PLAUR Recombinant Monoclonal Antibody

What is PLAUR and why are recombinant monoclonal antibodies against it significant for research?

PLAUR, also known as urokinase receptor (uPAR), CD87, or URKR, is a glycosylphosphatidylinositol-anchored cell surface protein that plays critical roles in cell adhesion, migration, and invasion processes . Recombinant monoclonal antibodies against PLAUR are significant for research because they can specifically inhibit uPAR-induced cell adhesion and invasion with high affinity (KD=115 pM) . This makes them valuable tools for studying the mechanisms of cancer progression, cardiovascular disorders, and immunological responses where PLAUR is implicated .

The significance of using recombinant antibodies rather than traditional antibodies lies in their increased sensitivity, confirmed specificity, high repeatability, excellent batch-to-batch consistency, sustainable supply, and animal-free production methods . These characteristics address the reproducibility issues that have plagued antibody-based research, while also reducing ethical concerns regarding animal use in antibody production .

How do recombinant monoclonal antibodies differ from traditional antibodies in PLAUR research applications?

Recombinant monoclonal antibodies differ fundamentally from traditional antibodies in several key aspects that impact their utility in PLAUR research. Traditional antibodies are generated through immunization of animals, followed by hybridoma technology or polyclonal antibody purification, which introduces variability between batches and raises ethical concerns about animal use .

In contrast, recombinant PLAUR antibodies are produced by cloning the antibody genes and expressing them in controlled expression systems like HEK293F cells . This recombinant approach offers several advantages:

• Standardization and reproducibility: The genetic encoding ensures identical molecular composition in every batch, eliminating the batch-to-batch variability common with hybridoma-derived antibodies .

• Engineered optimization: Recombinant antibodies can be designed with precise binding characteristics, as demonstrated by the high affinity (KD=115 pM) of anti-PLAUR antibodies that enable reliable detection and inhibition studies .

• Versatility for experimental design: Researchers can generate various forms of the same antibody (mouse, rabbit, or human variants) to accommodate different experimental designs and avoid cross-reactivity issues in multi-color immunostaining experiments .

• Animal-free production: The cell culture-based production methods eliminate the need for animals in the manufacturing process, addressing ethical concerns while maintaining high quality standards .

These differences make recombinant monoclonal antibodies particularly valuable for longitudinal studies where consistent reagent performance is critical for data interpretation and reproducibility.

What are the optimal applications for PLAUR recombinant monoclonal antibodies in cancer research?

PLAUR recombinant monoclonal antibodies have several optimal applications in cancer research, particularly due to their ability to inhibit uPAR-induced cell adhesion and invasion processes . Based on the available research data, the most effective applications include:

• Invasion and metastasis studies: These antibodies can be used to block uPAR function in cell migration assays, spheroid invasion models, or in vivo metastasis studies to elucidate the role of PLAUR in cancer progression .

• Plasminogen activation inhibition: As demonstrated in plasminogen activation inhibition assays (see Figure 1 from the Creative Biolabs data), anti-uPAR monoclonal antibodies can effectively inhibit this process, making them valuable for studying how cancer cells remodel their extracellular matrix during invasion .

• Immunofluorescence and flow cytometry: PLAUR antibodies can be used at defined concentrations (typically 0.6-2.1 μg/ml, depending on the specific antibody) for detecting PLAUR expression in tumor tissues and circulating tumor cells .

• Epitope-specific targeting: Western blotting analysis with anti-uPAR mAbs enables researchers to identify specific domains of uPAR that are crucial for its function in particular cancer types (as shown in Figure 2 of the epitope binding analysis) .

For optimal results in cancer research applications, recommended working concentrations range from 0.2-2.1 μg/ml for immunofluorescence applications, with specific concentrations depending on the particular antibody clone and experimental system .

How should researchers optimize immunofluorescence protocols when using PLAUR recombinant antibodies?

