinate them
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90
Q

What virus can hemagglutination inhibition be used to test for?

A
  • Rubella
    -influenza
    -respiratory syncytial virus)
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91
Q

What is the principle of direct agglutination?

A

-antigen is naturallyfound on a particle

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92
Q

Describe the results of direct agglutination

A

Agglutination indicates the presence of patient antibody

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93
Q

What is the principle of passive (indirect) agglutination?

A

Particles are coated with antigens not normally found on their surfaces

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94
Q

Describe the results of passive (indirect) agglutination

A

Agglutination indicates the presence of patient antibody

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95
Q

What is the principle reverse passive agglutination?

A

Particles are coated with reagent antibody

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96
Q

Describe the results of reverse passive agglutination

A

Agglutination indicates the presence of patient antigen

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97
Q

What is the principle of agglutination inhibition?

A
  • Haptens are attached to carrier particles.
  • particles compete with patient antigens for a limited number of antibody sites
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98
Q

Describe the results of agglutination inhibition

A

Lack of agglutination is a positive test, indicating the presence of patient antigen

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99
Q

What is the principle of hemagglutination inhibition?

A

RBCs spontaneously agglutinate in presence of certain viruses

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100
Q

Describe the results of hemagglutination inhibition

A

Lack of agglutination is a positive test, indicating the presence of patient antibody

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101
Q

Describe the monospot test

A

Used to detect the heterophile antibody characteristics of infections mononucleosis by its ability to agglutinate horse or bovine RBCs

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102
Q

Describe cold agglutins

A

-Produced by patients with certain autoimmune disorders, malignancies or infections, are antibodies that agglutinate human type O RBCs when incubated at cold temperatures

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103
Q

Describe PETINIA

A

“particle-enhanced turbidmetric inhibition immunoassay”
-patient sample is incubated with latex beads coated with analyte will prevent antibody turbidity results
-measures small analytes such as therapeutic drugs
- homogenous competitive immunoassay

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104
Q

Describe the results of PETINIA

A
  • If low amount of analyte is present in the sample, the reagent antibody will bind to the latex beads, resulting in aggregate formation andnigh level of turbidity
    -when concentration of analyteis high, analyte will bind to the reagent antibody and prevent it from binding to the latex beads, resulting is less turbidity
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105
Q

The amount of turbidity is ________ ________ to the concentration of the drug analyte in sample of PETINIA

A

Inversely proportional

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106
Q

How can most cross-reactivity be avoided?

A

Through use of monoclonal antibody directed against an antigenic determinant that is unique to antigen

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107
Q

What are advantages of agglutination reactions?

A
  • rapidity
  • easy to perform
  • no expensive equipment
  • relatively sensitive
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108
Q

When does maximum binding at antigen and antibody occur?

A

When the aggregate number of multivalent sites of antigen and antibody are approximately equal

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109
Q

Describe agglutination reaction with IgM

A
  • able to mediate lattice formation with antigen without additional enhancement
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110
Q

Describe the agglutination reaction with IgG

A

-requires enhancement techniques that vary physicochemical conditions and the addition of an anti-human immunoglobulin (Coombs reagent) in order to see a visible reaction

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111
Q

How does precipitation differ from agglutination?

A

Precipitation involves a soluble antigen, whereas agglutination involves a particulate antigen

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112
Q

What is imperative for optimal agglutination?

A
  • Avoid cross reactivity using monoclonal antibodies
  • store properly
    -check expiration dates
    -account for sensitivity and specificityforkits used
    -negative result does not rule out presence of the disease or antigen
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113
Q

Describe labeled immunoassays

A
  • designed for antigen and antibodies that may be small in size or present in very low concentrations
  • use a reactant labeled with a detection molecules to monitor the amount or specific binding that has taken place
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114
Q

What kind of things does labeled immunoassay detect?

A
  • microbial antigens
  • hormones
  • drugs
  • tumor markers
  • specific immunoglobulin
  • proteins
  • peptides
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115
Q

What is an analyte?

A

A biomarker

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116
Q

What are the varieties of labels that can be used for immunoassays?

A
  • Enzyme/colorimetric substrate
  • Chemiluminescent molecule/trigger solution
  • Fluorescent compound (fluorophore)
  • radioactive isotope (older methods)
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117
Q

How are labeled immunoassays differ from unlabeled techniques?

A

-a detection molecule (label) in the test system

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118
Q

What is a common technique to many labeled immunoassay?

