SPP1 Recombinant Monoclonal Antibody

What is SPP1 and why is it an important research target?

SPP1, also known as osteopontin, is a secreted arginine-glycine-aspartic acid (RGD)-containing glycoprotein originally isolated from bone. It plays critical roles in multiple biological processes including bone development, immune responses, inflammation, tissue repair, and oncogenesis. SPP1 has been found in kidney, vascular tissues, biological fluids, and various tumor tissues, where it interacts with integrins and CD44 to regulate cellular functions . Its elevated expression in various cancers, including colorectal cancer where it functions as an immune checkpoint, makes it a significant target for biomedical research .

What is the difference between traditional monoclonal and recombinant monoclonal antibodies against SPP1?

Traditional SPP1 monoclonal antibodies are produced through hybridoma technology, involving immunization of mice with recombinant human osteopontin, isolation of B cells from the immunized mice's spleen, fusion with myeloma cells, and purification of antibodies from mouse ascites using protein G affinity chromatography . In contrast, recombinant SPP1 monoclonal antibodies involve a four-step process: sequencing the SPP1 monoclonal antibody gene, cloning it into a plasmid vector, transfecting the recombinant vector into a host cell line (often HEK293F cells), and purifying the antibody from cell culture supernatant using affinity chromatography . The recombinant approach offers advantages in terms of reproducibility, lot-to-lot consistency, and elimination of animal-derived components in the final production stage .

What epitopes of SPP1 are commonly targeted by monoclonal antibodies?

SPP1 monoclonal antibodies can recognize distinct epitopes located in different regions of the protein. Some antibodies target the amino-terminal half of SPP1, while others recognize the carboxy-terminal half. For example, in studies with murine osteopontin, antibodies like OPN1.2 recognize the amino-terminal half, while OPN2.2, OPN2.3, and OPN3.1 recognize different regions of the carboxy-terminal half . Specific commercial antibodies, such as the 100D3 clone, react with both mouse and human osteopontin , while others like E4O2F target regions surrounding specific amino acids (e.g., Ala40 of mouse Osteopontin/SPP1 protein) .

Experimental Applications and Methodologies

For optimal performance, SPP1 antibodies should be stored at -20°C for long-term storage . During handling, they should be protected from light and freeze-thaw cycles should be avoided as they can degrade antibody quality . Most SPP1 antibodies are supplied in buffered solutions such as PBS (Phosphate Buffered Saline) with preservatives like sodium azide. For example, some preparations use "0.2 μm filtered solution in PBS, preservative free" , while others use "10mM Sodium phosphate, 150mM Sodium chloride, pH 7.4 + 0.2 Preservative: 0.05% Sodium azide" . Antibodies should not be aliquoted unless specifically recommended by the manufacturer .

What controls should be included when using SPP1 antibodies in experimental setups?

When using SPP1 antibodies, several controls should be implemented:

• Positive Control: Use tissues or cell lines known to express SPP1, such as kidney tissues where SPP1 expression has been confirmed .

• Negative Control: Include samples where SPP1 is absent or blocked.

• Isotype Control: Include an antibody of the same isotype (e.g., IgG1 for many SPP1 antibodies) but with irrelevant specificity.

• Secondary Antibody Control: Include a sample with only secondary antibody to check for non-specific binding.

• Blocking Peptide Competition: For validation, use a protocol where the SPP1 antibody is pre-incubated with the immunizing peptide to confirm specificity .

For ELISA specifically, duplicate serum samples should be included as quality control alongside blank controls to enable stability and accuracy of optical density (OD) values across plates .

How can cross-reactivity issues with SPP1 antibodies be identified and mitigated?

SPP1 antibodies may exhibit cross-reactivity between species, particularly between human and mouse SPP1 . To identify and mitigate cross-reactivity issues:

• Review Antibody Specifications: Check the manufacturer's documentation for known cross-reactivity. For example, some antibodies specifically state "Crossreacts with human and mouse OPN" .

• Perform Validation Tests:

  • Western blot analysis with recombinant proteins from different species

  • Competitive binding assays with peptides from different species

  • Pre-absorption experiments with the target antigen

• Mitigate Cross-Reactivity:

  • Use antibodies specifically validated for your species of interest

  • Increase antibody dilution to reduce non-specific binding

  • Modify blocking conditions to reduce background

  • Consider using highly specific recombinant antibodies that target unique epitopes

If working with multiple species, choosing an antibody that intentionally cross-reacts, such as the 100D3 clone that reacts with both mouse and human osteopontin , may be advantageous for comparative studies.

