Dengue Envelope-2 32kDa

What is Dengue Envelope-2 32kDa and what does it contain?

Dengue Envelope-2 32kDa is a recombinant protein derived from Dengue Virus Subtype 2, containing amino acids 45-297 of the viral envelope protein. This fragment encompasses domains I and II of the dengue envelope structure and is fused to a 6xHis Tag for purification purposes. The protein is expressed in Escherichia coli expression systems and purified using proprietary chromatographic techniques to achieve greater than 95% purity as determined by 12% PAGE with Coomassie staining. The final product is formulated in phosphate buffer .

This protein fragment represents a significant portion of the viral envelope that plays crucial roles in virus attachment, entry, and is a major target for neutralizing antibodies. The recombinant nature of this protein makes it particularly valuable for controlled laboratory investigations of dengue virus immunology and pathogenesis.

What are the optimal storage conditions for Dengue Envelope-2 32kDa?

For maximum stability and research reliability, Dengue Envelope-2 32kDa should be stored below -18°C. While the protein maintains stability at 4°C for approximately one week, long-term storage at refrigeration temperatures is not recommended as it can lead to protein degradation and loss of biological activity. It is critical to prevent freeze-thaw cycles, as repeated freezing and thawing causes protein denaturation and potential loss of antigenic properties .

When working with this protein for multiple experiments, researchers should aliquot the stock solution into single-use volumes prior to freezing to minimize the number of freeze-thaw cycles. For experiments requiring multiple uses of the same preparation, maintain the protein on ice during the experimental procedure rather than repeatedly freezing and thawing the stock.

What applications is Dengue Envelope-2 32kDa suitable for in research settings?

Dengue Envelope-2 32kDa is specifically designed for immunoassay applications in laboratory research settings. The primary applications include:

• Enzyme-linked immunosorbent assays (ELISAs) for detecting anti-dengue antibodies

• Western blot analyses for antibody characterization

• Lateral flow immunoassay development for rapid diagnostics

• Blockade of binding assays to evaluate polyclonal serum responses

• Epitope mapping studies for neutralizing antibodies

• Competition assays to determine antibody specificity

• Development of serological tests for epidemiological studies

It is important to emphasize that this protein is furnished for LABORATORY RESEARCH USE ONLY and must not be used for diagnostic purposes, therapeutic applications, drug development, agricultural products, pesticidal products, food additives, or household chemicals .

How can researchers verify the purity and integrity of Dengue Envelope-2 32kDa?

The purity of commercially available Dengue Envelope-2 32kDa is typically specified as >95% as determined by 12% PAGE with Coomassie staining. Researchers can independently verify this by:

• SDS-PAGE analysis: Run 1-5 μg of protein on a 12% polyacrylamide gel, followed by Coomassie staining to visualize a predominant band at approximately 32 kDa.

• Western blot analysis: Using anti-His tag antibodies to confirm the presence of the 6xHis tag and verify protein identity.

• Mass spectrometry: For precise molecular weight determination and sequence verification.

• Functional assays: Testing the protein's ability to bind known anti-dengue antibodies in ELISA or other immunoassay formats.

Any significant deviation from the expected molecular weight, the presence of multiple bands, or failure to react with appropriate antibodies may indicate protein degradation or contamination that could compromise experimental results .

How does Dengue Envelope-2 32kDa contribute to understanding neutralizing antibody responses?

Dengue Envelope-2 32kDa has been instrumental in elucidating the mechanisms of antibody neutralization against dengue virus serotype 2 (DENV2). Research using this protein has revealed that human neutralizing antibodies target at least two distinct sites on the DENV2 envelope protein. Studies demonstrate that antibodies raised following DENV2 infection or vaccination circulate as separate populations that neutralize by occupying different epitopes on the viral surface .

The protein serves as a valuable tool for characterizing antibody responses by:

• Enabling epitope mapping of neutralizing antibodies

• Facilitating competition assays between monoclonal antibodies and polyclonal sera

• Supporting the development of blockade of binding assays to quantify epitope-specific responses

• Providing a substrate for comparing antibody responses between natural infection and vaccination

These applications have contributed significantly to our understanding of protective immunity against DENV2 and informed vaccine development strategies by identifying key neutralizing epitopes .

What are the major epitopes on Dengue Envelope-2 32kDa targeted by neutralizing antibodies?

Research has identified at least two distinct epitopes on the DENV2 envelope protein that are targeted by strongly neutralizing antibodies:

Studies with convalescent immune sera from primary DENV2 cases have demonstrated that these individuals develop antibodies targeting both epitopes, though the ratio of antibodies varies between individuals. Some individuals show stronger responses to the 2D22 epitope, while others develop higher levels of antibodies targeting the 3F9 epitope. Importantly, control sera from DENV1 or DENV3 infections do not effectively block either 2D22 or 3F9 binding, confirming the serotype-specificity of these responses .

