CD274 Recombinant Monoclonal Antibody

What is CD274 and why are recombinant monoclonal antibodies against it significant?

CD274, also known as Programmed Death-Ligand 1 (PD-L1) or B7-H1, is a type I transmembrane glycoprotein that functions as an inhibitory immune checkpoint molecule. It is primarily induced by cytokines, most notably interferon gamma produced by T helper cells under inflammatory conditions . CD274 plays a crucial role in maintaining peripheral T-lymphocyte tolerance and regulating inflammation by binding to its receptor PD-1, thereby inhibiting T-cell activation, proliferation, and cytokine production . The significance of recombinant monoclonal antibodies against CD274 stems from their improved consistency, specificity, and reproducibility compared to conventional monoclonal antibodies, making them valuable tools for studying immune checkpoint inhibition mechanisms and developing cancer immunotherapies.

How do recombinant CD274 monoclonal antibodies differ from conventional monoclonal antibodies?

Recombinant CD274 monoclonal antibodies are produced using recombinant DNA technology, which offers several advantages over hybridoma-derived conventional antibodies. These recombinant antibodies demonstrate increased sensitivity, confirmed specificity, high repeatability, excellent batch-to-batch consistency, sustainable supply, and animal-free production . Unlike conventional monoclonal antibodies that may exhibit variability between production batches, recombinant antibodies are manufactured using proprietary expression systems, purified to homogeneity, and precisely dispensed to ensure robust and highly reproducible lot-to-lot consistency . This consistency is particularly valuable for long-term research projects where experimental reproducibility is critical.

What are the primary research applications for CD274 recombinant monoclonal antibodies?

CD274 recombinant monoclonal antibodies have diverse research applications as evidenced by published literature. The most common applications include:

These antibodies are validated across multiple cell types and tissues, including human placenta, lung cancer tissues, tonsillitis tissues, and various cell lines such as MDA-MB-231, HeLa, and A375 . The methodological approach should be optimized based on the specific experimental goals and sample types.

How should researchers optimize antigen retrieval for CD274 detection in immunohistochemistry?

Antigen retrieval optimization is critical for successful CD274 detection in fixed tissues. Based on validated protocols, two primary buffer systems have demonstrated effectiveness:

• TE buffer (pH 9.0) - This is the preferred method for most tissue types, particularly for human tonsillitis, heart, lung cancer, and placenta tissues .

• Citrate buffer (pH 6.0) - This serves as an alternative approach when TE buffer yields suboptimal results .

When optimizing antigen retrieval conditions, researchers should consider performing side-by-side comparisons of both buffer systems on the same tissue type. The effectiveness of antigen retrieval can be tissue-dependent, with certain tissues requiring longer incubation times or higher temperatures. For human samples, the optimal retrieval conditions typically involve heat-induced epitope retrieval (HIER) at 95-98°C for 20 minutes, followed by cooling to room temperature for 20 minutes. These parameters should be systematically tested and adjusted based on staining intensity and specificity for each new tissue type or fixation condition.

What factors influence the selection of specific CD274 recombinant antibody clones for research applications?

The selection of appropriate CD274 recombinant antibody clones should be guided by several critical factors:

• Epitope specificity - Different clones target distinct epitopes of CD274. For instance, clone 73-10 targets an epitope within the C-terminal region , which may be more accessible in certain experimental conditions.

• Cross-reactivity profile - Some antibodies demonstrate reactivity across multiple species (human, mouse, rat, pig), while others are species-specific . The cross-reactivity profile is particularly important for comparative studies across model organisms.

• Application compatibility - Certain clones perform optimally in specific applications. For example, based on validation data, clone RM320 shows excellent performance in IHC applications , while other clones may be optimized for flow cytometry or western blotting.

• Binding affinity - Higher-affinity antibodies may be required for detecting low-abundance CD274 expression in certain tissues or experimental conditions.

• Format and conjugation - Consider whether the experimental design requires unconjugated antibodies or those conjugated to specific reporter molecules (fluorophores, enzymes, etc.).

Systematic validation of multiple clones for the specific application and sample type is recommended to ensure optimal experimental outcomes.

How can researchers effectively validate CD274 antibody specificity in knockout/knockdown systems?

