ULBP1 Antibody, Biotin conjugated

What is ULBP1 and what is its physiological significance?

ULBP1 (UL16-binding protein 1) is a MHC class Ib protein that functions as a ligand for the NKG2D receptor expressed on natural killer (NK) cells, CD8+ T cells, and γδ T cells. It plays a critical role in immune surveillance by binding and activating the KLRK1/NKG2D receptor, thereby mediating natural killer cell cytotoxicity . ULBP1 is expressed on the surface of stressed or infected cells and is particularly important in tumor recognition by immune cells. Research has demonstrated that ULBP1 expression levels determine lymphoma susceptibility to γδ T cell–mediated cytolysis, with blockade of NKG2D significantly inhibiting lymphoma cell killing . ULBP1 has been identified as a nonredundant determinant in this process, highlighting its unique physiological relevance for tumor recognition by γδ T cells.

What are the molecular characteristics of ULBP1?

ULBP1 is a glycoprotein with the following characteristics:

• Calculated molecular weight: 28 kDa (244 amino acids)

• Observed molecular weight: 28 kDa in Western blot applications

• Subcellular location: Cell membrane

• Anchoring: GPI-linked to the membrane

• Structure: Possesses alpha 1 and alpha 2 Ig-like domains, but lacks the capacity to bind peptide or interact with beta 2-microglobulin

• Gene ID (NCBI): 80329

• UniProt ID: Q9BZM6

• Alternative names: N2DL1, RAET1I, ALCAN-beta, NKG2D ligand 1, Retinoic acid early transcript 1I

How do biotin-conjugated ULBP1 antibodies differ from unconjugated versions in experimental applications?

Biotin-conjugated ULBP1 antibodies offer several advantages over unconjugated versions:

• Detection flexibility: The biotin tag allows for secondary detection using various streptavidin-conjugated reporters (fluorophores, enzymes)

• Signal amplification: The high-affinity biotin-streptavidin interaction (Kd~10^-15 M) provides stronger signal amplification compared to direct antibody detection

• Multiplex compatibility: Allows for simultaneous detection of multiple targets in the same experiment when used with differently labeled streptavidin conjugates

• Stable storage: Biotin conjugation often results in antibodies with longer shelf-life than directly labeled antibodies with fluorophores

What are optimal experimental conditions for using biotin-conjugated ULBP1 antibodies in Western blotting?

Based on validated protocols for biotin-conjugated ULBP1 antibodies:

Sample preparation:

• Protein extraction should be performed using buffers containing protease inhibitors to prevent degradation

• For cell membrane proteins like ULBP1, detergent-based lysis buffers (e.g., RIPA buffer) are recommended

Western blot protocol:

• Recommended dilution: 1:500-1:2000 for most biotin-conjugated ULBP1 antibodies

• Blocking: 5% non-fat dry milk or 5% BSA in TBS-T for 1 hour at room temperature

• Primary antibody incubation: Overnight at 4°C

• Detection: Streptavidin-HRP (1:5000-1:10000) for 1 hour at room temperature

• Expected band size: 28 kDa

Positive controls:

• HEK-293 cells transfected with ULBP1

• Jurkat cells

• MOLT-4 human acute lymphoblastic leukemia cell line

Troubleshooting tips:

• If multiple bands appear, consider using more stringent washing or increasing blocking time

• For weak signals, extend exposure time or increase antibody concentration

• Ensure sample is not over-heated during preparation as ULBP1 may be heat-sensitive

How can flow cytometry protocols be optimized for biotin-conjugated ULBP1 antibodies?

For optimal flow cytometry results with biotin-conjugated ULBP1 antibodies:

Cell preparation:

• Use freshly harvested cells when possible

• Maintain cells at 4°C throughout the staining procedure to prevent internalization

• Fix cells with 2-4% paraformaldehyde if necessary

Staining protocol:

• Wash cells in cold PBS with 1-2% FBS

• Block Fc receptors with human serum or commercial Fc block

• Incubate with biotin-conjugated ULBP1 antibody (typically 10 μg/mL)

• Wash 2-3 times with cold PBS/FBS

• Incubate with streptavidin-conjugated fluorophore (PE, APC, or FITC)

• Wash again and analyze

Controls to include:

• Unstained cells

• Cells stained with isotype control-biotin plus streptavidin-fluorophore

• Positive control cell line (MOLT-4 or Jurkat cells)

Data analysis considerations:

• Use appropriate compensation when multiplexing

• Consider the bimodal distribution often seen with ULBP1 expression

• Compare mean fluorescence intensity (MFI) rather than just percent positive cells

As demonstrated in validated experiments, MOLT-4 human acute lymphoblastic leukemia cell line shows reliable staining with biotin-conjugated ULBP1 antibodies followed by streptavidin-PE or streptavidin-PerCP detection .

