KIF20B Antibody

What is KIF20B and why are antibodies against it valuable in research?

KIF20B (Kinesin Family Member 20B), originally named M-phase phosphoprotein 1 (MPP1), is a plus-end-directed kinesin-related protein that exhibits microtubule-binding and -bundling properties as well as microtubule-stimulated ATPase activity. It functions as a slow molecular motor that moves toward the plus-end of microtubules and is required for completion of cytokinesis . KIF20B plays crucial roles in neuronal development and cellular division.

Anti-KIF20B antibodies are valuable research tools because they:

• Enable visualization of KIF20B's dynamic subcellular localization throughout the cell cycle

• Serve as reference standards for anti-KIF20B autoantibody detection in clinical samples

• Facilitate studies on KIF20B's role in developmental cell biology and disease pathogenesis

• Allow for identification of specific cell populations expressing KIF20B in various tissues

The development of well-characterized antibodies like the monoclonal antibody 10C7 has addressed previous limitations in KIF20B research, providing reliable tools for investigating this important motor protein .

What are the different types of KIF20B antibodies available for research?

Several types of KIF20B antibodies have been developed for research applications:

• Monoclonal antibodies:

  • Mouse IgG1 monoclonal antibody 10C7, raised against amino acids 1732-1747 of human KIF20B (peptide sequence QPKRAKRKLYTSEISS)

  • Shows reactivity with human and monkey samples

• Polyclonal antibodies:

  • Rabbit polyclonal antibodies (e.g., 27269-1-AP) generated against KIF20B fusion proteins

  • Typically recognize various epitopes across the KIF20B protein

• Human autoantibodies:

  • Found in patient serum samples from individuals with idiopathic ataxia, SLE, and other conditions

  • Serve as valuable reference standards in certain research contexts

These different antibody types offer complementary advantages depending on the research application, with monoclonals providing high specificity and polyclonals often offering enhanced sensitivity through recognition of multiple epitopes.

How does KIF20B localize in cells throughout the cell cycle?

KIF20B exhibits distinct localization patterns that change dynamically throughout the cell cycle, as revealed by immunofluorescence studies with antibodies like 10C7:

• Interphase:

  • Mainly localized to a proportion of cell nuclei

• Metaphase:

  • Redistributed throughout the cytoplasm and perichromatin mass

• Telophase/Anaphase:

  • Specifically localized to the stem body and midzone of the midbody

This dynamic localization pattern is consistent with KIF20B's role in cytokinesis and cell division. Additionally, KIF20B shows remarkable staining in specific cell subsets within the cerebellum, ovary, and testis tissues, suggesting specialized functions in these tissues .

In cell lines such as HeLa, HEK293T, HEp2, and human leptomeningeal pericytes, KIF20B specifically localizes to G2-M phase mammalian nuclei and the intercellular bridge during cell division .

What are the recommended protocols for using KIF20B antibodies in Western blot analysis?

For optimal Western blot results with KIF20B antibodies, researchers should follow these methodological guidelines:

• Sample preparation:

  • Use cell lysates from lines known to express KIF20B (e.g., HT-29, HepG2, Jurkat cells, mouse brain tissue)

  • For enhanced signal, consider using HEK293T cells transfected with KIF20B expression vectors

• Gel electrophoresis:

  • Use low percentage gels (6-8%) to adequately resolve KIF20B (~214 kDa)

  • Load sufficient protein (20-50 μg per lane)

• Transfer:

  • Employ wet transfer methods for optimal transfer of large proteins

  • Consider extended transfer times (overnight at low voltage)

• Antibody incubation:

  • For polyclonal antibodies (e.g., 27269-1-AP), use dilutions of 1:1000-1:4000

  • For monoclonal antibody 10C7, follow manufacturer's recommendations

  • Incubate overnight at 4°C for best results

• Detection:

  • Use appropriate secondary antibodies (anti-mouse for 10C7, anti-rabbit for polyclonals)

  • Expected molecular weight: approximately 214 kDa

Always include positive controls from cells known to express KIF20B and consider using transfected cells as strong positive controls. The relatively large size of KIF20B requires careful optimization of transfer conditions for successful detection.

