Enterobacteria phage M13 III Monoclonal Antibody

What is Enterobacteria phage M13 III Monoclonal Antibody?

The Enterobacteria phage M13 III Monoclonal Antibody is a mouse-derived monoclonal antibody that specifically targets the attachment protein (pIII) of M13 bacteriophage. This antibody recognizes the minor coat protein (also called attachment protein G3P, Gene 3 protein, or G3P) located at the tip of the filamentous M13 phage structure. The antibody is developed using recombinant Enterobacteria phage M13 Attachment protein (19-424AA) as the immunogen and has an accession number of P69168 . This reagent is restricted for research use only and not intended for diagnostic procedures.

What validated applications are appropriate for M13 III Monoclonal Antibody?

The M13 III Monoclonal Antibody has been specifically validated for Enzyme-Linked Immunosorbent Assay (ELISA) and Western Blotting (WB) applications. These validated methods provide researchers with reliable approaches for detecting and quantifying M13 phage proteins in experimental samples . The antibody demonstrates consistent performance in immunoassay formats that rely on either colorimetric or chemiluminescent detection systems when used according to established protocols.

How does M13 phage structure influence antibody binding specificity?

The M13 bacteriophage has a filamentous structure with approximately 2,700 copies of the major coat protein (pVIII) and only 5 copies of the minor coat protein (pIII) located at one end of the phage. This distinctive 5:2700 molecular ratio between pIII and pVIII creates unique opportunities for signal amplification in immunoassays. The antibody specifically recognizes epitopes on the pIII protein, which plays a crucial role in bacterial infection and is often utilized as a display platform for foreign peptides in phage display technology . The structural arrangement of the pIII protein at the phage tip makes it particularly accessible for antibody binding.

What are the optimal conditions for using M13 III antibody in immunoassays?

For optimal performance in immunoassays, the M13 III Monoclonal Antibody should be used under the following conditions:

The antibody performs optimally in ELISA when used with proper blocking agents to minimize background signal. For Western blotting, overnight incubation at 4°C frequently yields better signal-to-noise ratios compared to shorter incubations .

How can researchers design M13 phage-based capture systems for analyte isolation?

Researchers can design effective M13 phage-based capture systems through several strategic approaches:

• Chemical conjugation: EDC/NHS reactions can be used to couple antibodies or other binding ligands to the surface proteins of M13 phage, creating a multivalent capture system. This approach provides relatively larger phage coverage but typically in a side-on manner.

• Genetic modification: By genetically modifying M13 phage to display 6His tags on the pIII protein, researchers can achieve end-on anchoring through 6His-NTA interactions. This method offers strong yet reversible assembly and facilitates the construction of bio-inspired architectures that mimic cellular threadlike structures.

• Enzymatic biotinylation: Using a 14-mer biotin acceptor peptide displayed on pIII protein, M13 phages can be biotinylated with biotin ligase (BirA), enabling end-on anchoring to streptavidin-functionalized surfaces or microbeads.

These phage-decorated capture systems have demonstrated superior performance compared to non-phage counterparts, with both enhanced sensitivity and selectivity. The improved performance is attributed to increased antibody loading capacity and enhanced molecular interactions facilitated by the flexible phage structure .

How can M13 phage antibodies be utilized in electrochemical biosensor development?

M13 phage antibodies can be integrated into electrochemical biosensors through the following methodological approach:

• Electrode surface preparation: Gold electrodes are typically modified with doped carbon quantum dots and graphene oxide to enhance conductivity and surface area.

• Phage functionalization: M13 phages are functionalized with 3-mercaptopropionic acid for immobilization on gold nanoparticle-deposited electrodes. This creates a stable sensing platform that maintains functionality across a wide pH range (3.0-10.0) and at elevated temperatures (up to 45°C).

• Antibody integration: Anti-M13 monoclonal antibodies conjugated with horseradish peroxidase (HRP) can be used as signal amplifiers, leveraging the multivalent structure of M13 phage.

• Electrochemical impedance spectroscopy (EIS): The phage-based EIS biosensors have demonstrated remarkable sensitivity, with limits of detection reaching 0.003-0.014 ng/mL for certain analytes.

