2.1 Enzyme Immunoassay Flashcards
Labeled immunoassays are designed for antigens and antibodies that may be?
- Small
- Present in very large concentration
- Present in very low concentration
a. 1,2,3
b. 1,2
c. 1,3
d. 1 Only
c
This is being determined in labelled immunoassays
a. Solute
b. analyte
c. enzyme
d. CPR
b
Analyte is being determined directly using a labeled reactant to detect whether or not specific binding has taken place
T or F?
F
Detection is done indirectly since instead of directly identifying the antigen and antibodies binding in their raw form, labeled reactant is used to label this binding to know if binding was taken place
ONE reactant, either the antigen or the antibody, is labeled with a marker so that the amount of binding can be monitored?
T or F
T
Labels used in the labeled immunoassay should not be reactive during the reaction.
T or F
T
All the reactants are mixed together simultaneously and labeled antigen competes with unlabeled patient antigen for a limited number of antibody-binding sites
a. Competitive assays
b. noncompetitive assays
c. Competitive Radioimmunoassays
d. Noncompetitive Radioimmunoassays
a
Which of the following is are the characteristics of competitive assays?
- The amount of bound label is inversely proportional to the concentration of the labeled antigen
- The more label detected, the less there is of patient antigen
a. 1 Only
b. 2 Only
c. Both
d. None of the above
c
Capture antibody is used in this assay?
a. Competitive assays
b. noncompetitive assays
c. Competitive Radioimmunoassays
d. Noncompetitive Radioimmunoassays
b
Capture antibody is first passivley absorbed by antigen in what state of matter?
a. Solid phase
b. Liquid phase
c. Gas phase
a
Sequence in noncompetitive assay?
1. Capture antibody is absorbed in solid phase antigen
2. after washing, label antibody is added
3. Unkown patient antigen is then reacted to captured antibody
a. 1,2,3
b. 2,3,1
c. 3,2,1
d. 1,3,2
d
The amount of label measured is directly proportional to the amount of patient antigen
(Noncompetitive assay)
T or F
T
The amount of labels measured is directly proportional to the amount of patient antigen
(Noncompetitive assay)
T or F
T
Antibodies used should be very specific since the higher the ________ for an antigen, the larger the amount of antigen bound to antibody and the more accurately specific binding can be measured
a. Affinity
b. Avidity
a
The sensitivity of the immunoassay depends largely on the?
a. Magnitude of attachment
b. Magnitude of avidity
c. Magnitude of affinity
d. Magnitude of the area
c
Labeled analytes that are made up in known concentrations of the substances to be measured
T or F
F
Unlabeled analytes
Significance of standards/Calibrators in immunoassays?
a. Establish relationship between labeled analyte measured and any unlabeled analyte
b. To know the amount of known concentration of label reagents
c. Both
d. none of the above
a
Separation method that involves a solid phase and require washing steps to remove unbound antigens and antibodies
a. Homogeneous immunoassays
b. Chemiluminescence
c. Titer
d. Heterogeneous immunoassays
d
Separation method that involves a solid phase and require washing steps to remove unbound antigens and antibodies
a. Homogeneous immunoassays
b. Chemiluminescence
c. Titer
d. Heterogeneous immunoassays
d
Separation method that consist Only liquid phase and do not require washing steps, less sensitive
a. Homogeneous immunoassays
b. Chemiluminescence
c. Titer
d. Heterogeneous immunoassays
a
Which of the following is true for heterogeneous immunoassays?
a. Faster and easier to automate
b. It can have a competitive and noncompetitive format
c. Liquid phase only
d. Does not require washing
b
Which of the following is true for Homogeneous immunoassays?