A

Enzyme-mediated catalysis of a reagent substrate to generate a light signal

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119
Q

Describe heterogenous immunoassays

A

-involve physical separation of bound and free components
-most commonly involve binding tu a solid phase (polystyrene reaction wells, microparticle beads, plastic tubes)
-magnetic separation or centrifugation maybe used.

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120
Q

Describe homogenous immunoassays

A

Do not require a physical separation step

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121
Q

Describe competitive immunoassays

A
  • All the reactants are mixed together simultaneously
    -labeled and unlabeled antigen compete for a limited number of binding sites on reagent antibody
    -have high specificity
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122
Q

In competitive immunoassays,the amount of bound labeled is ________ _______ to the concentration of the labeled antigen

A

Inversely proportional

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123
Q

What does competitive immunoassays measure?

A

measure small antigens that are relatively pure (drugs and hormones)

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124
Q

What is the steps of competitive immunoassay procedure?

A

①unknown concentration of analyte in patient sample competes with labeled analyte for binding sites on immobilized antibody
②wash to remove unbound materials
③after substrate is added, a colored product (signal) is generated with an intensity proportional to the amount of enzyme- labeled analyte boundto the antibody

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125
Q

What is the principle of noncompetitive immunoassays?

A
  • patients analyte is allowed to bind with an excess amount of labeled reagents
  • Also known as capture, sandwich or immunogenic assays
126
Q

What are the steps of noncompetitive immunoassay procedure?

A

① reagent antigen immobilized on solid phase binds antibody in patient sample
② Wash to remove unbound materials ( can be eliminated in one step assays)
③ add enzyme- labeled (detection) antibody that binds to human immunoglobulin
④ wash to remove unbound materials
⑤add substrate and measure signal. Signal is directly proportional to concentration of antibody in patient sample

127
Q

Describe radioimmunoassay (RIA)

A

-first immunoassay developed ‘
- uses radioactive labels- 125I is most popular
-emits gamma radiation, which is detected by a gamma counter
-is extremely sensitive and precise
-measures trace amounts of analyte (hormones, serum protein, and drugs) that are small in size

128
Q

In radioimmunoassay (competitive), the amount of label in the bound phase is _______ _______ to the amount of patient antigen present

A

Indirectly proportional

129
Q

What are disadvantages of radioimmunoassay?

A

① working with radioactive substance (hazardous)
② disposal of low- level radioactive waste
③ short shelf for some reagents
④ testing in clinical labs is limited

130
Q

Describe enzyme immunoassays (EIAs)

A

-highly sensitive assay that uses enzymes as labels, which react with suitable substrates to produce breakdown products that maybe chromogenic, fluorogenic, or luminescent
- available in competitive and noncompetitive formats

131
Q

What are commonly used enzymes in enzyme immunoassays?

A

-alkaline phosphate
-horseradish peroxidase
- glucose-6-phosphate dehygrogenease (G6PDH)
- beta-D- galactosidase

132
Q

Briefly describe alkaline phosphatase and horseradish peroxidase

A
  • Have the highest turnover (conversion of substrate) rates
  • have a high sensitivity and are easy to detect
133
Q

What are some substances produced and detected in EIAs?

A
  • Colored or visible light
  • ultraviolet light
  • fluorescent light
  • luminescence
134
Q

Give a brief description of heterogenous EIAs

A
  • Require a step to physically separate free analyte from bound analyte
135
Q

Describe competitive heterogenous EIAs

A
  • Involve enzyme-labeled antigens competing with unlabeled patient antigen for binding sites on antibody molecules
    -have high specificity
136
Q

What is competitive heterogenous EIAs used for?

A

For measuring small antigens that are relatively pure (drugs and hormones)

137
Q

Describe noncompetitive heterogenous EIAs

A

-offer high sensitivity and specificity, simplicity and low cost

138
Q

What type of immunoassay is ELISA considerat?

A

Noncompetitive heterogenous EIAs

139
Q

Describe the enzyme-linked immunosorbent assay (ELISA)

A

-used to measure antibody production to infectious agents that are difficult to isolate and form autoantibody testing
-easily applied to point of care and home testing
-detects HIV, Hep B and Hep C, rubella virus

140
Q

What is the principle of indirect ELISA?

A

Primary antibody specific for the antigen binds to the target, and a labeled secondary antibody against the host species of the primary antibody binds to the primary antibody

141
Q

In indirect ELISA, A signal is _______ _______ to concentration of antigen in patient sample

A

Directly proportional

142
Q

What are immunoassays that detect antibodies?

