SERPINF2 Antibody

Basic Research Questions

• What is SERPINF2/alpha-2-antiplasmin and why is it important in research?

  SERPINF2 (Serpin Family F Member 2), also known as alpha-2-antiplasmin (α2AP), is a member of the serpin superfamily that functions as the primary physiological inhibitor of the serine protease plasmin. The human serpin superfamily consists of at least 35 members that target not only serine proteases but also selected cysteine proteases and non-protease proteins . SERPINF2 plays a crucial role in regulating blood clotting by binding to plasmin's active site, resulting in a major conformational rearrangement that traps the enzyme in a covalent acyl-enzyme intermediate . Beyond blood clotting regulation, SERPINF2 is also involved in complement pathway modulation, extracellular matrix remodeling, and cell motility . Recent research has highlighted its significance in cerebrovascular and cardiovascular diseases, making it an important target for antibody-based research .

• What are the common applications of SERPINF2 antibodies in laboratory research?

  SERPINF2 antibodies are utilized across multiple research applications:

  • Western Blotting: For detecting SERPINF2 protein expression in tissue lysates and biological samples. This technique allows researchers to identify the protein based on molecular weight and confirm its presence in experimental models .

  • Immunohistochemistry (IHC): For visualizing SERPINF2 localization in tissue sections. For example, SERPINF2 has been detected in mouse intestine using goat anti-mouse SERPINF2 antibodies with HRP-DAB staining systems .

  • ELISA: For quantitative determination of SERPINF2 levels in biological samples. Sandwich ELISA systems using antibody pairs can detect human SERPINF2 with sensitivity as low as 31.25 pg/ml .

  • Immunoprecipitation: For isolating SERPINF2 protein complexes from biological samples to study protein-protein interactions .

  Each application requires specific optimization of antibody dilutions and conditions to ensure reliable results.

• What different types of SERPINF2 antibodies are available and how do they differ?

  Several types of SERPINF2 antibodies are available for research, each with distinct characteristics:

  • Polyclonal Antibodies: Typically raised in goats or rabbits against recombinant SERPINF2, these recognize multiple epitopes. For example, goat polyclonal antibodies against human SERPINF2 (Met28-Lys491) are available that show approximately 50% cross-reactivity with mouse SERPINF2 .

  • Monoclonal Antibodies: Derived from single B-cell clones, these target specific epitopes with high specificity. Clone 236122 is an example that shows no cross-reactivity with other human serpins (A1, A3, A4, A5) or mouse serpins (C1, D1, F2) .

  • Recombinant Monoclonal Antibodies: Engineered antibodies produced through recombinant DNA technology that offer high batch-to-batch consistency .

  • Variant-Specific Antibodies: Some antibodies specifically target particular variants of SERPINF2, such as Met(R6)-α2AP or Met(W6)-α2AP, which are important for studying polymorphic forms of the protein .

  The choice between these types depends on the research application, required specificity, and experimental design.

• How should researchers validate the specificity of SERPINF2 antibodies?

  Proper validation of SERPINF2 antibodies involves several critical steps:

  • Cross-reactivity Testing: Evaluate reactivity against other serpin family members. For example, some antibodies show less than 1% cross-reactivity with human Serpin A1, A3, A4, A5 and mouse Serpin C1, D1 .

  • Species Specificity Assessment: Determine cross-reactivity between species. For instance, AF1470 antibody shows approximately 50% cross-reactivity with mouse SERPINF2, while MAB1470 shows no cross-reactivity with mouse SERPINF2 .

  • Application-Specific Validation: Test the antibody in the intended application (Western blot, IHC, ELISA) using appropriate positive and negative controls.

  • Knockout/Knockdown Validation: When possible, use samples from knockout models or knockdown experiments as negative controls.

  • Multiple Antibody Approach: Use antibodies targeting different epitopes of SERPINF2 to confirm findings and reduce the risk of non-specific binding artifacts.

  These validation steps help ensure experimental results are specific to SERPINF2 and not confounded by cross-reactivity issues.

