Immunochemical Methods and Applications

Immunochemical Methods

Basic Terms

  • Immunochemical Methods: Techniques used to analyze or identify substances based on their interactions with antibodies (Ag-Ab interactions).

Blood Groups

  • Blood Groups Overview:

    • Classified according to antigens on erythrocytes’ surfaces.

    • Major systems include ABO and Rh.

    • Other examples include MNS, PIPK, Kell, Kidd, Duffy, Lutheran, Lewis.

  • ABO System:

    • Four blood groups defined by antigens A, B, and O (H).

    • Agglutinogens: Glycolipids (oligosaccharides) on surface.

    • Agglutinins: Antibodies against absent agglutinogens.

    • Genetics: hh genotype results in Bombay phenotype due to lack of antigen H.

    • % Distribution in Czechia: A (42%), O (39%), B (15%), AB (4%).

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C-Reactive Protein (CRP)

  • CRP Overview:

    • Full name: C-reactive protein, involved in innate immunity, serves as an opsonin.

    • Increases post-injury or infection: peaks in 24-48 hours.

    • Half-life: 24 hours.

    • Value Interpretation:

    • < 5 mg/l: Standard.

    • 6-30 mg/l: Viral infection.

    • 50-200 mg/l: Bacterial infection.

    • > 200 mg/l: Serious infection.

Antigens and Antibodies

  • Antigen Characteristics:

    • Macromolecules like proteins and polysaccharides recognized by the immune system.

    • Can be exoantigens (foreign) or autoantigens (self).

    • Complete antigens must have a carrier and an epitope.

    • Hapten: Small molecule that needs a carrier to induce an immune response.

  • Antibody Characteristics:

    • Glycoproteins produced by plasma cells with variable heavy and light chains.

    • Types of immunoglobulins: IgG, IgA, IgM, IgD, IgE.

    • Interaction Types: Non-covalent interactions, specificity, affinity, and avidity.

Types of Immunochemical Methods
Methods Without Markers

  • Immunoprecipitation: Relies on Ag-Ab interaction to isolate molecules. Variants include:

    • Immunoprecipitation in gels and solutions.

Immunoprecipitation Curves

  • Represents the relationship between antigen concentration and precipitate formation:

    • Excess Antibody Zone: Increasing antigen leads to greater precipitate.

    • Equivalence Zone: Optimal Ag-Ab interaction results in maximal precipitate.

    • Excess Antigen Zone: Precipitate decreases with increased antigen leading to more soluble complexes.

Immunochemical Methods With Markers

  • Use of markers to detect Ag-Ab interactions:

    • Types of markers:

    • Enzymes (ELISA).

    • Radioisotopes (RIA).

    • Fluorescent markers (FIA).

    • Luminescent markers (LIA).

    • Methods can be homogeneous or heterogeneous depending on whether they require washing.

Enzyme-Linked Immunoassays (ELISA)

  • Types of ELISA:

    • Direct ELISA: Fast, less prone to error, but reduced sensitivity due to non-specific binding.

    • Indirect ELISA: More sensitive due to secondary antibodies.

    • Sandwich ELISA: High sensitivity and specificity; allows for flexibility in detection.

    • Competitive ELISA: Robust, flexible design; suitable for low-abundance analytes.

Examples and Applications of Immunochemical Methods

  • Endocrinology: Measurement of hormones such as TSH, cortisol, and reproductive hormones.

  • Oncology: Tumor markers like PSA, AFP, and other biomarkers.

  • TDM (Therapeutic Drug Monitoring): Monitoring levels of various drugs in the body.

  • Cocaine Metabolites: Measurement via EMIT for detecting drug use.

  • Anti-Streptolysin O (ASLO): Indicates infection by controlling post-streptococcal diseases.

  • Anti-Müllerian Hormone (AMH): Indicates ovarian reserve and reproductive health.

Final Notes

  • Keep in mind the principles behind these immunochemical methods and their significance in clinical diagnostics, especially in light of various diseases and conditions.

    • Understanding how to interpret results and the importance of proper technique will greatly aid in practical training.