Electrophoresis and Chromatography Techniques

Introduction to Electrophoresis Techniques

  • Overview of electrophoresis techniques in chemistry and immunology.

  • Definition: Electrophoresis refers to the migration of charged solutes or particles in an electric field.

  • Explanation of Electric Field:

    • An electrical field is applied to a solution containing charged particles with oppositely charged electrodes:

    • Positive electrode is called the anode.

    • Negative electrode is called the cathode.

  • Movement of Charged Particles:

    • Cations (positively charged) move towards the cathode (negative electrode).

    • Anions (negatively charged) move towards the anode (positive electrode).

Gel Application in Electrophoresis

  • Charged particles are placed in a gel, typically an agarose gel slab.

  • Importance of Buffer:

    • Buffers used usually maintain a neutral pH around 8.6.

    • This pH helps achieve a slight positive charge on most particles, allowing them to migrate towards the negative electrode (cathode).

Serum Protein Electrophoresis (SPE)

  • Focus on serum, not plasma, for serum protein electrophoresis.

  • Purpose: To separate serum proteins into five distinct fractions within an agarose gel.

  • Five Fractions of Serum Proteins:

    1. Albumin

    2. Alpha one globulins

    3. Alpha two globulins

    4. Beta globulins

    5. Gamma globulins

Process of Serum Protein Electrophoresis

  • Collection of blood sample followed by clotting.

  • Serum is separated from the cells after clotting.

  • Application of serum onto the gel, specifically closer to the cathode.

  • Migration Behavior:

    • Most proteins exhibit slight negative charge, leading them to move towards the anode.

    • Order of Migration:

    • Albumin (farthest and fastest)

    • Alpha one and (occasionally) alpha two

    • Beta

    • Gamma

Visualization of Results

  • After electrophoresis, the gel is stained for visualization.

  • Typical pattern observed on the gel includes:

    • Highest concentration of albumin

    • Followed by alpha one peak, alpha two peak, beta peak, and gamma peak.

Importance of Serum Protein Electrophoresis in Disease Diagnosis

  • Patterns observed during SPE can indicate various disease states, focusing especially on the gamma region where immunoglobulins are located.

  • Significance of Gamma Region:

    • Spikes in gamma region may suggest multiple myeloma or increased immunoglobulin production by plasma cells.

Monitoring Abnormal Patterns

  • Initial pattern interpretation:

    • Normal SPE shows typical fraction peaks.

    • An increase in gamma globulins could indicate potential disorders; further inspection of patient history is necessary.

  • If spikes in gamma are detected:

    • Immunofixation is conducted to specify which immunoglobulin (IgG, IgA, IgM) is responsible for the increase.

Abnormal SPE Patterns

  • Different clinical conditions lead to changes in electrophoresis patterns:

    1. Alpha-One Antitrypsin Deficiency:

    • Reduced alpha one peak observed.

    1. **Nephrotic Syndrome:

    • Increase in alpha two and beta proteins; slight decrease in albumin.

    1. Inflammation (Acute Phase Reaction):

    • Increase in alpha one, alpha two, beta proteins, with a decrease in albumin.

    1. Cirrhosis:

    • Characteristic beta-gamma bridge observed with no distinct beta peak.

    1. Monoclonal Gammopathy (M Protein Spike):

    • Large increase in the gamma region, indicates potential monoclonal proliferation.

Immunofixation Electrophoresis

  • Purpose: To identify specific immunoglobulins causing monoclonal gammopathy.

  • Process: After running standard SPE, antibodies for each immunoglobulin are applied:

    • Anti-IgG, Anti-IgA, Anti-IgM, Anti-kappa, Anti-lambda.

  • Example Interpretations:

    • If a band appears after applying anti-IgG and anti-kappa:

    • Diagnosis: Monoclonal gammopathy producing IgG kappa antibodies.

    • If a band appears for anti-IgA and anti-lambda:

    • Diagnosis: Monoclonal gammopathy producing IgA lambda antibodies.

Capillary Electrophoresis

  • Definition: Similar technique to standard electrophoresis but on a micro-scale.

  • Benefits: High sensitivity, efficiency, and rapid results.

  • Comparison of Microchip and Conventional Capillary Electrophoresis:

    • Both techniques will not be a major focus for examinations but are acknowledged.

Introduction to Chromatography

  • Chromatography defined: Technique for the separation of molecules.

  • Phases involved:

    • Mobile Phase: Solvent moving through a mixture of solutes (e.g., buffers).

    • Solid Phase: Stationary phase that retains solutes being separated.

Types of Chromatography

  • Four main types of chromatography introduced:

    1. Gas Chromatography: Solute in the gaseous state.

    2. Liquid Chromatography: Solute in a liquid state.

    3. Flat Chromatography: Solute separation occurs on a static surface like paper.

    4. Column Chromatography: Solute moves through a column filled with stationary phase (such as beads).

Immunochromatography

  • Overview of immunochromatography process:

    • Typical structure includes:

    • Sample Pad: Where patient sample is added.

    • Conjugate Pad: Contains labeled biomolecules (e.g. antibodies or antigens).

    • Control Line: Ensures test accuracy.

  • Functionality: As sample moves through the pads, specific analytes bind to targeted antibodies along the test line, resulting in visual signals that confirm positive or negative results.