Immunological techniques Flashcards
• The difference between polyclonal antiserum and monoclonal antibodies • How antibodies are produced • How antibodies are used to determine the presence or concentration of substances/antigens and use in serology/immunoassays • How antibodies are used to visualise antigens - microscopy • How antibodies can neutralise biological activity – in research & therapy • Which immunological procedure is used to detect a particular pathogen/antigen
Antigen
Anything that is recognised by the immune
system as non-self
Antibody
proteins produced in response to an antigen. It
can only bind with the antigen that induced its formation – i.e. specificity
Epitope
the specific part of the antigen that binds to the antibody
Affinity
measure of binding strength between an epitope
and an antibody binding site. The higher the affinity the
stronger the interaction
Polycloncal/ monoclonal
Poly: lots of antibodies binding to lots of different shapes
One specific antibody binding to one antigen - monoclonal
Production of monoclonal antibodies (mAb) - usually mice used
- Mouse immunized with antigen
- Mouse produces Ab to Ag
- Spleen removed to get plasma cells
(NB. 1 plasma cell 1 Ab) - Plasma cells fused with immortal B
cells using polyethylene glycol to
produce immortal hybridomas - Cells are placed into 96-well plates
containing HAT (hypoxanthine,
aminopterin, thymidine). Kills off
non-fused cells so only hybridoma
cells alive. - Dilute so have only 1 hybridoma per
well – this will produce just a single
mAb with 1 specificity. - Hybridomas secreting high affinity
mAb selected using ELISA against
original Ag. - End up with a limitless supply of
high affinity mAb.
Production of monoclonal antibodies (mAb) - usually mice used
- Mouse immunized with antigen
- Mouse produces Ab to Ag
- Spleen removed to get plasma cells
(NB. 1 plasma cell 1 Ab) - Plasma cells fused with immortal B
cells using polyethylene glycol to
produce immortal hybridomas - Cells are placed into 96-well plates
containing HAT (hypoxanthine,
aminopterin, thymidine). Kills off
non-fused cells so only hybridoma
cells alive. - Dilute so have only 1 hybridoma per
well – this will produce just a single
mAb with 1 specificity. - Hybridomas secreting high affinity
mAb selected using ELISA against
original Ag. - End up with a limitless supply of
high affinity mAb.
Antibodies can be labelled (conjugated)
Unlabelled
Enzyme (horse radis, peroxidase alkaine-phosphatase)
Fluorescence (FITC, PE, many others)
Gold (electron microscopy)
Direct and indirect tests
Direct: plastic/ cell surface - Ag - mouse anti-human - tag
-generally we use this
Indirect: boosts the signal; plastic/ cell surface - Ag - mouse anti-human - rabbit anti-mouse - tag
Serological diagnosis
Use of Ab specificity to detect Ag
Not only do many serological assays give a Positive and
Negative result they can also quantitative the strength of Ab-Ag
interaction – TITRE
The TITRE of an Ab is defined as the LOEWEST DILUTION of the
sample that RETAINS a DETECTABLE ACTIVITY
Serological tests can be used to
- Diagnose Infections
- Identify Microorganisms
- Quantify proteins in the serum
- Type Blood – for blood banks and tissue transplantations
BUT - They are retrospective & only show that you have HAD an infection
Precipitation and immunodiffusion techniques
Not very often used these days
Relies on the ability of Ab to form complexes with Ag and precipitate
-antibody excess –> equivalence –> antigen excess
Immunoprecipitation: Ouchterlony diffusion test
•Ab and Ag are placed into well cut into agar gels.
•The Ab and Ag diffuse through the gel and form a precipitate at the
equivalence point (Usually visualised by staining).
•Was used to detect diphtheria toxin in serum – now PCR used to detect
bacteria
Immunoprecipitation Ouchterlony diffusion test results - Immunodouble diffusion
1) Precipitin band formed with a single antigen (identity)
2) Two independent Ag (a&b) react with their specific Ab (non-identity)
3) Ab (a&b) are specific for their Ag – (partial identity)
Single radial immunodiffusion
This technique involves the diffusion of Ag into an Ab-containing
gel. Precipitin rings indicate an immune reaction and the area of
the ring is proportional to the concentration of antigen
Immunoelectrophoresis (do not worry about this, not used anymore)
- Ag is placed in a well and separated by electrophoresis (electrical current).
