Exam 3_PQ 2

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Questions and Answers

What is the primary purpose of adding an enzyme-linked secondary antibody in an indirect ELISA?

To enhance the sensitivity of the assay

To serve as a substrate for color change

To bind directly to the antigen present in the sample

To indicate the presence of antibodies in the patient sample (correct)

Which statement accurately reflects the limitations of direct ELISA in diagnostics?

Direct ELISA is less cost-effective than some other methods. (correct)

Direct ELISA is less flexible compared to direct/indirect sandwich ELISAs. (correct)

Direct ELISA is more sensitive than indirect ELISA.

Direct ELISA can be used for broad antibody detection.

In the context of Lyme disease diagnosis, what is crucial for detecting the presence of antibodies?

Using a direct ELISA approach exclusively

Coating the wells with known antigens related to Lyme disease (correct)

Coating the wells with patient antibodies

Employing a colorimetric substrate without antibodies

What characteristic distinguishes an indirect ELISA from a direct ELISA?

Indirect ELISA involves a known antigen to detect antibodies. Signup and view all the answers

Which of the following best describes why the color change in an ELISA is significant?

It shows the quantity of patient antibody present in the sample. Signup and view all the answers

What is indicated by a color change in an ELISA test?

The amount of patient antibody Signup and view all the answers

Which of the following best describes the function of sandwich ELISAs?

To detect both anti-HIV antibodies and HIV-specific proteins Signup and view all the answers

What is a primary goal of lateral flow assays?

To offer rapid and easy point-of-care screening Signup and view all the answers

In the context of HIV testing, what role do anti-p24 antibodies play?

To bind to the HIV p24 antigen in a patient's blood Signup and view all the answers

Which of the following statements about indirect ELISA is true?

It requires the addition of a secondary antibody for detection. Signup and view all the answers

What distinguishes sandwich ELISA from other types of ELISA?

It can detect both antigens and antibodies simultaneously. Signup and view all the answers

Lyme disease diagnosis typically involves which of the following methods?

Sandwich ELISA for detecting Borrelia antigens Signup and view all the answers

What is the primary clinical application of ELISA in the context of infectious diseases?

To detect antibodies against various pathogens Signup and view all the answers

What is indicated by a color change in an indirect ELISA?

The amount of patient antibody Signup and view all the answers

What is the primary purpose of performing an indirect ELISA in the context of Lyme disease?

To detect anti-Borrelia antibodies in the patient Signup and view all the answers

Which step in a sandwich ELISA involves coating the wells with antibodies?

Coating the wells with a capture antibody Signup and view all the answers

Which of the following best describes an indirect ELISA?

Detects antibodies against a specific antigen Signup and view all the answers

What type of infection is primarily diagnosed through indirect ELISA?

Bacterial infections Signup and view all the answers

Which component is crucial for the substrate reaction in an indirect ELISA?

Colorimetric detection Signup and view all the answers

What is the relationship between the intensity of color change and patient antibody levels in an indirect ELISA?

Directly proportional Signup and view all the answers

In specific clinical tests, what does the term 'capture antibody' refer to?

An antibody that binds the target antigen in the assay Signup and view all the answers

Which of the following statements is true about sandwich ELISAs?

They can detect both antigens and antibodies. Signup and view all the answers

What is the main characteristic of an indirect ELISA?

It uses a labeled secondary antibody for detection. Signup and view all the answers

Which of the following best describes a Sandwich ELISA?

A technique where antigen is trapped between two antibodies. Signup and view all the answers

In a direct ELISA, which component serves as the detection antibody?

Labeled primary antibody. Signup and view all the answers

Which clinical application is most suited for an ELISA test?

Quantifying levels of antibodies against Lyme disease. Signup and view all the answers

What result would indicate a positive reading in a direct ELISA?

A significant color change is observed. Signup and view all the answers

What is the primary purpose of using labeled antibodies in ELISA?

To facilitate visualization or quantification of the antigen-antibody reaction. Signup and view all the answers

Which of the following statements about labeling antibodies is incorrect?

The use of fluorophores always results in a color change. Signup and view all the answers

In the context of immunological assays, what role do enzymatic reactions play?

They are used to cause a color change or light emission. Signup and view all the answers

Which of these methods is least likely to be used in a laboratory for antibody detection?

CT scan imaging Signup and view all the answers

What is a primary advantage of using serial dilutions in an ELISA?

To determine the titer of antibodies or antigens. Signup and view all the answers

What is the primary purpose of immunoassays?

To detect the presence and amount of antibodies or antigens Signup and view all the answers

Which type of immunoassay is commonly used for the detection of patient antibodies?

Indirect assay Signup and view all the answers

Which characteristic is typical of direct assays in immunoassays?

Detects patient antigen directly Signup and view all the answers

In the context of immunoassays, what does the term 'sandwich ELISA' refer to?

A type of assay that uses captured antibodies and detection antibodies Signup and view all the answers

What is a common clinical application of ELISA testing?

To diagnose viral infections Signup and view all the answers

Which of the following is a limitation of using indirect ELISA for antigen detection?

It primarily detects antibodies only Signup and view all the answers

Which antibody detection method would be most suitable for diagnosing Lyme disease?

Indirect ELISA Signup and view all the answers

Which statement about immunoassays is TRUE?

Antigens and antibodies may sometimes act as each other's targets. Signup and view all the answers

What role does signal amplification play in indirect assays?

It enhances the detection sensitivity Signup and view all the answers

In indirect ELISA, what is primarily being detected?

The concentration of antibodies specific to the antigen Signup and view all the answers

Which regions of a recombinant antibody are derived from human sources?

