ompA Antibody, Biotin conjugated

What is ompA and why is it targeted for antibody development?

OmpA (Outer membrane protein A) is a highly conserved protein found in the outer membrane of numerous gram-negative bacteria. It serves as an excellent target for antibody development due to its abundance, surface accessibility, and conservation across bacterial species. The protein plays critical roles in maintaining structural integrity of the bacterial outer membrane and can be involved in pathogenesis. For example, in Haemophilus influenzae, OmpA (also known as Outer membrane protein P5 or Fimbrin) spans amino acids 22-359 and has a UniProtID of P45996 . The high conservation of ompA across numerous gram-negative bacteria makes it particularly valuable as a universal target for detection, concentration, and potential therapeutic applications . This conservation has been confirmed through bioinformatics approaches analyzing sequences with more than 150 amino acid length and greater than 50% similarity to E. coli K12 OmpA sequence .

What are the key specifications of commercially available ompA Antibody, Biotin conjugated?

Most commercially available ompA Antibody, Biotin conjugated products are polyclonal antibodies raised in rabbits against recombinant ompA proteins. These antibodies typically have the following specifications:

• Host Species: Rabbit

• Clonality: Polyclonal

• Isotype: IgG

• Conjugate: Biotin

• Applications: Primarily ELISA

• Form: Liquid

• Buffer: Often preserved in 0.03% Proclin 300 with 50% Glycerol and 0.01M PBS (pH 7.4)

• Storage requirements: -20°C to -80°C, with recommendations to avoid repeated freeze-thaw cycles

For specific research applications, it's important to note that different products may have varying immunogens and reactivity profiles. For instance, some antibodies are developed using recombinant Haemophilus influenzae Outer membrane protein P5 protein (22-359AA) as the immunogen .

How does biotin conjugation benefit ompA antibody applications?

Biotin conjugation provides several methodological advantages for ompA antibody applications:

• Enhanced sensitivity: The strong affinity between biotin and streptavidin (Kd ≈ 10^-15 M) enables robust signal amplification in detection systems.

• Versatile detection options: Biotin-conjugated antibodies can be detected using various streptavidin-coupled reporters (enzymes, fluorophores, gold particles).

• Compatibility with immunomagnetic separation: When combined with streptavidin-coated magnetic beads, these antibodies facilitate bacterial concentration from dilute samples. This approach has been demonstrated to effectively concentrate bacterial pathogens at concentrations as low as 10-100 CFU/mL, reducing detection time by 8-24 hours compared to traditional culture methods .

• Multi-layered assay construction: Allows for building complex detection systems that leverage the biotin-streptavidin interaction for immobilization, purification, or signal enhancement.

The biotin conjugation is particularly valuable in research applications requiring high sensitivity or when working with complex sample matrices where signal amplification is beneficial .

What protocols are recommended for using ompA Antibody, Biotin conjugated in ELISA?

When using ompA Antibody, Biotin conjugated in ELISA, the following methodological approach is recommended:

Standard Direct ELISA Protocol:

• Coating: Add purified target antigen (typically recombinant ompA or bacterial lysates) to 96-well plates at 1-10 μg/mL in coating buffer (typically carbonate-bicarbonate buffer, pH 9.6). Incubate overnight at 4°C.

• Blocking: Block non-specific binding sites with 1-5% BSA or non-fat milk in PBS-T (PBS containing 0.05% Tween-20) for 1-2 hours at room temperature.

• Primary antibody: Apply ompA Antibody, Biotin conjugated at optimized dilutions (typically 1:500 to 1:5000) in blocking buffer. Incubate for 1-2 hours at room temperature or overnight at 4°C.

• Detection: Add streptavidin-coupled enzyme (typically streptavidin-HRP or streptavidin-alkaline phosphatase) diluted 1:1000 to 1:5000 in blocking buffer. Incubate for 30-60 minutes at room temperature.

• Substrate addition: Add appropriate substrate (TMB for HRP or pNPP for alkaline phosphatase) and monitor color development.

• Signal measurement: Measure absorbance at the appropriate wavelength using a microplate reader.

For bacterial detection in complex samples, researchers have successfully combined ompA antibodies with magnetic separation techniques to concentrate target bacteria before detection, which can reduce detection time by 8-24 hours when bacterial concentrations are between 10-100 CFU/mL .

How can researchers conduct competitive inhibition ELISA to identify epitopes recognized by ompA antibodies?

