OR23 Antibody

What is the tor23 antibody and what epitopes does it recognize?

Tor23 is a mouse monoclonal antibody (MIgG1 isotype) that recognizes acetylcholinesterase (ache), specifically targeting the presynaptic form. This antibody was generated by Pinky Kushner in 1984 as part of a library of monoclonal antibodies to Torpedo cholinergic synaptosomes . The immunogen used for generation was purified synaptosomes from Torpedo electric organ .

Biochemical characterization reveals that tor23 recognizes antigens with molecular weights of 180 kDa and 66.7 kDa . It demonstrates specific staining patterns in neural tissues, including spinal cord, cerebral cortex, and pyramidal cells in the hippocampal CA2 region. The antigen recognized by tor23 is also expressed in hNTERA-2 teratocarcinoma cells .

What experimental applications has the tor23 antibody been validated for?

Based on validation studies, the tor23 antibody has been confirmed effective for multiple research applications:

• ELISA (Enzyme-Linked Immunosorbent Assay)

• Immunofluorescence

• Immunohistochemistry

• Immunoprecipitation

• Western Blot

When designing experiments, researchers should consider that antibody performance may vary across applications. Implementation of proper controls is essential to confirm specificity, particularly when using the antibody in novel experimental contexts or with unstudied species.

What are the cross-species reactivity profiles of antibodies like tor23?

Antibody cross-reactivity is a critical consideration for comparative studies. The tor23 antibody has been experimentally validated to react with multiple species including:

• Human

• Rat

• Torpedo

• Xenopus

What methodological approaches ensure optimal antibody stability and performance?

For optimal experimental outcomes, proper antibody storage and handling are essential. Although many antibodies remain stable at 4°C for extended periods, shelf-life at this temperature varies considerably .

For short-term usage (≤2 weeks), 4°C storage is generally sufficient . For long-term preservation, antibody solutions should be divided into working aliquots to prevent repeated freeze-thaw cycles, which can cause protein denaturation and activity loss . When preparing dilutions for specific applications, researchers should use appropriate buffer systems that maintain protein stability while minimizing non-specific interactions.

How are monoclonal antibodies like tor23 generated for research applications?

The generation of monoclonal antibodies follows a systematic process:

• Immunization: Animals (typically mice) are immunized with the target antigen. For tor23, purified synaptosomes from Torpedo electric organ served as the immunogen .

• Hybridoma creation: B-lymphocytes from the immunized animal are fused with myeloma cells (NS1 strain for tor23) to create immortalized hybridoma cell lines.

• Selection and screening: Hybridomas are screened for antibody production against the target antigen using methods like ELISA.

• Clonal expansion: Positive hybridomas are subcloned to ensure monoclonality and expanded for antibody production.

• Characterization: The resulting antibodies are characterized for specificity, affinity, and application performance.

This process yields homogeneous antibody populations with consistent target recognition properties.

How do antibody-based diagnostic assays for bacterial resistance genes compare with conventional methods?

Antibody-based assays represent a significant advancement for rapid detection of antimicrobial resistance determinants. A prototype immunochromatographic lateral flow test (ICT) developed for OXA-23-producing carbapenem-resistant Acinetobacter demonstrated:

• 100% specificity in differentiating between OXA-23 subfamily-mediated carbapenem resistance and other OXA-mediated resistance mechanisms

• Turnaround time of only 20 minutes from culture plate to result

• Time savings of 12-48 hours compared to conventional diagnostic methods

This rapid detection capability enables earlier implementation of appropriate antimicrobial therapy and infection control measures, potentially improving patient outcomes and reducing resistance transmission . The methodology involves antibody pairs carefully selected for their ability to specifically recognize the target protein in the lateral flow format.

What factors influence antibody pair selection for different immunoassay platforms?

When developing antibody-based diagnostics, researchers have observed that optimal antibody combinations often differ between assay formats. Research on OXA-23 detection revealed that "the antibody pairs best suited for the sandwich ELISA format did not match the best pairs in the ICT format selected during the development process" .

This platform-dependent performance relates to multiple factors:

• Spatial configuration constraints within different assay matrices

• Antibody binding kinetics under varying buffer conditions

• Epitope accessibility differences between immobilized and solution-phase antigens

• Signal-to-noise considerations specific to each detection system

• Stability of antibody-antigen complexes under specific assay conditions

These observations highlight the necessity for empirical testing of multiple antibody combinations in the intended assay format rather than assuming transferability between platforms .

How do quantitative versus functional antibody assessments inform vaccine efficacy evaluation?

The relationship between antibody quantity and functionality provides crucial insights for vaccine development. Studies of pneumococcal vaccines in HIV-infected and uninfected individuals have demonstrated:

• Significant correlations between post-vaccination serotype-specific IgG levels and opsonophagocytic activity (OPA) titers across study groups

• Differential relationships between antibody isotypes and functional activity:

  • Strong correlation for serotype 23F in HIV+ individuals receiving PPV alone

  • Strong correlation for serotype 14 in HIV+ individuals receiving PCV/PPV

  • Moderate to strong correlations for both serotypes in HIV- individuals

• No significant correlations between post-vaccination IgM levels and OPA titers in any study group

These findings illustrate that quantitative antibody measurements alone may not fully predict protective immunity, necessitating functional assays for comprehensive vaccine assessment.

What methodological approaches measure functional antibody responses to encapsulated bacteria?

