omp25 Antibody

Introduction to Omp25 Antibody

Omp25 antibodies are immunoglobulins specifically developed to target Outer Membrane Protein 25 (Omp25), a major structural protein found in Brucella species. These antibodies have gained significant attention in both research and clinical contexts due to their high specificity and sensitivity in detecting Brucella infections. Omp25 is particularly valuable as a target because it is highly conserved across various types and subtypes of Brucellae and induces a strong immune response, making it an excellent candidate for serologic diagnosis of Brucella infections . Researchers have developed various forms of these antibodies, including monoclonal antibodies (mAbs) produced through hybridoma techniques and polyclonal antibodies, each with specific applications in diagnostic and research settings.

The significance of Omp25 antibodies stems from the serious nature of brucellosis itself. Diagnosing brucellosis through Brucellae cultures can take at least 10 days, and once the infection becomes chronic, patients may carry the bacteria for their entire lives. Therefore, rapid and effective diagnostic tools targeting Brucella antigens, such as those utilizing Omp25 antibodies, represent a crucial advancement in managing this persistent infection .

Structure and Function of Brucella Omp25

Omp25 is one of the major outer membrane proteins found in all Brucella species and plays a crucial role in the structural integrity and virulence of these bacteria. Research has demonstrated that Brucella species lacking Omp25 survive for shorter periods than wild-type strains in experimental models, highlighting its importance in bacterial pathogenesis . The protein consists of 213 amino acids and contains multiple epitopes that can be recognized by different antibodies.

As a structural protein, Omp25 is remarkably conserved across different Brucella species, biovars, and strains, making it an ideal target for diagnostic antibodies with broad reactivity against various Brucella pathogens . This conservation across species boundaries allows Omp25-targeting antibodies to detect multiple Brucella varieties, including B. melitensis, B. abortus, and B. suis, which are the most common species causing human infections.

Beyond its structural role, Omp25 significantly contributes to Brucella's virulence mechanisms. Studies have revealed that Omp25 is involved in inhibiting tumor necrosis factor alpha (TNF-α) production during infection of human macrophages . This immunomodulatory function represents a key mechanism through which Brucella evades host immune responses, as TNF-α is an essential cytokine for controlling bacterial infections. The expression of Omp25 correlates with the unusual absence of TNF-α release observed in human macrophages infected with Brucella species, demonstrating the protein's role in negative regulation of this critical immune response .

Types and Characteristics of Omp25 Antibodies

Researchers have developed multiple types of antibodies targeting Brucella Omp25, each with distinct characteristics and applications. The most extensively studied are monoclonal antibodies (mAbs), which provide high specificity for different epitopes of the protein.

Monoclonal Antibodies Against Omp25

Five notable monoclonal antibodies—2B10, 4A12, 4F10, 6C12, and 8F3—have been specifically developed and characterized for their reactivity with Brucella Omp25. These mAbs represent different immunoglobulin isotypes: two IgG1, two IgG2a, and one IgG2b . This diversity in isotypes provides varying functional properties that can be leveraged for different experimental and diagnostic applications.

These monoclonal antibodies demonstrate differential reactivity patterns with Brucella strains. Notably, mAbs 6C12, 8F3, and 4A12 show high reactivity with multiple Brucella species, including B. melitensis (M5-90), B. abortus (S19, 104M, and 2308), and B. suis strain (S2) . This cross-species reactivity makes these antibodies particularly valuable for broad-spectrum detection of Brucella infections regardless of the specific pathogenic species involved.

Importantly, specificity testing has confirmed that these antibodies do not cross-react with other gram-negative bacteria such as Yersinia enterocolitica O:9, Salmonella species, and Escherichia coli, thereby minimizing false-positive results in diagnostic applications .

Polyclonal IgY Antibodies Against Omp25

In addition to monoclonal antibodies, researchers have developed polyclonal antibodies against Omp25, including IgY antibodies produced in chickens. These IgY antibodies have been raised against synthetic peptides from Brucella abortus Omp25 protein and have demonstrated the ability to recognize B. abortus in multiple antibody binding assays (MABA) .

The production of IgY antibodies represents an alternative approach that offers certain advantages over traditional mammalian antibodies, including increased yield from egg extraction and reduced invasiveness in antibody collection procedures. Studies have confirmed that these IgY antibodies show good correlation between predictive immunogenicity studies and actual immunogenic responses in chickens .

Production and Purification of Omp25 Antibodies

The production of high-quality Omp25 antibodies involves sophisticated immunological techniques and careful characterization processes. The most common approach for monoclonal antibody production is the hybridoma technique, which yields highly specific antibodies capable of recognizing distinct epitopes on the Omp25 protein.

