GAA Antibody, Biotin conjugated
Description
Definition and Mechanism
GAA Antibody, Biotin Conjugated refers to a biotinylated antibody targeting lysosomal alpha-glucosidase (GAA), an enzyme critical for glycogen degradation. The biotin conjugation enables detection via streptavidin-based systems, enhancing sensitivity in immunoassays. This conjugate is primarily used in research applications such as Western blotting (WB), immunohistochemistry (IHC), and enzyme-linked immunosorbent assays (ELISA).
Detection Sensitivity and Specificity
Biotinylated GAA antibodies are validated in diverse assays:
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Western Blot: Detects GAA bands at ~76–110 kDa, confirming lysosomal localization .
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Immunohistochemistry (IHC): Used with streptavidin-biotin-peroxidase complexes (SABC) to visualize GAA in cancer tissues (e.g., lung, breast, prostate) .
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ELISA: Quantifies GAA levels in serum or lysates, leveraging biotin-streptavidin amplification .
Table: Validation Protocols
Conjugation Chemistry and Binding Efficiency
Biotin conjugation to GAA antibodies follows protocols similar to those for IFN-γ detection:
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Protein Purification: Antibodies are purified via Protein A/G affinity chromatography .
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Biotinylation: NHS-biotin or other reactive esters covalently link biotin to lysine residues or the Fc region .
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Validation: Dot blot assays confirm biotin accessibility post-conjugation. For example, 6HISMBP-mSA-CGC (colloidal gold-streptavidin) binds biotinylated IFN-γ, demonstrating robust conjugation .
Key Consideration: Free carboxylic acid groups in biotin are critical for transporter binding, but conjugation to antibodies often modifies this group, necessitating alternative detection strategies .
Comparative Analysis of Biotinylated vs. Non-Biotinylated Antibodies
| Parameter | Biotinylated GAA Antibody | Non-Biotinylated GAA Antibody |
|---|---|---|
| Detection System | Streptavidin-HRP, SABC, or colloidal gold conjugates | Direct HRP/alkaline phosphatase labeling |
| Sensitivity | Higher (amplified signal via biotin-streptavidin) | Moderate (direct detection) |
| Applications | ELISA, IHC, IF, multiplex assays | WB (primary detection) |
| Cross-Reactivity | Potential nonspecific binding if biotin is endogenous | Lower risk of background |
Challenges and Future Directions
Product Specs
**Constituents:** 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- PI-rhGAA holds potential as a valuable therapeutic option for improving the treatment of Pompe disease. PMID: 29102549
- The most prevalent mutation in Pompe disease is c.-32-13T, G. PMID: 29181627
- The narrow substrate-binding pocket of rhGAA is situated near the C-terminal ends of beta-strands within the catalytic (beta/alpha)8 domain. Its shape is influenced by a loop from the N-terminal beta-sheet domain and both inserts I and II. PMID: 29061980
- This study represents the first investigation of rhGAA to differentiate M6P glycans and identify their attachment sites, despite rhGAA's existing status as an approved drug for Pompe disease. PMID: 29274340
- GAA mutations are associated with Pompe disease. PMID: 28763149
- Enzyme activities (acid alpha-glucosidase (GAA), galactocerebrosidase (GALC), glucocerebrosidase (GBA), alpha-galactosidase A (GLA), alpha-iduronidase (IDUA), and sphingomyeline phosphodiesterase-1 (SMPD-1)) were assessed on approximately 43,000 de-identified dried blood spot (DBS) punches. Samples that screened positive were submitted for DNA sequencing to obtain genotype confirmation of disease risk. PMID: 27238910
- Enzyme replacement therapy (ERT) (alglucosidase alfa) stabilizes respiratory function and enhances mobility and muscle strength in late-onset Pompe disease. Lysosomal glycogen in muscle biopsies from treatment-naive LOPD patients exhibited reductions post-ERT (alglucosidase alfa). PMID: 27473031
- In adults with Pompe disease, antibody formation does not impede the efficacy of rhGAA in the majority of patients. It is linked to immune-related adverse events (IARs) and might be mitigated by the IVS1/delex18 GAA genotype. PMID: 27362911
