Immunoassays Flashcards
(139 cards)
1
Q
What is a competitive immunoassay?
A
An assay where an unlabelled analyte competes with a labelled analyte to bind to an antibody.
2
Q
What are the 2 types of competitive immunoassays? (select all that apply)
A
-Homogenous
-Heterogenous
3
Q
What is a homogenous competitive immunoassay? (select all that apply)
A
-A type of immunoassay where the analyte in a sample competes with a labelled analyte for binding sites on a specific antibody.
-The amount of the unbound, labelled analyte is measured, without the need for separation steps.
-This amount of labelled unbound analyte is measured. And is directly proportional to the sample analyte concentration.
4
Q
What is a heterogenous competitive immunoassay? (select all that apply)
A
-A method where a labelled analyte competes with an unlabeled analyte for binding sites on an antibody.
-This assay requires a physical separation (washing) step to distinguish between the antibody-bound and unbound fractions.
-The remaining labelled, bound analyte is measured. And is Inversely proportional to the sample analyte concentration.
5
Q
What is a non-competitive immunoassay? (select all that apply)
A
-Assays that have excess antibody binding sites and produce a signal directly proportional to the amount of analyte in the sample
-Unbound labelled antibodies are washed away. Bound labelled antibodies are measured.
6
Q
Whats is a two-site non-competitive immunoassay or sandwich assay?
A
Uses two antibodies to detect an analyte, with one antibody capturing the analyte and the other detecting it, resulting in a signal directly proportional to the analyte’s concentration.
7
Q
What are some types of specific immunoassays? (select all that apply)
A
-Enzyme-Linked Immunosorbent Assay (ELISA)
8
Q
-Enzyme Multiplied Immunoassay Technique (EMIT)
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9
Q
-Cloned Enzyme Donor Immunoassay (CEDIA)
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10
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-Fluorescence Polarization Immunoassay (FPIA)
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11
Q
What does ELISA stand for?
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Enzyme-Linked Immunosorbent Assay
12
Q
What type of immunoassay is ELISA?
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Non-competitive, heterogeneous immunoassay
13
Q
What is the primary purpose of an ELISA?
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A plate-based assay technique designed for detecting and quantifying soluble substances.
14
Q
What are some types of substances that can be detected using ELISA? (select all that apply)
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-Peptides
15
Q
-Proteins
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16
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-Antibodies
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17
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-Hormones
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18
Q
How is the antigen immobilized in the ELISA technique?
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The antigen is immobilized on a solid surface and then complexed with an antibody that is linked to a reporter enzyme.
19
Q
What type of plates are used to immobilize the antigen in the ELISA technique?
A
Polystyrene plates that passively bind protein.
20
Q
How is detection accomplished in ELISA?
A
By measuring the activity of the reporter enzyme via incubation with the appropriate substrate to produce a measurable product.
21
Q
What are some of the characteristics of the ELISA? (select all that apply)
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-It is a powerful method for detecting and quantifying a specific protein in a complex mixture.
22
Q
-It is the binding and immobilization of reagents that make ELISA’s easy to design and perform.
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23
Q
-Having the reactants immobilized to a plate makes it easy to separate bound from unbound material during the assay,
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24
Q
-This ability to use high-affinity antibodies and wash away non-specific bound materials makes ELISA uniquely suited to measuring specific analytes from crude preparations.
A
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What are the steps that all ELISA formats follow? (select all that apply)
-Coating/capture: direct or indirect immobilization of antigens to the surface of polystyrene microplate wells.
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-Plate blocking: Addition of irrelevant protein or other molecule to cover all unsaturated surface-binding sites of the microplate wells.
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-Probing/detection: incubation with antigen-specific antibodies that affinity-bind to the antigens
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-Signal Measurement: detection of the signal generated via the direct or secondary tag on the specific antibody
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What are the three ELISA formats? (select all that apply)
-Direct
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-Indirect
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-Sandwich
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What are the steps in a direct ELISA? (select all that apply)
-The first step is to coat a solid surface (like a microtiter plate well) with a capture antibody specific to the antigen you want to detect.
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-Add the sample containing the antigen to the coated surface, allowing the antigen to bind to the capture antibody.
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-Add an enzyme-labeled antibody that is specific to the same antigen as the capture antibody.
