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PHS2104 > Lecture 4: antibodies > Flashcards

Lecture 4: antibodies Flashcards

1
Q

What are antibodies?

A

Glycoproteins produced by plasma b cells that bind to and neutralise foreign antigens on bacteria and viruses.

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2
Q

What is an antigen?

A

A molecule or molecular structure that can be bound by an antigen specific receptor or B cell antigen receptor

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3
Q

What are the general uses of antibodies?

A
  1. Specific and precise therapeutics with minimal off target effects
  2. Excellent diagnostic tool for detecting and quantifying a
    broad array of targets.
  3. Protein purification and detection in manufacturing of protein biologics
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4
Q

How many polypeptide chains is an antibody composed of and what are they called?

A

4 polypeptide chains:
2 identical light chains
2 identical heavy chains

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5
Q

What joins the light chains and heavy chains together?

A

Interchain disulfide bonds

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6
Q

What is the Fc and Fab region of an antibody and what are their functions?

A
  1. Fab is the antigen binding region
  2. fc is the constant fragment: it is responsible for
    biological effector functions - Binds to macrophages, NK
    cells and neutrophils via Fc receptors; binds to complement
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7
Q

What are the 2 types of domains found on the light and heavy chains, and how many of those domains exist on each chain?

A

Constant and variable domain
Each light chain consists of 1 variable and 1 constant domain
Each heavy chain consists of 1 variable and 3 constant domains

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8
Q

What is the function of the variable regions of the antibody,and what is the complementarily-determining region?

A
  1. It is where the antigen binding site is
  2. Responsible for antigen specificity and affinity of antibodies
  3. CDR (complementarily-determining region) resides within Fv. It is the hypervariable region of Ig responsible for antigen specificity and affinity of antibody, and binds directly to the epitope.
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9
Q

What is an epitope?

A

It is the specific region of an antigen that interacts directly with an antibody

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10
Q

What are the effector functions once the antibody binds to the antigen? Which region of the antibody is responsible for this effect?

A

Fc region responsible
Effector functions:
1. Complement-dependent cytotoxicity (CDC). Fc binds to
components of complement system (complement fixation)
2. Antibody-dependent cellular cytotoxicity (ADCC). Fc
binds to Fc receptors on immune cells (phagocytic macrophages, neutrophils, NK cells, etc).

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11
Q

Can different antibodies share the same epitope?

A

Yes

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12
Q

What is affinity and avidity?

A
  1. Affinity: the strength of interaction between antibody and antigen at a single antigenic site
  2. Avidity: the strength of interaction between antibody and antigen at multiple epitopes
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13
Q

What are the different classes of antibodies?

A

IgM
IgG
IgD
IgA
IgE

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14
Q

What is the most abundant immunoglobulin?

A

IgG

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15
Q

Is IgG secreted in high quantities during primary or secondary exposure?

A

Secondary

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16
Q

What are the major functions of IgG?

A
  1. Neutralize microbes and toxins
  2. Opsonize antigens for phagocytosis
  3. Activate the complement system
  4. Protect the newborn (IgG can cross the placenta)
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17
Q

If you are measuring the amount of IgG in a patient’s blood, what can you conclude about the infection based on the levels of IgG found?

A
  1. 4-fold rise or fall indicates active infection
  2. A single positive sample indicates past exposure
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18
Q

Is IgM mostly secreted during primary or secondary exposure?

A

Primary

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19
Q

What are the major functions of IgM?

A

1.First antibody secreted during primary expose
2. Activates the complement system
3.. Used as a marker of recent infection (diagnostic testing)

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20
Q

What do different IgM levels indicate about infection?

A
  1. Presence of IgM in newborn means infection (since its the first antibody secreted + can’t pass through the placenta from the mother)
  2. Single positive sample in serum or CSF indicates recent or active infection
  3. Used to detect early phase of infection
21
Q

Do the high levels of IgM and IgG secreted in the body during infection persist?

A
  1. The IgM levels are high temporarily
  2. For IgG, higher levels persist in small amounts throughout life
22
Q

How does the class of antibodies produce by a B cell change through the course of an infection?

A
  1. During the primary response, the major class of antibody produced by B cells is IgM
  2. Class switching (gene rearrangement of constant regions of IgM) is induced.
  3. By the secondary response, the other classes (IgG, IgA,IgE) persist
23
Q

What is the process by which the affinity of IgG increases during the immune response and when does it most occur?

A

Affinity maturation –> gene reshuffling of the Fab regions which allows it to bind stronger
Most pronounced after the secondary exposure

24
Q

What is the difference between polyclonal and monoclonal antibodies?

