HAT Antibody

What is the Haemagglutination Test (HAT) for SARS-CoV-2 antibody detection?

The Haemagglutination Test (HAT) is a receptor-binding domain-specific assay developed for rapid screening of neutralizing antibodies against SARS-CoV-2. It serves as a surrogate marker for neutralizing antibodies, which provide protection against infection. The test is designed to be simple, inexpensive, and rapidly adaptable to emerging variants of concern (VOC), making it suitable for large-scale evaluation of potentially decreasing vaccine effectiveness .

HAT provides a visual readout that can be observed with the naked eye, eliminating the need for specialized equipment. The test has shown a specificity of >99% for detection of convalescent antibodies after PCR-confirmed infection .

What is the scientific principle behind HAT?

HAT operates on the principle of haemagglutination, which is the clumping together of red blood cells to form visible aggregates. In the HAT assay, haemagglutination is triggered by mixing a sample of the patient's blood with a synthetic protein reagent called IH4-RBD that coats the red blood cells with the receptor binding domain (RBD) of the virus spike protein, essentially "disguising" the cells as the virus .

If the patient's blood contains antibodies against the RBD, these antibodies will bind to the RBD domain presented by the red cells and cause them to clump, forming a visible red "button" of aggregated cells in the conical wells of the testing plate. In the absence of RBD-specific antibodies, the red cells will not agglutinate and will form a characteristic "teardrop" pattern when the wells are tilted .

What is the IH4-RBD reagent and how is it produced?

The IH4-RBD is the key reagent in the HAT assay, consisting of the receptor binding domain (RBD) of the SARS-CoV-2 spike protein linked to a monomeric anti-erythrocyte single domain nanobody (IH4). This fusion protein is designed to bind to red blood cells while presenting the RBD for antibody recognition .

For production, codon-optimized IH4-RBD sequences are expressed in Expi293F cells and purified using Ni-NTA chromatography via their C-terminal 6xHis tag. For variant testing, the IH4-RBD sequences are modified to include the specific amino acid changes found in various VOCs, such as:

• Alpha (B.1.1.7): N501Y

• Beta (B.1.351): K417N, E484K, N501Y

• Gamma (P.1): K417T, E484K, N501Y

• Delta (B.1.617.2): L452R, T478K

How is the standard HAT procedure performed?

The standard HAT procedure involves the following steps:

• Preparation of reagents in a final volume of 100 μl per well

• Addition of approximately 0.3 μl of packed RBCs per well (from 1 μl of 30% stock stored in Alsever's solution)

• Addition of IH4-RBD at a final concentration of 1 μg/ml (100 ng/well)

• Incubation of plates for 60 minutes at room temperature

• Visual assessment of agglutination patterns

For quantitative assessment, serial dilutions of serum or plasma can be performed before mixing with washed autologous RBCs or O-donor RBCs. The titration endpoint provides a quantitative measure of antibody levels .

What adaptations have been made to create the field-friendly HAT-field protocol?

The HAT-field protocol is an optimization of the original HAT designed specifically for use in resource-limited settings. Key improvements include:

• Quantification through titration of the IH4-RBD reagent rather than serum dilutions

• Use of PBN buffer (PBS containing 1% BSA and sodium azide), which:

  • Prevents nonspecific adsorption of IH4-RBD to plastic

  • Improves reagent stability across a range of temperatures

  • Eliminates the need for refrigeration

  • Enhances sensitivity by improving RBC settling

The HAT-field protocol requires minimal equipment: one lancet, one plastic Pasteur pipet, one plastic tube containing 300 μl of PBS-2 mM EDTA, 10 μl of whole blood, and one column of eight conical wells preloaded with 60 μl/well of a range of IH4-RBD concentrations .

How can sensitivity be improved in HAT assays?

Several methods can be employed to enhance HAT sensitivity:

• Prolonged incubation: Increasing incubation time from 60 minutes to 5 hours significantly improves sensitivity, with titration endpoints increasing by 2-3 dilution points compared to the standard 60-minute protocol .

• Centrifugation: A brief centrifugation step (100g for 1 minute) increases sensitivity to levels equivalent or superior to those achieved with 5-hour incubations. This centrifugation can be performed just 15 minutes after distributing diluted blood in the plate .

• PBN buffer: Using PBS containing 1% BSA and azide results in slightly increased sensitivity, likely due to improved settling of RBCs at the bottom of the wells .

• Increased reagent concentration: Using more IH4-RBD reagent can enhance sensitivity, though with potential trade-offs in specificity .

How does HAT compare with other serological testing methods?

