Unit 3 Flashcards
1
Q
What is precipitation?
A
Combine soluble antigen and stable antibody to produce insoluble complexes
2
Q
What is agglutination
A
Antigen -antibody complexes cross-link, causing clumping
3
Q
Why are precipitation and agglutination
Considered unlabeled immunoassays?
A
Because a marker label is not needed to detect the reaction
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
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
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
7
Q
What is serology?
A
Study of fluid components in the blood, especially antibodies
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
9
Q
What Color tube tops are serum collected in
A
Red top or tiger top
10
Q
What is the additive for the red tube top?
A
None
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
12
Q
What is the additive of the red and grey tiger top?
A
Gel barrier
13
Q
Why would a speciemen be rejected?
A
- hemolyzed
- clotted speciemen
- wrong tube or container
- tube not labeled
- inadequate quantity
- tube mislabeled
14
Q
What are the 3 phases of laboratory testing?
A
- preanalytical
-analytical
-postanalytical
15
Q
What are examples of preanalytical testing?
A
- Speciemen collection
- Transport
- processing
16
Q
What are some examples of analytical testing?
A
Testing
17
Q
What are some examples of postanalytical phase
A
- Testing results
- transmission
-interpretation
-follow up - retesting
18
Q
What phases of the laboratory testing constitutes for 90% of the errors that occur?
A
- Pre-analytical
- post-analytical
19
Q
What are the three phases of antigen/antibody complexes?
A
① primary phenomenon
② secondary phenomenon
③tertiary phenomenon
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
21
Q
Describe the secondary phenomenon phase of antigen/antibody complexes
A
- Precipitation, agglutination, and complement fixation
- easily detectable
- most serological tests based
22
Q
Describe the tertiary phenomenon phase of antigen/antibody complexes
A
-inflammation, phagocytosis, depression of immune complexes,immune adherence, chemotaxis (all in vivo)
23
Q
What is complement fixation?
A
Triggers the classical complement pathway to detect antibody or antigen in the patients serum
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
25
What does avidity measure?
Overall stability of an antigen- antibody complex
26
The more bonds that form between antigen and antibody, the higher the ________
Avidity
27
What is the law of mass action?
Free reactants are in equilibrium with bound
Reactants
28
What is the law of mass action equation?
K=[Ag/Ab]/[Ab][Ag]
29
In law of mass action, what does the Kvalue depend on?
The strength of binding between antibody and antigen
30
The higher the K value……
-the larger the amount of antigen-antibody complexes
- the more visible or easily detectable the reaction
31
In the law of mass action equation, what does [Ag/Ab] represent?
Concentration of the antigen- antibody complex (mol/L)
32
In the law of mass action equation, what does [Ab] represent?
Concentration of free antibody (mol/L)
33
In the law of mass action equation, what does [Ag] represent?
Concentration of free antigen (mol/L)
34
What does precipitation require antigens and antibodies to have?
- Multiple binding sitesfor one another
-equivalent concentrations
35
What are the 3 groups of the precipitation curve?
- zone of equivalence
- prozone
- postpone
36
For optimal precipitation, reactions must be run in what phase of the precipitation curve?
Zone of equivalent
37
What is the zone of equivalence of the precipitation curve?
- Number of multi-valent sites of antigen and antibody are approximately equal
38
Describe the prozone of precipitation curve
- Antibody excess
- antigen combine with one or two antibodies
- No cross linking
-
39
What could cause a false negative result in the prozone phase of the precipitation curve?
- High antibody concentration
40
What should be done if a false negative result is suspected in the prozone phase of precipitation curve?
- Diluting out the antibody and performing the test again may produce a positive result
41
Describe the postzone phase of the precipitation curve
-antigen excess
- small aggregates are surrounded by excess antigen
- No lattice network formed
42
What could cause a false negative result in the postzone phase of the precipitation curve?
-presence of a small amount of antibody
43
What should be done is false negative results occur in the postzone phase of the precipitation curve?
-test may be repeated a week later with another speciemen to give more time for antibody production
44
What are immunoturbidimetry and Nephleometry used to test for?
- Complement
- C-reactive protein
- immunoglobulins
- other serum protein
45
What is the sensitivity of immunoturbidity and nephelometry?
