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Immunology > Polyclonal and Monoclonal antibodies > Flashcards

Polyclonal and Monoclonal antibodies Flashcards

1
Q

What produces a polyclonal antibody?

A

Many different B cells responding to the same antigen.

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2
Q

What produces monoclonal antibodies?

A

Produced by population of indentical clonal B cells

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3
Q

A mixture of polyclonal antibodies contains?

A

Mixture of antibodies targetting different parts of the protein.

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4
Q

All monoclonal antibodies are…

A

Homogenous, recognising the same eptiope.

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5
Q

How is a polyclonal antibody produced?

A

Immunisation of animal with purified protein and adjuvant.
Cells are recruited, slow antigen release and active APCs via PRRs.
Whole sera is collected and purified.

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6
Q

In polyclonal antibody production, what does hapten require to elicit immune response?

A

Carrier protein

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7
Q

In polyclonal antibody production how are T cells needed?

A

CD40/CD40L interactions are needed for effective antibody production.
Cytokines.

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8
Q

How are monoclonal antibodies produced?

A

Immunisation of animal.
Isolate spleen cells.
Combine plasma cells and myeloma cells, hybridisation into hybridomas.
Select and clone hybridomas using limiting diltution (less than 1 cell per well) in 96 well plates.
Allow clones to expand.
Expand positive wells and test for production of antibody.
These hybridomas will produce the monoclonal antibodies.

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9
Q

Multiple boosts of polyclonal antibodies generate?

A

Higher tire and higher affinity antibodies.
Class switching + hypermutation generate high affinity IgG.

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10
Q

Uses of polyclonal antibodies

A
  • Understanding their generation is essential for vaccine design.
  • Research, ELISA, FACS, biopsy staining, functional blocking, western blots.
  • Diagnosis, ELISA, FACS, biopsy staining.
  • Anti-venom, passive immunity (rabies) prevention of Haemolytic Disease of the Newborn (anti-D)
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11
Q

Uses of monoclonal antibodies?

A
  • Research- tissue staining, western blot, ELISA.
  • Diagnostic/prognostic indicator: immunohistochemistry, flow cytometry, confocal microscopy.
  • Therapeutic antibodies (abzymes too).
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12
Q

What is Anti-D?

A

Rh- negative mother is sensitised to Rh+ foetus during 1st pregnancy. There is a production of IgG against Rh by memory.

B-cells during 2nd pregnacy destroy foetus.

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13
Q

How is anti-D treated?

A

Therapuetic anti-Rh antibody is given before birth preventing memory response?

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14
Q

Therapuetic uses of monoclonal antibodies: Rhematoid arthritis

A

Anti-TNF-alpha - Infliximab

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15
Q

Therapuetic uses of monoclonal antibodies: Crohn’s disease

A

anti-CD11a - Efalizumab

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16
Q

Therapuetic uses of monoclonal antibodies: B-cell leukaemia

A

Anti-CD20 - Rituximab

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17
Q

Therapuetic uses of monoclonal antibodies: Breast cancer

A

Anti-HER2/neu receptor- Herceptin

18
Q

Advantages of polyclonal antibodies:

A

Easy to obtain
Antibodies are against many eptitopes
More effective neutralisation

19
Q

Advantages of monoclonal antibodies

A

Constisten
Limitless supply of specific reagent
More easily tested for cross-reactivity
Optimised for application
Manipulated via recombinant tech.

20
Q

Disadvantages of polyclonal antibodies?

A

Difficult to manipulate due to mixture of antibodies.
Antisera preps different in specificity, avg affinity and cross reactive species.
Not optimised for application.
Limited supply

21
Q

What does ELISA stand for?

A

Enzyme linked immunosorbent assay

22
Q

ELISA function

A

Allows for specific, senstive and quantitative detection of soluble molecules within a complex mixture.

23
Q

Can multiplex ELISAs be performed?

A

Yes.

24
Q

Western Blot Analysis

A

Allows for specific detection from complex mixtures.
Provides size information but isn’t very quantitative.
(Primary antibody and secondary antibody+enz)

25
Q

Flow cytometry

A

Defines individual cells from mixture of cells.
Allows for analysis of multiple antigens at the same time on millions of cells.
Sensitive, quantitative and versatile.

26
Q

Immunohistochemistry/-fluoresence

A

Diagnostic/prognostic markers.
Confocal microscopy- cell colonisation.
Specific antibody linked to enzyme/flurophore.

27
Q

Lateral flow test (hCG test structure)

A

Monoclonal mouse anti-hCG enzyme conjugate: Test zone.
Fixed polyclonal anti hCG + substrate. On test line.
Fixed anti-mouse IgG + substrate: Control line

28
Q

Monoclonal therapy side effects can also be mediated by:

A

Fc region, complement activation, FcR activation.

29
Q

What is a scFv?

A

A single-chain variable fragment (scFv) is a fusion protein of the variable regions of the heavy (VH) and light chains (VL).

30
Q

What are the propeties of scFv

A

Less immunogenic
Lack Fc-mediated effects
Different bio half-lives
Greater tissue penetration

31
Q

Why is tissue penetration ability important?

A

Tumour or delivery to CNS.

32
Q

What are the problems with monoclonal mouse antibodies?

A

Immunogenic.
Need to house and kill mouse.
Once Ab is generated need to clone DNA seq before manipulation.

33
Q

Can antibodies be generated against very homologous proteins?

A

No.

34
Q

What is the solution to antibodies not being able to be generated against very homolohous proteins??

A

In vitro genration and phage display technology.

35
Q

What are magic bullets?

A

Not vibrators, but recombinant antibodies.

36
Q

Arming method of recombinant antibody: What is the effector mechanism of radioisotopes?

A

DNA damage, penetrates solid tumour

37
Q

Arming method of recombinant antibody: What is the effector mechanism of cytotoxic drugs?

A

Cytotoxicity. Drug must be internalised.

38
Q

Arming method of recombinant antibody: What is the effector mechanism of toxin?

A

Cytotoxicity. Toxin must be internalised.

39
Q

Arming method of recombinant antibody: What is the effector mechanism of antibody dimer?

A

More effective engagement of physiological killing mechanism.

40
Q

Arming method of recombinant antibody: What is the effector mechanism of enzyme?

A

Activation of prodrug.

41
Q

Arming method of recombinant antibody: What is the effector mechanism of bispecific antibody.

A

Engages cytotoxic T cells or NK cells.

42
Q

How is phage displayed done?

A

Rearranged Variable regions segments randomly recombined via recombinant tech, this produces large phage library of Abs/scFvs.

Bacteriophage library is used to infect E coli which displats antibody fragment on phage surfaces.

Panning selects binding phages.

Binding phage then isolated, re-infect E.coli.

Totally human derived Ab fragment with known sequence is now ready for manipulation

Immunology (12 decks)

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