- Reanalysis of the patient's DNA sample utilizing next-generation sequencing (NGS) of a panel of target genes associated with glycogen storage disorders revealed compound heterozygosity for a point mutation and an exonic deletion in the GAA gene. PMID: 28657663
- This study identified thirteen novel and two common GAA mutations. The allelic frequency of c.2662G > T (p.Glu888X) was 23.1% in northern Chinese patients and 4.2% in southern Chinese patients, while the allelic frequency of c.1935C >A (p.Asp645Glu) was 20.8% in southern and 3.8% in northern Chinese patients. PMID: 28394184
- This is the first report of the alpha-glucosidase inhibitory activity of compounds 20, 26, and 29. The findings support the significant role of Eremanthus species as novel sources of new drugs or herbal remedies for the treatment of type 2 diabetes. PMID: 27322221
- Compared to controls, GAA gene expression levels were significantly elevated in coronary artery disease (CAD) patients, suggesting a potential role for GAA in the development of CAD. PMID: 26580301
- This study presents clinical, biochemical, morphological, muscle imaging, and genetic findings of six adult Pompe patients from five unrelated families carrying the c.-32-13T>G GAA gene mutation in a homozygous state. All patients exhibited reduced GAA activity and elevated creatine kinase levels. PMID: 26231297
- Glycogen storage disease type II is caused by a deficiency of GAA activity stemming from mutations in the GAA gene. PMID: 26575883
- RT-PCR followed by DNA sequence analysis in patients with Pompe disease revealed a new variant in the GAA gene, resulting in an aberrant splicing event. PMID: 25243733
- Findings indicate that the GAA c.2238G > C (p.W746C) novel mutation is the most prevalent mutation in mainland Chinese late-onset Pompe patients, as observed in Taiwanese patients, expanding the genetic spectrum of the disease. PMID: 25526786
- This study showcases several alterations distributed along the GAA gene in a sample of Brazilian families. PMID: 25681614
- Mutations in the acid alpha-glucosidase gene are associated with Pompe disease. PMID: 25026126
- GAA deficiency leads to diminished mTORC1 activation, partially responsible for the skeletal muscle wasting phenotype. This can be ameliorated by leucine supplementation. PMID: 25231351
- The LO-GSDII phenotype with GAA mutations in Northern Italy appears not significantly different from other LO-GSDII populations in Europe or the USA. PMID: 24158270
- Data presents the largest informative family with late-onset Pompe disease described in the literature, demonstrating a unique complex set of GAA gene mutations that may partially elucidate the clinical heterogeneity observed in this family. PMID: 24107549
- 7 of 27 in: Gene. 2014 Mar 1;537(1) Novel GAA sequence variant c.1211 A>G reduces enzyme activity but not protein expression in infantile and adult onset Pompe disease. PMID: 24384324
- This study demonstrates that the c.-32-13T>G mutation of the GAA gene abrogates the binding of the splicing factor U2AF65 to the polypyrimidine tract of exon 2, and that several splicing factors influence exon 2 inclusion. PMID: 24150945
- This study describes two unrelated cases affected with classical early-onset Pompe disease, both originating from the same small Mexican region, with the same novel homozygous frameshift mutation at the GAA gene (c.1987delC). PMID: 24399866
- Mutations in the GAA gene are associated with glycogen storage disease type II. PMID: 23884227
- Adult patients with alpha-glucosidase mutations other than c.-32-13 T>G can exhibit very low alpha-glucosidase activity in fibroblasts but express higher activity in muscle and store less glycogen in muscle compared to patients with infantile Pompe disease. PMID: 23000108
- This study provides an update of the Pompe disease mutation database, incorporating 60 novel GAA sequence variants and additional investigations into the functional effects of 34 previously reported variants. PMID: 22644586
- Transcriptional response to GAA deficiency (Pompe disease) in infantile-onset patients. PMID: 22658377
- This report details genetic testing to identify GAA mutations in German patients with late-onset glycogen storage disease type II. PMID: 18607768
- This study defines a critical role for endoplasmic reticulum stress in the activation of autophagy due to the 546G>T acid alpha glucosidase mutation. PMID: 21982629
