PS/HR Antibody

PS-targeting antibodies:

Phosphatidylserine (PS) is a negatively charged phospholipid normally sequestered to the inner leaflet of the cell membrane in healthy cells. PS-targeting antibodies recognize PS when it becomes externalized during apoptosis or in pathological conditions like cancer and viral infections .

Primary applications include:

• Detection and imaging of apoptotic cells

• Cancer immunotherapy (blocking PS-mediated immunosuppression)

• Antiviral research (targeting PS-expressing viral envelopes)

• Studying immunomodulatory pathways

HR-targeting antibodies:

Anti-HR antibodies detect the protein HR lysine demethylase and nuclear receptor corepressor. In humans, the canonical protein has 1189 amino acid residues with a mass of 127.5 kDa . HR is a histone demethylase that specifically demethylates mono- and dimethylated 'Lys-9' of histone H3 .

Primary applications include:

• Studying epigenetic regulation

• Research related to alopecia (hair loss)

• Nuclear receptor signaling investigations

What experimental techniques are most commonly used with PS/HR antibodies?

PS/HR antibodies are versatile tools employed across multiple experimental platforms:

For PS-targeting antibodies specifically, techniques like flow cytometry for detecting apoptotic cells and in vivo imaging for tumor localization are particularly valuable .

How should PS/HR antibodies be properly stored and handled for optimal results?

Proper storage and handling of PS/HR antibodies is critical for maintaining their functionality:

• Store at -20°C or -80°C upon receipt

• Avoid repeated freeze-thaw cycles that can denature antibodies

• For the exemplar PS/HR antibody (CSB-PA321891ZA01VAA), the optimal storage buffer contains 0.03% Proclin 300 as preservative, 50% Glycerol, and 0.01M PBS at pH 7.4

• When working with the antibody, maintain cold chain integrity by keeping on ice

• For long-term experiments, consider aliquoting the antibody to minimize freeze-thaw cycles

• Document lot numbers and validation data for experimental reproducibility

• For prolonged work sessions, antibodies can be temporarily stored at 4°C (typically stable for 1-2 weeks)

The stability of PS-targeting antibodies specifically may be affected by formulation conditions, so testing buffer compatibility prior to critical experiments is recommended .

What controls are essential when using PS/HR antibodies in experimental design?

Rigorous experimental design requires appropriate controls:

Positive controls:

• Cell lines known to express the target protein (reference BioGPS and The Human Protein Atlas for information about relative abundance in different cell types)

• Recombinant protein standards

• For PS-targeting antibodies: apoptotic cells with confirmed PS externalization

• Samples treated with known inducers of the post-translational modification being studied

Negative controls:

• Cell lines lacking target expression

• Isotype-matched non-specific antibodies

• For PS antibodies: healthy cells without PS externalization

• Blocking peptide competition assays to confirm specificity

When studying antibody binding to PS in tumor microenvironments, researchers validated specificity by comparing endothelial cells in normal organs (negative) versus tumor blood vessels (positive) .

How can researchers optimize PS/HR antibody protocols for detecting post-translational modifications?

Detection of post-translationally modified proteins requires specific considerations:

• Targeted treatments: Apply specific treatments that activate the post-translational modification of interest. Resources like PhosphoSitePlus® provide information on treatments that modulate specific modifications in different cell models .

• Extraction conditions: Optimize lysis buffers to preserve the modification of interest (e.g., phosphatase inhibitors for phosphorylation studies).

• Gel selection: For HR protein (127.5 kDa), a 3-8% Tris-Acetate gel may provide better resolution than standard gels .

• Blocking conditions: Empirically determine optimal blocking conditions that don't interfere with antibody recognition of the modified epitope.

• Signal amplification: For low-abundance modifications, consider using signal amplification methods or more sensitive detection systems.

• Validation approach: Validate findings using complementary approaches:

  • Site-directed mutagenesis to confirm specific residues

  • Pharmacological inhibitors/activators of the modification

  • Parallel analysis with multiple antibodies recognizing different epitopes of the same modified protein

For studying HR lysine demethylase activity specifically, controls should include both mono- and dimethylated 'Lys-9' of histone H3 substrates .

What methodologies ensure PS/HR antibody specificity in complex biological samples?

