Expression and Evaluation of Wb-SXP-1 and Wb-123 Recombinant Antigens as Potential Diagnostic Biomarkers for Lymphatic Filariasis

Lymphatic filariasis (LF) remains a public health concern as it can cause permanent morbidity and disability to those infected. While the global elimination of LF in these endemic areas is ongoing through mass drug administration, there is the need to develop diagnostic tools that would be utilized to track the progress of total global eradication as well as perform surveillance for the recurrence of lymphatic filariasis transmission. Currently, approved LF diagnosis tools are faced with lack of specificity, low sensitivity, and periodicity dependence. Recombinant filarial antigen-based assays can address these drawbacks and offer practical instruments for LF diagnosis and surveillance. This present study, evaluated rWb-SXP-1 and rWb-123 antigens as potential diagnostic biomarker tools for Wuchereria banchrofti in human sera using microspheres-based multiplex serological assay. Based on statistical analysis using XLSTAT 2019 (Addinsoft) on data generated from multiplex technology assay, generated ROC curves for both rWb-SXP-1 and rWb-123 demonstrated 87.1% sensitivity to Wuchereria banchrofti human sera with rWb-SXP-1 antigens having the highest specificity of 96%. Indication that rWb-SXP-1 and rWb-123 antigens are capable of detecting immunoglobulin G4 (IgG4) antibodies in human sera synthesized specifically against W. banchrofti infections. Therefore, rWb-SXP-1 and rWb-123 antigens can be utilized to detect W. banchrofti infections by antibody profiling with excellent diagnostic sensitivity and specificity using microsphere-based multiplex serological tests. This method can be particularly practical for screening a large number of sera samples and/or for quick, extensive field-testing due to the high-throughput and quick formats applied.

STRUCTURE AND EXPRESSION OF EPSTEIN-BARR VIRUS MEMBRANE ANTIGEN IN RECOMBINANT VACCINIA VIRUS

The Epstein-Barr virus membrane antigen was constructed and inserted into vaccinia virus, Tian-tan strain in order to study the effect of this virus on EB infection and tumorogenesis. The EBV-derived membrane antigen was expressed under the control of a 7.5 K promoter of vaccinia virus. The antibody against the membrane antigen of EB virus was produced on rabbits vaccinated with recombinant vaccinia virus.

Synthesis of Novel Hydrophobic Media and Purification of Recombinant HBsAg

A novel hydrophobic medium with propyl as functional group and Sepharose 6B as matrix was designed and synthesized. The comparison of the hydrophobic medium synthesized with the commercial products was made by hydrophobic interaction chromatography(HIC) in isolating recombinant hepatitis B surface antigen(r HBsAg). r HBsAg was further purified to the final products by following a downstream procedure . The results indicate that the synthesized hydrophobic medium possesses a stable structure and desired physical and chemical properties. They were used to purify r HBsAg with a high yield and purity. Both the immunity and stability of hepatitis vaccine made by the r HBsAg products have reached the same level as other similar kinds of products.

A Sensitive and Specific IgM-ELISA for the Serological Diagnosis of Human Leptospirosis Using a rLipL32/1-LipL21-OmpL1/2 Fusion Protein

Objective To construct a lipL32//1-1ipL21-OmpL1//2 fusion gene and its prokaryotic expression system, and to establish an enzyme-linked immunosorbent assay （ELISA） using the rLipL32/1-LipL21-OmpL1/2 fusion antigen of Leptospira interrogans for sensitive and specific detection of IgM in the serum of patients with leptospirosis. Methods lipL32/1-1ipL21-OmpL1/2 fusion genes were constructed using a primer-linking PCFI. The target recombinant protein antigens, rLipL32/1, rLipL21, rOmpL1/2 and rLipL32/1-LipL21-OmpL1/2, were expressed and the purified antigens were then immobilized to the surface of microplate wells for ELISA-based detection of IgM in the sera of leptospirosis patients; Results Of 493 acute leptospirosis patients, 95.7% and 97.8% were positive by rLipL32/1-LipL21- OmpL1/2-1gM-ELISA using different serum dilutions, which was higher than the rLipL32/1-1gM-ELISA （93.1% and 90.3%）, rLipL21-1gM-ELISA （90.3% and 87.0%）, and rOmpLI-lgM-ELISA （85.6% and 81.1%） （P〈0.01）. All IgM-ELISAs tested negative against 56 non-leptospirosis patients with typhoid fever, hemorrhagic fever or dengue fever. Conclusion Trigeminal fusion antigen increases ELISA sensitivity and the rLipL32/1-LipL21-OmpL1/2- IgM-ELISA is a sensitive and specific serological diagnostic method for clinical leptospirosis.