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-Remove any unbound antibody by washing the surface.
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-Add a substrate that reacts with the enzyme, producing a detectable signal (e.g., a color change).
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-The intensity of the signal is detected with the labelled primary antibody and is proportional to the amount of antigen present in the sample.
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What are the steps in an indirect ELISA? (select all that apply)
-The wells of a microtiter plate are coated with the specific antigen that you want to detect.
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-A solution containing the primary antibody, which is specific for the antigen, is added to the wells.
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-The plate is incubated to allow the primary antibody to bind to the antigen. Unbound primary antibody is then washed away.
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-A secondary antibody, which is enzyme-conjugated and specific for the host species of the primary antibody, is added to the wells. This secondary antibody will bind to the primary antibody that is already bound to the antigen.
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-The plate is incubated again to allow the secondary antibody to bind to the primary antibody. Unbound secondary antibody is then washed away.
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-A substrate solution that reacts with the enzyme conjugated to the secondary antibody is added. The enzyme catalyzes a reaction with the substrate, producing a detectable signal (e.g., a color change).
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-Is detected indirectly with the labelled secondary antibody. The color change or other detectable signal is proportional to the amount of antigen present in the sample.
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What are the steps in a sandwich ELISA? (select all that apply)
-A capture antibody, which is specific for the target antigen, is bound to the wells of a microplate. This antibody acts as a "capture agent" to bind the antigen of interest.
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-To prevent non-specific binding of other molecules, the wells are blocked with a solution (e.g., BSA or milk).
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-The sample containing the target antigen is added to the wells. The antigen binds to the capture antibody on the plate.
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-Unbound sample components are washed away to ensure only the antigen bound to the capture antibody remains.
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-A detection antibody, which is also specific for the target antigen, is added to the wells. This antibody is often conjugated with an enzyme (e.g., horseradish peroxidase).
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-The detection antibody binds to the antigen that is already bound to the capture antibody, creating a "sandwich".
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-Unbound detection antibodies are washed away, ensuring that only the enzyme-labeled detection antibody remains bound to the antigen.
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-A substrate solution that reacts with the enzyme on the detection antibody is added. The enzyme converts the substrate into a detectable signal (e.g., a color change).
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-The signal is measured (e.g., using a spectrophotometer) to determine the concentration of the target antigen in the sample.
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Why is it called a Sandwich ELISA?
Because the analyte to be measured is bound between two primary antibodies, each detecting a different epitope of the antigen.
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What are the advantages of the Sandwich ELISA format?
It is highly used because of its sensitivity (signal amplification) and specificity.
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How does the Direct ELISA format measure concentration?
The detector binds directly to the unknown and is measured based on concentration or color.
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In an Indirect ELISA, how does the detection antibody interact with the antigen?
The detection antibody does not bind directly to the antigen.
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In a Sandwich ELISA, how is the unknown analyte detected?
It is sandwiched between a capture antibody bound to a solid support and a detection antibody based on enzyme detection.
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What does EMIT stand for?
Enzyme Multiplied Immunoassay Technique
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What type of immunoassay is EMIT?
Competitive homogeneous immunoassay
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What principle does EMIT work on?
Enzyme inhibition being proportional to the amount of analyte in the patient sample.
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What are the steps in EMIT? (select all that apply)
-Glucose-6-phosphate dehydrogenase (G6PDH) is conjugated to a known amount of antigen (usually a drug this is also the antigen that we are looking for)
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-When the antigen-specific antibody is added, which results in inhibition of this drug-enzyme complex.
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-Addition of patient sample that contains the drug results in competition for the antibody and therefore activation of the enzyme.
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-Free antigen-G6PDH conjugate is able to react with glucose-6-phosphate and NAD+ to produce a product that is measured colorimetrically.
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-Enzyme activity is directly proportional to the concentration of the sample antigen.
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What are some clinical applications of EMIT immunoassays?
Monitoring drug usage, drug abuse or therapy.
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What does 'homogeneous' in the context of the steps for immunoassay function mean?
It does not have a wash step.