- Ab is then placed in the trough and precipitin lines form as Ag and Ab
diffuse toward each other
Agglutinin tests
Commonly used in serology for many infections (& blood
typing)
Relies on polyclonal nature of serum
Relies on polyclonal serum to cross-link Ab (similar to
precipitation) but involves cells or beads
Influenza detection: Haemagglutination inhibition test purpose
This test is used to detect the presence of antibodies to influenza virus in a
patient’s serum or BAL fluid
Influenza detection: Haemagglutination inhibition test steps
1. Influenza has haemagglutinin molecules on their outer surface 2. Haemaglutinin binds the virus to red blood cells. 3. When virus particles are mixed with red blood cells they cause haemagglutination. This forms an aggregate 4. In the presence of specific Ab (anti-haemagglutinin Abs) binding of haemagglutinin to RBC is inhibited. RBC settle to bottom of tube
Haemophilus Influenzae Detection
Bacteria that can cause meningitis
1. Cerebrospinal fluid (CSF) sample taken from patient with meningitis
2. Sample mixed with a suspension of latex beads coated with specific
anti-H. Influenzae Ab
3. Interaction between Ag and Ab causes immediate agglutination of
beads which can be seen by eye – positive diagnosis for H. Influenzae
Detection of infection by Streptococcus bacteria
These Gram-positive bacteria are a group of oral & dermal pathogens
– soft tissue infections
They secrete Streptolysin O toxin – a protein that lyses RBC by
punching holes into them (a bit like complement MAC)
Detection of infection by Streptococcus bacteria: Anti-streptolysin O test
1. Serum taken from patient and diluted in tubes containing standard amount of sheep RBC and O toxin. 2. If patent has Ab to O toxin it will neutralise (inhibit) O toxin and stop it from lysing RBC (RBC settle to bottom of tube and tube is clear). 3. At low Ab concentration there is not enough Ab to stop RBC lysis (RBC bust releasing haemoglobin – red tubes). 4. This gives the Ab titre
Enzyme-Linked Immunosorbent Assay
•Used to detect levels of Ag, Ab or proteins in a sample
•Non-agglutination & non-precipitation test
•Extensively used in clinic and laboratory
•Used to accurately quantify levels of test molecule in a
sample using a standard curve
•Uses Ag or Ab bound to a solid phase (can be plastic or the
cell surface – for cell surface receptors)
1. Sandwich 2. Antigen
Sandwich ELISA steps
1. Capture Ab bound to plastic surface that is specific for desired Ag. 2. Add patient serum, CSF or supernatant (and standards to other wells) 3. Ab will bind specifically to Ag it is raised to. All other Ag are washed away. 4. Add Detection Ab – this has an Enzyme (usually HRP) conjugated to it. 5. Wash of excess detection Ab. 6. Add substrate for enzyme – this is colourless and turns blue in the presence of enzyme. 7. The more Ag the more colour is produced 8. Measure absorbance @450nm 9. Calculate concentration of Ag in the sample
Sandwich ELISA function - V IMPORTANT
Used to measure cytokines, virus, bacterial products etc in serum and lab
Antigen ELISA function
Used to measure conc. of human Ab in serum to bacteria - i.e. anti-Strep. Pneumoniae Ab etc.
Antigen ELISA steps
1. Bind Ag/bacteria/cell to solid phase 2. Wash off excess Ag 3. Add primary Ab specific for the Ag 4. Wash of excess primary Ab 5. Add secondary Ab. This is specific for the primary Ab and is conjugated with an enzyme (HRP) 6. Wash off excess secondary Ab 7. Add substrate 8. Substrate turns from colourless to blue. 9. Measure absorbance on spectrophotometer 10.Calculate concentration of Ag if have a std curve
Measuring cell-associated antigen
Techniques used to measure cell surface receptors or
things bound to the surface of cells.
