Reverse Passive Hemagglutination Assay

Ms. Neha Sharma

Introduction

  1. Reverse Passive Hemagglutination (RPHA) is a serological assay used to detect and quantify antigens in clinical and research settings.
  2. It is an adaptation of the passive hemagglutination (PHA) test, where instead of detecting antibodies, RPHA detects antigens using antibody-coated red blood cells (RBCs).
  3. This test is widely used for detecting viral, bacterial, and fungal antigens, including Hepatitis B surface antigen (HBsAg), Rotavirus, and Toxoplasma antigens.
  4. It is a sensitive, rapid, and cost-effective method for antigen detection and is commonly employed in diagnostic laboratories.

Principle

The RPHA assay relies on the ability of antibody-coated RBCs to agglutinate in the presence of specific antigens. This agglutination leads to a visible network of clumped RBCs, which indicates a positive result.

Steps in the Principle

  1. Sensitization of RBCs → Red blood cells (RBCs) are coated with specific antibodies against the target antigen.
  2. Antigen-Antibody Interaction → When a sample containing the target antigen is mixed with the coated RBCs, the antigen binds to the antibodies.
  3. Hemagglutination → This binding cross-links multiple RBCs, leading to a visible agglutination pattern in the test well.

The extent of hemagglutination correlates with the antigen concentration, allowing for qualitative and quantitative assessment. A lack of agglutination indicates a negative result (without antigen).

Procedure

A. Preparation of Sensitized RBCs

  1. Collect RBCs from a suitable source (e.g., sheep, human, or chicken blood).
  2. Wash RBCs thoroughly with phosphate-buffered saline (PBS) to remove plasma and unwanted proteins.
  3. Coat RBCs with specific antibodies against the target antigen using chemical linkers like glutaraldehyde or tannic acid.
  4. Store coated RBCs in a stabilizing solution at 4°C until use.

B. Test Setup

  1. In microtiter wells, prepare a serial dilution of the test sample (e.g., serum, urine, or cerebrospinal fluid).
  2. Add an equal volume of sensitized RBCs to each well.
  3. Incubate the plate at room temperature (usually 30–60 minutes).

C. Observation and Interpretation

  1. Check for agglutination:
    • Positive Result → RBCs form a diffuse mat across the well.
    • Negative Result → RBCs settle at the bottom, forming a tight button.
  2. Determine antigen concentration using the highest dilution showing visible agglutination.

Advantages

1. High Sensitivity

  • Can detect very low concentrations of antigens in biological samples.
  • More sensitive than traditional latex agglutination tests.

2. Rapid and Easy to Perform

  • Provides results within 1–2 hours.
  • Requires minimal equipment, making it suitable for point-of-care testing.

3. Cost-Effective

  • Uses simple reagents and does not require expensive instruments.
  • More economical than ELISA or PCR-based antigen detection.

4. Can Be Quantitative

  • By using serial dilutions, the antigen concentration can be estimated.
  • Useful for monitoring infection progression or treatment response.

5. Versatile

  • Can detect a wide range of bacterial, viral, and fungal antigens (e.g., HBsAg, Rotavirus, Toxoplasma).
  • Suitable for clinical diagnostics, food safety testing, and environmental monitoring.

Disadvantages 

1. False Positives Possible

  • Non-specific agglutination may occur due to cross-reactivity or contaminants in the sample.
  • Autoantibodies or rheumatoid factors may interfere with results.

2. Requires Standardization

  • Sensitivity depends on proper RBC coating and antibody quality.
  • Inconsistent coating may lead to variable results.

3. Short Shelf Life of Sensitized RBCs

  • Coated RBCs deteriorate over time, requiring frequent preparation and quality control.
  • Proper storage at 4°C is necessary to maintain stability.

4. Not Suitable for All Antigens

  • Some antigens may not bind effectively to RBC-bound antibodies.
  • Alternative tests (e.g., ELISA, PCR) may be more suitable for certain pathogens.

Limitations

1. Cannot Differentiate Antigen Subtypes

  • RPHA detects the presence of an antigen but does not differentiate antigenic variations or strains.
  • Further confirmatory tests (e.g., ELISA, Western blot, PCR) may be required.

2. Sample Interference

  • Hemolysis, lipemia, or contamination in biological samples may interfere with test results.
  • Pre-treatment of samples may be necessary for accuracy.

3. Requires Fresh and Standardized RBCs

  • RBCs must be properly maintained to prevent spontaneous agglutination or degradation.
  • Variations in RBC sources may lead to inconsistent sensitivity.

4. Limited Dynamic Range

  • While semi-quantitative, RPHA may not be as precise as ELISA or PCR for determining antigen load.