Immunological Techniques and Applications 10
Quantitative Precipitation Curve
Discovered in the 1950s by Heidelberger, and Kendall, the quantitative precipitation curve illustrates the dynamic interaction between antigens and antibodies.
Principle: Antigens possess specific epitopes that are recognized and bound by cognate antibodies. This interaction leads to immune complex formation, a principle used in various diagnostic applications.
Optimal Ratio: Accurate antibody quantification requires an optimal antigen-antibody ratio. An excess of either can lead to false negative results.
Immune Complex Formation: The interaction results in large immune complexes that precipitate out of solution. This precipitation is visible in the lab.
Procedure and Measurement
The procedure involves reacting a constant amount of antibodies (from patient serum) with varying amounts of antigen.
Reaction Conditions: The reaction can be accelerated using heat and cold or by allowing it to proceed over time.
Precipitation: Maximum precipitation occurs when all antigen is bound to all antibody.
False Negatives:
Antibody Excess: Too much antibody hinders complex formation due to insufficient antigen.
Antigen Excess: Too much antigen interferes with the formation of large complexes.
Spectrophotometry: The resulting immune complexes are measured using a spectrophotometer. A laser is directed through the sample, and the scattered light is measured as optical density.
Equivalence Point and Prozone
Equivalence Point: This is the point at which all antigen is bound to all antibody, resulting in maximum immune complex formation.
Prozone: An excess of either antigen or antibody leads to the prozone, resulting in false negative results. Healthcare workers need to consider the prozone effect to ensure the accuracy of test results.
Quantification: This test helps quantify specific antibodies against particular antigens.
Antigen Valency
The valency of an antigen refers to the number of antibodies each antigen can bind. This varies depending on the size and structure of the antigen.
Multiple Binding Sites: Large proteins may have multiple epitopes, allowing multiple antibodies to bind.
Variable Binding: Antibodies can bind to one or multiple antigens depending on their structure and the arrangement of epitopes.
Immunodiffusion Assays
Similar to ELISA, immunodiffusion assays are another method for quantifying antibody-antigen reactions.
A glass slide is coated with a smooth layer of agar with multiple evenly spaced wells cut out of the agarose.
The antibody diffuses out radially, and antigens in outer wells also diffuse radially.
At the zone of equivalence, a visible precipitation line forms, indicating a positive reaction. The distance from the well is correlated with concentration of antibody or antigen.
These precipitation lines reveals whether the test antibody is cognate for the antigens in the outer wells, giving a positive result for recognition of that tissues antigen.
Agglutination Reactions
These reactions are effective for assessing the cross-reactivity of blood groups.
Blood Transfusions: Patient serum is tested against donor red blood cells to detect potential incompatibilities.
Blood Group Antigens: Red blood cells have surface proteins (antigens) that determine blood type.
Antibody Response: Individuals produce antibodies against blood group antigens they lack.
Procedure: A constant number of red blood cells is added to each well of a plate, and serum is titrated, then serially diluted down the plate.
Hemagglutination: The assay looks for hemagglutination. The titre is the last well where agglutination is observed.
Immuno-Electrophoresis
This technique combines the movement of immune molecules using electricity with antigen-antibody interactions.
Procedure: A mixture of antigens is placed in a well and subjected to an electric field, which separates proteins by charge.
Migration: Negatively charged proteins move toward the anode, while positively charged proteins move toward the cathode.
Antibody Addition: After electrophoresis, antibodies are added to the trough, where they diffuse and interact with the separated antigens.
Precipitation: At the equivalence point, visible precipitation lines form, indicating specific antigen-antibody interactions. The size and shape of the precipitation patterns will depend on the specific protein, and their relative positions with respect to the proteins.
Can be used to detect monoclonal disorders and complement protein deficiencies.
Dyes can be added to show the presence by binding to any protein.
ELISA (Enzyme-Linked Immunosorbent Assay)
ELISA is a versatile assay for detecting and quantifying antigens or antibodies. It can be adapted for various applications.
Flexibility: Can be quantitative or qualitative, with different detection methods (colorimetric, fluorescent).
Applications: Detects antigens, specific antibodies, hormones, cytokines, tumor markers, autoantibodies, and allergen responses. Captures viruses or bacterial particles.
Procedure:
Plastic wells are coated with a protein (antigen, hormone, cytokine, etc.) overnight.
The protein binds irreversibly to the well.
