Immunological Techniques Flashcards
When will an Ab bind to an Ag?
ONLY IF the antigenic binding site is specific for that Ag (lock and key).
Why can many antibodies bind with the same Ag?
Because an Ag has many different antigenic determinants
Can an Ab bind with another Ab?
Yes, the variable region of an Ab can bind with the constant region of another Ab if it is recognized as a foreign protein.
How can we make Ab?
Polyclonal Ab
1. Inject immunogen into animal
2. Given secondary immunization to produce higher titers of Ab against particular Ag
3. After immunization, polyclonal Ab can be obtained straight from the serum or purified to obtain a solution which is free from other serum proteins
Monoclonal Ab
1. Inject Ag into animal, multiple times
2. Once develops immune response, B-lymphocytes are isolated from the animal’s spleen and fused with a myeloma cell line creating immortalized B cell-myeloma hybridomas
3. Hybdridomas, which are able to grow continuously in culture while producing Ab are then screened for desired mAb
What is the difference between monoclonal and polyclonal Ab?
Monoclonal Ab have monovalent affinity and only recognize the same epitope (single site) of an Ag.
Polyclonal Ab can recognize multiple sites
What are the immunology techniques?
- Immunocytochemistry/immunohistochemistry
- Immunoassays
- Western Blotting/Immunoblotting
- Agglutination
- Flow Cytometry and fluorescence-activated cell sorting (FACS)
What is immunocytochemistry and immunohistochemistry?
An immunology technique that qualitatively studies Ag localization in cells (cyto) and tissues (histo)
What do we rely on to detect Ab-Ag interactions?
- An Ab with specificity for the Ag of interest
- Virus/bacteria (in infected cell/tissue)
- Cluster of differentiation (CD) Ag on a leukocyte: tumor Ag, proteins - Means to visualize the binding reaction that occurs and quantify it/localize it
What are the two primary methods to detect antigens?
- Direct
- One PRIMARY Ab against Ag
- Simple, rapid - Indirect - Multiple Ab
- PRIMARY against Ag, SECONDARY against Primary Ab
- Sequential incubations
- More sensitive & specific bc multiple secondary Abs can bind primary and amplify signal
What are immunoassays used for?
Quantifying Ag or Ab concentrations (i.e., cytokine/protein or specific Ab produced)
What are the 2 types of immunoassays?
- Enzyme-linked immunosorbent assay (ELISA) color signal
- Rapid antigen test
What is the difference between direct and indirect ELISA?
Direct uses a primary conjugated Ab
Indirect ELISAs - an un-conjugated primary Ab binds to the Ag, then a labeled secondary Ab binds to the primary Ab
Is sandwich ELISA qualitative or quantitative?
Quantitative, it determines the amount of IL-2 in cell media against a standard curve of known concentrations
How does sandwich ELISA work?
- Immobilized capture Ab to Ag of interest (IL-2)
- Addition of cell media containing cytokines and growth factors - IL-2 binding during incubation
- Cell media is washed away
- Enzyme-conjugated Detection Ab is added-incubation: must recognize a different Ag determinant unbound detection Ab is washed away
- Substrate is added for time; reacts with enzyme to produce a coloured product
- Read on spectrophotometer
- The greater the concentration of Ag = increased amount of bound label = increased colour
- Can be quantified against a standard curve of known IL-2
What does the test efficacy of the rapid antigen test depend on?
- Appropriate window of time
- Appropriate amount of antigen
- Appropriate test sample
What antibody marks the primary response?
IgM
Differentiate between the RAT and RT-PCR.
RT-PCR
Speed - Slow (hours)
Cost - Expensive
Technical Difficulty - Complex (lab)
Sensitivity - Very high
RAT
Speed - Fast (15-30 min)
Cost - Cheap
Technical Difficulty - Simple (office)
Sensitivity - Moderate
What is western blot used for?
Detect proteins/Ab to proteins of specific MW
Allows one to determine the relative amount of protein/Ab to protein in a sample of cells
Example: testing for seropositivity (presence in serum of Abs) to HIV
Why is western blot useful?
- Proteins/Ab to proteins are up regulated in certain disease states
- Detecting the presence of this protein/Ab to proteins in combination with other immunology techniques can be diagnostic for the disease, determine prognosis and treatment
What is the use of Ab/Ag in clinic?
- Lyme neuroborreliosis - seropositivity distinguishes from non-Lyme-related symptoms and enables rapid treatment
- Efficacy of immunization (Hepatitis B/C Virus)
- Anti-HIV Ab suggest previous HIV infection - HIV viral proteins increase in # in host-cells may indicate disease activity
- SARS-CoV-2 Ag indicate active infection - Anti-SARS-CoV-2 Ab indicate previous infection
What are the steps of western blot to detect HIV?
- Lyse KNOWN HIV-infected cell line to extract HIV proteins
- Seperate proteins by gel electrophoresis by size and/or charge
- Incubate the HIV strip/blot with serum
- “+ serum” - HIV+ patient
- “- serum” - Healthy donor
- “test serum” - unknown patient - Incubate the blot with a secondary Ab (mouse anti-human Ab)
- Visualize with a marker
What is blood type determined by?
Presence/absence of proteins (Ag) on surface of RBCS
Why is blood typing important?
- Safe blood transfusions rely on compatability
- Incompatible blood results in severe acute hemolytic reaction = renal failure and shock = potentially fatal - Highly active Ab recognize “foreign” proteins on RBCs and bind components of the complements system to cause massive hemolysis of the transfused blood
How does agglutination happen?
RBC containing A and Rh Ag + Ab (anti-A or anti-Rh) = agglutination reaction
How do we type blood?
- A drop of blood is added to each reagent (Anti-A, Anti-Rh, Anti-B)
- The slide is gently rotated and examined for clumping-agglutination/immune complex precipitates
How does flow cytometry and fluorescence-activated cell sorting (FACS) work?
It provides rapid, quantitative, multi-parameter analyses on single living (or dead) cells based on measurement of light scattering ability and fluorescent light emission.
Distinguises leukocytes by scattering (size/granularity) or fluorescence intensity produced by fluorescent-labeled Ab or ligands that bind specific cell-associated molecules, CD4/CD8/B cell markers
What can flow cytometry be used for?
Leukemia diagnosis
- Analyze leukocyte subsets and determine which cell line is being produced abnormally
- Monoclonal Ab specific to several hematopoeitic cell surface Ag provide a profile
- Profile allows confirmation of diagnosis, prediction of the course of the disease and the likelihood of remission
How to assess immune status/lymphocyte function?
- Histology - examine lymph node that has very few lymphocytes/irregular structure suggests an immune deficiency/lymphoma
- Blood test - differential blood count would identify altered numbers of leukocytes/lymphocytes
- Flow cytometry - B-lymphoblastic lymphoma cells are CD19+, CD10 bright
- In vivo tests - a real response test
- Allergic response: panel of several common Ag
- Delayed type hypersensitivity (a red welt) - Lymphocyte activation ex vivo
- Any stimulus tht promotes T & B lymphocyte fcn
- Mitogen
- Mixed lymphocyte reaction: transplant rejection in a dish
How does the TB skin test work?
- A small amount of tuberculin is injected into the skin
- Individual returns 48-72 h to have rxn assessed
- +ve test indicated previous infection
- -ve test suggests TB disease not likely
How do we measure lymphocyte proliferation?
Carboxyfluorescein diacetate succinimidyl ester (CFSE) labelling
- Labelled cells are treated with a mitogen that mimics Ag
