HA-33 Antibody, HRP conjugated

Basic Research Questions

• What is HA-33 and why is it significant in botulinum research?

  HA-33 (Hemagglutinin-33) is a 33 kDa protein component of the botulinum neurotoxin complex from Clostridium botulinum. It plays a critical role in protecting the structural integrity of the neurotoxin and increases internalization of the neurotoxin into the host bloodstream. The protein is involved in binding to the small intestine through interactions with glycolipids and glycoproteins containing sialic acid moieties .

  This protein is significant in botulinum research as it represents a target for detection and neutralization strategies. Understanding HA-33's structure and function provides insights into botulinum toxin pathogenesis and can inform the development of improved diagnostic tools and therapeutic approaches.

• What are the optimal experimental conditions for using HA-33 Antibody, HRP conjugated in ELISA assays?

  For optimal performance in ELISA assays, HA-33 Antibody, HRP conjugated should be used with the following methodological considerations:

  • Working dilution should be experimentally determined for each specific protocol, though starting dilutions of 1:5000-1:50000 are recommended based on similar HRP-conjugated antibodies

  • Buffer composition should match the antibody's storage buffer (PBS with 50% Glycerol, 0.03% Proclin 300, pH 7.4)

  • Blocking solutions containing 0.5-1% BSA are compatible with the antibody formulation

  • Optimal incubation times typically range from 1-2 hours at room temperature or overnight at 4°C

  • Thorough washing steps with PBS-T (0.05% Tween-20) should be included to minimize background

  • TMB or other HRP-compatible substrates should be used for detection

  The assay sensitivity can be further enhanced by optimizing sample concentration, incubation temperature, and detection exposure times.

• How should HA-33 Antibody, HRP conjugated be stored to maintain optimal activity?

  Proper storage is critical for maintaining antibody activity and shelf life. The recommended storage protocols are:

  • Store at -20°C or -80°C for long-term storage

  • Avoid repeated freeze-thaw cycles as they can compromise antibody structure and function

  • When received, consider aliquoting into single-use volumes to prevent repeated freezing and thawing

  • Keep protected from light, especially important for the HRP conjugate which can be light-sensitive

  • The storage buffer (50% Glycerol, 0.03% Proclin 300 in PBS, pH 7.4) helps stabilize the antibody during freeze-thaw cycles

  • The antibody remains stable for approximately one year when properly stored

  Research demonstrates that antibodies stored under these conditions maintain >90% of their original activity compared to those subjected to multiple freeze-thaw cycles.

• What controls should be included when using HA-33 Antibody, HRP conjugated?

  Proper experimental controls are essential for result interpretation. When using HA-33 Antibody, HRP conjugated, researchers should implement:

  • Positive control: Purified recombinant HA-33 protein (AA 2-286) from Clostridium botulinum

  • Negative control: Samples known to be negative for HA-33

  • Isotype control: Non-specific rabbit IgG, HRP-conjugated, to assess non-specific binding

  • Secondary antibody control: Reaction without primary antibody to evaluate background signal

  • Blocking control: Wells with blocking solution only to establish baseline signal

  • Cross-reactivity controls: Testing with related Clostridium species to verify specificity

  These controls help distinguish specific binding from background noise and validate experimental findings.

• What is the reactivity spectrum of HA-33 Antibody, HRP conjugated?

  The HA-33 Antibody, HRP conjugated shows specific reactivity against:

  • Primary target: Clostridium botulinum HA-33 protein (AA 2-286)

  • UniProt accession: P46084

  • Minimal to no cross-reactivity with other bacterial species has been reported

  • Binding specificity is directed against amino acids 2-286 of the HA-33 protein

  The antibody has been validated for ELISA applications with recombinant and native HA-33 protein. When designing experiments, researchers should consider that the antibody recognizes the full-length protein rather than specific epitopes, making it valuable for general detection but potentially limiting for structural or functional studies targeting specific domains.

Advanced Research Questions

• How can thermostabilization techniques be applied to enhance anti-HA33 antibody performance?

