Block A - Immunoassay Flashcards
What is an immunoassay?
An immunoassay uses antibodies or antigens to detect or quantify molecules (antibody)
True or False: Immunoassays can only detect proteins.
False: Immunoassays can detect a variety of substances, including proteins, hormones, drugs, and nucleic acids.
Fill in the blank: The two main types of immunoassays are _____ and _____ assays.
qualitative, quantitative
What is the primary purpose of using antibodies in immunoassays?
The primary purpose of using antibodies in immunoassays is to specifically bind to the target analyte for detection and quantification.
Which of the following is a common application of immunoassays?
(A) Blood glucose monitoring
(B) DNA sequencing
(C) MRI imaging
A) Blood glucose monitoring
What does ELISA stand for?
Enzyme-Linked Immunosorbent Assay
True or False: Immunoassays can be used for both research and clinical diagnostics.
True
Name one advantage of using immunoassays.
One advantage of using immunoassays is their high specificity and sensitivity for detecting low concentrations of substances.
What is the role of the substrate in an enzyme-linked immunoassay?
The substrate reacts with the enzyme linked to the antibody to produce a measurable signal, often a color change.
Fill in the blank: In a competitive immunoassay, the amount of _____ is inversely proportional to the concentration of the analyte.
signal
True or False: Immunoassays can only be performed in laboratory settings.
False: Some immunoassays can be performed in point-of-care settings.
What is a monoclonal antibody?
A monoclonal antibody is a produced by immortalizing a specific plasma cell (hybridoma) molecule to target a specific antigen. it can only bind to a specific epitope
Multiple choice: Which type of immunoassay is typically faster?
(A) Indirect ELISA
(B) Sandwich ELISA
(C) Lateral flow assay
C) Lateral flow assay
What is the main difference between direct and indirect immunoassays?
Direct immunoassays use a labeled antibody that binds directly to the target epitope, while indirect immunoassays use a secondary labeled antibody that binds to the primary antibody.
Fill in the blank: The sensitivity of an immunoassay refers to its ability to detect _____ concentrations of an analyte.
low
True or False: Immunoassays require a complex sample preparation process.
False: Some immunoassays can utilize simple sample preparations.
What is the primary disadvantage of immunoassays?
The primary disadvantage of immunoassays is potential cross-reactivity, which can lead to false positives.
Which of the following is NOT a component of an immunoassay?
(A) Analyte
(B) Antibody
(C) PCR enzyme
C) PCR enzyme
What does the term ‘cross-reactivity’ mean in the context of immunoassays?
Cross-reactivity refers to the ability of an antibody to bind to similar but non-target antigens, potentially causing inaccurate results.
Fill in the blank: Immunoassays can be classified into _____ and _____ assays based on the format of antigen-antibody interaction.
homogeneous; heterogeneous
What is the significance of the calibration curve in an immunoassay?
The calibration curve is used to determine the concentration of an analyte in a sample by correlating the signal to known concentrations.
True or False: The specificity of an immunoassay is determined by the affinity of the antibody for the antigen.
True
What type of sample is commonly used in immunoassays?
Common samples include blood, serum, plasma, urine, and tissue extracts.
Multiple choice: Which type of immunoassay is most commonly used for pregnancy testing?
(A) Western blot
(B) Lateral flow assay
(C) Radioimmunoassay
B) Lateral flow assay
What is the purpose of blocking agents in immunoassays?
Blocking agents are used to prevent non-specific binding of antibodies to the assay surface.
Fill in the blank: In a sandwich immunoassay, the analyte is captured between two _____.
antibodies
True or False: Immunoassays can utilize both colorimetric and fluorescent detection methods.
True
What is a key factor that influences the performance of an immunoassay?
The choice of antibodies used in the assay is a key factor that influences its performance.
Multiple choice: Which of the following is a disadvantage of using radioimmunoassay?
(A) High sensitivity
(B) Need for special disposal of radioactive waste
(C) Cost-effectiveness
B) Need for special disposal of radioactive waste
What is the role of a secondary antibody in an indirect immunoassay?
The secondary antibody binds to the primary antibody and is typically labeled for detection.
