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IMMUNOLOGY PART 1 DEN303 > Immunology techniques > Flashcards

Immunology techniques Flashcards

1
Q

Define antigen

A

Anything recognised by the immune system as non-self

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2
Q

Define antibody

A

Proteins produced in response to an antigen. Can only bind wth antigen that induced its formation.

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3
Q

Define epitope

A

Specific part of antigen that binds to the antibody

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4
Q

Define affinity

A

Measure of binding strength between an epitope and antibody binding site. Higher affinity = stronger the interaction

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5
Q

How is polyclonal antiserum formed?

A
  1. Based on vaccination principle of Ab production to foreign Ag
  2. Injec Ag into antimal, leave for 4 weeks for primary Ab response
  3. Give booster injec of same Ag
  4. Leave 4 weeks for secondary Ab response which is bigger due to memory
  5. Collect blood and centrifuge to isolate serum
  6. Collect serum and check for specificity - ELISA
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6
Q

Production of monoclonal antibodies?

A
  1. Mouse immunised with antigen
  2. Mouse produce Ab to Ag
  3. Spleen removed to get plasma cells
  4. Plasma cells fused with immortal B cells using polyethylene glycol to produce immortal hybridomas
  5. Cells are placed into 96-well plates containing HAT - kills off non-fused cells so only hybridoma cells aliva
  6. Dilute so have only 1 hybridoma per well = produces single mAB with 1 specificity
  7. Hybridomas secreting high affinity mAB selected using ELISA against original Ag
  8. = Limited supply of high affinity mAB
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7
Q

How are antibodies labelled (conjugated)?

A

Unlabelled, Stick enzyme, fluorescence or gold on FC region

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8
Q

Serological diagnosis - what is it?

A

Use of Ab specificity to detect Ag

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9
Q

What is a titre of an Ab?

A

The lowest dilution of the sample that retains a detectable activity

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10
Q

What can serological tests be used to do?

A

Diagnose infecs, identify microorganisms, quantify proteins in the serum, type blood - for blood banks and tissue transplants
ONLY SHOW THAT YOU HAVE HAD AN INFECTION

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11
Q

What do ppt and immunodiffusion techniques rely on?

A

Ability of Ab to form complexes with Ag and ppt

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12
Q

Immunoprecipitation - Ouchterlony diffusion test?

A

Ab and Ag diffuse through agar gel and form a ppt at the equivalence point
Used to detect diphtheria toxin in serum - now PCR used to detect bac

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13
Q

Immunodouble diffusion steps?

A
  1. Preciptinin band formed with single antigen
  2. Two independent Ag react with their specific Ab
  3. Ab are specific for their Ag
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14
Q

Single radial immunodiffusion?

A

Involves diffusion of Ag into Ab-containing gel. Precipitin rings indicate an immune rxn and the area of the ring is proportional to the conc of antigen (larger ring = more ag)

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15
Q

Immunoelectroporesis?

No longer used

A
  1. Ag placed in a well and separated by electrophoresis

2. Ab is laced in trough and precipitin lines form as Ag and Ab diffuse toward each other

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16
Q

Agglutinin tests?

A

Used in serology for infecs, relies on polyclonal nature of serum, relies on polyclonal serum to cross-link Ab but involves cells or beads

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17
Q

How to detect influenza?

A

Haemagglutination inhibition test;

  1. Influenza has haemagglutinin molecules on outer surface
  2. Haemagglutinin binds the virus to red blood cells
  3. When virus particles are mixed with red cells they cause haemagglutination = forms aggregate
  4. In presence of specific Ab, binding of haemagglutinin to RBC is inhibited. RBC settle to bottom of tube.
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18
Q

Haemophilus influenzae detection?

A
  1. CSF sample taken from pt with meningitis
  2. Sample mixed with 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 of H influenzae
19
Q

Detection of infection by streptococcus bacteria?

A

Anti-streptolysin O test;

  1. Serum taken from pt and diluted in tubes containing standard amount of sheep RBC and O toxin
  2. If pt has Ab to O toxin = it will neutralise O toxin and stop it from lysin RBC (tube clear)
  3. At low Ab conc there is not enough Ab to stop RBC lysis
  4. = Gives the Ab titre
20
Q

What do streptococcus bac secrete?

A

Streptolysin O toxin = lyses RBC by punching holes into them

21
Q

When can blood be transfused?

A

When no agglutination occurs

22
Q

Enzyme linked immunosorbent assay uses?

MOST IMPORTANT

A
  • Detects level of Ag, Ab or proteins in sample
  • Non-agglutination and non-ppt test
  • Accurately quantifies levels of test molecules in a sample using a standard curve
  • Uses Ag or Ab bound to a solid phase
23
Q

Enzyme linked immunosorbent assay - ELISA types?