Ensuring antibody specificity is critical for obtaining reliable results:

Analytical validation approaches:

• Combine computational-experimental approaches: As demonstrated in glycan-binding antibody research, integrate high-throughput techniques for characterizing structure and specificity with computational modeling :

  • Quantitative glycan microarray screening to determine KD values

  • Site-directed mutagenesis to identify key residues in antibody combining sites

  • STD-NMR to define glycan-antigen contact surface

  • Computational docking and molecular dynamics simulation

• Antibody validation hierarchy:

  • Genetic approaches: Test against knockout/knockdown samples

  • Orthogonal methods: Verify findings with independent techniques

  • Independent antibodies: Use multiple antibodies targeting different epitopes

  • Expression of tagged proteins: Compare antibody detection with tag detection

  • Immunoprecipitation followed by mass spectrometry

• Advanced specificity testing:

  • For PS antibodies: Competitive binding assays with annexin V (which binds PS via a different epitope)

  • Testing against physiologically relevant competing molecules

Research shows that PS-targeting antibodies like 9D2 and annexin V do not compete with one another for PS, indicating different binding epitopes, and 9D2 binding to PS does not require calcium, unlike annexin V .

How are PS-targeting antibodies utilized to study tumor microenvironments?

PS-targeting antibodies have become valuable tools in cancer research due to the aberrant exposure of PS in tumor microenvironments:

Research applications:

• Tumor vasculature targeting: PS is abnormally exposed on tumor endothelial cells. PS-targeting antibodies like 3G4 and 2aG4 localize specifically to tumor blood vessels but not normal tissue endothelium .

• Blocking immunosuppression: PS exposure contributes to immunosuppressive signals in tumors. PS-targeting antibodies can block PS-mediated immunosuppression and potentially enhance anti-tumor immune responses .

• Modulating macrophage phenotype: Treatment with 3G4 caused tumor-associated macrophages to adopt an M1-like phenotype, which is more anti-tumorigenic .

• Enhancing effects of standard therapies: PS exposure increases after radiation and chemotherapy. Studies in animal models showed enhanced anti-tumor responses when PS-targeting antibodies were combined with standard chemotherapies for breast, hepatocellular, and pancreatic cancers .

• Combination with radiation therapy: In A549 human lung tumor models (which are relatively radio-resistant), mice treated with 2aG4 and radiation had significantly slower tumor growth compared to either treatment alone .

Methodological considerations:

• Include proper imaging controls when assessing PS exposure in tumors

• Consider the timing of antibody administration relative to other treatments (radiation, chemotherapy)

• Monitor immune cell infiltration and phenotype changes (particularly dendritic cell maturation)

What are the recent advances in using PS-targeting antibodies for viral detection and inhibition?

PS-targeting antibodies show promise in antiviral research:

Mechanistic insights:

• Viral infection and PS exposure: Surface exposure of PS occurs as a consequence of viral infection through virus-induced apoptosis, resulting in translocation of PS from the inner to the outer layer of infected cells' plasma membranes .

• Antibody-mediated viral clearance: Antibodies binding exposed PS can limit viral infections by:

  • Initiating removal of enveloped viruses from the bloodstream

  • Inducing antibody-dependent cellular cytotoxicity (ADCC) against virus-infected cells

• Advantages over direct antiviral antibodies:

  • Specificity for infected cells rather than viral proteins

  • Independence from virus escape mutations that change viral epitopes

Research applications:

• PGN401, a PS-targeting antibody, has been studied with hepatitis C virus and reached clinical trials

• Efficacy demonstrated in guinea pig models of Pichindé virus (a model for Lassa fever)

• Binding to Ebola virus (EBOV) virions and EBOV-infected cells has been evaluated

Experimental considerations:

• Include appropriate controls for distinguishing PS exposure due to viral infection versus other causes of apoptosis

• Consider calcium-dependence of different PS-binding reagents (some require calcium, others don't)

• Use combination of virological and immunological readouts to fully assess efficacy

How can high-throughput antibody screening methods be integrated with PS/HR antibody research?

Modern antibody research increasingly incorporates high-throughput methods:

Functional screening approaches:

• Genotype-phenotype linked screening: A novel method compatible with Next-Generation Sequencing (NGS) to rapidly identify antigen-specific clones has been developed. This approach:

  • Allows functional screening by selecting strong binders

  • Enables narrowing down large numbers of clones to identify those with desired properties

  • Can be applied to discover antibodies with high affinity (demonstrated with influenza virus where a strong affinity of Kd ≃ 5.66×10^-10 M was achieved)

• Integration with computational methods:

  • Combine high-throughput experimental data with computational modeling

  • Use computational screening of selected antibody 3D-models against relevant glycomes to validate specificity

• Multi-platform assessment:

  • Analyze antibody performance across multiple experimental platforms simultaneously

  • Apply machine learning to predict antibody properties from sequence data

Practical implementation:

• Automation of experiments can overcome limitations of well-based systems

• Robotic systems enable processing of larger cell numbers and increase throughput

• For infectious disease research, automation helps overcome biosafety limitations

This combined approach accelerates the discovery of useful monoclonal antibodies for various diseases, with broad implications for vaccine development .

