CoV-2 S1 (16-685), Biotin

What is SARS-CoV-2 S1 (16-685), Biotin and how is it produced?

SARS-CoV-2 S1 (16-685), Biotin is a recombinant protein comprising the S1 subunit of the SARS-CoV-2 Spike protein, specifically encompassing amino acids 16-685. This protein is enzymatically biotinylated using Avi-Tag™ technology. The production process typically involves expressing the protein with a C-terminal tag (either an Fc fusion or His-tag) followed by an Avi-Tag™, which allows for site-specific biotinylation using biotin ligase (BirA) . The protein is then affinity purified to ensure high purity before use in experimental applications. This specific region contains the receptor binding domain (RBD) that interacts with human angiotensin-converting enzyme 2 (hACE2), making it particularly valuable for studying virus-host interactions and antibody neutralization.

What are the structural differences between Fc Fusion and His-Tag versions of S1 (16-685), Biotin?

The key structural difference lies in the C-terminal fusion partner:

The Fc fusion version offers advantages for certain detection methods and potentially enhanced stability due to dimerization through the Fc region, while the His-tagged version may be preferred for applications where the Fc region might interfere with experimental outcomes .

How does biotinylation affect the functionality of the S1 protein in research applications?

Biotinylation of the S1 (16-685) protein significantly enhances its utility in multiple research applications without compromising its functional integrity. Enzymatic biotinylation via the Avi-Tag™ ensures site-specific modification, minimizing potential disruption to protein folding and binding sites. This modification enables strong, non-covalent binding to streptavidin (Kd ≈ 10^-15 M), allowing for:

• Improved immobilization on streptavidin-coated surfaces for binding assays

• Enhanced detection sensitivity through streptavidin-conjugated reporter systems

• Oriented presentation of the protein, ensuring accessibility of functional domains

In surrogate virus neutralization tests (sVNTs), biotinylated S1 proteins coupled with HRP-conjugated streptavidin demonstrate improved performance compared to directly HRP-conjugated RBD proteins, offering enhanced sensitivity and specificity for detecting neutralizing antibodies . The biotinylation efficiency is typically verified to be ≥90%, ensuring consistent performance across experimental applications .

How can S1 (16-685), Biotin be utilized in surrogate virus neutralization tests?

S1 (16-685), Biotin serves as a key component in developing biotin-based surrogate virus neutralization tests (sVNTs), which offer significant advantages over traditional virus neutralization assays:

• Methodology: Biotinylated S1 proteins are immobilized on streptavidin-coated plates or beads. Serum samples containing potential neutralizing antibodies are pre-incubated with the biotinylated S1, followed by addition of hACE2. The degree of inhibition of S1-hACE2 binding indicates neutralizing activity .

• Detection System: The biotin-based sVNT utilizes biotinylated RBD and HRP-conjugated streptavidin instead of directly HRP-conjugated RBD, enabling enhanced signal amplification and improved sensitivity .

• Performance Metrics: Recent studies demonstrate that biotin-based sVNTs strongly correlate with both FDA-approved commercial kits (R² = 0.8521) and pseudovirus neutralization tests (R² = 0.9006) .

• Variant Testing: The system has been successfully applied to test neutralizing antibodies against multiple SARS-CoV-2 variants, including Omicron BA.2, providing valuable data on vaccine efficacy and antibody persistence .

This approach offers significant advantages for high-throughput screening of neutralizing antibodies in BSL-2 facilities, without requiring live virus manipulation.

What temporal patterns of neutralizing antibody responses can be detected using S1 (16-685), Biotin-based assays?

Biotin-based sVNTs utilizing S1 (16-685) have revealed important temporal patterns in neutralizing antibody responses following vaccination:

These temporal patterns highlight the waning of neutralizing antibodies over time and differential responses to various vaccine platforms, with mRNA vaccines (BNT162b2) potentially offering longer-lasting protection than adenoviral vector vaccines (AZD1222) . This information is crucial for determining optimal timing for booster vaccinations and assessing population-level immunity.

How do neutralizing antibodies interact with S1 (16-685) compared to other viral proteins in immune responses?

Research comparing antibody responses to different SARS-CoV-2 proteins reveals distinct patterns that impact our understanding of immune protection:

• Differential Antibody Production: Serological analyses demonstrate that serum antibody titers to the nucleocapsid (N) protein are substantially higher than those to the S protein in most COVID-19 patients. For example, patients ZD004 and ZD006 showed minimal antibody responses to the S protein while exhibiting much higher titers to the N protein .

