Module 05 Flashcards
What does ELISA stand for?
Enzyme-linked immunosorbent assay
What is ELISA used for in health care?
Broadly-applicable technique which can be modified to detect and quantify substances such as peptides, proteins, antibodies, hormones, and other molecules
How does ELISA work? (4 steps)
1- Bottom of wells are coated with an antigen that is specifically recognized by the antibody you wish to measure
WASH -remove excess antigen
2- Sample containing antibody to be measure is added to the well. Primary antibodies, if present, will bind to the antigens atteched to the bottom of the well
WASH - remove excess primary antibody not attached to antigen
3- Enzyme-conjugated secondary antibody is added to the well. This will bind to the Fc Portion of the primary antibodies already present in the well - specific to animal
WASH - remove excess secondary antibody
4- Substrate of the enzyme attached to the secondary antibody is added to the well. Reaction of the substrate and the enzyme produces a coloured product which can be measure by absorbance
What does ELISA measure?
Coloured reaction product buy absorbance by a spectrophotometric plate reader.
Data measured correlates with the presence of an antibody or an antigen
– USed to detect presence of viral disease
What is an indirect ELISA?
Detects/Quantifies antibody
What is the difference between Detection and Quantification
Detection: IF substance is present or absent
Quantification: evaluates the concentration of substance
How can flow cytometry be used in the diagnosis of HIV/AIDS?
Diagnosis of AIDS is made based upong the number of CD4+ T-cells found in a patient’s serum. Flow cytometry can be used to identify and count these specific T-cells in a blood sample
What does flow cytometry measure?
Physical properties of a cell eg specific antigen on or inside a cell
What detection tool is used in CBC (complete blood counts)?
Flow cytometry
How does flow cytometry work?
1- Narrow stream of cells in single file is passed through a laser light source – each cell uniquely scatter laser light
- Forward and Side scattered light is measured which helps in the discrimination by cell size
What is a clinical application of flow cytometry?
Diagnosis of cancer
What is the advantage of using monoclonal antibodies in contrast to polyclonal antibodies?
Monoclonal antibodies are a homogenous population of antibodies with exquisite specificity for a single antigen whereas polyclonals could recognize multiple antigens
What does Monoclonal antibodies measure?
It is not a specific technique of measurement - tool useful in many applications
What are the 2 types of monoclonal antibodies?
1- immunotoxins
2- Radiolabelled antibodies
What is immunotoxins and what is it used for?
Tumour-specific monoclonal antibody attached to a deadly toxin.
This technique is still under investigation but shows promise in targeting and eliminating tumour cells and treat cancer
What is Radiolabelled antibodies and what is it used for?
Monoclonal antibodies tagged with a radioactive isotope. Can bind to antigens on a tumour thereby allowing precise location
Can be used for the diagnosis of tumour earlier than other methods.
How does Monoclonal Antibodies work
Monoclonal antibodies are produced in the laboratory by hybridomas, immortal cells that produce unlimited qties of one identical antibody
Hybridomas are the results of fusion between a plasma cell and a cancerous cell. - share properties of both types
Who developed Monoclonal antibodies?
George Kohler and César Milan
What are 2 clinical application of monoclonal antibodies?
Defence against specific diseases and even cancer
Why are vaccines preventative in protecting against infection?
Vaccines are made up of fragments/entire pathogen that is modified (killed or attenuated) allowing immunological response to develop without contracting the disease. This results in immunological memory
What are the 4 types of vaccines?
- Live-attenuated
- Killed-Inactivated
- Toxoid
- Subunit
What are live attenuated vaccines?
Contains a modified strain of the disease-causing agent which has lost its pathogenic ability, but retains its capacity to replicate within the hose
What are the advatange (1) and disadvantages (2) of live attenuated vaccines?
Ad:
Provides a prolonged exposure to the pathogen and is suitable to generate cell-mediated immunity
Dis:
- Potential to revert to a virulent form
- Requires specific storage and transport conditions (refrigeration)
Small pox vaccine, oral poliovirus vaccine and measles vaccine are example of which type of vaccine?
Live attenuated
Give 3 examples of live-attenuated vaccines?
Smallpox vaccine
Oral poliovirus vaccine
Measles vaccine
What are killed-inactivated vaccines?
Contains a strain of the pathogen that has been inactivated by heat, chemical or radiation. It has the ability to generate an immune response but unable to replicate
What are the advantages (2) and disadvantages (2) of killed-attenuated vaccine?
Ad:
- Safer Option as it cannot mutate back to virulent form
- Easy to store and transport
Dis:
-Generally requires multiple booster doses to maintain immunity
- Generally must be administered by injection
Rabies vaccine and flu vaccine are examples of what type of vaccine?