Fill in the blank: The limit of detection (LOD) is the lowest concentration of an analyte that can be reliably _____ by an immunoassay.
detected
True/ False: polyclonal antibodies bind to a specific epitope
false, polyclonal antibodies can recognize multiple epitopes on an antigen
How are polyclonal antibodies produced?
produced in animals and are then harvested
What are 2 disadvantages of using polyclonal antibodies? (2 marks)
variability and possible cross-reactivity
what are 4 advantages of monoclonal antibodies
can be produced in high quantities, specificity to one epitope, consistency in Ig class, and highly pure
What’s an advantage of polyclonal antibodies
cheaper
What is the direct type of ELISA?
uses one labeled antibody
What is the indirect type of ELISA?
uses a primary antibody and a labeled secondary antibody
What is the sandwich type of ELISA?
captures antigens between two antibodies
What is the competitive type of ELISA?
measures antigen/antibody concentration by inhibition
What is serology?
Detects antibodies (IgM, IgG) which are produced during infections.
In serology, what does IgM indicate?
indicates recent infection
In serology, what does IgG indicate?
indicates past or chronic infection
What is conjugation assay?
where antibodies are tagged with detectable molecules
Give me 3 examples of detectable molecules that they tag antibodies with in a conjugation assay?
-enzymes
-fluorochromes
-radioactive isotopes
Which of the following is NOT a common application of immunoassays?
A) Detecting infections
B) Identifying tumor markers
C) Measuring soil pH
D) Developing therapeutic antibodies
C
What is the role of monoclonal antibodies in immunoassays?
A) Recognizing multiple epitopes
B) Providing high specificity and consistency
C) Being produced from polyclonal serum
D) Functioning only in research, not diagnostics
B
Serology primarily involves:
A) Measuring antigen concentrations using ELISA.
B) Detecting microbial DNA in serum.
C) Using antibodies to detect antigens in tissues.
D) Detecting antibodies in serum produced during infections.
D
Which fluorochrome is commonly used in fluorescence-based immunoassays?
A) Propidium iodide
B) FITC (Fluorescein Isothiocyanate)
C) Horseradish peroxidase
D) Hematoxylin
B
In flow cytometry, what does the intensity of fluorescence indicate?
A) Size of the cell
B) Granularity of the cell
C) Presence of a specific marker on the cell
D) Number of cells in a population
C
Confocal microscopy is used to:
A) Analyze cell viability using light scattering.
B) Visualize fluorescence in thin tissue sections with high resolution.
C) Quantify enzyme activity in living cells.
D) Separate different immune cell populations.
B
Which method is commonly used to isolate peripheral blood mononuclear cells (PBMCs)?
A) Western blot
B) Ficoll density centrifugation
C) ELISA
D) Immunoprecipitation
B
What is a hemocytometer used for?
A) Measuring fluorescence intensity in cells
B) Counting and calculating the concentration of cells in a suspension
C) Isolating specific cell subsets using antibodies
D) Analyzing antigen-specific responses
B
Which assay measures cell proliferation by detecting mitochondrial enzyme activity?
A) CFSE assay
B) MTT assay
C) Propidium iodide assay
D) H-thymidine incorporation
B
What is the main advantage of in vivo immunoassays over in vitro techniques?
A) They are more cost-effective.
B) They provide information in a reductionist, controlled environment.
C) They maintain the structural and cellular interactions of the immune system.
D) They require less technical expertise.
C
Genetic engineering in immunoassays is used to:
A) Visualize immune interactions in tissue samples.
B) Produce polyclonal antibodies for therapeutic use.
C) Add or remove genes to create models of specific diseases.
D) Measure cytokine production in cell culture.
C
Targeted therapeutic antibodies, such as those against TNFα, are used in:
A) Asthma treatment
B) Autoimmune diseases like arthritis
C) Detecting infections in patients
D) Flow cytometry assays
B
A researcher wants to detect a specific antigen at extremely low concentrations in a complex sample. Which of the following modifications to an immunoassay would most improve its sensitivity?
A) Using polyclonal antibodies for detection
B) Employing a monoclonal antibody conjugated with a fluorochrome
C) Increasing the concentration of the substrate in an ELISA
D) Adding a competitive inhibitor to reduce background noise
B
In serology, a pregnant woman tests positive for IgM against Toxoplasma gondii. What is the most likely clinical implication of this result?
