pof5 Antibody

What is PFDN5 and what biological functions does it serve?

PFDN5 (prefoldin subunit 5) is a component of the prefoldin hexameric protein complex exclusively found in archaea and eukaryotes. It functions primarily as a molecular chaperone that binds to and stabilizes unfolded target polypeptides, subsequently delivering them to group II chaperonins to complete the folding process. This action prevents misfolding of newly synthesized polypeptides . PFDN5 is expressed in a wide variety of tissues, including neuronal cells, and plays a protective role against cell death. Genetic disruption of PFDN5 in mice has been shown to cause retinal degeneration, highlighting its importance in maintaining ocular tissue integrity . Additionally, PFDN5 protects cells from aggregated protein-induced cell death, suggesting its role in cellular stress responses and homeostasis .

How are anti-PFDN5 antibodies detected in research settings?

The primary method for detecting anti-PFDN5 antibodies in research settings is Enzyme-Linked Immunosorbent Assay (ELISA). A standardized protocol involves:

• Coating 96-well plates with 1 μg/mL of recombinant human PFDN5

• Adding diluted serum samples (typically 1:200 dilution)

• Incubating for 2 hours

• Adding appropriate secondary antibodies (such as goat anti-human IgG)

• Measuring optical density at 450 nm absorbance

For detecting PFDN5 protein itself (rather than antibodies against it), commercially available ELISA kits can be used. The methodology allows for quantitative assessment of anti-PFDN5 antibody levels, enabling comparison between different patient groups or experimental conditions.

What are the primary research applications of PFDN5 and anti-PFDN5 antibodies?

The primary research applications include:

• Biomarker identification: Anti-PFDN5 antibodies serve as potential biomarkers for uveitis in ankylosing spondylitis patients

• Disease prediction: Elevated levels may predict future development of uveitis in susceptible populations

• Cellular protection studies: Investigating the protective role of PFDN5 against apoptosis in retinal cells

• Protein folding research: Understanding the chaperone functions of prefoldin complexes

• Autoimmune disease research: Exploring autoantibody signatures in various inflammatory conditions

These applications span from clinical diagnostics to fundamental cellular biology research, making PFDN5 and its antibodies valuable tools in multiple research domains.

In which biological samples can PFDN5 and anti-PFDN5 antibodies be detected?

PFDN5 and anti-PFDN5 antibodies can be detected in:

• Serum: The most common sample type for measuring both PFDN5 protein and anti-PFDN5 antibodies in human studies

• Ocular tissues: PFDN5 expression has been observed in retinal cells and ocular lesions in animal models

• Cell cultures: In vitro studies using cell lines such as ARPE-19 (retinal pigment epithelium cells) can be used to investigate PFDN5 expression and function

• Animal model tissues: Expression has been studied in tissues from curdlan-treated SKG mice, particularly in ocular tissues

The detection methods vary depending on the sample type, with ELISA being preferred for serum samples and immunohistochemistry or western blotting being more suitable for tissue samples.

How do anti-PFDN5 antibody levels correlate with uveitis in ankylosing spondylitis?

Research has demonstrated a significant correlation between anti-PFDN5 antibody levels and the presence of uveitis in ankylosing spondylitis (AS) patients. In a US-based cohort, high-density protein microarray analysis revealed increased antibodies to PFDN5 specifically in AS patients with uveitis compared to those without uveitis and patients with other autoimmune diseases . This finding was confirmed in a Korean cohort using ELISA, which showed significantly higher levels of anti-PFDN5 antibodies in AS patients with uveitis compared to those without uveitis .

The area under curve (AUC) of anti-PFDN5 reactivity (cut-off value: 28.95) was 1.00 when comparing AS patients with and without uveitis, suggesting excellent discrimination ability . Importantly, not only anti-PFDN5 antibodies but also PFDN5 protein itself showed elevated serum concentrations in AS patients with uveitis .

This correlation suggests that anti-PFDN5 antibodies could serve as a specific biomarker for uveitis in AS patients rather than as a general marker of AS disease activity, as no correlation was found between anti-PFDN5 levels and the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) .

What are the optimal experimental conditions for ELISA detection of anti-PFDN5 antibodies?

For optimal ELISA detection of anti-PFDN5 antibodies, researchers should consider the following protocol specifications:

• Plate coating: Use 1 μg/mL of recombinant human PFDN5 as the coating antigen

• Sample dilution: Dilute serum samples 1:200 in appropriate buffer

• Incubation time: Incubate diluted samples for 2 hours at room temperature

• Secondary antibody: Use goat anti-human IgG for human samples

• Detection: Measure optical density at 450 nm absorbance

For PFDN5 protein detection, commercially available ELISA kits (e.g., Product No. SEE738Hu, USCN Life Science Inc.) can be used according to manufacturer's instructions .

