fam19a5a Antibody

What is FAM19A5 and what are its primary biological functions?

FAM19A5 (also known as TAFA5) is a novel secretory protein predominantly expressed in the brain. Recent research has elucidated that FAM19A5 plays a crucial role in synapse elimination by binding to LRRC4B, a postsynaptic adhesion molecule. When FAM19A5 binds to LRRC4B, it inhibits the interaction between LRRC4B and PTPRF (a presynaptic adhesion molecule), leading to synapse elimination . This mechanism is particularly significant in the context of Alzheimer's disease (AD), where excessive synapse loss contributes to cognitive decline. Additionally, research has demonstrated that FAM19A5 is potentially associated with the progression of AD, although its precise pathogenic role remains under investigation .

Where is FAM19A5 primarily expressed in the body, and how does this impact antibody targeting?

The tissue distribution of FAM19A5 has been a subject of scientific debate. Comprehensive studies using knockout and knockin mouse models have demonstrated that FAM19A5 transcript levels in the central nervous system are significantly higher than in any peripheral tissues . While some researchers have suggested that FAM19A5 is also highly expressed in adipose tissue and functions as an adipokine , contradictory evidence indicates that FAM19A5 protein levels in adipose and reproductive tissues were below detectable limits for Western blot analysis and enzyme-linked immunosorbent assay (ELISA) . This predominantly central nervous system expression pattern has important implications for antibody targeting, as therapeutic antibodies must cross the blood-brain barrier (BBB) to effectively engage with FAM19A5 in the brain.

What are the currently available types of FAM19A5 antibodies for research applications?

Several types of FAM19A5 antibodies have been developed for research purposes:

• Monoclonal Antibodies:

  • NS101: A high-affinity monoclonal antibody that targets specific epitopes (Arg52, Pro57, Arg58, and Arg59) of FAM19A5, with a KD value of 111 pM .

  • N-A5-Ab and C-A5-Ab: Chimeric chicken/human monoclonal antibodies that recognize epitopes formed at the N-terminal and C-terminal regions of FAM19A5, respectively .

• Polyclonal Antibodies:

  • Sheep anti-mouse FAM19A5 polyclonal antibody targeting amino acids 26-125 .

• Commercial ELISA Kits:

  • Sandwich ELISA kits for human FAM19A5 detection in various sample types including cell culture supernatant, cell lysate, plasma, serum, and tissue lysate .

What methodological approaches are effective for generating specific anti-FAM19A5 antibodies?

Generation of high-quality anti-FAM19A5 antibodies involves several sophisticated methods:

• Chicken Immunization Approach:

  • Immunization of chickens (Gallus gallus domesticus) with purified recombinant N-terminal His-tagged FAM19A5 protein.

  • RNA extraction from spleen, bursa of Fabricius, and bone marrow of immunized chickens.

  • cDNA synthesis and construction of a single-chain variable fragment (scFv) library using pComb3X-SS vector system.

  • Phage display technique for antibody selection with helper phage VCM13.

  • Multiple rounds of biopanning using recombinant N-HIS-FAM19A5-coated magnetic beads to identify high-affinity antibodies .

• Antibody Optimization Process:

  • Linking human Cĸ gene to anti-FAM19A5 antibody sequence for light-chain variable region.

  • Addition of CH1, CH2, and CH3 genes of human immunoglobulin isotype IgG1 to the heavy-chain variable region.

  • Transfection of vectors encoding anti-FAM19A5-IgG1 antibody into HEK293F cells.

  • Purification using Protein A beads .

  • Further deimmunization and optimization by amino acid substitution to generate improved antibodies (e.g., NS101 and SS01) .

• Epitope Mapping Strategy:

  • Dividing the FAM19A5 linear sequence into multiple groups (e.g., F1 to F6).

  • Alanine substitution mutagenesis to identify crucial residues for binding.

  • Custom synthesis of mutant peptides to elucidate key residues in the binding domain .

How can researchers validate the specificity and affinity of FAM19A5 antibodies?

Comprehensive validation of FAM19A5 antibodies requires multiple complementary approaches:

• Specificity Assessment:

  • Testing binding exclusively to FAM19A5 among the FAM19A family members using ELISA .

  • Using FAM19A5 knockout tissues as negative controls for antibody validation .

  • Performing Western blot analysis with recombinant FAM19A5 protein and tissue lysates.

  • Conducting immunoprecipitation experiments followed by mass spectrometry analysis.