A) She has immunity to toxoplasmosis and requires no treatment.
B) She has an acute infection and is at risk of vertical transmission to the fetus.
C) She has a chronic infection and needs prophylactic antibiotics.
D) She does not have a Toxoplasma infection but may be at risk of future exposure.
B
When performing flow cytometry, cells labeled with two fluorochromes (FITC and PE) are analyzed. The results show overlapping fluorescence signals. What is the most likely explanation for this?
A) Improper fixation of the cells
B) High cell granularity affecting scatter detection
C) Spectral overlap of FITC and PE emission wavelengths
D) Insufficient washing leading to non-specific binding
C
Which of the following is a key advantage of confocal microscopy over standard fluorescence microscopy?
A) It can measure dynamic changes in fluorescence over time.
B) It eliminates out-of-focus light, improving resolution in thick samples.
C) It allows quantification of cytokine secretion in live cells.
D) It uses radioactive labeling for increased sensitivity.
B
A scientist is studying T cell proliferation in response to a specific antigen. Which of the following methods would allow the most precise quantification of the number of cell divisions in the population?
A) MTT assay measuring mitochondrial activity
B) CFSE labeling and flow cytometry analysis
C) Trypan blue staining for viable cells
D) Radioactive thymidine incorporation into DNA
B
When isolating PBMCs using Ficoll density centrifugation, what is the primary factor that separates these cells from other blood components?
A) Electrical charge differences between cell types
B) Differential buoyant density in a gradient medium
C) Binding specificity to antibodies coated on magnetic beads
D) Adhesion of PBMCs to the walls of the centrifuge tube
B
Genetic engineering allows the creation of mouse models with specific gene deletions to study immune responses. Which of the following diseases is most likely to be studied using a TNFα-knockout mouse?
A) Type 1 diabetes
B) Rheumatoid arthritis
C) Malaria
D) Influenza
B
In an in vivo experiment, a researcher observes that antigen-specific T cells proliferate and migrate to a lymph node. Which of the following techniques would most likely have been used to track these cells in the living organism?
A) Flow cytometry of the lymph node
B) Immunohistochemistry on lymph node sections
C) Whole-body fluorescence imaging using labeled T cells
D) ELISPOT assay to detect cytokine production
C
A new immunoassay is designed to detect low levels of cytokines secreted by a single T cell. Which method would provide the best sensitivity for this application?
A) ELISA
B) ELISPOT
C) Flow cytometry
D) Western blot
B
What is a key disadvantage of polyclonal antibodies compared to monoclonal antibodies in advanced immunoassays?
A) High specificity leading to a narrow range of detection
B) Variability in antibody composition between batches
C) Limited ability to recognize conformational epitopes
D) Requirement for hybridoma technology to produce them
B
Antibody specificity is critical for immunoassays. Which of the following factors primarily determines an antibody’s specificity for its target?
A) The concentration of the antigen in the sample
B) The structure of the antibody’s Fc region
C) The amino acid sequence of the antigen-binding site
D) The presence of adjuvants during immunization
C
A researcher uses a polyclonal antibody in an assay but notices inconsistent results. Which is the most likely explanation?
A) Polyclonal antibodies lack the ability to recognize multiple epitopes.
B) Polyclonal antibodies are susceptible to denaturation at high temperatures.
C) Polyclonal antibodies have batch-to-batch variability due to their heterogeneous nature.
D) Polyclonal antibodies require a hybridoma for production.
C
In a competitive ELISA, which of the following outcomes would indicate a high concentration of antigen in the sample?
A) Increased fluorescence intensity in the final reaction
B) Reduced binding of labeled antibodies to the plate
C) Greater color change due to higher enzyme activity
D) Increased absorbance at the end of the assay
B, because it will outcompete the labelled antigens and occupy most of the binding sites
The primary advantage of using a sandwich ELISA over a direct ELISA is:
A) Higher specificity due to two antibodies recognizing the antigen.
B) Reduced time required to complete the assay.
C) Elimination of the need for a secondary antibody.
D) Its ability to detect smaller-sized antigens.
A
During an ELISA, why is it important to include a blocking step before adding the sample?
A) To prevent the denaturation of antibodies.
B) To ensure antigen binding to the plate surface.
C) To minimize non-specific binding to the plate.