Critical experimental variables to control include:

• Consistent plate coating concentration

• Standardized incubation times and temperatures

• Inclusion of appropriate positive and negative controls

• Uniform washing procedures to minimize background

• Parallel standard curves for quantification

These conditions ensure reproducible and reliable results when measuring anti-PFDN5 antibody levels across different experimental groups.

What animal models are suitable for studying PFDN5-related pathologies?

The curdlan-induced SKG mouse model has been validated for studying PFDN5-related pathologies, particularly in the context of uveitis. This model offers several advantages:

• Disease progression: Following curdlan injection, SKG mice develop anterior uveitis by week 8 post-injection, with all treated mice showing histologically confirmed uveitis by week 16

• Biomarker correlation: The levels of anti-PFDN5 antibodies increase over time in sera of curdlan-treated SKG mice, correlating with disease progression

• Tissue expression: Increased expression of PFDN5 and apoptosis is observed in ocular lesions of treated mice

• Imaging compatibility: The model is suitable for advanced imaging techniques such as PET-MRI scanning to monitor disease progression

Key experimental considerations for this model include:

• Timing of assessments (early anterior uveitis at week 8, progressing to pan-uveitis by week 16)

• Appropriate controls (PBS-treated SKG mice)

• Serial sampling for monitoring antibody level changes over time

• Combined ophthalmic examination and histologic confirmation

While this model primarily develops anterior uveitis (similar to typical AS patients), inflammation may extend to the posterior segment by week 16, resulting in pan-uveitis, which should be considered when interpreting results .

How does PFDN5 expression relate to cellular apoptosis in ocular tissues?

Research indicates that PFDN5 plays a protective role against apoptosis in ocular tissues. In vitro experiments have demonstrated that knockdown of PFDN5 in ARPE-19 cells (a human retinal pigment epithelial cell line) results in increased apoptosis, suggesting that PFDN5 normally functions to protect against cell death .

This protective mechanism is consistent with the known functions of the prefoldin complex:

• PFDN binds to and stabilizes unfolded target polypeptides

• It delivers these polypeptides to group II chaperonins to complete proper folding

• This process prevents protein misfolding and aggregation

• PFDN protects cells from aggregated protein-induced cell death

In the curdlan-induced SKG mouse model of uveitis, increased expression of PFDN5 was observed in ocular lesions, suggesting that PFDN5 expression may be upregulated in response to inflammatory stress as a protective mechanism . The elevated PFDN5 levels detected in serum of AS patients with uveitis further support this hypothesis .

These findings suggest that PFDN5 upregulation may represent a compensatory mechanism to protect retinal cells from apoptosis in the context of inflammatory eye disease.

How can researchers differentiate between cause and effect when studying elevated anti-PFDN5 antibodies in disease?

Differentiating between causative roles and secondary effects when studying elevated anti-PFDN5 antibodies presents several methodological challenges that researchers should address:

• Temporal studies:

  • In the SKG mouse model, anti-PFDN5 antibody levels increased over time, correlating with disease progression

  • By week 8, some mice had not yet developed visible uveitis, while all showed histological evidence by week 16

  • The increasing antibody levels preceding full disease manifestation suggest potential predictive value

• Functional studies:

  • Knockdown experiments of PFDN5 in ARPE-19 cells resulted in increased apoptosis

  • This indicates PFDN5 has a protective role against cell death rather than a pathogenic one

  • The presence of anti-PFDN5 antibodies may interfere with this protective function

• Experimental approaches to establish causality:

  • Passive transfer experiments with purified anti-PFDN5 antibodies

  • Neutralization studies using recombinant PFDN5 protein

  • PFDN5 knockout or overexpression in animal models

  • Time-course studies correlating antibody appearance with disease onset

When interpreting results, researchers should consider that elevated anti-PFDN5 antibodies might represent: (1) a causative factor in disease pathogenesis, (2) a secondary response to PFDN5 release during tissue damage, or (3) a biomarker without direct pathogenic involvement.

What validation steps are essential before using anti-PFDN5 antibodies in research?

Before using anti-PFDN5 antibodies in research, the following validation steps are essential:

• Specificity testing:

  • Western blot analysis to confirm binding to PFDN5 of appropriate molecular weight

  • Competitive inhibition with recombinant PFDN5 protein

  • Testing against knockout/knockdown samples as negative controls

  • Cross-reactivity assessment with related proteins in the prefoldin family

• Sensitivity determination:

  • Establish detection limits using serial dilutions of recombinant PFDN5

  • Determine optimal working dilutions for different applications (ELISA, Western blot, immunohistochemistry)

  • Validate sensitivity across different sample types (serum, tissue lysates)

• Reproducibility assessment:

  • Perform intra-assay and inter-assay variability tests

  • Evaluate lot-to-lot consistency if using commercial antibodies

  • Test stability under various storage conditions

• Application-specific validation:

  • For ELISA applications: optimize coating concentration, blocking conditions, and detection systems

  • For immunoprecipitation: verify efficient pull-down of PFDN5 from complex mixtures

  • For immunohistochemistry: confirm specific staining pattern in known PFDN5-expressing tissues

• Reference standard establishment:

  • Create standard curves using purified recombinant PFDN5

  • Develop positive and negative control samples for each experimental run

Thorough validation ensures reliable results and minimizes the risk of false positives or negatives in PFDN5-related research.

