lECTURE 6 - Serology Flashcards

(10 cards)

1
Q

What are antibody detection techniques, and how are they used in viral diagnostics?

A

Purpose:
* Used to detect antibody titers following infection or vaccination.
* Commonly employed in serosurveillance studies and to monitor vaccine efficacy.

Immune Response:
* Host produces antibodies in response to infection or vaccination, which can be detected in serological tests.
* Understanding the immune response is critical for accurate interpretation of results.

Limitations:
* Serological tests for anti-viral antibodies have limitations and are not always perfect.
* Subject to errors that require cautious and informed interpretation of results.

Key Consideration:
* While these assays can be useful for detecting past infections or vaccine response, they cannot always confirm active infection and may give false positives or false negatives.

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2
Q

What is ELISA, and how is it used in diagnostic testing?

A

Definition:
* ELISA is a laboratory technique used to detect antigens or antibodies in a sample.
* It is commonly used for immune response testing, especially to detect antibody titers.

Configurations:
* There are many configurations of ELISA, each tailored for specific uses (e.g., antigen detection, antibody detection).

Applications:
* Primarily used to detect immune responses (antibodies) following infection or vaccination.
* Can also be used for hormone and hapten detection.

Advantages:
* Highly sensitive and specific, allowing for detection of even small amounts of the target substance.
* Widely validated and used in diagnostic laboratories worldwide.

Limitations:
* While ELISA is useful, it is crucial to interpret results with caution due to potential cross-reactivity or interference.

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2
Q

What is Capture Antibody ELISA, and how is it used in diagnostic testing?

A

Purpose:
* Used to detect specific antigens in a sample by using a capture antibody that binds the antigen.

How it Works:
* Step 1: A capture antibody is coated onto a solid surface (e.g., well of a plate), which binds the target antigen from the sample.
* Step 2: A secondary (indicator) antibody that binds to the captured antigen is then added.
* Step 3: An enzyme-linked secondary antibody is used to generate a detectable signal, such as a color change when a substrate is added.

Key Factors Affecting Specificity:
* Capture antibody specificity: The assay’s specificity depends heavily on the capture antibody’s ability to bind only to the target antigen.
* Monoclonal antibodies are often used because they provide a highly specific capture of the antigen.
* The indicator antibody should not cross-react with the capture antibody to avoid false results.

Advantages:
* Highly specific for the antigen of interest, especially when using monoclonal antibodies.
* Can be used to detect low levels of antigens in complex samples.

Limitations:
* The specificity of the capture antibody is critical; cross-reactivity can lead to false positives.
* Must ensure that the capture and indicator antibodies do not interfere with each other.

SEE DIAGRAM IN LECTURE 6!!!

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2
Q

What is indirect ELISA, and how is it used for antibody testing?

A

Indirect ELISA – Antibody Testing:

Purpose:
* Used to detect antibodies in a sample, specifically for immune response following infection or vaccination.

How it Works:
* Step 1: The antigen is coated onto a solid surface (e.g., well of a plate).
* Step 2: The sample (serum) containing antibodies is added. If the antibodies are present, they will bind to the antigen.
* Step 3: A secondary antibody, which is conjugated to an enzyme, is added. This secondary antibody binds to the primary antibody.
* Step 4: A substrate is added that reacts with the enzyme, causing a color change, indicating the presence of specific antibodies.

Advantages:
* High sensitivity and can detect low levels of antibodies.
* Commonly used for serological surveys or determining previous infections.

Applications:
* Frequently used to test for specific antibodies against pathogens or to monitor vaccine efficacy.

Limitations:
* Potential for false positives due to cross-reactivity or non-specific binding.
* May not detect recent infections if antibody levels are too low.

SEE DIAGRAMS IN LECTURE 6!

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3
Q

What is competitive ELISA, and how does it differ from other types of ELISA?

A

Purpose:
* Used to measure the concentration of an antigen in a sample, particularly when the antigen is small or difficult to measure directly.

How it Works:
* Step 1: The sample antigen (unknown concentration) is incubated with a known quantity of labeled antigen (usually conjugated to an enzyme).
* Step 2: The capture antibody (specific to the antigen) is coated onto the solid surface (e.g., well of a plate).
* Step 3: The sample antigen and the labeled antigen compete for binding to the capture antibody.
* Step 4: The amount of signal produced (color change or fluorescence) is inversely proportional to the amount of antigen in the sample (i.e., less signal means more antigen in the sample).

Key Feature:
* The amount of signal produced is inversely proportional to the concentration of antigen in the sample. The more antigen in the sample, the less labeled antigen will bind to the capture antibody, resulting in a lower signal.

Advantages:
* Can be used to detect low concentrations of antigens.
* Useful for detecting small antigens that may not work well in other types of ELISA.

Limitations:
* Requires careful optimization of antigen and antibody concentrations for accurate results.
* Cross-reactivity with other substances in the sample can interfere with the assay.
SEE DIAGRAM IN LECTURE 6!!