A

Indirect ELISA

143
Q

What are the immunoassays that detect antigens?

A

Capture immunoassays

144
Q

Why is it referred to as “indirect” ELISA?

A

Because the enzyme-labeled reagent does not participate in the initial antigen-antibody reaction

145
Q

Describe capture immunoassays

A

-EIAs that detect antigen in patient sample
-best suited for antigens that have multiple determinants ( cytokines,proteins, tumor marker, microbial antigens)
- can be used to detect immunoglobulins of a particular class

146
Q

What is the principle of capture immunoassay?

A

Capture the antibody to an ELISA plate and allow the analyte of interest to bind to the capture antibody, then secondary antibody is added and binded

147
Q

In the capture immunoassay, enzyme activity is ______ ______ to the amount of antigen in the test sample

A

Directly proportional

148
Q

Describe biotin-avidin labeling

A
  • Biotin → vitamin B7 (vitamin H)
  • streptavidin→bacterial protein that has a strong affinity for biotin
    -biotin can be complexed to antibody and streptavidin to solid phase material to increase signaling and sensitivity in ELISAs and capture immunoassays
149
Q

What are some possible interferences with immunoassays?

A

-maybe caused by properties of the specimen, antigen interference or antibody interference
-high dose biotin supplements can cause interference in assays using biotin-SAv labeling
-false-positive and false-negatives can occur

150
Q

Describe the high dose hook effect

A
  • Antigen interference
  • excess patient antigen causes falsely decreased detection
    -analyte concentration appears to below or normal when it is actually high
151
Q

What is another name for the high-dose hook effect?

A

Postpone effect

152
Q

When does the high dose hook effect occur?

A

When there are not enough capture antibody sites for antigen binding because the majority of binding sites are filled, so the remainder of patient antigen has no place to bind and gets removed during the Wash step

153
Q

What are some interferences of antibodies?

A
  • Autoantibodies → rheumatoid factor can cause a false positive. Produced in vivo
  • heterophile antibodies→ usually cause false-negative results. Example: human and mouse antibodies (HAMA)
154
Q

What false result does cross-reactivity cause?

A

False positive

155
Q

Describe homogenous EIAs

A
  • Less sensitive than heterogenous assays
  • rapid and simple to perform
  • include EMIT and CEDIA
  • does not require washing or separation step
    -when antibody binds to specific determinant sites on the antigen, the active site on the enzyme is blocked, resulting in a measurable loss of activity
156
Q

What are homogenous EIAs used for?

A

To determine low-molecular weight analytes in serum and urine: hormones, therapeutic drugs, and drugs of abuse

157
Q

In homogenous EIAs, enzyme activity is ______ ______ to the concentration of patient antigen or hapten present in the test solution

A

Directly proportional

158
Q

Describe enzyme multiplied immunoassay technique (EMIT)

A
  • Original homogenous design
  • enzyme active site is blocked when antibody binds to enzyme.
  • does not allow substrate to react with enzyme
  • based on the principle of change in enzyme activity as specific antigen-antibody interaction occurs in solution.
159
Q

What are the steps of EMIT?

A

① reagent antigen bound to an enzyme tag, commonly G6PDH
② when reagent antibody binds to specific sites on the enzyme- antigen pair, the active site on the enzyme is blocked. Results in loss of activity

160
Q

In EMIT, the resulting enzyme activity and signal generated are ______ ______ to the concentration of patient antigen or hapten present in test solution

A

Directly proportional

161
Q

Describe cloned-enzyme donor immunoassay (CEDIA)

A
  • produce the beta-galatosidase enzyme in two parts: acceptor and donor
    -when the acceptor-donor pair combine, enzymatic activity is restored, and reagent substrate is catalyzed to generate a product signal that is measured by photometry.
162
Q

In CEDIA, enzymatic activity and signal are ______ ______ to patient antigen Ag concentration.

A

Directly proportional

163
Q

Describe chemiluminescent immunoassays

A
  • Highly sensitive
  • heterogeneous or homogeneous
  • automated immunoassays
  • detects antibodies or antigens (therapeutic drugs, steroid hormones)
  • the emission of light caused by a chemical reaction, typically an oxidation reaction, producing an excited molecule that decays back to its original ground state
164
Q

What are the chemiluminiscent molecules?