Competitive inhibition ELISA is a valuable technique for epitope mapping of ompA antibodies. The methodology is as follows:

• Plate preparation: Coat 96-well plates with purified ompA antigen (typically 1-5 μg/mL) and block as in standard ELISA.

• Competitor addition: Add serial two-fold dilutions of polyclonal serum (100 μL) to wells (rows 1-11) as competitor.

• Biotinylated MAb addition: Add a pre-selected quantity of biotinylated monoclonal antibody (100 μL) that produces an ELISA absorbance of 0.5-1.0 optical density units. Mix and incubate overnight at 4°C.

• Detection: Wash plates and develop with streptavidin-alkaline phosphatase (or streptavidin-HRP) and appropriate substrate.

• Analysis: Calculate percent inhibition by comparing the signal in test wells to control wells (no competitor). Significant inhibition indicates that the competitor antibodies recognize the same or overlapping epitopes as the biotinylated MAb.

This method has been successfully used to determine whether different anti-ompA MAbs recognize the same epitope. For instance, research has shown that when the binding of each biotinylated MAb is decreased by the presence of other, non-biotinylated MAbs, it suggests these antibodies recognize a common epitope .

What approaches can be used to validate the specificity of ompA antibodies?

Validating the specificity of ompA antibodies requires a multi-faceted approach:

• Cross-reactivity testing against multiple bacterial species:

  • Test antibody binding to a panel of both target and non-target gram-negative bacteria

  • Include closely related species to assess discrimination capacity

  • Quantify binding using ELISA, flow cytometry, or immunofluorescence

• Protein-level validation:

  • Western blot analysis using bacterial lysates to confirm binding to protein of expected molecular weight

  • Competitive inhibition using purified recombinant ompA protein

  • Peptide mapping using overlapping peptides spanning the ompA sequence

• Genetic validation approaches:

  • Testing against wild-type and ompA knockout strains (where available)

  • Testing against bacteria expressing variant ompA sequences

  • Expression of recombinant ompA fragments to map binding sites

• Functional validation:

  • Assess antibody's ability to detect bacteria in complex matrices (e.g., food samples, clinical specimens)

  • Compare results with established molecular methods (e.g., PCR detection)

One study demonstrated the effectiveness of their anti-LAMOA-1 antibody (targeting a conserved ompA sequence) by showing it could effectively recognize 12 different foodborne bacterial species, validating its broad-spectrum reactivity . Proper validation should include both positive and negative controls and quantitative measurements of binding affinity and specificity.

How can ompA antibodies be combined with magnetic separation for bacterial concentration?

Immunomagnetic separation using ompA antibodies offers a powerful approach for concentrating bacteria from dilute samples:

Methodological Protocol:

• Preparation of antibody-conjugated beads:

  • Activate magnetic beads through carbodiimide chemistry (typically using EDC/NHS activation)

  • Directly conjugate purified anti-ompA antibodies to activated beads

  • Block remaining active sites with BSA or other blocking proteins

  • Wash and resuspend beads in storage buffer (typically PBS with 0.1% BSA and preservative)

• Sample processing:

  • Mix antibody-conjugated beads with sample (often 10-50 μL beads per mL of sample)

  • Incubate with gentle agitation for 30-60 minutes at room temperature

  • Apply magnetic field to separate bead-bacteria complexes

  • Wash complexes 2-3 times with buffer containing 0.05% Tween-20

  • Resuspend in appropriate buffer for downstream analysis

• Analysis of captured bacteria:

  • Direct plating on selective media for viable counting

  • Molecular detection methods (PCR, isothermal amplification)

  • Microscopy or flow cytometry with appropriate staining

This approach has been shown to effectively concentrate bacteria when present at low concentrations (10-100 CFU/mL) in test samples, reducing detection time by 8-24 hours compared to traditional enrichment methods. The success of this method hinges on using antibodies that target conserved epitopes accessible on the bacterial surface, such as those developed against the LAMOA-1 consensus sequence derived from multiple ompA variants .

What is the potential for single-cell analysis in developing ompA-specific antibodies?

