Immunological Techniques lecture 16

Immunological Techniques

Immunological techniques are widely used in diagnostic labs and research, not only for immunology but for all sorts of research involving detection using antibodies. The principles underlying these tests are based on immune responses and reactions.

Definition of Immunological Techniques

Immunological techniques are laboratory methods based on the principles of the immune system. The fundamental principle of the immune system, especially the adaptive immune system, is to differentiate foreign substances from self. This specificity, based on interactions between antibodies and antigens, is crucial for the functionality of immunological tests.

ELISA (Enzyme-Linked Immunosorbent Assay)

ELISA is a sensitive method used extensively in labs, including diagnostic labs, often performed to amplify signals for detection of substances present in small amounts. The multiple layers of antibodies involved rely on specific antibody-antigen interactions.

Advantages of ELISA

1. Sensitivity: Multiple layers of antibodies amplify the signal, making it suitable for detecting low-concentration substances.

2. Specificity: Nonspecific binding substances can be washed away due to the high specificity of antibody-antigen interaction. Washing steps are crucial to remove nonspecifically bound materials from the solution.

3. Automation: ELISA can be easily automated, which is particularly important for diagnostic labs.

Procedure and Mechanism

1. The secondary antibody used in ELISA is labeled with an enzyme.

2. This enzyme hydrolyzes a substrate, producing a product that absorbs and reflects light, resulting in a color change.

3. The color density is proportional to the concentration of the substance being measured.

4. A standard curve with different concentrations must be generated for accurate measurements by measuring the absorption of specific wavelengths.

Applications of ELISA

• Diagnostic Labs

  • Detecting antibodies to infectious agents in patient serum.

  • Diagnosing autoimmune diseases by measuring antibodies that react to human substances (e.g., RNA, DNA, proteins).

  • Measuring IgE levels for diagnosing allergic diseases.

• Research

  • Studying inflammatory responses by measuring cytokine levels in cell culture supernatants.

Example: Dengue Virus Serology

In diagnosing a patient with a fever after traveling to a tropical area where dengue fever is endemic, clinicians may perform dengue virus serology to detect antibodies against the dengue virus.

1. Coating the ELISA Plate: The ELISA plate is pre-coated with dengue viral antigen.

2. Adding Patient Serum: Patient serum is added to the wells, allowing antibodies (if present) to interact with the dengue viral antigen.

3. Washing: Nonspecific antibodies and proteins are washed away, leaving only the specifically bound antibodies.

4. Adding Secondary Antibody: A secondary antibody (developed in animals like mice or rabbits), labeled with an enzyme, is added to bind to the human immunoglobulin.

5. Washing: Unbound secondary antibodies are washed away.

6. Adding Substrate: The enzyme hydrolyzes the substrate to produce a colored product.

Interpretation of Results

• IgM: An increase in IgM indicates a recent infection.

• IgG: Presence of IgG indicates a past infection or vaccination.

Enzymes Used

Common enzymes used include horseradish peroxidase and alkaline phosphatase, each requiring specific substrates to produce a detectable color change.

Research Example: Eromonas Veronae

Research on Eromonas Veronae, an emerging enteric pathogen, uses ELISA to measure inflammatory cytokines produced by macrophages in response to the bacteria. This helps determine if the bacterium induces more inflammation compared to other bacteria like E. coli.

Nephelometry

Nephelometry is used for measuring high concentrations of proteins, such as serum proteins, immunoglobulin, and complement components. It's particularly useful for acute-phase proteins like C-reactive protein (CRP).

Procedure

1. Antibody specific to the target protein (e.g., CRP) is added to the patient's serum.

2. The antibody-antigen complex forms, scattering light when a light beam is passed through the sample.

3. The amount of scattered light is measured by a detector, which is proportional to the concentration of the protein.

Clinical Significance of CRP

CRP is produced by the liver in response to interleukin-6 during inflammation. Measuring CRP levels helps clinicians detect inflammation in the body, although it does not specify the cause (e.g., infection or autoimmune disease).