1-10 ug AB/ML
46
What is the principle of immunoturbidity and Nephelometry?
Light that is scattered at an angle is measured, indicating the amount of antigen or antibody present
47
What is radial immunodiffusion used to test for?
- Complement
- immunoglobulins
48
What is the sensitivity of radial immunodiffusion?
10-50 ug AB/ML
49
What is the principle of radial immunodiffusion?
Antigen diffuses out into gel that is infused with antibody. Measurement of the radius indicates concentration of antigen
50
What is Ouchterlony double diffusion used to test for?
- Complex antigens, such as fungal antigens
51
What is the sensitivity of the Ouchterlony double diffusion?
20-200 ug AB/ML
52
What is the principle of Ouchterlony double diffusion?
Both antigen and antibody diffuse out from wells in a gel. The lines of precipitate formed indicate the relationship of antigens
53
What does immunoelectrophoresis test for?
Differentiation of serum proteins
54
What is the sensitivity of immunoelectrophoresis?
20-200 ug AB/ML
55
What is the principle of immunoelectrophoresis?
Electrophoresis of serum is followed by diffusion of antibody from the wells
56
What is immunofixation electrophoresisuse to test for?
Over or under production of antibody
57
What is the sensitivity of immunofixation electrophoresis?
Varies
58
What is the principle of immunofixation electrophoresis
Electrophoresis of serum is followed by direct application of antibody to the gel
59
What machines could be used for immunoturbidity?
- Spectrophotometer
-automated clinical chemistry analyzer
60
What machine is used for nephelometry?
Nephelotometer
61
Describe a nephelotometer
- 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
62
What does agarose gel do?
- Helps stabilize the diffusion process and allow visualization of the preciptin band
63
What is passive immunodiffusion?
-when no electric currentis used to speed up the migration of the reactants
64
What can affect the rate of diffusion in passive immunodiffusion?
- Size of particle
- temperature
- gel viscosity
65
What is the end point (Mancini) method?
Reaction goes to completion
66
What is the kinetic (Fahey) method?
-faster
-measurements taken before reaction is complete
67
What is the RID end point method results?
The square of the diameter is proportional to the antigen concentration
68
What are the three possible patterns of Ouchterlony diffusion?
- Identity (arc)
- partial identity (one line extends)
- nonidentity (line cross X)
69
Immunofixation electrophoresis (IFE) is
Performed to visualize what?
Increased or decreased productionof antibody classes and to differentiate monoclonal and polyclonal immunoglobulins
70
What are agglutins?
- Produced by antibodies
71
What are the two steps of agglutination?
① sensitization
② lattice formation
72
Describe the sensitization step of agglutination
- initial bonding
- antigen and antibody unite through bonding of antigenic determinant sites
- fast and reversible
73
Describe the lattice formation of agglutination
- Creation of large aggregates
-visible aggregates form as multiple antigen and antibody molecules bind to create a stable lattice
74
What is sensitization of agglutination affected by?
-by nature of the antigens on agglutinating ' particles
- RBC and bacterial cells have a slightly negative surface charge
- class of immunoglobulin
75
What occurs if epitopes are sparse in the sensitization step of agglutination?
- Less likely to interact with antibody
76
What is Coombs reagent?
- Anti-human immunoglobulin
- frequently used in blood bank
- binds to Fc portion of IgG
77
Briefly describe immunoturbidity
- automated methods
- measures reduction in light intensity
78
Briefly describe nephelometry
- Automated methods
-measures light scatter as immune complexes form
79
What are the 4 types of agglutination reactions?
① direct agglutination
② passive agglutination
③ reverse passive agglutination
④ agglutination inhibition
80
Describe direct agglutination
- Uses particles with naturally occurring antigens to test for antibodies in patient serum
81
What does direct agglutination test for?
- Bacteria (Widal test)
- RBCs
82
Describe the Widal test
- A rapid screening test used to determine the possibility of typhoid fever
- uses Salmonella O (somatic) and H (flagellar) antigens
- direct agglutination
83
What test are ran for RBCs?
- Hemagglutination
- ABO typing
(direct agglutination)
84
Describe passive agglutination
-uses particles that are coated with antigens not normally found on their surfaces: erythrocytes, gelatin, latex, and silicates
85
What does passive agglutination test for?