A

-acridinium esters
-ruthenium derivatives
-nitrophenol oxalates

165
Q

Describe chemiluminiscent microparticle immunoassay (CMIA)

A
  • Heterogenous assay in which patient antigen competes with chemiluminescent antigen for antibody- coated microparticles; magnets attract the particles for physical separation and washing
  • contains acridinium esters
166
Q

Describe electrochemiluminissent immunoassay (ECLIA)

A

-ruthenium label undergoes a chemical reaction at the surface of an electrode

167
Q

In CMIA, The resulting signal is ______ ______ to the antigen concentration in patient sample

A

Indirectly proportional

168
Q

Describe Fluorescent immunoassays

A
  • Use flurochrome as the label
  • these compounds absorb energy from incident light and convert it to a longer wavelength electrons return to the ground state
169
Q

What are some examples of fluorescent immunoassays?

A

-fluorescein
-rhodamine
(Isothiocyanates)

170
Q

Describe fluorescein

A

Absorbs light at 490-495 nm and emits green light at 520 nm

171
Q

Describe rhodamine

A

Absorbs light at 550 nm and emits red lights at 585 nm

172
Q

Describe fluorochromes

A

Fluorescent compound that can absorb energy from an incident source and convert that energy into light of a longer wavelength and lower energy as excited electrons return to ground state

173
Q

What is direct immunofluorescence assay procedure?

A

① antibody with a fluorescent tag is added to tissue or cells fixed onto a slide
② after incubation and a wash step, the slide is read using a fluorescence microscopic

174
Q

Describe direct immunofluorescence assays

A
  • used to identify pathogens in patient samples
  • fluorescent- labeled antibodies bind to CD antigens
  • can be used to identify lymphocytes and other cells by flow cytometry
  • used for cells or tissues
175
Q

What is the principle of indirect immunofluorescence assay?

A

① patient serum is incubated with microscopic slide to which a known antigen is attached
② The slide is washed, then an anti-human Immunoglobulin labeled with a fluorescent tag is added
③ A sandwich chis formed with the first antibody,which localizes the fluorescence
* can be used to identify patient antibody (ANAs)

176
Q

What are indirect immunoflnorescence assay used to detect?

A
  • Antibody ID
  • syphilis testing (treponemal antibodies, viral antibodies, and autoantibodies)
177
Q

Describe multiplex immunoassay (MIA)

A
  • Fluorescent immunoassay that allows multiple antibodies or antigens to be detected simultaneously
178
Q

What is the principle of multiplex immunoassay (MIA) ?

A

①to detect antibodies, patient serum is incubated with polystyrene beads conjugated to different antigens
② antibody binding is detected by addition of a fluorescent-tagged anti-human immunoglobulin
③ beads containing bound antibody are identified by flow cytometry and can be distinguished by their unique shade of red

179
Q

What is a benefit of MIA?

A
  • Allows for multiple antibodies to be detected simultaneously
180
Q

How can MIA be used?

A
  • ANA testing
    -detection of antibodies in transplant patients donor antigens
181
Q

Describe fluorescence polarization immunoassay (FPIA)

A
  • determines concentrations of therapeutic drugs and hormones
  • based on change in polarization of fluorescent light emitted from a labeled molecule when it is bound by antibody
  • if the labeled molecule is bound to antibody, the molecule emits increased amount of polarized light
    -homogenous
182
Q

Describe rapid immunoassays

A
  • membrane-based
  • single-use
  • based on immunochromatography
  • easy to perform, with fast turnaround time; ideal for point-of-care testing
183
Q

What is the rapid immunoassay procedure?

A

① patient sample is added to the test membrane and combines with labeled antigen or antibody conjugated to colored latex or colloidal gold particles
② immune complexes are formed that migrate across the membrane to produce a colored reaction

184
Q

What speciemen type is used for rapid immunoassay?

A

Urine or serum

185
Q

Describe immunochromatography

A
  • Combine an methods of rapid immunoassays into one step
186
Q

How do heterogenous immunoassays differ from homogenous immunoassays?

A

Heterogenous immunoassays require a separation step

187
Q

Why is immunefluorescence assays may be difficult to interpret?

A
  • Autofluorence of substances in serum
  • nonspecific bindingto serum proteins
  • subjectivity in reading results
188
Q

What is the principle of FPIA?

A

-fluorescent -labeled antigen competes with patient antigen for a limited number of soluble antibody-binding sites

189
Q

Why are molecular diagnostic techinques performed?

A

-to gain informationto aid in diagnosis, prognosis, and monitoring of disease as well as treatment decisions
-detect specific nucleicacid sequences in microorganisms or cells before antibody detection is possible

190
Q

What do nucleic acids do?