Development of Multiple ELISAs for the Detection of Antibodies against Classical Swine Fever Virus in Pig Sera

The major immunogenic proteins （Ems, E2 and NS3） of classical swine fever virus （CSFV） （Shimen strain） were expressed in E. coli and purified by affinity chromatography. The recombinant antigens were applied to develop multiple enzyme-linked immunosorbent assays （ELISAs） for the detection of specific antibodies in pig sera. Optimum cut-off values were determined by receiver operating characteristic （ROC） analysis after testing 201 sera of vaccinated pigs and 64 negative sera of unvaccinated piglets. The multiple ELISAs were validated with 265 pig sera yielding high sensitivity and specificity in comparison with the virus neutralization results. The results demonstrated that multiple ELISAs can be a valuable tool for the detection of CSFV infection and serological surveys in CSFV-free countries or for the evaluation of the antibody responses in pigs induced by a live attenuated C-strain vaccination

Identification of a gene engineering antibody against cystic echinococcosis in liver

OBJECTIVE: To identify a gene engineering antibody against cystic echinococcosis in liver. METHODS: A single chain of variable fragment of human antibodies (ScFvs) was selected from the library by using affinity selection technique with the recombinant antigen on solid surface. The positive clones were demonstrated by ELISA and their DNA sequences were also determined. RESULTS: The DNA sequence data showed that the antibody gene is composed of 768bp. In addition, a specific combination capacity with recombinant Echinococcus granulosus antigen B (r-EgB) was demonstrated by ELISA. CONCLUSION: The obtained gene engineering antibody against r-EgB may have potential implications in immunological treatment and drug targeting delivery.

Application of antigenic biomarkers for Mycobacterium tuberculosis

The study and characterization of biomolecules involved in the interaction between mycobacteria and their hosts are crucial to determine their roles in the invasion process and provide basic knowledge about the biology and pathogenesis of disease.Promising new biomarkers for diagnosis and immunotherapy have emerged recently.Mycobacterium is an ancient pathogen that has developed complex strategies for its persistence in the host and environment,likely based on the complexity of the network of interactions between the molecules involved in infection.Several biomarkers have received recent attention in the process of developing rapid and reliable detection techniques for tuberculosis.Among the most widely investigated antigens are CFP-10(10-kDa culture filtrate protein),ESAT-6(6-kD a early secretory antigenic target),Ag85 A,Ag85 B,CFP-7,and PPE18.Some of these antigens have been proposed as biomarkers to assess the key elements of the response to infection of both the pathogen and host.The design of novel and accurate diagnostic methods is essential for the control of tuberculosis worldwide.Presently,the diagnostic methods are based on the identification of molecules in the humoral response in infected individuals.Therefore,these tests depend on the capacity of the host to develop an immune response,which usually is heterogeneous.In the last 20 years,special attention has been given to the design of multiantigenic diagnostic methods to improve the levels of sensitivity and specificity.In this review,we summarize the state of the art in the study and use of mycobacterium biomolecules with the potential to support novel tuberculosis control strategies.

Effect of chicken egg anti-F4 antibodies on performance and diarrhea incidences in enterotoxigenic Escherichia coli K88^+-challenged piglets

The aim was to evaluate the effects of dietary supplementation of spay-dried whole egg containing antiF4 antibodies(SDWE) against recombinantly produced F4 antigens in enterotoxigenic Escherichia coli K88^+(ETEC)-challenged piglets. Twenty-seven 21-d-old and individually housed piglets were randomly allotted to 3 treatments consisting of a wheat-soybean meal basal diet containing either 0(control egg powder; CEP), 0.1%(SDWE1) or 0.4%(SDWE2) SDWE. After a 7-d adaptation period, blood samples were collected from all pigs,and pigs were weighed and orally challenged with an ETEC inoculum. Blood was sampled at 24 and 48 h post-challenge, and diarrhea incidences and scores were recorded. On d 14, all pigs were weighed and then euthanized to obtain intestinal tissue samples for histomorphology measurement. During the pre-challenge period, pigs fed the SDWE showed a linear improvement(P < 0.05)in average daily gain(ADG) and gain to feed ratio(G:F), but there were no differences among treatments in growth performance during the post-challenge period. Diarrhea incidences and scores, fecal shedding of ETEC, plasma urea nitrogen content and intestinal histo morphology were similar among treatments.The results show that 0.4% SDWE supported greater piglet performance before challenge although such benefits were not evident during the post-challenge period at either 0.1% or 0.4% supplementation.