Optimizing immunofluorescence protocols for PLAUR recombinant antibodies requires attention to several critical parameters:

• Fixation method: For PLAUR detection, perform pre-extraction in PHEM buffer (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, 4 mM MgSO4, pH 7.0) followed by 0.5% Triton X-100 lysis for 5 minutes. Then fix cells with freshly prepared 4% paraformaldehyde in PHEM buffer at 37°C for 20 minutes .

• Blocking and antibody dilution: Block with 10% boiled donkey serum (BDS) in PHEM buffer for 1 hour at room temperature. Dilute primary antibodies in 5% BDS for optimal signal-to-noise ratio .

• Antibody concentration optimization: Different PLAUR antibody clones require different working concentrations for optimal results. For example:

  • rMAb-Hec1 ms: 1.5 μg/ml

  • rMAb-Hec1 rb: 0.2 μg/ml

  • rMAb-Hec1 hu: 1 μg/ml

• Species selection considerations: If conducting multi-color immunofluorescence with other primary antibodies, select PLAUR antibodies with appropriate species specificity to avoid cross-reactivity with secondary antibodies. For example, if using another mouse antibody, select the rabbit or human variant of the PLAUR antibody .

• Wash steps: Perform 3 × 5 minute washes with PHEM-T buffer (PHEM + 0.1% Triton X-100) after fixation and between antibody incubations to reduce background .

For advanced multi-color immunofluorescence applications, consider using directly conjugated antibody fragments (scFv or Fab) or full-length antibodies with different species specificities to overcome limitations in available secondary antibody combinations .

What analytical techniques are most appropriate for characterizing PLAUR recombinant antibodies?

The characterization of PLAUR recombinant antibodies requires a multi-faceted analytical approach to ensure their specificity, purity, and functional properties. Based on the current research, the most appropriate techniques include:

• Chromatographic methods:

  • Reversed-Phase Liquid Chromatography (RPLC/RP-HPLC): Optimal for assessing antibody purity and detecting modifications like oxidation .

  • Size Exclusion Chromatography (SEC): Particularly useful for analyzing antibody aggregation and monitoring MAb oxidation variants .

  • Cation Exchange Chromatography: Effective for charge variant analysis, which is critical for validating consistent production of PLAUR antibodies .

• Mass Spectrometry coupled techniques:

  • RPLC-MS: Provides detailed molecular characterization and is applicable to a wide range of monoclonal antibodies, making it suitable for PLAUR antibody analysis .

  • RP-UPLC-MALS (Multi-Angle Light Scattering): Particularly valuable for bispecific antibodies and structural variants of IgG1 and IgG2 isotypes .

• Functional binding assays:

  • Plasminogen activation inhibition assays: Essential for confirming the inhibitory function of anti-PLAUR antibodies, as demonstrated in the line graph showing inhibitory effects of fifteen anti-uPAR monoclonal antibodies .

  • Epitope binding analysis: Western blotting can be used to confirm specific domain targeting of the PLAUR antibody, as shown in Figure 2 of the epitope binding analysis .

• Affinity determination:

  • Surface Plasmon Resonance (SPR): For determining binding kinetics and affinity constants, such as the KD value of 115 pM reported for high-affinity anti-PLAUR antibodies .

These analytical techniques should be selected based on the specific research question and combined to provide comprehensive characterization of PLAUR recombinant antibodies before their application in experimental systems.

How can researchers modify the species specificity of PLAUR recombinant antibodies for multi-color immunofluorescence?

Modifying the species specificity of PLAUR recombinant antibodies is a sophisticated approach to expand experimental capabilities, particularly for multi-color immunofluorescence. The methodology involves genetic engineering of the antibody constant regions while preserving the antigen-binding domains:

• Sequence-based engineering approach:

  • First, isolate the DNA sequences encoding only the variable regions of both heavy chains (HC) and light chains (LC) from the original PLAUR antibody .

  • Design PCR fragments corresponding to the constant regions of the target species (mouse, rabbit, human) for both HC and LC .

  • Use Gibson assembly method to combine the variable regions with the new species' constant regions in an appropriate expression vector (such as modified pEGFP-N1) .

• Expression and validation:

  • Co-express the modified HC and LC plasmids in HEK293 suspension culture cells (such as Expi293F cells) .