D) To enhance enzyme activity for signal generation.
C
Phage display technology offers several advantages in antibody production. Which of the following is NOT true about phage display?
A) It allows rapid screening of large antibody libraries.
B) It relies on bacterial expression systems for antibody production.
C) It produces antibodies that always have correct folding for mammalian systems.
D) It can identify antibodies with high affinity for a specific antigen.
C
Which of the following is an advantage of nanobodies over traditional monoclonal antibodies?
A) They are larger in size, allowing better epitope coverage.
B) They require hybridomas for production.
C) They are less likely to induce an immune response in vivo.
D) They cannot be used in imaging applications.
C
Why are monoclonal antibodies preferred for therapeutic applications over polyclonal antibodies?
A) Monoclonal antibodies recognize multiple epitopes, providing broader coverage.
B) Monoclonal antibodies are produced by injecting animals with antigens.
C) Monoclonal antibodies have consistent specificity and reduced batch variability.
D) Monoclonal antibodies do not require purification before use.
C
What is the most significant limitation of polyclonal antibodies in diagnostic immunoassays?
A) They are only available in limited quantities.
B) They require multiple animals for production.
C) They contain antibodies that may bind to unintended targets, causing cross-reactivity.
D) They cannot be conjugated to enzymes or fluorochromes.
C
What is the primary reason for purifying antibodies before use in sensitive assays?
A) To increase their concentration for storage.
B) To remove contaminating proteins that may interfere with the assay.
C) To ensure that only polyclonal antibodies are present.
D) To change the antibody isotype for better detection.
B
Which of the following best explains the concept of antibody avidity in the context of immunoassays?
A) The ability of a single Fab fragment to bind to an epitope with high affinity.
B) The overall binding strength of multiple Fab fragments on the same antibody interacting with multiple epitopes.
C) The variability in binding strength between monoclonal and polyclonal antibodies.
D) The rate at which an antibody binds to and dissociates from its target antigen.
B
When designing an immunoassay, why is it critical to avoid antigenic cross-reactivity in polyclonal antibodies?
A) Cross-reactivity enhances detection of all potential analytes in a sample.
B) It introduces non-specific binding, reducing assay specificity and leading to false positives.
C) Cross-reactivity occurs only with monoclonal antibodies, making it irrelevant to polyclonal antibodies.
D) It increases the stability of antibodies under harsh conditions.
B
A sandwich ELISA is being performed to quantify a low-abundance protein in human serum. Which of the following modifications would most improve the assay’s dynamic range and sensitivity?
A) Use a competitive format to eliminate high-affinity binding biases.
B) Employ a pair of monoclonal antibodies with non-overlapping epitopes.
C) Replace the enzyme label with a fluorescent label to reduce signal variability.
D) Pre-treat the serum with high-salt buffer to improve antigen solubility.
B
In a direct ELISA, a researcher observes a high background signal even in negative control samples. Which of the following is the most likely cause?
A) Antigen degradation during the incubation step.
B) Non-specific binding of the enzyme-labeled antibody to the plate surface.
C) Insufficient washing after substrate addition.
D) Incorrect coating concentration of the capture antibody.
B
Competitive ELISAs often show reduced signal intensity as antigen concentration increases. What is the mechanistic explanation for this inverse relationship?
A) Increased antigen concentrations inhibit substrate binding to the enzyme label.
B) Higher antigen concentrations compete with labeled antigens for limited antibody binding sites.
C) Excess antigen increases non-specific binding of the detection antibody.
D) Competitive assays have lower sensitivity, making this observation irrelevant.
B
Why might antibodies generated using phage display libraries sometimes require further modification before clinical application?
A) Phage-derived antibodies are limited to bacterial systems and lack mammalian post-translational modifications.
B) Phage-derived antibodies exhibit low affinity and cannot bind their target effectively.
C) Phage libraries produce polyclonal antibodies unsuitable for therapeutic use.
D) Phage display is incompatible with high-throughput screening technologies.
A
Nanobodies derived from camelids are gaining popularity in immunoassays. Which of the following statements is correct regarding their unique properties?
A) Nanobodies contain a heavy chain and a light chain, which improve antigen binding.
B) Their small size allows better tissue penetration and reduces steric hindrance in complex assays.