Opsonophagocytic activity (OPA) assays represent the gold standard for evaluating functional antibody responses to encapsulated bacteria like Streptococcus pneumoniae. The methodology employs:

• Standardized protocols using target bacteria, antibody-containing serum, complement source, and phagocytic cells

• Quantification of bacterial killing, defined as "the reciprocal of the serum dilution that killed 50% of target bacteria (compared to serum-free control) during 45 minutes of incubation at 37°C"

• Data analysis using specialized software (e.g., Opsotiter1)

• Classification of positive responses as ≥4-fold increases from baseline

This functional assessment provides critical information about protective potential beyond antibody concentration, particularly relevant for evaluating vaccines in immunocompromised populations.

How do HIV infection and aging affect antibody responses to polysaccharide versus conjugate vaccines?

Research comparing pneumococcal vaccine responses in aging HIV-infected and uninfected individuals has revealed nuanced differences:

Key findings include:

• HIV+ groups (regardless of vaccine regimen) showed similar post-vaccination IgG levels and OPA titers

• Post-vaccination IgM levels for serotype 23F were significantly higher in HIV+ individuals receiving PCV/PPV compared to PPV alone

• Compared to HIV- individuals, HIV+ individuals receiving PCV/PPV demonstrated significantly reduced:

  • IgG and IgM levels for serotype 14

  • OPA titers for serotype 23F

These findings suggest that the recommended PCV/PPV sequential regimen may not substantially improve antibody responses in HIV-infected individuals compared to PPV alone, highlighting the need for alternative vaccination strategies in this population .

What strategies optimize antibody development for diagnostic applications?

Developing antibodies for diagnostic applications requires systematic optimization:

• Immunogen selection: Carefully designed immunogens that present relevant epitopes in native conformation improve antibody specificity.

• Sequential screening: For the OXA-23 diagnostic test, antibodies were "screened sequentially for their ability to bind native OXA-23" , ensuring recognition of the naturally occurring protein.

• Pair optimization: Testing multiple antibody combinations empirically in the intended assay format rather than relying on theoretical predictions .

• Validation with diverse samples: Employing "well-characterized collections" of samples with defined properties to ensure diagnostic accuracy .

• Performance assessment: Evaluating specificity, sensitivity, and time-to-result under realistic testing conditions .

This systematic approach resulted in a diagnostic test with 100% specificity and significant time advantages over conventional methods .

How should researchers document antibody characteristics in scientific publications?

Proper documentation of antibody reagents is essential for research reproducibility. Publications should include:

• Complete antibody identification: Clone name/number, isotype, and registry information (e.g., Antibody Registry ID: AB_2722484 for tor23)

• Source attribution: Acknowledge both the original developer and repository (e.g., "tor23 was deposited to the DSHB by Harland, Richard")

• Validation details: Applications tested, species reactivity confirmed, and any known limitations

• Epitope information: When available, include epitope sequence or location

• Experimental conditions: Dilutions, incubation parameters, and detection methods used

This comprehensive documentation enables other researchers to accurately replicate experimental conditions and interpret results appropriately.

How can monoclonal antibodies contribute to rapid diagnostic platforms for infectious diseases?

Monoclonal antibodies offer significant advantages for infectious disease diagnostics:

• Specificity: The ability to distinguish between closely related bacterial resistance mechanisms, as demonstrated by the OXA-23 ICT which achieved "100% specificity" in differentiating between OXA-23 subfamily-mediated carbapenem resistance and other OXA-mediated resistance mechanisms

• Speed: Rapid results (20 minutes from culture plate to result) compared to conventional methods that require 12-48 additional hours

• Ease of use: Lateral flow formats enable point-of-care testing without specialized equipment

• Clinical impact: Earlier detection enables "earlier intervention to control the transmission of OXA-23-producing carbapenem-resistant Acinetobacter isolates to other patients and healthcare workers"

These advantages position antibody-based diagnostics as valuable tools for antimicrobial stewardship and infection control programs.

What are the challenges in developing antibodies that distinguish between closely related protein variants?

Developing antibodies with high specificity for related protein variants presents several challenges:

• Epitope identification: Identifying unique epitopes that differentiate between closely related proteins like different OXA subfamilies

• Cross-reactivity assessment: Thorough testing against related proteins to ensure specificity, as performed for the OXA-23 ICT against other OXA variants

• Native protein recognition: Ensuring antibodies recognize naturally occurring proteins rather than just recombinant or denatured forms

• Format-specific performance: Accounting for the observation that antibody pairs optimal in one format may perform poorly in another

• Validation with diverse clinical isolates: Testing with "well-characterized collections" to confirm real-world performance

Addressing these challenges requires iterative optimization and comprehensive validation to ensure diagnostic accuracy.

How might antibody technology address challenges in detecting emerging resistance mechanisms?

Future applications of antibody technology for antimicrobial resistance detection may include:

• Multiplexed detection systems: Simultaneous identification of multiple resistance determinants from a single sample

• Enhanced sensitivity: Detection of resistance markers directly from clinical specimens without culture steps

• Quantitative capacity: Development of semi-quantitative tests that correlate with resistance levels

• Integration with digital platforms: Smartphone-based readers for objective result interpretation and data sharing

• Expanded target range: Development of antibodies against emerging resistance determinants

These advancements could further reduce diagnostic time, improve antimicrobial stewardship, and enhance surveillance of resistance patterns.

What novel approaches might improve vaccine responses in immunocompromised populations?

Research on pneumococcal vaccines in HIV-infected individuals highlights the need for innovative approaches:

• Alternative adjuvant formulations: Development of adjuvants specifically designed to enhance immune responses in immunocompromised hosts

• Targeted epitope selection: Identification of immunodominant epitopes that elicit protective responses even in compromised immune systems

• Novel delivery platforms: Exploration of alternative delivery methods that might bypass specific immune defects

• Personalized vaccination schedules: Tailoring vaccination timing based on individual immune status indicators

• Combination approaches: Integration of passive immunization strategies with active vaccination