Development Of Conventional Real-Time And Thermostabilised Multiplex PCR For The Detection Of Toxoplasma Gondii

Toxoplasma gondii is an important pathogen in veterinary and human medicines. It is widely distributed throughout the world and has a broad range of hosts that includes warm-blooded animals as intermediate hosts and felid (cat) as the definitive host. Infection with this parasite is usually asymptomatic in healthy individuals but can cause morbidity and mortality in immunocompromised patients and in congenitally infected infants. Molecular biology is increasingly being used as a diagnostic method for diagnosis of toxoplasmosis, especially for specimens collected from amniotic fluid, cerebrospinal fluid, placenta tissue, blood and eye fluid. However, the existing PCRbased methods for the detection of Toxoplasma DNA suffer from variations in performance among the laboratories. Indeed, the use of PCR-based assay for diagnosis of Toxoplasma is still uncommon in Malaysia. The aim of this study is to develop rapid, specific and sensitive PCR-based assays to detect T. gondii DNA for diagnosis of toxoplasmosis. Three PCR-based assays namely conventional multiplex PCR, realtime multiplex PCR and thermostabilised PCR for the detection of T. gondii DNA were developed. All the primers and probes used were newly designed and optimized. All three PCR assays showed 100% specificity whereby no cross-reactivity with other organisms was observed

Construction Of Human Lymphatic Filariasis Antibody Phage Display Library And Production Of Recombinant Monoclonal Antibodies Against BmR1 And BmSXP Filarial Antigens

A Global Programme for Elimination of Lymphatic Filariasis aims to eliminate the disease as a public health problem by the year 2020. Brugia Rapid and PanLF Rapid are rapid diagnostic tools that are being used in this programme. They are based on the detection of recombinant proteins BmR1 and BmSXP. The quality of these tests should be well-maintained at all time or even improved as the GPELF progresses and a sensitive and specific test may be needed at the end of the programme and for surveillance post-certification. To address this need, the availability of recombinant monoclonal antibodies to the two recombinant proteins would be very useful

Antibody-Based Protective Immunity against Helminth Infections: Antibody Phage Display Derived Antibodies against BmR1 Antigen

Helminth parasite infections are significantly impacting global health, with more than two billion infections worldwide with a high morbidity rate. The complex life cycle of the nematodes has made host immune response studies against these parasites extremely difficult. In this study, we utilized two phage antibody libraries; the immune and naïve library were used to identify single chain fragment variable (scFv) clones against a specific filarial antigen (BmR1). The V-gene analysis of isolated scFv clones will help shed light on preferential VDJ gene segment usage against the filarial BmR1 antigen in healthy and infected states. The immune library showed the usage of both lambda and kappa light chains. However, the naïve library showed preferential use of the lambda family with different amino acid distributions. The binding characteristics of the scFv clones identified from this work were analyzed by immunoassay and immunoaffinity pull down of BmR1. The work highlights the antibody gene usage pattern of a naïve and immune antibody library against the same antigen as well as the robust nature of the enriched antibodies for downstream applications

Potential Effects of Chrysin on MDA-MB-231 Cells

This study aims to elucidate the effects of chrysin on human ER-negative breast cancer cell line, MDA-MB-231. The study demonstrated that treatment of MDA-MB-231 cells with 20 μM chysin for 48 h significantly inhibited the growth of MDA-MB-231 cells and induced cytoplasmic lipid accumulation in the cells, but that the observed of cell death was not caused by apoptosis. The expression of PPARalpha mRNA in chrysin-treated MDA-MB-231 cells was significantly increased, which was likely associated to the proliferation of the cells post chrysin treatment

Antibody-Based Protective Immunity against Helminth Infections: Antibody Phage Display Derived Antibodies against BmR1 Antigen

Helminth parasite infections are significantly impacting global health, with more than two billion infections worldwide with a high morbidity rate. The complex life cycle of the nematodes has made host immune response studies against these parasites extremely difficult. In this study, we utilized two phage antibody libraries; the immune and naïve library were used to identify single chain fragment variable (scFv) clones against a specific filarial antigen (BmR1). The V-gene analysis of isolated scFv clones will help shed light on preferential VDJ gene segment usage against the filarial BmR1 antigen in healthy and infected states. The immune library showed the usage of both lambda and kappa light chains. However, the naïve library showed preferential use of the lambda family with different amino acid distributions. The binding characteristics of the scFv clones identified from this work were analyzed by immunoassay and immunoaffinity pull down of BmR1. The work highlights the antibody gene usage pattern of a naïve and immune antibody library against the same antigen as well as the robust nature of the enriched antibodies for downstream applications

Isolation and Production of Human Monoclonal Antibody Proteins against a Toxocara canis Excretory–Secretory Recombinant Antigen

Toxocariasis is a widespread zoonotic parasitic disease with a significant socioeconomic impact, particularly on underprivileged communities. Limitations of existing diagnostic tools and vague presenting symptoms may lead to misdiagnosis, thus underestimating the actual global impact of the disease. The present study describes the isolation and production of novel recombinant monoclonal antibodies against Toxocara canis recombinant TES-26 antigen (rTES-26) utilizing a human helminth scFv phage display library. The isolated antibody clones were characterized based on their gene sequences and binding characteristics. Three clones representing unique gene families (clone 48: IgHV3-LV1; clone 49: IgHV3-LV3; clone 50: IgHV6-LV3) were isolated, but only clones 48 and 49 showed successful insertion of the full-length scFv antibody sequence after sub-cloning. Both clones produced antibody proteins of good solubility and satisfactory yield and purity. Binding assays via Western blot and ELISA using rTES-26 and Toxocara canis native protein showed that both monoclonal antibodies were highly specific and sensitive to the target antigen. A preliminary antigen detection ELISA showed the diagnostic potential of the monoclonal antibody proteins. The proteins can also be useful in studying host–parasite interactions and therapeutic applications

Production of Toxocara cati TES-120 Recombinant Antigen and Comparison with its T. canis Homolog for Serodiagnosis of Toxocariasis

Photograph of a scene at Ed Galloway's Totem Pole Park

A Pentaplex Real-Time Polymerase Chain Reaction Assay for Detection of Four Species of Soil-Transmitted Helminths

Soil-transmitted helminth infections remain a major public health burden in low- and middle-income countries. The traditional diagnosis by microscopic examination of fecal samples is insensitive and time-consuming. In this study, a pentaplex real-time polymerase chain reaction (PCR) was evaluated for the simultaneous detection of Ancylostoma, Necator americanus, Ascaris lumbricoides, and Strongyloides stercoralis. The results were compared with those obtained by conventional parasitological diagnostic methods. Real-time PCR was positive in 48 of 77 samples (62.3%) and microscopic examination was positive in six samples (7.8%) only (P < 0.05). In conclusion, the real-time PCR assay described in this study provides a specific and sensitive diagnostic tool for the detection of these four helminth species in epidemiological studies and monitoring of treatment programs