To accurately determine the antibodies responsible for a disease or a condition, the following immunoassays immunological applications have been devised:
- ELISA Assays
ELISA or Enzyme-Linked Immunosorbent assay is the most common type of assay carried out to detect the presence and the amount of antibodies or other analytes. ELISA assays are efficient in determining if the level of antibodies raised in an immune reaction. The process involves use of a microtiter well-plate with about 96 holes immobilized with an antigen. The serum with the suspected antibodies specific to the antigen in the plates will bind via adsorption or through the introduction of a capture antibody-specific to the antigen.
The assays can be done in four different ways; the direct, indirect, competitive, and the sandwich ELISA assay. The type of ELISA testing carried out depends on the antibodies tested and the specificity of the antigens and antibodies.
- Immunofluorescence (IF)
Immunofluorescence assays are important in detecting the presence of an antibody. The antibody binds to a particular protein in a sample, which can be on tissue sections or fixed cells. The binding of the antibody and the proteins (antigens) is visualized using fluorescent detection. The visualization also gives the location of the proteins in the sample.
The staining can be primary, using primary antibodies conjugates in the flourochromes, or indirect where secondary antibodies are labelled to the flourochromes.
- Immunohistochemistry (IHC)
This is a histological procedure used to detect the presence of a protein in a tissue through its binding with an antibody introduced in the sample. Biomarkers and morphological abnormalities such as those resulting from cancers are diagnosed using this procedure. The antibody binding provided information about presence and location of the biological markers or abnormalities in the tissue.
The most common types of IHC testing kits have the primary antibodies directly conjugated on the fluorescent labels.
- Flow Cytometry
This scientific and biochemical procedure measures and characterizes cells in a fluid as the cells pass through lasers. The cells are fluorescently labelled using conjugated antibodies. Light of different wavelengths is emitted as the antibodies pass through the laser.
The three common aspects of the cells measured include the granularity, the relative fluorescence, and the size of the cells. Flow cytometry is an important procedure for cell counting, sorting, detection of biomarkers, and in protein engineering. Hematopoietic disorders, whether malignant or benign are determined using this procedure. Lymphomas and Leukemia are detected using flow cytometry.
- Western Blotting
Western blotting is also called protein blotting and it is an essential cell and molecular biology procedure. It detects proteins in complex protein mixtures. The procedure depends on three elements: gel electrophoresis for separation of the protein mixtures, transfer of the proteins to a solid support, and detection of the target protein. Visualization of the proteins is done on an X-ray film, imaging system, or a blotting membrane.
Western blots are carried out alongside other procedures like ELISA, or IHC in scientific and clinical tests.
In this procedure, the proteins of interest are captured when the antibodies are cross-linked to sepharose, agarose or magnetic beads. The results obtained are used for identification of post-translational modifications and characterization of the proteins.
In conclusion, immunohistochemistry, immunofluorescence, ELISA assays, flow cytometry, immunoprecipitation, and western blots are carried out in various scientific and clinical settings to determine presence or absence of a disease or a condition and in other cases, the severity of the condition.