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Questions and Answers
What is the primary purpose of adding an enzyme-linked secondary antibody in an indirect ELISA?
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?
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?
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?
What characteristic distinguishes an indirect ELISA from a direct ELISA?
Which of the following best describes why the color change in an ELISA is significant?
Which of the following best describes why the color change in an ELISA is significant?
What is indicated by a color change in an ELISA test?
What is indicated by a color change in an ELISA test?
Which of the following best describes the function of sandwich ELISAs?
Which of the following best describes the function of sandwich ELISAs?
What is a primary goal of lateral flow assays?
What is a primary goal of lateral flow assays?
In the context of HIV testing, what role do anti-p24 antibodies play?
In the context of HIV testing, what role do anti-p24 antibodies play?
Which of the following statements about indirect ELISA is true?
Which of the following statements about indirect ELISA is true?
What distinguishes sandwich ELISA from other types of ELISA?
What distinguishes sandwich ELISA from other types of ELISA?
Lyme disease diagnosis typically involves which of the following methods?
Lyme disease diagnosis typically involves which of the following methods?
What is the primary clinical application of ELISA in the context of infectious diseases?
What is the primary clinical application of ELISA in the context of infectious diseases?
What is indicated by a color change in an indirect ELISA?
What is indicated by a color change in an indirect ELISA?
What is the primary purpose of performing an indirect ELISA in the context of Lyme disease?
What is the primary purpose of performing an indirect ELISA in the context of Lyme disease?
Which step in a sandwich ELISA involves coating the wells with antibodies?
Which step in a sandwich ELISA involves coating the wells with antibodies?
Which of the following best describes an indirect ELISA?
Which of the following best describes an indirect ELISA?
What type of infection is primarily diagnosed through indirect ELISA?
What type of infection is primarily diagnosed through indirect ELISA?
Which component is crucial for the substrate reaction in an indirect ELISA?
Which component is crucial for the substrate reaction in an indirect ELISA?
What is the relationship between the intensity of color change and patient antibody levels in an indirect ELISA?
What is the relationship between the intensity of color change and patient antibody levels in an indirect ELISA?
In specific clinical tests, what does the term 'capture antibody' refer to?
In specific clinical tests, what does the term 'capture antibody' refer to?
Which of the following statements is true about sandwich ELISAs?
Which of the following statements is true about sandwich ELISAs?
What is the main characteristic of an indirect ELISA?
What is the main characteristic of an indirect ELISA?
Which of the following best describes a Sandwich ELISA?
Which of the following best describes a Sandwich ELISA?
In a direct ELISA, which component serves as the detection antibody?
In a direct ELISA, which component serves as the detection antibody?
Which clinical application is most suited for an ELISA test?
Which clinical application is most suited for an ELISA test?
What result would indicate a positive reading in a direct ELISA?
What result would indicate a positive reading in a direct ELISA?
What is the primary purpose of using labeled antibodies in ELISA?
What is the primary purpose of using labeled antibodies in ELISA?
Which of the following statements about labeling antibodies is incorrect?
Which of the following statements about labeling antibodies is incorrect?
In the context of immunological assays, what role do enzymatic reactions play?
In the context of immunological assays, what role do enzymatic reactions play?
Which of these methods is least likely to be used in a laboratory for antibody detection?
Which of these methods is least likely to be used in a laboratory for antibody detection?
What is a primary advantage of using serial dilutions in an ELISA?
What is a primary advantage of using serial dilutions in an ELISA?
What is the primary purpose of immunoassays?
What is the primary purpose of immunoassays?
Which type of immunoassay is commonly used for the detection of patient antibodies?
Which type of immunoassay is commonly used for the detection of patient antibodies?
Which characteristic is typical of direct assays in immunoassays?
Which characteristic is typical of direct assays in immunoassays?
In the context of immunoassays, what does the term 'sandwich ELISA' refer to?
In the context of immunoassays, what does the term 'sandwich ELISA' refer to?
What is a common clinical application of ELISA testing?
What is a common clinical application of ELISA testing?
