SARS Nucleocapsid PT3851

What is SARS Nucleocapsid PT3851 and what is its primary application?

PT3851 is a mouse monoclonal antibody that targets the nucleocapsid protein of SARS-CoV-2. It is specifically designed as a biotin-conjugated antibody primarily used in ELISA applications. The antibody is generated against a recombinant protein fragment corresponding to amino acids 1-49 of the SARS nucleoprotein . This antibody serves as a valuable research tool for detecting SARS-CoV-2 nucleocapsid protein in experimental samples, particularly in studies involving viral detection and immune response analysis. The high specificity of this IgG2b isotype antibody makes it suitable for sensitive detection methods in coronavirus research applications .

What is the structure of the SARS-CoV-2 nucleocapsid protein that PT3851 targets?

The SARS-CoV-2 nucleocapsid protein consists of two major domains: the N-terminal domain (N-NTD) and C-terminal domain (N-CTD), which have been crystallized at 1.8 Å and 1.5 Å resolution, respectively . The N-NTD, which contains the epitope targeted by PT3851 (amino acids 1-49), plays a crucial role in RNA binding. The N-CTD forms a tight homodimer with a rectangular slab shape, with each protomer exhibiting a C-shaped structure composed of five α-helices, two β-strands, and two 3₁₀ helices . The β-hairpin from one protomer inserts into the cavity of the other, creating a four-stranded antiparallel β-sheet at the dimer interface. This dimerization buries a substantial surface area of 2,590 Ų and is stabilized by extensive hydrogen bond interactions and hydrophobic interactions .

How sensitive and specific is nucleocapsid antibody detection compared to spike protein detection?

Research demonstrates that antibodies against the SARS-CoV-2 nucleocapsid protein show superior sensitivity compared to spike protein antibodies in serological diagnostics. Specifically, 15 or more days after symptom onset, anti-nucleocapsid antibody tests achieved 100% sensitivity and 100% specificity, while anti-spike antibody detection showed 91% sensitivity and 100% specificity . This makes nucleocapsid-based antibody detection a more reliable marker for confirming previous SARS-CoV-2 infection, particularly in diagnostic and epidemiological studies. The higher sensitivity of nucleocapsid antibody detection is consistent with findings from previous SARS-CoV-1 research, where anti-nucleocapsid antibodies were also found to be the most sensitive target for serological diagnosis .

What are the recommended storage conditions for PT3851 antibody?

The PT3851 antibody should be shipped at 4°C. For short-term storage (up to 2 weeks), the antibody can be kept at 4°C. For long-term storage, it is recommended to store the antibody at -20°C . The antibody is supplied in Dulbecco's Phosphate Buffered Saline with 1% BSA and 0.1% Proclin 950 at a concentration of 1 mg/ml . Proper storage conditions are essential to maintain the antibody's functionality and specificity, ensuring reliable experimental results in research applications.

How is PT3851 antibody purified and what is its formulation?

The PT3851 antibody is purified using Protein A chromatography, resulting in a purity of >90% as determined by SDS-PAGE analysis . The purified antibody is provided in liquid form at a concentration of 1 mg/ml. The formulation consists of Dulbecco's Phosphate Buffered Saline supplemented with 1% BSA (bovine serum albumin) and 0.1% Proclin 950 as a preservative . This formulation helps maintain antibody stability and prevents microbial growth during storage, ensuring consistent performance in experimental applications.

What structural characteristics make the nucleocapsid protein a suitable target for antibody detection and drug development?

The nucleocapsid protein contains several conserved structural features that make it an excellent target for both antibody detection and potential drug development. Crystal structure analysis reveals that the SARS-CoV-2 N-NTD contains a conserved positively charged groove that serves as an RNA-binding site . Additionally, the N-CTD forms stable dimers with distinctive electrostatic potential surfaces that differ between coronavirus species. For instance, MERS-CoV N-CTD displays a positively charged central region, while SARS-CoV-2 and SARS-CoV structures show a negatively charged region .

The nucleocapsid protein contains binding sites for small molecules that could potentially inhibit viral infection by blocking RNA-binding activity or disrupting normal oligomerization. One such binding site involves key residues (S51, F53, Y109, Y111, and R149 in SARS-CoV-2 N-NTD) that are conserved across coronavirus species, making it a promising drug target . The compound PJ34, which mimics AMP binding to N-NTD, has been shown to fit within the ribonucleotide-binding pocket of the nucleocapsid protein in HCoV-OC43, and this binding site is conserved in SARS-CoV-2 .

How does the timing of nucleocapsid antibody development compare between different patient populations?

The timing of nucleocapsid antibody development also appears to be slightly earlier than or concurrent with spike protein antibody development. This timing information is crucial for understanding the dynamics of the immune response to SARS-CoV-2 infection and for optimizing the timing of serological testing in clinical settings .

How do structural differences in nucleocapsid proteins among coronavirus species affect antibody cross-reactivity?

These structural differences may influence the specificity of antibodies targeting the nucleocapsid protein and should be considered when evaluating potential cross-reactivity of PT3851 with other coronavirus species. Understanding these structural variations is crucial for developing specific diagnostic tests and for interpreting serological data in regions where multiple coronavirus species may circulate.

What impact does heat inactivation have on nucleocapsid antibody detection?

