sdf4 Antibody

Introduction to SDF4 Antibody

SDF4 antibodies are immunoglobulins specifically designed to recognize and bind to Stromal Cell Derived Factor 4 (SDF4), also known as Cab45 (45 kDa calcium-binding protein). These antibodies serve as valuable tools in various research applications, including western blotting, immunoprecipitation, immunofluorescence, and enzyme-linked immunosorbent assays (ELISA) . SDF4 antibodies are available in different formats, including monoclonal and polyclonal variants, with capabilities to detect SDF4 protein across multiple species including human, mouse, and rat samples .

The target protein, SDF4, belongs to the C-X-C or α chemokine family, characterized by a pair of cysteine residues separated by a single amino acid. This family primarily functions as chemo-attractants for neutrophils and includes other members such as IL-8, NAP-2, MSGA, and stromal cell derived factor-1 (SDF-1) . Understanding the structure, function, and biological relevance of SDF4 is essential for appreciating the significance of SDF4 antibodies in research and clinical applications.

Molecular Structure and Isoforms

SDF4 is a member of the CREC (Cab45/reticulocalbin/ERC45/calumenin) protein family . The protein consists of 361 amino acids and contains six EF-hand calcium-binding motifs and a HEEF motif at the C-terminal . SDF4 has a signal sequence but no membrane-anchor sequences, allowing it to function as a soluble protein primarily within the Golgi lumen .

Alternative splicing produces three distinct isoforms of SDF4, each with different localization patterns and functions:

1. Cab45-G (Golgi-localized variant): The primary isoform that localizes to the Golgi complex .

2. Cab45-C (Cytosolic variant): Lacks the signal sequence and is localized in the cytosol. It contains only five EF-hands, lacking the sixth EF-hand and HEEF sequence .

3. Cab45-S (Secreted variant): Located in the endoplasmic reticulum and is secreted. Like Cab45-C, it lacks the sixth EF-hand and HEEF motif .

Cellular Localization and Function

SDF4 was initially identified as a novel 45-kD protein from mouse 3T3-L1 adipocytes . It is ubiquitously expressed across tissues and primarily localizes to the Golgi lumen, where it plays critical roles in calcium-dependent cellular processes . SDF4 was the first calcium-binding protein identified in the lumenal portion of a post-ER compartment and the first known soluble protein resident in the Golgi lumen .

The primary functions of SDF4 include:

1. Regulation of calcium-dependent activities in the endoplasmic reticulum lumen or post-ER compartment .

2. Involvement in cargo sorting at the trans-Golgi network (TGN) .

3. Participation in zymogen granule exocytosis (specifically the Cab45-C isoform) .

4. Promotion of cancer cell mobility, proliferation, and migration .

5. Interaction with C-X-C chemokine receptor type 4 (CXCR4) to induce VEGFD expression for angiogenesis by phosphorylating ERK and p38 pathways .

Monoclonal SDF4 Antibodies

Monoclonal antibodies against SDF4 are produced from identical immune cells derived from a unique parent cell, ensuring high specificity and consistency. These antibodies recognize specific epitopes on the SDF4 protein. Commercial examples include:

1. SDF-4 Antibody (E-12): An IgM κ mouse monoclonal antibody that detects SDF4 protein of mouse, rat, and human origin by western blot, immunoprecipitation, immunofluorescence, and ELISA .

2. Anti-SDF4 (Human) mAb (148B10H3): A mouse IgG2aκ monoclonal antibody generated from human SDF4-expressed Ba/F3 transfectants .

3. SDF4 Mouse Monoclonal Antibody (BF8500): A mouse IgG1 monoclonal antibody purified via affinity-chromatography that reacts with human samples and is applicable for western blot and immunohistochemistry .

Polyclonal SDF4 Antibodies

Polyclonal antibodies against SDF4 are produced by multiple B cell lineages in immunized animals, resulting in antibodies that recognize various epitopes on the SDF4 protein. Examples include:

1. SDF4 antibody (AA 37-362) (FITC): A rabbit polyclonal antibody conjugated to FITC that recognizes amino acids 37-362 of human SDF4 .

2. Anti-SDF4 Goat Polyclonal Antibody: Purified from goat serum by ammonium sulphate precipitation followed by antigen affinity chromatography using an immunizing peptide corresponding to amino acids 161-175 of human SDF4 .

3. SDF4 Rabbit Polyclonal Antibody (CAB15443): Generated in rabbits against a recombinant fusion protein containing a sequence corresponding to amino acids 37-200 of human SDF4 .

Comparison of Available SDF4 Antibodies

Table 1 provides a comprehensive comparison of commercially available SDF4 antibodies, highlighting their key properties and applications:

Antibody Generation Techniques

SDF4 antibodies are generated through several established immunological techniques:

1. Hybridoma Technology: Used for monoclonal antibody production, this approach involves immunizing animals (typically mice) with SDF4 protein or peptides, followed by fusion of antibody-producing B cells with myeloma cells to create hybridomas that secrete SDF4-specific antibodies .

