Immunology chapter 9

Chapter 9: Electrophoresis Techniques and Chromatography - Part 1

Introduction to Electrophoresis

Definition:

Electrophoresis is the process of migration of charged particles in an electrical field, a critical separation technique in molecular biology and biochemistry. It operates under the principle that charged particles move in response to an electric field, allowing for the separation of various biomolecules based on their size, shape, and charge.

Applications:

• Separation of Biological Molecules: Electrophoresis is utilized to separate proteins, nucleic acids (DNA and RNA), antibodies, hemoglobins, and lipids for analysis and research.

• Clinical Diagnostics: It plays a pivotal role in clinical diagnostics, particularly in assessing serum samples, identifying specific proteins, and diagnosing conditions like multiple myeloma and other blood disorders.

Basics of Electrophoresis

Separation Mechanism:

• Migration Characteristics: The movement of substances is influenced by their size and electrical charge. Larger molecules experience greater friction and thus migrate slower than smaller molecules.

• Mass to Charge Ratio: The velocity of migration is determined by the mass-to-charge ratio of the particle; this ratio is crucial for accurate separations.

Types of Charged Particles:

• Cations: Positively charged particles that migrate toward the cathode (negatively charged electrode) under the influence of an electrical field.

• Anions: Negatively charged particles that migrate toward the anode (positively charged electrode), reflecting the nature of their charge.

Focus on Serum Protein Electrophoresis (SPE)

Purpose:

SPE is specifically designed to separate and quantify proteins in serum samples, which is vital for diagnosing and monitoring various diseases and conditions affecting protein levels in the blood.

Instruments:

• Densitometer: A device that measures the concentration of proteins in bands after electrophoresis, providing quantitative data critical for analyzing serum protein profiles.

Mediums for Separation:

• Cellulose Acetate or Agar Gel: The choice of medium influences the resolution and efficiency of protein migration, with factors like concentration and pH affecting separation.

Fractions/Bands in Serum Protein Electrophoresis:

SPE typically results in the separation of proteins into five distinct fractions:

1. Albumin: The most abundant protein, reflecting nutritional status and liver function, also includes prealbumin.

2. Alpha-1 Globulin: Includes proteins involved in transport functions, such as alpha-1 antitrypsin.

3. Alpha-2 Globulin: Contains haptoglobin and ceruloplasmin, involved in binding hemoglobin and copper, respectively.

4. Beta Globulin: Involves transferrin and complement proteins,

5. Gamma Globulin: Comprises immunoglobulins (antibodies) which are crucial for immune responses.

Staining and Visualization

Staining Agents:

• Sulfasalicylic Acid: Commonly used to stain the protein bands in SPE; the intensity of staining correlates directly with the quantity of protein present in the sample, enabling visualization of protein patterns.

Electrophoresis Chamber Setup:

The electrophoresis chamber is essential for the process, containing components such as gel, electrodes, buffer solutions, and sample wells, enabling the efficient migration of proteins across the gel matrix when an electric field is applied.

Results Interpretation

Analyzing Densitometer Output:

• Normal Results: Typically show a higher concentration of albumin compared to globulins, reflecting a normal protein balance in serum. The normal pattern indicates that albumin is usually two to three times more concentrated than the combined globulin fractions.

Abnormal Results:

• Liver Cirrhosis: Characterized by decreased levels of albumin and an increased level of immunoglobulins, often with elevated IgG levels due to compensatory mechanisms and chronic inflammation.

• Monoclonal Gammopathy: Observed as an increased gamma peak indicating excessive production of specific immunoglobulin (an M spike), which is significant in diseases such as multiple myeloma.

Clinical Applications

Identifying Abnormal Proteins:

• Monitoring Protein Levels: Abnormal protein levels can indicate disease states, and thus measuring total protein allows for the calculation of globulin fractions (total protein minus albumin).

• Quantitative Assays: Specific immunoglobulin levels may require more detailed assays such as nephelometry or turbidimetry.

• Additional Tests: Tests like immunofixation electrophoresis (IFE) and immunoassays provide deeper insights into protein abnormalities and identification of specific protein types.