Cell-based ELISA can be used
- But this is mainly for lab research
Flow cytometry
Widely used to analyse cells and cell surface receptors in clinic and
research
Uses Ab that are conjugated with fluorescent tags (can be direct or
indirect)
A laser is used to excite the FL tags
The emission intensity given by the FL tags is recorded
More emission intensity the more receptors on the cell
Up to 12 colours can be detected simultaneously – so you could
measure expression of 12 cell surface receptors all at the same
time. Can also measure the size and granularity of cells
How fluorescence works
- The fluorescent molecule is excited by a laser (usually 488nm
but now have 4 lasers per machine) – This provides
electromagnetic energy that excites an electron in the
fluorescent molecule . - The electron moves to an excitation state at the next energy
level. - Energy is released in the form of a photon of light
(fluorescence) and the electron moves back down to the lower
energy level.
Flow cytometry
Fluorescently-activated cell sorter - FACS
Use of flow cytometry in HIV
HIV infects CD4 T cells (T helper cells)
The number of CD4 T cells compared to CD8 T cells is determined
using Flow Cytometry
In HIV patients the number of CD4 cells is important as low CD4
numbers can mean progression to AIDS
Use of Ab in Microscopy
Used to localise Ag in tissue
Used in diagnosis of cancer and auto-immune diseases
2 main types:
Immunohistochemistry – staining of sections of
tissues (wax or frozen)
Immunocytochemistry – staining of cells
Immunohistochemistry - wax
- Tissue biopsy taken from patient/animal
- Tissue sample fixed in 10% Formalin
- Tissue sample processed (though alcohols & xylene) & then
paraffin embedded - 5µM sections cut using a microtome and placed onto glass
slides - Tissue section de-waxed with xylene, processed though
alcohol gradient to water - Antigen retrieval (like performing voodoo)
- Primary Ab added
- Secondary Ab added (with enzyme – HRP or Alkaline
Phosphatase) - Use substrate (colourless to brown, red or purple – brown
(DAB mostly used) to visualise Ab location
Immunohistochemistry - frozen
- Tissue biopsy taken from patient/animal
- Tissue sample snap frozen in liquid nitrogen
- 5µM sections cut using a microtome and placed onto glass slides
- Primary Ab added (with Fluorescent tag – for direct)
- Secondary Ab added (with Fluorescent tag – for indirect)
Pros of frozen immunohistochemistry
Wax preserves the tissue architecture
BUT not as many Ab can be used due to antigen retrieval
Immunohistochemistry - frozen - auto-immune disease
Pemphigoid
Immunocytochemistry
Staining of cells (not tissue)
Can be 1. Cells grown in culture
2. Cells deposited on a glass slide - Cytospin
3. Cells taken from a px - cytology (oral, vaginal)
Immunoblotting (used in research) steps
- Proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
- Electroblotted onto nitrocellulose (NC) paper
- Incubated with antigen-specific 1°Ab (mouse anti-human) and then rabbit anti-mouse HRP
conjugated antiserum (2°Ab). - Add substrate and this is converted to light (not colour)
- Develop on light-sensitive paper to identify if protein is there.
Use of therapeutic Ab
Monoclonal Ab specifically bind to Ag it is raised against
Monoclonal Ab can block the activity of proteins if they bind to its active site –
remember the Streptolysin O test!
They can be used to block proteins binding to receptors on cells or viruses binding to
and entering cells
Use of therapeutic Ab - problems
Most monoclonal Ab are raised in mice
These are NOT human (not-self)
The human immune system will raise Ab to the therapeutic Ab and will inactivate it.
Use of therapeutic Abs - solution
Make humanised antibodies.
These are Ab where only the Ab binding site is mouse, the rest has been engineered to
be human. Enough of the therapeutic Ab is human for it not to be recognised as nonself
Example of therapeutic Ab
Trastuzumab (trade name Herceptin)
All drugs that are monoclonal Ab end in mab
-breast, ovarian, stomach and endometrial cancer
Titre
Patient serum dilution
Equivalence point where there is enough antibody for the complex to form (influenza detection)
Therapeutic Ab steps
Therapeutic Ab taken into cell via endocytosis It enters the lysosome where the proteolytic enzymes and pH release the drug inside the cell The drug gets transported to the nucleus where it affects cell division. Other cytotoxic molecules can be used to kill cells directly