Research demonstrates that heat inactivation of samples does not significantly reduce either antibody levels or the rate of seropositivity when detecting nucleocapsid antibodies . This finding has important practical implications for laboratory safety protocols when handling potentially infectious samples from COVID-19 patients. Heat inactivation can be employed as a safety measure to reduce the risk of live virus in blood samples without compromising the sensitivity of nucleocapsid antibody detection assays .

The stability of nucleocapsid antibodies under heat inactivation conditions contributes to the robustness of serological assays using PT3851, making these tests reliable even when samples require pre-treatment for safety reasons.

How can PT3851 be optimized for use in multiplex serological assays?

PT3851, being a biotin-conjugated antibody, is particularly well-suited for multiplex serological assays due to the versatility of biotin-streptavidin coupling systems. For multiplex optimization, researchers should consider the following methodological approaches:

• Determine optimal antibody concentration through titration experiments to establish the minimum concentration that yields maximum signal-to-noise ratio.

• Evaluate potential cross-reactivity with other coronavirus nucleocapsid proteins to ensure specificity, particularly important in multiplex settings where multiple targets are being detected simultaneously.

• Establish appropriate blocking conditions to minimize non-specific binding, which becomes more critical in multiplex formats where multiple reagents increase background potential.

• Validate the performance in the presence of other detection antibodies to identify any interference effects that might occur in the multiplex environment.

• Implement appropriate positive and negative controls, including the Mouse IgG2b [MPC-11] (Biotin) (A86393) isotype control, to accurately interpret results .

What structural data table parameters are important when analyzing nucleocapsid protein crystal structures?

The following crystallographic parameters are critical when analyzing nucleocapsid protein structures:

These parameters, derived from the crystal structures of SARS-CoV-2 nucleocapsid protein domains, provide the foundation for structural analyses that inform both antibody development and drug design efforts . The high resolution (1.8 Å for N-NTD and 1.39 Å for N-CTD) allows for detailed visualization of potential binding sites and structural motifs that can be targeted by therapeutic interventions or diagnostic reagents like PT3851.

What are the recommended protocols for using PT3851 in ELISA for nucleocapsid protein detection?

For optimal ELISA performance using PT3851, researchers should follow these methodological guidelines:

• Coating: Apply recombinant nucleocapsid protein or patient samples to high-binding ELISA plates at 1-10 μg/ml in carbonate buffer (pH 9.6) overnight at 4°C.

• Blocking: Block non-specific binding sites with 1-5% BSA in PBS for 1-2 hours at room temperature.

• Primary antibody: Dilute PT3851 to 1-10 μg/ml in blocking buffer and incubate for 1-2 hours at room temperature or overnight at 4°C.

• Detection: Utilize streptavidin-HRP conjugate (1:1000-1:5000) to bind to the biotin on PT3851, followed by TMB substrate development.

• Analysis: Measure absorbance at 450 nm with 620 nm reference wavelength. Calculate results using appropriate controls, including the recommended Mouse IgG2b [MPC-11] (Biotin) (A86393) isotype control .

The biotin conjugation of PT3851 provides flexibility in detection systems, allowing for amplification of signal through streptavidin-based secondary detection systems, which can enhance sensitivity for detecting low levels of nucleocapsid protein.

How can PT3851 be utilized in studying the kinetics of antibody response to SARS-CoV-2?

PT3851 can be employed in competitive ELISA formats to study the kinetics of anti-nucleocapsid antibody development in patient samples. This methodological approach involves:

• Establishing a baseline assay using PT3851 to detect recombinant nucleocapsid protein with maximal signal.

• Adding patient serum samples at different dilutions to compete with PT3851 for binding to the nucleocapsid protein.

• Measuring the reduction in PT3851 binding signal as an indicator of patient anti-nucleocapsid antibody levels.

• Collecting longitudinal samples from patients to track the development of antibody responses over time, particularly focusing on the critical 8-14 day window after symptom onset when nucleocapsid antibodies typically appear .

• Comparing the kinetics in different patient populations, such as immunocompetent versus immunocompromised individuals, to understand variations in immune response timing and magnitude .

This approach allows researchers to quantitatively track the emergence and evolution of the antibody response, providing valuable insights into SARS-CoV-2 immunology and potentially identifying correlates of protection.

What validation steps should researchers perform when using PT3851 in new experimental systems?

When implementing PT3851 in new experimental systems, researchers should conduct the following validation steps:

• Antibody titration: Determine the optimal concentration of PT3851 by testing serial dilutions to identify the concentration that provides maximum specific signal with minimal background.

• Specificity testing: Confirm that PT3851 specifically recognizes SARS-CoV-2 nucleocapsid protein by:

  • Testing against recombinant nucleocapsid proteins from various coronavirus species

  • Using Western blot analysis to verify recognition of the expected 46 kDa nucleocapsid protein

  • Performing competitive inhibition with unlabeled antibodies or recombinant nucleocapsid protein

• Sensitivity assessment: Establish the lower limit of detection using purified recombinant nucleocapsid protein at varying concentrations.

• Reproducibility evaluation: Perform replicate experiments to determine inter-assay and intra-assay coefficients of variation, which should ideally be below 15%.

• Comparison with reference methods: Benchmark PT3851 performance against established nucleocapsid detection methods to ensure comparable or improved results.

These validation steps ensure robust and reliable research outcomes when incorporating PT3851 into experimental workflows.