A
  1. Polyclonal antibodies are secreted by different B
    cell lineages while monoclonal antibodies are secreted by one B cell lineage
  2. Polyclonal antibodies are a collection of immunoglobulin molecules that react against a specific antigen, each identifying a different epitope, while monoclonal antibodies just react against one epitope of a specific antigen (monovalent affinity)
  3. Polyclonal antibodies have a higher potential for cross reactivity than monoclonal antibodies
  4. Monoclonal antibodies are mostly used for biopharmaceuticals
25
Q

How do polyclonal antibodies arise?

A

B cell antigen receptors (surface Ig) recognize only one specific region (epitope) on an antigen molecule. However, if the antigen has multiple epitopes, it can activate more than one clone of B cells, each specific to a different epitope on the antigen.
Hence they all bind to the same antigen, just different epitopes

26
Q

Do monoclonal antibodies exist naturally in the body?

A

No they are artificially produced

27
Q

What are the advantages of polyclonal antibodies in their applications?

A
  1. They can recognise multiple epitopes, so they are more tolerant of small changes in the nature of the antigen, so they provide more robust detection and they are the preferred choice for the detection of denatured proteins
  2. They can be generated in a variety of animal species - rabbit, goat, sheep, donkey, chicken etc –> so there are many options in experimental design
  3. The antiserum containing a heterogenous mixture of Igs can be used to treat certain diseases.
28
Q

What are the advantages of monoclonal antibodies in their applications?

A

Since they only recognise 1 epitope of 1 antigen:
1. They possess high specificity, which is useful for
– detecting antigens in cells and tissues (immunocytochemistry, immunohistochemistry)
– antigen purification (immunoaffinity chromatographic purification of proteins)
2. They have a high homogeneity, so their effects are highly reproducible, making them useful for
– Commercial development into diagnostic test kits.
– Commercial development into therapeutic molecules.
– Many experimental research techniques

29
Q

What is the process by which monoclonal antibodies are produced by the hybridoma technology?

A
  1. The mouse is immunized with antigen X, and mouse spleen produces plasma cells that secrete antibodies against that antigen.
  2. Myeloma cells that are unable to produce antibodies or HGRPT are selected (because they are cancerous and can proliferate indefinitely)
  3. The mouse’s spleen is removed, and the plasma cells from the spleen are mixed with myeloma cells –> cell fusion is induced to produce hybridoma cells
  4. The hybridoma cells are transferred to a HAT medium, where the unfused plasma cells die
  5. The hybridomas that produce antibodies specific to antigen X are selected and grown in bulk
30
Q

Why are the hybridoma cells transferred to a HAT medium?

A
  1. HAT is hypoxanthine-aminopterin-thymidine
  2. The de novo pathway of DNA synthesis starts with dihydrofolate
  3. Aminopterin blocks the DNA synthesis pathway from dihydrofolate by inhibiting dihydrofolate reductase
  4. To salvage this, dGTP and dATP can be synthesized from hypoxanthine and dTTP can be synthesized from thymidine.
  5. dGTP and dATP synthesis needs HGRPT. Unfused myeloma cells lack HGPRT and can’t synthesize nucleotides so they die.
  6. Unfused plasma cells can synthesize nucleotides but they aren’t immortal so they eventually die off
  7. Fused hybridoma cells can synthesize nucleotides and proliferate forever, so they are the only ones that remain.
31
Q

What is the process of cell culture once the hybridoma cells have been made?

A
  1. The hybridoma cells are diluted to one-cell per flask
  2. Monoclonal antibody in individual flask
  3. Propagate the monoclonal antibodies from each clone in cell culture
    Can you explain what this means?
32
Q

What are the limitations of monoclonal antibodies produced with hybridomas?

A
  1. They may induce immunogenecity
  2. They may fail to trigger a number of effector functions
  3. They have shorter half lives of around 30-40 h
33
Q

How are chimeric monoclonal antibodies different from monoclonal antibodies directly from mice? Why are they produced that way?

A
  1. Chimeric Mabs are 75% human
  2. The constant regions of the light and heavy chains predominantly contribute to immunogenicity, and so their amino acid sequences are replaced with the human sequences
  3. The antigen binding sequences are preserved
34
Q

How are humanized monoclonal antibodies different from monoclonal antibodies directly from mice? Why are they produced that way?

A
  1. They are more than 90% human , so even less immunogenicity than chimeric Mab
  2. All mouse amino acid sequences are replaced with human sequences, except for the hypervariable complementarity determinizing regions of the variable region (the part that binds to the epitope)
35
Q

What are the suffixes used for the different types of therapeutic antibodies?

A
  1. Murine: -omab
  2. Chimeric: -ximab
  3. Humanised: -zumab
  4. Human: -umab
36
Q

How are human monoclonal antibodies produced?