High-throughput single-cell analysis represents a cutting-edge approach for isolating and characterizing ompA-specific antibodies:

Technical Approach Using the Berkeley Lights Beacon System:

• Immunization strategy:

  • Immunize mice with purified OMPs (typically 50 μg per dose)

  • Administer prime and boost injections (days 0, 14, and 21)

  • Collect serum to determine antibody titers via ELISA

  • Select highest-titer animals for cell harvesting

• Antigen preparation for screening:

  • Biotinylate purified OMPs

  • Couple biotinylated OMPs to streptavidin-coated assay beads

  • Mix conjugated beads with fluorescently labeled anti-mouse secondary antibody

• Cell processing and screening:

  • Load antibody-secreting cells onto OptoSelect chips

  • Culture at 25°C in specialized survival medium

  • Encapsulate single cells in NanoPens using OptoElectroPositioning technology

  • Perform on-chip fluorescence-based assay to identify antibody-secreting cells

  • Export positive cells for sequencing

• Antibody characterization:

  • Amplify VH and VL sequences using RT-PCR

  • Perform sequence analysis and determine CDR3 regions

  • Express recombinant antibodies for functional testing

This approach has been successfully used to isolate antibodies against Omp38 of Acinetobacter baumannii, with the entire process identifying 47 OMP-specific antibody-producing cells from over 10,600 screened cells . The resulting monoclonal antibodies demonstrated therapeutic potential in mouse models of A. baumannii infection, suggesting similar approaches could be valuable for developing ompA-targeted therapeutics.

How does ompA sequence conservation impact antibody design and applications?

The conservation pattern of ompA across bacterial species significantly influences antibody design strategies and applications:

Sequence Analysis Approach:

• Comprehensive sequence collection:

  • Download bacterial ompA protein sequences with >150 amino acid length from NCBI

  • Filter sequences based on similarity to reference (e.g., E. coli K12 OmpA)

  • Exclude sequences with <50% similarity to reference

• Multiple sequence alignment:

  • Align sequences using tools like CLUSTALW 2.1

  • Identify conserved regions across diverse bacterial species

  • Generate consensus sequences representing conserved epitopes

• Structural analysis:

  • Predict spatial conformational sequences exposed on the outer membrane

  • Identify regions that maintain structural conservation despite sequence variation

  • Select regions that are accessible to antibodies in intact bacteria

• Consensus antigen design:

  • Design consensus antigens (like LAMOA-1) based on alignment results

  • Express and purify recombinant consensus proteins

  • Use for immunization to generate broadly reactive antibodies

This approach led to the development of LAMOA-1, a consensus ompA sequence that elicited antibodies capable of recognizing multiple bacterial species. The resulting anti-LAMOA-1 antibodies effectively recognized 12 different foodborne bacterial species, demonstrating the value of targeting conserved epitopes .

The conservation analysis also enables:

• Prediction of cross-reactivity profiles

• Design of species-specific versus broad-spectrum antibodies

• Development of antibody panels targeting different ompA epitopes

• Selection of optimal immunogens for therapeutic antibody development

What are common causes of inconsistent results when using ompA antibodies in ELISA?

Several technical and biological factors can lead to inconsistent ELISA results with ompA antibodies:

• Antibody-related factors:

  • Degradation due to improper storage or repeated freeze-thaw cycles

  • Lot-to-lot variation in commercial antibodies

  • Suboptimal working dilution or concentration

• Antigen presentation issues:

  • Variation in ompA expression levels between bacterial cultures

  • Conformational changes in ompA during sample processing

  • Epitope masking by other bacterial components

  • Strain-to-strain variation in ompA sequence or accessibility

• Technical variables:

  • Inconsistent blocking or washing procedures

  • Temperature fluctuations during incubation steps

  • Suboptimal streptavidin-enzyme conjugate concentration

  • Substrate degradation or light exposure

• Buffer and reagent concerns:

  • pH variations affecting antibody-antigen binding

  • Detergent concentration affecting membrane protein solubility

  • Presence of interfering substances in complex samples

  • Incompatible preservatives or stabilizers

To address these issues, researchers should:

• Establish detailed standardized protocols with precise timing and temperature controls

• Include multiple controls (positive, negative, background, and cross-reactivity)

• Perform validation with known bacterial samples before testing unknowns

• Consider using recombinant ompA fragments as standards to ensure consistency

• Store antibodies according to manufacturer recommendations (typically -20°C to -80°C)

How can researchers distinguish between specific and non-specific binding in ompA antibody assays?

