micro 20.2 pt 4

Immunoelectrophoresis (IEP)

A technique that combines protein electrophoresis with antibody-based precipitation to identify specific proteins in a sample.

Type of electrophoresis performed at the beginning of immunoelectrophoresis

Polyacrylamide gel electrophoresis (PAGE).

Purpose of PAGE in immunoelectrophoresis

It separates the proteins in a sample based on their electrophoretic properties before they are identified with antibodies.

What is added after the proteins have been separated during immunoelectrophoresis?

Antisera against selected serum proteins are added to troughs running parallel to the electrophoresis track.

What forms when antibodies in immunoelectrophoresis interact with their corresponding proteins?

Precipitin arcs form.

What do the precipitin arcs in immunoelectrophoresis allow scientists to identify?

They allow identification of specific proteins in the sample.

Medical condition immunoelectrophoresis is particularly useful for diagnosing

Multiple myeloma, a cancer of antibody-secreting cells.

What happens to antibody production in multiple myeloma?

The abnormal cells produce monoclonal proteins (M proteins) instead of normal antibodies.

M proteins

Abnormal monoclonal immunoglobulin proteins produced by the cancerous cells of multiple myeloma.

What happens to serum protein levels in patients with multiple myeloma?

They typically have elevated serum protein levels with an abnormal concentration of immunoglobulins.

What does a protein electrophoresis pattern look like in multiple myeloma?

It shows a distinct band in the gamma-globulin region and a sharp M-protein spike rather than the normal broad gamma-globulin pattern.

Significance of a sharp M-protein spike

It indicates an unusually large amount of a single type of immunoglobulin, which can suggest multiple myeloma.

How can antibodies against heavy and light chains help identify M proteins?

They produce skewed precipitin arcs corresponding to one class of heavy chain and one class of light chain.

Discovery about antibodies from protein electrophoresis

It helped show that antibodies migrate primarily in the gamma-globulin region of serum proteins.

Western blot

An immunoblot technique used to identify specific proteins in a complex mixture using antibodies.

First step in a Western blot

Proteins are separated by PAGE according to their size.

What happens to the proteins after PAGE during a Western blot?

The proteins are transferred from the gel onto a nitrocellulose membrane and immobilized there.

Primary antibody in a Western blot

An antibody that specifically binds to the protein of interest on the membrane.

Function of the secondary antibody in a Western blot

It binds to the primary antibody and carries a detectable molecular label.

Types of labels attached to secondary antibodies in a Western blot

Enzymes or fluorophores.

How an enzyme label allows detection in a Western blot

A colorless chromogenic substrate is added; the enzyme converts it into a colored product at the location of the target protein.

Fluorophore

A molecule that produces fluorescence when excited by light.

Visible band on a Western blot represents

The location of a specific protein recognized by the antibody.

Types of antibodies typically used in Western blot assays

Polyclonal antibodies are typically used.

Why polyclonal antibodies are more sensitive than monoclonal antibodies in Western blots

They can bind to multiple epitopes on the target protein, generally producing a stronger signal.

Why monoclonal antibodies might still be used in Western blots

They can provide highly specific detection, although they are generally more expensive and less sensitive because they recognize only one epitope.

Southwestern blot

Used to identify DNA-protein interactions. Proteins are separated, transferred to a membrane, allowed to renature, and then probed with labeled DNA.

Far-western blot

Used to identify protein-protein interactions between immobilized proteins and non-antibody protein probes.

Basic principle of a complement fixation test

If antibodies in patient serum bind the tested antigen, they fix/consume complement, leaving none available to lyse subsequently added red blood cells.

Interpretation of results in a complement fixation test: clear vs pink solution

Clear = positive: antibodies against the antigen are present and complement was fixed. Pink = negative: the antibodies are absent, so complement lysed the red blood cells.