Exam 3 Flashcards
Which detection enzyme results in the formation of a final reaction product by the removal of a phosphate group
Alkaline phosphatase
What is true of the LSAB detection system
secondary antibody is biotinylated
This alkaline phosphatase substrate is most sensitive since it continues to develop over many hours
BCIP/NBT
Doesn’t affect primary incubation time
pH
One advantage of using fragmented secondary antibodies is that they
Penetrate tissue more efficiently
Which of the following will help to reduce unavoidable aggregation and polymerization of antibodies to the storage container
BSA
Which of the following diluents would be a logical choice for immunofluorescence (IF) on a frozen section
PBS
Which of the following could be used as an enhancer for alkaline phosphatase detection
Magnesium chloride
When a primary antibody conjugated with an enzyme is used, the technique is called
Direct
Which of the following is an example of a DAB enhancer
Copper sulfate
An IHC labeling method in which a large avidin-biotin complex is directly labeled with an enzyme is called
ABC
An advantage of polymer detection over LSAB is
Decreased nonspecific staining
This visualization method provides the best microscopic image quality
Fluorophore
A common fluorophore used in immunohistochemistry is
Fluorescein/FITC
Which of the following is a disadvantage of RTU antibodies
Shorter shelf life
If the pipette tip is immersed too far into the reagent this can occur
More liquid than intended will be aspirated
A disadvantage of DAB chromogen is that
False positives may occur without endogenous peroxidase blocking
Which of the following is an AP substrate
Naphthol AS-MX
In regards to concentrated antibodies, which would be the best practice
Aliquot into smaller amounts to avoid repeated freezing and thawing
This type of polymer is most susceptible to steric hindrance
Dextran polymer
You should pause with the pipette tip in the liquid for at least one second after aspirating or this may result
An inaccurate amount may be aspirated
Prediluted antibodies are typically stored at
2 to 8°C
The shelf life of an antibody is directly linked to
Proper storage
Which of the following antibody diluents has a higher risk for non-specific background staining
PBS
In which instance would AEC be preferred over DAB
When evaluating a melanocytic lesion
Which of the following is a substrate/chromogen used in alkaline phosphatase detection
BCIP/NBT
An increased antibody concentration would have what effect on the primary incubation
Decreased time
Which antibody format can be used as it is received from the vendor
RTU
The compact polymer is considered more sensitive than its dextran counterpart due to the
Smaller size
Which of the following reagents would be added to diluents to decrease surface tension
Tween 20
Which factor determines the optimal primary incubation time
Point of saturation or equilibrium
With this type of detection system, the enzyme and secondary antibody can be conjugated on the same complex
Polymer
The streptavidin complex (LSAB) method incorporates
streptavidin-biotin complex bound to a biotin labeled secondary antibody
Fluorochrome-conjugated primary antibodies are an example of
Direct detection
Low affinity primary antibodies may require a(n)
Overnight incubation
Which of the following is a substrate/chromogen used with horseradish peroxidase
DAB
When using the direct method for immunofluorescence
Fluorochrome-labeled primary antibody is applied
Which is one of the two most important considerations with antibody storage and handling
Temperature
An advantage of direct detection is the
Shorter staining time
A disadvantage of fluorescent visualization is that
Photo-bleaching can occur
In the indirect immunohistochemistry method
Unlabeled primary antibody is used
Which of the following is a chromogen used in HRP immunohistochemistry that is soluble in alcohol
AEC
The concentration of antibody in serum is referred to as the
Titer
The antibody dilution is the proportion of antibody to:
Diluent
Which of the following lists the sensitivity of indirect detection methods from least sensitive to most sensitive
ABC, LSAB, dextran polymer, compact polymer
The antibody diluent pH should be between
7-9
Which is true of the ABC method
Large complex which can hinder specificity
What is the most common host species of secondary antibodies
Goat
The secondary antibody must
Be directed against the host species of the primary
The average primary incubation time is
10-30 mins
Turbidity or cloudiness of IHC reagents likely indicates
Contamination
Which of the following best describes the method shown in the image
Indirect polymer
Which of the following factors governs the optimal antibody dilution
Intrinsic affinity
Which type of detection system will allow for conjugation of both anti-mouse and anti-rabbit secondary antibodies on the same complex
Polymer
Alkaline phosphatase is isolated from
Calf intestine
The peroxidase anti-peroxidase (PAP) staining method visualizes the antibody-antigen interaction with a(n)
Tertiary antibody directed against an enzyme
The most preferred regulatory status of antibodies used in a clinical setting is
IVD
Antibodies within this regulatory status are not intended for diagnostic use
RUO
Red chromogen may be chosen over brown (DAB) for which of the following reasons
Provides good contrast in melanocytic lesions
This type of visualization would most likely be used to demonstrate antigens in frozen tissue sections
FITC
Which of the following is NOT an inhibitor of the AP detection enzyme
Excess hydrogen peroxide
Which of the following antibody diluents would provide the best staining results
TBS
Horseradish peroxidase requires the presence of this to catalyze the reaction with DAB
Hydrogen peroxide
With which antigen retrieval technique would you likely need to lengthen the primary antibody incubation time
HIER using a water bath
Which of the following would be an advantage of concentrated antibodies
Extended shelf life
What is the relationship between the antibody dilution and concentration
The higher the dilution, the lower the concentration
Diluent is combined with a/n ____________________ to create a working dilution
Antibody
The primary antibody is goat anti-mouse. What would be the optimal choice for the secondary antibody
Bovine anti-goat
Which of the following is NOT a factor when selecting a chromogen
Molecule size
In relation to antibody storage, this diluent component serves as a stabilizing or carrier protein
BSA
Which of the following is not a factor affecting the optimal dilution of an antibody
The regulatory status of the antibody
A low salt concentration is added to diluents to
Decrease nonspecific interaction
