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MICB 202 > Chapter 7- Immu > Flashcards

Chapter 7- Immu Flashcards

1
Q

Antibodies as tools in research

A

Antibodies are useful tools in microbiology, immunology and clinical laboratories
- they are proteins that recognize distinct molecular structures and bind to these structures with high affinity

This property can be exploited for

  • Visualization of protein in permeabilized cell - relative amounts of expression, location of expression
  • Purification of protein from a mixed solution
  • Diagnostic: detection of cancers, measuring hormones levels (eg pregnancy, thyroid), identification of pathogen in clinical samples
  • Isolation of specific cell type from a mixed population of cells
  • Therapeutics: neutralization of pathogens, treatment of some types of Cancers, treatment of some inflammatory conditions
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2
Q

Antibodies I

A
  • The aa sequence of variable region of antibodies are expected to be different, (eg large vurys/bacteria, more different epitopes to binds to)
  • the aa sequence of the constant region will be different across different species (same within the same species)
  • The constant region of the heavy chain of antibodies from different species are immunogenic (the C terminus, bottom part)
  • antibodies from another species can be antigens and antibodies against the antibodies are made
  • eg. injected dog anti-rabies antibodies into a human –> human produce human anti-dog antibody antibodies
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3
Q

Antibodies of Antibodies - naming

A

Who made it - the antigen (made against what) - what was made

eg - Human- anti dog antibody - antibodies

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4
Q

Antibodies II

A
  • Antibodies are roughly raised in sheep, goats, rabbits or horses against human Ig or mouse iG
  • most antibody raised in these animals will recognize the constant region of the heavy chain of the human of mouse Ig
  • Antibodies can be tagged with enzymes of fluorescent compounds and are readily available from scientific supply companies
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5
Q

Application in Research - Antibodies III: How can animal “anti human antibody” antibodies be useful to researcher

A
  • Antibodies against antibodies can be used as detectors
  • This allows the researchers to detect the binding of an antibody to the antigen by conversion of substrate to colored product or by fluorescent under specific wavelength of light
  • use animal anti-human antibody antibodies to bind to human antibody binding to antigen (see how many and bounded) eg sorting etc
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6
Q

Monoclonal and Poly clonal Antibodies

A

B cell differentiation:

  1. Antigen recognition induces expression of effector molecules by T cell, which activates the B cells
  2. B cell proliferation
  3. differentiation to resting memory cells and antibody secreting plasma cells
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7
Q

Polyclonal Antibodies

A
  • Most antigens have many epitopes - when that antigen is injected into a mouse/human many different B cells will be activated-each one with a BCR that recognizes a different epitope
  • When B cells differentiate into plasma cell, they secrete antibody into the blood plasma
  • Polyclonal serum - mix of different antibodies in serum
  • Not only will there be antibodies of different specificities (epitopes), there will be antibodies of different affinity
  • Polyclonal = many clones
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8
Q

Polyclonal Antiserum

A
  • Polyclonal serum = mix of different antibodies
  • depending on what you need to use the antibodies fo this could be or or not so good

Pros: There will be a mix of proteins, so if you want to use protein Y, it is possible that some of the antibodies may bind to protein Y

Cons: When supply is gone, it is gone. If you were to take another blood sample from the mouse a few weeks later, its immune system has changes –> may not see high concentrations of antibodies to produce protein Y

aka a variety but only small copies of each

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9
Q

Monoclonal Antibodies

A

Monoclonal antibodies = antibodies produced by descendants of ONE close of B cells - they are maintained in a cell culture (in vitro)

  • Freshly isolated B cells are mortal - they survive about a week in cell culture
  • Myelomas : They are B cell that have lost its ability to make/secrete antibodies but are immortal
  • If we take a single B cell and fuse it with a myeloma –> hybridoma
  • these are immortal
  • Hybridomas and all its descendants will all make the same antibody as the original B cell (monoclonal) so antibodies will all bind the same epitope
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10
Q

Making monoclonal antibodies - 1

A
  1. immunize mice with specific antigens to stimulate antibody production
    - take blood sample - check it for presence of antibodies to antigen X by ELISA
    a. If no antibodies can be detected or the response is weal - re immunize mouse with antigen X
    b. If positive, remove spleen - source of antibody producing B cells
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11
Q

