Comparison of BP26, Omp25 and Omp31 and a Multi-Epitope Based Fusion Protein in Serological Detection of Canine Brucellosis

Background Brucellosis is one of the most important zoonotic diseases in the world. Canine brucellosis caused mainly by B. canis is seriously neglected and there is lack of accurate diagnostic tools. Methods In this study, using 34 brucellosis positive dog sera and 62 negative control sera, the Brucella outer membrane protein of Omp31, BP26, Omp25 and a multi-epitope based fusion protein were evaluated by iELISA for their potential use as antigens in serological diagnosis for canine brucellosis. Results The result showed that multi-epitope based fusion protein performed best in distinguishing brucellosis positive and negative dog sera, with positive predictive value (PPV) was 100% and the negative predictive value (NPV) was 98.41%. BP26 and Omp31 showed excellent sensitivity in detecting brucellosis positive dog sera, but their cross reaction to sera infected with Vibrio parahaemolyticus and Listeria monocytogenes may hinder their application as diagnostic reagent. While Omp25 was lack of sufficient sensitivity and just showed limited ability in distinguishing positive and negative dog sera. Conclusion The multi-epitope based fusion protein could accelerate the development of diagnostic kit for canine brucellosis currently urgently needed in China.

Assessing the antigenicity of different VP3 regions of infectious bursal disease virus in chickens from South Brazil

Background Infectious bursal disease (IBD), also known as Gumboro disease, is a viral infection that causes mortality and immunosuppression in chickens (Gallus gallus). VP2 and VP3 are the major structural viral capsid components and are the most immunogenic proteins of IBD virus (IBDV). Reliable diagnostic tests using VP2 and VP3 produced in heterologous systems are important tools to control this infection. One advantage of an IBD diagnostic based on VP3, over those that use VP2, is that VP3 has linear epitopes, enabling its production in bacteria. Results We tested the suitability of recombinant VP3 (rVP3) as a diagnostic reagent in an enzyme-linked immunosorbent assay (ELISA). Compared with a commercial test, rVP3 ELISA showed high sensitivity and specificity as a diagnostic tool for vaccinated animals. In addition, rVP3, but not the commercial ELISA, was able to detect antibodies in nonvaccinated chickens, probably developed against circulating IBDV strains. It was possible the assessment of VP3 regions antigenicity using chicken antisera. Conclusions The full-length recombinant VP3 can be used to assess post vaccination immunological status of chickens and its production is feasible and inexpensive. The evaluation of VP3 regions as candidates for general use in the diagnosis of IBD in chickens should be conducted with caution. Our work was the first to identify several regions of VP3 recognized by chicken antibodies.

Development of an NT-ProBNP Assay Reagent Based on High Specific Activity Alkaline Phosphatase CmAP and an Improved Coupling Method

(1) Background: Chemiluminescent enzyme immunoassay (CLEIA) is an efficient analytical method. Alkaline phosphatase (ALP) with high specific activity is the basis for CLEIA to achieve high sensitivity. In this study, a high specific activity Cobetia marina ALP (CmAP) and an improved coupling method were used to develop an N-terminal pro-B-type natriuretic peptide (NT-proBNP) diagnostic reagent. (2) Methods: The purification method of CmAP was improved and the related enzyme activities were assessed. The enzyme and magnetic beads were coupled only to the Fc region of the detection antibody and the capture antibody, respectively, by using a specially improved method. The NT-proBNP in human serum was assessed. (3) Results: The specific activity of the purified CmAP was found to be 13,133 U/mg. No loss in the enzyme activity was observed after its storage at room temperature for 4 months. The sensitivity of the in vitro diagnostic reagents was found to be 0.58 ng/L. (4) Conclusions: CmAP can be applied as a substitute for the commercial ALP. Analytical parameters indicated that the chemiluminescence diagnostic reagent for NT-proBNP is adequately sensitive and reliable for detecting the serum NT-proBNP, which suggests that both the enzyme and coupling method are suitable for the CLEIA.

Interactions of Paraoxonase-1 with Pharmacologically Relevant Carbamates

Mammalian paraoxonase-1 hydrolyses a very broad spectrum of esters such as certain drugs and xenobiotics. The aim of this study was to determine whether carbamates influence the activity of recombinant PON1 (rePON1). Carbamates were selected having a variety of applications: bambuterol and physostigmine are drugs, carbofuran is used as a pesticide, while Ro 02-0683 is diagnostic reagent. All the selected carbamates reduced the arylesterase activity of rePON1 towards the substrate S-phenyl thioacetate (PTA). Inhibition dissociation constants (Ki), evaluated by both discontinuous and continuous inhibition measurements (progress curves), were similar and in the mM range. The rePON1 displayed almost the same values of Ki constants for Ro 02-0683 and physostigmine while, for carbofuran and bambuterol, the values were approximately ten times lower and two times higher, respectively. The affinity of rePON1 towards the tested carbamates was about 3–40 times lower than that of PTA. Molecular modelling of rePON1-carbamate complexes suggested non-covalent interactions with residues of the rePON1 active site that could lead to competitive inhibition of its arylesterase activity. In conclusion, carbamates can reduce the level of PON1 activity, which should be kept in mind, especially in medical conditions characterized by reduced PON1 levels.

Development of a horseradish peroxidase-nanobody fusion protein for visual detection of ochratoxin A by dot immunoassay

The horseradish peroxidase-nanobody fusion protein was developed as a promising immunological diagnostic reagent for toxic low-molecular-weight compounds in food.

Gastric cancer indocyanine green lymph node navigation surgery: systematic review

Sentinel lymph node (LN) biopsy is a common practice to determinate if a lymphadenectomy is needed in various malignancies. Recent studies have investigated the possibilities to extend sentinel LN biopsy in gastric cancer. Indocyanine green (ICG) is a diagnostic reagent recently introduce in sentinel LN biopsy field. This review aims to determinate the feasibility to used ICG to detect sentinel LN in gastric cancer.