GAA Antibody, HRP conjugated

What is the GAA enzyme and why study it with HRP-conjugated antibodies?

Acid alpha-glucosidase (GAA) is a lysosomal enzyme encoded by the GAA gene that breaks down glycogen to glucose. Mutations in this gene cause Pompe disease, characterized by glycogen accumulation in tissues, particularly skeletal and cardiac muscle . HRP-conjugated GAA antibodies provide a sensitive detection method for studying GAA expression, localization, and function in research settings. The HRP conjugation enables chromogenic or chemiluminescent detection in western blots, immunohistochemistry, and ELISAs, making it versatile for multiple experimental applications .

What are the recommended dilutions for GAA antibody, HRP conjugated across different applications?

Based on manufacturer recommendations, the following dilutions are typically used for GAA antibody, HRP conjugated:

These dilutions serve as starting points and should be optimized for specific experimental conditions and sample types .

How does HRP conjugation affect the functionality of GAA antibodies?

HRP conjugation to GAA antibodies occurs at surface-exposed lysine residues. This process may potentially affect antibody binding activity if lysine residues are present in or near the antigen-binding site . When using GAA antibody, HRP conjugated, researchers should be aware that:

• The conjugation chemistry can influence antibody performance

• Not all antibodies maintain full activity after conjugation

• Validation with positive and negative controls is essential before experimental use

• Performance may vary between different batches of conjugated antibodies

For optimal results, researchers should validate the conjugated antibody's specificity for their particular application before conducting critical experiments .

How can GAA antibody, HRP conjugated be used to monitor antibody formation in enzyme replacement therapy studies?

Enzyme replacement therapy (ERT) is a standard treatment for Pompe disease, but its efficacy can be limited by anti-rhGAA antibody formation, particularly in CRIM-negative patients . GAA antibody, HRP conjugated can be instrumental in monitoring:

• Development of anti-rhGAA antibodies following ERT administration

• Antibody titers over time to assess immune response dynamics

• Correlation between antibody formation and treatment efficacy

• Comparison of antibody formation between different treatment protocols

In mouse models, researchers have observed that lentiviral gene therapy can prevent anti-rhGAA antibody formation during ERT, with titers remaining below 1:300 (considered background levels) . This prevention was achieved even at subtherapeutic lentiviral doses, suggesting immune tolerance induction. The antibody can be used in ELISA assays to detect even low antibody titers, as demonstrated in studies where titers of 1:3,000 up to 1:1,000,000 were detected .

What methodological considerations are important when using GAA antibody, HRP conjugated in Pompe disease mouse models?

When using GAA antibody, HRP conjugated in Pompe disease mouse models like the Gaa−/− knockout or the CRISPR-generated Gaa em1935C>A knock-in model, several methodological considerations are crucial:

• Sample preparation: Tissue-specific protocols may be required for different affected tissues (skeletal muscle, cardiac muscle, diaphragm)

• Background control: Use age-matched wild-type controls to establish baseline signals

• Cross-reactivity: Verify antibody specificity for both endogenous mouse GAA and recombinant human GAA if using in ERT studies

• Detection sensitivity: Optimize protocols for detecting varying levels of GAA expression, particularly in gene therapy studies where expression levels may vary between tissues

The Gaa em1935C>A knock-in mouse model has near-abolished GAA enzymatic activity and demonstrates markedly increased tissue glycogen storage with concomitantly impaired autophagy . These characteristics should be considered when interpreting antibody signals in experimental contexts.

How can researchers distinguish between endogenous GAA and therapeutic GAA in gene therapy experiments?