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-Enzyme labelled antigen is the antigen were trying to detect and emit it is genetically engineered when the antibody binds to the enzyme it blocks the antibody of the enzyme and blocks the colour
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-The competition takes place when we add the patient sample with the drug.... When mixed the serum sample with the drug we are trying to amplify will compete to bind to the monoclonal antibody that we add to the immunoassay, when we add the binding between substrate and enzyme we get a colour reaction.
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-Antibody and analyte + patient sample with drug is mixed first to promote binding between the drug in the sample serum.
This image portrays how EMIT works, using this image describe how this process is done. (select all that apply)
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In EMIT why does the strength of the sample get a reaction that is directly proportional?
Due to the enzyme-catalyzed reaction, where more analyte leads to more enzyme-substrate complex formation and thus a stronger signal.
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What is Cloned Enzyme Donor Immunoassay (CEDIA)?
A competitive homogeneous immunoassay.
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What principle does CEDIA work on?
It works on the principle of enzyme fragments assembling into a fully functional enzyme based on the presence of an analyte.
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What are the steps for CEDIA? (select all that apply)
-One enzyme fragment is attached to the analyte of interest.
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-The other enzyme fragment exists by itself.
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-These fragments can spontaneously combine to form active enzyme.
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-Formation of the functional enzyme is inhibited by the addition of an antibody that binds to the analyte-enzyme complex.
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-If a patient sample that contains the analyte of interest is added to our mixture, some of the antibodies will bind to the patient analyte, releasing the analyte-enzyme complex and allowing it to form a fully functional enzyme(based on the enzyme beta galactosidase)
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-Enzyme activity is directly proportional to the amount of analyte in the patient sample
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-Enzyme activity can be measured colorimetrically
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What is the enzyme involved in CEDIA?
The enzyme beta-galactosidase.
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What is unique about the enzyme in CEDIA?
The enzyme is engineered into two active enzymes (polypeptides).
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What is the Cloned Enzyme Donor Immunoassay (CEDIA)?
A type of immunoassay that uses two inactive fragments of a genetically modified β-galactosidase enzyme, which reassemble to form an active enzyme upon binding to a substrate, allowing for the detection and quantification of analytes
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How is β-galactosidase engineered for CEDIA? (select all that apply)
-A β-galactosidase protein was engineered into a large polypeptide (an enzyme acceptor) and a small polypeptide (an enzyme donor).
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-A unit of enzyme acceptor and a unit of enzyme donor were assembled to form an enzymatically active tetrameric molecule of β-galactosidase.
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-The β-galactosidase catalyzes the galactopyranoside substrate to produce a coloured product that can be measured by spectrophotometry.
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-The assay is initiated when a patient sample is added to a reagent containing an anti analyte antibody, and enzyme donor-analyte conjugate, and an enzyme acceptor.
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-If the analyte is present in the patient sample, then the antibody will bind to this analyte and the enzyme donor will be free to form active enzymes with the enzyme acceptor.
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-This reaction serves to modulate the amount of active β-galactosidase formed.
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-The signal generated by the conversion of substrate to product by the enzyme is directly proportional to the analyte concentration in the patient serum.
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-EA and ED are the enzymes that we add
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-What happens if theres no unknown in the sample? We get no binding of the two components mentioned together^^
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-What happens when we do? They compete for an antibody and we have a fully functional enzyme (directly proportional relationship between our enzyme unknown and the donor)(double check)
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-The more intense the colour reaction the higher the concentration
This image portrays how CEDIA works, using this image describe how this process is done. (select all that apply)
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What type of immunoassay is Fluorescence Polarization Immunoassay (FPIA)?
Competitive homogenous immunoassay
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What principle does Fluorescence Polarization Immunoassay (FPIA) work on?
Works on the principle of fluorescence polarization where a fluorescent molecule is exposed to fluorescent light that is polarized.
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What steps are considered the principals of how FPIA functions? (select all that apply)
-Fluorescence happens when a photon of a particular wave length excites an electron and sends it to a higher state of energy. This electron eventually returns to its ground state by releasing this energy in the form of heat and light.
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-The light that is released upon excitation from a polarized light source is also polarized.
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-If the molecule is rotating quickly then the time it takes between excitement and emission is long enough to cause the emitted light to be directed in a direction that is not detected.
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-If an antibody were to bind to the rotating fluorescent molecule, it would slow down its rotational velocity due to conservation of momentum.