A

-they carry genetic informationthat codes for protein structure

191
Q

What are some primary characteristics of nucleic acids?

A

-nuclei acid complementary
- melting temperature

192
Q

What is an advantage to molecular testing?

A

Advantageous because nucleic acids can be detected earlier than antibodies during the course of an illness

193
Q

What does molecular diagnostics make important contributions to?

A
  • Therapeutic decisions
  • transplant selection
  • drug efficacy
  • forensics
    -diagnosis of infections
    -disease prognosis
194
Q

Describe DNA

A
  • Nucleic acid that carries the primary genetic informationwithin chromosomes
  • contains sugar deoxyribose
195
Q

What are the purine bases of DNA?

A

Adenine
Guanine

196
Q

What are the pyrimidine bases of DNA?

A

Cytosine
Thymine

197
Q

Describe RNA

A
  • Helps convert the genetic information encoded within DNA into proteins
  • contains sugar ribose
198
Q

What are the purine bases of RNA?

A

Adenine
Guanine

199
Q

What are the pyrimidine bases of RNA?

A

Cytosine
Uracil

200
Q

What are the types of RNA?

A
  • messenger RNA (mRNA)
  • transfer RNA (tRNA)
  • ribosomal RNA (rRNA)
  • noncoding RNAs
201
Q

What are nucleotides composed of?

A
  • Deoxyribose OR ribose sugar
  • nitrogen base
  • a phosphate group
202
Q

Describe nucleotides

A

-units that -make DNA and RNA

203
Q

What does the nitrogen base (of DNA) attach to?

A

-The 1’ carbon of deoxyribose sugar
- deoxyribose 5’ carbon may be bound to one, two, or three phosphate groups
- deoxyribose 3’ carbon carries a hydroxyl group (OH)

204
Q

What does the nitrogen base (of RNA) attach to?

A
  • ribose sugar→ carries hydroxyl groups on both the 2’ and 3’ carbons
205
Q

What are some natural modifications to the nucleotide structure?

A
  • Methylation
  • deanimation
  • additions
  • substitutions
  • other chemical modifications
    -nucleotide modifications
206
Q

What are the two strands of the DNA double helix are held together by?

A

Hydrogen bonds between their nucleotide bases.

207
Q

What is complementary to guanine in DNA?

A

Cytosine

208
Q

What is adenine complementary to in DNA?

A

Thymine

209
Q

How many hydrogen bonds are between guanine and cytosine?

A

3

210
Q

How many hydrogen bonds are between adenine and thymine?

A

2

211
Q

What is the length of double stranded DNA macromolecule measured in?

A

Base pairs

212
Q

What is the length of a single stranded RNA measured in?

A

Bases

213
Q

What is a megabase pair/megabase?

A

When there is one million base pairs/bases

214
Q

What is a chromosome?

A
  • Double helix of DNA
215
Q

How many chromosomes are in a cells nucleus?

A

-46
-two copies each of the 22 autosomes or non-sex chromosomes
-plus two copies of X chromosomes in females OR one X chromosome and Y chromosome for males

216
Q

What are genes?

A
  • Sequences of nucleotides in chromosomes that carry information for either a protein or non coding genes making up the diploid genome
217
Q

What is genome?

A

Entirety of DNA in the cell

218
Q

What is a microbiome?

A

Population of microorganisms in a host

219
Q

Describe DNA replication

A
  • The two DNA strands separate ‘’
  • each strand is a template for synthesis of a complementary strand
    -DNA polymerase is needed for synthesis
    -each new strand is synthesized in the 5’ to 3’ direction as the original double strand unwinds
    -two identical double-stranded molecules results
220
Q

What is the “origin of replication” for DNA

A
  • Defined sequence of nucleotides that start DNA replication
221
Q

DNA replication proceeds through the chromosomes and is followed by what?

A

Binary fission of the bacterial wall

222
Q

What is the cell cycle?

A

While cells divide, they undergo a series of events in which they increase in size and duplicate their DNA

223
Q

What are the four stages of the cell cycle?

A

G1
S
G2
M

224
Q

Describe S phase of the cell cycle

A
  • DNA replication occurs here
225
Q

Describe the G2 phase of the cell cycles

A

DNA complement of the cell are doubled

226
Q

Describe the M phase of the cell cycle

A

One complement of chromosomes is divided into each of two daughter cells

227
Q

Describe the G1 phase of the cell cycle

A
  • Each of the new daughter cells are here
  • movement from G1 to the S phaseis strictly regulated
228
Q

What is the lagging strand?