  • Validate the species-modified antibodies using immunofluorescence at optimized concentrations:

    • Mouse PLAUR antibodies: typically 1.5-2.1 μg/ml

    • Rabbit PLAUR antibodies: typically 0.2-1.9 μg/ml

    • Human PLAUR antibodies: typically 0.6-1.12 μg/ml

• Experimental advantages:

  • This approach enables simultaneous use of multiple antibodies that would otherwise have cross-reactivity issues due to shared species origin.

  • For example, a mouse anti-PLAUR antibody can be converted to rabbit specificity, allowing its use alongside other mouse antibodies in the same immunofluorescence experiment .

This methodology provides significantly greater flexibility in experimental design, allowing researchers to overcome the limitations imposed by available secondary antibody combinations, particularly in complex co-localization studies involving multiple proteins in the same cellular compartment.

What strategies exist for generating antibody fragments from PLAUR recombinant monoclonal antibodies?

Generating antibody fragments from PLAUR recombinant monoclonal antibodies offers researchers enhanced flexibility for specialized applications. Three main strategies have been developed for this purpose:

• scFvC (single chain variable fragment plus truncated constant region) generation:

  • Design a single polypeptide chain containing the variable regions of HC and LC connected by a flexible linker, attached to specific HC constant regions (CR2 + CR3).

  • The resulting fragment is approximately 60 kDa in mass as a monomer and 120 kDa after dimerization.

  • Implementation requires designing a single plasmid encoding the entire scFvC fragment, then expressing it in appropriate cell systems .

• scFv (single chain variable fragment) production:

  • Similar to scFvC but without the constant regions, resulting in a smaller fragment (~25-30 kDa).

  • These fragments retain antigen binding capability while providing better tissue penetration.

  • The smaller size makes them particularly valuable for super-resolution microscopy and applications where steric hindrance is a concern .

• Fab (antigen binding fragment) development:

  • Requires separate expression of the variable region plus CH1 domain of the HC and the complete LC.

  • These fragments retain the binding specificity of the original antibody but with approximately one-third the molecular weight.

  • Fab fragments eliminate potential Fc-mediated interactions, reducing background in certain experimental systems .

Each fragment type offers distinct advantages depending on the experimental objective:

• scFvC fragments maintain some effector functions while reducing size

• scFv fragments provide minimal size while preserving binding specificity

• Fab fragments offer a balance between stability and reduced size

These strategies significantly expand the researcher's toolkit beyond conventional full-length antibodies, allowing for optimization of PLAUR detection in specialized experimental contexts such as super-resolution microscopy, FRET applications, or in vivo imaging.

How does post-translational modification analysis impact PLAUR antibody performance in research applications?

Post-translational modifications (PTMs) of PLAUR recombinant monoclonal antibodies can significantly impact their performance in research applications, warranting careful analytical consideration:

• Critical PTMs affecting antibody function:

  • Glycosylation patterns affect antibody stability, half-life, and effector functions

  • Oxidation of methionine residues can alter binding affinity and specificity

  • Deamidation of asparagine residues may reduce thermal stability

  • C-terminal lysine processing influences charge heterogeneity

• Analytical approaches for PTM characterization:

  • Mixed-mode Size Exclusion Chromatography (SEC) can detect and quantify oxidation variants in monoclonal antibodies, which is crucial for maintaining consistent inhibitory function in plasminogen activation assays .

  • Cation exchange chromatography coupled with mass spectrometry (CEX-MS) is particularly valuable for distinguishing PLAUR antibodies with varying charges resulting from different PTMs .

  • Reversed-Phase Liquid Chromatography (RPLC) can be used to detect subtle modifications that might affect the high-affinity binding (KD=115 pM) reported for anti-PLAUR antibodies .

• Impact on experimental reliability:

  • Inconsistent PTMs between antibody batches can lead to variable results in functional assays such as the plasminogen activation inhibition assay.

  • For PLAUR immunofluorescence applications, where antibodies are used at specific concentrations (0.2-2.1 μg/ml), consistent glycosylation is essential for reproducible staining intensity and pattern recognition .

