ELISA (Enzyme-Linked Immunosorbent Assay) in Hematological Disorders

 


1. Objective

The objective of the experiment was to detect and quantify disease-specific antigens or antibodies in hematological disorders using the ELISA technique.

2. Principle

The ELISA technique was based on antigen-antibody interaction. Either the antigen or antibody was immobilized on a solid surface (usually a microtiter plate), and a corresponding enzyme-labeled antibody was used for detection. Upon addition of a chromogenic substrate, a color change occurred, which was measured spectrophotometrically and correlated with the concentration of the target molecule.

3. Materials Used

  • Microtiter ELISA plate

  • Patient serum or plasma samples

  • Known standards and controls

  • Enzyme-conjugated antibodies (e.g., anti-FVIII for Hemophilia A)

  • Substrate (e.g., TMB – Tetramethylbenzidine)

  • Stop solution (e.g., sulfuric acid)

  • Plate washer and reader

  • Pipettes and sterile tips

4. Procedure

  1. The microtiter plate was coated with capture antibody and incubated.

  2. Wells were blocked with blocking buffer to prevent non-specific binding.

  3. Patient samples and standards were added and incubated.

  4. After washing, enzyme-linked detection antibody was added.

  5. Wells were washed again, and substrate was added to initiate color change.

  6. After incubation, the stop solution was added.

  7. Optical density (OD) was measured using an ELISA reader at appropriate wavelength (e.g., 450 nm).

  8. Concentrations were determined by comparing sample OD values with the standard curve.

5. Result Interpretation

  • A high OD value indicated elevated levels of the target antibody/antigen.

  • In hematological disorders, this could indicate the presence of:

    • Anti-FVIII antibodies in Hemophilia A

    • Anti-platelet antibodies in Immune Thrombocytopenia (ITP)

    • Cytokines (e.g., IL-6, TNF-α) in leukemias/lymphomas

  • The standard curve allowed for quantitative analysis.

6. Applications in Hematology

  • Diagnosis of Hemophilia inhibitors

  • Detection of antibodies in autoimmune hematological diseases

  • Monitoring cytokine profiles in blood cancers

  • HIV antigen/antibody testing in blood donors

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