• Mutation Patterns: Antibodies directed against the S1 region show minimal somatic hypermutation (SHM) from germline sequences (most had 0-1 mutations), indicating a primary antibody response. In contrast, N protein-reactive antibodies display relatively high mutation frequencies (mean of 5.7%), suggesting stronger antigen stimulation and potentially more mature immune responses .

• Functional Differences: While S1-targeting antibodies (particularly those binding the RBD) typically exhibit neutralizing activity, N protein antibodies may serve other roles, including curbing complement hyperactivation induced by the N protein, which contributes to inflammatory lung injury .

• Temporal Aspects: Despite shorter hospitalization periods (e.g., 9 days for patient ZD006), convalescent patients can develop mature antibody responses to the N protein while maintaining germline-like responses to the S protein, suggesting differential kinetics of immune response development .

These findings highlight the complex interplay between different viral antigens in stimulating protective immunity and potential therapeutic interventions beyond direct viral neutralization.

What are the optimal experimental conditions for using S1 (16-685), Biotin in binding and neutralization assays?

Optimizing experimental conditions is crucial for reliable results when working with S1 (16-685), Biotin:

When developing assays for variant comparison, researchers should maintain identical conditions across all variant proteins to ensure valid comparisons of binding or neutralization potency. Additionally, including appropriate controls (positive sera with known neutralizing activity, negative sera, and buffer-only controls) is essential for valid interpretation of results .

How can researchers develop biotin-based surrogate virus neutralization tests with improved sensitivity and specificity?

Developing high-performance biotin-based sVNTs requires careful consideration of several key factors:

• Optimized Signal Amplification: Utilizing biotinylated S1 proteins with HRP-conjugated streptavidin creates an improved detection system compared to directly HRP-conjugated RBD, enhancing sensitivity through signal amplification. The biotin-streptavidin system allows for controlled stoichiometry and optimized signal-to-noise ratios .

• Substrate Selection: For enhanced detection sensitivity in C2 internal quenched fluorescent peptide-based analysis, researchers can use fluorescence substrate (2Abz-SLGRKIQI-Lys(Dnp)-NH₂) to monitor complement activation. This approach enables precise measurement of initial reaction rates (V₀) and determination of steady-state reaction constants (Vmax and Km) .

• Sample Preparation Protocol:

  • Collect serum samples with known C3 serologic values

  • Standardize pre-incubation times between sera and S1 proteins

  • Implement consistent washing procedures to reduce background

  • Utilize calibrated positive controls to normalize results across experiments

• Validation Metrics: New assays should be validated against:

  • FDA-approved commercial kits (e.g., cPass sVNT)

  • Pseudovirus neutralization tests (pVNT)

  • When possible, plaque reduction neutralization tests (PRNT) as the gold standard

• Variant Adaptation: For testing multiple variants, researchers should express and biotinylate S1 proteins representing key variants of concern, maintaining identical production and quality control processes to ensure comparable results .

Following these methodological considerations can yield highly sensitive and specific assays that correlate strongly with established neutralization tests (R² values >0.85) while offering advantages in throughput and biosafety.

How can S1 (16-685), Biotin be used in B cell immunoprofiling studies?

S1 (16-685), Biotin serves as an effective tool for isolating and characterizing SARS-CoV-2-specific B cells, providing insights into humoral immune responses:

• Antigen-Specific B Cell Sorting Protocol:

  • Label S1 (16-685), Biotin with different fluorophores (e.g., PE-Cy7 and BV421) through streptavidin conjugation

  • Use dual-color labeling to minimize false positives in flow cytometry

  • Sort single CD19⁺/CD27⁺ memory B cells or CD19⁺/CD20ˡᵒʷ⁻ⁿᵉᵍ/CD27ʰⁱ/CD38ʰⁱ plasma cells showing elevated fluorescence for both fluorophore-labeled S1

  • Perform single-cell RT-PCR to amplify immunoglobulin variable regions

• Analytical Parameters for B Cell Response Characterization:

  • VH gene usage and mutation frequency from germline sequences

  • CDR3 length analysis (similar average length observed between S- and N-reactive antibodies)

  • Isotype distribution and class-switching patterns

  • Binding affinity assessment of recombinantly expressed monoclonal antibodies

From previous studies, S1-reactive antibodies typically display germline-like sequences with minimal mutations (0-1/300), suggesting a primary antibody response. This contrasts with antibodies targeting other viral proteins like nucleocapsid, which show higher mutation frequencies (mean 5.7%) more consistent with secondary immune responses .