How can researchers validate the specificity of KIF20B antibodies?

Validating antibody specificity is crucial for ensuring reliable research results. For KIF20B antibodies, consider these methodological approaches:

• Multiple detection methods:

  • Confirm reactivity across different applications (WB, IHC, immunofluorescence)

  • Consistent results across various techniques increase confidence in specificity

• Recombinant protein controls:

  • Test against purified recombinant KIF20B protein

  • Evaluate reactivity with specific protein domains or peptides

  • The 10C7 antibody showed high reactivity (MFI: 17953) against recombinant protein and even higher reactivity (MFI: 24038) against the immunizing peptide

• Genetic approaches:

  • Compare staining between wildtype and KIF20B-depleted samples (siRNA, CRISPR)

  • Assess enhanced signal in KIF20B-overexpressing systems

• Peptide competition assays:

  • Pre-incubate antibody with immunizing peptide before application

  • Specific signals should be substantially reduced

• Cell cycle-dependent localization:

  • Verify expected subcellular localization patterns throughout the cell cycle

  • KIF20B should show nuclear localization in interphase, cytoplasmic distribution in metaphase, and midbody concentration during telophase/anaphase

Table 1: Validation data showing specificity of 10C7 antibody compared to controls

What immunoassay platforms are available for detecting anti-KIF20B autoantibodies?

Several immunoassay platforms have been developed and validated for detecting anti-KIF20B autoantibodies in research and clinical samples:

• Immunoprecipitation using in vitro transcription/translation:

  • Early reference method for anti-KIF20B detection

  • High specificity but labor-intensive and low-throughput

• Addressable Laser Bead Immunoassay (ALBIA):

  • Quantitative detection with results expressed as median fluorescence intensity (MFI)

  • Positivity thresholds: >500 MFI for lysates, >250 MFI for synthetic peptides

  • Allows multiplexing with other autoantigens

  • Used on platforms like Luminex MAGPIX

• ELISA:

  • Shows excellent agreement with immunoprecipitation results (Cohen's κ >0.8)

  • Higher throughput and standardization potential

  • Suitable for larger cohort studies

• Indirect Immunofluorescence:

  • Provides insights into subcellular localization patterns

  • Less quantitative but offers pattern recognition advantages

  • Useful for distinguishing antibodies against different epitopes

When selecting an immunoassay platform, researchers should consider required throughput, need for quantitative results, equipment availability, and specific research questions. For clinical studies, standardized methods with established cutoff values are essential for reliable results and cross-study comparability.

How do anti-KIF20B autoantibodies contribute to neurological disorders?

The pathogenic mechanisms by which anti-KIF20B autoantibodies may contribute to neurological disorders remain under investigation, but several plausible pathways have been proposed:

• Disruption of neuronal development:

  • KIF20B plays essential roles in cerebral cortex growth and neuronal polarization

  • It participates in the mobilization of SHTN1 and accumulation of PIP3 in neuronal growth cones

  • Autoantibodies could potentially interfere with these developmental processes

• Interference with neuronal migration:

  • KIF20B cooperates with SHTN1 to promote the transition from multipolar to bipolar stage in neurons

  • It supports radial migration of cortical neurons from the ventricular zone toward superficial layers

  • Antibody-mediated disruption could affect proper neuronal positioning

• Impact on axonal transport:

  • As a microtubule motor protein, KIF20B may be involved in cargo transport along axons

  • Antibodies could potentially disrupt transport of essential vesicles, organelles, or signaling molecules

• Specific vulnerability of cranial nerves:

  • Strong association with cranial neuropathy in SLE (OR 5.2, 95% CI 1.4, 19.1)

  • May reflect differential expression or accessibility of KIF20B in cranial nerve structures

The remarkable staining of specific cell subsets in the cerebellum with anti-KIF20B antibodies further supports the potential relevance of these antibodies to cerebellar dysfunction in ataxia. These antibodies have been reported in up to 25% of patients with idiopathic ataxia , suggesting a significant clinical association.