  Thermostabilization of anti-HA33 antibodies can significantly improve their utility in demanding research applications. Based on established research methodologies:

  • CDR grafting onto stabilized frameworks can increase thermostability by approximately 10°C while maintaining or improving binding affinity

  • Framework stabilizing mutations can be incorporated into the variable domains, including:

    • Heavy chain mutations: L5V, R19I, S49C, I69C (in IgHV3-23)

    • Light chain mutations: M4L, P12A, T14L, F36Y, R46L, Y87F (in IgKV2D-30)

  In a published study, an anti-HA33 antibody underwent stabilization through CDR grafting into a stabilized human framework, resulting in a melting temperature (Tm) increase from 82.1°C to 92.1°C . This approach maintained binding affinity with only a modest 1.5-fold reduction (from 6 nM to 9 nM Kd) .

  Antibody	Description	Mutations	Fab Tm (°C)	KD
  APE1136	Original mouse Fab	NA	82.1	6 nM
  APE1148	Chimeric antibody	NA	85.9	6 nM
  APE1196	Partial framework mutations	VH (Q5V, G49C, I69C) and VL (M4L)	89	6 nM
  APE1146	Full CDR graft to stabilized framework	NA	92.1	9 nM
  APE1553	Affinity matured variant	HC: H35N, A53L, Q64R; LC: N50D, G66E	88.2	30 pM
  APE1854	Modified affinity matured variant	Same as APE1553 without G66E in LC	92	45 pM

  This data demonstrates that optimized antibodies can maintain 100% activity after 1 hour at 70°C, while the original antibody showed complete loss of activity under the same conditions .

• What methodologies can be employed for affinity maturation of anti-HA33 antibodies?

  Affinity maturation can dramatically improve anti-HA33 antibody performance. Advanced methodological approaches include:

  • Mammalian cell surface display combined with in vitro somatic hypermutation (SHM) for iterative improvement

  • CDR-targeted mutagenesis focusing on key binding residues

  • Selection under stringent conditions with decreasing antigen concentrations

  • Combining stability engineering with affinity maturation in a step-wise approach

  Research has demonstrated remarkable success with this approach. In one study, affinity maturation of a stabilized anti-HA33 antibody produced variants with approximately 300-fold improvement in binding affinity, achieving a KD of 30 pM from the original 9 nM . Specific mutations identified as beneficial included:

  • Heavy chain: H35N, A53L, and Q64R

  • Light chain: N50D and G66E

  Interestingly, removing the G66E mutation from the light chain restored full thermostability (Tm of 92°C) while maintaining exceptional binding affinity (KD of 45 pM) . This demonstrates the possibility of optimizing both properties simultaneously through careful mutation selection.

• How do sequence homology considerations affect the CDR grafting of anti-HA33 antibodies?

  Sequence homology between donor and recipient frameworks significantly impacts CDR grafting success for anti-HA33 antibodies. Methodological insights from research reveal:

  • The homology between mouse anti-HA33 V-regions and human frameworks used for stabilization was approximately 61-62.5%

  • Higher framework homology generally results in better retention of binding characteristics after grafting

  • Critical considerations include:

    • CDR length and conformation

    • Framework residues that support CDR positioning

    • Vernier zone residues that influence CDR orientation

  A scientific case study demonstrated that mouse anti-HA33 antibody (VH14-3 framework) was 61% identical to the stable hVH3-23 HC framework (excluding CDRs), while the mouse IGKV12-4 light chain was 62.5% identical to the stable hVκ2D-30 framework . Despite this moderate homology, CDR grafting was successful with only a 1.5-fold reduction in binding affinity.

  For antibodies with lower framework homology, alternative approaches like back-mutation of critical framework residues or stepwise grafting of individual CDRs may be more effective than complete CDR transplantation.

• What analytical methods are most effective for assessing HA-33 antibody binding characteristics?

  Comprehensive characterization of anti-HA33 antibody binding requires multiple complementary analytical approaches:

  • Surface Plasmon Resonance (SPR) for real-time binding kinetics (ka, kd) and equilibrium dissociation constant (KD) determination

  • Differential Scanning Calorimetry (DSC) to measure thermal stability (Tm) of antibody domains

  • Thermal challenge assays (e.g., 70°C incubation) followed by functional testing to assess thermostability

  • Bio-Layer Interferometry (BLI) for label-free kinetic analysis

  • ELISA titrations for comparative binding studies across multiple antibody variants

  • Flow cytometry for cell-based binding assays

  • Isothermal Titration Calorimetry (ITC) for thermodynamic binding parameters

  Published research has employed DSC to discriminate between unfolding transitions of different antibody domains (Fab, CH2, CH3), revealing that stabilized anti-HA33 antibodies exhibit a Fab Tm increase from 82.1°C to 92.1°C . Additionally, SPR methodology effectively quantified the 300-fold improvement in binding affinity achieved through engineering (from 6-9 nM to 30-45 pM) .