• Affinity Determination:

  • Surface Plasmon Resonance (SPR) measurements to determine binding kinetics (association and dissociation rates) and equilibrium dissociation constant (KD) .

  • Competitive binding assays to evaluate relative affinities of different antibodies.

  • Isothermal titration calorimetry to characterize thermodynamic parameters of antibody-antigen interactions.

• Functional Validation:

  • Assessing the antibody's ability to inhibit FAM19A5-LRRC4B complex formation through competition assays .

  • Evaluating antibody effectiveness in disrupting the biological function of FAM19A5 in cell-based assays.

  • Testing antibody activity in relevant animal models of neurological disorders .

What are the pharmacokinetic properties of anti-FAM19A5 antibodies in animal models?

Understanding the pharmacokinetic behavior of anti-FAM19A5 antibodies is crucial for therapeutic development:

In a study with APP/PS1 mice (an Alzheimer's disease model), the FAM19A5 antibody exhibited the following pharmacokinetic properties after intravenous (IV) administration at 10 mg/kg:

• Plasma Pharmacokinetics:

  • Peak levels occurred immediately after administration.

  • Gradual decrease with a half-life of 6.8 days.

  • Prolonged systemic exposure facilitated delivery across the blood-brain barrier .

• Brain Pharmacokinetics:

  • Peak brain levels achieved approximately 1.25 days (30 hours) after IV injection.

  • Gradual decline over 28 days with a half-life of 17.3 days in the brain.

  • Evidence of successful penetration across the blood-brain barrier .

• Brain-to-Plasma Ratio:

  • The extended brain half-life (17.3 days) compared to plasma half-life (6.8 days) suggests brain accumulation and/or retention.

  • This favorable pharmacokinetic profile enables less frequent dosing regimens in preclinical efficacy studies .

What ELISA protocols are most effective for detecting FAM19A5 in biological samples?

A robust ELISA protocol for FAM19A5 detection typically involves:

• Plate Preparation:

  • Coating 96-well microplates with LRRC4B (453-576) protein diluted in 50 mM carbonate buffer (pH 9.6) to a final concentration of 1 µg/ml.

  • Overnight incubation at 4°C.

  • Washing twice with washing buffer (PBS with 0.05% Tween 20).

  • Blocking with 200 μl of blocking buffer (PBS with 1% BSA and 0.05% Tween 20) per well at 37°C for 1 hour .

• Sample Processing:

  • Adding 100 μl of standard solution and samples to each well.

  • Incubating at room temperature for 90 minutes.

  • Washing five times with washing buffer .

• Detection System:

  • Adding 100 μl of HRP-conjugated C-A5-Ab (anti-FAM19A5 antibody) diluted in blocking buffer to a final concentration of 0.2 μg/ml.

  • Incubating at 37°C for 1 hour.

  • Adding 100 μl of TMB solution and incubating at room temperature for 20 minutes.

  • Stopping the reaction with 100 μl of 1 N sulfuric acid.

  • Measuring optical density at 450 nm using a microplate reader .

• Modifications for Different Applications:

  • For measuring CSF NS101, using a pair of rabbit anti-human IgG heavy chain antibodies and HRP-conjugated goat α-human IgG kappa light chain antibodies.

  • For assessing binding affinity between FAM19A5 and LRRC4 family proteins, using various LRRC4/4B/4C deletion proteins as capture agents .

How can immunohistochemistry and immunocytochemistry be optimized for FAM19A5 detection?

Optimized protocols for FAM19A5 immunostaining include:

• Cell Preparation for Immunocytochemistry:

  • Seeding HEK293 cells onto fibronectin-coated plastic film coverslips at a density of 3 × 10^4 cells per well.

  • Co-transfecting cells with 150 ng of FLAG-tagged LRRC4B and FAM19A5 after 24-hour incubation.

  • Washing with ice-cold DPBS and fixing with 4% paraformaldehyde (PFA) in PBS for 20 minutes .

• Blocking and Antibody Incubation:

  • Blocking with buffer containing 3% bovine serum albumin (BSA) and 0.1% Triton X-100 in PBS for 30 minutes.

  • Incubating with primary antibodies overnight at 4°C.

  • Washing with DPBS and incubating with secondary antibodies and Hoechst solution for 1 hour at room temperature.

  • For antibody treatment studies, applying NS101 and recombinant proteins containing the FB domain for 30 minutes prior to fixation .