A
  • Copied discontinously toward the replication fork
229
Q

What is the leading strand?

A

Copied continuously in the direction of replication

230
Q

Describe RNA synthesis

A
  • Can start de novo, without primer
  • is catalyzed by RNA polymerase
  • occurs throughout the cell cycle
    -mRNA codes for the amino acid sequence of a protein
231
Q

Describe the protein synthesis and translation steps

A

① mRNA is transcribed from DNA using RNA polymerase
② The mRNA delivers the information to ribosomes, where protein synthesis takes place
③ as each amino acid is added, the peptide chain continues to grow
④this translation process is accomplished with help of tRNA,which brings in individual amino acids

232
Q

What is a phenotype?

A

Observable properties of an organism (blue eyes, height)

233
Q

What is a codon?

A

3 base recognition sequence

234
Q

Describe mutations and variants

A
  • Changes in the nucleotide sequence of DNA
  • some are associated with disease
235
Q

What is the difference between a mutation and variant?

A

-variants are inherited
-mutations are spontaneous changes in somatic cells

236
Q

Describe polymorphisms

A
  • Alterations in DNA or protein sequences shared by at least 2% of the population
    -MHC is a highly polymorphic region in the human genome
237
Q

Describe electrophoresis

A
  • involves movement of particles under the force or an electrical current
  • electrophoresis of nucleic acids commonly takes place in a semisolid matrix called a gel
  • nuclei’s acids are negatively charged and migrate toward the positive pole (anode)
  • smaller nucleic acid chains migrate faster than large chains
238
Q

What are the two gels used in electrophoresis?

A

Agarose
Polyacrylamide

239
Q

In electrophoresis, the distance from the loading well to the band will be ______ ______ to the size of the nuclei acid

A

Inversely proportional

240
Q

Describe capillary electrophoresis

A
  • A more sensitive, semi-automated method that separates particles in a gas, liquid, or gel
    -DNA chains carry a fluorescent label that is excited by a laser to produce peaks of fluorescence
241
Q

Describe molecular analysis

A
  • Nucleic acid test designed to detect changes (mutations and polymorphism) in the DNA sequence
242
Q

What are the four main approaches to nucelic acid analysis?

A

① strand cleavage methods
② hybridization methods
③ amplification methods
④ sequencing methods

243
Q

What do the strand cleavage methods use to cleave DNA at specific locations?

A

Restriction endonuclease enzymes

244
Q

Describe the strand cleavage methods of molecular analysis

A

-Separate cleaved fragments by size and charge using electro-phoresis, revealing potential variations
-used to investigate small genomes, such as those of microorganisms or plasmids
- detect mutations and polymorphisms

245
Q

Describe CRISPR-Cas9

A

-newer method that uses an enzyme to alter DNA at user-defined locations identified by a small guide RNA molecule.
- used for DNA analysis, gene therapy, and genome editing
- A strand cleavage method

246
Q

What was the first method for analysis of DNA?

A

Restriction enzyme mapping

247
Q

Describe the hybridization methods of molecular analysis.

A

-involves the binding of two complements nucleic acids, most notably a template and probe
-used on large, complex genomes

248
Q

What are some examples of hybridization methods of molecular analysis?

A
  • Southern blot
    -microarray
    -bead array
    -in situ hybridization
249
Q

What are probes?

A

Short nucleic acids that bind to complementary sequences

250
Q

What is hybridization?

A

Involves binding of two complementary strands of nucleic acids

251
Q

Describe western blot

A

-detection of proteins and protein modifications
-separated by gel electrophoresis and blotted to membrane
-probes: polyclonal or monoclonal antibodies specific for the proteins of interest

252
Q

Describe microarrays

A
  • Allow for multiple targets or sample t be analyzed simultaneously
  • The test sample is labeled with a fluorescent dye and added to a glass slide containing thousands of specific unlabeled probes
  • fluorescent spots appear where complementary binding occurs
253
Q

What are the types of microarrays?

A
  • Comparative genomic array
  • RNA expression arrays
  • SNP arrays
254
Q

Describe comparative genomic arrays

A
  • detect amplifications or deletions in DNA
255
Q

Describe RNA expression arrays

A

-detect genes that have been actively transcribed into mRNA

256
Q

Describe SNP arrays

A

Detect single nucleotide differences in test samples, some of which may be associated with a particular disease

257
Q

What is genomics?