How can Dengue Envelope-2 32kDa be used in competitive binding assays to evaluate polyclonal responses?

Competitive binding assays using Dengue Envelope-2 32kDa provide valuable insights into the epitope specificity of polyclonal antibody responses. These assays, also known as blockade of binding (BOB) assays, can be implemented using the following methodology:

• Coat ELISA plates with purified Dengue Envelope-2 32kDa (typically 1-2 μg/ml)

• Block plates with appropriate blocking buffer (BSA or non-fat milk)

• Pre-incubate test sera at various dilutions with labeled monoclonal antibodies (e.g., 2D22 or 3F9)

• Add the pre-incubated mixture to the coated plates

• Wash and detect bound monoclonal antibodies

• Calculate percent inhibition relative to controls without competing sera

This approach has revealed that convalescent immune sera from primary DENV2 cases effectively block the binding of both 2D22 and 3F9 to their respective epitopes, whereas control DENV1 or DENV3 sera do not. Interestingly, the ratio of antibodies targeting each epitope differs across individuals, with some showing stronger blocking of 2D22 and others more effectively blocking 3F9 .

This methodology can also be applied to evaluate vaccine-induced responses, as demonstrated by studies showing that DENV2 monovalent vaccine sera also block both 2D22 and 3F9 binding to their respective epitopes .

How does antibody binding to Dengue Envelope-2 32kDa compare between natural infection and vaccination?

Studies comparing antibody responses to Dengue Envelope-2 32kDa between natural infection and vaccination have revealed both similarities and differences in the epitope targeting patterns:

These findings suggest that both natural infection and monovalent DENV2 vaccination elicit antibodies targeting the same key epitopes on the envelope protein. The observation that neither natural infection nor vaccination leads to complete inhibition of monoclonal antibody binding suggests a limit to the concentration of epitope-specific antibodies in the sera .

The similarities in epitope targeting between natural infection and vaccination are encouraging for vaccine development, suggesting that vaccines can induce antibodies with specificities similar to those generated during natural infection, which are known to be protective against subsequent DENV2 infection.

What methodological considerations are important when using Dengue Envelope-2 32kDa in structural studies?

When using Dengue Envelope-2 32kDa for structural studies, researchers should consider several important methodological factors:

For advanced structural studies, researchers may need to consider alternative expression systems (such as mammalian or insect cells) that can provide proper post-translational modifications for a more accurate representation of the native envelope structure.

How can Dengue Envelope-2 32kDa be integrated into reverse genetics approaches?

Dengue Envelope-2 32kDa can be effectively integrated into reverse genetics approaches to study viral epitopes and antibody interactions through several strategies:

• Structure-guided immunogen design: Using the amino acid sequence and structural information from Dengue Envelope-2 32kDa (residues 45-297) to design mutations in infectious clones.

• Epitope transplantation: Creating recombinant viruses containing amino acid alterations and epitope transplants between different serotypes to map antibody binding sites.

• Domain swapping: Exchanging domains between different dengue serotypes to create chimeric viruses for evaluating domain-specific neutralization.

Implementation of these approaches has successfully identified epitopes targeted by type-specific neutralizing antibodies, demonstrating that human DENV2 neutralizing antibodies target two distinct regions on the viral surface .

When implementing these methods, researchers should consider that modifications to the envelope protein may affect viral fitness and viability, potentially requiring compensatory mutations. Additionally, the effects of such modifications on virus maturation, assembly, and entry should be carefully evaluated to ensure that the engineered viruses accurately represent the immunological properties of interest.

What are the key experimental parameters for using Dengue Envelope-2 32kDa in immunoassays?

For optimal use of Dengue Envelope-2 32kDa in immunoassays, consider the following technical parameters:

These parameters should be optimized for each specific experimental setup, as optimal conditions may vary based on the specific antibodies, detection systems, and experimental goals .

How can researchers troubleshoot inconsistent results when working with Dengue Envelope-2 32kDa?

When encountering inconsistent results with Dengue Envelope-2 32kDa, researchers should systematically evaluate:

• Protein quality: Verify protein integrity by SDS-PAGE and Western blot before use. Multiple freeze-thaw cycles or improper storage can lead to degradation.

• Buffer compatibility: Ensure that the phosphate buffer in which the protein is formulated is compatible with your assay system. In some cases, buffer exchange may be necessary.

• Antibody specificity: Validate antibody specificity using appropriate controls, including other dengue serotypes or related flaviviruses.

• Blocking conditions: Optimize blocking conditions to minimize background without interfering with specific binding.

• Assay sensitivity: Determine the detection limits of your assay system and ensure that protein concentrations are within the linear range.

• Conformational epitopes: Be aware that some antibodies may recognize conformational epitopes that could be disrupted under certain experimental conditions.

Systematic optimization and validation of each experimental parameter will help ensure reproducible results when working with this protein .