Researchers should implement systematic PTM analysis as part of their quality control process to ensure consistent antibody performance across experiments, particularly for longitudinal studies where batch-to-batch variation could confound results interpretation.

What are common pitfalls in PLAUR antibody experiments and how can researchers address them?

Researchers working with PLAUR recombinant monoclonal antibodies frequently encounter several challenges that can compromise experimental outcomes. Here are the most common pitfalls and their methodological solutions:

• Inconsistent staining patterns in immunofluorescence:

  • Problem: Variable signal intensity or subcellular localization patterns.

  • Solution: Implement standardized fixation protocols using PHEM buffer with 0.5% Triton X-100 for pre-extraction, followed by 4% paraformaldehyde fixation at 37°C. Optimize antibody concentration based on specific clones - for example, rMAb-Hec1 requires different concentrations depending on species (mouse: 1.5 μg/ml; rabbit: 0.2 μg/ml; human: 1 μg/ml) .

• Cross-reactivity issues in multi-color immunostaining:

  • Problem: Secondary antibody cross-reactivity leading to false co-localization signals.

  • Solution: Utilize species-swapped variants of PLAUR antibodies or consider antibody fragments (scFvC, scFv) to overcome secondary antibody limitations. For example, if using other mouse primary antibodies, select the rabbit or human variant of the PLAUR antibody .

• Variable inhibitory efficacy in functional assays:

  • Problem: Inconsistent inhibition of plasminogen activation or cell adhesion.

  • Solution: Characterize each antibody batch using appropriate analytical techniques like RP-HPLC or cation exchange chromatography to ensure consistent quality and post-translational modifications. Validate functional activity using standardized plasminogen activation inhibition assays before experimental application .

• Background signal in Western blotting:

  • Problem: Non-specific bands obscuring PLAUR-specific signals.

  • Solution: Perform epitope binding analysis to confirm antibody specificity, as demonstrated in Figure 2 of the epitope binding analysis by Western blotting. Select antibody clones that show clean, specific binding patterns to PLAUR epitopes .

Through careful attention to these methodological details, researchers can significantly improve the reliability and reproducibility of their PLAUR antibody-based experiments.

How should researchers validate the specificity of their PLAUR recombinant antibodies?

Comprehensive validation of PLAUR recombinant antibody specificity requires a multi-faceted approach using complementary methodologies:

• Knockout/knockdown controls:

  • Generate PLAUR knockout cell lines using CRISPR/Cas9 technology

  • Alternatively, create transient knockdowns using validated siRNA against PLAUR

  • Compare antibody staining patterns between wild-type and knockout/knockdown samples across multiple applications (immunofluorescence, Western blotting, flow cytometry)

• Epitope-specific validation:

  • Perform epitope binding analysis using Western blotting with denatured and non-denatured protein samples to confirm epitope recognition, similar to the approach shown in Figure 2 of the epitope binding analysis

  • Use competitive binding assays with free peptides corresponding to the targeted epitope to confirm binding specificity

• Cross-platform concordance:

  • Compare results across multiple detection methods (ELISA, Western blotting, immunofluorescence, flow cytometry)

  • Verify that the antibody performs consistently across different applications at appropriate concentrations:

    • For immunofluorescence: 0.2-2.1 μg/ml depending on the specific clone

    • For Western blotting and ELISA: Typically higher concentrations may be required

• Multiple antibody comparison:

  • Test multiple anti-PLAUR antibodies targeting different epitopes of the protein

  • For example, compare antibody clones such as 13C4, PABL-355, 8B12, C042M, and 7G1 (as listed in the Creative Biolabs product table)

  • Consensus results across multiple antibodies provide stronger evidence of specificity

• Recombinant expression systems:

  • Express tagged versions of PLAUR in cell systems and confirm co-localization of the anti-PLAUR antibody with the tag-specific antibody

  • This approach can distinguish true targets from potential cross-reactive proteins

Thorough validation using these complementary approaches ensures that experimental results reflect genuine PLAUR biology rather than artifacts of non-specific antibody interactions.

