Entamoeba histolytica Antigen ELISA test


1. Objective

To detect specific antigens of Entamoeba histolytica in stool samples using the Enzyme-Linked Immunosorbent Assay (ELISA) technique, helping to diagnose amebiasis.

2. Principle

The test is based on the antigen-antibody interaction.
Specific monoclonal antibodies against E. histolytica are coated on a microtiter plate. When a stool sample containing E. histolytica antigen is added, it binds to these antibodies. A secondary enzyme-linked antibody then binds to the complex. After adding a chromogenic substrate, a color change occurs if the antigen is present. The intensity of the color (measured spectrophotometrically) corresponds to the antigen concentration.

3. Materials

  • Stool sample (fresh or preserved)

  • Entamoeba histolytica Antigen ELISA kit:

    • Microtiter plate pre-coated with anti-E. histolytica antibodies

    • Sample diluent

    • Wash buffer

    • Enzyme-conjugated detection antibody

    • Substrate solution (e.g., TMB)

    • Stop solution

    • Positive and negative controls

  • Micropipettes and sterile tips

  • Microplate washer (manual or automatic)

  • ELISA plate reader (typically at 450 nm)

  • Waste disposal container

  • Personal protective equipment (gloves, coat, etc.)

4. Procedure

  • Prepare samples: Dilute stool sample in sample diluent as per kit instructions.

  • Add samples and controls to designated wells.

  • Incubate the plate (usually 30–60 minutes at room temperature).

  • Wash the wells to remove unbound material.

  • Add enzyme-conjugated antibody to each well.

  • Incubate again and wash thoroughly.

  • Add substrate solution (e.g., TMB) and incubate for color development.

  • Add stop solution to halt the reaction.

  • Read absorbance in an ELISA plate reader at 450 nm.

5. Result

  • Positive: High absorbance reading; presence of E. histolytica antigen.

  • Negative: Low or baseline absorbance; antigen not detected.

  • Cut-off value: Provided in the kit to differentiate positive and negative samples.

6. Uses

  • To confirm invasive intestinal amebiasis.

  • Differentiates E. histolytica from nonpathogenic species like E. dispar and E. moshkovskii (which are morphologically identical).

  • Ideal for epidemiological surveys and clinical diagnostics in both symptomatic and asymptomatic individuals.

7. Conclusion

The Entamoeba histolytica Antigen ELISA is a sensitive, specific, and reliable method for detecting intestinal amebiasis. It surpasses traditional microscopy in accuracy, especially in distinguishing pathogenic E. histolytica from nonpathogenic species.

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