A

Analysis of hundreds to thousands of targetsor whole genomes, rather than single genes

258
Q

Describe Bead arrays

A
  • used for multiplex detection of proteins and nuclei’s acids
    -tissue typing
    -respiratory virus panels
  • beads may have antibodies or single-stranded oligionucleotides attached
259
Q

Describe In Situ hybridization

A
  • labeled probes hybridize to tissues or cells on glass sides
260
Q

What are types of in Situ hybridization?

A
  • immunochemistry
  • fluorescence In Situ hybridization
261
Q

Describe immunohistochemistry

A
  • uses enzyme-labeled antibodies to identity protein targets
262
Q

Describe fluorescence In Situ hybridization (FISH)

A
  • Uses fluorescent labeled probes to detect specific DNA sequences
  • detects chromosome abnormalities (microdeletion and gene amplification)
263
Q

What are examples of the amplification method?

A

-PCR
- target amplification
- transcription based amplification
- strand displacement amplification
- signal amplification

264
Q

Describe the polymerase chain reaction (PCR) procedure

A

① denaturation → DNA is separated into single strands by heat (95°C)
② annealing → primer attach (60°C)
③ extension→ complementary nucleotides are added to 3’ end (~70°C)

265
Q

Describe Target amplification

A

-amplification of PCR products, under optimal conditions, each cycle results in doubling product
-in this manner, a target sequence present that are fewer than 100 copies can be detected
-resulting DNA fragments (amplicons) are detected using various methods

266
Q

Today, what is used to detect amplicons?

A

Labeled nucleic acid probes

267
Q

Describe PCR

A

-in vitro DNA replication procedure

268
Q

What is key for specificity of a PCR reaction?

A

Oligonucleotide primers→ synthetic single-stranded nuclei acids, usually 18 to 30 b in length

269
Q

What are some premixed PCR reagents?

A
  • Deoxyribonucleotides, primers, and buffer
270
Q

What is the instrument used to carry out the amplification program?

A

Thermal cycler
- chamber-based
- block-based

271
Q

What is a common approach used to detect mutations and polymorphisms?

A

SSP-PCR

272
Q

Describe quantitative PCR (qPCR)

A

-determines the amount of a specific sequence in the original sample
- Both DNA and RNA target can be measured by qPCR

273
Q

What are the four probe types of qPCR?

A
  • Fluorescence resonance energy transfer (FRET)
  • Taqman
  • molecular beacon
  • scorpion probes
274
Q

What are some commonly used application of qPCR?

A

-detection of microorganisms, especially viruses and other pathogens, tumor associated gene expression and tissue typing

275
Q

Describe reverse- transcriptase PCR (RT-PCR)

A
  • Amplifies cDNA made from an RNA template
  • HIV, HCV
276
Q

Describe digital droplet PCR

A
  • Provides absolute quantification of PCR product
277
Q

How is digital PCR applied?

A
  • Analysis of gene copy number variation
  • detecting rare mutations
  • infections disease
278
Q

Describe transcription-based amplification (TMA)

A

-RNA is the target as well as the primary product
-product detected by chemiluminescence with acridinium ester
- isothermal process
- useful in testing for RNA viruses (HIV), chlamydia trachomata’s and cytomegalovirus
- temperature cycling not required

279
Q

Describe probe amplification

A

-number of target nucleic acid sequences does not change
- instead, primers are extended or ligated into many copies of detectable probes

280
Q

What are some examples of probe amplification

A
  • Strand displacement amplification (SDA)
  • loop-mediated isothermal amplification (LAMP)
    -molecular inversion probe (MIP)
281
Q

Describe strand displacement amplification (SDA)

A
  • after initial denaturation step,the reaction proceeds at one temperature
  • nick formed in strand by restriction endonucleus enzyme
282
Q

Describe loop-mediated isothermal amplification (LAMP)

A
  • high specifics and sensitivity for target DNA
  • PCR primers carry sequences at 5’ end that will self hybridize, forming loops that self prime in cyclic manner
  • advantage→ shortened Run time
283
Q

What is LAMP used to detect?

A
  • HIV
  • cytomegalovirus
  • staphylococcus aureus
  • E. coli
284
Q

Describe molecular inversion probe (MIP)

A

-highly sensitive
- probe ends bind to target sequences so that, in the presence of target, probes ends are brought together and ligated to form circles

285
Q

What is MIP used to detects?