• Visualization and Analysis:

  • Capturing fluorescence images using a confocal microscope.

  • Mounting coverslips with mounting solution.

  • Processing images using Las X software.

  • Quantifying synaptic markers and their colocalization using ImageJ with the colocalization plugin .

What are the best practices for measuring FAM19A5-LRRC4B binding inhibition?

To effectively measure FAM19A5-LRRC4B binding inhibition:

• Plate Preparation:

  • Coating plates with LRRC4B(453-576) protein.

  • Adding the test sample (inhibitor) and FAM19A5 protein.

• Detection and Quantification:

  • Determining the degree of binding inhibition by detecting bound FAM19A5 using HRP-conjugated C-A5-Ab.

  • Calculating IC50 values to compare inhibitory potencies of different compounds (e.g., NS101 has an IC50 of 0.2 nM vs. FB protein with an IC50 of 8 nM) .

• Comparative Analysis:

  • Testing PTPRF-LRRC4B binding inhibition by coating plates with LRRC4B(36-576) protein.

  • Adding test samples and PTPRF protein.

  • Detecting bound PTPRF-hFc using an anti-human IgG Fc antibody .

How do different FAM19A5 antibodies affect cognitive function in Alzheimer's disease models?

FAM19A5 antibodies have demonstrated significant cognitive improvements in multiple AD mouse models:

How can researchers resolve contradictory findings regarding FAM19A5 expression in peripheral tissues?

Addressing contradictory findings regarding FAM19A5 expression requires systematic methodological approaches:

• Employ Multiple Detection Methods:

  • Combine transcript analysis (qPCR), protein detection (Western blot, ELISA), and immunohistochemistry.

  • Use multiple antibodies targeting different epitopes of FAM19A5.

  • Validate findings with genetic models (knockout/knockin) .

• Include Appropriate Controls:

  • Utilize FAM19A5 knockout tissues as negative controls.

  • Include brain tissue (known high expression) as positive control.

  • Perform antibody validation with recombinant FAM19A5 protein .

• Address Methodological Differences:

  • Some studies report high FAM19A5 expression in adipose tissue and characterize it as an adipokine , while others find minimal expression outside the brain .

  • Differences may arise from:

    • Antibody specificity issues (cross-reactivity with related proteins).

    • Different detection thresholds across methods.

    • Potential post-translational modifications affecting antibody recognition.

    • Sample preparation variables affecting protein stability or recovery .

• Quantitative Comparative Analysis:

  • Conduct side-by-side comparisons of FAM19A5 levels in brain versus peripheral tissues.

  • Perform absolute quantification using recombinant protein standards.

  • Evaluate expression ratios across tissues rather than isolated measurements .

What are the technical considerations for developing FAM19A5 antibodies as therapeutic agents?

Development of FAM19A5 antibodies as therapeutics requires careful consideration of several technical aspects:

• Blood-Brain Barrier Penetration:

  • Anti-FAM19A5 antibodies must cross the BBB to reach their target in the brain.

  • Studies have shown that systemically administered FAM19A5 antibodies can successfully penetrate the BBB, with peak brain levels achieved approximately 30 hours after IV injection .

  • Engineering strategies to enhance BBB penetration may include:

    • Utilizing receptor-mediated transcytosis mechanisms.

    • Reducing antibody size (e.g., single-chain variable fragments, nanobodies).

    • Conjugation with BBB shuttle peptides or molecules.

• Epitope Selection and Antibody Engineering:

  • Target epitopes that are critical for FAM19A5 function (e.g., NS101 targets Arg52, Pro57, Arg58, and Arg59, which are key residues for FAM19A5 binding to LRRC4B) .

  • Consider antibody format (full IgG vs. fragments) based on tissue penetration requirements.

  • Engineer for optimal pharmacokinetic properties, including half-life and tissue distribution.

  • Humanize or fully human antibodies to minimize immunogenicity in clinical applications .

• Dosing Regimen Optimization:

  • In mouse models, cognitive improvements have been observed with periodic IV administration of FAM19A5 antibodies.

  • The extended brain half-life of 17.3 days suggests potential for less frequent dosing.

  • Dose-response studies are essential to determine minimal effective dose .

• Target Engagement and Efficacy Markers:

  • Develop assays to monitor target engagement in the brain.

  • Identify peripheral biomarkers that correlate with CNS effects.

  • Consider combining with imaging techniques to visualize effects on synaptic density .