Reference Range

Diagnostic labs provide a reference range for CRP, typically derived from testing healthy individuals. For example, a CRP reference range of 10 mg/L means 95% of healthy people have CRP levels below this.

Immunofluorescence

Immunofluorescence utilizes antibody-antigen reactions with secondary antibodies labeled with fluorescent dyes. When excited by light, these dyes emit light at different wavelengths, producing different colors.

Procedure

1. Cells (live or fixed) or biopsy samples on slides are incubated with patient serum.

2. If autoantibodies are present, they bind to their respective antigens in the cells.

3. A secondary antibody labeled with a fluorescent dye is added to bind to the autoantibodies.

4. After washing, the slides are examined under a fluorescent microscope.

Application: Diagnosis of Autoimmune Diseases

Immunofluorescence is crucial in diagnosing autoimmune diseases by detecting antinuclear antibodies (ANA) that target components like DNA or histones.

Antinuclear Antigen (ANA) Patterns

Different autoimmune diseases are associated with specific ANA patterns. For example, a homogenous pattern with high titer antibodies may indicate systemic lupus erythematosus (SLE).

1. Homogeneous Pattern: In this pattern, the entire nucleus stains evenly. It is often associated with:

   • Systemic Lupus Erythematosus (SLE)

   • Drug-induced Lupus

2. Speckled Pattern: This pattern shows a speckled appearance within the nucleus, with varying sizes and distribution of speckles. It is associated with:

   • Mixed Connective Tissue Disease (MCTD)

   • Systemic Lupus Erythematosus (SLE)

   • Sjögren's Syndrome

   • Systemic Sclerosis (Scleroderma)

3. Nucleolar Pattern: This pattern shows staining primarily in the nucleoli (dense structures within the nucleus). It is often associated with:

   • Systemic Sclerosis (Scleroderma)

4. Centromere Pattern: This pattern shows discrete speckles corresponding to the centromeres of chromosomes. It is strongly associated with:

   • Limited Cutaneous Systemic Sclerosis (CREST syndrome)

Flow Cytometry

Flow cytometry is a technique used to analyze and sort cells based on their characteristics. Cells are stained with multiple antibodies labeled with fluorescent dyes (fluorochromes).

Procedure

1. Cells are stained with antibodies labeled with various fluorochromes.

2. The cells are loaded into a flow chamber where a sheath fluid ensures they pass through a laser beam in a single file.

3. As cells pass through the laser beam, several parameters are measured:

   • Forward Scatter (FSC): Light scattered by the size of the cell.

   • Side Scatter (SSC): Light scattered based on the internal complexity or granularity of the cell.

   • Fluorescence: Emission from the fluorochromes attached to antibodies, indicating the presence of specific markers.

Cell Sorting

Flow cytometry can also be used for cell sorting. After analysis, cells with specific characteristics can be separated by charging them negatively or positively and directing them into different collection tubes.

Clinical Applications

Flow cytometry is used for diagnosis and monitoring treatment, especially in cancer patients, to track specific cell populations and assess treatment effectiveness.

Case Study: Immunodeficiency in a Six-Month-Old Boy

A six-month-old boy with repeated infections was suspected of having an immune deficiency. Clinicians performed immunoglobulin measurements and lymphocyte analysis using immunophenotyping.

1. Immunoglobulin Measurement: Nephelometry showed very low levels of IgG, IgA, and IgM, indicating potential immunoglobulin deficiency.

2. Lymphocyte Analysis: Flow cytometry was used to analyze lymphocyte populations.

Gating Strategy

1. Identifying Lymphocytes: Lymphocytes are identified based on forward scatter (size) and side scatter (complexity) or using a CD45 marker (pan-leukocyte marker).

2. T Cell and B Cell Identification:

   • T cells are stained with anti-CD3 antibody.

   • B cells are stained with anti-CD19 antibody.

   • NK cells do not bind to either CD3 or CD19.

Results

In the patient, flow cytometry revealed the absence of B cells, indicating a primary immunodeficiency known as B cell deficiency. This case illustrates the importance of immunotechnology in diagnosing immune disorders.