How can researchers optimize immunohistochemistry protocols for biotin-conjugated ULBP1 antibodies?

For effective immunohistochemistry using biotin-conjugated ULBP1 antibodies:

Tissue preparation:

• Use formalin-fixed, paraffin-embedded (FFPE) sections (4-6 μm thick)

• Heat-mediated antigen retrieval in EDTA buffer (pH 8.0) is recommended

Staining protocol:

• Deparaffinize and rehydrate tissue sections

• Perform antigen retrieval (critical for ULBP1 detection)

• Block endogenous peroxidase with 3% H₂O₂

• Important: Include biotin blocking step using commercially available kits

• Block non-specific binding with 10% normal goat serum

• Incubate with biotin-conjugated ULBP1 antibody (2 μg/ml) overnight at 4°C

• Incubate with streptavidin-HRP or use Streptavidin-Biotin-Complex (SABC)

• Develop with DAB and counterstain with hematoxylin

Validated positive controls:

• Human mammary cancer tissue has shown consistent ULBP1 expression

• Human testis tissue can also be used as a positive control

Optimization steps:

• Titrate antibody concentration (1:100 to 1:500 dilution range)

• Adjust incubation times based on signal strength

• Consider signal amplification systems for low-expressing samples

How can biotin-conjugated ULBP1 antibodies be used to investigate immune evasion mechanisms in cancer?

Biotin-conjugated ULBP1 antibodies are valuable tools for studying cancer immune evasion through multiple methodologies:

Multiparametric flow cytometry approach:

• Analyze ULBP1 expression on tumor cells alongside other NKG2D ligands (MICA/B, ULBP2-6)

• Simultaneously assess NK cell activation markers (CD69, CD107a)

• Correlate ULBP1 expression with NK cell cytotoxicity using degranulation assays

In vitro functional assays:

• Use biotin-conjugated ULBP1 antibodies to block ULBP1-NKG2D interactions selectively

• Compare effects with pan-NKG2D blocking to determine ULBP1's specific contribution

• Measure NK cell cytotoxicity against tumor cells before and after ULBP1 blockade

Research has demonstrated that ULBP1 expression levels have a wide spectrum in primary biopsies from lymphoma and leukemia patients, suggesting this variation impacts responsiveness to γδ T cell-based immunotherapy . Importantly, specific loss-of-function studies have shown that ULBP1's role in tumor recognition is nonredundant, highlighting its unique physiological relevance .

Data from cytotoxicity assays indicate that SV40-infected cells with downregulated ULBP1 show significantly decreased susceptibility to NK cell killing. This reduction in killing is specifically due to reduced NKG2D recognition, as demonstrated by equivalent killing when NKG2D is blocked .

What methodologies exist for investigating viral immune evasion through ULBP1 downregulation?

Viruses have evolved mechanisms to evade immune surveillance by downregulating ULBP1. Biotin-conjugated ULBP1 antibodies can be employed in these research methodologies:

Kinetic analysis of ULBP1 downregulation:

• Infect target cells with virus at various MOIs (1-100)

• Harvest cells at different time points post-infection

• Analyze ULBP1 expression by flow cytometry using biotin-conjugated antibodies

• Simultaneously analyze viral protein expression

Mechanistic investigation using viral mutants:

• Generate viral mutants lacking specific genes

• Compare ULBP1 downregulation between wild-type and mutant viruses

• Correlate findings with NK cell functional assays

Research with SV40 virus has revealed that:

• ULBP1 downregulation occurs in an MOI-dependent manner

• ULBP1 mRNA levels are reduced at 72 hours post-infection while ULBP2/3 mRNA remains unchanged

• ULBP1 is not shed from the cell surface during viral infection

• SV40 microRNAs, capsid proteins, and agnoprotein are not responsible for ULBP1 downregulation

NK cell functional assays:

• NK cytotoxicity assays can be performed using primary bulk human NK cells

• CD107a degranulation assays confirm differences in NK cell activation

• Blocking NKG2D can determine if reduced killing is specifically due to ULBP1 downregulation

How can biotin-conjugated ULBP1 antibodies be incorporated into multiplexed assays for comprehensive immune monitoring?