Validation of CD274 antibody specificity using genetic knockout or knockdown approaches is essential for confirming signal authenticity. A comprehensive validation strategy should include:

• CRISPR/Cas9-mediated CD274 knockout cell lines - Several studies have utilized CD274 knockout validation, confirming the specificity of antibody binding . The knockout approach provides the most definitive evidence of antibody specificity.

• siRNA or shRNA knockdown systems - When knockout models are unavailable, RNA interference approaches can reduce CD274 expression levels. Signal reduction proportional to knockdown efficiency provides evidence of specificity.

• Isotype control comparisons - Parallel staining with matched isotype controls (e.g., Mouse IgG1 for clone 66248-1-Ig or Rabbit IgG for 82719-15-RR) should show minimal background signal .

• Peptide competition assays - Pre-incubation of the antibody with excess immunizing peptide should significantly reduce specific signal in positive samples.

• Multi-antibody concordance - Signal concordance using antibodies targeting different epitopes of CD274 provides additional validation evidence.

For IHC and IF applications, researchers should include positive control tissues with known CD274 expression (e.g., human placenta, tonsillitis tissue) and negative control tissues or cells . The validation parameters should be reported comprehensively in publication methods to ensure reproducibility.

What are the optimal detection strategies for CD274 in different subcellular compartments?

CD274 exhibits dynamic localization patterns that require tailored detection strategies:

• Cell membrane localization - CD274 predominantly localizes to the plasma membrane in normal physiological conditions. For membrane detection:

  • Flow cytometry using live, non-permeabilized cells with antibodies targeting extracellular domains

  • Immunofluorescence with minimal permeabilization using 0.1% Triton X-100 or digitonin

  • Cell surface biotinylation followed by immunoprecipitation

• Cytoplasmic localization - Under certain conditions, CD274 may localize to cytoplasmic compartments:

  • Immunofluorescence with standard permeabilization (0.2-0.5% Triton X-100)

  • Subcellular fractionation followed by western blotting

  • Proximity ligation assays to detect interactions with cytoplasmic proteins

• Nuclear localization - Some studies report nuclear translocation of CD274 fragments:

  • Confocal microscopy with z-stack analysis to confirm intranuclear signal

  • Nuclear and cytoplasmic fractionation followed by western blotting

  • Chromatin immunoprecipitation if associated with DNA

Different fixation methods significantly impact detection sensitivity: paraformaldehyde (2-4%) typically preserves membrane epitopes while methanol fixation may enhance detection of some intracellular epitopes. Co-staining with organelle markers (plasma membrane, endoplasmic reticulum, Golgi) can help confirm subcellular localization patterns in immunofluorescence studies.

What strategies can address inconsistent CD274 detection in western blotting applications?

Inconsistent CD274 detection in western blotting is a common challenge that can be addressed through systematic optimization:

• Sample preparation considerations:

  • Ensure complete denaturation by heating samples at 95-100°C for 5-10 minutes in reducing buffer

  • Include protease inhibitors to prevent degradation

  • For membrane proteins like CD274, use RIPA or specialized membrane protein extraction buffers

• Gel electrophoresis parameters:

  • CD274 has a calculated molecular weight of 33 kDa but is observed at 45-50 kDa due to glycosylation

  • Use 10-12% polyacrylamide gels for optimal resolution

  • Consider gradient gels (4-15%) when analyzing both high and low molecular weight proteins simultaneously

• Transfer optimization:

  • Use semi-dry transfer for 15-30 minutes or wet transfer for 1-2 hours

  • PVDF membranes generally provide better results than nitrocellulose for CD274 detection

  • Transfer in Towbin buffer with 10-20% methanol at constant voltage (25V) overnight at 4°C for difficult-to-transfer proteins

• Blocking and antibody incubation:

  • Test different blocking agents (5% BSA often works better than milk for phosphorylated targets)

  • Optimize primary antibody dilution (1:2000-1:10000 is recommended for most CD274 antibodies)

  • Extend primary antibody incubation to overnight at 4°C

• Signal enhancement strategies:

  • Use high-sensitivity ECL substrates for low-abundance detection

  • Consider signal accumulation using longer exposure times with lower antibody concentrations

When inconsistencies persist, comparison of multiple CD274 antibody clones targeting different epitopes can help confirm band specificity and resolve discrepancies.

