Ab characterization and Ab based techniques

Question 1

what is the general principle for Precipitation techniques?

Answer 1

Formation of high-molecular weight antigen-antibody
complexes results in insoluble precipitate.
Such insoluble precipitates are explored and exploited in
precipitation techniques.

Pros
- Simple, basic technologies
- Cheap (used in clinical laboratories)
- No instrument needed
Cons
- “too” basic
- sample dependent (size, valence etc)
- Antibody (Ab) dependent (mAb vs. pAb)

Question 2

The amount of precipitate is dependent on:

Answer 2

• Amount of antigen and antibody
• Molar ratio antigen:antibody
• Valence

Question 3

Valence

Answer 3

the maximum number of ab that can bind ag.

e.g. small antigen with 2 epitopes has a valence =2

intermediate antigen, 6 epitopes,antigen valence= 4

large antigen 10 epitopes, antigen valence = 8

Question 4

single radial immunodiffusion

Answer 4

• Quantitative technique
• the antibody is casted in a gel
• the sample is applied in wells (punched holes) in the gel
• the sample diffuses out in the gel and forms
  precipitates (that can be detected)

With increased amount of antigen, the further out the
antigen has to diffuse before equivalence is reached

Question 5

how do you find the conc of ag using single radial immunodiffusion

Answer 5

1. Measure the diameter of the precipitate
2. Plot against known conc. (std curve)
3. Able to estimate conc. of unkown sample

Question 6

rocket electrophoresis

Answer 6

• Quantitative technique
• Increased resolution
• A gel including the antibodies is casted
• Ag added in wells, and the
  electrophoresis is run
• Precipitates are formed, with the surface area (height) proportional to the amount of antigen
  NB!
  The pH of the buffer is chosen so that the antibodies in the gel do not move, while the antigen moves, and then formes agab.

Question 7

Immunochromatography – affinity chromatography

Answer 7

• Affinity interaction (e.g. ab-ag) exploited
• Can be used to purify antibodies
• Can be used to purify other proteins based
  on the specificity of antibodies

Question 8

Purification of antibodies

Answer 8

• Antigen chromatography
• Other affinity chromatography (e.g. protein A/G)
  The choice of method depending on the
• properties (e.g. specificity, stability etc)
• down-stream application

Question 9

Purification of antibodies binding to a specific antigen

Answer 9

• Antigen chromatography
• Antigen immobilized on beads
• Pack the beads into a column
• Antibody preparation added (specific ab binds)
• Wash away un-bound material (ie non-specific ab)
• Elute specifically bound antibodies
  (e.g. lowered/increased pH, free ag)

Issues
- non-specific binding (to beads, to protein)
- Elution
lowered pH – risk?
free antigen – risk?

Question 10

Purification of antibodies (not based on the binding specificity)

Answer 10

• Other affinity chromatography
• Protein A or Protein G column
• Antibody preparation added (binds)
• Wash away un-bound material
• Elute specifically bound antibodies
  (e.g. lowered pH)
  Protein A and G bacterial proteins capable of
  binding antibodies.
  Binds to Fc part of the antibodies.
  Antibodies can be eluted with
  “mild” conditions (lowered pH)

Question 11

Polyclonal antibody preparations may have to be purified from fraction(s) of antibodies with unwanted reactivity, describe the method

Answer 11

solid-phase absorption:
In a column you immobilize protein and those antibodies you want to get rid of will bind to this protein.
In this manner, antibodies displaying unwanted reactivity
against e.g. similar proteins from other species can be
eliminated.
Example: You want antibodies that bind to rabbit-IgG (antirabbit-IgG), but these antibodies must not bind to IgG from other species
(i.e should not cross react with e.g. mouse IgG. Important in
many assays)

Question 12

What can happen in
this assay if the
swine anti-rabbit is
not correctly
absorbed? (mouse antibody
against antigen, antigen, rabbit antibody against antigen, Enzyme labelled
swine anti-rabbit IgG)

Answer 12

If the swine anti-rabbit IgG is not properly absorbed, it may contain antibodies that can bind nonspecifically to components other than the intended rabbit antibody. This nonspecific binding can increase background noise in the assay, making it difficult to distinguish between specific and nonspecific signals.

Binding to Mouse Antibodies: Swine anti-rabbit IgG might cross-react with mouse antibodies (the initial antibodies against the antigen). This can lead to false positive signals because the enzyme-labeled swine anti-rabbit IgG could bind to the mouse antibodies instead of exclusively binding to the rabbit antibodies.

Binding to Antigen or Other Proteins: Swine anti-rabbit IgG might also cross-react with the antigen or other proteins present in the assay. This can further contribute to nonspecific signals and reduce the specificity of the assay.

Question 13

Purification of other proteins using Abs

Answer 13

Purified antibodies can be immobilized on various
chromatographic supports (e.g. Sepharose) and be
used for purification of antigen

Question 14

Affinity chromatography – problems and solution ?

Answer 14

The high binding strength of ag-ab – drastic elution conditions may be
required (e.g. pH 2.5) which may lead to irreversible denaturation

Potential solution:
- Other elution conditions (e.g. high pH)
- Develop antibodies (e.g. via phage display) that dissociates from ag at mild conditions (e.g. pH 6)

Question 15

Purification of antibody fragments

Answer 15

Molecular biology approaches often involves production of
antibody fragments e.g. scFv or Fab

His-tag (6 histidins in a row) is suitable for purification, since it has affinity for e.g. Ni2+ and can be used in so-called immobilised metal chelate affinity chromatography
OBS! mild elution conditions (high conc. of immidazole)!

Question 16

Immunoblotting techniques

Answer 16

-Immunoblotting (western blotting)
- Immune precipitation

Question 17

immunoblotting (western blot)

Answer 17

Western blotting, also known as immunoblotting, is a widely used analytical technique to detect specific proteins in a sample. The process involves the following main steps: protein separation, transfer to a membrane, blocking, antibody probing, and detection.

Question 18

immunoprecipitation

Answer 18

technique used to isolate a specific antigen (protein) from a mixture, such as a cell lysate, using a specific antibody.

1. cell lysed in detergent
2. antibodies added on beads
   Add either Ab or Ab+protein to answer:
   * Ab: which protein does it bind to?
   * Ab+Protein: Which protein(s) does the Ab-bound protein bind to?
3. other proteins not bound are washed away
4. proteins eluted and separated by SDS-PAGE

Question 19

Agglutination techniques

Answer 19

Agglutination is the clumping of particles (note, difference to
precipitation: Agglutination are larger particles)

Principle:
Antibodies agglutinates antigen-coated particles
Example of particles:
Erythrocytes (röda blodkroppar) (heme agglutination)
Example of applications:
Blood group determination.

Question 20

Blood group determination

Answer 20

Erythrocytes express different
cell surface markers
- can be used for determination of blood group.
RH system (+ or -)
(rhesus antigen pos or neg)
(Rh + accept both + and -,
while RH- only accept -)
ABO + RH combined
= used system

Question 21

what Importance does the bloodtypes have for blood transfusion?

Answer 21

If a unit of incompatible blood is transfused between a donor and recipient, a severe acute immunological reaction, hemolysis (RBC destruction),
renal failure and shock are likely to occur, and death is a possibility.