- RA
- antibodies to Group A streptococcus antigens
- antibodies to viruses such as rubella
- heterophile antibody in infections mononucleosis
86
Describe the reverse passive agglutination process
① antibody is attached to the carrier particle
② agglutination occurs is antigenis present in the patient sample
87
What can reverse passive agglutination test for?
- 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
88
Describe agglutination inhibition
-based on competitionbetween particle and soluble antigens for limited antibody- combining sites
- used to detect haptens antigens (illicit drugs)
- lack of agglutination= positive reaction
89
What is an example of an agglutination inhibition procedure?
- hemagglationation inhibition → uses RBCs
-used to detect antibodies to certain viruses that can bind to RBCs and agglutinate them
90
What virus can hemagglutination inhibition be used to test for?
- Rubella
-influenza
-respiratory syncytial virus)
91
What is the principle of direct agglutination?
-antigen is naturallyfound on a particle
92
Describe the results of direct agglutination
Agglutination indicates the presence of patient antibody
93
What is the principle of passive (indirect) agglutination?
Particles are coated with antigens not normally found on their surfaces
94
Describe the results of passive (indirect) agglutination
Agglutination indicates the presence of patient antibody
95
What is the principle reverse passive agglutination?
Particles are coated with reagent antibody
96
Describe the results of reverse passive agglutination
Agglutination indicates the presence of patient antigen
97
What is the principle of agglutination inhibition?
- Haptens are attached to carrier particles.
- particles compete with patient antigens for a limited number of antibody sites
98
Describe the results of agglutination inhibition
Lack of agglutination is a positive test, indicating the presence of patient antigen
99
What is the principle of hemagglutination inhibition?
RBCs spontaneously agglutinate in presence of certain viruses
100
Describe the results of hemagglutination inhibition
Lack of agglutination is a positive test, indicating the presence of patient antibody
101
Describe the monospot test
Used to detect the heterophile antibody characteristics of infections mononucleosis by its ability to agglutinate horse or bovine RBCs
102
Describe cold agglutins
-Produced by patients with certain autoimmune disorders, malignancies or infections, are antibodies that agglutinate human type O RBCs when incubated at cold temperatures
103
Describe PETINIA
"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
104
Describe the results of PETINIA
- 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
105
The amount of turbidity is ________ ________ to the concentration of the drug analyte in sample of PETINIA
Inversely proportional
106
How can most cross-reactivity be avoided?
Through use of monoclonal antibody directed against an antigenic determinant that is unique to antigen
107
What are advantages of agglutination reactions?
- rapidity
- easy to perform
- no expensive equipment
- relatively sensitive
108
When does maximum binding at antigen and antibody occur?
When the aggregate number of multivalent sites of antigen and antibody are approximately equal
109
Describe agglutination reaction with IgM
- able to mediate lattice formation with antigen without additional enhancement
110
Describe the agglutination reaction with IgG
-requires enhancement techniques that vary physicochemical conditions and the addition of an anti-human immunoglobulin (Coombs reagent) in order to see a visible reaction
111
How does precipitation differ from agglutination?
Precipitation involves a soluble antigen, whereas agglutination involves a particulate antigen
112
What is imperative for optimal agglutination?
- 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
113
Describe labeled immunoassays
- 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
114
What kind of things does labeled immunoassay detect?
- microbial antigens
- hormones
- drugs
- tumor markers
- specific immunoglobulin
- proteins
- peptides
115
What is an analyte?
A biomarker
116
What are the varieties of labels that can be used for immunoassays?
- Enzyme/colorimetric substrate
- Chemiluminescent molecule/trigger solution
- Fluorescent compound (fluorophore)
- radioactive isotope (older methods)
117
How are labeled immunoassays differ from unlabeled techniques?
-a detection molecule (label) in the test system
118
What is a common technique to many labeled immunoassay?
Enzyme-mediated catalysis of a reagent substrate to generate a light signal
119
Describe heterogenous immunoassays
-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.
120
Describe homogenous immunoassays
Do not require a physical separation step
121
Describe competitive immunoassays
- 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
122
In competitive immunoassays,the amount of bound labeled is ________ _______ to the concentration of the labeled antigen
Inversely proportional
123
What does competitive immunoassays measure?
measure small antigens that are relatively pure (drugs and hormones)
124
What is the steps of competitive immunoassay procedure?