How can researchers address variability in CD274 immunohistochemical staining across different tissue types?

Variability in CD274 immunohistochemical staining is a significant challenge that requires systematic approach:

• Tissue fixation standardization:

  • Standardize fixation time (24-48 hours in 10% neutral buffered formalin)

  • Consider testing alternative fixatives for sensitive epitopes

  • Process tissues consistently to minimize pre-analytical variables

• Antigen retrieval optimization by tissue type:

  • For lymphoid tissues: TE buffer (pH 9.0) at 98°C for 20 minutes is typically optimal

  • For lung and placental tissues: Comparative testing of TE buffer (pH 9.0) and citrate buffer (pH 6.0)

  • For highly fibrotic tissues: Extended retrieval times or enzymatic pre-treatment may improve epitope accessibility

• Blocking endogenous activities:

  • Dual peroxidase/alkaline phosphatase block for highly vascular tissues

  • Extended hydrogen peroxide treatment (3% H₂O₂, 10-15 minutes) for tissues with high endogenous peroxidase

  • Biotin/avidin blocking for tissues with high biotin content when using biotin-based detection systems

• Detection system selection based on expression level:

  • For low CD274 expression: Polymer-based or tyramide signal amplification systems

  • For constitutive expression: Standard ABC or polymer detection

  • For quantitative analysis: Chromogenic multiplexing or spectral imaging

• Validation with multiple positive controls:

  • Include human tonsillitis tissue as a positive control showing characteristic membrane staining pattern

  • Human placenta tissue exhibits reliable CD274 expression

  • Lung cancer tissues with known PD-L1 status serve as reference standards

Antibody titration should be performed systematically for each new tissue type, beginning with manufacturer-recommended dilutions (1:5000-1:20000 for IHC) and adjusting based on signal-to-noise ratio.

What are the methodological considerations for developing CD274 blocking antibodies for immunotherapy research?

Developing effective CD274 blocking antibodies for immunotherapy research requires careful consideration of multiple parameters:

• Epitope selection strategy:

  • Target epitopes within the PD-1 binding domain of CD274 for direct blocking activity

  • Structural biology approaches using X-ray crystallography or cryo-EM to identify critical binding interfaces

  • Epitope mapping using peptide arrays or hydrogen-deuterium exchange mass spectrometry

• Antibody format optimization:

  • Full IgG formats (especially human IgG1) provide extended half-life and potential ADCC/CDC activity

  • Fab or F(ab')2 fragments offer improved tissue penetration but reduced half-life

  • Single-domain antibodies (VHH) provide access to recessed epitopes with reduced immunogenicity

• Functional screening methodology:

  • PD-1/PD-L1 binding inhibition assays using ELISA, AlphaScreen, or TR-FRET

  • Cell-based reporter assays measuring T-cell activation upon PD-1/PD-L1 blockade

  • Mixed lymphocyte reactions to assess functional restoration of T-cell proliferation

  • Cytokine release assays measuring IFN-γ, IL-2, or TNF-α production

• In vitro validation approaches:

  • Co-culture systems with CD274-expressing tumor cells and tumor-specific T cells

  • 3D organoid models incorporating immune components

  • Ex vivo human tumor explant cultures to assess tumor microenvironment effects

• In vivo assessment strategies:

  • Syngeneic mouse models with murine cross-reactive antibodies

  • Humanized mouse models for human-specific antibodies

  • Pharmacokinetic/pharmacodynamic correlations with target engagement and biological activity

When generating recombinant monoclonal antibodies for blocking applications, researchers should employ rapid antibody generation workflows from single antigen-specific antibody secreting cells, which can reduce development time to under 10 days .

How should researchers design experiments to investigate CD274 expression heterogeneity in the tumor microenvironment?

