CA1 Antibody

What is Carbonic Anhydrase I and why are antibodies against it significant in research?

Carbonic Anhydrase I (CA1) is a critical enzyme that catalyzes the reversible hydration of carbon dioxide (CO₂ + H₂O ⇌ HCO₃⁻ + H⁺). It also possesses the ability to hydrate cyanamide to urea . CA1 antibodies enable researchers to detect, localize, and quantify this enzyme in various tissues and experimental contexts, providing insights into carbon dioxide metabolism, pH regulation, and related physiological processes.

What are the common applications for anti-CA1 antibodies in laboratory research?

Anti-CA1 antibodies are versatile research tools primarily utilized in:

• Western Blotting (WB): Detecting CA1 protein in cell and tissue lysates at approximately 28-30 kDa

• Immunohistochemistry (IHC): Localizing CA1 in formalin-fixed, paraffin-embedded tissues

• Immunofluorescence (IF): Visualizing cellular distribution of CA1

• ELISA: Quantifying CA1 protein levels

• Immunoprecipitation (IP): Isolating CA1 protein complexes

What is the expected molecular weight when detecting CA1 using antibodies in Western blot analysis?

When performing Western blot analysis, researchers should expect to observe CA1 at approximately 28-30 kDa under reducing conditions . The protein may appear slightly diffuse due to post-translational modifications. For example, when using R&D Systems' anti-CA1 antibody (AF2180) on PVDF membranes with human liver or colon tissue lysates, CA1 appears as a specific band at approximately 30 kDa . Similar results are observed with other anti-CA1 antibodies, such as Abcam's ab109755, which detects CA1 at its predicted molecular weight of 28 kDa in human liver lysates .

How should researchers optimize antibody dilutions for various experimental applications?

Optimal antibody dilution is application-dependent and requires systematic titration:

Researchers should establish optimal conditions for each experimental system through titration experiments, particularly when switching between different tissue types or experimental conditions.

What are the critical considerations for validating the specificity of CA1 antibodies?

Rigorous validation of CA1 antibodies should include:

• Positive control tissues: Include known CA1-expressing tissues such as human erythroleukemic cell lines (HEL 92.1.7), human liver, or human colon tissue

• Negative controls: Include tissues with minimal CA1 expression or use blocking peptides

• Molecular weight verification: Confirm detection at the expected 28-30 kDa size on Western blots

• Cross-reactivity testing: Verify specificity across species if working with non-human samples (many CA1 antibodies react with human, rat, and mouse CA1)

• Knock-down/knock-out validation: Use CA1 siRNA or CRISPR-mediated knockout samples to confirm specificity

How do monoclonal and polyclonal CA1 antibodies differ in their research applications?

Why might researchers observe multiple bands or non-specific signals in Western blots using CA1 antibodies?

Multiple bands or non-specific signals may result from:

• Post-translational modifications: CA1 undergoes modifications that can alter migration patterns

• Proteolytic degradation: Incomplete protease inhibition during sample preparation

• Cross-reactivity: Antibody binding to other carbonic anhydrase isoforms (CA2-CA15)

• Non-specific binding: Insufficient blocking or too high primary antibody concentration

• Secondary antibody issues: Cross-reactivity with sample proteins

Methodological solution: Optimize blocking conditions (5% BSA or 5% non-fat milk), reduce antibody concentration, add 0.1% Tween-20 to wash buffers, and ensure complete protein denaturation. Using fresh samples with complete protease inhibitor cocktails also helps maintain protein integrity .

What are the essential controls for CA1 antibody-based experiments?

What is the CA1 antibody in SARS-CoV-2 research and how does it differ from anti-Carbonic Anhydrase I antibodies?

In SARS-CoV-2 research, "CA1" refers to a specific human monoclonal antibody isolated from COVID-19 patients that targets the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein . This is entirely distinct from antibodies targeting the Carbonic Anhydrase I enzyme. The CA1 anti-SARS-CoV-2 antibody:

• Was isolated from a COVID-19 convalescent patient through B-cell sorting techniques

• Binds to the SARS-CoV-2 RBD with high affinity (KD = 4.68 ± 1.64 nM)

• Shows neutralizing activity against SARS-CoV-2 in vitro

• Functions through blocking the interaction between viral RBD and the ACE2 receptor

What methods are used to evaluate the binding affinity and neutralization potential of the CA1 anti-SARS-CoV-2 antibody?

Scientists employ multiple complementary techniques to characterize CA1:

• Surface Plasmon Resonance (SPR): Measures binding kinetics and affinity (KD) between CA1 and SARS-CoV-2 RBD, revealing a KD value of 4.68 ± 1.64 nM

• Pseudovirus Neutralization Assays: Assesses CA1's ability to prevent viral entry into target cells such as Huh7, Calu-3, and HEK293T cells, quantified as neutralization dose (ND50)

• Live Virus Neutralization: Evaluates CA1's ability to neutralize infectious SARS-CoV-2, showing an ND50 of 0.38 μg/ml (less potent than the related CB6 antibody at 0.036 ± 0.007 μg/ml)

• Octet-Based Binding Competition Assays: Determines whether CA1 competes with other antibodies or ACE2 for binding to the RBD

• Computational Stability Analysis: Predicts how RBD mutations might affect CA1 binding, identifying immune-escaping hotspots that could disrupt antibody recognition

How do RBD mutations in SARS-CoV-2 variants impact CA1 antibody binding and neutralization efficacy?

Computational and experimental analyses have identified several mutation-sensitive regions:

These findings highlight why continuous monitoring of emerging SARS-CoV-2 variants is essential for assessing the continued efficacy of therapeutic antibodies and vaccines.

How does the binding stoichiometry of CA1 compare to other SARS-CoV-2 neutralizing antibodies?

Different anti-SARS-CoV-2 antibodies exhibit varying binding stoichiometries that don't necessarily correlate with neutralization potency:

• CA1 binding stoichiometry data is limited in the provided search results

• Comparative examples show that antibodies like COVA2-15 exhibit a preference for 2:1 stoichiometry (antibody:spike protein), while COVA1-18 displays a preference for 1:1 binding

• These different binding patterns appear uncorrelated to both affinity and neutralization potency, as both antibodies show similar neutralization efficacy despite their distinct binding stoichiometries

Understanding these molecular binding patterns is critical for developing effective antibody cocktails that target non-overlapping epitopes to minimize viral escape.

What emerging technologies might enhance the utility of CA1 antibodies in both contexts?

Several cutting-edge approaches could improve CA1 antibody applications:

• Single-cell antibody sequencing: Generating more diverse and specific CA1 antibody variants from B cell repertoires of multiple donors

• Cryo-EM structural analysis: Providing atomic-level resolution of antibody-antigen interactions to guide epitope-based design

• CRISPR-based validation: Creating precise knockout systems for validating CA1 (enzyme) antibody specificity

• Multiplexed imaging technologies: Combining CA1 antibody detection with other markers for spatial analysis of protein interactions

• AI-driven antibody engineering: Optimizing affinity, specificity, and stability of both anti-CA1 enzyme antibodies and anti-SARS-CoV-2 CA1 therapeutic antibodies