What is the relationship between KIF20B antibodies and disease activity in SLE?

Research has established important correlations between anti-KIF20B antibodies and systemic lupus erythematosus (SLE) disease activity:

• Association with SLEDAI-2K scores:

  • SLE Disease Activity Index-2000 (SLEDAI-2K) was significantly higher in anti-KIF20B-positive SLE patients (p = 0.013)

  • In multivariate regression analysis, anti-KIF20B positivity remained significantly associated with high SLEDAI-2K scores (p = 0.003), independent of anti-ssDNA and anti-dsDNA antibodies

• Prevalence in SLE:

  • Anti-KIF20B antibodies were found in approximately 20% of SLE patients

  • Significantly higher prevalence in SLE patients compared to healthy controls (18/89 vs. 3/46, p = 0.045)

• Neuropsychiatric manifestations:

  • Strong association with cranial neuropathy (70.0% vs. 29.3% positivity; OR 5.2, 95% CI 1.4, 18.5)

  • This association remained significant after adjusting for confounding variables

• Association with specific ACR criteria:

  • In a large international cohort, anti-KIF20B+ patients showed higher rates of:

    • Arthritis (75.5% vs. 68.3%; difference 7.2%, 95% CI 0.5%, 13.9%)

    • Immunological disorder (84.8% vs. 77.4%; difference 7.4%, 95% CI 1.7%, 13.1%)

    • Hypocomplementemia (50.2% vs. 39.7%; difference 10.6%, 95% CI 2.7%, 18.4%)

    • Anti-double-stranded DNA positivity (81.1% vs. 70.3%; difference 10.9%, 95% CI 4.1%, 17.6%)

These findings suggest that anti-KIF20B antibodies may serve as useful biomarkers for disease monitoring in SLE, particularly in patients with neuropsychiatric manifestations. The independent association with disease activity indicates potential pathogenic relevance beyond being a simple epiphenomenon.

How can epitope mapping of KIF20B be performed for antibody characterization?

Epitope mapping of KIF20B is crucial for understanding antibody specificity and potential functional effects. Several methodological approaches can be employed:

• Peptide-based approaches:

  • Synthetic peptide arrays with overlapping peptides spanning the KIF20B sequence

  • The monoclonal antibody 10C7 was developed against peptide QPKRAKRKLYTSEISS (amino acids 1732-1747)

  • Testing reactivity of various antibodies against this defined epitope

• Protein fragment approaches:

  • Expression of different domains of KIF20B as recombinant fragments

  • Testing antibody reactivity against each domain

  • Using recombinant partial-length protein (e.g., KIF20B amino acids 1671-1780)

• Competition assays:

  • Compete binding between characterized and uncharacterized antibodies

  • Determine if antibodies recognize overlapping or distinct epitopes

Data from addressable laser bead immunoassay (ALBIA) has revealed interesting epitope recognition patterns:

Table 2: Comparison of reactivity patterns suggesting different epitope recognition between monoclonal and human autoantibodies

These data suggest that human autoantibodies may recognize different epitopes than the monoclonal antibody 10C7, as evidenced by the variable reactivity to the immunizing peptide. Understanding these epitope differences may provide insights into pathogenesis and guide development of more specific diagnostic tests.

How can researchers establish reliable cutoff values for anti-KIF20B antibody positivity?