C) Nanobodies are exclusively produced in mammalian cell lines due to their instability in bacteria.
D) Their high immunogenicity makes them unsuitable for in vivo therapeutic use.
B
A researcher aims to produce monoclonal antibodies targeting a human protein for therapeutic use. What is the most critical step to reduce immunogenicity of the antibody when used in humans?
A) Generate the monoclonal antibody using phage display.
B) Ensure the antibody is humanized by replacing mouse constant regions with human sequences.
C) Use hybridoma technology to produce a mouse monoclonal antibody and inject it directly into patients.
D) Immunize mice with the human protein and purify the resulting polyclonal antibody pool.
B
A novel immunoassay uses a monoclonal antibody paired with a nanobody for antigen capture and detection. What is the most likely advantage of this design over traditional monoclonal-monoclonal pair systems?
A) Increased cross-reactivity ensures broader antigen coverage.
B) Enhanced signal strength due to dual monoclonal specificity.
C) Improved tissue penetration and reduced steric hindrance at the detection site.
D) Simplified assay preparation as nanobodies do not require conjugation.
C
In an advanced ELISA, a researcher uses a fluorescent label for detection instead of an enzyme label. Which of the following best describes a limitation of this approach?
A) Fluorescent signals cannot be quantified accurately due to their dynamic range.
B) Fluorescent assays require specialized equipment for signal detection.
C) Fluorescent labels are more prone to degradation during assay preparation.
D) Fluorescent detection eliminates the ability to multiplex assays.
B
A diagnostic immunoassay for a rapidly mutating virus requires high sensitivity and adaptability. Which strategy is most effective in ensuring reliable antigen detection across multiple viral strains?
A) Use a cocktail of monoclonal antibodies targeting conserved epitopes.
B) Employ a single high-affinity polyclonal antibody.
C) Utilize phage display to identify strain-specific antibodies after every mutation.
D) Replace the antibody detection system with a nucleic acid-based method.
A
A researcher designs an immunoassay to differentiate between active and latent tuberculosis infections using IgG and IgM detection. Which of the following assay results would most accurately suggest active infection?
A) High IgG and low IgM levels
B) High IgM levels with negligible IgG
C) Equal concentrations of IgG and IgM
D) Low levels of both IgG and IgM
B
Which of the following would most likely improve the specificity of an ELISA used to diagnose autoimmune diseases?
A) Use monoclonal antibodies with broad cross-reactivity.
B) Select polyclonal antibodies to maximize epitope coverage.
C) Pre-block nonspecific binding sites on the plate with a relevant serum.
D) Increase the antigen concentration during the coating step.
C
A flow cytometry experiment involves staining immune cells with FITC, PE, and APC-labeled antibodies. Unexpectedly, high background fluorescence is observed in the PE channel. What is the most likely cause of this issue?
A) Autofluorescence from the cells overlaps with the PE emission spectrum.
B) Fluorescence quenching of FITC interferes with PE detection.
C) Cross-reactivity between APC and PE-labeled antibodies.
D) Suboptimal washing, leaving unbound FITC contributing to the PE signal.
A
Which of the following experimental designs would provide the most reliable analysis of multiple cytokines in a single sample using fluorescence-based immunoassays?
A) Use a single fluorochrome to sequentially detect cytokines in separate reactions.
B) Conjugate each cytokine-specific antibody to a spectrally distinct fluorochrome and analyze using flow cytometry.
C) Combine all cytokine-specific antibodies into one reaction and analyze using a fluorescence microscope.
D) Perform an ELISA for each cytokine using a fluorogenic enzyme-substrate system.
B
A confocal microscope is used to visualize fluorochrome-labeled antibodies binding to antigens in tissue sections. Which of the following adjustments would improve resolution in thick samples?
A) Increase the laser power to enhance fluorescence signal intensity.
B) Use a lower numerical aperture objective to increase the field of view.
C) Apply optical sectioning to acquire images at different depths.
D) Replace the fluorochromes with enzyme-based colorimetric detection.
C
During cell isolation using Ficoll density centrifugation, why is it crucial to minimize mechanical mixing after the gradient is formed?
A) To prevent cell rupture, which could release inhibitory molecules.
B) To maintain the density gradient required for cell separation.
C) To reduce contamination by serum proteins.