- Faster and easier to automate
- Competitive format only
- Liquid phase only
- More sensitive
a. 1,2,3,4
b. 1,3,4
c. 1,2,3
d. 1,2,4
c
What is the last step common to all immunoassays?
a. Detection of labeled analyte
b. Detection of unlabeled analyte
c. Separation of labeled and unlabeled analyte
d. None of the above
a
Measurement used in radioactivity?
a. Spectophotometry
b. Scintillation counter
c. Radiogram
b
Measurement used in Enzyme, fluorescence or chemiluminescence?
a. Spectophotometry
b. Scintillation counter
c. Radiogram
a
What is being measured in spectophotometry?
a. absorbance
b. concentration
c. Density
d. Volume
a
Running blank tube for every test and a NC and a high and a low LP control should be run in addition for Quality control
T or F
T
Radioimmunoassays is pioneered by?
Yalow and Berson in 1950s
It was used to determine the level of insulin-anti-insulin complexes in diabetic patients
Radioimmunoassays
Which of the following labels are gamma counter for Radioimmunoassays?
- 131I (Iodine)
- 125I (Iodine)
- 3H (Hydrogen)
a. 1,2,3
b. 2,3
c. 1,2
d. 1,3
c
Most common label in radioimmunoassays?
a. 131I
b. 125I
c. 3H
b
Which of the following labels are beta counter for Radioimmunoassays?
a. 131I
b. 125I
c. 3H
c
What is used in testing for radioimmunoassay?
- Thyroid stimulating hormone
- Total serum IgE
- Total serum IgG
- Cerebral Spinal Fluid
a. 1,2,3,4
b. 3,4
c. 2,4
d. 1,2
d
The analyte being detected competes with radiolabeled analyte for a limited number of binding sites on a high -affinity
a. Competitive assays
b. noncompetitive assays
c. Competitive Radioimmunoassays
d. Noncompetitive Radioimmunoassays
c
Radioactivity in the solid phase is inversely proportional to the antigen concentration?
T or F
T
The advantage of Radioimmunoassays is extremely sensitive and precise in detecting analytes in small size
T or F
T
The following are disadvantage of radioimmunoassays
1. Health hazard
2. Expensive equipment
a. Only 1
b. Only 2
c. Both
d. None
c
Are naturally occuring molecule that catalyze certain biochemical reactions
Enzymes
The following are advantages of enzyme immunoassays:
- Cheap
- long shelf life
- High sensitivity
- easily adapted to automation
T or F
T
The following labels are part of enzyme immunoassays:
- Horseradish peroxidase (HRP)
- Glucose-6-phosphate dehydrogenase
- Alkaline phosphatase
- Beta-D-galactosidease
- Muraminidase
a. 1,2,3,4
b. 2,3,4,5
c. 3,4,5
d. 1,3,4,5
a
To be used in an EIA, an enzyme must be:
- High degree of stability
- Extreme specificity
- Presence of antigen and antibody
- No alteration by inhibitor
Which of the following does not belong?
a.1
b.2
c.3
d.4
c
Absence from the antigen or antibody
Enzyme-labeled antigen competes with unlabeled patient antigen
Typically used for measuring small antigens that are relatively pure, such as insulin and estrogen
a. Competitive enzyme immunoassays
b. noncompetitive enzyme immunoassays
c. Competitive Radioimmunoassays
d. Noncompetitive Radioimmunoassays
a
In Competitive enzyme immunoassays, Enzyme activity is inversely proportional to the concentration of the test substance
T or F
T
One of the most frequenlty used immunoassays in the clinical laboratory due t its sensitivity, specificity, simplicity, and low cost
Use to detect HIV, HAV, HCV, EBV
a. ELISA
b. PCR
c. TITER
a
Noncompetitive enzyme immunoassays is often referred to as?
Indirect enzyme-linked immunosorbent assays
Noncompetitive enzyme immunoassays, the amount of enzyme label detected is directly proportional to the amount of antibody in the specimen
T or F
T
If antibody is bound to the solid phase, these assays are often called?