How might emerging antibody engineering techniques impact future PLAUR research?

Emerging antibody engineering techniques are poised to revolutionize PLAUR research in several significant ways:

• Multispecific antibodies for pathway analysis:

  • Bispecific or trispecific antibody formats could simultaneously target PLAUR and its binding partners (like uPA or integrins)

  • These engineered antibodies would enable more sophisticated studies of PLAUR-mediated signaling networks by modulating multiple pathway components simultaneously

  • The techniques used for generating various antibody fragments (scFvC, scFv, Fab) provide the foundation for creating these multi-specific constructs

• Intracellular antibodies (intrabodies):

  • Engineering cell-penetrating PLAUR antibodies or expressing intracellular antibody fragments

  • This approach would allow real-time visualization of PLAUR trafficking and processing within living cells

  • The methodologies for generating scFv fragments could be adapted for intracellular expression with appropriate localization signals

• Antibody-drug conjugates for targeted therapy research:

  • Development of PLAUR-targeted antibody-drug conjugates for precision medicine applications

  • These tools would enable studies of selective targeting of PLAUR-expressing cells in complex models

  • The high-affinity binding (KD=115 pM) of anti-PLAUR antibodies makes them excellent candidates for conjugate development

• Nanobody and single-domain antibody development:

  • Engineering smaller (15-25 kDa) antibody formats derived from camelid antibodies

  • These would provide superior tissue penetration and reduced immunogenicity for in vivo imaging

  • The expertise gained from producing scFv fragments provides a technological foundation for nanobody development

• Computationally designed antibodies:

  • AI-assisted antibody design to create PLAUR antibodies with predetermined properties

  • This approach could yield antibodies with precisely engineered binding kinetics, thermal stability, and specificity

  • The analytical techniques currently used for antibody characterization (chromatography, spectroscopy) would be essential for validating these designed antibodies

These emerging technologies will significantly expand the research toolkit available for PLAUR studies, enabling more sophisticated investigations of its role in cancer, cardiovascular disease, and immunological disorders.

What are the most promising applications of PLAUR recombinant antibodies in translational research?

The unique properties of PLAUR recombinant monoclonal antibodies position them for several high-impact translational research applications:

• Cancer biomarker development and companion diagnostics:

  • PLAUR expression correlates with invasive and metastatic potential in multiple cancer types

  • Recombinant antibodies with consistent batch-to-batch performance are critical for developing standardized diagnostic assays

  • The high affinity (KD=115 pM) and specificity of anti-PLAUR antibodies make them ideal candidates for detecting low abundance biomarkers in liquid biopsies

• Targeted therapy development:

  • PLAUR's role in cancer, cardiovascular disease, and immunological disorders makes it an attractive therapeutic target

  • Recombinant antibodies that inhibit uPAR-induced cell adhesion and invasion provide proof-of-concept for therapeutic development

  • The ability to modify antibody fragments and species specificity enables creation of humanized versions for clinical translation

• Precision medicine approaches:

  • Stratification of patients based on PLAUR expression patterns using standardized immunohistochemical protocols

  • The specificity and reproducibility of recombinant PLAUR antibodies address a critical need for reliable biomarkers

  • Multiple antibody formats (full-length, scFv, Fab) provide flexibility for different diagnostic platforms

• In vivo imaging development:

  • Smaller antibody fragments (scFv, Fab) with preserved PLAUR specificity are ideal for developing molecular imaging agents

  • These could enable non-invasive monitoring of PLAUR-expressing tumors or inflammatory lesions

  • The methodologies for generating and characterizing these fragments are directly applicable to imaging probe development

• Drug delivery systems:

  • PLAUR-targeted nanoparticles or liposomes for selective delivery of therapeutic agents

  • The high binding affinity of anti-PLAUR antibodies (KD=115 pM) would facilitate efficient targeting

  • Various antibody fragments provide options for optimizing circulation time and tissue penetration

The translation of these applications from bench to bedside will require rigorous validation using the analytical techniques described previously, ensuring consistent performance across different experimental and clinical contexts.