HTRA1 Antibody, Biotin conjugated

What is HTRA1 and why is it a significant target for biotin-conjugated antibodies?

HTRA1 (HtrA Serine Peptidase 1) is a serine protease involved in regulating the availability of insulin-like growth factors by cleaving IGF-binding proteins and repressing signaling by TGF-beta family members. It has significant research interest due to its implications in several pathological conditions, including age-related macular degeneration type 7 (ARMD7) and cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) . Biotin conjugation enhances detection sensitivity without interfering with the antibody's binding specificity, making it particularly useful for amplifying signals in complex biological matrices.

What are the primary applications for biotin-conjugated HTRA1 antibodies?

Biotin-conjugated HTRA1 antibodies have been validated for multiple applications with specific recommended dilutions:

These applications allow researchers to detect HTRA1 in various experimental contexts, from protein expression analysis to cellular and tissue localization studies .

How should researchers optimize detection protocols using biotin-conjugated HTRA1 antibodies in Western blots?

For optimal Western blot results with biotin-conjugated HTRA1 antibodies:

• Sample preparation: Use appropriate lysis buffers that preserve HTRA1's native conformation (typically PBS with protease inhibitors).

• Loading control: Include protein samples with known HTRA1 expression (e.g., hTERT-RPE1 cells, HeLa cells) as positive controls .

• Blocking: Use 5% BSA in PBS to minimize background (avoid milk-based blockers which contain biotin).

• Primary antibody incubation: Apply the biotin-conjugated HTRA1 antibody at 0.2-2μg/mL concentration (1:250-2500 dilution) for optimal signal-to-noise ratio .

• Detection: Utilize streptavidin-HRP conjugates rather than secondary antibodies, as biotin binds streptavidin with exceptional affinity.

• Expected band size: The observed molecular weight for HTRA1 is approximately 51 kDa .

This protocol maximizes specificity while minimizing background interference common with biotin-conjugated reagents.

What experimental conditions affect the specificity of biotin-conjugated HTRA1 antibodies in immunohistochemistry?

Several factors influence the specificity of biotin-conjugated HTRA1 antibodies in IHC:

• Antigen retrieval method: Heat-induced epitope retrieval in citrate buffer (pH 6.0) typically yields optimal results for HTRA1 detection.

• Endogenous biotin blocking: Critical step using avidin/biotin blocking kit to prevent false positives, especially in biotin-rich tissues like brain samples where HTRA1 colocalizes with amyloid deposits .

• Antibody dilution: Begin with 5-20μg/mL (1:25-100) and titrate as needed .

• Incubation conditions: Optimal results typically require 1-2 hour incubation at room temperature or overnight at 4°C.

• Detection system: Employ streptavidin-HRP followed by 3,3′,5,5′-tetramethylbenzidine (TMB) or diaminobenzidine (DAB) visualization .

• Validation controls: Include HtrA1-knockout samples when available as negative controls.

These considerations are particularly important when studying HTRA1 in human brain tissues where it colocalizes with β-amyloid deposits, requiring careful distinction from background signals .

How can biotin-conjugated HTRA1 antibodies be utilized to investigate HTRA1's role in neurodegenerative disease mechanisms?

Biotin-conjugated HTRA1 antibodies offer several strategic approaches for investigating HTRA1's role in neurodegenerative conditions:

• Dual immunofluorescence studies: Combine biotin-conjugated HTRA1 antibodies with antibodies against amyloid-β to examine colocalization patterns in brain tissue. This approach has revealed that HTRA1 colocalizes with β-amyloid deposits in human brain samples .

• Ex vivo proteolytic activity assays: Utilize biotin-conjugated HTRA1 antibodies to immunoprecipitate native HTRA1 from tissue lysates, then assess its ability to cleave potential substrates such as amyloid precursor protein fragments. Research has demonstrated that HtrA1 degrades various APP fragments including Aβ .

• Activity-based probe coupling: Pair biotin-conjugated antibodies with fluorescent activity-based probes to simultaneously track HTRA1 localization and enzymatic activity. This approach has been validated using HtrA1-directed activity-based probes with diphenyl phosphonate reactive groups .