• What are the optimal conditions for SERPINF2 antibody storage and handling?

  To maintain antibody activity and specificity, the following storage and handling practices are recommended:

  • Long-term Storage: Store at -20 to -70°C for up to 12 months from the date of receipt .

  • After Reconstitution:

    • For short-term use (up to 1 month): Store at 2 to 8°C under sterile conditions

    • For longer storage (up to 6 months): Store at -20 to -70°C under sterile conditions

  • Freeze-Thaw Cycles: Use a manual defrost freezer and avoid repeated freeze-thaw cycles, which can degrade antibody quality .

  • Working Dilutions: Prepare fresh working dilutions on the day of use or store at 4°C for up to one week.

  • Reconstitution Medium: Follow manufacturer recommendations for the appropriate buffer system for reconstitution.

  Proper storage and handling are essential for maintaining antibody functionality and experimental reproducibility.

Advanced Research Questions

• How can SERPINF2 antibodies be used to study the role of alpha-2-antiplasmin in disease models?

  SERPINF2 antibodies have been instrumental in elucidating the role of alpha-2-antiplasmin in various disease models:

  • Cerebrovascular Disease: Research has demonstrated that SerpinF2-binding antibodies that reduce SerpinF2 activity can be used in methods for preventing or treating brain hemorrhage. This approach is based on the finding that reduction of alpha2-AP levels was responsible for the decreased hemorrhage observed with microplasmin treatment . In this context, antibodies can serve as both therapeutic agents and research tools.

  • Rheumatoid Arthritis: Studies have identified functional autoantibodies against serpins (including serpin E2) in RA patients that interfere with the inhibitory activity of these proteins, potentially facilitating joint destruction . SERPINF2 antibodies can be used to investigate similar mechanisms involving alpha-2-antiplasmin.

  • Fibrotic Conditions: Proteomic analysis using SERPINF2 antibodies has revealed that losartan treatment can reverse fibrotic changes in disease models, as validated by Western blotting of skin lysates from wild-type, untreated, and losartan-treated mice .

  • Cancer Metastasis: Research has shown that serpins can promote cancer metastasis by remodeling the tumor microenvironment. Antibodies targeting specific serpins can help elucidate these mechanisms .

  These applications demonstrate how SERPINF2 antibodies can advance our understanding of disease pathogenesis and potential therapeutic approaches.

• What methodological considerations are important when studying SERPINF2 polymorphic variants?

  When investigating polymorphic variants of SERPINF2 (such as Met(R6)-α2AP versus Met(W6)-α2AP), researchers should consider:

  • Variant-Specific Antibodies: Develop or select antibodies with demonstrated specificity for particular variants. Previous assays were limited to ELISAs that could only measure Met(R6)-α2AP, lacking reactivity against Met(W6)-α2AP .

  • Recombinant Protein Standards: Generate recombinant proteins expressing the different variants (e.g., Met(R6)-α2AP, Met(W6)-α2AP, and Asn-α2AP) to use as standards for assay development and validation .

  • Screening Methodology: Implement rigorous screening procedures to identify antibodies with the desired variant specificity. For example, using non-competitive ELISAs with plates coated with different α2AP variants .

  • Genotyping Correlation: When using these antibodies in human studies, correlate antibody-based measurements with genotyping data to confirm variant-specific detection.

  • Functional Assays: Complement antibody-based detection with functional assays to determine how polymorphic differences affect SERPINF2 activity, such as its rate of conversion by antiplasmin cleaving enzyme (APCE) .

  These considerations are crucial for accurately studying the functional consequences of SERPINF2 polymorphisms in research and clinical settings.

• How can antibody-based approaches be used to study SERPINF2-protease interactions?

  Antibody-based approaches offer valuable methods for investigating SERPINF2-protease interactions:

  • Epitope-Specific Antibodies: Antibodies recognizing specific structural determinants involved in protease binding can be used to block or modulate these interactions. For example, researchers produced a serpinE2-specific antibody (Ab11) recognizing a 12 amino acid peptide that is essential for LRP-mediated internalization of serpin/protease complexes .