D) To enhance the yield of granulocytes in the final preparation.
B
In a functional assay measuring T cell killing of tumor cells, which of the following methods would most accurately quantify tumor cell death?
A) Trypan blue exclusion assay to detect live cells.
B) CFSE staining to measure tumor cell proliferation.
C) Annexin V and propidium iodide staining analyzed by flow cytometry.
D) MTT assay to detect changes in mitochondrial activity of tumor cells.
C
A cell proliferation assay uses CFSE labeling to measure T cell division. After stimulation, a flow cytometry analysis shows no CFSE dilution. Which of the following explanations is the most likely?
A) T cells are undergoing necrosis due to overstimulation.
B) The antigen used to stimulate T cells lacks MHC compatibility.
C) CFSE staining was too weak to detect changes.
D) Flow cytometry gating excluded the proliferating cell population.
B
A TNFα-knockout mouse model is used to study immune responses to bacterial infection. Which of the following findings would be most consistent with the role of TNFα in immunity?
A) Reduced production of IgG antibodies against the bacteria.
B) Impaired recruitment of neutrophils to the site of infection.
C) Enhanced T cell activation and cytokine production.
D) Increased phagocytosis of bacteria by macrophages.
B
Which of the following in vivo techniques would best allow tracking of antigen-specific T cells following vaccination?
A) ELISPOT to detect cytokine production in draining lymph nodes.
B) Flow cytometry of blood samples for fluorescently labeled T cell markers.
C) Bioluminescence imaging of luciferase-expressing T cells.
D) Western blot to measure T cell receptor expression.
C
In an in vivo therapeutic study, a humanized monoclonal antibody is tested in mice for its effect on a chronic autoimmune disease. Which of the following would most likely limit its efficacy?
A) Development of mouse anti-human antibodies neutralizing the therapeutic.
B) Rapid clearance of the antibody due to its high binding affinity.
C) Insufficient activation of mouse complement by the human Fc region.
D) Cross-reactivity of the monoclonal antibody with non-target mouse antigens.
A
When administering immunisation injections to produce antibodies, what should we do to the scheduling to increase the amount and affinity of the Ab.
multiple immunisations and at intervals, results in class-switching (more likely to be IgG/IgA)
What type of antibodies are produced from immunised cow?
polyclonal
Why might polyclonal antibodies have a higher chance of cross reactivity?
because the harvested serum may contain other proteins, which can cause problem in sensitive assays.
What can we do to reduce the chance of cross reactivity when using polyclonal antibodies
we can purify the antibodies to remove the other proteins
What’s a nanobody?
a antibody found in camelids (camels, llamas, alpaca) consisting of only a single variable heavy chain domain, making them much smaller while retaining high antigen-binding specificity and affinity.
EQ. Compare and contrast nanobodies and conventional antibodies in terms of their structure, function, advantages, and limitations in biomedical applications. (14 marks)
-Conventional antibodies such as IgG are large (∼150 kDa) Y-shaped proteins composed of two heavy and two light chains.
-and have a large antigen-binding site, often requiring surface epitopes for recognition.
-Nanobodies (∼15 kDa) are derived from the variable domain (VHH) of heavy-chain-only antibodies found in camelids and lack light chains.
-and have a smaller, single-domain binding region, allowing them to target hidden or concave epitopes, such as enzyme active sites.
-Antibodies require mammalian cell expression systems, making them costly and complex to produce
-Nanobodies can be produced in bacterial or yeast systems, which are more cost-effective. They are also highly stable under extreme pH and temperature conditions.
-Antibodies are widely used in immunotherapy, diagnostics, and vaccine development, with applications like monoclonal antibody treatments.
-they are valued for their longer half-lives but have poor tissue penetration due to their large size.
-Nanobodies are advantageous for targeted drug delivery, imaging, and passing biological barriers (e.g., crossing the blood-brain barrier).
-its highly hydrophilic framework reduces aggregation and precipitation at high temperatures, which often destabilizes conventional antibodies
-while conventional antibodies rely on inter-chain disulphide bonds between its heavy and light chains, nanobodies can remain stable even if its disulphide bonds are broken due to its intrinsically stable core structure that doesn’t depend on these bonds for proper folding
-they can also maintain functionality at high temperatures, as high as 90C, and has an ability to refold after denaturation
-nanobodies also exhibit low immunogenicity as their framework is highly similar to human antibody variable domains, and their lack of a Fc region compared to conventional antibodies reduces a potential immune system activation.
