LCN2 Antibody Pair

What is LCN2 and why is it significant in scientific research?

Lipocalin-2 (LCN2) is a 25-kDa protein belonging to the Lipocalin family, characterized by its distinctive eight-stranded, anti-parallel, symmetrical β-barrel fold structure. In humans, the canonical protein comprises 198 amino acid residues with a molecular mass of approximately 22.6 kDa . LCN2 is known by several other names including NGAL (Neutrophil gelatinase-associated lipocalin), p25, 24p3, and Siderocalin .

LCN2 plays diverse biological roles that make it a valuable research target. Initially thought to function primarily as a transporter of small lipophilic molecules due to its lipocalin fold, studies in Lcn2 null mice have revealed its bacteriostatic properties and crucial role in innate immunity through sequestration of bacterial iron siderophores . The protein has since been implicated in multiple biological processes including cell differentiation, energy expenditure, apoptosis, chemotaxis, and cell migration .

LCN2 is predominantly expressed in neutrophils, but can be found in cytoplasmic vesicles, cytoplasm, and as a secreted protein in various biological fluids . Its involvement in inflammatory responses, innate immunity, and renal development has established it as an important biomarker and functional protein across immunology, nephrology, and oncology research fields.

What are the established applications of LCN2 antibody pairs in research protocols?

LCN2 antibody pairs serve as essential tools in various immunoassay formats, with applications spanning multiple research areas:

a) Enzyme-Linked Immunosorbent Assay (ELISA):

• Primary application for quantitative measurement of LCN2 in biological samples

• Typically formatted as sandwich ELISA with capture and detection antibodies

• Enables detection across a broad concentration range (typically 156-10,000 pg/ml)

b) Additional Immunoassay Platforms:

• Chemiluminescent Immunoassay (CLIA) for enhanced sensitivity

• ELISPOT for detecting LCN2-secreting cells at the single-cell level

• Luminex/multiplex assays for simultaneous detection with other analytes

• Immunochromatography for rapid point-of-care detection

c) Sample Compatibility:

• Cell culture supernatants for in vitro secretion studies

• Serum and plasma (particularly heparin-treated) for clinical research

• Urine for nephrology investigations

• Saliva for non-invasive sampling approaches

These applications enable researchers to investigate LCN2's role in multiple contexts including inflammatory responses, kidney injury, neutrophil activation, infection and immunity, iron sequestration, and cancer models. The versatility of antibody pair applications makes them foundational tools for both mechanistic studies and translational research involving LCN2.

What strategy should be used to validate the specificity of LCN2 antibodies?

A comprehensive validation strategy for LCN2 antibodies should incorporate multiple approaches to ensure specificity and reliability:

The search results specifically caution that many commercially available antibodies fail rigorous validation tests despite manufacturer claims of specificity. The literature emphasizes that "uncritical usage of antibodies directed against LCN2 receptors might be a source for faulty data" . A particularly concerning validation gap is the reliance on inadequate negative controls in immunohistochemistry, where authors often only omit the primary antibody rather than using proper isotype controls .

Researchers should maintain detailed documentation of all validation steps and provide comprehensive antibody information (including catalog number, lot number, dilution, and validation strategy) in publications to enable reproducibility.

What sample types are compatible with LCN2 antibody pairs and what are their handling requirements?

LCN2 antibody pairs can be used with various sample types, each requiring specific handling and preparation considerations:

Each sample type requires optimization for the specific antibody pair being used. The search results indicate that performance can be "sample-dependent," necessitating titration "in each testing system to obtain optimal results" . The manufacturer guidance frequently recommends checking validation data for specific sample types and performing titration experiments to determine optimal dilution ranges.

For immunohistochemical applications, the search results specifically mention antigen retrieval conditions: "suggested antigen retrieval with TE buffer pH 9.0; alternatively, antigen retrieval may be performed with citrate buffer pH 6.0" , highlighting the importance of optimization for each specific application and sample type.

What molecular weight patterns should be expected when detecting LCN2 in Western blot analysis?

When detecting LCN2 via Western blot or other size-separation methods, researchers should anticipate specific molecular weight patterns that reflect both the core protein and its post-translational modifications:

Several factors can influence the observed molecular weight pattern:

• Sample preparation conditions (reducing vs. non-reducing)

• Degree of glycosylation, which may vary by cell/tissue type

• Species differences (though human, mouse, and rat orthologs have similar sizes)

• Potential proteolytic processing

When evaluating a new antibody, researchers should assess whether the observed molecular weight aligns with expectations, considering both the core protein and potential modifications. The search results specifically mention that LCN2 is subject to post-translational modifications, including glycosylation , which accounts for the difference between calculated and observed molecular weights.