  • Complex Formation Analysis: Antibodies can be used to detect and quantify the formation of SERPINF2-protease complexes in biological samples through techniques like co-immunoprecipitation or proximity ligation assays.

  • Functional Modulation Studies: Antibodies that neutralize SERPINF2 can be used to study the consequences of reduced inhibitory activity. For instance, Fab-4H9 (a Fab fragment of a murine monoclonal antibody) has been used to neutralize murine alpha2-AP, confirming that alpha2-AP depletion affects fibrinolytic activity .

  • Conformational Change Detection: Specialized antibodies that recognize conformational changes in SERPINF2 upon protease binding can provide insights into the mechanics of these interactions.

  • In vivo Imaging: Labeled antibodies can be used to track SERPINF2-protease interactions in vivo, providing spatial and temporal information about these processes in disease models.

  These approaches enable researchers to dissect the complex interactions between SERPINF2 and its target proteases in physiological and pathological contexts.

• What are the technical challenges and solutions in generating monoclonal antibodies against specific forms of SERPINF2?

  Generating monoclonal antibodies against specific forms of SERPINF2 presents several technical challenges:

  • Challenge: Producing Recombinant Variant Proteins
    Solution: Express recombinant forms of SERPINF2 variants in appropriate expression systems, such as Drosophila S2 cells, which have been successfully used to express Met(R6)-α2AP, Met(W6)-α2AP, and Asn-α2AP .

  • Challenge: Immunization Strategy for Variant-Specific Responses
    Solution: Employ strategic immunization protocols, such as using a mixture of variant forms (e.g., Met(R6)-α2AP and Met(W6)-α2AP) followed by specific boosting schedules. For example, subcutaneous injection of 10 μg α2AP in complete Freund's adjuvant, followed by intraperitoneal injection of 10 μg α2AP in incomplete Freund's adjuvant two weeks later, and final boosting with 10 μg α2AP in saline .

  • Challenge: Hybridoma Screening for Variant Specificity
    Solution: Implement parallel screening using one-site non-competitive ELISAs with plates coated with different variants to identify clones with the desired specificity profile .

  • Challenge: Cross-Reactivity Assessment
    Solution: Conduct comprehensive cross-reactivity testing against related serpins and SERPINF2 from different species using direct ELISAs and Western blots .

  • Challenge: Ensuring Functional Relevance
    Solution: Validate antibodies in functional assays to confirm they recognize biologically relevant epitopes and potentially modulate SERPINF2 activity.

  Addressing these challenges is essential for developing high-quality, variant-specific antibodies for SERPINF2 research.

• How can computational approaches complement antibody-based research on SERPINF2-antigen complexes?

  Computational approaches increasingly enhance antibody-based SERPINF2 research:

  • Sequence-to-Structure Prediction: Tools like AlphaFold-Multimer and RoseTTAFold can predict antibody-antigen complex structures from sequences, though with limitations. Studies show that better models exhibit more common PDB-like TERtiary Motifs (TERMs) at the antibody-antigen interface than poorer models .

  • Structural Bias Analysis: Computational analysis can identify structural biases in antibody-antigen interactions. For instance, analysis of TERMs can reveal whether predicted interactions resemble those found in non-antibody containing structures from the Protein Data Bank .

  • Epitope Mapping: Computational epitope mapping can guide antibody development by identifying accessible, stable regions on SERPINF2 that might serve as good targets for antibody binding.

  • Multiple Sequence Alignment (MSA) Analysis: Although MSA richness has been reported to produce better antibody-antigen models in some cases, research shows little correlation between MSA quality and model accuracy for antibody-antigen complexes (R² of 0.07 for paired MSA correlation) .

  • Contact Prediction: Computational methods can predict residues involved in contacts between antibodies and SERPINF2, informing experimental design and interpretation.

  These computational approaches, when integrated with experimental antibody-based research, provide a more comprehensive understanding of SERPINF2-antibody interactions and their functional implications.

