METHODS: Recombinant PPDK (rPPDK) was expressed, purified and evaluated by Western blot. In parallel, recombinant galactose-and-N-acetyl-D-galactosamine inhibitable lectin (Gal/GalNAc lectin) was produced and tested similarly. The protein identity was confirmed by analysis using MALDI-TOF/TOF. A lateral flow dipstick (LFD) test using rPPDK was subsequently developed (rPPDK-LFD) and evaluated for serodiagnosis of ALA.
RESULTS: rPPDK was expressed as soluble protein after 4 hours of induction with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 30°C. Purification using nickel-nitrilotriacetic acid (Ni-NTA) resin yielded 1.5 mg of rPPDK from 1 L of culture with estimated molecular mass of 98 kDa on SDS-PAGE. Western blots using sera from patients with ALA, healthy individuals and other diseases probed with anti-human IgG4-HRP showed the highest sensitivity (93.3%) and specificity (100%); as compared to blots using IgG and IgG1 as secondary antibodies. Moreover, rPPDK showed better specificity when compared to rGal/GalNAc lectin. In the development of the LFD test, the optimum amount of rPPDK was 0.625 μg per dipstick and the optimum working concentration of colloidal gold conjugated anti-human IgG4 was optical density (OD) 5 (1.7 μg of anti-human IgG4). Evaluation of rPPDK-LFD using ALA patients and controls serum samples showed 87% diagnostic sensitivity and 100% specificity.
CONCLUSION: The developed rPPDK-LFD showed good potential for rapid diagnosis of ALA, and merit further multicentre validation using larger number of serum samples.
METHODS: An indirect enzyme-linked immunosorbent assay (ELISA) was developed to evaluate the usefulness of USM.TOXO1 antigen for the detection of IgG antibodies against Toxoplasma gondii in human sera. Whereas the reactivity of the developed antigen against IgM antibody was evaluated by western blot and Dot enzyme immunoassay (dot-EIA) analysis.
RESULTS: The diagnostic performance of the new antigens in IgG ELISA was achieved at the maximum values of 85.43% and 81.25% for diagnostic sensitivity and specificity respectively. The USM.TOXO1 was also proven to be reactive with anti- T. gondii IgM antibody.
CONCLUSIONS: This finding makes the USM.TOXO1 antigen an attractive candidate for improving the toxoplasmosis serodiagnosis and demonstrates that multiepitope antigens could be a potential and promising diagnostic marker for the development of high sensitive and accurate assays.
RESULTS: cSC binds > 0.06 μg/ml of purified human and mouse pIgA with negligible cross-reactivity against IgM and IgA. Virus-specific pIgA was significantly higher in serum of acute HAV (n = 6) and HEV (n = 12) patients than uninfected samples (HEV: p
RESULTS: Of the 368 cats that were evaluated in this study, 12.2% (45/368; 95% CI = 8.88 - 15.58) were positive for FeLV p27 antigen, 31.3%, (115/368; 95% CI = 26.51 - 35.99) were seropositive to FIV antibodies, and 4.3% (16/368; 95% CI = 2.27 - 6.43) had evidence of both viruses. Factors found to significantly increase the risk for FeLV seropositivity include sex, age, behaviour, sickness, and living in a multi-cat household. Seropositive response to FIV was significantly associated with sex, neuter status, age, behaviour, and health status.
CONCLUSIONS: The present study indicates that FeLV and FIV are common among domestic cats in peninsular Malaysia, and that factors related to cat demographics and health such as age, sex, behaviour, health status and type of household are important predictors for seropositive status to FeLV or FIV in peninsular Malaysia. High prevalence of FeLV or FIV observed in our study is of concern, in view of the immunosuppressive potentials of the two pathogens. Specific measures for control and prevention such as screening and routine vaccination are needed to ensure that FeLV and FIV are controlled in the cat population of peninsular Malaysia.