What methodological approaches can address cross-reactivity issues with LCN2 antibodies?

Cross-reactivity represents a significant challenge with LCN2 antibodies, requiring rigorous methodological strategies to ensure specificity:

a) Epitope Selection and Analysis:

• Choose antibodies targeting unique regions with minimal sequence homology to related proteins

• Analyze the immunogen sequence for potential cross-reactive epitopes

• The search results specifically identify immunogen information: "Lipocalin-2/NGAL fusion protein Ag25715"

b) Genetic Model Validation:

• Employ knockout/knockdown models as definitive controls

• The search results specifically highlight issues with antibodies failing testing against genetic models

• Include comparative analysis of wild-type and knockout samples using the same protocols

c) Orthogonal Detection Methods:

• Verify findings using techniques based on different principles

• Mass spectrometry can provide unbiased protein identification

• Consider using multiple antibodies targeting different epitopes

d) Specificity Testing Protocol:

• Competitive binding assays with recombinant LCN2

• Pre-adsorption experiments for polyclonal antibodies

• Analysis of reactivity across multiple sample types

The search results contain explicit warnings about cross-reactivity issues, particularly with antibodies against LCN2 receptors. One publication states: "missing proper controls, nonspecific binding, or using wrong antibodies for a specific antigen provoke erroneous and inconsistent results" . A specific example cited describes a study using an antibody purportedly against the LCN2 receptor NGALR that was actually directed against an entirely different protein (ACKR3/CXCR7/GPR159) , underscoring the critical importance of independent validation.

Many antibodies marketed for multiple applications (e.g., WB, IHC, ELISA) may not perform equally well across all methods. The search results note that some antibodies advertised as "specific for several species and applicable for immunohistochemistry, Western blot analysis, flow cytometry, and ELISA testing" failed rigorous testing .

What experimental design considerations are critical when investigating LCN2 in immune response models?

When studying LCN2 in immune response contexts, several methodological considerations are essential for robust experimental design:

a) Model System Selection:

• Comparative studies using wild-type and Lcn2-/- mice reveal LCN2's immunomodulatory functions

• The search results describe findings that "LCN2 protected from excessive weight loss and improved survival" in influenza infection models

• Experimental models should include both physiological and pathological states

b) Microbiome Considerations:

• The search results reveal that "LCN2 profoundly affects gut microbial composition"

• Co-housing experiments between WT and Lcn2-/- mice can distinguish direct immune effects from microbiome-mediated effects

• Antibiotic treatment studies demonstrated that "LCN2-related effects are mediated by the microbiome"

c) Comprehensive Immune Cell Analysis:

• Flow cytometry panels should include markers for multiple cell populations

• The search results specifically noted LCN2-dependent differences in:

  • CD4+ T helper cells and CD8+ cytotoxic T cells

  • CD69+ activated T cells

  • IFNγ-expressing and antigen-specific CD8+ T cells

  • Influenza-specific serum IgG antibody levels

d) Antigen Presentation Assessment:

• The search results describe that "LCN2 negatively regulates antigen presentation of CD103+ DCs to CD8+ T cells"

• Consider both in vivo and in vitro antigen presentation assays

• T cell proliferation assays with BMDCs should include appropriate controls

e) Time-Course Analysis:

• The search results describe assessment at multiple time points (7, 9, and 16 days post-infection)

• Include temporal analysis to capture dynamic immune response changes

These design elements are critical for understanding LCN2's complex immunomodulatory roles. The search results describe sophisticated experimental approaches including "T cell chimerism and in vitro T cell proliferation assays" that enabled researchers to determine that "improved antigen processing by CD103+ DCs, rather than T cell intrinsic effects of LCN2, contribute to the exacerbated T cell response" .

What are the critical parameters and optimization strategies for LCN2 detection in ELISA?

Optimizing antibody pairs for LCN2 detection in ELISA requires systematic parameter adjustment to achieve maximum sensitivity and specificity:

The search results emphasize that reagents "should be titrated in each testing system to obtain optimal results" , highlighting the importance of optimization for each specific research context. A systematic approach using a checkerboard titration (varying both capture and detection antibody concentrations) allows identification of the optimal combination that provides the highest signal-to-noise ratio while maintaining standard curve linearity.

For sample analysis, researchers should include appropriate controls including known positive samples. The search results mention specific cell types known to express LCN2 (e.g., HT-29 cells, human saliva) that could serve as positive controls.

How should knockout/knockdown models be utilized for rigorous validation of LCN2 antibodies?