A key advantage of these biosensors is their regenerative capacity—they can be regenerated by elution with Glycine-HCl (pH 2.2) and reused for up to 6 cycles without significant loss of sensitivity . This approach provides a significant advantage over traditional immunosensors in terms of cost-effectiveness and sustainability.

What strategies can enhance specificity in phage-based peptide library screening?

To enhance specificity in phage-based peptide library screening, researchers should implement a multi-tiered approach:

• Library design optimization: Construct cyclic 8-, 9-, or 10-residue peptide libraries using phagemid systems like pComb-pVIII, which achieve high-density peptide display while preserving library diversity.

• Biopanning strategy refinement: Employ blended biopanning of cyclic peptide libraries with stringent washing steps to isolate phages with high target specificity. This approach has successfully yielded phages with exceptional binding characteristics for various targets including toxins and cellular receptors.

• Counter-selection steps: Include negative selection rounds against similar but non-target molecules to eliminate cross-reactive phage clones.

• Sensitivity validation: Test isolated phage clones in both competitive and non-competitive phage ELISA formats to ensure their practical utility in analytical applications.

• Signal amplification: Utilize the structural advantage of M13 (5:2700 ratio of pIII:pVIII) to achieve signal amplification by conjugating the phage with nanoparticles or reporter enzymes.

This methodological approach has successfully generated highly specific peptides against diverse targets including clothianidin and prostate-specific antigen, with demonstrated limits of detection reaching 0.16 ng/mL .

How can researchers minimize non-specific binding in M13 phage-based immunoassays?

Non-specific binding can significantly compromise the reliability of M13 phage-based immunoassays. Researchers can implement the following evidence-based strategies to minimize this issue:

• Gold layer coating: Studies have demonstrated that coating phage-decorated microbeads with a thin gold layer substantially reduces non-specific binding in immunoassays.

• Blocking optimization: Employ a dual blocking approach using both protein-based blockers (5% BSA) and mild detergents (0.05% Tween-20) in all assay buffers.

• Surface chemistry modification: When immobilizing M13 phages on electrode surfaces, consider the roughness of the electrode surface, as increased roughness can contribute to biofouling issues.

• Anti-fouling peptide display: Genetically modify M13 phages to display anti-fouling peptides alongside target-binding peptides to reduce non-specific interactions with complex biological matrices.

• Elution pH optimization: For regenerable biosensors, determine the optimal elution pH (typically Gly-HCl at pH 2.2) that removes bound analytes without damaging the immobilized phage.

Implementation of these strategies has enabled researchers to develop phage-based sensing platforms with exceptional selectivity and minimal background interference, particularly important when working with complex biological samples .

What are common issues when using M13 phage III antibodies in Western blotting?

When utilizing M13 phage III antibodies in Western blotting, researchers frequently encounter several technical challenges:

Researchers should validate antibody specificity using purified M13 phage as a positive control and an unrelated phage (such as fd or f1) as a negative control. The addition of non-ionic detergents (0.1% Triton X-100) to washing buffers can significantly improve signal-to-noise ratio when using these antibodies .

How should results from M13 phage antibody-based assays be quantified and validated?

Proper quantification and validation of M13 phage antibody-based assays require a structured analytical approach:

• Standard curve generation: Prepare serial dilutions of purified M13 phage (quantified by plaque assay or spectrophotometry) to create a standard curve. The relationship between signal intensity and phage concentration typically follows a sigmoidal curve that can be modeled using a four-parameter logistic equation.

• Linear range determination: Identify the linear portion of the standard curve (typically 10^7-10^11 phage particles/mL) for accurate quantification. Samples should be diluted to fall within this range.

• Statistical analysis: Calculate the coefficient of variation (CV) for technical replicates (acceptable range: <10% for intra-assay, <15% for inter-assay). Use appropriate statistical tests (t-test, ANOVA) to determine significant differences between experimental groups.

• Assay validation parameters:

• Method comparison: Validate results by comparing with an orthogonal method (e.g., qPCR for phage quantification) to ensure consistency and accuracy of the immunoassay approach .