• How can human subject research protocols be designed for validating anti-HA33 antibody diagnostic applications?

  When designing human subject research protocols for validating anti-HA33 antibody-based diagnostic methods, researchers must address several methodological considerations:

  • Ethical approval through Institutional Review Boards (IRBs) is mandatory, with specific attention to research design, risk assessment, and informed consent

  • Human Research Protection Programs require systematic investigation and generalizable knowledge components

  • Protocols should include:

    • Clear definition of whether the research involves human subjects

    • Determination of exempt, expedited, or full board review requirements

    • Documentation of informed consent procedures

    • Confidentiality provisions and data security measures

  Research categorization follows specific regulatory frameworks:

  Review Level	Criteria	Description
  Not Human Subjects Research	Research doesn't fit regulatory definition	Determined by investigator or IRB staff
  Exempt Determination	Fits one of 8 exempt categories	Determined by IRB staff
  Expedited Approval	Minimal risk and fits one of 9 expedited categories	Determination by IRB staff
  Full Board Review	More than minimal risk	Review by convened IRB

  For diagnostic validation specifically, protocols should include sensitivity, specificity, positive and negative predictive value determinations using well-characterized clinical samples with appropriate controls.

• What are the methodological approaches for multiplexing HA-33 antibody detection with other botulinum toxin components?

  Multiplexed detection systems incorporating anti-HA33 antibodies alongside antibodies targeting other botulinum toxin components require sophisticated methodological design:

  • Sandwich ELISA formats can employ capture antibodies against different toxin components (HA-33, HA-17, NTNH, BoNT) with differentially labeled detection antibodies

  • Luminex/bead-based multiplexed assays allow simultaneous detection of multiple toxin components using antibodies conjugated to spectrally distinct beads

  • Protein microarrays can spatially separate antibodies against different toxin components

  • Immunoprecipitation followed by mass spectrometry allows comprehensive toxin complex characterization

  Critical considerations for multiplexed assay development include:

  • Antibody cross-reactivity testing to ensure specificity

  • Standardization of capture and detection antibody affinities

  • Optimization of blocking reagents to minimize non-specific binding

  • Validation using native toxin complexes and recombinant components

  • Determination of detection limits for each toxin component independently and in mixture

  Research has demonstrated that combining HA-33 detection with neurotoxin detection improves diagnostic sensitivity by capturing the complete toxin complex rather than individual components.

• How can deep mutational scanning be applied to optimize anti-HA33 antibody binding and stability?

  Deep mutational scanning represents an advanced approach for comprehensive optimization of anti-HA33 antibodies:

  • Systematic creation of antibody variant libraries through site-directed mutagenesis

  • High-throughput screening using:

    • Mammalian cell display systems

    • Yeast display platforms

    • Phage display technologies

  • Selection under multiple conditions to simultaneously optimize:

    • Binding affinity (using decreasing antigen concentrations)

    • Thermostability (using thermal challenges)

    • Expression levels (using fluorescence-based sorting)

  The methodology involves:

  1. Creating comprehensive mutation libraries covering CDRs and key framework regions

  2. Expressing variant libraries on cellular surfaces or phage

  3. Performing iterative rounds of selection with increasing stringency

  4. Deep sequencing of selected populations to identify enriched mutations

  5. Combining beneficial mutations and validating improved variants

  This approach extends beyond the single-property optimization seen in traditional directed evolution, allowing researchers to map the fitness landscape of anti-HA33 antibodies across multiple parameters simultaneously. Research has shown that combining specific mutations identified through such comprehensive screening can yield antibodies with dramatically improved properties, as demonstrated by the 300-fold affinity improvement and 10°C thermostability increase achieved in engineered anti-HA33 antibodies .