Investigating CD274 expression heterogeneity in the tumor microenvironment requires integrated multi-parameter approaches:

• Spatial profiling technologies:

  • Multiplex immunohistochemistry (mIHC) with panels including CD274, immune cell markers (CD3, CD8, CD4, CD68), and tumor markers

  • Multiplex immunofluorescence with spectral unmixing for up to 8-10 markers simultaneously

  • Imaging mass cytometry or CODEX for highly multiplexed spatial profiling (30-40 markers)

  • Digital spatial profiling for quantitative assessment of protein and RNA in defined regions

• Single-cell analysis methodologies:

  • Single-cell RNA sequencing of tumor dissociates to identify cell populations with variable CD274 expression

  • Single-cell proteomics using mass cytometry (CyTOF) with metal-conjugated CD274 antibodies

  • Flow cytometry with panel design including markers for tumor cells, immune cell subsets, and activation status

• Longitudinal assessment approaches:

  • Sequential biopsies before and during treatment

  • Window-of-opportunity clinical trial designs

  • Patient-derived xenograft models with serial sampling

• Data integration frameworks:

  • Computational methods linking spatial information with single-cell transcriptomics

  • Machine learning algorithms for pattern recognition in heterogeneous expression

  • Systems biology approaches to understand regulatory networks controlling CD274 expression

• Analytical validation requirements:

  • Clone-to-clone comparison using multiple validated CD274 antibodies

  • Positive and negative control tissues in each experimental batch

  • Quantitative scoring systems with continuous variable analysis rather than arbitrary cutoffs

For optimal analysis of expression heterogeneity, immunohistochemical staining should utilize antibody clones validated for membranous detection, such as clone RM320 or 73-10 , at dilutions optimized for specific tumor types (typically 1:200-1:800 for IHC) .

What methodological approaches are recommended for investigating post-translational modifications of CD274?

Post-translational modifications (PTMs) of CD274 significantly influence its stability, localization, and function, requiring specialized methodological approaches:

• Glycosylation analysis:

  • CD274 exhibits significant N-linked glycosylation, resulting in observed molecular weights of 45-50 kDa despite a calculated weight of 33 kDa

  • Enzymatic deglycosylation using PNGase F or Endo H followed by western blotting

  • Lectin blotting to characterize glycan structures

  • Mass spectrometry-based glycopeptide mapping for site-specific glycan characterization

  • Monosaccharide compositional analysis by HPAEC-PAD

• Phosphorylation assessment:

  • Phospho-specific antibody development targeting known regulatory sites

  • Phos-tag SDS-PAGE to separate phosphorylated from non-phosphorylated forms

  • IP-Mass spectrometry with titanium dioxide enrichment of phosphopeptides

  • In vitro kinase assays to identify responsible kinases

• Ubiquitination detection:

  • Co-IP under denaturing conditions using ubiquitin antibodies

  • Tandem Ubiquitin Binding Entities (TUBEs) for capturing ubiquitinated proteins

  • Mass spectrometry with diGly remnant antibody enrichment

  • Cell-based ubiquitination assays with proteasome inhibitors

• Other PTM analysis approaches:

  • SUMOylation analysis using SUMO-specific antibodies or SUMO-trap technologies

  • Acetylation detection using pan-acetyl-lysine antibodies followed by mass spectrometry

  • S-palmitoylation assessment using click chemistry-based approaches (Acyl-RAC or ABE)

• Integrated PTM analysis:

  • Multimodal PTM mapping using advanced mass spectrometry

  • Correlation of PTMs with functional outcomes in cell-based assays

  • Computational modeling of PTM crosstalk and hierarchical relationships

For comprehensive PTM characterization, researchers should combine site-directed mutagenesis of putative modification sites with functional assays measuring CD274 stability, localization, and PD-1 binding capacity.

What are the considerations for applying CD274 recombinant antibodies in single-cell analysis techniques?