Constant regions Signup and view all the answers

Which mechanism of action does Rituxan® NOT utilize?

T cell proliferation Signup and view all the answers

When using Fab fragments as antidotes for drug toxicity, what is their primary function?

Neutralize the drug Signup and view all the answers

Which statement is true regarding trifunctional antibodies?

They have two different Fab regions. Signup and view all the answers

Which monoclonal antibody is entirely human-derived?

Golimumab Signup and view all the answers

In an ELISA assay targeting the spike protein for Covid-19, what type of secondary antibody should be used?

Anti-rabbit conjugated to horse radish peroxidase Signup and view all the answers

What is a notable feature of bispecific antibodies like Hemlibra?

They can bind to two different antigens simultaneously. Signup and view all the answers

What unique property do Fab fragments possess when used as antidotes?

They lack the Fc region, limiting their effector functions. Signup and view all the answers

Which of the following is a common misconception about trifunctional antibodies?

They have only one type of Fab region. Signup and view all the answers

What regions of a recombinant antibody are derived from humans?

Variable regions Signup and view all the answers

Which mechanism of action can Rituxan® possibly engage in?

Neutralization Signup and view all the answers

When Fab fragments are used as antidotes for drug toxicity, what is their primary function?

Neutralize the drug Signup and view all the answers

Trifunctional antibodies have the ability to bind to how many different antigens?

Two Signup and view all the answers

Which monoclonal antibody is noted to be 100% human?

Golimumab Signup and view all the answers

In an ELISA assay, if the primary antibody is derived from rabbits, which secondary antibody is appropriate?

Anti-rabbit conjugated to horse radish peroxidase Signup and view all the answers

What differentiates bispecific antibodies from traditional monoclonal antibodies?

They can bind to two different antigens Signup and view all the answers

Which of the following statements about Fab fragments is incorrect?

They contain an Fc region Signup and view all the answers

What is a key characteristic of humankind-derived variable regions in recombinant antibodies?

They are responsible for antigen specificity Signup and view all the answers

Which statement regarding the composition of monoclonal antibodies is true?

Some monoclonal antibodies contain murine components Signup and view all the answers

Study Notes

Immunoassays

Immunoassays are used to detect both antigen & antibody in a sample.
The test is designed to detect either an antigen or an antibody in a patient sample.
Most immunoassays are categorized as direct or indirect.

Direct Immunoassays

Direct assays often detect the presence of patient antigen in a sample.
Patient antibodies can also be antigens.

Indirect Immunoassays

Indirect assays are often used to detect patient antibodies in a sample.
Indirect assays amplify the signal during detection of patient antigen in a sample.

ELISA

ELISAs are also known as an enzyme-linked immunosorbent assay.
ELISAs utilize enzyme-labeled antibodies to detect and quantify antigens or antibodies in a patient sample.
ELISAs are often used to determine titer(s).
ELISAs can be categorised as direct, indirect or sandwich.

Direct ELISAs

Direct ELISAs detect patient antigen.
Direct ELISAs utilize labeled primary antibodies.

Indirect ELISAs

Indirect ELISAs detect patient antibodies.
Indirect ELISAs utilize labeled secondary antibodies.

Sandwich ELISAs

Sandwich ELISAs are a specific type of ELISA that can be set up as either a direct or indirect assay.

HIV Testing

HIV testing often uses sandwich ELISAs.
HIV testing aims to detect both HIV antibodies and HIV-specific proteins.

Lyme Disease Diagnosis

The initial test performed to diagnose Lyme disease is often an indirect ELISA to detect anti-Borrelia antibodies.

Common Labels

Common labels for immunoassays include enzymes, fluorophores and radioactive nuclides.

Enzyme Labels

Commonly used enzymes include horseradish peroxidase and alkaline phosphatase.

Fluorophore Labels

Commonly used fluorophores include fluorescein isothiocyanate (FITC) and rhodamine.

Radioactive Nuclide Labels

Commonly used radioactive nuclides include 125I and 3H.

Lateral Flow Assays

Lateral flow assays are modified ELISAs.
Lateral flow assays are rapid and easy to use.
Lateral flow assays are often used as point of care screening assays.

Direct ELISA Steps

Wells are coated with a patient sample.
A known enzyme-linked primary antibody is added.
A substrate for the enzyme is added; the substrate cleavage causes a colour change.
The intensity of the colour change is proportional to the amount of patient antibody.

Indirect ELISA Steps

Wells are coated with a known antigen.
Patient sample is added, allowing the patient antibodies to bind to the known antigen.
An enzyme-linked secondary antibody is added, which binds to the patient's (primary) antibody.

Visualisation & Quantification

Labeling an antibody or antigen allows for visualization and quantification of the antigen-antibody reaction with the use of proper instruments.

Recombinant Antibodies

Variable regions are human-derived in recombinant antibodies.

Rituxan® Mechanism of Action

Rituxan® can activate complement, leading to the destruction of B lymphocytes.

Fab Fragments as Antidotes

Fab fragments can neutralize drugs by directly binding to them.

Trifunctional Antibodies

Trifunctional antibodies have two different Fab regions, allowing them to bind to two different antigens.

100% Human Monoclonal Antibodies

Monoclonal antibodies with a "u" in front of "mab" are 100% human, for example, Golimumab.

ELISA Assay for COVID-19

Anti-rabbit conjugated to horseradish peroxidase is the appropriate secondary antibody for an ELISA using a rabbit monoclonal antibody as the primary antibody.

Hemlibra: Bispecific Antibody without Fc Function

Bispecific antibodies are engineered to have two different antigen binding sites, allowing them to target multiple antigens simultaneously.