Which of the following is a limitation of using indirect ELISA for antigen detection?
Which of the following is a limitation of using indirect ELISA for antigen detection?
Which antibody detection method would be most suitable for diagnosing Lyme disease?
Which antibody detection method would be most suitable for diagnosing Lyme disease?
Which statement about immunoassays is TRUE?
Which statement about immunoassays is TRUE?
What role does signal amplification play in indirect assays?
What role does signal amplification play in indirect assays?
In indirect ELISA, what is primarily being detected?
In indirect ELISA, what is primarily being detected?
Which regions of a recombinant antibody are derived from human sources?
Which regions of a recombinant antibody are derived from human sources?
Which mechanism of action does Rituxan® NOT utilize?
Which mechanism of action does Rituxan® NOT utilize?
When using Fab fragments as antidotes for drug toxicity, what is their primary function?
When using Fab fragments as antidotes for drug toxicity, what is their primary function?
Which statement is true regarding trifunctional antibodies?
Which statement is true regarding trifunctional antibodies?
Which monoclonal antibody is entirely human-derived?
Which monoclonal antibody is entirely human-derived?
In an ELISA assay targeting the spike protein for Covid-19, what type of secondary antibody should be used?
In an ELISA assay targeting the spike protein for Covid-19, what type of secondary antibody should be used?
What is a notable feature of bispecific antibodies like Hemlibra?
What is a notable feature of bispecific antibodies like Hemlibra?
What unique property do Fab fragments possess when used as antidotes?
What unique property do Fab fragments possess when used as antidotes?
Which of the following is a common misconception about trifunctional antibodies?
Which of the following is a common misconception about trifunctional antibodies?
What regions of a recombinant antibody are derived from humans?
What regions of a recombinant antibody are derived from humans?
Which mechanism of action can Rituxan® possibly engage in?
Which mechanism of action can Rituxan® possibly engage in?
When Fab fragments are used as antidotes for drug toxicity, what is their primary function?
When Fab fragments are used as antidotes for drug toxicity, what is their primary function?
Trifunctional antibodies have the ability to bind to how many different antigens?
Trifunctional antibodies have the ability to bind to how many different antigens?
Which monoclonal antibody is noted to be 100% human?
Which monoclonal antibody is noted to be 100% human?
In an ELISA assay, if the primary antibody is derived from rabbits, which secondary antibody is appropriate?
In an ELISA assay, if the primary antibody is derived from rabbits, which secondary antibody is appropriate?
What differentiates bispecific antibodies from traditional monoclonal antibodies?
What differentiates bispecific antibodies from traditional monoclonal antibodies?
Which of the following statements about Fab fragments is incorrect?
Which of the following statements about Fab fragments is incorrect?
What is a key characteristic of humankind-derived variable regions in recombinant antibodies?
What is a key characteristic of humankind-derived variable regions in recombinant antibodies?
Which statement regarding the composition of monoclonal antibodies is true?
Which statement regarding the composition of monoclonal antibodies is true?
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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.
Recombinant Antibodies
- Variable regions are human derived in recombinant antibodies
Rituxan® (Rituximab)
- Rituxan® is a therapeutic monoclonal antibody
- Rituxan® targets CD20 on B lymphocytes
- Rituxan®'s mechanism of action involves the activation of complement
Fab Fragments
- Fab fragments are antibody fragments used to neutralize drugs
- Fab fragments lack the Fc region so cannot activate complement
Trifunctional Antibodies
- Trifunctional antibodies have two different Fab regions with different antigen binding sites.
100% Human Monoclonal Antibody
- Monoclonal antibodies with "u" in front of "mab" are 100% human
ELISA for Covid-19
- ELISA is a technique used to diagnose Covid-19
- A monoclonal antibody targeting the spike protein is used as the primary antibody in an ELISA assay
- Anti-rabbit conjugated to horse radish peroxidase is used as the secondary antibody
Hemlibra (Emicizumab)
- Hemlibra is a bispecific antibody that lacks the Fc region
- Hemlibra binds to two different antigens
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