2. Recombinant Antibody Production: Modern approaches involve expressing antibody genes in various expression systems including mammalian cells, bacteria, yeast, or insect cells .

3. Polyclonal Antibody Generation: Animals (commonly rabbits, goats, or chickens) are immunized with SDF4 protein or peptides, and antibodies are harvested from serum. For example, the generation of an anti-Cab45 antibody involved rabbits immunized with full-length recombinant Cab45 protein prepared with TiterMax Gold Adjuvant liquid .

Purification Strategies

The purification of SDF4 antibodies typically involves several techniques:

1. Affinity Chromatography: Utilizes the specific binding between SDF4 antibodies and their antigens. For instance, the Anti-SDF4 Goat Polyclonal Antibody is purified from goat serum by ammonium sulphate precipitation followed by antigen affinity chromatography using the immunizing peptide .

2. Protein A/G Affinity Chromatography: Exploits the binding of antibodies to bacterial proteins A or G immobilized on a solid support .

3. Ion Exchange Chromatography: Separates antibodies based on their charge properties. Anion exchange chromatography uses positively charged stationary phases to attract negatively charged molecules, while cation exchange chromatography employs negatively charged stationary phases .

4. Size Exclusion Chromatography: Separates antibodies from contaminants based on molecular size, serving as a crucial polishing step in antibody purification .

A typical purification protocol for SDF4 antibodies involves:

• Preparation of buffers and column setup

• Dilution of serum and application to the column

• Washing to remove non-specific proteins

• Elution of bound antibodies

• Dialysis and concentration of purified antibodies

Research Applications

SDF4 antibodies serve various research purposes:

1. Protein Detection and Quantification: Western blotting using SDF4 antibodies allows for the detection and quantification of SDF4 protein in cell and tissue lysates. For example, SDF4 expression has been analyzed in Jurkat, HeLa, and U251 cell lysates .

2. Localization Studies: Immunofluorescence and immunohistochemistry with SDF4 antibodies reveal the subcellular localization of SDF4. Studies have shown that SDF4 predominantly localizes to the Golgi apparatus in cells such as U2OS .

3. Protein-Protein Interaction Studies: Immunoprecipitation with SDF4 antibodies facilitates the investigation of proteins that interact with SDF4, helping to elucidate its functional networks.

4. Flow Cytometry: SDF4 antibodies enable the analysis of SDF4 expression in individual cells. For instance, flow cytometry has been used to detect SDF4 expression in A431 cells .

Diagnostic Applications

Emerging research highlights the potential of SDF4 antibodies in diagnostics:

1. Cancer Biomarker Detection: Serum SDF4 levels, detected using antibody-based assays, have demonstrated promise as a diagnostic biomarker for gastric cancer and potentially other malignancies with high sensitivity (89%) and specificity (99%) .

2. Tissue Pathology: Immunohistochemistry using SDF4 antibodies can detect SDF4 expression in cancer tissues, contributing to cancer classification and potentially prognosis. SDF4 expression has been observed in the cytoplasm of gastric cancer cells but not in tumor stroma or normal tissues .

Specificity Characteristics

The specificity of SDF4 antibodies—their ability to bind exclusively to SDF4 rather than to other proteins—varies depending on the antibody:

1. Epitope Recognition: Different SDF4 antibodies target distinct regions of the protein. For example, the Anti-SDF4 Goat Polyclonal Antibody recognizes amino acids 161-175 of human SDF4 , while the SDF4 Rabbit Polyclonal Antibody (CAB15443) targets amino acids 37-200 .

2. Cross-Reactivity: Some SDF4 antibodies demonstrate cross-reactivity across species. The SDF-4 Antibody (E-12) detects SDF4 protein from mouse, rat, and human origins , whereas others are species-specific.

3. Isoform Detection: Depending on the epitope, antibodies may recognize specific isoforms of SDF4 (Cab45-G, Cab45-C, or Cab45-S) or all isoforms.

Sensitivity Parameters

The sensitivity of SDF4 antibodies—their ability to detect low concentrations of SDF4—is crucial for both research and diagnostic applications:

1. Detection Limits: Various SDF4 antibodies exhibit different detection thresholds. For instance, the SDF4 Polyclonal Antibody from Thermo Fisher Scientific demonstrates a detection limit dilution of 1:64,000 in peptide ELISA .

2. Signal-to-Noise Ratio: High-quality SDF4 antibodies provide clear signals with minimal background, enabling reliable detection of SDF4 even at low expression levels.

3. ELISA Sensitivity: In the context of diagnostic applications, the sensitivity of SDF4 ELISA kits is particularly important. The Chicken SDF4 ELISA Kit from Abbexa exhibits a sensitivity of 0.94 pg/ml , making it suitable for detecting minimal quantities of SDF4 in biological samples.

