New Arrival | East-Mab Bio Launches Brucellosis BP26 Protein – Clinical Sample Specificity Reaches 99.4%!

What is Brucellosis?

Brucellosis (also known as "undulant fever") is a zoonotic infectious disease caused by infection with Brucella bacteria. In China, it is primarily endemic in northern regions,however, in recent years, the prevalence has also increased in southern areas, with sporadic outbreaks occurring in certain areas. Currently, 12 species of Brucella bacteria have been identified in China.

Global Brucella Infection Status

International
According to a survey by Laine CG et al. in September 2023 [1], 82.3% (144/175) of countries worldwide and 43.2% (3.2 billion / 7.4 billion) of the global population are considered at risk for brucellosis. Regionally, the risk distribution is as follows:

l Africa: 92.5% (49/53) of countries and 57.5% (690 million / 1.2 billion) of the population;

l Asia: 85.7% (42/49) of countries and 47.7% (2.1 billion / 4.4 billion) of the population;

l Americas: 80.6% (25/31) of countries and 19.4% (190 million / 980 million) of the population;

l Europe: 66.7% (28/42) of countries and 24.3% (180 million / 740 million) of the population;

(Note: A global heatmap predicting the annual incidence rate of human brucellosis per million people at risk would be described here).

Target Selection for Brucellosis Detection

Diagnostic targets for brucellosis are divided into three categories: Lipopolysaccharide (LPS), Outer Membrane Proteins (OMPs), and other bacterial proteins [2]:

1. Lipopolysaccharide (LPS)

Brucella LPS consists of three regions: Lipid A, core oligosaccharide, and O-antigen or O-chain. The O-chain portion contains the vast majority of antigenic epitopes on the surface of Brucella and serves as its most prominent surface antigen. Based on the presence or absence of the O-chain, Brucella is classified into rough (R) and smooth (S) phenotypes. LPS is the most commonly used target for brucellosis diagnosis and plays an irreplaceable role in its prevention and control. Literature reports on whole-cell proteins, crude outer membrane proteins, salt-extracted proteins, phenol-salt extracted proteins—all primarily contain LPS as the major component, as evidenced by their extraction methods.

2. Outer Membrane Proteins (OMPs)

Outer Membrane Proteins (OMPs) are a unique and critical class of proteins in Gram-negative bacteria. They contribute to membrane permeability, maintain membrane structural integrity, and, importantly, some OMPs act as immunogenic molecules capable of stimulating immune responses. This makes them important diagnostic targets in immunological studies. In Brucella, over a dozen OMPs have been explored as diagnostic targets, the main ones being BCSP31, BP26, Omp10, Omp25, and Omp31. The major proteins are described in detail below:

(1) BCSP31: approximately 31 kDa in size, is a Brucella outer membrane protein. It is highly antigenic, conserved yet polymorphic, with high interspecies homology—studies have confirmed that BCSP31 genes in Brucella strains infecting cattle, sheep, and pigs share 100% homology. The BCSP31 coding gene is also a primary target for PCR diagnosis of brucellosis. However, some studies using protein microarrays for Brucella immunodetection found that antibodies against BCSP31 were only present in sera from infected goats and not in any of the 62 human serum samples confirmed to be Brucella-positive. This suggests that BCSP31 may not be suitable as a diagnostic target in human brucellosis detection.

(2) Omp10: a 10 kDa outer membrane lipoprotein and an important immunogenic protein found in all known Brucella species and biovars. It is associated with Brucella virulence, and studies have shown that a 138 bp sequence at the 3′ end of its coding gene is homologous to HemH (ferrochelatase), suggesting a possible role in iron utilization and biosynthesis. Research on its application as a diagnostic target is still limited.

(3) Omp16: a homolog of Peptidoglycan-Associated Lipoprotein (Pals). To date, no omp16 mutants have been generated in any Brucella strain and little is known about the function or pathogenic mechanisms. It possesses a conserved Pal domain and is highly conserved within Brucella. Attempts to delete omp16 in the B. suis vaccine strain S2 were unsuccessful, indicating Omp16 is essential for Brucella survival.

