Immunological techniques Flashcards
antigen
anything that is recognised by the immune system as non self
antibody
proteins produced in response to an nation
can only bind to the antigen that induced its formation i.e. specificity
epitope
specific part of the antigen that binds to the antibody
affinitiy
measure of binding strength between an epitope and an antibody binding site
- the higher the affinity the stronger the interactio
antibodies havw
Fc
Fab
regions
what binds to the antigen
Fab region binds to antigen
- but only a small part of the antigen (known as epitope)
polyclonal
mixture of antibodies
monoclonal
single antibody
specificity
production of polyclonal antiserum
Usually use Rabbit, Goat or other large animals
1) inject antigen into the animal
2) primary antibody response
3) leave for 4 weeks, the give booster of the same antigen
4) production of many antibodies against the antigen, will be polyclonal
5) Then collect the blood, centrifuge to isolate serum
6) can check for Ab specificity and affinity
issues with production of polyclonal antibodies
- lots of antibodies in the serum directed for other things
- less specific
- background binding
- this creates polyclonal antibodies
production of monoclonal antibodies
1) antigen of interest with 2 epitopes
2) inject antigens into small animals
3) same procedure as above
4) then instead of taking blood, mouse is killed and spleen is taken
5) Plasma cells taken from the spleen
6) idea is that one plasma cell will secrete one specific antibodies
7) Plasma cells fused with immortal B cells using polyethylene glycol to produce immortal hybridomas
8) To isolate the hybridomas, cells are placed into 96 well plates containing HAT (hypoxanthine, aminopterin, thymidine)
- this kills off the non fused cells so only hybridoma cells survive
9) dilute so only one hybridoma per well to produce a single mAb with 1 specificity
10) then determine the specific of the hybridomas with high affinity mAb selected using ELISA against original Ag
issues with production of mono clonal antibodies
Issues
- plasma cells taken from the spleen die very quickly
To remove this issue
- we fuse it with a B cell immortalised from another mouse
outcomes of monoclonal antibody production
1) Plasma cells fused with the B cells
- hybridomas
2) plasma cells don’t fuse
3) immortalised cells which don’t fuse
- die due to the HAT
how can antibodies be labelled/with what
1) unlabelled
2) Enzyme
- horse radish
- Peroxidase
- alkaline-phosphatase
3) fluorescence
- FITC
- PE
- others
4) Gold
- electron
- microscopy
direct antibody tests
- antibody binds to the antigen
1) surface with antigen sticking up from
2) epitope at the top of the Ag
3) add antibody (mouse antihuman) which is specific to the epitope (may have a tag)
indirect antibody tests
Indirect – boosts the signal
1) surface with antigen sticking up from
2) Antibody binds to the Ag
3) boosted by a rabbit anti mouse
4) boost the signal as there are several binding
titre of antibodies
- titre of an Ab is defined as the lowest dilution of the sample that retains a detectable activity
what do serological assays give/provide/reason to do them
Serological assays give
- positive and negative results
- quantitative strength of Ab-Ag interaction
uses for serological assay tests
1) diagnose infections
2) identify microorganisms
3) quantify proteins in the serum
4) type blood – for blood banks and tissue transplantations
do serological assay test show youve had an infection during before or after
after
- only show you’ve had an infecion
reterospective
what does precipitation and immunodiffusion techniques rely on
ability of Ab to form complexes with Ag and precipitate
immunoprecipitation - diffusion test and precipitation marks
- Ag and Ab are placed into a well cut into agar gels
- 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)
Precipitation marks:
1) precipitin band formed with single antigen (identity)
2) Two independent Ag (a and b) react with their specific Ab (non identity)
3) Ab (a+b and B alone) are specific for their Ag (partial identity)
single radial immunodiffusion
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
- lots of antigen gives bigger diameter ring size
what is agglutination test used for and relies on
Commonly used in serology for many infections (and blood typing)
- Relies on polyclonal nature of the serum
- Relies on polyclonal serum to cross link Ab(similar to precipitation) but involves cells or beads
ELISA tests uses
Used to detect levels of Ag, Ab or proteins in a sample
- extensively used in clinical and laboratory
- Used to accurately quantify levels of test molecule in a sample using a standard curve
- used Ag or Ab bound to a solid phase (can be plastic or the cell surface – for cell surface receptors)
main types of ELISA test
1) sandwich ELISA
2) antigen ELISA
sandwich ELISA use and how
Used to measure cytokines, virus, bacterial products etc in serum and lab
1) capture Ab specific for desired Ag on surface
2) specific to TNF alpha, Fc region will only bind to the anti TNF alpha epitope
3) detection antibody added, will only bind to TNF alpha, specifically to the epitope on the other side of the Ab
4) this antibody has an enzyme, presence of a substrate turns blue to a colour product (previously colourless)
5) the more Ag the more colour is produced
6) measure absorbance @ 450nm
7) calculate concentration of Ag in sample
use of standads
use of the known concentrations to compare
antigen ELISA
Used to measure concentration of human Ab in serum to a bacteria – antibodies to Strep.pneumoniae ect
1) Bind Ag/bacteria to solid phase
2) Wash off excess Ag
3) Add primary Ab specific for the Ag
4) wash off excess primary Ab
5) Add secondary Ab which is specific for the primary Ab and is conjugated with an enzyme (HRP)
6) wash off secondary Ab
7) add substrate (Which will turn from colourless to blue if positive)
8) measure absorbance on spectrophotometer
9) Calculate concentration of Ag if have an std curve
Flow cytometry use
analyse cells and cell surface receptors
- uses Ab that are conjugated with a fluorescent tag
- laser excite the FL tag
- Emission intensity recorded
- can also measure size and granularity of cells
how does fluoresce work in flow cytometry
- have a fluorescent molecule
- stimulated by laser
- molecule goes to higher energy state (unstable)
- emits photon of light to return to ground state
- can measure photon given off as fluorcence
Laser has a light at 488nm - FITC excited at 488, emits green light
- PerCP emits red light
excited at the same time but emit different wavelengths, helps us look at different colours
side scatter measures
granularity
forward scatter measures
measure of cell size
use of Ab in microscopy
- Used to localised Ag in tissues
- Used in diagnosis of cancer and autoimmune diseases
2 types of Ab use in microscopy
immunohistochemistry
immunocytochemistry
immunohistochemistry
- staining of sections of tissues (wax or frozen)
- ab added with fluorescent tag
immunocytochemistry and wha cells
staining of cells
1) cells grown in culture - (human endothelial cells grown in culture and stained for Von Willebrand factor (factor VIII) using fluorescently labelled mouse monoclonal antibody
2) cells deposited on a glass slide – cytospin
3) cells taken from a patient – cytology
what are therapeutic antibodies based on
Monoclonal Ab specifically binds to Ag it is raised against
They can be used to block proteins binding to receptors on cells or viruses binding to and entering cells
therapeutic antibodies example
Trastzumab (trade name = Herceptin)
All drugs that are monoclonal end in mab
Cell has HER2 or ErbB2 (protein
- ligand binds to cell
- causes proliferation (which can lead to cancer)
- antibody will bind to the protein, prevents ligand interacting
- limits cancer progression
