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BDS 3 immunology > Immunological techniques > Flashcards

Immunological techniques Flashcards

1
Q

antigen

A

anything that is recognised by the immune system as non self

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2
Q

antibody

A

proteins produced in response to an nation

can only bind to the antigen that induced its formation i.e. specificity

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3
Q

epitope

A

specific part of the antigen that binds to the antibody

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4
Q

affinitiy

A

measure of binding strength between an epitope and an antibody binding site
- the higher the affinity the stronger the interactio

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5
Q

antibodies havw

A

Fc
Fab
regions

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6
Q

what binds to the antigen

A

Fab region binds to antigen

- but only a small part of the antigen (known as epitope)

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7
Q

polyclonal

A

mixture of antibodies

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8
Q

monoclonal

A

single antibody

specificity

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9
Q

production of polyclonal antiserum

A

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

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10
Q

issues with production of polyclonal antibodies

A
  • lots of antibodies in the serum directed for other things
  • less specific
  • background binding
  • this creates polyclonal antibodies
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11
Q

production of monoclonal antibodies

A

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

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12
Q

issues with production of mono clonal antibodies

A

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

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13
Q

outcomes of monoclonal antibody production

A

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

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14
Q

how can antibodies be labelled/with what

A

1) unlabelled
2) Enzyme
- horse radish
- Peroxidase
- alkaline-phosphatase
3) fluorescence
- FITC
- PE
- others
4) Gold
- electron
- microscopy

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15
Q

direct antibody tests

A
  • 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)
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16
Q

indirect antibody tests

A

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

17
Q

titre of antibodies

A
  • titre of an Ab is defined as the lowest dilution of the sample that retains a detectable activity
18
Q

what do serological assays give/provide/reason to do them

A

Serological assays give

  • positive and negative results
  • quantitative strength of Ab-Ag interaction
19
Q

uses for serological assay tests

A

1) diagnose infections
2) identify microorganisms
3) quantify proteins in the serum
4) type blood – for blood banks and tissue transplantations

20
Q

do serological assay test show youve had an infection during before or after

A

after
- only show you’ve had an infecion
reterospective

21
Q

what does precipitation and immunodiffusion techniques rely on

A

ability of Ab to form complexes with Ag and precipitate

22
Q

immunoprecipitation - diffusion test and precipitation marks

A
  • 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)

23
Q

single radial immunodiffusion

A

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
24
Q

what is agglutination test used for and relies on

A

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
25
Q

ELISA tests uses

A

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)
26
Q

main types of ELISA test

A

1) sandwich ELISA

2) antigen ELISA

27
Q

sandwich ELISA use and how

A

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

28
Q

use of standads

A

use of the known concentrations to compare

29
Q

antigen ELISA

A

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

30
Q

Flow cytometry use

A

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
31
Q

how does fluoresce work in flow cytometry

A
  • 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
32
Q

side scatter measures

A

granularity

33
Q

forward scatter measures

A

measure of cell size

34
Q

use of Ab in microscopy

A
  • Used to localised Ag in tissues

- Used in diagnosis of cancer and autoimmune diseases

35
Q

2 types of Ab use in microscopy

A

immunohistochemistry

immunocytochemistry

36
Q

immunohistochemistry

A
  • staining of sections of tissues (wax or frozen)

- ab added with fluorescent tag

37
Q

immunocytochemistry and wha cells

A

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

38
Q

what are therapeutic antibodies based on

A

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

39
Q

therapeutic antibodies example

A

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

BDS 3 immunology (8 decks)

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