Killed-attenuated
Give 2 examples of killed attenuated vaccines?
Rabies, Flu
What are toxoid vaccines?
Contain an inactivated toxin which is a product from the pathogen
What are the advantages (2) and disadvantage (1) of toxoid vaccines?
Ad:
- Safe as it is not a living organism that can divide, spread and/or revert
- Stable as they are less susceptible to changes in temp, humidity and light
Dis:
- May require several doses and usually need an adjuvant
What is an adjuvant?
A substance that enhances the body’s immune response to an antigen
Tetanus vaccine and diptheria vaccine are examples of what type of vaccine?
Toxoid
Give 2 examples of toxoid vaccines
Tetanus & diphteria
What are Subunit Vaccines?
Contains only a small part of fragment of the pathogen
What are the advantage (1) and disadvantage (1) of subunit vaccine?
Ad:
- Safest type of vaccine = can be used on everyone, including immunocompromised, pregnant, and elderly
Dis:
-Rarely successful at inducing long-lasting immunity, which means it will require multiple booster doses to maintain immunity and might even need to be conjugated to a carrier
Name an example of subunit vaccine
Hep B
Hepatitis B is an example of what type of vaccine?
Subunit
What are VLPs?
Virus like particles are composed of the structural proteins of the virus, which can self assemble into particles that resemble the natural virus both structural and immunologically. As they do not contain viral DNA, VLPs are not infectious
HPV is a type of what kind of vaccine?
virus like particles (VLPs)
What are the 3 authorized HPV vaccines in Canada?
Cervarix, Gardasil, Gardasil 9
What is the efficacy reported for the HPV vaccine?
approaching 100%
What is the safety profile of the HPV vaccine
92% of adverse effects have been categorized as non-serious
What did the EBOV vaccine used to make its vaccine?
glycoproteins from Ebola virus and putting then into a live-attenuated recombinant vesicular stomatitis virus (VSV) that expresses the transmembrane glycoproteins of EBOV and MARV
What’s a modern Vaccine failure story and why?
Genital Herpes vaccine
Subunit vaccine was developed and only showed 20% efficacy
What are the 6 stages of vaccine development?
1- Lab studies 2- Preclinical trial 3- Clinical Phase 1 4- Clinical phase 2 5- clinical phase 3 6- Health canada approval
Describe the laboratory studies phase of Vaccine Development (3 characteristics)
- Identify disease causing agent and selecting a strain relevant to target population
- Development and testing of the manufacturing process according to good manufacturing practice
- Dependent on laboratory research
Describe the preclinical studies phase of Vaccine Development
- Research carried out in animal models to evaluate pharmacological aspects of the product; demonstrate immunogenicity and safety profile
Describe the Clinical phase 1 studies phase of Vaccine Development
Small scale trial in humans (10-100 individuals) to assess vaccine safety by evaluating local and systemic reactions after administration + preliminary data on immunogenicity in humans
Describe the Clinical phase 2 studies phase of Vaccine Development
Bigger scale trials in humans (50-500) to collect data on the safety, side-effects, and efficacy of the vaccine + evaluates dosage requirements
Describe the Clinical phase 3 studies phase of Vaccine Development
- Multiple geographic sites with many hundreds of subjects (300-30000) to evaluate efficacy under natural disease conditions
- Researchers MUST demonstrate the efficacy in target populations and complete safety assessment for the vaccine
Describe the Health Canada Approval phase of Vaccine Development
Submission to Health Canada to be considered for approval with sufficient scientific, and clinical evidence to show that it is safe, efficacious, and of suitable quality
What is the “cold chain”?
Live-attenuated vaccines need to be stored in specific conditions which often include refrigeration - if cold chain is broken the vaccine risks losing potency or becoming completely unusable
What are some challenges that faces vaccine development?
- Cost
- Cold chain
- Continuous monitoring (mutation/new strain)
- Gold standard (new vaccines must be as good or better than older ones)
What are the specific challenges that come with the production of a one-for-all flu vaccine?
Structural nature of the virus; there are 11 NA subtypes and 8 HA subtypes = 198 potential strains
Why is it important to vaccinate everyone who can be vaccinated?
Herd Immunity; form of indirect protection from an infectious disease that occurs when a large proportion of the population is immune to an infectious agent - this slows or completely stops the spread of the disease
Very important for immunocompromised patients
What is cancer
Disease characterized by an error in either cell or cell death which can result in out of control growth of cells
What are 5 characteristics of cancer?
- Cancer cells behave differently than normal cells, they do not need specific growth factors to divide and don’t respond to the signals that cause normal cells to stop dividing
- Cancer cells continue to divide and grow ultimately forming a tumour (abnormal mass of tissue)
- Role of the immune system in cancer development is under intensive research
- Immunotherapies are treatments based on secreted of cellular components of the immune system aiming at enhancing anti-tumour immune responses
- Cancer Cells are resilient
In the field of immunology how are cancer cells viewed?