- No common mutation has been found in association with low levels of acid alpha-glucosidase activity in late-onset Pompe disease. Most patients produce unprocessed forms of GAA protein compared to patients exhibiting higher GAA activity. PMID: 21484825
- Mutation analysis of the GAA gene revealed the p.D645E in all patients with Pompe disease, suggesting it as the most prevalent mutation in the Thai population. PMID: 21039225
- The enzymatic screening of Pompe disease is justifiable in patients with myopathies of unknown etiology, as demonstrated in this report of a Mexican patient with late-onset glycogen-storage disease type 2. PMID: 20350966
- Data shows that the p.R1147G missense mutation impaired glucosidase activity. PMID: 19834502
- Homozygosity for multiple contiguous single-nucleotide polymorphisms as an indicator of large heterozygous deletions: identification of a novel heterozygous 8-kb intragenic deletion (IVS7-19 to IVS15-17) in a patient with glycogen storage disease type II. PMID: 11854868
- Novel target of the Notch-1/Hes-1 signaling pathway. PMID: 12065598
- Two novel mutations of the acid alpha-glucosidase gene, P361L and R437C, were found in a juvenile-onset glycogen storage disease type II (GSDII) 16-year-old Chinese patient. The asymptomatic 13-year-old brother of the proband is also a compound heterozygote. PMID: 12601120
- Mutations in the alpha glucosidase gene are associated with infantile onset glycogen storage disease type II. PMID: 12923862
- Childhood Pompe disease demonstrating phenotypic variability of p.Asp645Asn. PMID: 15145338
- Data indicates that the mature forms of GAA characterized by polypeptides of 76 or 70 kDa are, in fact, larger molecular mass multicomponent enzyme complexes. Peptides released during proteolytic processing remained tightly associated with the major species. PMID: 15520017
- Two novel mutations (Ala237Val and Gly293Arg) were found in the acid alpha-glucosidase gene in a Pompe disease patient with vascular involvement. PMID: 15668445
- Acid-alpha-glucosidase activity and specific activity, along with lysosomal glycogen content, are useful predictors of age of onset in Pompe disease. PMID: 15993875
- Complete molecular analysis of the GAA gene in patients with late onset glycogen storage disease type II reveals missense mutations and splicing mutations. PMID: 16917947
- From 14 Argentinean patients diagnosed with either infantile or late-onset disease, we identified 14 distinct mutations in the acid alpha-glucosidase (GAA) gene, including nine novel variants. PMID: 17056254
- Two new missense mutations (p.266Pro>Ser and p.439Met>Lys) were identified as causing late onset GSD II. PMID: 17092519
- Patients with the same c.-32-13T-->G haplotype (c.q. GAA genotype) may exhibit first symptoms at different ages, suggesting that secondary factors can significantly influence the clinical course of patients with this mutation. PMID: 17210890
- This study demonstrated a significant increase (1.3-7.5-fold) in GAA activity after imino sugar treatment in fibroblasts from patients carrying the mutations L552P (three patients) and G549R (one patient). PMID: 17213836
- N-glycans of recombinant human GAA were expressed in the milk of transgenic rabbits. PMID: 17293352
- The role of autophagy in Pompe disease was examined by analyzing single muscle fibers. PMID: 17592248
- Mutations in glucosidase alpha are associated with glycogen storage disease type II. PMID: 17616415
Q&A
What are the primary applications of biotin-conjugated GAA antibodies in academic research?
Biotin-conjugated GAA antibodies are optimized for signal amplification in immunoassays. Common applications include:
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Western blotting: Detection of lysosomal α-glucosidase (GAA) with enhanced sensitivity via streptavidin-HRP conjugates .
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Immunohistochemistry (IHC): Localization of GAA in paraffin-embedded tissues (e.g., lung, breast, prostate cancers) using biotin-avidin systems .
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ELISA: Quantification of GAA via biotin-streptavidin amplification, particularly in glycogen storage disease research .
Methodological Tip: For IHC, combine biotin-conjugated primary antibodies with streptavidin-biotin complex (SABC) or HRP-conjugated streptavidin for chromogenic detection .