Hybridoma Technique for Monoclonal Antibody Production

In the development of anti-Omp25 monoclonal antibodies, researchers typically begin with the production of recombinant Omp25 (rOmp25) as an immunogen. This recombinant protein is purified to approximately 95% purity using techniques such as Ni-NTA Agarose column chromatography . The purified rOmp25 is then used to immunize mice, stimulating an immune response against the protein.

Following immunization, B cells are harvested from the mice and fused with myeloma cells to create hybridomas—immortalized cells capable of producing specific antibodies. These hybridoma cells are screened for reactivity with rOmp25 and native membrane proteins (NMPs) of Brucella species using enzyme-linked immunosorbent assay (ELISA) . Positive hybridoma clones are then selected and expanded to produce larger quantities of the desired monoclonal antibodies.

IgY Antibody Production Using Synthetic Peptides

For polyclonal IgY antibody production, researchers have utilized synthetic peptide constructs derived from the Omp25 sequence. These synthetic peptides are designed based on computational predictions of antigenic regions and are used to immunize chickens . The antibodies are then extracted from egg yolks, representing a less invasive method of antibody production compared to traditional approaches requiring animal bleeding.

The binding activity and specificity of these IgY antibodies have been verified through Western blot analysis using cell extracts from B. abortus. Studies have demonstrated that Omp25 peptide constructs are effective candidates for producing specific IgY anti-peptide antibodies capable of recognizing proteins from sonicated B. abortus strain S19 .

Epitope Mapping and Reactivity Patterns

Understanding the specific epitopes recognized by different Omp25 antibodies is crucial for their effective application in diagnostics and research. Detailed epitope mapping has revealed important differences in the binding characteristics of various monoclonal antibodies.

Linear and Conformational Epitopes

Studies have identified both linear and semi-conformational epitopes within the Omp25 protein. Through testing with peptides derived from the 213 amino acid sequence of B. melitensis Omp25, researchers found that four monoclonal antibodies (4A12, 8F3, 4F10, and 2B10) react with three different peptides, indicating recognition of linear epitopes . The specific amino acid sequences of these linear epitopes have been characterized as 19-20 mers peptides designated P2, P4, and P7.

In contrast, mAb 6C12 demonstrates a distinct binding pattern—it doesn't react with any of the isolated peptides but does bind to denatured native Omp25, suggesting recognition of a semi-conformational epitope . This distinction in epitope recognition has important implications for the application of these antibodies in various detection methods, as conformational epitopes may be preserved differently depending on the sample preparation technique.

Comparative Reactivity with Various Brucella Species

The reactivity of Omp25 monoclonal antibodies with different Brucella species has been thoroughly evaluated. Testing with soluble sonicated proteins (SSPs) from multiple Brucella strains, including 2308, 104M, S19, S2, and M5-90 strains from B. melitensis, B. abortus, and B. suis, has revealed differential reactivity patterns .

When used in paired combinations for double-antibody sandwich ELISA (DAS-ELISA), certain mAb pairs demonstrate superior performance. Specifically, the combinations 8F3/6C12-HRP and 8F3/4A12-HRP have been identified as particularly effective for detecting various Brucella species . These findings highlight the importance of selecting optimal antibody combinations for maximum sensitivity and specificity in diagnostic applications.

Diagnostic Applications of Omp25 Antibodies

Omp25 antibodies have demonstrated significant potential for diagnosing and monitoring Brucella infections through various immunoassay techniques. These applications represent important advancements in managing brucellosis, a disease that remains challenging to diagnose definitively.

Flow Cytometry for Detecting Intracellular Brucellae

One of the most promising applications of Omp25 antibodies is the detection of intracellular Brucellae in peripheral blood mononuclear cells (PBMCs) using flow cytometry (FCM). This technique, particularly using mAb 6C12 conjugated with phycoerythrin (PE), has proven effective for identifying infected monocytes in blood samples from brucellosis patients .

In clinical evaluations, the FCM technique established a threshold where <1.0% of stained monocytes indicates lower levels or negative intracellular Brucellae-infected cells, while ≥1.0% indicates higher levels of infection . When tested on 28 brucellosis patients and 20 healthy blood donors, the FCM method revealed that 46.4% (13/28) of brucellosis patients carried intra-PBMCs Brucellae (≥1.0%), while none of the blood donors exceeded this threshold . The detection results from FCM analysis of clinical samples are summarized in the table below:

These results suggest that FCM with mAb 6C12 represents a practical assay for determining the frequency of intracellular Brucellae-infected PBMCs in individual brucellosis patients, potentially providing a valuable tool for monitoring treatment efficacy .

Enzyme-Linked Immunosorbent Assays (ELISA)

Various ELISA formats utilizing Omp25 antibodies have been developed for detecting Brucella species. Double-antibody sandwich ELISA (DAS-ELISA) using pairs of monoclonal antibodies has shown particular promise. Five specific pairs of mAbs—4A12/6C12-HRP, 4A12/8F3-HRP, 6C12/8F3-HRP, 8F3/4A12-HRP, and 8F3/6C12-HRP—have demonstrated reactivity with recombinant Omp25 .