Making monoclonal antibodies - 2

A
  1. Isolation of antibody secreting Plasma cells

Myelomas

  • immortal
  • no longer secretes antibodies
  • Drug sensitive
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12
Q

Making monoclonal antibodies - 3

A

3) Fusion and generation of Hybridomas

Hybridomas secrete antibodies, they are drug resistant

  • these hybridomas thus have both properties of secreting antibodies and drug resistant
  • Grow cells in an in vitro culture system - use drug selection to select cells that have specific properties of both B cells and myeloma
  • the cells that survive
    Example of monoclonal antibody: Humeria - for Anti TNF
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13
Q

Making monoclonal antibodies - 4

A
  1. Growth in vitro and ELISA screening
  • put all the hybridomas that secrete antibody into a microtiter plate
  • select the hydribomas that produces antibodies of desired specificity
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14
Q

Making monoclonal antibodies - 5

A
  1. Expansion of selected hybridoma to produce monoclonal antibodies
    - Grow the desired hybridoma in cell culture and harvest monoclonal antibodies
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15
Q

Using Monoclonal antibodies

A
  • Monoclonal antibodies such as Humira are prescribed to patients for a variety of medical conditions
  • they are directed against TNf-alpha
  • they are anti-TNF antibody
  • it is originally made using b cells isolated from mice that has repeatedly been immunized with human TNF - alpha
  • one possible problem is that the immune system will attach the Fc chain of the mouse antibodies - serum sickness
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16
Q

Using Monoclonal Antibodies - II

How is it that patient can repeatedly administer this product to control their medical condition?

A
  • these hybridoma cells producing the antibodies are genetically modified using molecular bio techniques
  • the mouse’s H chain region are replaced with human H chain region in the DNA

Another method: make the antibodies in a Xenomouse which produce antibodies that are 1–% human

17
Q

Why would anti TNF - alfa medication be inadvisable if patient had an infectious disease

A
  • TNF-alpha are secreted by macrophage and monocytes to establish inflammatory response
18
Q

ELISA - Enzyme Linked Immunosorbent Assay

A

ELISA is a very useful protocol for measuring molecules in solution
- eg. proteins, hormones and immunoglobulins

  • 2 basic variations of ELISA, both rely on specific antigen antibody binding

Version 1 useful for detection of soluble antibodies in solution

Version 2 - useful for detection of any other soluble molecule types (eg antigen) but not antibody

  • ELISA cannot be used with cells because of the instrument used to measure the result cannot tell the difference between light that has been absorbed or light that has been scattered
19
Q

ELISA - I Application: developmental of monoclonal antibodies

A
  • during proceedure to make hybridomas that secrete monoclonal antibodies we are interested in , we had to constuct many clones in order to have a reasonable chance that we will succeed in making hybridoma to protein X
  • that means tht you have 1000s of clones that might be secreting antibodys of something we are not interested in
  • now you need a quick easy way to test culture from all the 10000 hybridomas to identify which one is making monoclonal antibody that bind to protein X

Solution: perform ELISA using the antigen
the test sample (which may contain primary antibody) and secondary antibody tagged with an enzyme (this antibody binds to the Fc region of primary antibody)

20
Q

Screening Hybridomas

A
  • Hybridomas are initially cultured in a 96 well microtiter plate, each well containing 0.3 mL of culture media
  • using multi channel pipettor, transfer the culture supernatant from each well into and ELISA plate to perform ELISA
21
Q

ELISA V.1 - detects antibodies

A
  1. Antigens for specific (wanted) antibodies are added to wells and coats the bottom of the walls - unbounded antigens are washed away
  2. Sample is added to the well, if the antibody is present it binds to the antigen
    - These are the primary antibodies - excess unbound antibodies are washes away
  3. the secondary antibodies ( with an enzyme covalently bonded to its Fc region) i are added
    - if there is an antibody bound to the antigen this antibody will bind to it
  4. a colorless substate is added and converted to a colored product by enzyme on the secondary antibody.
    The concentration of the colored product can be measured and is proportional to amount of secondary antibody present which is proportional to the amount of primary antibody present
22
Q