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
125
What is the principle of noncompetitive immunoassays?
- patients analyte is allowed to bind with an excess amount of labeled reagents
- Also known as capture, sandwich or immunogenic assays
126
What are the steps of noncompetitive immunoassay procedure?
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
Describe radioimmunoassay (RIA)
-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
In radioimmunoassay (competitive), the amount of label in the bound phase is _______ _______ to the amount of patient antigen present
Indirectly proportional
129
What are disadvantages of radioimmunoassay?
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
Describe enzyme immunoassays (EIAs)
-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
What are commonly used enzymes in enzyme immunoassays?
-alkaline phosphate
-horseradish peroxidase
- glucose-6-phosphate dehygrogenease (G6PDH)
- beta-D- galactosidase
132
Briefly describe alkaline phosphatase and horseradish peroxidase
- Have the highest turnover (conversion of substrate) rates
- have a high sensitivity and are easy to detect
133
What are some substances produced and detected in EIAs?
- Colored or visible light
- ultraviolet light
- fluorescent light
- luminescence
134
Give a brief description of heterogenous EIAs
- Require a step to physically separate free analyte from bound analyte
135
Describe competitive heterogenous EIAs
- Involve enzyme-labeled antigens competing with unlabeled patient antigen for binding sites on antibody molecules
-have high specificity
136
What is competitive heterogenous EIAs used for?
For measuring small antigens that are relatively pure (drugs and hormones)
137
Describe noncompetitive heterogenous EIAs
-offer high sensitivity and specificity, simplicity and low cost
138
What type of immunoassay is ELISA considerat?
Noncompetitive heterogenous EIAs
139
Describe the enzyme-linked immunosorbent assay (ELISA)
-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
What is the principle of indirect ELISA?
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
In indirect ELISA, A signal is _______ _______ to concentration of antigen in patient sample
Directly proportional
142
What are immunoassays that detect antibodies?
Indirect ELISA
143
What are the immunoassays that detect antigens?
Capture immunoassays
144
Why is it referred to as “indirect" ELISA?
Because the enzyme-labeled reagent does not participate in the initial antigen-antibody reaction
145
Describe capture immunoassays
-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
What is the principle of capture immunoassay?
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
In the capture immunoassay, enzyme activity is ______ ______ to the amount of antigen in the test sample
Directly proportional
148
Describe biotin-avidin labeling
- 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
What are some possible interferences with immunoassays?
-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
Describe the high dose hook effect
- Antigen interference
- excess patient antigen causes falsely decreased detection
-analyte concentration appears to below or normal when it is actually high
151
What is another name for the high-dose hook effect?
Postpone effect
152
When does the high dose hook effect occur?
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
What are some interferences of antibodies?
- 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
What false result does cross-reactivity cause?
False positive
155
Describe homogenous EIAs
- 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
What are homogenous EIAs used for?
To determine low-molecular weight analytes in serum and urine: hormones, therapeutic drugs, and drugs of abuse
157
In homogenous EIAs, enzyme activity is ______ ______ to the concentration of patient antigen or hapten present in the test solution
Directly proportional
158
Describe enzyme multiplied immunoassay technique (EMIT)
- 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
What are the steps of EMIT?
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
In EMIT, the resulting enzyme activity and signal generated are ______ ______ to the concentration of patient antigen or hapten present in test solution
Directly proportional
161
Describe cloned-enzyme donor immunoassay (CEDIA)
- 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
In CEDIA, enzymatic activity and signal are ______ ______ to patient antigen Ag concentration.
Directly proportional
163
Describe chemiluminescent immunoassays
- 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
What are the chemiluminiscent molecules?
-acridinium esters
-ruthenium derivatives
-nitrophenol oxalates
165
Describe chemiluminiscent microparticle immunoassay (CMIA)
- 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
Describe electrochemiluminissent immunoassay (ECLIA)
-ruthenium label undergoes a chemical reaction at the surface of an electrode
167
In CMIA, The resulting signal is ______ ______ to the antigen concentration in patient sample
Indirectly proportional
168
Describe Fluorescent immunoassays
- 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
What are some examples of fluorescent immunoassays?