Biotin-conjugated ULBP1 antibodies offer unique advantages for multiplexed immune monitoring:

Multi-parameter flow cytometry panels:

• Use biotin-conjugated ULBP1 antibody with streptavidin-conjugated fluorophores that fill spectral gaps

• Include markers for:

  • Additional NKG2D ligands (MICA/B, ULBP2-6)

  • NK cell receptors (NKG2D, KIRs, NCRs)

  • Activation/inhibition markers (CD69, PD-1, TIM-3)

  • Functional markers (IFN-γ, granzyme B, perforin)

Mass cytometry (CyTOF) applications:

• Conjugate ULBP1 antibodies to biotin

• Use metal-labeled streptavidin for detection

• Create comprehensive panels with 30+ parameters for deep immune profiling

Spatial profiling of ULBP1 in tissue microenvironment:

• Use biotin-conjugated ULBP1 antibodies in multiplexed immunofluorescence

• Detect with streptavidin-fluorophores or quantum dots

• Combine with antibodies against immune cell markers (CD8, CD56)

• Use cyclic immunofluorescence methods for extended multiplexing

Research has shown that analyzing ULBP1 alongside other immune checkpoints provides more comprehensive understanding of immune evasion. Multiplexed assays have revealed that the expression pattern of ULBP1 can be distinct from other NKG2D ligands in various tumor types, highlighting the importance of monitoring multiple ligands simultaneously .

How should researchers address endogenous biotin interference when using biotin-conjugated ULBP1 antibodies?

Endogenous biotin can cause significant background issues when using biotin-conjugated antibodies, particularly in tissues with high biotin content:

Pre-analytical strategies:

• Implement avidin/biotin blocking steps:

  • Incubate samples with avidin solution (10-20 μg/mL) for 15 minutes

  • Wash thoroughly

  • Follow with biotin solution (2 μg/mL) for 15 minutes

  • Wash again before applying biotin-conjugated primary antibody

• For immunohistochemistry, consider using specialized blocking kits:

  • Commercial avidin/biotin blocking kits

  • Streptavidin/biotin blocking kits (higher affinity than avidin)

Alternative detection strategies:

• For tissues with persistent high background:

  • Consider unconjugated ULBP1 antibodies with non-biotin detection systems

  • Use biotin-free detection systems (e.g., polymeric detection systems)

• Validation controls to include:

  • Secondary-only controls

  • Isotype control-biotin plus detection reagent

  • Comparison of staining patterns between biotin-conjugated and unconjugated antibodies

Cell-specific considerations:
For cells known to have high endogenous biotin (e.g., liver, kidney, adipose tissue), additional background reduction steps include:

• Extended blocking times (2+ hours)

• Higher concentrations of blocking reagents

• Pre-absorption of detection reagents with tissue powder

What strategies can address non-specific binding when using biotin-conjugated ULBP1 antibodies?

Non-specific binding can complicate interpretation of results with biotin-conjugated ULBP1 antibodies:

Optimizing blocking conditions:

• For Western blot:

  • Test different blocking agents (5% BSA vs. 5% non-fat milk)

  • Extend blocking time to 2 hours at room temperature

  • Add 0.1-0.3% Tween-20 to blocking buffer

• For flow cytometry:

  • Include 2% FBS or BSA in staining buffer

  • Add 10% serum from the species of secondary reagent

  • Use commercial Fc receptor blocking reagents

• For immunohistochemistry:

  • Use 10% normal goat serum as demonstrated in validated protocols

  • Increase blocking time to 1 hour at room temperature

  • Consider adding 0.1-0.3% Triton X-100 for intracellular targets

Antibody validation strategies:

• Confirm specificity using:

  • ULBP1 knockout/knockdown cells as negative controls

  • Transfected cells overexpressing ULBP1 as positive controls

  • Pre-absorption with recombinant ULBP1 protein

• Cross-reactivity testing:

  • Test against related proteins (ULBP2-6) to ensure specificity

  • Verify results with a second ULBP1 antibody recognizing a different epitope

Research shows that ULBP1 antibodies such as clone 170818 have been validated in multiple cell lines including MOLT-4, Jurkat, and transfected HEK-293 cells .

How should researchers interpret variations in ULBP1 detection across different experimental platforms?

Variations in ULBP1 detection across platforms (flow cytometry, Western blot, IHC) can occur for multiple reasons:

Platform-specific considerations:

Biological variables affecting detection:

• Cell stress conditions: ULBP1 is upregulated under various stress conditions

• Cell cycle phase: Expression may vary with cell cycle

• Intracellular trafficking: Surface vs. intracellular pools of ULBP1

• Post-translational modifications: Variations in glycosylation patterns

Quantitative comparisons across platforms:

• Always include the same positive controls across platforms

• Consider relative expression rather than absolute values

• Use multiple antibody clones when available

• Validate key findings with functional assays (e.g., NK cytotoxicity)

Research has demonstrated that ectopic expression of SV40 large T antigen can induce ULBP1 expression (approximately 3-fold elevation in MFI) , while ULBP1 expression is reduced following viral infection. This highlights the importance of considering the biological context when interpreting ULBP1 detection results.