How do I validate a biotin-conjugated GAA antibody for my specific application?
Validation requires multifaceted testing:
Troubleshooting: If background persists, use reduced antibody concentrations or enhanced blocking agents (e.g., 5% BSA instead of milk) .
What are the advantages of using biotin-conjugated antibodies over direct HRP-conjugated variants?
Biotin conjugation enables flexible signal amplification:
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Sensitivity: Streptavidin-HRP systems amplify signals 10–100× compared to direct HRP .
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Experimental Control: Biotin allows sequential addition of reagents (e.g., primary antibody → streptavidin-HRP), enabling optimization of each step .
Limitation: Risk of endogenous biotin interference in tissues (e.g., liver, kidney). Mitigate with biotin-blocking kits or avidin/biotin pre-treatment .
How do I resolve cross-reactivity issues in biotin-conjugated GAA antibody assays?
Cross-reactivity often arises from epitope homology or non-specific binding. Solutions include:
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Epitope Mapping: Use antibodies targeting distinct regions (e.g., AA 541–640 vs. AA 1–952) .
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Stringent Washes: Employ 0.1% Tween-20 in TBS for WB/IHC to reduce non-specific interactions .
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Competitive Blocking: Incubate samples with unconjugated GAA peptide (1:100 ratio) to saturate non-specific binding sites .
Example: For rodent models, verify predicted reactivity (e.g., dog, sheep) via species-specific controls (e.g., mouse GAA lysates) .
What conjugation methods are used for biotin-GAA antibodies, and how do they impact performance?
Biotin conjugation typically employs N-hydroxysuccinimide (NHS) ester chemistry or diazo coupling (for antigens with aromatic amines) .
Key Insight: NHS ester conjugation is preferred for GAA antibodies due to preservation of lysosomal epitopes critical for binding .
How can I optimize the biotin-avidin system for GAA detection in low-abundance samples?
For ultrasensitive detection, implement:
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Signal Amplification: Use polymer-based streptavidin-HRP (e.g., SuperSignal West Pico) for enhanced enzymatic activity .
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Antibody Concentration: Optimize primary antibody (0.5–1 µg/mL for WB) and streptavidin-HRP (1:1,000–1:5,000) .
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Substrate Choice: Employ chemiluminescent substrates (e.g., ECL) over chromogenic substrates for WB .
Example Protocol:
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WB: Block with 5% BSA → Primary antibody (0.5 µg/mL) → Streptavidin-HRP (1:2,000) → ECL detection .
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ELISA: Use biotinylated GAA capture antibodies with streptavidin-alkaline phosphatase for linear signal amplification .
What are the reported performance metrics for biotin-conjugated GAA antibodies?
Note: Performance varies by antibody isotype (polyclonal vs. monoclonal) and epitope specificity .
How does biotin conjugation affect antibody stability and storage?
Biotin-conjugated antibodies require gentle storage:
Troubleshooting: Aggregation may occur if conjugation efficiency exceeds 10 biotin/antibody. Use HABA assay to quantify biotin loading .
How do I integrate biotin-GAA antibodies into multiplex assays?
For multiplex IHC/WB, implement:
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Sequential Labeling: Use biotin-GAA with streptavidin-Cy3 and another primary antibody with Alexa Fluor 488 .
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Blocking Steps: Apply avidin/biotin blocking kits between rounds to prevent cross-talk .
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Validation: Include single-plex controls to confirm specificity .
Example: Co-detect GAA and LAMP2 in lysosomal membranes using biotin-GAA + streptavidin-Alexa Fluor 647 and LAMP2-Alexa Fluor 488 .
What are the limitations of biotin-GAA antibodies in studying glycogen storage disease (GSD II)?
Key challenges include:
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Epitope Masking: Propeptide cleavage in lysosomes may obscure antibody binding sites .
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Cross-Tissue Reactivity: GAA is expressed in multiple tissues (e.g., liver, muscle), complicating IHC specificity .
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Enzyme Activity Assays: Biotin conjugation may sterically hinder GAA’s enzymatic activity in functional studies .