Summary

The evidence gathered from electrophoresis techniques is crucial for the detection, diagnosis, and monitoring of conditions associated with abnormal protein levels, making it an indispensable tool in clinical laboratories.

Chapter 9: Electrophoresis Techniques and Chromatography - Part 2

Immunofixation Electrophoresis (IFE)

Process Overview:

IFE consists of two primary phases: the electrophoresis phase, where proteins are separated based on size and charge, and the fixation phase, where specific antisera are used to bind to anticipated antigens (e.g., IgG, IgA, IgM, kappa light chains, lambda light chains).

IFE Methodology

Electrophoresis Phase:

This phase solely focuses on the effective separation of proteins, allowing for the individual bands to form based on their respective charge and size.

Fixation Phase:

In this phase, antisera specific to the proteins of interest interact with the bands formed during electrophoresis, allowing for enhanced visualization of those proteins that have reacted.

Interpretation:

Results from IFE are primarily qualitative and rely on visual observation of the banding pattern produced, where normal patterns typically show characteristic heights and intensities for different immunoglobulin types.

Results in IFE

Patterns Analysis:

• Normal Patterns: Expected findings include a pronounced dark band for IgG, a lighter band for IgA, and absent or faint IgM bands. Kappa bands usually appear two times darker than lambda bands, reflecting the normal kappa-to-lambda ratio ( approximately 2:1).

Abnormalities:

• Monoclonal Gammopathy: Unusually characterized by an increased presence of specific proteins (M protein), signaling potential plasma cell disorders such as multiple myeloma or lymphoma as diagnostic entities.

Clinical Follow-Up for IFE

Regular Monitoring:

Patients diagnosed with monoclonal gammopathy require ongoing monitoring through IFE post-diagnosis and treatment due to the potential for disease progression or relapse.

• Frequency of Testing: IFE tests are recommended every three months following treatment to assess response and ensure disease stability.

Chapter 9: Chromatography Overview - Part 3

Introduction to Chromatography

Definition:

Chromatography is an analytical technique that separates complex mixtures based on diverse physical interactions of components present within the mixture. This method is central in various fields such as chemistry, biology, and environmental science.

Qualitative Technique:

Chromatography primarily identifies the presence and characteristics of various substances. When coupled with mass spectrometry, it allows for precise quantification of components, enhancing its analytical capabilities.

Components of Chromatography

Basic Setup:

Chromatography typically includes:

• Mobile Phase: The solvent (either liquid or gas) that carries the mixture through the stationary phase.

• Stationary Phase: The phase that remains fixed in the column or plate; interactions between the stationary phase and analytes lead to separation.

• Column or Plate: The physical structure where the separation occurs, impacting resolution and analysis speed.

Types of Chromatographic Methods:

Numerous techniques are available, including:

• Paper Chromatography: Utilizes a stationary phase on paper for separation based on capillary action.

• Thin Layer Chromatography (TLC): Conducts separations on a thin layer of adsorbent, ideal for identifying the number of components.

• High-Performance Liquid Chromatography (HPLC): A sophisticated method that allows for high resolution and speed, often used for pure compound isolation and analysis.

Immunochromatography

Definition:

Immunochromatography merges chromatography with immunochemical reactions, providing a method for rapid antigen detection, commonly seen in diagnostic tests.

Flow Types:

• Lateral Flow Assays (LFA): Allow for rapid results using simple device formats, e.g., home pregnancy tests.

• Vertical Flow Assays (VFA): Often utilized in laboratory settings for more complex applications.

Mechanism:

In immunochromatography, a sample is absorbed onto a functionalized surface, where it interacts to form an antigen-antibody complex. This interaction results in a visual signal, often indicating presence or absence, enabling quick diagnostic interpretations.

Practical Example: Rapid Strep Test

The Rapid Strep Test exemplifies immunochromatography by detecting antigens from streptococcal bacteria through antibody interactions, providing immediate results in clinical settings.

Conclusion

Immunofixation Electrophoresis (IFE) offers a more sensitive differentiation of proteins compared to traditional Serum Protein Electrophoresis. This sensitivity is vital for detailed monitoring of conditions such as multiple myeloma and other related protein abnormalities, thus enhancing diagnostic accuracy and patient management in clinical practice.