-fluorescein
-rhodamine
(Isothiocyanates)
170
Describe fluorescein
Absorbs light at 490-495 nm and emits green light at 520 nm
171
Describe rhodamine
Absorbs light at 550 nm and emits red lights at 585 nm
172
Describe fluorochromes
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
What is direct immunofluorescence assay procedure?
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
Describe direct immunofluorescence assays
- 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
What is the principle of indirect immunofluorescence assay?
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
What are indirect immunoflnorescence assay used to detect?
- Antibody ID
- syphilis testing (treponemal antibodies, viral antibodies, and autoantibodies)
177
Describe multiplex immunoassay (MIA)
- Fluorescent immunoassay that allows multiple antibodies or antigens to be detected simultaneously
178
What is the principle of multiplex immunoassay (MIA) ?
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
What is a benefit of MIA?
- Allows for multiple antibodies to be detected simultaneously
180
How can MIA be used?
- ANA testing
-detection of antibodies in transplant patients donor antigens
181
Describe fluorescence polarization immunoassay (FPIA)
- 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
Describe rapid immunoassays
- membrane-based
- single-use
- based on immunochromatography
- easy to perform, with fast turnaround time; ideal for point-of-care testing
183
What is the rapid immunoassay procedure?
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
What speciemen type is used for rapid immunoassay?
Urine or serum
185
Describe immunochromatography
- Combine an methods of rapid immunoassays into one step
186
How do heterogenous immunoassays differ from homogenous immunoassays?
Heterogenous immunoassays require a separation step
187
Why is immunefluorescence assays may be difficult to interpret?
- Autofluorence of substances in serum
- nonspecific bindingto serum proteins
- subjectivity in reading results
188
What is the principle of FPIA?
-fluorescent -labeled antigen competes with patient antigen for a limited number of soluble antibody-binding sites
189
Why are molecular diagnostic techinques performed?
-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
What do nucleic acids do?
-they carry genetic informationthat codes for protein structure
191
What are some primary characteristics of nucleic acids?
-nuclei acid complementary
- melting temperature
192
What is an advantage to molecular testing?
Advantageous because nucleic acids can be detected earlier than antibodies during the course of an illness
193
What does molecular diagnostics make important contributions to?
- Therapeutic decisions
- transplant selection
- drug efficacy
- forensics
-diagnosis of infections
-disease prognosis
194
Describe DNA
- Nucleic acid that carries the primary genetic informationwithin chromosomes
- contains sugar deoxyribose
195
What are the purine bases of DNA?
Adenine
Guanine
196
What are the pyrimidine bases of DNA?
Cytosine
Thymine
197
Describe RNA
- Helps convert the genetic information encoded within DNA into proteins
- contains sugar ribose
198
What are the purine bases of RNA?
Adenine
Guanine
199
What are the pyrimidine bases of RNA?
Cytosine
Uracil
200
What are the types of RNA?
- messenger RNA (mRNA)
- transfer RNA (tRNA)
- ribosomal RNA (rRNA)
- noncoding RNAs
201
What are nucleotides composed of?
- Deoxyribose OR ribose sugar
- nitrogen base
- a phosphate group
202
Describe nucleotides
-units that -make DNA and RNA
203
What does the nitrogen base (of DNA) attach to?
-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
What does the nitrogen base (of RNA) attach to?
- ribose sugar→ carries hydroxyl groups on both the 2' and 3' carbons
205
What are some natural modifications to the nucleotide structure?
- Methylation
- deanimation
- additions
- substitutions
- other chemical modifications
-nucleotide modifications
206
What are the two strands of the DNA double helix are held together by?
Hydrogen bonds between their nucleotide bases.
207
What is complementary to guanine in DNA?
Cytosine
208
What is adenine complementary to in DNA?
Thymine
209
How many hydrogen bonds are between guanine and cytosine?
3
210
How many hydrogen bonds are between adenine and thymine?
2
211
What is the length of double stranded DNA macromolecule measured in?
Base pairs
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What is the length of a single stranded RNA measured in?
Bases
213
What is a megabase pair/megabase?
When there is one million base pairs/bases
214
What is a chromosome?
