Novel IgG and IgY Sandwich Immunoassays for Rapid and Low-Cost Ebola Virus Detection

• Author / Creator

  Singh, Bharti

• Ebola Virus Disease (EVD) is a major public health concern with a high mortality rate in infected individuals. Overlapping symptoms of EVD with other diseases and lack of sensitive diagnostics increases mortality. Thus, there is an urgent need to develop a rapid, reliable, inexpensive and sensitive diagnostic to control the spread and manage the disease at the initial stages. Therefore, early detection of VP40 in 7 days is required to increase survivability. VP40 is the abundant protein consisting of 40% of total protein in Ebola virus. Our research aims to develop immunoassay (DAS-ELISA) based on IgY, biotin labelled monoclonal antibody (bmAb) and bispecific antibodies (bsAb) for the detection of VP40 protein in in-vitro test with lowered limit of detection (LOD) than prevailing assays. The VP40 protein was expressed in E.coli and purified using immobilized metal ion chromatography. The recombinant VP40 (rVP40) was used to immunize chickens for the development of specific IgY antibodies. Anti-VP40 hybridoma cells were cultured and second generation bispecific antibodies were developed by quadroma technology. Standardized combinations of IgY, bmAb and bsAb has been used to optimize Ebola detection assay and LOD was calculated for each DAS-ELISA format. VP40 protein expression and purity was confirmed by western blot. The yield of purified rVP40 obtained was 12 mg/l of bacterial culture. Three Abs (IgY, mAbs and bsAb) were produced in sterile conditions. Purity and specificity of the antibodies was confirmed by SDS-PAGE and western blot. The different parameters of DAS-ELISA was optimized (i) DAS-ELISA-mAb-IgY : capture antibody as mAb (cAb) 4μg/ml, detecting antibody (dAb) IgY 8 μg/ml, the optimized dilution of anti-chicken IgY at 1:10,000; (ii) DAS-ELISA-mAb-bmAb: cAb 4 μg/ml, bmAb 31.25 ng/ml, the optimized dilution of streptavidin-HRP at 1:10,000; (iii) DAS-ELISA-mAb-bsAb: cAb 2 μg/ml and bsAb 14 ng/ml. The LOD of VP40 antigen in PBS for each format was (i) 33 ng/ml, (ii) 23 ng/ml, and (iii) 9.72 ng/ml. In DAS-ELISA-mAb-bmAb, the LOD for VP40 protein spiked in serum samples of human was found to be 71.25 ng/ml whereas for DAS-ELISA-mAb-bsAb it was 6.28 ng/ml when the serum sample was two-fold diluted. DAS-ELISA-mAb-bsAb showed higher sensitivity (10 times) to detect VP40 in spiked human serum samples compared to DAS-ELISA-mAb-bmAb. A student t-test was performed for the statistical analysis. In conclusion, the formats of DAS-ELISA developed have specificity and sensitivity to detect VP40 protein in nanogram levels. DAS-ELISA-mAb-bsAb showed the highest sensitivity among the three different formats. The developed assays has the potential to be used in clinical settings for screening suspected individuals in the early stage of the infection.

• Subjects / Keywords

  • Immunoassay
  • Ebola virus
  • ELISA

• Graduation date

  Fall 2016

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/R3X63BM14

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.