How should researchers interpret conflicting anti-PFDN5 antibody data from different patient cohorts?

When faced with conflicting anti-PFDN5 antibody data from different patient cohorts, researchers should implement a systematic approach to interpretation:

• Methodological comparisons:

  • Assess differences in antibody detection methods (protein microarrays vs. ELISA)

  • Compare coating antigens (recombinant sources, purification methods)

  • Evaluate cut-off value determination methods

  • Examine differences in sample processing and storage

• Cohort characteristic analysis:

  • Compare demographic factors (age, sex, ethnicity)

  • Assess disease duration and severity

  • Evaluate concurrent medications and treatments

  • Consider genetic background variations (particularly HLA-B27 status in AS patients)

• Disease heterogeneity consideration:

  • Uveitis in AS can vary in presentation and severity

  • Consider anterior vs. pan-uveitis distinctions

  • Evaluate acute vs. chronic uveitis cases

  • Assess uveitis activity at the time of sampling

• Statistical approaches:

  • Meta-analysis of multiple cohorts when possible

  • Stratification of patients based on relevant clinical parameters

  • Multivariate analysis to identify confounding factors

  • Power calculations to ensure adequate sample sizes

• Validation in independent cohorts:

  • The correlation between anti-PFDN5 antibodies and uveitis in AS was validated in both US-based and Korean cohorts despite potential population differences

  • This cross-validation strengthens the reliability of the finding

What experimental controls are essential for anti-PFDN5 antibody research?

Robust experimental controls are crucial for reliable anti-PFDN5 antibody research:

• Positive controls:

  • Serum samples from confirmed AS patients with uveitis known to have high anti-PFDN5 antibody levels

  • Recombinant PFDN5 protein at known concentrations for standard curves

  • Tissue samples with validated PFDN5 expression (for immunohistochemistry)

• Negative controls:

  • Serum from healthy individuals

  • Samples from AS patients without uveitis

  • Isotype-matched irrelevant antibodies

  • Secondary antibody-only controls to assess non-specific binding

• Disease specificity controls:

  • Samples from patients with other autoimmune diseases (e.g., rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis)

  • Samples from patients with non-AS uveitis to determine specificity to AS-associated uveitis

• Procedural controls:

  • For ELISA: blank wells (no sample), substrate-only wells

  • For immunoprecipitation: pre-immune serum, non-specific antibodies of the same isotype

  • For tissue staining: absorption controls using recombinant PFDN5

• Validation controls:

  • PFDN5 knockdown or knockout samples

  • Dose-response experiments with recombinant PFDN5 inhibition

  • Cross-validation using different detection methods

Implementing these controls helps ensure that observed differences in anti-PFDN5 antibody levels are truly related to the biological phenomenon being studied rather than technical artifacts or non-specific effects .

What is the current understanding of PFDN5 as a biomarker in inflammatory diseases?

Current research positions PFDN5 and anti-PFDN5 antibodies as promising biomarkers in inflammatory diseases, particularly in ankylosing spondylitis-associated uveitis:

These findings represent a significant advance in biomarker discovery for AS-associated uveitis, for which no diagnostic biomarkers had previously been identified. Further research is needed to determine the broader applicability of PFDN5-related biomarkers across different inflammatory and autoimmune conditions.

How does PFDN5 expression compare across different autoimmune and inflammatory conditions?

The comparative analysis of PFDN5 expression and anti-PFDN5 antibody levels across different autoimmune and inflammatory conditions provides important insights into disease specificity:

• Ankylosing spondylitis with uveitis:

  • Significantly elevated levels of anti-PFDN5 antibodies

  • Significantly elevated serum PFDN5 protein levels

  • Increased expression of PFDN5 in ocular tissues (based on animal model data)

• Ankylosing spondylitis without uveitis:

  • Low levels of anti-PFDN5 antibodies

  • Low serum PFDN5 protein levels

  • Comparable to healthy controls

• Other autoimmune conditions:

  • Rheumatoid arthritis: Low anti-PFDN5 antibody levels

  • Juvenile idiopathic arthritis: Low anti-PFDN5 antibody levels

  • Psoriatic arthritis: Low anti-PFDN5 antibody levels

  • Pulmonary arterial hypertension: Low anti-PFDN5 antibody levels

Based on human protein microarray analysis of the MADGC cohort, anti-PFDN5 antibody reactivity specifically distinguished AS patients with uveitis from those with other diseases, including AS without uveitis .