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

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

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

  • Patient samples and standards were added and incubated.

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

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

  • After incubation, the stop solution was added.

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

  • 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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