Further testing revealed that two mAb pairs, 8F3/6C12-HRP and 8F3/4A12-HRP, exhibit superior reactivity for detecting various Brucella species . These findings suggest that 8F3 is particularly suitable for solid-phase capture, while 6C12 or 4A12 work well when conjugated with HRP for detection of Brucella Omp25 in ELISA formats .

Immunostaining Techniques

Omp25 antibodies have also proven effective in various immunostaining applications, including immunofluorescence staining (IFS) and immunochemical staining (ICS). These techniques allow for visual detection of Brucella in infected cells or as intact bacteria.

Three monoclonal antibodies—6C12, 2B10, and 4F10—have demonstrated strong fluorescent reactivity with lentivirus-Omp25 transduced cells in IFS applications . Additionally, IFS with mAb 6C12 has successfully detected visible fluorescent intracellular Brucellae in infected PBMCs from patients with brucellosis, while showing negative results with samples from healthy blood donors .

For ICS applications, cultured Brucella can be visualized microscopically after staining with HRP-conjugated antibodies 6C12, 8F3, or 4A12 on glass slides . These staining techniques provide valuable tools for direct visual confirmation of Brucella infection in clinical and research settings.

Research Applications and Findings

Beyond diagnostic applications, Omp25 antibodies have contributed significantly to understanding Brucella pathogenesis and host-pathogen interactions. Several research findings highlight the importance of these antibodies in elucidating the functions of Omp25 in Brucella virulence and immune evasion.

Role of Omp25 in TNF-α Suppression

Research utilizing Omp25 antibodies has revealed that this protein plays a crucial role in inhibiting tumor necrosis factor alpha (TNF-α) production during infection of human macrophages . Studies with Δomp25 Brucella suis and Δomp31 B. suis mutants demonstrated that the expression of Omp25 correlates with the unusual absence of TNF-α release observed in human macrophages infected with Brucella species .

These findings suggest that Omp25 is involved in the negative regulation of TNF-α production, which may represent a key mechanism through which Brucella evades host immune responses. By suppressing this important pro-inflammatory cytokine, Brucella can potentially establish more persistent infections in host cells.

Omp25's Role in Interferon Signaling Pathway

Recent research has uncovered another important function of Brucella Omp25 in modulating host immune responses—specifically, its ability to inhibit type I interferon (IFN) production. Studies have shown that Omp25 suppresses the production of IFN-β and its downstream IFN-stimulated genes induced by various DNA viruses or IFN-stimulatory DNA in multiple species' monocyte/macrophages or peripheral blood mononuclear cells .

The mechanism behind this inhibition involves Omp25 promoting cyclic guanosine monophosphate–adenosine monophosphate synthase (cGAS) degradation via the proteasome-dependent pathway . This leads to decreased cyclic guanosine monophosphate–adenosine monophosphate production and reduced downstream signaling activation upon DNA virus infection or IFN-stimulatory DNA stimulation.

Further mapping of the functional domains of Omp25 identified that amino acids 161 to 184 are required for Omp25-induced cGAS degradation, with five specific residues (R176, Y179, R180, Y181, and Y184) being essential for its inhibitory effect on IFN-β induction . These findings provide valuable insights into the molecular mechanisms through which Brucella modulates host immune responses.

Vaccine Development Utilizing Omp25

The high immunogenicity of Omp25 has made it an attractive candidate for vaccine development against brucellosis. Several innovative approaches have been explored to utilize this protein in creating effective vaccines.

Chimeric Omp25-Omp31 Vaccines

Researchers have designed chimeric antigens combining Omp25 with other Brucella outer membrane proteins, particularly Omp31. One study described a new chimeric OMP25-OMP31 antigen formulated in Chitosan nanoparticles and evaluated its protective efficiency in vivo .

This chimeric protein was produced using spliced overlap extension by polymerase chain reaction, and its 3D structure and antigenic ability were predicted using computational tools. Immune response analysis showed that the chimeric rOMP25–OMP31 antigen induced higher titers of IFN-γ and TNF-α cytokines, while producing the lowest amount of IL-4 . This cytokine profile suggests a strong Th1-type immune response, which is generally considered beneficial for protection against intracellular pathogens like Brucella.

Lactococcus-Based Delivery Systems

Another innovative approach involves using Lactococcus lactis as a delivery vehicle for Brucella abortus Omp25 antigen . This recombinant oral antigen delivery system takes advantage of the fact that most Brucella infections occur at mucosal membranes, making mucosal vaccination potentially crucial for managing brucellosis.

The development of such oral vaccine candidates represents an important avenue for creating more accessible and effective vaccines against brucellosis, particularly in endemic regions where resources for traditional vaccination programs may be limited .