Immunological techniques

Question 1

Antigen

Answer 1

Anything that is recognised by the immune

system as non-self

Question 2

Antibody

Answer 2

proteins produced in response to an antigen. It

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

Question 3

Epitope

Answer 3

the specific part of the antigen that binds to the antibody

Question 4

Affinity

Answer 4

measure of binding strength between an epitope
and an antibody binding site. The higher the affinity the
stronger the interaction

Question 5

Polycloncal/ monoclonal

Answer 5

Poly: lots of antibodies binding to lots of different shapes

One specific antibody binding to one antigen - monoclonal

Question 6

Production of monoclonal antibodies (mAb) - usually mice used

Answer 6

1. Mouse immunized with antigen
2. Mouse produces Ab to Ag
3. Spleen removed to get plasma cells
   (NB. 1 plasma cell 1 Ab)
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 (hypoxanthine,
   aminopterin, thymidine). Kills off
   non-fused cells so only hybridoma
   cells alive.
6. Dilute so have only 1 hybridoma per
   well – this will produce just a single
   mAb with 1 specificity.
7. Hybridomas secreting high affinity
   mAb selected using ELISA against
   original Ag.
8. End up with a limitless supply of
   high affinity mAb.

Question 7

Production of monoclonal antibodies (mAb) - usually mice used

Answer 7

1. Mouse immunized with antigen
2. Mouse produces Ab to Ag
3. Spleen removed to get plasma cells
   (NB. 1 plasma cell 1 Ab)
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 (hypoxanthine,
   aminopterin, thymidine). Kills off
   non-fused cells so only hybridoma
   cells alive.
6. Dilute so have only 1 hybridoma per
   well – this will produce just a single
   mAb with 1 specificity.
7. Hybridomas secreting high affinity
   mAb selected using ELISA against
   original Ag.
8. End up with a limitless supply of
   high affinity mAb.

Question 8

Antibodies can be labelled (conjugated)

Answer 8

Unlabelled
Enzyme (horse radis, peroxidase alkaine-phosphatase)
Fluorescence (FITC, PE, many others)
Gold (electron microscopy)

Question 9

Direct and indirect tests

Answer 9

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

Question 10

Serological diagnosis

Answer 10

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

Question 11

Serological tests can be used to

Answer 11

1. Diagnose Infections
2. Identify Microorganisms
3. Quantify proteins in the serum
4. Type Blood – for blood banks and tissue transplantations
   BUT - They are retrospective & only show that you have HAD an infection

Question 12

Precipitation and immunodiffusion techniques

Answer 12

Not very often used these days
Relies on the ability of Ab to form complexes with Ag and precipitate
-antibody excess –> equivalence –> antigen excess

Question 13

Immunoprecipitation: Ouchterlony diffusion test

Answer 13

•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

Question 14

Immunoprecipitation Ouchterlony diffusion test results - Immunodouble diffusion

Answer 14

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)

Question 15

Single radial immunodiffusion

Answer 15

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

Question 16

Immunoelectrophoresis (do not worry about this, not used anymore)

Answer 16

1. Ag is placed in a well and separated by electrophoresis (electrical current).
2. Ab is then placed in the trough and precipitin lines form as Ag and Ab
   diffuse toward each other

Question 17

Agglutinin tests

Answer 17

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

Question 18

Influenza detection: Haemagglutination inhibition test purpose

Answer 18

This test is used to detect the presence of antibodies to influenza virus in a
patient’s serum or BAL fluid

Question 19

Influenza detection: Haemagglutination inhibition test steps

Answer 19

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

Question 20

Haemophilus Influenzae Detection

Answer 20

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

Question 21

Detection of infection by Streptococcus bacteria

Answer 21

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)

Question 22

Detection of infection by Streptococcus bacteria: Anti-streptolysin O test

Answer 22

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

Question 23

Enzyme-Linked Immunosorbent Assay

Answer 23

•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

Question 24

Sandwich ELISA steps

Answer 24

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

Question 25

Sandwich ELISA function - V IMPORTANT

Answer 25

Used to measure cytokines, virus, bacterial products etc in serum and lab

Question 26

Antigen ELISA function

Answer 26

Used to measure conc. of human Ab in serum to bacteria - i.e. anti-Strep. Pneumoniae Ab etc.

Question 27

Antigen ELISA steps

Answer 27

``` 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 ```

Question 28

Measuring cell-associated antigen

Answer 28

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

Question 29

Flow cytometry

Answer 29

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

Question 30

How fluorescence works

Answer 30

1. 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 . 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.

Question 31

Flow cytometry

Answer 31

Fluorescently-activated cell sorter - FACS

Question 32

Use of flow cytometry in HIV

Answer 32

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

Question 33

Use of Ab in Microscopy

Answer 33

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

Question 34

Immunohistochemistry - wax

Answer 34

1. Tissue biopsy taken from patient/animal 2. Tissue sample fixed in 10% Formalin 3. Tissue sample processed (though alcohols & xylene) & then paraffin embedded 4. 5µM sections cut using a microtome and placed onto glass slides 5. Tissue section de-waxed with xylene, processed though alcohol gradient to water 6. Antigen retrieval (like performing voodoo) 7. Primary Ab added 8. Secondary Ab added (with enzyme – HRP or Alkaline Phosphatase) 9. Use substrate (colourless to brown, red or purple – brown (DAB mostly used) to visualise Ab location

Question 35

Immunohistochemistry - frozen

Answer 35

1. Tissue biopsy taken from patient/animal 2. Tissue sample snap 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)

Question 36

Pros of frozen immunohistochemistry

Answer 36

Wax preserves the tissue architecture | BUT not as many Ab can be used due to antigen retrieval

Question 37

Immunohistochemistry - frozen - auto-immune disease

Answer 37

Pemphigoid

Question 38

Immunocytochemistry

Answer 38

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)

Question 39

Immunoblotting (used in research) steps

Answer 39

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 (mouse anti-human) 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.

Question 40

Use of therapeutic Ab

Answer 40

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

Question 41

Use of therapeutic Ab - problems

Answer 41

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.

Question 42

Use of therapeutic Abs - solution

Answer 42

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

Question 43

Example of therapeutic Ab

Answer 43

Trastuzumab (trade name Herceptin) All drugs that are monoclonal Ab end in mab -breast, ovarian, stomach and endometrial cancer

Question 44

Titre

Answer 44

Patient serum dilution | Equivalence point where there is enough antibody for the complex to form (influenza detection)

Question 45

Therapeutic Ab steps

Answer 45

``` 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 ```