HAT has been extensively compared with other antibody detection methods, with the following key observations:

While HAT is not as sensitive as ELISA, CLIA, or FACS, it correlates well with neutralizing antibody assays and offers significant advantages in terms of accessibility, cost, and simplicity .

How can HAT be adapted to detect antibodies against SARS-CoV-2 variants of concern?

One of the key advantages of HAT is its adaptability to emerging variants. This is achieved by modifying the IH4-RBD reagent to incorporate the specific mutations present in variant RBDs:

• Identify the key amino acid changes in the RBD of the variant of interest

• Design codon-optimized IH4-RBD sequences containing these modifications

• Express and purify the variant-specific IH4-RBD proteins

• Validate the new reagents against control sera

This process allows for rapid development of variant-specific HAT assays, enabling researchers to evaluate potential changes in antibody recognition and vaccine effectiveness against new variants .

What is the correlation between HAT titers and neutralizing antibody levels?

Studies have demonstrated that HAT titers correlate well with neutralizing antibody levels as measured by pseudotype, microneutralization, and virus neutralization assays. This correlation has been validated in multiple cohorts, including:

• Convalescent sera from naturally infected individuals

• Sera from mRNA vaccine recipients (BNT162b2)

• Populations of different age groups (1-89 years for convalescent, 23-77 and 80-99 years for vaccinees)

This correlation establishes HAT as a valuable surrogate marker for neutralizing antibodies, potentially allowing for the establishment of a correlate of protection (COP) similar to the haemagglutination inhibition (HAI) titer of 40 for 50% protection from influenza infection .

What are the key limitations of HAT and how can they be addressed?

Despite its utility, HAT faces several limitations that researchers should consider:

• Lower sensitivity: HAT is less sensitive than ELISA, CLIA, or FACS. This can be addressed through:

  • Prolonged incubations (5 hours)

  • Brief centrifugation (100g for 1 minute)

  • Using PBN buffer

  • Increasing IH4-RBD concentration

• Time constraints: Long incubations are impractical for field settings. The HAT-field protocol with centrifugation can reduce total assay time to under 30 minutes .

• Equipment needs: While minimal, some procedures like centrifugation require basic equipment. In field settings, this can be addressed using adapted "salad-spinners" as low-tech centrifuges .

• Background seroprevalence: The increasing seroprevalence due to vaccination makes finding truly negative controls challenging. Using pre-pandemic samples or carefully screened donors can address this issue .

What methodological considerations should researchers take into account when interpreting HAT results?

Researchers should consider several factors when interpreting HAT results:

• Incubation time effects: Longer incubations increase sensitivity but may decrease specificity, potentially resulting in false positives .

• Buffer composition: The choice of buffer affects both sensitivity and reagent stability. PBN (PBS with 1% BSA and azide) is recommended for optimal performance .

• Quantification approach: Different quantification methods (serum dilution vs. reagent titration) may yield slightly different results. Researchers should maintain consistency within studies .

• Sample type considerations: Whole blood from finger pricks can be used directly in HAT-field, while standard HAT typically uses washed RBCs from venous blood. The sample source should be considered when comparing results .

• Reading time: Results should be read at consistent time points, as agglutination patterns can evolve over time .

How can HAT be employed in large-scale seroprevalence studies?

HAT and HAT-field offer several advantages for large-scale seroprevalence studies:

• Cost-effectiveness: The minimal reagent requirements and simple equipment needs make HAT highly cost-effective for population studies .

• Field applicability: The HAT-field protocol can be performed in remote locations without sophisticated laboratory infrastructure .

• Rapid results: With the optimized protocol, results can be obtained in under 30 minutes, facilitating high-throughput screening .

• Variant screening: The adaptability of HAT allows for assessment of population immunity against multiple variants simultaneously .

HAT has already been successfully deployed in seroprevalence studies, such as measuring seropositivity rates in Sri Lanka, where it compared favorably with sensitive ELISA methods .

How can HAT contribute to monitoring vaccine effectiveness against emerging variants?

HAT provides a valuable tool for monitoring vaccine effectiveness against emerging variants through:

• Rapid adaptation: New variant-specific HAT assays can be developed quickly following the emergence of new variants .

• Comparative analysis: By testing the same samples against different variant HATs, researchers can quantify potential reductions in antibody recognition .

• Population screening: Large-scale testing can identify demographic groups with potentially reduced protection against new variants .

• Longitudinal monitoring: Tracking antibody levels over time using quantitative HAT can help determine the durability of vaccine-induced immunity and identify optimal timing for booster doses .

This application is particularly valuable given the ongoing evolution of SARS-CoV-2 and the need to continually assess vaccine effectiveness against new variants .