How might FAM19A5 antibodies be integrated with existing AD therapeutics?

The potential for combining FAM19A5 antibodies with current AD therapeutics offers promising avenues for enhanced treatment efficacy:

• Combination with Anti-Amyloid Antibodies:

  • Anti-amyloid antibodies like aducanumab have shown promise in reducing amyloid plaque burden but may have limited effects on restoring lost synapses.

  • FAM19A5 antibodies focus on preserving and restoring synapses through a different mechanism.

  • The combination could address both pathological protein accumulation and synaptic dysfunction simultaneously .

• Complementary Mechanisms with Tau-Targeted Therapies:

  • FAM19A5 levels increase in association with aging and tau accumulation.

  • Combining tau-targeted approaches with FAM19A5 inhibition could provide synergistic benefits by addressing both the cause and consequence of synaptic loss .

• Rational Timing of Combination Therapy:

  • Sequential therapy might be optimal, with initial amyloid/tau clearance followed by synaptic restoration via FAM19A5 inhibition.

  • Alternatively, simultaneous administration could prevent ongoing synapse loss while clearing pathological proteins .

• Biomarker-Guided Combination Strategies:

  • FAM19A5 levels in cerebrospinal fluid increase proportionally with aging and tau accumulation.

  • This biomarker could potentially guide decisions on when to initiate FAM19A5 antibody therapy in combination regimens .

What methodological advances are needed to better characterize the role of FAM19A5 in synapse elimination?

Advancing our understanding of FAM19A5's role in synapse elimination requires sophisticated methodological approaches:

• Advanced Imaging Techniques:

  • Super-resolution microscopy to visualize FAM19A5-LRRC4B interactions at synapses.

  • Live-cell imaging to track synapse elimination dynamics in the presence of FAM19A5.

  • Correlative light and electron microscopy to connect molecular interactions with ultrastructural changes .

• Single-Cell Analysis Approaches:

  • Single-cell transcriptomics to identify cell populations most affected by FAM19A5.

  • Spatial transcriptomics to map FAM19A5 expression patterns in relation to synaptic markers.

  • Patch-clamp electrophysiology combined with molecular profiling to link FAM19A5 activity with functional synaptic changes .

• In Vivo Monitoring Systems:

  • Development of FAM19A5 biosensors for real-time monitoring of protein levels and activity.

  • Integration with in vivo electrophysiology to correlate FAM19A5 levels with synaptic function.

  • PET ligands targeting FAM19A5 for non-invasive imaging in animal models and humans .

• Systems Biology Integration:

  • Network analysis to place FAM19A5 within broader synapse elimination pathways.

  • Computational modeling of FAM19A5-LRRC4B-PTPRF interactions to predict intervention points.

  • Multi-omics approaches to characterize downstream effects of FAM19A5 inhibition .

What are the challenges in developing sensitive assays for monitoring FAM19A5 levels in clinical settings?

Development of clinical assays for FAM19A5 faces several technical challenges:

• Assay Sensitivity Requirements:

  • FAM19A5 may be present at low concentrations in accessible biofluids (e.g., blood, CSF).

  • Ultra-sensitive detection methods such as Single Molecule Array (Simoa) or immuno-PCR may be required.

  • Sample preparation protocols must minimize protein loss and degradation .

• Specificity Considerations:

  • Cross-reactivity with other FAM19A family members must be eliminated.

  • Potential interference from binding partners (e.g., LRRC4B) in biological samples.

  • Validation using samples from FAM19A5 knockout models as negative controls .

• Sample Matrix Challenges:

  • Matrix effects from blood or CSF can affect assay performance.

  • Pre-analytical variables (collection, processing, storage) must be standardized.

  • Reference ranges need to be established for different age groups and disease states .

• Clinical Validation Requirements:

  • Correlation of FAM19A5 levels with disease severity and progression.

  • Evaluation of FAM19A5 as a pharmacodynamic biomarker for anti-FAM19A5 therapies.

  • Assessment of FAM19A5 levels in different neurological and metabolic conditions to establish specificity for AD .

Table 1: Comparison of Key FAM19A5 Antibodies and Their Properties

Table 2: Pharmacokinetic Properties of FAM19A5 Antibody in APP/PS1 Mice

Table 3: Cognitive Effects of FAM19A5 Antibody Treatment in AD Mouse Models

Table 4: ELISA Protocol Components for FAM19A5 Detection