What methodological considerations apply when using PS/HR antibodies across different formulation conditions?

Antibody performance can vary significantly across formulation conditions:

Buffer compatibility issues:

• Common formulation challenges: While PBS (pH 7.4) is commonly used for antibody work, histidine-based buffers are the most prevalent in therapeutic antibody formulations, followed by acetate, with pH values around 5.7 ± 0.4 .

• Stabilization strategies: For assays like Affinity-capture self-interaction nanoparticle spectroscopy (AC-SINS), histidine buffer induces gold particle aggregation of both unconjugated AuNP and immunogold conjugates. Solutions include:

  • Using PEG2000 as a stabilizing reagent (1 μg/mL at quenching step after conjugation)

  • Additional supplementation (0.1 μg/mL) in final reaction mixtures

• Optimization across multiple conditions: Recent advances allow screening antibodies in various formulation conditions:

  • Histidine buffer at pH 6.0

  • Citrate buffer at pH 6.0

  • Acetate buffer at pH 5.0

Practical recommendations:

• Test antibody performance in the specific buffer system that will be used for the final application

• Consider how buffer conditions might affect epitope accessibility or antibody binding kinetics

• For PS antibodies specifically, calcium requirements may vary depending on the antibody clone

• Document buffer composition, pH, and ionic strength in experimental reports for reproducibility

These methodological considerations are particularly important when transitioning from research applications to therapeutic development, where formulation conditions become critical for stability and efficacy .

How do antibody responses against PS differ between symptomatic and asymptomatic SARS-CoV-2 infections?

Understanding antibody responses in different COVID-19 severity groups provides valuable insights:

Key findings from clinical studies:

• Antibody dynamics in hospitalized patients:

  • Patients produce strong antibody responses to SARS-CoV-2 with high correlation between different viral antigens (spike protein and nucleoprotein)

  • Antibody classes (IgA, IgG, IgM and neutralizing antibodies) show coordinated responses

  • Antibody peak is reached by 3 weeks from hospital admission followed by a sharp decrease

  • No difference was observed in any antibody parameter, including neutralizing antibodies, between recovered patients and those with fatal outcomes

• Asymptomatic response characteristics:

  • Only few asymptomatic subjects developed antibodies at detectable levels

  • At hospital admission, 35.7% of patients were negative for S1 IgM, 26.2% for S1 IgA, 30.9% for S1 IgG

  • By day 6 of sample collection, 97.6% of subjects were positive to all assays

• Neutralizing antibody dynamics:

  • Neutralizing antibodies were found in all patients, with titers ranging from 10 to 5120

  • At baseline, titers were 40.8 (95%CI 1.3–1296.4) in recovered patients vs 24.4 (95%CI 0.2–3093.8) in deceased patients

  • By day 6, titers increased to 427.9 (95% CI 29.0–6321.5) in recovered patients vs 226.3 (95% CI 12.1–4228.2) in those deceased

  • In recovered patients, titers plateaued until day 18-20, then declined by day 27-30

Methodological considerations:

• Use appropriate serological tests: ELISA-based assays with different coatings (S1, S1+S2, S1-S2, RBD) or nucleoprotein

• Complement with virus neutralization assays (considered gold standard for measuring functional antibodies)

• Statistical analysis should account for time course variations in antibody levels

• Consider ethical approval requirements for such studies

What considerations apply when using PS-targeting antibodies for UTI diagnostics research?

While traditional PS/HR antibody applications focus on cancer and viral infections, researchers are exploring novel applications in urinary tract infection (UTI) diagnostics:

Research challenges in UTI detection:

• Multi-organism infections: UTIs often involve multiple pathogens simultaneously. Traditional diagnostics may identify only the dominant organism, leading to incomplete treatment and recurrent infections .

• Need for comprehensive detection: Advanced diagnostic approaches targeting PS exposure on bacteria or infected epithelial cells could potentially offer more complete pathogen identification .

• Antibiotic resistance profiling: Specialized testing that identifies both the infecting organisms and their antibiotic sensitivities/resistances is crucial for effective treatment .

Methodological approaches:

• Develop multiplex antibody panels targeting PS and other bacterial markers

• Incorporate resistance gene detection into antibody-based diagnostic workflows

• Validate findings through multiple complementary detection methods

• Consider serial testing to monitor treatment response and confirm infection clearance

While direct evidence for PS-targeting antibodies in UTI diagnostics is limited in the provided search results, the principles of PS externalization during cellular stress make this an intriguing area for further investigation .