In gene therapy experiments for Pompe disease, distinguishing between endogenous GAA and therapeutic GAA is critical for evaluating treatment efficacy. Strategies using GAA antibody, HRP conjugated include:

• Epitope tags: When possible, use therapeutic GAA constructs with epitope tags (such as the Xpress epitope) that can be detected with specific antibodies like Anti-Xpress-HRP

• Species-specific antibodies: Use antibodies that specifically recognize human GAA when human GAA is used therapeutically in mouse models

• Quantitative comparison: Compare GAA signal in treated versus untreated tissues, accounting for the significantly reduced or absent endogenous expression in disease models

• Molecular weight differentiation: Some therapeutic GAA variants may have slightly different molecular weights that can be resolved on western blots

Lentiviral gene therapy studies have demonstrated the importance of distinguishing therapeutic from endogenous GAA, particularly when evaluating immune tolerance and treatment efficacy over time .

What is the recommended protocol for using GAA antibody, HRP conjugated in western blot applications?

For optimal western blot results with GAA antibody, HRP conjugated:

• Sample preparation:

  • Lyse tissues in RIPA buffer with protease inhibitors

  • Centrifuge at 14,000×g for 15 minutes at 4°C

  • Quantify protein concentration using Bradford or BCA assay

• Gel electrophoresis and transfer:

  • Load 20-50 μg protein per lane on 8-10% SDS-PAGE gels

  • Transfer to PVDF membrane at 100V for 1 hour or 30V overnight

• Antibody incubation:

  • Block membrane with 5% non-fat milk in PBST for 1 hour at room temperature

  • Dilute GAA antibody, HRP conjugated at 1:500 in blocking buffer

  • Incubate membrane overnight at 4°C or 2 hours at room temperature

  • Wash 3× with PBST, 5 minutes each

• Detection:

  • Add HRP substrate (ECL) directly to membrane

  • Image using chemiluminescence detection system

  • Expected molecular weight for GAA: ~110 kDa precursor, ~95 kDa and ~76 kDa processed forms

For troubleshooting weak signals, consider increasing antibody concentration, extending incubation time, or using enhanced chemiluminescence substrates .

How should researchers optimize ELISA protocols for detecting anti-GAA antibodies in serum samples?

For detecting anti-GAA antibodies in serum samples, the following ELISA protocol can be optimized:

• Plate coating:

  • Coat 96-well plates with 50 μl of purified recombinant GAA (1-10 μg/ml in PBS)

  • Incubate overnight at 4°C

  • Wash 3× with PBST

• Blocking and sample addition:

  • Block with 200 μl PBSTM (PBS with 0.05% Tween-20 and 2% milk) for 1-2 hours at room temperature

  • Add diluted serum samples (starting at 1:100 dilution with serial 3-fold dilutions)

  • Incubate for 2 hours at room temperature

  • Wash 3× with PBST

• Secondary antibody:

  • Add GAA antibody, HRP conjugated (1:1000 in PBSTM)

  • Incubate for 1-2 hours at room temperature

  • Wash 3× with PBST

• Detection:

  • Add 50 μl TMB substrate

  • Incubate for 5-30 minutes until color develops

  • Stop reaction with 50 μl 2N H₂SO₄

  • Read absorbance at 450 nm

This protocol can detect antibody titers ranging from 1:300 (background levels) to 1:1,000,000 as observed in gene therapy studies . To ensure accuracy, include positive controls (serum from immunized animals) and negative controls (pre-immune serum) .

What are the common pitfalls when using GAA antibody, HRP conjugated and how can they be addressed?

Several common pitfalls can occur when using GAA antibody, HRP conjugated:

It's important to note that some antibodies may have lysine residues in their antigen-binding sites, and conjugation may affect binding activity. If experiencing persistent problems with a particular conjugate, contacting technical support is recommended .

How can GAA antibody, HRP conjugated be used to evaluate immune tolerance in gene therapy approaches for Pompe disease?