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4
Q

What is antigen ELISA, and how is it used in diagnostic testing?

A

Purpose:
* Used to detect the presence of a specific antigen (usually viral or bacterial) in a sample, such as serum, urine, or tissue.

How it Works:
* Step 1: The capture antibody (specific to the antigen) is coated onto a solid surface (e.g., a microplate well).
* Step 2: The sample is added. If the antigen is present, it will bind to the capture antibody.
* Step 3: A secondary antibody linked to an enzyme (usually horseradish peroxidase or alkaline phosphatase) is added. This antibody binds to the antigen.
* Step 4: A substrate is added, and the enzyme catalyzes a reaction that produces a color change or fluorescent signal, indicating the presence of the antigen.

Key Features:
* The presence of antigen is directly measured, with the color change correlating to the amount of antigen present in the sample.
* Sensitivity and specificity depend on the quality of the capture antibody and the assay’s conditions.

Advantages:
* Simple, quick, and cost-effective for detecting specific antigens.
* Widely used for diagnosis of infections, such as viral or bacterial diseases.

Limitations:
* False negatives can occur if the antigen concentration is too low or the antibody doesn’t bind well.
* May have cross-reactivity with other similar antigens, leading to false positives.
SEE DIAGRAM IN LECTURE 6!!!

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5
Q

What is the Serum Neutralization Test (SNT) used for in virus serotyping, and how is it performed?

A

Purpose:
* Used to identify the serotype of a known virus when the serotype is unknown.
* Can be used to determine the presence of specific antibodies against different serotypes of a virus.

How it Works:
* Step 1: The virus is known but the serotype is unknown.
* Step 2: Neutralizing antibodies that are specific to a known serotype (e.g., Dengue 1-4, BTV serotypes) are used.
* Step 3: The serum is incubated with the virus at a constant concentration in the presence of the serotype-specific antibody.
* Step 4: If the antibody neutralizes the virus, the virus will be unable to infect the cells, preventing the cytopathic effects (CPE). This indicates a match between the antibody and the virus serotype.

Key Features:
* This test inhibits virus infectivity in cell culture, providing evidence of neutralizing antibodies.
* Serotype-specific antibodies are used to identify the virus serotype by neutralizing the infectivity of a known virus.

Advantages:
* Highly specific for identifying and confirming the serotype of viruses.
* Crucial for understanding the epidemiology of infections and vaccine development.

Limitations:
* Requires access to serotype-specific antibodies.
* May be time-consuming and require specialized facilities and expertise.

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5
Q

What is the Viral Neutralization Test (VNT), and how is it used in diagnostic virology?

A

Purpose:
* VNT is used to detect neutralizing antibodies in serum, which can inactivate a specific virus.
* It helps quantify the immune response to a virus, often used for diagnosing viral infections and assessing vaccine efficacy.

How it Works:
* Step 1: Serum sample is mixed with a known virus in vitro.
* Step 2: If the serum contains neutralizing antibodies, these antibodies will bind to the virus and prevent it from infecting host cells.
* Step 3: The mixture is then added to host cells. If neutralizing antibodies are present, there will be no cytopathic effects (CPE) or viral replication in the cells.
* Step 4: The amount of neutralization is quantified based on the highest dilution of serum that prevents viral infectivity.

Key Features:
* The test measures the ability of antibodies to neutralize a virus and prevent it from infecting host cells.
* The titer is determined by the serum dilution at which 50% of cells are protected or show no CPE.

Advantages:
* Considered a gold standard for assessing the presence of neutralizing antibodies.
* Essential for understanding immune responses to infections and vaccines, and determining vaccine efficacy.

Limitations:
* Labor-intensive and time-consuming, requiring specialized laboratory facilities.
* The test is virus-specific and requires a known virus for each test.

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6
Q

What is the difference between sensitivity and specificity in laboratory diagnostics?

A

Sensitivity:

Laboratory Sensitivity:
* Refers to the ability of a test to detect even small amounts of the target, such as a reagent (e.g., antibody).
* High sensitivity means the test can detect very low levels of the target substance or organism.

Clinical Sensitivity:
* Refers to the ability of a test to correctly identify infected animals or individuals.
* High clinical sensitivity means the test is good at identifying those who are truly infected (low false negatives).

Specificity:

Refers to the test’s ability to avoid detecting non-target cases, i.e., it should not detect samples that are negative or do not contain the target.

High specificity means the test correctly identifies non-infected or negative cases, minimizing false positives.

Compromise Between Sensitivity and Specificity:

There is often a trade-off between sensitivity and specificity.

Increasing sensitivity may decrease specificity and vice versa, requiring a balance depending on the purpose of the test (e.g., screening vs. confirmatory testing).

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7
Q

How does the progress of disease affect diagnostic approaches?

A

SEE DIAGRAM IN LECTURE 6 SLIDE 26

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