Applying CD274 recombinant antibodies in single-cell analysis techniques requires specialized optimization:

• Flow cytometry optimization parameters:

  • Cell fixation protocols significantly impact epitope accessibility; paraformaldehyde (2-4%) typically preserves membrane epitopes

  • Permeabilization agents (Triton X-100, saponin, methanol) should be selected based on epitope location

  • Fluorophore selection considering spectral overlap in multiparameter panels

  • Titration experiments to determine optimal signal-to-noise ratio (typically 1:50-1:500 for flow cytometry)

  • Controls including FMO (Fluorescence Minus One) and isotype controls

• Mass cytometry (CyTOF) considerations:

  • Metal conjugation chemistry should preserve antibody binding capacity

  • Antibody stability testing post-conjugation

  • Panel design accounting for signal spillover and oxidation states

  • Barcoding strategies for batch processing

  • Optimized fixation protocols for epitope preservation

• Single-cell RNA-protein co-detection:

  • CITE-seq compatibility testing of antibodies

  • Oligonucleotide conjugation protocols preserving antibody function

  • Validation of detection sensitivity compared to conventional flow cytometry

  • Background assessment in negative control populations

• Imaging-based single-cell analysis:

  • Imaging flow cytometry optimization for membrane vs. cytoplasmic signal

  • Confocal microscopy with deconvolution for subcellular localization

  • Super-resolution techniques (STORM, PALM, STED) for nanoscale distribution

  • Live-cell imaging compatibility for dynamics studies

• Data analysis considerations:

  • Dimensionality reduction approaches (tSNE, UMAP) for visualizing heterogeneity

  • Clustering algorithms for identifying distinct expression patterns

  • Trajectory analysis for developmental or activation-related expression changes

  • Integration with transcriptomic or other proteomic datasets

When applying recombinant antibodies in these techniques, it is essential to validate clone performance in each specific application, as antibodies that perform well in conventional applications may show differential performance in single-cell techniques due to fixation, permeabilization, or conjugation effects.

What quality control measures are essential when working with CD274 recombinant antibodies across different experimental platforms?

Implementing rigorous quality control measures is critical for ensuring reliable results with CD274 recombinant antibodies:

• Antibody validation requirements:

  • Multi-application validation across Western blot, IHC, and flow cytometry

  • Knockout/knockdown validation as the gold standard for specificity

  • Lot-to-lot consistency assessment through side-by-side testing

  • Epitope mapping or epitope tag validation for confirmation of binding site

• Standard operating procedures for common applications:

  • Detailed protocols with critical steps identified

  • Standardized positive and negative controls for each application

  • Technical replicates and biological replicates requirements

  • Quantification methods standardization

• Application-specific quality controls:

  • For Western blotting: Molecular weight markers, loading controls, positive control lysates

  • For IHC/IF: Tissue microarrays with known positive and negative samples

  • For flow cytometry: Fluorescence minus one (FMO) controls, compensation controls

  • For IP: IgG control, input control, non-specific binding assessment

• Reporting standards implementation:

  • Complete antibody identification (catalog number, clone, lot)

  • RRID (Research Resource Identifier) inclusion (e.g., AB_2756526 or AB_3086518)

  • Detailed methodological documentation including incubation times, temperatures, and buffer compositions

  • Raw data preservation and accessibility

• Cross-platform validation strategies:

  • Orthogonal method confirmation (e.g., IF findings confirmed by flow cytometry)

  • Multiple antibody clones targeting different epitopes

  • Integration of protein and transcript data when possible

When working with CD274 recombinant antibodies, researchers should maintain detailed records of antibody performance across different lots, storage conditions, and experimental parameters to facilitate troubleshooting and ensure reproducibility.

How can researchers effectively compare data generated using different CD274 antibody clones in published literature?

Effectively comparing data generated with different CD274 antibody clones requires systematic evaluation:

• Epitope mapping comparison:

  • Identify the specific epitope region targeted by each antibody clone

  • Assess whether epitopes are in functionally distinct domains (e.g., PD-1 binding domain vs. cytoplasmic region)

  • Consider how conformational changes might affect epitope accessibility

• Cross-reactivity profile assessment:

  • Document species cross-reactivity for each clone

  • Note any reported non-specific binding issues

  • Evaluate performance in tissues vs. cell lines

• Application-specific performance analysis:

  • Compare sensitivity and specificity metrics by application

  • Note optimal working concentrations for each application

  • Identify application-specific limitations

• Standardization approaches:

  • Use of reference standard materials when available

  • Quantitative comparison using calibration curves

  • Digital image analysis with standardized parameters

• Integration frameworks:

  • Meta-analysis methods for combining multiple antibody datasets

  • Statistical approaches to normalize for clone-specific sensitivities

  • Consensus scoring systems for IHC interpretation