Knockout/knockdown models represent the gold standard for antibody validation, providing definitive negative controls that establish specificity. A comprehensive validation approach using these models should include:

a) Genetic Model Verification:

• Confirm knockout/knockdown at the genetic level (PCR genotyping)

• Verify at the transcript level (qRT-PCR)

• Validate at the protein level using previously validated antibodies if available

b) Systematic Multi-application Testing:

• Test the antibody across all intended applications with paired WT and KO/KD samples

• Western blot: Should show complete absence of specific bands

• IHC/ICC: Should show no specific staining above background

• Flow cytometry: Should show no signal above isotype control

• ELISA: Should yield readings at background level

c) Quantitative Assessment Protocol:

• For knockdown models, quantify the relationship between knockdown efficiency and signal reduction

• Compare signals across a range of antibody concentrations

• Document results with unmodified images showing both positive and negative controls

d) Rescue Experiment Design:

• Re-express LCN2 in knockout models to restore detection

• Use expression vectors with tags distinguishable from endogenous protein

• Demonstrate concentration-dependent signal recovery

The search results specifically discuss the use of Lcn2-/- mice as critical negative controls . One publication explicitly describes "enhanced adaptive immune response... in Lcn2-/- mice" , demonstrating how these models not only serve for antibody validation but also reveal functional insights.

A significant critique in the literature notes that many commercially available antibodies fail rigorous validation against genetic models despite claims of specificity. The search results emphasize that proper documentation of validation using genetic models should be included in publications to ensure reproducibility.

How do epitope targeting strategies influence LCN2 detection outcomes in different applications?

Epitope selection significantly impacts detection outcomes across applications due to LCN2's complex structure and interactions:

a) Structural Accessibility Considerations:

• LCN2's eight-stranded, anti-parallel, symmetrical β-barrel fold creates epitopes with varying accessibility

• Applications using denatured protein (Western blot) versus native conformation (ELISA) require different epitope targeting strategies

• Consider how sample preparation affects epitope exposure

b) Functional Domain Recognition:

• LCN2 functions as an iron-trafficking protein

• Antibodies targeting the siderophore-binding pocket may show differential binding based on iron/siderophore occupancy

• The search results note different binding affinities between "apo-LCN2" and "LCN2/ferric-enterobactin" to LCN2 receptors

c) Post-translational Modification Effects:

• Glycosylation can mask epitopes or alter antibody binding

• Consider using antibodies recognizing different regions when studying modified forms

• The difference between calculated (22.6 kDa) and observed (25 kDa) molecular weights is attributed to glycosylation

d) Multi-epitope Strategies for Different Applications:

• For sandwich ELISA:

  • Capture and detection antibodies must recognize non-competing epitopes

  • Optimal pairs detect LCN2 in native conformation

• For Western blot:

  • Target linear epitopes that remain accessible after denaturation

  • Consider N-terminal versus C-terminal targeting

e) Species Cross-reactivity Considerations:

• Different epitopes have varying conservation across species

• For studies spanning multiple species, target highly conserved regions

• The search results specifically discuss human LCN2, with some antibodies showing reactivity in mouse and rat models

Epitope selection is particularly critical for comprehensive detection of all LCN2 forms. The search results mention alternative splicing yielding two different isoforms , requiring careful epitope selection to either detect all variants or discriminate between them. Using antibodies targeting different epitopes can provide complementary information and increase confidence in experimental findings.

What technical approaches can address tissue-specific challenges in LCN2 detection?

Detecting LCN2 across different tissue types presents unique challenges requiring tissue-specific technical strategies:

a) Fixation and Antigen Retrieval Optimization:

• The search results specify particular retrieval conditions:

  • "Suggested antigen retrieval with TE buffer pH 9.0"

  • "Alternatively, antigen retrieval may be performed with citrate buffer pH 6.0"

• Systematic comparison of fixation methods (formalin, paraformaldehyde, alcohol-based)

• Optimization of retrieval parameters (buffer, pH, temperature, duration)

b) Tissue-specific Background Reduction:

• The search results emphasize issues with non-specific binding

• Implement tissue-specific blocking protocols

• For autofluorescent tissues, consider:

  • Sudan Black B treatment

  • Spectral unmixing

  • Photobleaching protocols

c) Tissue-specific Control Selection:

• Positive controls: The search results mention specific tissues where detection has been verified:

  • "Human colon cancer tissue, human stomach cancer tissue" for IHC

• Negative controls: Beyond omission of primary antibody

  • Use "serum or isotype-specific immunoglobulins in the same protein concentration as the primary antibody"

  • Include genetic model tissues when available

d) Signal Amplification Strategies:

• For low-expressing tissues, consider:

  • Tyramide signal amplification

  • Polymer-based detection systems

  • Higher antibody concentrations with optimized incubation conditions

e) Multi-staining Approaches:

• Co-localization with cell-type specific markers

• Sequential staining protocols for challenging combinations

• Spectral imaging for separating closely overlapping signals

f) Quantification Methodology:

• Standardized image acquisition parameters

• Appropriate analysis algorithms for different tissue morphologies

• Normalization strategies for cross-tissue comparisons

The search results highlight that antibody performance often varies across tissues, requiring independent validation for each new tissue type. A specific caution notes that some antibodies produce "unspecific, un-interpretable background signals" and that nuclear staining patterns can appear when membrane staining would be expected for receptors, emphasizing the need for careful interpretation.

How do post-translational modifications of LCN2 impact experimental detection and interpretation?

Post-translational modifications (PTMs) of LCN2 significantly influence detection methods and data interpretation, requiring specific methodological considerations:

a) Glycosylation Effects on Detection:

• Impacts molecular weight: Calculated 22.6 kDa vs. observed 25 kDa

• May mask epitopes near glycosylation sites

• Creates heterogeneity in protein migration patterns

b) Methodological Approaches for PTM Analysis:

• Enzymatic treatments:

  • PNGase F for N-glycan removal

  • O-glycosidase for O-glycan removal

  • Compare treated vs. untreated samples on Western blots

• Lectin-based analyses:

  • Lectin blotting to identify glycan types

  • Lectin affinity chromatography for enrichment

• Recombinant comparison:

  • Bacterial expression systems typically produce non-glycosylated proteins

  • Mammalian expression systems (NS0 mentioned in search results ) may produce glycosylated forms

c) PTM Heterogeneity Considerations:

• LCN2 may exist as a mixture of differently modified forms

• Different tissues/conditions may alter PTM patterns

• Consider enrichment strategies to isolate specific forms

d) PTM Impact on Biological Function:

• Modified forms may have altered:

  • Receptor binding (search results discuss multiple receptors with varying affinities )

  • Protein stability and half-life

  • Cellular localization

e) Technical Approaches for Comprehensive Analysis:

• Mass spectrometry for definitive PTM characterization

• 2D electrophoresis to separate PTM variants

• Site-directed mutagenesis to assess PTM site importance

Understanding PTM effects is essential for accurate data interpretation, particularly when comparing results across different tissues, conditions, or detection methods. The search results specifically mention glycosylation as a PTM affecting LCN2 , accounting for the difference between calculated and observed molecular weights, but researchers should consider the potential for additional modifications that may influence detection and function.

What strategies ensure successful development of multiplex assays incorporating LCN2 detection?

Developing multiplex assays that include LCN2 alongside other analytes requires careful consideration of several technical factors:

a) Antibody Selection and Validation:

• Prioritize antibodies with minimal cross-reactivity

• The search results caution that many LCN2 antibodies show cross-reactivity issues

• Validate each antibody individually before multiplexing

• Test antibody pairs in the multiplex context against single-plex performance

b) Signal Resolution Optimization:

• For fluorescence-based systems:

  • Select fluorophores with minimal spectral overlap

  • Include compensation controls

  • Consider quantum dot labeling for narrow emission spectra

• For enzymatic detection:

  • Use orthogonal substrates with distinct spectral properties

  • Implement sequential detection if necessary

c) Dynamic Range Harmonization:

• LCN2 concentrations vary widely across sample types

• The search results mention an assay range of 156 pg/ml - 10,000 pg/ml

• Develop sample dilution protocols to bring all analytes into measurable range

• Consider multiple sample dilutions to capture out-of-range analytes

d) Buffer and Reagent Compatibility:

• Identify compatible buffer conditions for all antibodies

• The search results mention specific storage buffers:

  • "PBS with 0.02% sodium azide and 50% glycerol pH 7.3"

• Optimize blocking reagents to minimize background across all analytes

e) Platform-specific Considerations:

• The search results mention LCN2 antibody pairs for various platforms:

  • ELISA, CLIA, ELISPOT, Luminex, Immunochromatography

• Each platform has unique multiplexing requirements:

  • Bead-based systems: Bead classification and stability

  • Planar arrays: Spot morphology and density

  • Microfluidic platforms: Flow dynamics and surface chemistry

f) Standard Curve Design:

• Generate reliable multi-analyte standard curves

• Address potential standard cross-reactivity

• Consider recombinant protein cocktails as calibrators

g) Validation Strategy:

• Compare multiplexed results with single-plex gold standards

• Assess spike recovery across all analytes

• Determine limits of detection/quantification in the multiplex format

A comprehensive development and validation approach will ensure that multiplexed assays incorporating LCN2 provide reliable data while maximizing the information obtained from precious samples.