GAA antibody, HRP conjugated plays a crucial role in evaluating immune tolerance in gene therapy approaches for Pompe disease:

• Monitoring antibody formation: After lentiviral gene therapy and subsequent ERT administration, GAA antibody, HRP conjugated can be used in ELISA to detect anti-rhGAA antibodies, with titers reflecting the degree of immune response

• Assessing immune tolerance thresholds: Research has shown that prevention of anti-rhGAA antibody formation was achieved using lentiviral gene therapy at both therapeutic (MOI = 20) and subtherapeutic (MOI = 2) doses, indicating immune tolerance induction at different thresholds

• Evaluating long-term tolerance: Studies demonstrated that HSPC-mediated lentiviral gene therapy prevented antibody formation for extended periods (10+ weeks) during weekly ERT injections

• Investigating tolerance mechanisms: By comparing antibody titers between different treatment intervals and conditioning regimens, researchers identified that preconditioning intensity affects immune tolerance, with reduced conditioning (2 Gy vs. 6 Gy) failing to prevent antibody formation

These applications have revealed that a minimum interval between gene therapy and ERT administration may be necessary for establishing immune tolerance, with shorter intervals (1 week) resulting in low antibody titers (1:3,000) compared to longer intervals (6-12 weeks) which completely prevented antibody formation .

What role does GAA antibody, HRP conjugated play in assessing novel therapeutic approaches for correcting GAA splicing defects?

GAA antibody, HRP conjugated is particularly valuable in evaluating novel therapeutic approaches targeting GAA splicing defects:

• Validation of splice correction: In studies of late-onset Pompe disease (LOPD) caused by the common c.-32-13T>G mutation, which affects splicing, GAA antibody can detect restored protein expression following therapeutic intervention

• Quantification of therapeutic efficacy: By measuring GAA protein levels via western blot using GAA antibody, HRP conjugated, researchers can quantitatively assess how effectively a splice-correcting therapy restores GAA expression

• Correlation with enzyme activity: Comparing protein expression levels (detected via antibody) with functional enzyme activity assays provides a comprehensive assessment of therapeutic efficacy

• Tissue-specific evaluation: The antibody allows researchers to evaluate splice correction efficacy across different tissues, which is crucial as splicing efficiency can vary between tissue types

Recent research has developed therapeutic approaches designed to correct splicing defects caused by the c.-32-13T>G mutation in LOPD patients. These approaches aim to restore proper GAA production by correcting the splicing process. GAA antibody, HRP conjugated provides a direct method to verify increased GAA protein levels resulting from these interventions .

How does GAA antibody, HRP conjugated performance compare across different species samples?

GAA antibody, HRP conjugated performance varies across species samples, with important implications for cross-species research:

Researchers should consider species-specific validation, particularly when:

• Comparing human GAA expression in mouse models after gene therapy

• Translating findings between model organisms and human samples

• Studying differences in GAA processing between species

• Evaluating antibody responses to human GAA in animal models

The commercially available GAA rabbit polyclonal antibody, HRP conjugated has demonstrated reactivity to human, mouse, and rat samples , making it versatile for comparative studies across these species.

How can GAA antibody, HRP conjugated be used to assess GAA processing in different cellular compartments?

GAA undergoes complex processing from a 110 kDa precursor to mature forms of approximately 76 kDa and 70 kDa. GAA antibody, HRP conjugated can be used to track this processing in different cellular compartments:

• Subcellular fractionation analysis:

  • Separate cellular fractions (cytosol, endoplasmic reticulum, lysosome)

  • Run western blots with GAA antibody, HRP conjugated

  • Compare molecular weight patterns across fractions to track processing stages

• Immunofluorescence co-localization (using de-conjugated primary antibody):

  • Use GAA antibody in combination with organelle markers

  • Quantify co-localization coefficients

  • Assess processing efficiency across cellular compartments

• Pulse-chase analysis:

  • Metabolically label newly synthesized GAA

  • Immunoprecipitate with GAA antibody at different time points

  • Track processing through molecular weight changes

This approach is particularly valuable in studying GAA processing in Pompe disease models where mutations like c.1935C>A (p.Asp645Glu) in Southern Han Chinese patients can affect GAA processing and trafficking to lysosomes .