Single Radial Hemolysis Assay

Ms. Neha Sharma

Introduction

  1. The Single Radial Hemolysis (SRH) Assay is a serological technique for detecting virus-specific antibodies, particularly against hemolytic viruses.
  2. It is widely used in virology, immunology, and vaccine research to evaluate immune responses.
  3. This assay is based on antigen-antibody interactions, where antibodies in the test serum bind to a viral antigen embedded in an agarose gel containing complement and red blood cells (RBCs).
  4. If antibodies are present, complement activation leads to hemolysis (lysis of RBCs), creating a clear zone around the sample well.
  5. The size of the hemolytic zone is proportional to the antibody concentration, allowing for both qualitative and quantitative assessment of the immune response.
  6. SRH is commonly used for Influenza, Measles, and Rubella serology and is a reliable method for evaluating vaccine efficacy.

Principle

The Single Radial Hemolysis (SRH) Assay is based on three key mechanisms:

  1. Antigen-Antibody Binding – The viral antigen is incorporated into an agar gel. If specific antibodies are present in the serum sample, they bind to the antigen.
  2. Complement Activation – The presence of complement proteins in the gel leads to antibody-mediated activation of the complement cascade, which causes RBC lysis.
  3. Hemolysis Formation – Lysis of RBCs results in forming a clear zone around the well, indicating the presence and quantity of virus-specific antibodies.

Interpretations

  • Larger Hemolysis Zone → Higher antibody concentration.
  • Smaller or No Hemolysis Zone → Low or absent antibodies.

This principle makes SRH a sensitive and reliable method for assessing immunity after infection or vaccination.

Procedure

A. Preparation of Hemolysis Agar Plate

  1. Prepare agar medium containing:
    • Buffered saline solution (PBS)
    • Red Blood Cells (RBCs) (e.g., sheep or human RBCs)
    • Viral antigen (e.g., Influenza, Measles, or Rubella antigens)
    • Complement proteins (e.g., guinea pig complement)
  2. Pour the mixture into Petri dishes and allow the agar to solidify.

B. Sample Application and Incubation

  1. Punch small wells into the agar (approximately 3 mm in diameter).
  2. Add test serum samples (10-20 µL per well) into the wells.
  3. In a humid chamber, incubate the plates at 37°C for 18-24 hours.

C. Result Interpretation

  1. After incubation, observe the hemolysis zones under light.
  2. Measure the diameter of the clear zones using a ruler or caliper.
  3. Compare results with a standard antibody concentration curve for quantification.

Advantages of Single Radial Hemolysis

1. Rapid and Simple

  • The assay provides quick results within 24 hours and does not require complex equipment.
  • It is easy to perform and interpret, making it suitable for large-scale screening.

2. Quantitative and Qualitative

  • The size of the hemolytic zone correlates with antibody concentration, allowing quantification of immune response.
  • It helps in monitoring vaccination efficacy and natural infection immunity.

3. High Sensitivity and Specificity

  • SRH is highly sensitive for detecting IgG antibodies against hemolytic viruses.
  • It is less prone to non-specific reactions than other serological tests like ELISA.

4. No Need for Live Virus Handling

  • Unlike viral culture methods, SRH does not require handling live infectious viruses, making it safer for laboratory personnel.

5. Useful for Vaccine and Immunity Studies

  • SRH is widely used to evaluate vaccine-induced immunity, especially for Influenza and Rubella vaccines.
  • It helps in determining seroprotection levels in populations.

Disadvantages of Single Radial Hemolysis

1. Requires Complement Proteins

  • The assay depends on functional complement proteins, which must be carefully sourced and stored to prevent degradation.

2. Limited to Hemolytic Viruses

  • Only useful for viruses that trigger hemolysis, such as Influenza, Measles, and Rubella.
  • It cannot detect non-hemolytic viruses (e.g., HIV, Hepatitis B).

3. Requires Standardization

  • The hemolysis zone measurement must be standardized, and variations in RBC concentration or antigen quality can affect results.
  • Requires comparison with a standard antibody reference curve.

4. Possible False Negatives

  • Low antibody titers may not generate detectable hemolysis, leading to false-negative results.
  • Poor complement activity or degraded antigen may reduce test sensitivity.

5. Need for Skilled Interpretation

  • Measuring hemolysis zones requires experience, and minor variations in incubation conditions may affect results.

Limitations of Single Radial Hemolysis

1. Not Suitable for All Viruses

  • The SRH assay is restricted to viruses that cause complement-mediated hemolysis.
  • For non-hemolytic viruses (e.g., Hepatitis B, Dengue, or SARS-CoV-2), ELISA or PCR-based tests are preferred.

2. Sensitivity Affected by Complement Source

  • Complement proteins degrade over time, and their effectiveness may vary depending on the source and storage conditions.

3. Does Not Differentiate Antibody Types

  • SRH primarily detects total antibodies (IgG, IgM, or IgA) but does not differentiate between recent infection (IgM) and past exposure (IgG).

4. Requires Fresh and Standardized RBCs

  • RBC stability is crucial for accurate results, and variations in RBC quality can affect hemolysis zones.
  • Freshly prepared standardized RBC suspensions are necessary for reproducibility.

Leave a Reply

Your email address will not be published. Required fields are marked *