Research Applications in Disease Models

• How can SERPINF2 antibodies be utilized to study the role of alpha-2-antiplasmin in cerebrovascular diseases?

  SERPINF2 antibodies offer powerful tools for investigating alpha-2-antiplasmin's role in cerebrovascular pathologies:

  • Therapeutic Potential Assessment: SerpinF2-binding antibodies that reduce SerpinF2 activity have shown promise in preventing or treating brain hemorrhage. This approach was developed based on the finding that reduction of alpha2-AP levels was the mechanism underlying the beneficial effects of microplasmin in reducing hemorrhage .

  • Mechanism Elucidation: Research has confirmed that "the reduction of haemorrhage reported in document D8 was due to the reduction of alpha2-AP levels," and that an "antibody capable of neutralising alpha2-AP would be suitable" as an alternative to microplasmin to achieve the same clinical effect .

  • Pathway Analysis: Alpha2-AP depletion, whether achieved through plasmin/miniplasmin or through neutralizing antibodies like Fab-4H9, leads to similar effects, confirming that the mechanism is primarily through alpha2-AP reduction rather than other proteolytic activities .

  • Validation Studies: Experiments with neutralizing antibodies have confirmed the hypothesis that "FII reduction was the result of alpha2-AP depletion and not of the proteolytic activity of injected plasmin" .

  • Translational Research: Board-reviewed evidence indicates that "the skilled person starting from the disclosure in document D8 and seeking an alternative means to deplete alpha2-AP (the mechanism underlying the effects of microplasmin), would have learned from document D3 that an antibody neutralising alpha2-AP (i.e., a SerpinF2-binding antibody that reduces SerpinF2 activity) would be suitable" .

  These applications demonstrate the critical role of SERPINF2 antibodies in advancing our understanding and treatment of cerebrovascular diseases.

• What techniques can be employed to study SERPINF2 expression and localization in tissue samples?

  Multiple antibody-based techniques can effectively analyze SERPINF2 expression and localization:

  • Immunohistochemistry (IHC): This technique reveals the spatial distribution of SERPINF2 in tissue sections. For example, SERPINF2 has been detected in mouse intestine using 5 μg/mL goat anti-mouse SERPINF2 antibody with HRP-DAB staining and hematoxylin counterstaining . Optimization typically involves:

    • Fixation method selection (perfusion fixed frozen sections vs. FFPE)

    • Antigen retrieval optimization

    • Antibody concentration titration (typically 1-10 μg/mL)

    • Appropriate detection system selection

  • Immunofluorescence: Offers higher resolution and multiplexing capabilities for co-localization studies of SERPINF2 with other proteins. Typical dilutions range from 1:50-1:100 for optimal staining .

  • In Situ Hybridization: Can be combined with immunohistochemistry to correlate mRNA expression with protein localization, particularly useful in tissues like liver, kidney, muscle, intestine, central nervous system, and placenta where SERPINF2 is expressed .

  • Quantitative Image Analysis: Modern digital pathology tools allow quantification of SERPINF2 staining intensity and distribution across different tissue compartments.

  • Laser Capture Microdissection: Combined with immunostaining, this technique enables isolation of SERPINF2-expressing cells for downstream molecular analysis.

  These techniques provide complementary information about SERPINF2 expression patterns in normal and pathological tissues.

• How can researchers develop ELISAs for specific detection of SERPINF2 variants?

  Developing variant-specific SERPINF2 ELISAs requires careful consideration of several factors:

  • Recombinant Protein Production: Generate purified recombinant forms of specific SERPINF2 variants (e.g., Met(R6)-α2AP, Met(W6)-α2AP, and Asn-α2AP) using appropriate expression systems like Drosophila S2 cells .

  • Antibody Selection/Generation: Previous assays were limited to an ELISA that could only measure Met(R6)-α2AP, lacking reactivity against Met(W6)-α2AP. To overcome this limitation, researchers generated monoclonal antibodies with selective reactivity against different variants .