What factors influence the detection of different SPP1 isoforms and post-translational modifications?

SPP1 exists in multiple isoforms and undergoes extensive post-translational modifications that can affect antibody recognition. Key considerations include:

• Epitope Location: Antibodies targeting different regions of SPP1 may have different capabilities in detecting various isoforms. For example, OPN1.2 and OPN2.2 can recognize thrombin-cleaved osteopontin, whereas OPN2.3 and OPN3.1 cannot .

• Post-translational Modifications:

  • Glycosylation: SPP1 is heavily glycosylated, resulting in a molecular weight of 58-75 kDa (compared to its ~33 kDa core protein)

  • Phosphorylation: Affects protein conformation and epitope accessibility

  • Proteolytic processing: Thrombin cleavage creates distinct fragments with different biological activities

• Sample Preparation: Denaturing conditions in Western blotting versus native conditions in ELISA may affect epitope accessibility and antibody binding.

• Antibody Selection: Choose antibodies validated for detecting specific isoforms or modifications of interest. For example, some antibodies specifically recognize the C-terminal (a.a. 167-314) of osteopontin .

How can SPP1 antibodies be optimized for detecting low abundance targets in complex samples?

For detecting low abundance SPP1 in complex samples:

• Signal Amplification Systems:

  • Use polymer-based detection systems in IHC

  • Employ tyramide signal amplification (TSA)

  • Consider chemiluminescent substrates with enhanced sensitivity for Western blotting

• Sample Enrichment:

  • Perform immunoprecipitation before analysis

  • Use fractionation techniques to concentrate the target protein

  • Consider using SPP1 antibodies specifically validated for immunoprecipitation (e.g., at 1:200 dilution)

• Protocol Optimization:

  • Extend primary antibody incubation time (overnight at 4°C)

  • Optimize blocking conditions to reduce background

  • Use highly sensitive detection reagents

  • Increase antigen retrieval efficiency for fixed tissues

• Antibody Selection: Choose antibodies with demonstrated high sensitivity. For example, some SPP1 antibodies have been validated for detecting endogenous levels of the protein .

How can SPP1 antibodies be utilized in cancer biomarker studies?

SPP1 antibodies play a crucial role in cancer biomarker research, as demonstrated in studies of esophageal squamous cell carcinoma (ESCC):

• Tissue Expression Analysis: Immunohistochemistry using SPP1 antibodies revealed that SPP1 protein is significantly overexpressed in ESCC tissues compared to adjacent normal tissues, correlating with pathological grades (G1, G2, and G3) and PDL1 expression .

• Serum Autoantibody Detection: ELISA using recombinant SPP1 protein as a coating antigen can detect anti-SPP1 autoantibodies in patient sera. In one study, the positive frequency of autoantibody to SPP1 was 45.16% in ESCC patients versus only 16.13% in normal human sera .

• Diagnostic Value Assessment:

  • ROC analysis demonstrated that autoantibody to SPP1 could distinguish ESCC patients from normal controls with AUC values of 0.653 and 0.739 in discovery and validation groups, respectively

  • Sensitivity of 45.16% and specificity of 83.87% were achieved

• Validation Methods: Combine ELISA results with western blotting to confirm the occurrence of immunoreactivity to SPP1 in cancer sera .

These approaches demonstrate how SPP1 antibodies can be used to develop novel diagnostic biomarkers for cancer detection.

What strategies exist for using SPP1 antibodies in functional blocking studies?

SPP1 blocking antibodies can be used to investigate the functional roles of SPP1 in various biological processes:

• Tumor Immunity Studies: The 100D3 antibody is a blocking antibody that has been shown to increase the efficacy of tumor-specific CTLs in killing colon tumor cells in vitro and suppress colon tumor growth in tumor-bearing mice in vivo .