-however they have a short half-life and may require PEGylation or fusion to albumin for extended circulation time
What are 3 features of nanobodies?
-small size which allows it to penetrate tissues and cross biological barriers more effectively than traditional antibodies
-very stable, resistant to extreme PH, temperature
-high specificity & affinity, similar to full size antibodies but only a fraction of the size.
briefly name the 5 steps in an ELISA for IgG
-surface coated with antigen
-add serum containing IgG
-add anti-IgG
-add substrate
-stop substrate and measure
Which step in ELISA would u change to change the type of label on Abs
substrate
briefly name the 5 steps in an ELISA for a pathogen
-surface coated with anti-pathogen Ab
-add sample containing pathogen
-add anti-pathogen
-add substrate
-stop reaction and measure
What are the main 3 differences between ELISPOT and ELISA?
-ELISpot measures the number of cells secreting a specific cytokine/antibody while ELISA measures the total concentration of a antibody/antigen in a sample.
-ELISpot uses live cells while ELISA uses biological fluids
-detects individual spots, results are quantified as spot forming units (SFU) while ELISA is measured with absorbance (OD) values to represent total antigen/ antibodies in the sample.
what do we use a haemocytometer for?
we use it to count the number of cells, so we can calculate the concentration of cells per millilitre of suspension and the total number of ells.
after we’ve calculated the concentration of cells in a suspension, what do we use to identify how many of those cells are T cells and B cells.
flow cytometry
what is a mitogen-induced response
mitogens activate cells in non-specific manner, driving proliferation
give 3 examples of a mitogen that would induce a immune response and state the responding cells
-PHA - T cells
-ConA - T cells
-LPS - B cells
Briefly describe an MTT assay.
-relies on enzymes in mitochondria converting salt into coloured product
-amount of colour is proportional to the number of cells
-which is proportional to the amount of cell proliferation
Briefly describe an 3H-Thymidine incorporation assay.
-add radioactive thymidine to cell culture
-as cells proliferate, they incorporate this into the DNA of daughter cells, resulting in radioactive cells
-the amount of cell-associated radioactivity, proportional to the amount of cell division
What does RT-PCR detect in SARS-CoV-2 testing?
Viral RNA
Why is RT-PCR considered the gold standard for SARS-CoV-2 diagnosis?
It has high sensitivity (~95-99%) and specificity, detecting even low viral loads.
What are the main limitations of RT-PCR?
Expensive, requires specialized lab equipment, and takes 4-6 hours for results.
What do antigen tests detect?
SARS-CoV-2 viral proteins (e.g., nucleocapsid (N) or spike (S) proteins).
What are the 3 main types of antigen tests?
Lateral Flow Immunoassay (LFIA) (Rapid test)
ELISA
CLIA
What are 3 advantages of antigen tests?
Fast results (15 min - 3 hrs)
Cheap and easy to use
Good for mass screening
What are the 3 disadvantages of antigen tests?
Lower sensitivity (~50-80%) than RT-PCR
Less reliable in asymptomatic cases
False negatives possible
When are antigen tests most effective?
Early in infection when viral load is high.
What do antibody tests detect?
Host IgM, IgG, or IgA antibodies against SARS-CoV-2.
What are the 3 main types of antibody tests?
Lateral Flow Immunoassay (LFIA)
ELISA
CLIA
What 3 things are antibody tests used for?
Identifying past infections
Epidemiological studies
Vaccine response monitoring
What are the 2 advantages of antibody tests?
Good for detecting past infections (~2-3 weeks post-infection)
Higher sensitivity than antigen tests later in infection
What are the disadvantages of antibody tests?
Not useful for early diagnosis (delayed antibody response)
Cannot distinguish infection from vaccine-induced immunity
Potential cross-reactivity with other coronaviruses
Which test is best for early SARS-CoV-2 detection?
RT-PCR (most sensitive, detects viral RNA).
Which test is best for rapid screening?
Antigen tests (fast, cheap, but lower sensitivity).
Which test is best for determining past infection?
Antibody tests (detects immune response, not current infection).