Fundamental Cell Biology Studies

Research employing SDF4 antibodies has contributed significantly to our understanding of cellular processes:

1. Discovery of Cab45: In 1996, Scherer et al. identified and characterized Cab45 (SDF4) as a novel 45-kD protein from mouse 3T3-L1 adipocytes. This groundbreaking study established Cab45 as the first calcium-binding protein localized to the lumenal portion of a post-ER compartment and the first known soluble protein resident in the Golgi lumen .

2. Secretory Cargo Sorting Mechanism: Crevenna et al. (2016) used SDF4 antibodies to investigate the mechanism by which secretory cargoes are segregated at the trans-Golgi network. They demonstrated that Ca²⁺-dependent changes in Cab45 oligomerization mediate sorting of specific cargo molecules at the TGN .

3. GARP Complex Function: A 2022 study utilized SDF4 antibodies to show that the GARP complex controls Golgi physiology by stabilizing COPI vesicle machinery. The researchers found that GARP deficiency leads to displacement of SDF4 into other compartments, likely causing its missorting and degradation .

Cancer Research Applications

SDF4 antibodies have provided valuable insights into cancer biology:

1. Angiogenesis in Cancer: Kuo et al. (2021) employed SDF4 antibodies to demonstrate that the fibroblast CEBPD/SDF4 axis responds to chemotherapy-induced stress, promoting angiogenesis. They found that SDF4 interacts with CXCR4 to induce VEGFD expression through the activation of ERK1/2 and p38 pathways in endothelial cells .

2. Cancer Biomarker Identification: A 2023 study by Osumi et al. used SDF4 antibodies in both ELISA and immunohistochemistry to validate SDF4 as a liquid biopsy-based diagnostic biomarker for gastric cancer. Their results showed that serum SDF4 levels could distinguish healthy controls from gastric cancer patients with remarkable accuracy (area under the curve value of 0.973) .

Table 2 summarizes key research findings utilizing SDF4 antibodies:

Cancer Diagnostics

Recent research has highlighted the potential of SDF4 as a cancer biomarker:

1. Gastric Cancer Detection: A 2023 study demonstrated that serum SDF4 levels, measured using antibody-based ELISA, could distinguish healthy controls from gastric cancer patients with remarkable accuracy (area under the curve value of 0.973, sensitivity of 89%, and specificity of 99%) .

2. Early Cancer Detection: Importantly, serum SDF4 levels were significantly elevated in patients with early-stage gastric cancer compared to healthy subjects, suggesting potential utility in early cancer screening .

3. Broad Cancer Applicability: The same study found elevated serum SDF4 levels in patients with various solid cancers compared to healthy controls, indicating potential utility as a general cancer screening tool .

Prognostic Value

SDF4 expression has been linked to cancer prognosis:

1. Breast Cancer Prognosis: SDF4 expression levels have been associated with the prognosis of human breast cancer, though detailed mechanisms remain to be fully elucidated .

2. Tumor Characteristics: Immunohistochemistry studies using SDF4 antibodies have shown that SDF4 expression patterns in gastric cancer tissue do not significantly differ between stages, suggesting its expression may be an early event in carcinogenesis .

Table 3 presents data on the diagnostic performance of SDF4 compared to conventional biomarkers in gastric cancer:

Technical Challenges

Despite their utility, SDF4 antibodies face several technical challenges:

1. Isoform Specificity: The existence of three SDF4 isoforms (Cab45-G, Cab45-C, and Cab45-S) complicates antibody development and application. Antibodies may not distinguish between these isoforms unless specifically designed to target unique regions.

2. Cross-Reactivity: Some SDF4 antibodies may cross-react with related proteins in the CREC family, such as reticulocalbin and ERC55, due to sequence homology outside the EF-hand motifs.

3. Optimization Requirements: Applications like immunohistochemistry and immunofluorescence often require significant optimization of SDF4 antibody dilutions and staining protocols to achieve specific and sensitive results.

Research Limitations

Current research using SDF4 antibodies has several limitations:

Emerging Research Areas

Several promising research directions for SDF4 antibodies include:

1. Therapeutic Applications: Development of therapeutic antibodies targeting SDF4 or its signaling pathways, particularly in cancers where SDF4 promotes angiogenesis and tumor growth.

2. Multi-Cancer Screening: Exploration of SDF4 antibody-based assays for screening multiple cancer types beyond gastric cancer, potentially as part of cancer screening panels.

3. Personalized Medicine: Investigation of SDF4 expression patterns in tumors to guide personalized treatment approaches, particularly for therapies targeting angiogenesis.

Technological Advancements

Technological innovations will likely enhance SDF4 antibody applications:

1. Single-Cell Analysis: Integration of SDF4 antibodies with single-cell technologies to understand heterogeneity in SDF4 expression and function at the individual cell level.

2. Multiplexed Imaging: Application of multiplexed immunofluorescence or mass cytometry using SDF4 antibodies to simultaneously visualize multiple proteins in the SDF4 signaling network.

3. Point-of-Care Diagnostics: Development of rapid, antibody-based point-of-care tests for SDF4 detection to facilitate early cancer screening in resource-limited settings.