How can researchers investigate the differential regulation of ULBP1 compared to other NKG2D ligands?

Understanding the unique regulation of ULBP1 versus other NKG2D ligands requires specialized approaches:

Transcriptional regulation analysis:

• Use reporter constructs with ULBP1 promoter regions

• Employ ChIP-seq to identify transcription factors binding to the ULBP1 promoter

• Compare with other NKG2D ligand promoters (MICA/B, ULBP2-6)

• Analyze expression data from forward genetic screens that have identified novel factors supporting ULBP1 expression

Post-transcriptional regulation:

• Assess mRNA stability through actinomycin D chase experiments

• Investigate microRNA regulation of ULBP1 versus other ligands

• Examine RNA-binding proteins that may differentially regulate ULBP1

Post-translational regulation:

• Analyze protein half-life using cycloheximide chase assays

• Investigate ubiquitination patterns using immunoprecipitation

• Examine trafficking pathways using confocal microscopy with biotin-conjugated antibodies

• Study shedding mechanisms using ELISA assays for soluble ULBP1

Research using forward genetic screens has identified several factors that specifically support ULBP1 expression . Additionally, studies with SV40 infection demonstrated differential regulation of ULBP family members, where ULBP1 mRNA was substantially reduced at 72 hours post-infection while ULBP2 and ULBP3 mRNA levels remained unchanged .

What methodologies can assess the functional significance of ULBP1 in immune surveillance against different cancer types?

To assess ULBP1's specific role in immune surveillance across cancer types:

Primary sample analysis:

• Analyze ULBP1 expression across tumor biopsies using flow cytometry or IHC

• Correlate with clinical outcomes and treatment response

• Compare expression patterns between primary tumors and metastases

Functional assays:

• NK cell degranulation assays:

  • Measure CD107a expression on NK cells co-cultured with cancer cells

  • Compare degranulation with and without ULBP1 blockade

  • Assess the contribution of ULBP1 versus other NKG2D ligands

• Cytotoxicity assays with specific blockade:

  • Use biotin-conjugated ULBP1 antibodies to specifically block ULBP1-NKG2D interaction

  • Compare with isotype controls and pan-NKG2D blockade

  • Calculate the percentage of killing attributable specifically to ULBP1

• 3D tumor models:

  • Employ engineered three-dimensional tumor models to study NK cell suppression

  • Analyze ULBP1's role in spatial context of tumor microenvironment

Research has revealed a very wide spectrum of ULBP1 expression levels in primary biopsies from lymphoma and leukemia patients, suggesting this variation impacts responsiveness to γδ T cell-based immunotherapy . Furthermore, studies have demonstrated that the role of ULBP1 is nonredundant in lymphoma recognition, highlighting its unique physiological relevance .

A reduction in NK killing of SV40-infected cells compared to mock-infected cells was observed, resulting from reduced NKG2D recognition. When NKG2D was blocked, killing of all cells was equivalent, confirming ULBP1's specific contribution to immune recognition .

How can researchers develop improved detection systems for ULBP1 in complex biological samples?

Advanced detection systems for ULBP1 in complex samples can enhance research capabilities:

Proximity ligation assays (PLA):

• Combine biotin-conjugated ULBP1 antibodies with antibodies against NKG2D

• Visualize and quantify ULBP1-NKG2D interactions in situ

• Assess spatial distribution of interactions within the immune synapse

Mass spectrometry-based approaches:

• Use biotin-conjugated ULBP1 antibodies for immunoprecipitation

• Employ quantitative proteomics to identify ULBP1-interacting proteins

• Analyze post-translational modifications of ULBP1 in different contexts

Single-molecule detection techniques:

• Employ quantum dot-labeled streptavidin with biotin-conjugated ULBP1 antibodies

• Use total internal reflection fluorescence (TIRF) microscopy

• Track single ULBP1 molecules on cell surfaces to understand dynamics

Multiplex digital detection platforms:

• Incorporate biotin-conjugated ULBP1 antibodies into digital ELISA platforms

• Achieve ultra-sensitive detection of soluble ULBP1 in serum/plasma

• Correlate with disease progression and treatment response

Validated data from recombinant human ULBP1 binding studies show that:

• Human KLRK1 protein captured on COOH chip can bind Human ULBP1 protein with an affinity constant of 2.27 nM as detected by LSPR Assay

• Immobilized KLRK1 at 10 μg/ml can bind human Biotinylated ULBP1, with EC₅₀ of 4.254-7.295 ng/ml