A
  1. Similar to the approach used to manufacture recombinant proteins - CHO cells
  2. Use the DHFR MTX to amplify the cells that have successfully taken up the vector (maybe revise that part pls its on the doc)
37
Q

What are the challenges associated with production of human recombinant monoclonal antibodies?

A
  1. The light chain and heavy chain genes are inserted into separate vectors , which are then transfected into the cells simultaneously.
  2. Due to the separate vectors used for the light and heavy chains, the resulting cell population can be more heterogeneous, meaning there’s an increased chance of cells not having both recombinant genes localized with the amplification gene (DHFR). This heterogeneity can impact the efficiency and consistency of antibody production.
38
Q

What are the quality control steps done during the production of human recombinant monoclonal antibodies?

A
  1. Binding assays of the antibody to defined regions of antigens to determine the affinity, avidity and immunoreactivity
  2. Study and document the unintended reactivity with human tissues
  3. Define the epitope that the antibody binds to, and where that epitope is found
39
Q

What are the application of monoclonal antibodies?

A
  1. As in vitro diagnostic agents
  2. Protein purification
  3. In vivo diagnostic tools
  4. As therapeutic molecules for different diseases
40
Q

What kind of antibody is pembrolizumab?

A

A humanized monoclonal antibody

41
Q

How is pembrolizumab used to treat cancer?

A
  1. Cancer cells express PD-L1 which binds to PD-1 found on T cells that suppresses T cell activation and proliferation
  2. Pembrolizumab blocks PD-1 and prevents binding of PD-L1 , which prevents the inactivation of T cells by cancer cells so the cancer cell is killed by the T cell
42
Q

What are the different types of antibody derivatives?

A
  1. Canonical antibodies
  2. Antibody drug conjugates
  3. Bispecific
  4. Fragments
43
Q

In what cases may we not need the presence of the Fc region on the antibody derivative?

A
  1. Antagonism of enzyme actions (bind to active site).
  2. To neutralize receptor ligands such as hormones or cytokines. To counteract overproduction of cytokines (cytokine storm).
  3. Neutralize toxins. Eg. Snake venom.
    (here only the antigen binding action is needed to neutralise)
44
Q

What is a bispecific antibody? Give an example of it.

A
  1. It is an artificial antibody that can simultaneously bind to 2 different types of antigen
  2. Example: Bi-specific T-cell engagers (BiTEs) which act as a link between T cells and tumor cells. This causes T cells to exert cytotoxic activity on tumor cells.
45
Q

What are the applications of bispecifics?

A
  1. Bridging cells (allow effector cell to bind to target cell via an antibody)
  2. Receptor inhibition (target 2 for increased specificity)
  3. Receptor activation (same principle as before)
46
Q

What are antibody drug conjugates used for ?

A
  1. They are a class of biopharmaceutical drugs designed as a targeted therapy for treating cancer.
  2. Unlike chemotherapy, ADCs target and kill tumor cells while sparing healthy cells.
  3. The antibody targets a specific tumor antigen, and it has an active cytotoxic drug conjugated to it.
47
Q

How does CAR T cell therapy work?

A
  1. Blood is taken from the patient. The white blood cells, including the T cells are separated out and the rest of the blood is retuned to the patient.
  2. The cells are engineered to find and kill cancer cells
    - AN inactive virus is used to insert genes into the T cells.
    - The genes cause the T cells to make CAR receptors on their surfaces
    - The cells are multiplied
  3. The CART cells are put back into the patient
  4. The CAR receptors allow the T cells to identify tumor cells and kill them.
48
Q

What is the process by which the Rapid COVID Antibody test works?

A
  1. The whole blood, serum, or plasma clinical specimen is added to the well.
  2. The combined sample flows down to the sample pad.
  3. Capillary action/lateral flow will move the sample across the test.
  4. Conjugation pad: contains the COVID-19 antigen conjugated to gold nanoparticles that bind antibodies specific for COVID-19 antigen antigen. Control: Non specific rabbit IgG conjugated to gold nanoparticles
  5. Nitrocellulose membrane: three test lines: IgG, IgM and control.
  6. M line: immobilized antibody that recognizes human IgM. Any IgM antibodies will bind here. However, only human IgM antibody/COVID-19 antigen/gold nanoparticle complexes will produce a visible coloured line.
  7. G Line: immobilized antibody that recognizes Human IgG.
    Control line: immobilised antibody that recognises Rabbit IgG (control). To serve as a procedural control, a coloured line should always appear in the control line region, indicating that the proper volume of specimen has been added and lateral flow has occurred.
  8. After 10 minutes, the results of the test can be read.
49
Q
A

PHS2104 (15 decks)

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