Why don’t we simply put the enzyme on the primary antibody instead

A
  • might not have primary antibody at all

- faster and safer

23
Q

ELISA V.2

A
  1. Antibody specific to antigen is coated to the well. Excess antibody is washed away
  2. Sample is added to the well, if the soluble antigen is present, it binds to the primary antibody and the excess is washed away
  3. Secondary antibody with an enzyme covalently bounded to Fc is added
    - it recognizes a different epitopes on the antigen and it does not bind to the primary antibody
  4. A colorless substrate is added and converted to a colored product by enzyme on the secondary antibody
    - the concentration of the product can be measured and is proportional to the amount of antigen captured by the first antibody
24
Q

V1 va V2

A
  • in 1, antigen is used to coat, while in 2 antibody coat the well
25
Q

Technologies that apply concept of ELISA

A
  • pregnancy kits - look from hCGs, a hormone secreted by developing placenta
26
Q

Immunofluorescence

A

Fluorescent Labelled antibodies can be used to detect:

  • the expression of a protein- can tract them
  • change in expression : can tract them increased/decreased - time coursed studies
  • distribution (location of protein in cell)
  • movement of a protein inc ell (eg for secreted protein)

If target is on the cell surface, the labelled antibodies can be incubated with the sample directly

if target is inside the cell, the cells are first treat with a weak detergent solution to permeabilize the cells

27
Q

Direct Immunofluorescence

A
  • Fluorescent labeled antibodies can label cell surface proteins
  • cell surface proteins recognize by an antibody tagged with fluorochrome
  • incubate with antibody tagged fluorochrome, wash away unbounded excess antibody
28
Q

Infect Immunofluorescence

A
  • Immobilized antigen A
  • Primary antibody : rabbit antibody directed against antigen A
  • Secondary antibodies: marker coupled antibodies directed against rabbit antibodies
29
Q

Fluorescent Microscopy

A

We expose it to UV light of the correct wavelength to excite the fluorochrome,

  • we can then detect the emitted light with microscope
  • we take different images with different wavelength then combine image
30
Q

FACs - Fluorescence Activated Cell Sorting

A

FACs allowed us to

  • SORT mixtures of cells into different populations based on what proteins they are expressing
  • COUNT/determine the number of cells in a sample

Example: cells from lymph node can be separated into populations

  • B and T cells
  • CD4, CD6 and non T cells

To do this we need an antibody that specifically binds to the desired cel types
- this antibody is tagged with fluorescent molecule

31
Q

FACs

Protocol: separate cells from lymph node

A
  1. Mix lymph node cells with anti-CD4 antibodies
    - allow antibody to bind
    - wash away excess
    - some cells are labeled with the antibody - green
  2. Add anti CD8 antibodies
    allow antibody to bind
    - wash away excess
    - some cells are labeled with the antibody - red
  3. program FACs machine to sort the cells into 4 groups - green only, red only, green+ red, no color
32
Q

FACs machinery

A

FACs machines inspect each cell individually to see what fluorochromes are attached to it

A vibrating nozzle ensures that droplets leaving the nozzle contains single cell

The FACs machine sort the cells into separate containers depending on what is detected

The machine also counts the number of cells going into each container

33
Q

FACs proceedure

A
  1. target cell type within a mixture is fluorescenly labelled
  2. Cell mixture leaves nozzle in droplets
    - laser beans strikes each droplets
    - FSC detectors identifies cell size
    - SCC detector identifies fluorescence/granularity
    - Electrodes assign +/- charge
    • charge cells move close to negative charged plates (visa vera)
  4. separate cells are collected in different tubes
34
Q

FACs ploting

A
  • After sorting a counting cells, the computer generates a four quadrant plot
  • experiment thresh-hold are indicated by green and red lines (as axis)
  • if amounts of fluorescent detected exceed that threshold is considered positive
35
Q

Plot example - Th and CTL cells

A

there are no cells in both positive quadrant:
- because mature T cells decide in thymus whether it would be a CTp of TH cells - in lymph node already matured/ differentiates

  • both negative cells are non T cells eg structural cells

MICB 202 (19 decks)

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