A
  1. Sandwich ELISA (most important)

2. Antigen ELISA

24
Q

What is sandwich ELISA?

V IMPORTANT

A
  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 off 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

Used to measure cytokines, virus, bac products in serum

25
Q

What is antigen ELISA?

A

Used to measure conc of human Ab in serum to
bacteria – ie anti-Strep. Pneumoniae Ab

  1. Bind Ag/bac/cell to solid phase - wash off excess Ag
  2. Add primary Ab specific for Ag. Wash excess primary Ab.
  3. Add 2ndry Ab specific to primary Ab and is conjugated with an enzyme
  4. Wash excess 2ndry Ab
  5. Add substrate = turns from colourless to blue
  6. Measure absorbance on spectrophometer
  7. Calculate Ag conc
26
Q

Measuring cell-associated antigen?

A

By cell based ELISA

27
Q

Flow cytometry?

A
  • Analyses cells and cell surface receptors in clinic and research
  • Uses Ab conjugated with fluorescent tags
  • Laser excites FL tags
  • Emission intensity from the FL tags is recorded
  • More emission intensity = more receptors on cell
28
Q

How does fluorescence work?

A
  1. Fluorescent molecule is excited by a laser = provides electromagnetic energy that excites an electron in the
    fluorescent molecule .
  2. The electron moves to an excitation state at the next energy
    level.
  3. Energy is released in the form of a photon of light
    (fluorescence) and the electron moves back down to the lower
    energy level.
29
Q

What does HIV infect?

A

CD4 T cells (T helper cells)

30
Q

How does flow cytometry work in HIV?

A

Flow cytometry determines no. of CD4 T cells compared to CD8 T cells
In HIV = low CD4 can progress to AIDS

31
Q

Uses of Ab in microscopy?

A

Used to localise Ag in tissue

Used in cancer and auto-immune disease diagnosis

32
Q

Types of Ab in microscopy?

A

Immunohistochemistry - staining of sections of tissues

Immunocytochemistry - staining of cells

33
Q

How does immunohistochemistry of wax occur?

A
  1. Tissue biopsy from patient/animal
  2. Tissue fixed in 10% Formalin
  3. Tissue processed then paraffin embedded
  4. 5µM sections cut using a microtome and placed onto glass
    slides
  5. Tissue de-waxed with xylene, processed though alcohol gradient to water
  6. Antigen retrieval
  7. Primary Ab added
  8. Secondary Ab added (with enzyme – HRP or Alkaline
    Phosphatase)
  9. Use substrate (colourless to brown, red or p
34
Q

How does immunohistochemistry of frozen samples occur?

A
  1. Tissue biopsy taken from patient/animal
  2. Tissue frozen in liquid nitrogen
  3. 5µM sections cut using a microtome and placed onto glass slides
  4. Primary Ab added (with Fluorescent tag – for direct)
  5. Secondary Ab added (with Fluorescent tag – for indirect)
35
Q

Pros and cons of immunohistochemistry - frozen?

A 
Pros = wax preserves tissue architecture better
Cons = not as may Ab can be used due to antigen retrieval
36
Q

In immunohistochemistry -frozen, what are green and white cells? What is blue?

A 
Green = tumour associated macrophages stained with anti-CD68 
White = endothelial cells (BV) stained with anti-CD31
Blue = cell nuclei
37
Q

What is immunoblotting?

A
  1. Proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
  2. Electroblotted onto nitrocellulose (NC) paper
  3. Incubated with antigen-specific 1°Ab and then rabbit anti-mouse HRP
    conjugated antiserum (2°Ab).
  4. Add substrate and this is converted to light (not colour)
  5. Develop on light-sensitive paper to identify if protein is there.
38
Q

Use of therapeutic Ab?

A
  • 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
  • They can be used to block proteins binding to receptors on cells or viruses binding to
    and entering cells
39
Q

Problems with therapeutic Ab?

A
  • 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
40
Q

Solutions for therapeutic Ab?

A
  • Make humanised antibodies = 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
41
Q

What do all drugs that are monoclonal Ab end in?

A

Mab

42
Q

Example of therapeutic Ab?

A

Trastuzumab (trade name = Herceptin)

43
Q

Actions of therapeutic Ab?

A 
Therapeutic Ab taken
into cell via endocytosis
= enters the lysosome
= proteolytic enzymes and pH release the drug inside the cell
Drug gets
transported to the
nucleus = affects
cell division.
Other cytotoxic
molecules can be used to
kill cells directly

IMMUNOLOGY PART 1 DEN303 (5 decks)

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