Distinguishing specific from non-specific binding requires systematic controls and validation:

Methodological Approach:

• Essential controls:

  • Isotype control: Use biotin-conjugated rabbit IgG (non-immune) at the same concentration

  • Blocking controls: Perform pre-incubation with unlabeled antibody before adding biotin-conjugated antibody

  • Antigen competition: Pre-incubate antibody with purified ompA protein before adding to assay

  • Cross-reactivity panel: Test against ompA-negative bacteria or other membrane proteins

• Signal validation techniques:

  • Dose-response testing: Perform serial dilutions of antibody to demonstrate proportional signal reduction

  • Western blot confirmation: Verify binding to protein of expected molecular weight

  • Competitive inhibition ELISA: As detailed in section 2.2, to confirm epitope specificity

• Statistical approaches:

  • Calculate signal-to-noise ratios (specific signal vs. background)

  • Establish clear cut-off values based on negative controls

  • Use replicate testing to ensure reproducibility

• Advanced validation:

  • Cross-adsorption against related bacterial species to remove cross-reactive antibodies

  • Epitope mapping using peptide arrays or competitive binding with defined fragments

  • Flow cytometry to confirm binding to intact bacteria

In published research, competitive binding assays have demonstrated that certain anti-ompA MAbs recognize the same epitope, as shown by decreased binding of biotinylated MAbs in the presence of non-biotinylated MAbs . Such approaches provide robust evidence for binding specificity.

What are the optimal storage conditions for maintaining ompA antibody activity?

Preserving ompA antibody activity requires careful attention to storage conditions:

Recommended Storage Protocol:

• Long-term storage:

  • Store at -20°C to -80°C as recommended by manufacturers

  • Aliquot antibodies upon receipt to minimize freeze-thaw cycles

  • Use sterile, low-protein binding tubes for storage

  • Include proper labeling with date, dilution, and lot information

• Buffer composition:

  • Typical storage buffers contain 50% glycerol to prevent freezing damage

  • 0.01M PBS at pH 7.4 maintains proper ionic strength and pH

  • Preservatives like 0.03% Proclin 300 prevent microbial contamination

  • Some formulations may include carrier proteins for additional stability

• Working solution handling:

  • Keep on ice when in use

  • Return to -20°C promptly after use

  • Avoid more than 5 freeze-thaw cycles

  • Consider adding protein stabilizers (BSA, gelatin) to diluted working solutions

• Critical precautions:

  • Avoid repeated freeze-thaw cycles as explicitly warned by manufacturers

  • Protect biotinylated antibodies from strong light exposure

  • Maintain sterile handling to prevent contamination

  • Monitor for signs of degradation (precipitation, color change, loss of activity)

For biotin-conjugated antibodies specifically, additional considerations include avoiding prolonged exposure to avidin/streptavidin-containing reagents or samples prior to use in assays, as this can lead to premature binding and reduced available antibody.

What is the therapeutic potential of ompA-targeted antibodies in bacterial infections?

Emerging research indicates significant therapeutic potential for antibodies targeting bacterial outer membrane proteins:

Therapeutic Mechanisms and Evidence:

• Direct antimicrobial activities:

  • Complement-mediated bacterial killing

  • Opsonization enhancing phagocytosis

  • Inhibition of bacterial adhesion to host cells

  • Neutralization of virulence-associated functions

• Evidence from animal models:

  • Monoclonal antibodies against Omp38 significantly improved survival rates in mouse models of lethal A. baumannii infection

  • Reduced bacterial loads in infected tissues following antibody administration

  • Decreased inflammatory cytokine production in sublethal infection models

  • Reduced immune cell infiltration at infection sites

• Advantages over conventional antibiotics:

  • Lower likelihood of promoting antimicrobial resistance

  • More targeted approach reducing potential adverse effects

  • Potential for combination with conventional antibiotics for synergistic effects

  • Extended half-life compared to small-molecule antibiotics

• Challenges and considerations:

  • Need for epitope conservation across clinically relevant strains

  • Potential for antigenic variation in target pathogens

  • Optimization of antibody formulation and delivery

  • Cost-effectiveness compared to conventional therapeutics

Research with Omp38-specific monoclonal antibodies has demonstrated that intravenous administration significantly improved survival rates and reduced bacterial loads in mice infected with A. baumannii. Flow cytometry and ELISA analyses confirmed reduced immune cell infiltration and decreased cytokine production, indicating the antibodies facilitated bacterial clearance while minimizing inflammatory response and host damage .

How can novel high-throughput technologies advance ompA antibody development?