Self cells that have been altered to escape the normal growth-regulating mechanisms as the results of DNA alterations
What is a benign tumour?
Tumour that is NOT cancerous, and thus, is unable to grow indefinitely or invade surrounding tissues
What is a malignant tumour?
Cancerous tumour that has the ability to metastasize, they continue to grown and become progressively invasive
What is metastasis?
Colonization by tumour cells of sites different from their primary site of origin - small clusters of cancerous cells will break off and invade the surrounding blood or lymphatic vessels travelling to different areas of the body where they can proliferate
What is resilience?
A cell’s ability to cope with environmental distrubances, and possibly even adapt for future exposure
What is stress?
Umbrella term for the harsh environmental exposures on tumour cells
What are the 7 steps of the cancer immunity cycle?
1- Release of cancer Cell Antigens
2- Cancer antigen presentation
3- Priming and activations (APCs & T-cells)
4- Trafficking of T-cells to tumours (CTLs)
5- Infiltration of T-cells into tumours (CTLs, endothelial cells)
6- Recognition of cancer cells by T-cells (CTLs, cancer cells)
7- Killing of cancer cells (immune and cancer cells)
What is the theory of tumour immunosurveillance?
Tumour cells are identified and kept under control by the immune system of healthy individuals
What is immunoediting?
Dynamic process which describes the connection between the tumour cells and the immune system in the context of surveillance and tumour progression. Immune system kills growing cancer cells, which promote tumour growth which have the ability to evade immunosurveillance It has 3 phases: Elimination Equilibrium Escape
Describe the 3 phases of immunoediting
1- Elimination
a. When a tumour arises in a tissue, the immune system can quickly act to remove it.
b. NK cells, cytotoxic cells, and helper T-cells can recognize altered cell
2- Equilibirum
a. Survivor tumour cells can enter a state of equilibrium where their proliferation is matched by cell killing
b. Short time or many years
3- Escape
a. Tumour cells are no longer recognized by immune system and so avoid elimination
b. Able to grow uncontrolled and eventually forms a tumour
What are 2 mechanisms through which cancer cell evades the immune system?
Reduced MHC Expression
Poor costimulatory molecules
Describe how reduced MHC expression help cancer cells evade the immune response
Tumour cells display low levels of MHC Class I molecules on their cell surface = inhibiting their recognition by CTLs
Describe how poor costimulatory molecules help cancer cells evade the immune response
Recall that T-cells require both expression of MHC and costimulatory molecules to become activated
Thus, tumour cells lacking these costimulatory molecules = poor immunogenicity due to only partially activated T-cells
What functions of the immune system do you think would be important in treating cancer?
Recognition of abnormal/potentially cancerous cells
Stronger immune response to abnormal/potentially cancerous cells
Compare Immunotherapy and traditional methods in the treatment of cancer
Traditional: Use specific poison or targeted drugs to selectively kill cancer cells, but not the host cells (selective toxicity)
Immunotherapy: Designed to “flag” cancer cells to the immune system, so that the body’s own defences can eliminate the cancerous cells
Why does immunotherapy have the potential to be highly effective to treat cancer? (4)
- Attack of cancerous cells through all organs in the body
- Allows immune system to specifically target and eliminate cancer cells without damaging healthy cells – fewer side effects
- Takes advantage of immunological memory, allowing for the possibility of long term protection
- Applied to almost all types of cancer
What is a hot tumour?
Solid tumours that are T-cell inflamed
What is a cold tumour?
solid tumours that are T-cell non-inflamed
What are the 2 types of tumours?
T-cell inflamed tumours (hot)
T-cell non-inflamed tumor (cold)
Described T-cell inflamed tumours (4)
- Higher immune activity vs cold tumours
- High numbers of CD8+ TILs
- High levels of Interferon genes
- Usually responds well to treatment
Describe T-cell non-inflamed tumours (4)
- lower immune activity vs hot tumours
- low numbers of CD8+ TILs
- Low levels of interferon genes
- Usually inferior response to treatment
What is one way to enhance cold tumours response to treatment?
By converting it to a hot tumour though the stimulation of the tumour’s interferon activity
What are TILs
Tumour Infiltrating Lymphocytes, can be both T-cells or B-cells
What are the used of TILs in cancer diagnosis?
Suggested to be prognostic biomarker in some cases
What is the workflow for determining an immunoscore?
1- Separating the tumours in the central (CT) and peripheral (IM) regions
2- Staining T-cells and conducting digital pathology
3- Assigning a score tot he tumour to relate it with an associated diagnosis or risk attirbution