A
  • staphylococcus aureus
  • streptococcus mutans
  • influenza virus
  • RNA typing
286
Q

Describe signal amplification

A
  • large amounts of signals are bound to the target sequences
    -example: branched DNA
287
Q

Describe branched DNA (bDNA)

A
  • has been used to detect certain viruses
  • series of short, single stranded DNA probes are used to capture the target nucleic acid and to bind to multiple reporter molecules, loading target nucleic acid with signal ‘
288
Q

Describe DNA sequencing

A
  • Direct determination of the order of ‘nucleotides in a DNA chain
  • The most specific method of identifying genetic mutations or polymorphisms
  • methods include Sanger sequencing, prosequencing, and next- generation sequencing (NGS)
289
Q

Describe Sanger sequencing

A
  • uses all components of PCR plus fluorescent- labeled dideoxynucleatides (ddNTPs) to synthesize DNA complementary to the target
  • when correct ddNTPs is added, DNA synthesis stops and fragments of varying sizes are produced
  • results are read by gel or capillary electrophoresis
    -Less sensitive than other methods
290
Q

What is another name for Sanger sequencing?

A

Chain termination sequencing

291
Q

Describe pyrosequencing

A
  • Based on the generation of light when nucleotides are added to a growing strand of DNA
    -not as versatile as Sanger sequencing, but less labor intensive
292
Q

Describe next-generation sequencing (NGS)

A
  • Can sequence large numbers of templates simultaneously (massively parallel sequencing)
  • using bioinformatics, the short sequences are assembled to determine the complete sequence, which is compared to known database
  • more error prone than Sanger sequencing
293
Q

What are applications of next-generation sequencing (NGS)?

A
  • ID of genetic variations in inherited diseases and tumor cells
  • HLA allele typing
  • analysis of mixed microbial populations
294
Q

Describe ion conductance sequencing

A

Nucleotide order is determined through release of hydrogen ions by DNA synthesis

295
Q

Describe reversible arse terminator technology

A
  • Libraries carry sequences complementary to ‘ immobilized primers on a solid support ( flow cell)
    -after introduction of flow cell, libraries are amplified by bridge PCR, forming clusters of templates across flow cell
296
Q

Describe whole exonerated sequencing

A

-used to identify variants responsible for inherited disease conditions

297
Q

Describe whole genome sequencing

A

-Primarily research tools
-massive parallel sequencing has the capacity to investigate all known genetic loci alterations

298
Q

Describe targeted libraries (sequencing)

A

Selected genes or gene regions

299
Q

Describe bioinformatics

A

Uses information technology to analyze the vast amount of data generated by NGS testing and interprets the data for clinical relevance by comparison with uncrown database

300
Q

What is the basis of all molecular diagnostic testing?

A

The high specificity of detection of nucleic acid sequences through complementary base pairing

301
Q

.between promoter, cytosine, S phase, andprimer, which one is associated with RNA synthesis only?

A

Promoter

302
Q

What is the speed of a nuclei acids migrating in gel electrophoresis determined by?

A

Site of the nucleic acid

303
Q

What is the function of restriction endonucleuses?

A
  • The cleave DNA at specific sites
304
Q

What technique uses RNA-guided enzymes?

A

CRISPR

305
Q

To what does in Situ hybridization refer?

A

Probes react with intact cells within tissues

306
Q

What is the purpose of an amplification control in qPCR

A

To avoid false negatives

307
Q

In NGS, what is coverage?

A

The number of times a region is sequenced

308
Q

What are some hybridization techniques?

A

Microarray technology
Fluorescent In Situ hybridization

309
Q

What is the principle of homogenous EIA?

A

-patient antigen and labeled antigen react with reagent antibody in solution. Enzyme label is inactivated when reagent antigen binds to antibody
- No physical separation step

310
Q

What is the principle of heterogenous EIA?

A
  • Requires solid-phase material to allow for antigen or antibody binding. Includes both competitive and non n competitive immunoassay designs
311
Q

What is the principle of EMIT?

A

-reagent antigen is bound to an enzyme tag. If reagent antibody binds to the enzyme-antigen pair, enzyme activity is blocked.otherwise, enzyme is active -active enzyme catalyzes reduction of NAD+ to NADH

312
Q

What is the principle of rapid immunochromatographic?

A

-patient sample is added to test strip and migrates through the strip. Patient antigen complexes to antibody-labeled particles or competes with antigen- labeled particles across individual test lanes at unique detection zones

MLT immunology (7 decks)

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