What control strategies are critical for LCN2 receptor studies given the challenges of receptor antibody specificity?

Establishing appropriate controls for LCN2 receptor studies is particularly critical given the challenges highlighted in the search results regarding receptor antibody reliability:

a) Genetic Control Implementation:

• Receptor knockout models:

  • CRISPR/Cas9-generated cell lines

  • Conditional knockout animals

• Transient knockdown approaches:

  • siRNA with appropriate scrambled controls

  • Inducible shRNA systems

• The search results specifically discuss multiple LCN2 receptors (NGALR, LRP2, MC4R)

b) Expression System Controls:

• Overexpression models:

  • Transiently transfected cells with receptor constructs

  • The search results mention using "transient overexpressed NGALR" as controls

• Tagged receptor constructs:

  • Allow detection via tag antibodies rather than receptor antibodies

  • Enable pull-down experiments for binding studies

c) Antibody-specific Controls:

• Comprehensive negative controls:

  • Isotype-matched irrelevant antibodies

  • The search results emphasize using "serum or isotype-specific immunoglobulins in the same protein concentration as the primary antibody"

  • They specifically caution against using only "no primary antibody" controls

• Epitope blocking:

  • Pre-incubate antibodies with immunizing peptides

  • Should abolish specific signal

d) Functional Validation Approaches:

• Signaling response measurement:

  • Assess downstream pathway activation

  • Compare wild-type receptor with function-blocking mutations

• Direct binding assays:

  • The search results provide dissociation constants (Kd) for different receptors:

    • NGALR: ~92 pM in one study, 7-10 μM in another

    • LRP2: ~60 nM

    • Multiple MC receptors: 51-86 nM

The search results contain explicit warnings about LCN2 receptor antibodies, noting that "many antibodies available for NGALR, LRP2 and MC4R that have not undergone strict quality controls" and that some marketed as "specific for several species" failed validation tests. A specific case described an antibody supposedly targeting NGALR that actually recognized an entirely different protein (ACKR3/CXCR7) , highlighting the critical importance of rigorous controls.

What experimental approaches can reliably characterize LCN2-receptor interaction dynamics?

Studying LCN2-receptor interactions requires multiple complementary approaches to establish binding specificity, affinity, and functional consequences:

a) Quantitative Binding Analysis:

• Surface Plasmon Resonance (SPR):

  • Provides real-time binding kinetics

  • The search results report varied dissociation constants (Kd):

    • NGALR: ~92 pM or 7-10 μM (varying reports)

    • LRP2: ~60 nM

    • MC receptors: 51-86 nM

• Isothermal Titration Calorimetry (ITC):

  • Provides thermodynamic parameters of binding

  • Label-free approach for unmodified proteins

• Microscale Thermophoresis (MST):

  • Requires minimal sample amounts

  • Measures interactions in solution

b) Cellular Binding Studies:

• Flow Cytometry:

  • Quantifies receptor expression and ligand binding

  • The search results mention flow cytometry applications for LCN2

• Confocal Microscopy:

  • Visualizes binding, internalization, and trafficking

  • Enables co-localization studies with cellular markers

c) Structural Analysis Approaches:

• Mutagenesis Studies:

  • Identify critical binding residues

  • Create binding-deficient controls

• Domain Mapping:

  • Determine specificity-determining regions

  • Generate domain-specific antibodies

d) Functional Characterization:

• Signaling Studies:

  • Assess downstream pathway activation

  • Compare signaling between different receptor types

• Gene Expression Analysis:

  • Examine transcriptional responses to receptor engagement

  • The search results mention transcriptome analysis revealing "differential regulation of antigen processing and presentation and antiviral immunity pathways"

e) Ligand Form Considerations:

• Compare binding of different LCN2 forms:

  • Iron-bound vs. iron-free states

  • The search results note different affinities for "apo-LCN2" vs. "LCN2/ferric-enterobactin"

  • Glycosylated vs. non-glycosylated forms

f) In Vivo Approaches:

• Receptor Knockout Models:

  • Assess physiological relevance of specific interactions

  • The search results describe studies in Lcn2-/- mice revealing immunomodulatory functions

• Competitive Binding Studies:

  • Determine receptor selectivity in physiological contexts

  • Identify potential endogenous competitors

Multiple complementary approaches provide a more complete understanding of LCN2-receptor interactions, with each method offering distinct advantages. The search results highlight the importance of comparing findings across different methodologies, as receptor binding characteristics can vary significantly depending on experimental conditions.