• Inhibitor studies: Use biotin-conjugated HTRA1 antibodies to quantify HTRA1 levels in conjunction with specific HtrA1 inhibitors. In astrocyte cell cultures, HtrA1 inhibition causes accumulation of Aβ in culture supernatants, suggesting HTRA1's role in Aβ clearance .

These methods provide multidimensional insights into HTRA1's mechanistic role in protein quality control and proteostasis relevant to neurodegenerative conditions.

What are the current methodologies for using biotin-conjugated HTRA1 antibodies in quantitative proteomics studies?

For quantitative proteomics applications, biotin-conjugated HTRA1 antibodies can be employed in several sophisticated approaches:

• Substrate identification workflow:

  • Immunoprecipitate HTRA1 using biotin-conjugated antibodies

  • Perform in vitro proteolysis assays with potential substrate proteins

  • Analyze cleavage products via mass spectrometry

  • Compare with catalytically inactive HTRA1 controls

• N-terminomics approach:

  • Apply N-terminomic proteomic techniques to identify specific cleavage sites

  • Analyze amino termini of proteins after incubation with HTRA1 vs. catalytically inactive HTRA1

  • This method has successfully identified the consensus cleavage site of HTRA1

• Activity-based protein profiling:

  • Combine biotin-conjugated HTRA1 antibodies with activity-based small-molecule probes

  • Use activity-based probes containing Val and Leu at P1 and P2 positions to direct reactivity against HTRA1

  • This approach has demonstrated improved selectivity for HtrA1 in complex biological matrices like vitreous humor

• Pharmacodynamic biomarker assessment:

  • Utilize ELISA systems with biotin-conjugated HTRA1 antibodies for detecting HtrA1 in conditioned media

  • Standard procedure involves coating plates with monoclonal α-HtrA1, followed by sample incubation, then detection with polyclonal α-HtrA1 and biotin-conjugated secondary antibodies

These methodologies have enabled identification of physiologically relevant HTRA1 substrates and pharmacodynamic biomarkers applicable to both preclinical models and clinical studies.

What are common sources of background signal when using biotin-conjugated HTRA1 antibodies and how can they be minimized?

Several factors contribute to background signal with biotin-conjugated HTRA1 antibodies:

• Endogenous biotin interference:

  • Problem: Tissues rich in biotin (brain, kidney, liver) can produce false-positive signals

  • Solution: Pre-block sections with avidin/biotin blocking kit before antibody application

  • Validation: Include biotin-blocked control sections for comparison

• Non-specific binding:

  • Problem: Biotin-conjugated antibodies sometimes exhibit off-target binding

  • Solution: Increase blocking stringency (5% BSA in PBS with 0.3% Triton X-100)

  • Optimization: Titrate antibody concentration starting from 5-20μg/mL

• Cross-reactivity with related proteases:

  • Problem: HtrA family includes multiple members with sequence homology

  • Solution: Select antibodies targeting unique epitopes (e.g., antibodies against AA 23-248)

  • Validation: Test specificity using western blots with recombinant HTRA1, HTRA2, and HTRA3

• Fixation artifacts:

  • Problem: Overfixation can mask epitopes and increase non-specific binding

  • Solution: Optimize fixation time and perform suitable antigen retrieval

  • Approach: Compare heat-induced versus enzymatic antigen retrieval methods

• Detection system sensitivity issues:

  • Problem: Excessive amplification can increase background

  • Solution: Use diluted streptavidin-HRP (1:500) and short development times

  • Alternative: Consider tyramide signal amplification for specific applications requiring higher sensitivity

These strategies should be systematically evaluated to achieve optimal signal-to-noise ratios in each experimental system.

How can researchers validate the specificity of biotin-conjugated HTRA1 antibodies across different experimental systems?