- Double helix of DNA
215
How many chromosomes are in a cells nucleus?
-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
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What are genes?
- Sequences of nucleotides in chromosomes that carry information for either a protein or non coding genes making up the diploid genome
217
What is genome?
Entirety of DNA in the cell
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What is a microbiome?
Population of microorganisms in a host
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Describe DNA replication
- 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
What is the “origin of replication” for DNA
- Defined sequence of nucleotides that start DNA replication
221
DNA replication proceeds through the chromosomes and is followed by what?
Binary fission of the bacterial wall
222
What is the cell cycle?
While cells divide, they undergo a series of events in which they increase in size and duplicate their DNA
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What are the four stages of the cell cycle?
G1
S
G2
M
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Describe S phase of the cell cycle
- DNA replication occurs here
225
Describe the G2 phase of the cell cycles
DNA complement of the cell are doubled
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Describe the M phase of the cell cycle
One complement of chromosomes is divided into each of two daughter cells
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Describe the G1 phase of the cell cycle
- Each of the new daughter cells are here
- movement from G1 to the S phaseis strictly regulated
228
What is the lagging strand?
- Copied discontinously toward the replication fork
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What is the leading strand?
Copied continuously in the direction of replication
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Describe RNA synthesis
- 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
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Describe the protein synthesis and translation steps
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
What is a phenotype?
Observable properties of an organism (blue eyes, height)
233
What is a codon?
3 base recognition sequence
234
Describe mutations and variants
- Changes in the nucleotide sequence of DNA
- some are associated with disease
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What is the difference between a mutation and variant?
-variants are inherited
-mutations are spontaneous changes in somatic cells
236
Describe polymorphisms
- 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
Describe electrophoresis
- 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
What are the two gels used in electrophoresis?
Agarose
Polyacrylamide
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In electrophoresis, the distance from the loading well to the band will be ______ ______ to the size of the nuclei acid
Inversely proportional
240
Describe capillary electrophoresis
- 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
Describe molecular analysis
- Nucleic acid test designed to detect changes (mutations and polymorphism) in the DNA sequence
242
What are the four main approaches to nucelic acid analysis?
① strand cleavage methods
② hybridization methods
③ amplification methods
④ sequencing methods
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What do the strand cleavage methods use to cleave DNA at specific locations?
Restriction endonuclease enzymes
244
Describe the strand cleavage methods of molecular analysis
-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
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Describe CRISPR-Cas9
-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
What was the first method for analysis of DNA?
Restriction enzyme mapping
247
Describe the hybridization methods of molecular analysis.
-involves the binding of two complements nucleic acids, most notably a template and probe
-used on large, complex genomes
248
What are some examples of hybridization methods of molecular analysis?
- Southern blot
-microarray
-bead array
-in situ hybridization
249
What are probes?
Short nucleic acids that bind to complementary sequences
250
What is hybridization?
Involves binding of two complementary strands of nucleic acids
251
Describe western blot
-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
Describe microarrays
- 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
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What are the types of microarrays?
- Comparative genomic array
- RNA expression arrays
- SNP arrays
254
Describe comparative genomic arrays
- detect amplifications or deletions in DNA
255
Describe RNA expression arrays
-detect genes that have been actively transcribed into mRNA
256
Describe SNP arrays
Detect single nucleotide differences in test samples, some of which may be associated with a particular disease
257
What is genomics?
Analysis of hundreds to thousands of targetsor whole genomes, rather than single genes
258
Describe Bead arrays
- 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
Describe In Situ hybridization
- labeled probes hybridize to tissues or cells on glass sides
260
What are types of in Situ hybridization?
- immunochemistry
- fluorescence In Situ hybridization
261
Describe immunohistochemistry
- uses enzyme-labeled antibodies to identity protein targets
262
Describe fluorescence In Situ hybridization (FISH)
- Uses fluorescent labeled probes to detect specific DNA sequences
- detects chromosome abnormalities (microdeletion and gene amplification)
263
What are examples of the amplification method?
-PCR
- target amplification
- transcription based amplification
- strand displacement amplification
- signal amplification
264
Describe the polymerase chain reaction (PCR) procedure
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
Describe Target amplification
-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
Today, what is used to detect amplicons?
