Capacity building permitting comprehensive monitoring of a severe case of Lassa hemorrhagic fever in Sierra Leone with a positive outcome. Part 2
Human Subjects: Suspected LF patients, close contacts, and healthy volunteers were eligible to participate in these studies as outlined in Tulane University’s Institutional Review Board (IRB) protocol for this project, National Institutes of Health/National Institutes of Allergy and Infectious Diseases guidelines governing the use of human subject for research, and Department of Health and Human Services/National Institutes of Health/National Institute of Allergy and Infectious Diseases Challenge and Partnership Grant Numbers AI067188 and AI082119. This project was approved by the Tulane University IRB. Adult patients in this manuscript have given written informed consent for the publication of their case details. Written informed consent was obtained from the adult guardian of patient G-1180 for publication of this case report.
Normal and positive control sera: Serum from one Sierra Leonean and two Caucasian American volunteers were used in these studies as normal controls. A serum sample collected from a 20-year-old pregnant woman who succumbed to Lassa fever in the KGH Maternity Ward on August 29, 2010 was used as a positive control. A single serum sample was collected from this subject before expiration, and assigned the coded designation G-1177.
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Detection of Lassa virus antigen and Lassa virus-specific antibodies: Serum levels of Lassa nucleoprotein (NP)-specific antigen were first determined using LFI modules currently under pre-clinical development by Corgenix Medical Corp., Broomfield, CO, U.S.A., and the Lassa fever consortium (see acknowledgements). The LFI modules utilize LASV NP specific murine monoclonal antibodies in a capture line and gold-conjugated detection reagent. Downstream of the capture line is an anti-murine polyclonal control line. Serum samples are added to a sample well followed by buffer solution to initiate lateral flow through the reagent pads and across the capture and control lines. The formation of Lassa NP antigen immune-complexes by the reagents produces a red signal due to gold conjugate deposition, allowing visual interpretation or measurement by reflectance. The red signal can be seen within 3-5 minutes with full signal development between 15-25 minutes. The positive Lassa fever diagnosis is then confirmed with a sensitive antigen-capture ELISA employing either a murine monoclonal or caprine polyclonal capture antibody, followed by a peroxidase-labeled caprine reagent and tetramethylbenzidine (TMB) substrate. A standard curve was generated with recombinant LASV NP in order to quantify serum levels of virus-associated NP.
IgM and IgG levels to recombinant LASV proteins (NP alone and NP – glycoprotein 1 (GP1) – glycoprotein complex (GPC) combination) ELISA: Individual LASV proteins and combinations optimized for detection of virus-specific IgM and IgG levels in serum were coated in stripwell plates, blocked, dried, and packaged with desiccating packs (Corgenix Medical Corp.). For analysis, sera were diluted 1:100 in a proprietary sample dilution buffer and incubated in wells for 30 minutes at room temperature, washed, and incubated with optimized HRP-labeled anti-human IgG or IgM conjugates for an additional 30 minutes. After washing, detection was performed with TMB substrate for 10 minutes at room temperature, stopped with sulphuric acid, and read at A450 in a BioTek ELISA plate reader.