Why might an antigen test require RT-PCR confirmation?
Lower sensitivity means false negatives are common.
Which of the following is the primary reason for the lower sensitivity of rapid antigen tests compared to RT-PCR?
A) Antigen tests detect host immune response, which takes longer to develop.
B) Antigen tests require a higher viral load for detection.
C) RT-PCR has lower specificity, leading to more false positives.
D) Antigen tests are unable to detect SARS-CoV-2 nucleocapsid proteins.
B. Antigen tests require a higher viral load, meaning they are less effective in detecting early or low-level infections.
In an outbreak investigation, researchers want to determine how many people in a population have been previously exposed to SARS-CoV-2. Which test is the most appropriate?
A) RT-PCR
B) Rapid antigen test
C) IgG ELISA
D) Nasopharyngeal swab antigen test
C. IgG ELISA is the best option because it detects past infections by identifying IgG antibodies that develop weeks after exposure.
Which of the following factors does NOT significantly impact the accuracy of a SARS-CoV-2 rapid antigen test?
A) Timing of sample collection
B) Patient’s viral load
C) Presence of cross-reacting antibodies
D) Presence of SARS-CoV-2 RNA mutations
D. RNA mutations affect RT-PCR, but not antigen tests, since antigen tests detect proteins, not genetic material.
A patient presents with COVID-19 symptoms, but their RT-PCR test is negative. Which scenario is the most likely explanation?
A) The patient has a high viral load but poor sample collection affected results.
B) The patient is at a late stage of infection, and viral RNA levels are low.
C) The patient has an early-stage infection, and RT-PCR lacks sensitivity.
D) The patient’s immune response has cleared the virus, leaving only intact viral RNA.
B. In the late stage of infection, viral RNA levels drop, making RT-PCR results negative, even if symptoms persist.
Why do serological antibody tests have limited use in diagnosing acute SARS-CoV-2 infection?
A) They only detect active viral replication.
B) They cannot distinguish between viral antigens and host proteins.
C) Antibody production occurs too late to be useful for early diagnosis.
D) They require nasopharyngeal samples, which are difficult to obtain.
C. Antibodies (IgM/IgG) appear days to weeks after infection, making them unsuitable for early diagnosis
In a low-prevalence population, what is the major drawback of using SARS-CoV-2 antibody tests for widespread screening?
A) Increased likelihood of false positives due to low pre-test probability
B) Decreased specificity leading to more false negatives
C) The test becomes completely ineffective
D) Antibody tests are only useful in symptomatic patients
A. In low-prevalence populations, false positives become more common due to Bayes’ theorem, reducing test reliability.
Which of the following best explains why antigen tests are preferred over RT-PCR for large-scale rapid screening?
A) Higher specificity ensures fewer false positives.
B) Faster turnaround time and ease of use.
C) Greater sensitivity ensures early detection.
D) Ability to detect viral RNA mutations more effectively.
B. Antigen tests are fast, cheap, and easy to use, making them ideal for mass screening, despite lower sensitivity.
Which of the following modifications would most likely improve the sensitivity of a SARS-CoV-2 antigen test?
A) Using a higher dilution factor in the sample preparation
B) Targeting viral RNA instead of proteins
C) Increasing the affinity of the capture antibodies for viral antigens
D) Eliminating the wash steps in ELISA testing
C. Increasing antibody affinity improves antigen capture efficiency, enhancing sensitivity.
A COVID-19 antigen test shows a positive result in an asymptomatic individual. Which step is most appropriate next?
A) Assume the person is infected and begin isolation immediately.
B) Perform a confirmatory RT-PCR test to rule out a false positive.
C) Perform an antibody test to check for past infection.
D) Repeat the antigen test multiple times to ensure consistency.
B. Antigen tests have lower specificity, so RT-PCR confirmation is needed, especially in asymptomatic individuals.
A new SARS-CoV-2 variant has multiple mutations in the spike protein but no changes in the nucleocapsid protein. Which diagnostic test is most likely to remain effective?
A) RT-PCR targeting the spike protein gene
B) ELISA detecting spike protein antigens
C) Rapid antigen test targeting nucleocapsid protein
D) Whole-genome sequencing of the viral RNA
C. Nucleocapsid-targeting antigen tests remain effective if mutations occur only in the spike protein.