A highly specific antibody is used to detect the captured molecule. Unbound antibodies are washed away.
A secondary antibody, linked to an enzyme, is added. It binds to the primary antibody.
A substrate is added, which is converted by the enzyme to produce a color change.
Intracellular ELISA assay (ELISpot) -The ELISA protocol can be modified.
Peripheral blood taken from patient, and plasma cells stick to the bottom of a plate.
The plate is washed to remove any unbound cells.
Any antibody being secreted from plasma cells will be captured on the plate.
The plate can be stained to visualise the antibody
Variations of ELISA
Direct ELISA: Detects the antigen directly with a labeled antibody. Requires a high concentration of antigen for detection.
Indirect ELISA: Uses a primary antibody and a labeled secondary antibody. Enhances the signal. colour change is observed
Sandwich ELISA: Captures the antigen with a coating antibody and detects it with another antibody.
Competitive ELISA: Assesses the strength of binding by competing with other solutions. Reveals information about binding affinity and specificity.
Flow Cytometry
Flow cytometry is a powerful technique for analyzing individual cells in a heterogeneous population.
Principle: Cells are stained with fluorescently labeled antibodies specific for different surface molecules.
Procedure:
Immune cells (leukocytes or lymphocytes) are extracted from blood.
Cells are stained with fluorescent antibodies against specific surface markers (e.g., CD4, CD8).
The cells are passed in a single file through a laser beam.Fluorescent tags deflect the signal toward a specific sensor depending on its specific wavelength.
Detectors capture fluorescent signals, which are then plotted on a graph. This measures how many molecules a single cell has relative to others.
Data Analysis:
Dot Plots: Two-dimensional plots showing the expression of two different markers (e.g., CD4 vs. CD8).
Histograms: Single-parameter plots showing the distribution of a single marker.
*The location of dots on the plot signifies relative quantity of the two surface proteins of interest (e.g. CD4 and CD8).
The intensity of color staining will represent the number of cells present at a particular location of the plot.
Boxes, called quadrants, may be drawn around a region of the plot, and flow cytometry can calculate the percentage of all cells present inside of each of these boxes.
Immunohistochemistry and Immunocytochemistry
These techniques visualize antigens in tissues (immunohistochemistry) or cells (immunocytochemistry).
Procedure:
Tissue biopsies are taken and either frozen or fixed in formaldehyde.
Sections are cut using a microtome and mounted on glass slides.
Sections are stained with antibodies linked to fluorescent markers.
The staining pattern is visualized under a fluorescent microscope.
Applications:
Testing for protein expression (e.g., Her2 in breast cancer).
Detecting autoantibodies in patient serum.
Identifying complement deposits in tissues.
Passive Immunization
Providing preformed antibodies to an individual for immediate protection.
Immune prophylaxis: antibodies that are administered to prevent infection or disease.
Examples:
Intravenous immunoglobulin (IVIg) for antibody deficiencies.
Maternal IgG transfer across the placenta.
Antibodies against rabies, botulism toxin, and snake venom.
This can only provide short term immunity from weeks to at most months.
Active Immunization
Vaccines stimulate the body to produce its own antibodies and memory cells.
Mechanism:
The initial exposure to a weakened or fractionated organism primes B cells to produce specific antibodies.
A booster shot stimulates memory B cells, resulting in a rapid and robust antibody response.
The goal is to achieve a threshold level of neutralizing antibody that prevents infection from causing disease.
Types of Vaccines:
Live Attenuated: Weakened forms of the pathogen that elicit a strong immune response.
Killed Inactivated: Inactivated pathogens that are safe but may require boosters.
Toxoids: Inactivated toxins that generate a strong antibody response (e.g., tetanus, diphtheria).
DNA and RNA Based: Stimulate cellular responses and antibody responses.
We ideally want memory cells to become present, and this refers to B, T helper, and cytotoxic T cells
Cancer immunotherapy
Antibodies (Ab) can be used to target tumour with pro-drug or radioactivity or cytokine
Abs can be blocking can be blocking antibodies that prevent ligand binding to receptor
Abs can be engineered to be cell specific, to improve binding, to induce the required response
vaccines can be made of ex vivo activated immune cells, or can be cancer-lysing viruses.
immunosuppressive drugs inhibit the immune cell signalling responses, usually broadly supressive, prolongs transplant survival eg corticosteroids, ciclosporin, tacrolimus, sirolimus.