Antibodies as Tools of Discovery

Final post for biochemistry!

Definitions

First of all I'm going to start with some definitions for you:

• Antigen- A foreign substance that elicits an immune response.
• Antibody- An immunoglobulin protein that binds to an antigen.
• Epitope- A part of the antigen where an antibody binds.
• Polyclonal antibodies- A mixture of different antibodies. Different antibodies may bind to different epitopes on the same antigen.
• Hapten- A small molecule that doesn't elicit an immune response by itself, though it may elicit an immune response when bound to some other protein. Some of the antibodies raised against a hapten-protein complex may also bind to the hapten on its own.

Immunoglobulin G (IgG)

As I mentioned before, antibodies are immunoglobulin proteins. There are five types of immunoglobulin proteins, but for now we're only going to focus on one: IgG.

IgG is kind of Y-shaped. It is made up of two heavy chains, as well as two light chains inside the two small arms of the Y. The light and heavy chains are bound to each other by disulfide bonds, and the heavy chains are also bound to each other by disulfide bonds. Additionally, there are intrachain disulfide bonds that afford greater stability of the protein. Each L-chain consists of two Ig-fold domains, whereas each H-chain consists of four Ig-fold domains. These Ig-fold domains are simply beta barrels (see my earlier post for more information on how proteins fold).

There are many different IgGs that can attack different epitopes on different antigens. All IgGs have a constant sequence, taking up the long arm of the Y as well as half of each of the small arms. The tips of the small arms of the antibodies, where antigens bind, are variable sequences. Antigens bind to these areas quite tightly, with a dissociation constant usually less than 10^-9 M (that's really small).

It is possible to use the protease papain to cleave IgG just below the fork of the Y. This creates two F_ab fragments, each of which can still bind an antigen. This is useful when you want small antigen-binding molecules.

Creating Diverse Antibodies

There are millions of antibodies, so it wouldn't be very efficient if there were different genes coding for every single antibody. Thankfully, as I mentioned above, IgGs all have constant sequences, which is rather helpful.

First, let's look at light chains, or more specifically at κ-type light chains, which are the main light chains in humans and mice. In the genome, there is an "L_κ + V_κ Library," which contains pairs of L_κ (leader/Targeting sequences- ensure that the mRNA is translated on the endoplamic reticulum) and V_κ (variable region coding sequences). These are then spliced with one of five J_κ (joining sequences) and finally with the one and only C_κ (constant region coding sequence). There are roughly 100 L_κ/V_κ pairs, and five J_κs, resulting in over 500 combinations (additional combinations are created by random deletions when things get spliced together).

Heavy chains are pretty similar, but instead of the sequences being called L_κ, V_κ etc., they're called L_H, V_H etc. There is also one extra step: L_H/V_H sequences are spliced with one of 30 D_H (diversity sequences), then to one of six J_Hs, and finally to C_H. Random addition or deletion of nucleotides can create even more combinations.

Note that I only talked about variations within chains. There is even more variation when you take into account that different light chains can pair up with different heavy chains, and so on and so forth.

Antibody Production

To use antibodies as effective tools, we need a way to produce them. Here's how:

• An animal (e.g. mouse) is injected with an antigen, and spleen cells, some of which are making antibodies to the antigen, are extracted
• These spleen cells are fused with mutant mouse myeloma cells that are unable to grow on a medium called HAT. This results in the production of three kinds of cells:
  1. Unfused spleen cells that die not long after being out of the animal
  2. Unfused myeloma cells that die when on HAT
  3. Fused cells that produce antibodies and don't die
• Single cells are cultured in separate well. Each cell produces monoclonal antibodies but doesn't die

Uses for Antibodies

There are plenty of uses for antibodies, one of which (ELISA) you have already seen. Here are some other uses:

• Western Blotting- kinda like Northern/Southern blotting but for proteins. Antibodies are used to detect the protein of interest.
• Immunocytochemistry- using fluorescently tagged antibodies to detect proteins within the cell. Requires cells to be fixed and pores created in the cell membrane.
• Drug targeting- Antibodies against a cell surface marker can be coupled with cytotoxic or radioactive reagents- helpful for killing off cancerous cells.