  • Hybridoma Screening Strategy: Implement a screening procedure using one-site non-competitive ELISAs with plates coated with different SERPINF2 variants to identify clones with the desired specificity profile .

  • Sandwich ELISA Development: Pair capture and detection antibodies that together provide specificity for particular variants. Commercial antibody pairs can detect human SERPINF2 with sensitivity as low as 31.25 pg/ml and assay ranges of 31.25-2000 pg/ml .

  • Validation with Clinical Samples: Validate the ELISA using samples from individuals with known genotypes. With an allele frequency of 81% for the R allele, approximately 66% of study samples can be measured with Met(R6)-α2AP-specific ELISAs .

  • Cross-Reactivity Testing: Ensure the developed ELISA doesn't cross-react with other serpin family members or with SERPINF2 from other species unless desired .

  These methodological considerations are crucial for developing ELISAs that can accurately distinguish between SERPINF2 variants for research and potential clinical applications.

• What is the significance of functional autoantibodies against serpins in autoimmune diseases?

  Functional autoantibodies against serpins play important roles in autoimmune pathology:

  • Interference with Inhibitory Function: In rheumatoid arthritis (RA), autoantibodies against serpin E2 have been shown to interfere with its inhibitory activity toward serine proteases. Anti-serpin E2 autoantibodies isolated from RA sera reversed the inhibitory activity of serpin E2 by 70% .

  • Correlation with Disease Activity: Levels of anti-serpin E2 autoantibodies correlate with urokinase plasminogen activator (uPA) activity in vivo, suggesting a mechanistic link to disease progression .

  • Diagnostic Potential: Significantly higher levels of anti-serpin E2 autoantibodies were present in synovial fluid (28%) and serum (22%) from RA patients compared with osteoarthritis patients (0% and 6%, respectively) or healthy individuals (6% of sera) .

  • Joint Destruction Mechanism: By neutralizing the inhibitory function of serpins, these autoantibodies may permit unchecked proteolytic activity that contributes to joint destruction in RA .

  • Research Methods: These autoantibodies can be characterized using techniques including:

    • Modified SEREX technique with cDNA from disease tissues

    • Enzyme-linked immunosorbent assay for quantification

    • Functional assays measuring the impact on serpin inhibitory activity

    • In vivo correlation with protease activity

  Understanding these autoantibodies provides insights into disease mechanisms and potential therapeutic targets in autoimmune conditions.

• How can SERPINF2 antibodies contribute to therapeutic development for cerebrovascular diseases?

  SERPINF2 antibodies offer promising avenues for therapeutic development:

  • Target Validation: SERPINF2 antibodies have helped establish alpha-2-antiplasmin as a valid therapeutic target. Evidence shows that "the reduction of haemorrhage reported was due to the reduction of alpha2-AP levels" .

  • Mechanism-Based Drug Design: Research using antibodies has confirmed that "depletion of alpha2-AP can be obtained with both plasmin and miniplasmin... and alternatively with a Fab fragment neutralising murine alpha2-AP (Fab-4H9)" . This mechanistic understanding enables rational design of therapeutics.

  • Alternative Therapeutic Approaches: Studies demonstrate that "the skilled person seeking an alternative agent to microplasmin to achieve the same clinical effect would have learned... that an antibody capable of neutralising alpha2-AP would be suitable" .

  • Proof-of-Concept Studies: Experiments with neutralizing antibodies confirm the hypothesis that "FII reduction was the result of alpha2-AP depletion and not of the proteolytic activity of injected plasmin" , providing proof-of-concept for therapeutic antibody development.

  • Target Specificity: Unlike broader-acting agents, antibodies can specifically target alpha-2-antiplasmin without affecting other serpins, potentially reducing off-target effects .

  • Therapeutic Antibody Engineering: The knowledge gained from research with SERPINF2 antibodies informs the development of therapeutic antibodies with optimized properties for clinical applications.

  These applications highlight how SERPINF2 antibodies are not just research tools but also serve as stepping stones toward novel therapeutics for cerebrovascular diseases.