(4) Omp25: a 25 kDa protein plays a crucial role in maintaining the structural stability of the Brucella outer membrane. Its coding gene is highly conserved among Brucella species, biovars, and strains. Omp25's amino acid sequence is also the most specific among bacteria causing cross-reactivity in brucellosis tests. Boigegrain et al., reported that Omp25 was also secreted extracellularly when analyzing the proteins secreted by Brucella under acid stress, suggesting its association with the Brucella’s membrane secretion system.

(5) BP26 (also known as Omp28): a 26kDa Brucella periplasmic protein. It is present in all Brucella strains and is a dominant immunogen in infections of cattle, sheep, goats, and humans. Numerous reports indicate its high accuracy as a target for serological detection. Researchers using protein microarrays found it to be the only target within the Brucella proteome that specifically reacts with sera from infected humans and sheep. Recently, two linear epitopes on BP26 have been confirmed (93DRDLQTGGI101 and 104QPIYVYPD111), providing a basis for further immunological analysis of this protein.

(6) Omp31: a 25 kDa outer membrane protein exposed on the bacterial surface, typically existing in oligomeric form. It possesses the ability to renature from SDS denaturation at temperatures below 60°C. It is highly conserved among different Brucella species and is expressed in all species except B.abortus. There are reports on immunological detection methods for brucellosis based on OMP31. However, recent protein microarray studies failed to detect antibodies against this protein in sera from infected humans or goats, suggesting further research is needed to validate it as a detection target.

(7) Omp2b: an important structural component of the Brucella outer membrane with high immunogenicity. Bioinformatics software was used to analyze the protein structure, predict dominant T-cell and B-cell epitopes, and T-B combined epitopes. ELISA was used to detect specific IgG antibody levels against Omp2b T-B combined peptides in brucellosis patient sera. Analysis identified a potential T-B cell combined antigenic epitope segment (196-216). Patients showed significantly higher numbers of IFN-γ–secreting cells (SFU) compared to healthy controls (F = 25.413, P < 0.01), along with increased serum levels of IgG, perforin, and granzyme B (F = 13.653, P < 0.01).

(8) VirB12: a cell surface protein that can induce antibody production during animal infection, making it a potential target for serological diagnosis. Studies show recombinant VirB12 protein reacts strongly with sera from brucellosis patients. Compared with current ELISA kits used in clinical settings, VirB12-based ELISA demonstrated the following performance metrics: accuracy 90.0%, specificity 94.0%, sensitivity 87.8%, negative predictive value 80.0%, and positive predictive value 96.6%. These results indicate that VirB12 is antigenic and a promising candidate target for human brucellosis diagnosis.

3. Other Proteins

In additional to OMPs,several other immunogenic proteins in Brucella have also attracted attention as potential diagnostic targets. These include nucleoprotein L7/L12, chaperone proteins GroEL and GroES, heat shock protein DnaK, and superoxide dismutase (SOD). However, the amino acid sequence similarity of these proteins with homologous proteins in other cross-reactive bacteria is generally above 60%, which may compromise their specificity as diagnostic markers for Brucella.

East-Mab Bio Launches Brucellosis Antigens

In recent years, East-Mab Bio has focused on developing antigens and antibodies related to respiratory and infectious diseases. They have newly launched Brucella target proteins BP26, Omp16, and VirB12 for brucellosis detection. They have already received positive feedback from multiple customers with particularly strong performance observed for the BP26 antigen—showing a positive detection rate of over 80% in confirmed clinical serum samples and a specificity as high as 99.4%.

① Evaluation of Positive Detection Rate using Confirmed Brucellosis-Positive Sera:

② Evaluation of Specificity using Clinical Serum Samples:

u 177 clinical serum samples were tested.

u IgG detection: 4 false positives and 1 weak positive (RBPT and SAT both positive) were found resulting in an IgG specificity of 97.7% .

u IgM detection: 1 false positive (weak positive) was found resulting in an IgM specificity of 99.4%.

(Above data sourced from customer validation using BP26 protein for detection)

[1]Laine CG, Johnson VE, Scott HM, Arenas-Gamboa AM. Global Estimate of Human Brucellosis Incidence. Emerg Infect Dis. 2023 Sep;29(9):1789-1797. doi: 10.3201/eid2909.230052. PMID: 37610167; PMCID: PMC10461652.

[2]夏淑婷,王鹏,宋志忠.布鲁氏菌病血清学诊断靶点的研究进展[J].中国人兽共患病学报,2014,30(03):324-329.

Brucellosis Antigens I East-Mab Bio