To establish confidence in biotin-conjugated HTRA1 antibody specificity:

• Epitope validation:

  • Compare antibodies targeting different HTRA1 regions (N-terminal vs. C-terminal)

  • Available epitope options include AA 23-248, AA 116-147, AA 381-412, or AA 141-480

  • Confirm consistent detection pattern across antibodies recognizing different epitopes

• Knockout/knockdown controls:

  • Use HTRA1-knockout mice tissues or HTRA1-silenced cell lines as negative controls

  • Antibodies showing signals in knockout samples indicate non-specificity issues

• Competing peptide assay:

  • Pre-incubate antibody with excess recombinant HTRA1 protein or immunizing peptide

  • Signal disappearance confirms specificity for the target epitope

• Cross-species reactivity assessment:

  • Test antibody across samples from different species where sequence homology is known

  • Confirm reactivity aligns with epitope conservation

  • Many HTRA1 antibodies show reactivity with human, mouse, rat, pig, and rabbit samples

• Activity-based probe competition:

  • Perform activity inhibition assays using antibodies and fluorescent activity-based probes

  • Specific antibodies should inhibit probe binding to active HTRA1

  • This approach has been validated using HtrA1 ABP in rabbit vitreous humor

These validation approaches should be documented and reported alongside experimental results to ensure reproducibility and reliability of findings.

How are biotin-conjugated HTRA1 antibodies contributing to therapeutic development for age-related macular degeneration?

Biotin-conjugated HTRA1 antibodies play crucial roles in AMD therapeutic development through several mechanisms:

• Biomarker validation:

  • HTRA1 antibodies enable identification of potential pharmacodynamic biomarkers

  • One key substrate, Dickkopf-related protein 3, has been validated as a robust biomarker for anti-HtrA1 activity in both preclinical animal models and clinical studies in geographic atrophy patients

• Therapeutic antibody development:

  • Biotin-conjugated antibodies facilitate screening for therapeutic antibodies with inhibitory properties

  • Example: Clone 15H6 showed strong inhibition of human HtrA1 enzymatic activity and was subsequently humanized through hypervariable region grafting

• Activity monitoring:

  • Using activity-based probes coupled with biotin-conjugated antibodies allows monitoring of:

    • Target engagement (whether therapeutic antibodies bind to HTRA1)

    • Functional inhibition (whether binding reduces proteolytic activity)

  • This approach enabled development of potent anti-HtrA1 Fab inhibitors of HtrA1 proteolytic activity in the retina

• Disease mechanism investigation:

  • Studies using HTRA1 antibodies revealed that variations in the promoter region of HTRA1 are the cause of susceptibility to age-related macular degeneration type 7 (ARMD7)

  • This insight guided development of targeted therapeutic approaches

These applications demonstrate how biotin-conjugated HTRA1 antibodies serve as crucial tools throughout the therapeutic development pipeline from basic mechanistic understanding to clinical biomarker development.

What are the current methodological advances in using biotin-conjugated HTRA1 antibodies for multiplexed imaging applications?

Recent methodological advances in multiplexed imaging with biotin-conjugated HTRA1 antibodies include:

• Sequential multiplexed immunofluorescence:

  • Biotin-conjugated HTRA1 antibodies can be paired with streptavidin-conjugated quantum dots

  • Different quantum dot emissions allow simultaneous visualization of multiple antigens

  • This approach enables co-localization studies of HTRA1 with amyloid deposits and other disease markers

• Mass cytometry adaptation:

  • Conjugation of HTRA1 antibodies with biotin followed by metal-tagged streptavidin

  • Allows integration into CyTOF (Cytometry by Time-Of-Flight) panels

  • Enables single-cell analysis of HTRA1 expression in heterogeneous cell populations

• Proximity ligation assays:

  • Combining biotin-conjugated HTRA1 antibodies with antibodies against potential binding partners

  • Identifies protein-protein interactions through rolling circle amplification

  • Useful for studying HTRA1's interactions with substrates like insulin-like growth factors and their binding proteins

• Expansion microscopy compatibility:

  • Biotin-conjugated antibodies retain functionality after sample expansion

  • Provides super-resolution visualization of HTRA1 localization

  • Particularly valuable for examining HTRA1's association with subcellular structures

• Live-cell imaging approaches:

  • Cell-permeable, minimally disruptive detection systems using split-biotin complementation

  • Allows tracking of HTRA1 dynamics in living cells

  • Facilitates studies of HTRA1 trafficking and activity regulation in real-time

These advanced imaging approaches are expanding our understanding of HTRA1's subcellular localization and functional interactions in both normal and pathological states.