Capture assays
Antigens captured in the capture assays must have multiple?
a. Epitope
b. Paratope
c. FAB
d. FC
a
In Capture assays, Enzymativ activity is directly proportional to the amount of antigen in the test sample
T or F
T
Easy to perform and give reproducible results
Rapid immunoassays
Designed primarily for point-of-care or home testing, but have been modified for increased sensitivity and can be made semiquantitative for use in a clinical laboratory
Typically single use, disposable
Membrane-based cassette assays
What are the membrane-based cassette assays made of especially in plastic cartridge?
Nitrocellulose
Advantages of enzyme immunoassays
No health hazard
higher sensitivity and shorter incubation time
Cheap
Disadvantage of enzyme immunoassays
Some specimen may contain natural enzyme inhibitors
Size of enzyme label may be a limiting factor in the design
Nonspecific protein binding
Who demonstrated Fluorescent immunoassays?
Albert Coons in 1941
What do you call the fluorescent compounds?
Fluorophores / fluorochromes
Most commonly used fluorescent immunoassay
Fluorescein isothiocyanate
Fluorescein absorbs maximally at?
490 to 495nm
What absrobance Fluorescein emits green color?
517 to 520 nm
Fluorochrome that can absorbs 550nm
Tetramethylrhodamine (Rhodamine)
Rhodamine emits red light at what absorbance?
580 to 585nm
First used for histochemical localization of antigen in tissues through a technique called?
A term restricted to qualitative observations involving the use of a fluorescence microscope
Immunofluorescent assay
This use for diagnosis of viral diseases such as HSV, CMV, EBV, detect cell surface antigen, Chlamydial antigen, flow cell cytometry
Antigen + Fluoresceinated antibody = Fluorescence
Direct immunofluorescent or single layer technique
Solid phase antigen + antibody + Fluoresceinated anti-immunogobulin
Uses in FANA, FTA-ABS
Indirect immunofluorescent or Double layer technique
The amount of fluorescence is directly proportional to the amount of patient antibody present
T
What is the advantage of fluorescent immunoassays?
High sensitivity and versatile
What is the disadvantage of fluorescent immunoassays?
Light sensitive (Need to be in a Dark room)
Nonspecific binding to substance in serum can cause quenching or diminishing of the signal and change the amount of fluorescence generated
Separation of the signal on the label from autofluorescence produced by different organic substances normally present in serum
Emission of light caused by a chemical reaction, typically an oxidation reaction, producing an excited molecule that decays back to its original ground state
Chemiluminescent immunoassay
Most commonly used labels in chemiluminescent?
Luminol
Acridinium esters
Ruthenium derivatives
Nitrophenyl oxalates
What is the measurement used in chemiluminescent immunoassay?
Photomultiplier tube
What is the advantage of Chemiluminescent?
Excellent sensitivity, comparable to EIA and RIA
Stable and nontoxic reagents
Inexpensive and fast
What is the disadvantage of Chemiluminescent?
Lack of precision in injection of the hydrogen peroxide or if some biological materials such as urine or plasma cause quenching of the light emission
Semiconductors nanocrystals used as fluorescent labeling reagents for biological imaging
Quantum dots
Use of superconducting quantum interference device to detect the tagged antigen-antibody complex
Used in detection of Listeria monocytogenes
Squid technology
Based on using donor bead and receptor bead
Used in RNA determination
Luminescent Oxygen-channeling immunoassay
Tyramide signal amplification
Detection of B cells in tissues
Signal Amplification Technology
Uses high resolution magnetic recording technology
For DNA tests
Magnetic Labeling Technology
Measurement is done after a certain period to exclude background interference
Uses europium-labeled antibodies
Time-resolved Fluoroimmunoassay
Binding to a large antibody molecule slows down the rate of rotation and increases the degree of polarization
Polarization is determined by its rate of rotation
Fluorescence Polarization immunoassay
Uses fluorescent molecules to brightly “paint” genes or chromosomes
Uses recombinant DNA technology
Fluorescence in Situ Hybridization (FISH)