Establishing reliable cutoff values for anti-KIF20B antibody positivity is crucial for clinical research. Several methodological approaches can be employed:

• Reference population-based approach:

  • Test a large cohort of healthy controls

  • Calculate mean + 2 or 3 standard deviations as cutoff

  • For ALBIA, positivity has been defined as >500 MFI for lysates and >250 MFI for synthetic peptides, set at three standard deviations above values observed in healthy controls

• ROC curve analysis:

  • Test both disease and control populations

  • Plot sensitivity versus (1-specificity) for different cutoff values

  • Select optimal cutoff based on desired sensitivity/specificity balance

• Validation against reference methods:

  • Compare new assay with established reference method

  • In one study, ELISA cutoff was established using 59 samples previously tested by immunoprecipitation

  • Agreement was assessed using Cohen's kappa coefficient (κ >0.8 indicates excellent agreement)

• Clinical correlation:

  • Correlate antibody levels with disease severity or specific manifestations

  • May identify clinically relevant thresholds distinct from statistical cutoffs

In the Systemic Lupus International Collaborating Clinics (SLICC) inception cohort study, anti-KIF20B testing was performed using ALBIA, with 29.8% of 795 SLE patients testing positive at baseline . The clear definition of positivity threshold enabled robust analysis of associations with clinical manifestations, particularly cranial neuropathy.

Researchers should clearly document their methodology for establishing cutoffs, as this significantly impacts reported prevalence rates and clinical associations.

What is the significance of anti-KIF20B antibodies in cranial neuropathy associated with SLE?

The association between anti-KIF20B antibodies and cranial neuropathy (CN) in SLE represents a significant finding with potential diagnostic and pathophysiological implications:

• Strength of association:

  • In the SLICC inception cohort study, anti-KIF20B positivity was significantly higher in SLE patients with CN versus without CN (70.0% vs 29.3%)

  • This represented a robust association with an odds ratio of 5.2 (95% CI 1.4, 18.5)

  • The association remained significant (OR 5.2, 95% CI 1.4, 19.1) after adjusting for age, gender, race/ethnicity, and relevant ACR criteria

• Specificity of the association:

  • Anti-KIF20B was specifically associated with CN, rather than with all neuropsychiatric manifestations

  • This specificity suggests a potential role in the pathogenesis of CN in particular

  • CN patients were also more likely to fulfill ACR hematological (90.0% vs 66.1%) and ANA (100% vs 95.7%) criteria

• Biological plausibility:

  • KIF20B plays crucial roles in neuronal development and function

  • As a microtubule motor protein, disruption of KIF20B function could potentially affect axonal transport in cranial nerves

This association introduces anti-KIF20B as a potential novel biomarker for CN in SLE and opens new avenues for understanding the pathophysiology of neuropsychiatric SLE . Future research should investigate whether anti-KIF20B antibodies are directly pathogenic and whether antibody titers predict development of CN or response to treatment.

What are the emerging applications of KIF20B antibodies in cancer research?

While much research has focused on anti-KIF20B autoantibodies in autoimmune conditions, emerging evidence suggests important applications in cancer research:

• Association with specific cancers:

  • KIF20B has been associated with bladder cancer

  • Acts as an oncogene promoting bladder cancer cell proliferation, inhibiting apoptosis, and enhancing carcinogenic progression

• Immunohistochemistry applications:

  • KIF20B antibodies show positive staining in human colon cancer tissue

  • May serve as potential diagnostic or prognostic markers

• Cell proliferation studies:

  • KIF20B is involved in positive regulation of cell population proliferation

  • Antibodies can help visualize KIF20B's role in cancer cell division

• Potential therapeutic implications:

  • Understanding KIF20B's role in cancer may lead to novel therapeutic targets

  • Antibodies serve as valuable tools for validating such targets

• Research tools:

  • Commercial antibodies like 27269-1-AP have been validated for use in cancer cell lines (HT-29, HepG2) and cancer tissues

  • Enable investigation of KIF20B's role in oncogenic pathways

KIF20B's designation as Cancer/testis antigen 90 (CT90) further underscores its potential relevance to cancer biology. As research progresses, KIF20B antibodies may prove valuable not only for basic cancer research but also for potential diagnostic, prognostic, or therapeutic applications.