Topic 7: Antibodies As Tools In Medicine And Biology

Front 1

Define: Hybridoma

Back 1

Hybridoma are cells that have been engineered to produce a desired antibody in large amounts.

Front 2

How are antibodies produced?

Back 2

Immunizing animals or by hybridomas

Front 3

What are examples of medical uses of Abs?

Back 3

1) Detection of pathogens (bacteria, viruses, toxins) in patient samples
2) Detection of ABs in blood which indicate exposure to an Ag (HIV Test)
3) Detection and measurement of hormone levels (thyroid hormones, insulin pregnancy tests)
Iv) Analysis of blood cells and immune cells (blood typing, AIDS progression)

Front 4

What is the theory behind polyclonal antisera?

Back 4

Animal (rabbit, goat) can be immunized with an antigen (a virus) and antibodies can be isolated from the animal's serum
-Serum is the liquidd portion of blood remaining after it has been allowed to clot
-Serum contains mixture of antibodies that recognize different epitopes on the protein, virus, etc. that was injected

Front 5

Why is it called polyclonal antiserum?

Back 5

Recall that each antibody is made by different B cell clone
-B cell clones arise when a single B cell binds antigen, become activated, proliferates and differentiates into plasma cells or memory B cells
-Most antigens have more than one epitope and will activate several different B cells each with a different Ab specificity
-Each B cell will give rise to a clone of plasma cells that secrete antibodies
-Since there are many plasma cell clones secreting different antibodies it is called a polyclonal antiserum

Front 6

What is an antiserum?

Back 6

Plural = antisera
-It is defined as a serum from an immunized animal which contains Abs

Front 7

What are the weaknesses of polyclonal antisera?

Back 7

1) It is not a standardized reagent. The serum from one rabbit may contain a different set of Abs than that from another rabbit
2) Limited supply (Rabbit may die)
3) Sometimes it is necessary to detect only one epitope. For example if you want to detect the presence of a mutant virus strain that differs from the wild type virus strain in only one epitope
-A polyclonal antiserum that recognized all the epitopes on the virus wouldn't be able to distinguish the mutant virus from the wild type virus

Front 8

What is the most desirable antibody proudction scheme?

Back 8

To have an infinite supply of identical antibodies that recognizes only one epitope

Front 9

What are monoclonal antibodies?

Back 9

1) It is a process to isolate B cell clones that grow indefinitely in cultural dishes and produce a single Ab of desired specificity

Front 10

What are the preparotory work that you must do to prepare monoclonal antibodies?

Back 10

1) Mouse must be repeatedly immunized with an antigen in order to get a good secondary immune response
-B cells specific for different epitopes on the antigen will be come activated, proliferate and differentiate into clones of antibody-secreting plasma cells

Front 11

What are the steps in making monoclonal antibodies?

Back 11

Immortalization of the antibody-secreting plasma cells:
-1) REmove the mouse's spleen which contains many of the Ab-secreting plasma cells. These cells can live in culture and make Abs for only a few days before they die
2) To make plasma cells immortal you can fuse them with a type of tumor cell that can grow forever in culture
-The type of tumor cell used is called a myeloma cell
-It was once a plasma cell but has lost the ability to make its own Abs
-Chemical called polyethylene glycol allows the plasma cells and myeloma cells to fuse and become a hybrid cell called a hybridoma
3) Hybridoma can grow and divide indefinitely in tissue culture dishes. It secretes large amounts of the same Ab that the plasma cell was making
4) Since the plasma cells that were fused to the myeloma cells were a mixture of different clones, you now have a mixture of immortal hybridomas making Abs against different epitopes. There are also some unfused mylemoma cells but these can be killed off with special drugs

Front 12

What are the steps in making monoclonal antibodies?

Back 12

Isolation of the hybridoma clone which is making antibody to the desired epitope
1) To obtain clones derived from single cells put one cell in each well of a 96 well tissue culture plate. The cells in each well will divide and form clones of identical cells
2) Select the hybridoma clones that are making antibodies against the desired epitope. To do this take a little bit of the culture medium from each well
-Use an ELISA assay to see if antibodies that were secreted into the medim by the hybridoma cells bind to the epitope of interest

Front 13

What is ELISA

Back 13

-Enzyme linked immunoadsorbent assay
-It is a sensitive method for detecting the presence of Abs or Ags in fluids and determining their concentration

Front 14

What are three examples of ELISA?

Back 14

1) Hybridoma screening
2) HIV test
3) Using ELISA to determine concentrations of antigens

Front 15

How is ELISA involved in Hybridoma screening?

Back 15

Purpose: To determine which of the hybridomas in the 96 well plates are making an antibody against the epitope of interest

Front 16

What are the steps in Hbridoma screening using ELISA

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1) Coat the wells of a 96 well ELISA plate with the antigen or epitope you want a antibody against
-Plastic of the ELISA plate has been specifically treated so that proteins will bind to it
2) Take a small amont of culture medium from each well of the 96 well plates containing the hybridoma clones
-Transfer this culture medium to the wells of the antigen-coated ELISA plate
-You can use a plate replicator to pick up a small amount of medium from all 96 hbridoma wells at the same time and transfer them to the ELISA plate
-Thus, well A1 of the ELISA plate recieves medium from one hybridoma clone in well A1 of the hybridoma plate
3) After 1 hour pour out fluid from ELISA plate and wash the well
-ONly Abs that bind to the antigen coated onto the ELISA plate will remain bound to well
-Abs against other epitopes will be washed away so that no Ab will be bound in those well
4) Determine which wells have antibodies that are bound to antigen
-How can you detect the presence of Abs? The antibodies produced by the hybridomas are mouse IgG antibodies since the hybridomas are derived from mouse B cells
-To detect the mouse IgG bound to the wells, you use a secondary antibody, an Ab that recognizes the constant region of the AB you want to detect in this case mouse IgG
-These aAbs are made by immunizing rabbits with mouse IgG. The mouse IgG is foreign to htese animals and they will make antibodies against it
-2ndary Ab is covalently linked to an enzyme (hence the enzyme-linked) in ELISA) which allows you to detect the presence of the Abs bound to the Ag in the well
-These enzyme linked secondary Abs can be bought from companies
=Add the secondary antibodies to the wells, incubate 1 hour then dump out the plate and wash out the wells. 2ndary antibody will only bind to wells in which mouse IgG from the hybridoma medium had bound to the antigen coated onto the well
-Add the substrate for the enzyme that is linked to 2ndary antibodies. Type of enzyme that is linked to secondary abs turns a colorless substrate into a clored product
-Thus only the wells that had antibodies bound to the antigen will turn color

Front 17

How do we use ELISA when dealing with HIV tests?

Back 17

-We use it to detect presence of Abs
-Only people who have been exposed to HIV have antibodies to HIV in their blood
1) Coat wells of 96 well plate with purified HIV protein
2) Add diluted patient serum to the wells
3) After 1 hour, pour out the fluid and wash the wells- Only antibodies that bind to the HIV protein will remain bound to well. Wells with serum from someone who has not been exposed to HIV will have no antibodies that bind
4) Add an enzyme linked rabbit anti human IgG antibody to dtect the human IgG bound to the wells
5) Incubate 1 hour. Dump out the fluid and wash out hte wells. Secondary antibody will only bind to wells in which IgG proteins from the patient serum had bound to the HIV protein cotaed onto the well
6) Add the enzyme substrate (colorless) A colored product will only be produced in the wells that had human IgG bound to the HIV protein