How can researchers reconcile contradictory results in M13 phage antibody experiments?

When confronted with contradictory results in M13 phage antibody experiments, researchers should implement a systematic troubleshooting approach:

• Antibody characterization verification: Confirm the specificity of the antibody using Western blot against purified M13 phage proteins. The anti-pIII antibody should recognize a protein of approximately 42.5 kDa.

• Experimental variable isolation: Systematically examine key variables that might influence results:

  • Phage preparation method (PEG precipitation vs. cesium chloride gradient)

  • Storage conditions (fresh vs. stored preparations)

  • Buffer composition (pH, salt concentration, detergents)

  • Blocking agents (BSA vs. casein vs. commercial blockers)

• Control implementation: Include positive controls (purified M13 phage), negative controls (related but distinct phages), and method controls (spike-in recovery) in each experiment.

• Cross-validation with multiple techniques: If ELISA and Western blot results conflict, employ a third method such as immunofluorescence or immunoprecipitation to resolve the discrepancy.

• Batch effects analysis: Determine if contradictions occur between different lots of antibody or between different experimental days, suggesting systematic rather than random error.

By implementing this structured approach, researchers can identify the source of contradictory results and establish reliable protocols for M13 phage detection and characterization .

What are emerging applications for M13 phage antibodies in biosensing technologies?

M13 phage antibodies are poised to revolutionize biosensing technologies through several innovative approaches:

• Surface-Enhanced Raman Spectroscopy (SERS): M13 phages displaying highly specific peptide sequences (such as RKIVHAQTP) can be conjugated with silver nanoparticles to form phage-AgNPs networks for label-free Raman analysis. This approach enables detection of specific cell types and pathogens with exceptional sensitivity.

• Electrochemical Impedance Spectroscopy (EIS): M13 phage-based EIS sensors functionalized with 3-mercaptopropionic acid demonstrate remarkable stability across wide pH ranges (3.0–10.0) and elevated temperatures (45°C). These sensors can be regenerated and reused for up to 6 measurement cycles.

• Competitive immuno-electrochemical sensing: By decorating phages with anti-M13 mAb-HRP conjugates, signal amplification can be achieved for detecting small molecules like organophosphorus pesticides, reaching limits of detection as low as 0.003–0.014 ng/mL.

• Phage-display derived peptide libraries: The continued development of cyclic 8-, 9-, and 10-residue peptide libraries using phagemid systems like pComb-pVIII enables high-density display of peptides while preserving library diversity, opening new avenues for target discovery and validation.

These emerging applications leverage the unique structural features of M13 phage and the specificity of anti-M13 antibodies to create next-generation biosensing platforms with unprecedented sensitivity and selectivity .

What methodological advancements would improve M13 phage-based analyte separation?

To advance M13 phage-based analyte separation methods, researchers should focus on these promising methodological improvements:

• End-on anchoring optimization: Further refinement of end-on phage anchoring strategies through genetic modification of pIII protein to display 6His tags could enhance conjugation with functional molecules and facilitate the construction of bio-inspired architectures mimicking cellular threadlike structures.

• Biotinylation enhancement: Development of improved biotinylation strategies using biotin ligase (BirA) for site-specific modification of pIII protein could increase the efficiency of phage immobilization on streptavidin-functionalized substrates.

• Multifunctional phage engineering: Genetic modification of M13 phage to simultaneously display different functional peptides on distinct coat proteins (e.g., target-binding peptides on pIII and reporter/anchor peptides on pVIII) would create more versatile separation platforms.

• Surface chemistry innovations: Development of novel coupling strategies to immobilize M13 phage on magnetic beads or other solid substrates could improve analyte capture efficiency while maintaining the native conformation and functionality of the phage.

• Regeneration protocol optimization: Refinement of regeneration conditions, such as elution with Glycine-HCl (pH 2.2), could extend the reusability of phage-based separation systems beyond the current 6-cycle limit without compromising performance.

These methodological advances would significantly enhance the utility of M13 phage-based systems for analyte isolation and separation in complex biological samples, addressing current limitations in sensitivity, selectivity, and reusability .