• Experimental Design Considerations:

  • Use isotype-matched control antibodies to validate specificity of effects

  • Determine optimal antibody concentration through dose-response studies

  • Consider timing of administration in relation to biological processes being studied

• Applications in Cell Culture:

  • Pre-incubate cells with blocking antibodies before adding stimuli

  • Add antibodies directly to culture medium for continuous blocking

  • Use in combination with genetic approaches (siRNA, CRISPR) for validation

• In Vivo Applications:

  • Administer at therapeutically relevant doses (price ranges suggest various size options are available for in vivo studies, from $172.00 to $4,494.00)

  • Consider pharmacokinetics and tissue distribution

  • Validate target engagement in tissues of interest

How can SPP1 antibodies be integrated into multiplexed imaging and flow cytometry studies?

Integrating SPP1 antibodies into multiplexed detection systems requires careful planning:

• Panel Design for Multiplexed Imaging:

  • Select SPP1 antibodies raised in different host species than other target antibodies

  • Choose fluorophores with minimal spectral overlap

  • Validate antibodies individually before combining in multiplexed panels

  • Consider using recombinant antibodies for improved reproducibility and reduced batch-to-batch variation

• Flow Cytometry Applications:

  • Ensure antibodies are validated for flow cytometry (e.g., 1:25-1:100 dilution)

  • Titrate antibodies to determine optimal concentration

  • Include appropriate compensation controls

  • Consider fixation and permeabilization requirements for intracellular SPP1 detection

• Spatial Analysis in Tissues:

  • Combine SPP1 antibodies with other markers for cell identity and function

  • Use sequential immunostaining protocols if antibody species conflict

  • Employ multispectral imaging systems to separate closely overlapping fluorophores

  • Consider tyramide signal amplification for improved sensitivity

• Data Analysis Approaches:

  • Implement machine learning algorithms for pattern recognition

  • Use spatial statistics to quantify co-localization with other markers

  • Perform cluster analysis to identify cell populations based on multiple markers

How are SPP1 antibodies being utilized in studying the role of SPP1 in immune checkpoint regulation?

Recent research has identified SPP1 as a potential immune checkpoint, particularly in colorectal cancer . Approaches using SPP1 antibodies in this context include:

• Tumor Microenvironment Analysis:

  • Spatial profiling of SPP1 expression relative to immune cell infiltrates

  • Co-staining with established immune checkpoint markers (PD-1, PD-L1, CTLA-4)

  • Correlation of SPP1 expression with immune cell function and phenotype

• Functional Studies:

  • Using blocking antibodies like 100D3 to assess effects on T cell activation and tumor killing

  • Combining SPP1 blockade with established checkpoint inhibitors

  • Investigating mechanisms of SPP1-mediated immune suppression

• Predictive Biomarker Development:

  • Correlating SPP1 expression/autoantibody levels with response to immunotherapy

  • Stratifying patients based on SPP1 status for clinical trials

  • Developing combinatorial biomarker panels including SPP1

• Therapeutic Potential Assessment:

  • Evaluating SPP1 blocking antibodies as potential immunotherapeutic agents

  • Investigating combination approaches with existing therapies

  • Assessing safety and efficacy in preclinical models

What novel technologies are enhancing the specificity and utility of SPP1 recombinant antibodies?

Advanced technologies improving SPP1 recombinant antibody development include:

• Antibody Engineering Approaches:

  • Single-chain variable fragments (scFvs) for improved tissue penetration

  • Bispecific antibodies targeting SPP1 and another relevant molecule

  • Humanized antibodies for reduced immunogenicity in therapeutic applications

  • Antibody fragments with enhanced stability and reduced non-specific binding

• Production Advancements:

  • Cell-free expression systems for rapid antibody production

  • Continuous manufacturing processes for improved consistency

  • Site-specific conjugation technologies for creating antibody-drug conjugates

  • Synthetic biology approaches for designing novel binding domains

• Validation Technologies:

  • Super-resolution microscopy for precise epitope localization

  • Enhanced validation protocols confirming specificity through multiple methods

  • CRISPR/Cas9 knockout validation to confirm antibody specificity

  • Competitive binding assays with defined peptide fragments

• Application Innovations:

  • Proximity ligation assays for studying SPP1 interactions with binding partners

  • Mass cytometry (CyTOF) for highly multiplexed protein detection

  • Spatial transcriptomics combined with protein detection for correlating SPP1 mRNA and protein expression

  • In vivo imaging using labeled SPP1 antibodies for biodistribution studies