Cutting-edge technologies are revolutionizing the development of ompA-targeted antibodies:

Advanced Technological Approaches:

• Berkeley Lights Beacon optofluidic system:

  • Enables high-throughput screening of thousands of single B cells

  • Uses OEP (OptoElectroPositioning) technology to manipulate individual cells

  • Performs on-chip fluorescence-based assays to identify antigen-specific cells

  • Allows export of selected cells for sequencing and antibody production

  • Successfully applied to isolate OMP-specific antibody-producing cells with a high degree of precision

• Single B-cell sequencing and antibody reconstruction:

  • Direct sequencing of paired heavy and light chain variable regions

  • Analysis of CDR3 sequences and somatic mutations using databases like IMGT

  • Codon optimization and synthesis of antibody genes

  • Expression in mammalian systems for recombinant antibody production

  • Enables rapid transition from B-cell isolation to functional antibody testing

• Computational approaches for epitope prediction:

  • Algorithm-based analysis of ompA sequence conservation across bacterial species

  • Prediction of surface-exposed epitopes using structural modeling

  • Design of consensus immunogens to generate broadly reactive antibodies

  • In silico assessment of antibody cross-reactivity potential

  • Has led to development of consensus antigens like LAMOA-1 that elicit broadly reactive antibodies

• Microfluidic systems for antibody characterization:

  • Rapid assessment of binding kinetics using surface plasmon resonance

  • High-throughput analysis of cross-reactivity against multiple bacterial strains

  • Functional testing in simulated physiological conditions

  • Real-time visualization of antibody-bacteria interactions

These technologies have significantly accelerated the development process, as demonstrated by the successful isolation of 47 OMP-specific antibody-producing cells from over 10,600 screened cells in a single experiment using the Beacon system .

What are the comparative advantages of polyclonal versus monoclonal ompA antibodies?

Selecting between polyclonal and monoclonal ompA antibodies requires careful consideration of their respective advantages and limitations:

What factors influence the selection of immunogens for developing broadly reactive ompA antibodies?

Developing broadly reactive ompA antibodies requires careful immunogen design:

Critical Factors in Immunogen Selection:

• Sequence conservation analysis:

  • Comprehensive alignment of ompA sequences from target bacterial species

  • Identification of highly conserved regions across diverse species

  • Selection of segments with >50% sequence identity to reference strains

  • Phylogenetic analysis to understand evolutionary relationships

• Structural considerations:

  • Prediction of surface-exposed regions accessible to antibodies

  • Analysis of conformational epitopes versus linear epitopes

  • Selection of regions that maintain structural conservation despite sequence variation

  • Consideration of post-translational modifications or processing

• Immunogen design strategies:

  • Full-length recombinant ompA versus specific fragments

  • Synthetic consensus peptides based on multiple alignments

  • Chimeric constructs combining conserved epitopes from multiple species

  • Carrier protein conjugation for enhanced immunogenicity

• Production and purification methods:

  • Expression systems (bacterial, mammalian, cell-free)

  • Purification approaches that maintain native conformation

  • Quality control for endotoxin contamination

  • Stability assessment under storage conditions

The successful development of LAMOA-1, a consensus OmpA analog, exemplifies this approach. Researchers analyzed OmpA protein sequence conservation in gram-negative bacteria and predicted spatial conformational sequences exposed on the outer cell membrane. The resulting recombinant protein elicited antibodies that effectively recognized 12 different foodborne bacterial species, demonstrating the value of this approach for generating broadly reactive antibodies .

How can researchers optimize immunomagnetic separation using ompA antibodies for maximum bacterial recovery?

Optimizing immunomagnetic separation (IMS) with ompA antibodies requires systematic refinement of multiple parameters:

Optimization Protocol:

• Antibody conjugation parameters:

  • Determine optimal antibody density on bead surface (typically 5-20 μg antibody per mg of beads)

  • Compare different conjugation chemistries (carbodiimide, maleimide, streptavidin-biotin)

  • Evaluate orientation-controlled conjugation to maximize antigen binding

  • Test various blocking agents to minimize non-specific binding

• Capture conditions optimization:

  • Systematically vary incubation time (15-60 minutes)

  • Test different temperatures (4°C, room temperature, 37°C)

  • Optimize sample-to-bead ratio (typically 10-50 μL beads per mL sample)

  • Evaluate buffer composition (ionic strength, pH, additives)

• Separation and washing parameters:

  • Compare different magnetic separation times (1-10 minutes)

  • Optimize number and volume of wash steps

  • Test various wash buffer compositions (detergent concentration, salt concentration)

  • Evaluate gentle mixing methods during incubation

• Elution and downstream processing:

  • Compare direct analysis versus elution from beads

  • Test various elution methods (pH shift, competitive elution, enzymatic treatment)

  • Optimize elution volume for maximum concentration

  • Evaluate compatibility with downstream detection methods