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-This decrease in rotation would increase our ability to measure the fluorescence.
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What is polarized light?
Light that passes through a filter and moves in only one direction.
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What are the steps in FPIA? (select all that apply)
-The Assay uses fluorescein which is a fluorescent molecule which is excited at 490nm(incident wavelength of light) and reemits fluorescence at 520nm.(longer wavelength)
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-The assay begins with the addition of the patient sample to an analyte-specific antibody.
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-If analyte is present in the sample, it will bind to the antibody.
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-Next, an antigen-fluorescein labelled reagent is added to the mixture.
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-Patient antigen and antigen-fluorescein complex will compete for the antibody.
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-Any antigen-fluoresceine complex bound to an antibody will have a low rotational velocity and can therefore be measured by the polarized light detector.
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-The amount of polarized fluorescence emitted is measured
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-The quantity of analyte in the serum sample is inversely proportional to the amount of polarized fluorescence emitted
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What happens if the If analyte is not present in FPIA? (select all that apply)
-Unable to measure the fluorescence of the molecule because it is moving to quickly
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-concentration of the unknown in this case will be inversely proportional because as the concentration of our unknown goes up the signal is low.
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What is another name for the hook effect?
Prozone effect
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What are required for accurate results in immunoassays? (select all that apply)
-There must be an excess of antibodies, both capture and enzyme conjugates, relative to the analyte being detected.
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-It is only under these conditions that the dose response curve is positively sloped and the assay provides quantitative results.
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What happens to immunocomplex formation at very high concentrations of antibody or analyte?
It is impaired.
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What occurs to the dose response curve when analyte concentration exceeds antibody concentration? (select all that apply)
-As the concentration of the analyte begins to exceed the amount of antibody present, the dose response curve will flatten.
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-With further increase in analyte concentration, the curve may become negatively sloped.
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What is the phenomenon called when the dose response curve becomes negatively sloped due to high analyte concentration?
Hook effect
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What must be done to validate questionable sample results that may have high analyte concentrations?
Use a dilution protocol to test linearity.
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What does the dilution protocol establish regarding patient results?
Whether the results lie on the positively sloped region of the linearity plot.
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What can result from failing to validate the potential for the hook effect?
Severe underestimation of the true analyte.
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What are heterophile antibodies? (select all that apply)
-Heterophile antibodies are antibodies produced against poorly defined antigens, leading to false positive reactions.
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-Generally weak with multi-specific activities.
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What causes the development of human anti-animal antibodies? (select all that apply)
-As a result of treatment with animal immunoglobulins, such as monoclonal antibodies.
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-Antibodies with strong avidities, produced against well defined antigens on animal antibodies.
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What happens if a patient is treated with monoclonal antibodies? (select all that apply)
-The patient's immune system will develop antibodies against those antibodies.
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-Heterophile antibodies crosslink the capture and detection antibodies and gives a false positive reaction.
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What is a method to control for anti-animal antibodies in testing?
Dilution can be used to control for anti-animal antibodies.
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What is the mechanism by which heterophile antibodies interfere with immunoassays?
Heterophile antibodies interfere with immunoassays by a non-competitive mechanism.
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How do heterophile antibodies cause interference in immunoassays?
Heterophiles can bind to the conjugate, enzyme, or other parts of the detection system and cause interference.
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What is routinely used to avoid interference from heterophile antibodies?
Blocking agents are routinely used.
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What do human anti-animal antibodies bind to in immunoassays?
To animal antibodies commonly used in immunoassays.
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What is a characteristic of human anti-animal antibodies in terms of their binding?
They show strong binding to a particular antigen and are expressed in such high titres that they compete with the test antigen by cross reacting with reagent antibodies.
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What is the most common type of human anti-animal antibody? (select all that apply)
-Most commonly human anti-mouse antibodies (HAMA).
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-Also include antibodies to rats, rabbits, goats, sheep, cattle, etc.
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What percentage of patient samples report the presence of human anti-animal antibodies?
Human anti-animal antibodies are reported to occur in 30-40% of patient samples.
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How can the interference caused by anti-animal antibodies be eliminated?
The interference caused by anti-animal antibodies can be eliminated by sample pre-treatment or assay redesign.