This detailed immunoprofiling approach provides valuable insights into the development of protective immunity and can guide vaccine design by identifying optimal epitopes and understanding antibody maturation processes.

How can S1 (16-685), Biotin contribute to monitoring vaccine efficacy against emerging variants?

S1 (16-685), Biotin serves as a powerful tool for longitudinal monitoring of vaccine efficacy against emerging SARS-CoV-2 variants:

• Comparative Variant Analysis: By developing parallel biotin-based sVNTs using S1 proteins from multiple variants, researchers can directly compare neutralizing antibody potency across variants in vaccinated populations. Recent studies demonstrated that neutralizing antibodies against SARS-CoV-2 variants in second vaccination sera decreased after a median of 141.5 days, highlighting the importance of temporal monitoring .

• Vaccine Platform Comparison: Research utilizing S1 (16-685), Biotin has revealed that mRNA vaccines (BNT162b2) maintain neutralizing antibodies for longer periods compared to adenoviral vector vaccines (AZD1222). This information is crucial for optimizing vaccination strategies and timing booster doses .

• Breakthrough Infection Risk Assessment: Quantitative measurements of neutralizing antibody titers using S1-based assays help establish correlates of protection and identify population subgroups that may require additional boosters due to waning immunity.

• Standardized Cross-Study Comparisons: The strong correlation between biotin-based sVNTs and established neutralization assays (R² > 0.85) enables standardized comparisons across different studies and laboratories, facilitating global surveillance of vaccine efficacy .

This approach offers significant advantages over conventional plaque reduction neutralization tests (PRNT) in terms of throughput, biosafety requirements, and ability to simultaneously test multiple variants, making it ideal for large-scale vaccine efficacy monitoring programs.

What role can S1 (16-685), Biotin play in understanding complement hyperactivation in COVID-19?

S1 (16-685), Biotin can serve as a valuable control in studies examining the role of SARS-CoV-2 proteins in complement activation:

• Differential Complement Activation: While the nucleocapsid (N) protein has been identified as a potent activator of complement through binding to mannan-binding lectin (MBL)-associated serine protease 2 (MASP-2), the S1 protein's role in complement activation requires further investigation. Using biotinylated S1 in parallel with N protein allows researchers to distinguish protein-specific effects on complement pathways .

• Enzymatic Analysis Methodology: Using the C2 internal quenched fluorescent peptide-based analysis approach, researchers can assess complement activation levels in the presence of different viral proteins. The system measures fluorescence signal from cleaved C2 synthetic peptide substrates in reaction mixtures containing complement and viral proteins with or without neutralizing antibodies .

• Therapeutic Antibody Screening: S1 (16-685), Biotin can be used to identify antibodies that specifically target S1 without affecting complement pathways, distinguishing them from antibodies that might curb complement hyperactivation induced by other viral proteins.

By employing these approaches, researchers can develop a more comprehensive understanding of the molecular mechanisms underlying COVID-19 pathogenesis, potentially leading to targeted therapeutic strategies that address both viral neutralization and immunopathology.

How can researchers use S1 (16-685), Biotin to study the dynamics of antibody responses following vaccination and infection?

S1 (16-685), Biotin enables detailed analysis of antibody response dynamics through several methodological approaches:

• Longitudinal Serum Profiling: Serial sampling from vaccinated or infected individuals allows tracking of:

  • Neutralizing antibody titers over time

  • Antibody affinity maturation

  • Epitope specificity shifts

  • Cross-variant neutralization breadth

• B Cell Repertoire Analysis: Combining S1 (16-685), Biotin with B cell sorting and sequencing techniques reveals:

  • Clonal expansion patterns

  • Somatic hypermutation trajectories

  • Memory B cell persistence

  • Germinal center reaction duration

• Competitive Binding Assays: S1 (16-685), Biotin can be used in competition assays to map epitope specificities and determine if vaccination and infection induce antibodies targeting different regions of the S1 domain.

• Affinity Maturation Assessment: By measuring binding kinetics at multiple timepoints, researchers can track the evolution of antibody affinity, which correlates with neutralization potency and durability of protection.

Recent studies demonstrate significant differences in antibody persistence between vaccine platforms, with BNT162b2 recipients maintaining higher neutralizing antibody levels than AZD1222 recipients after a median of 141.5 days. This approach is particularly valuable for determining optimal booster timing and composition for different populations .