Labeled nucleic acid probes
267
Describe PCR
-in vitro DNA replication procedure
268
What is key for specificity of a PCR reaction?
Oligonucleotide primers→ synthetic single-stranded nuclei acids, usually 18 to 30 b in length
269
What are some premixed PCR reagents?
- Deoxyribonucleotides, primers, and buffer
270
What is the instrument used to carry out the amplification program?
Thermal cycler
- chamber-based
- block-based
271
What is a common approach used to detect mutations and polymorphisms?
SSP-PCR
272
Describe quantitative PCR (qPCR)
-determines the amount of a specific sequence in the original sample
- Both DNA and RNA target can be measured by qPCR
273
What are the four probe types of qPCR?
- Fluorescence resonance energy transfer (FRET)
- Taqman
- molecular beacon
- scorpion probes
274
What are some commonly used application of qPCR?
-detection of microorganisms, especially viruses and other pathogens, tumor associated gene expression and tissue typing
275
Describe reverse- transcriptase PCR (RT-PCR)
- Amplifies cDNA made from an RNA template
- HIV, HCV
276
Describe digital droplet PCR
- Provides absolute quantification of PCR product
277
How is digital PCR applied?
- Analysis of gene copy number variation
- detecting rare mutations
- infections disease
278
Describe transcription-based amplification (TMA)
-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
Describe probe amplification
-number of target nucleic acid sequences does not change
- instead, primers are extended or ligated into many copies of detectable probes
280
What are some examples of probe amplification
- Strand displacement amplification (SDA)
- loop-mediated isothermal amplification (LAMP)
-molecular inversion probe (MIP)
281
Describe strand displacement amplification (SDA)
- after initial denaturation step,the reaction proceeds at one temperature
- nick formed in strand by restriction endonucleus enzyme
282
Describe loop-mediated isothermal amplification (LAMP)
- 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
What is LAMP used to detect?
- HIV
- cytomegalovirus
- staphylococcus aureus
- E. coli
284
Describe molecular inversion probe (MIP)
-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
What is MIP used to detects?
- staphylococcus aureus
- streptococcus mutans
- influenza virus
- RNA typing
286
Describe signal amplification
- large amounts of signals are bound to the target sequences
-example: branched DNA
287
Describe branched DNA (bDNA)
- 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
Describe DNA sequencing
- 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
Describe Sanger sequencing
- 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
What is another name for Sanger sequencing?
Chain termination sequencing
291
Describe pyrosequencing
- 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
Describe next-generation sequencing (NGS)
- 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
What are applications of next-generation sequencing (NGS)?
- ID of genetic variations in inherited diseases and tumor cells
- HLA allele typing
- analysis of mixed microbial populations
294
Describe ion conductance sequencing
Nucleotide order is determined through release of hydrogen ions by DNA synthesis
295
Describe reversible arse terminator technology
- 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
Describe whole exonerated sequencing
-used to identify variants responsible for inherited disease conditions
297
Describe whole genome sequencing
-Primarily research tools
-massive parallel sequencing has the capacity to investigate all known genetic loci alterations
298
Describe targeted libraries (sequencing)
Selected genes or gene regions
299
Describe bioinformatics
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
What is the basis of all molecular diagnostic testing?
The high specificity of detection of nucleic acid sequences through complementary base pairing
301
.between promoter, cytosine, S phase, andprimer, which one is associated with RNA synthesis only?
Promoter
302
What is the speed of a nuclei acids migrating in gel electrophoresis determined by?
Site of the nucleic acid
303
What is the function of restriction endonucleuses?
- The cleave DNA at specific sites
304
What technique uses RNA-guided enzymes?
CRISPR
305
To what does in Situ hybridization refer?
Probes react with intact cells within tissues
306
What is the purpose of an amplification control in qPCR
To avoid false negatives
307
In NGS, what is coverage?
The number of times a region is sequenced
308
What are some hybridization techniques?
Microarray technology
Fluorescent In Situ hybridization
309
What is the principle of homogenous EIA?
-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
What is the principle of heterogenous EIA?
- Requires solid-phase material to allow for antigen or antibody binding. Includes both competitive and non n competitive immunoassay designs
311
What is the principle of EMIT?
-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
What is the principle of rapid immunochromatographic?
-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
