The ReEBOV RDT was also cross-reactive to Sudan and Bundibugyo EBOV species at 1.0 103C1.0 104 PFU/mL which equated to 2.2 109C2.5 1010 GEq/mL from in vitro viral stocks (Table ?(Table4).4). tested with 5 replicates. Test strips were incubated a minimum of 15 minutes before visual scoring. The preliminary LOD was considered the lowest dilution in which all 5 test strips were positive by visual interpretation. Verification of the preliminary LOD was performed by testing 20 replicates of diluted rVP40 or Zaire EBOV at and near the preliminary LOD dilution levels. Repeatability Thirteen volunteers donated venous whole blood collected in ethylenediaminetetraacetic acid (EDTA)Clined vacutainers (BD Medicine). Randomized whole-blood samples were spiked with rVP40 antigen at a LOD of 9.4 ng/test and a 2-fold increases series up to 600 ng/test. Extra samples were spiked with rVP40 at the LOD and 2 times the LOD. A total of 75 replicates of unspiked whole blood were tested for assay specificity. All samples were blinded and tested according to instructions in the package insert. After incubation for 15 minutes, each dipstick was evaluated by 3 professionals. Scores were tabulated based on agreement of 3 professionals. Interfering Substances Interfering substances screening was conducted in accordance with Clinical Laboratory Standards Institute approved guideline EP7-A2 . The rVP40 antigen was diluted in normal control serum at a concentration near the LOD of 0.2 g/mL (6.0 ng/test). Substances and their concentrations to be tested were found in EP7-A2, appendix C or D . For substances not listed, the equivalent of a 20X stock for the normal adult dose diluted in 5 L (blood volume) was prepared. Prior to testing, the 20X material stock was diluted in an aliquot of the rVP40 sample and normal control serum without antigen. Each material and solvent control was tested in 5 replicates. Additional levels of select substances were also requested by the FDA. A material was considered noninterfering if the antigen-spiked median score did not differ from that of the solvent control or if it did not generate a false-positive signal in normal control serum. Specificity and Sensitivity To evaluate the specificity of the ReEBOV RDT, normal US serum panels developed from commercial donor serum were used (Plasma Services Group, Huntingdon Valley, Pennsylvania). A total of 4 panels representing 138 individual serum donors were tested. Each sample was tested according to its package insert and scored individually. During fresh donor blood collection, 20 healthy US volunteers consented to finger-stick screening. Each volunteer provided duplicate finger sticks. One full drop (approximately 30 L) was allowed to develop around the finger and then was transferred directly to the plasma separation sample pad by touching the dipstick directly to the blood drop. This was repeated to collect a second sample from each volunteer. Dipsticks were immediately inserted into test tubes containing sample buffer to initiate development of ReEBOV RDT. After incubation for 15 minutes, results were scored blinded by a second technician. Thirteen donor whole-blood samples were collected in EDTA-lined vacutainers for ReEBOV RDT specificity and contrived sensitivity testing in a blinded, randomized design. Six Cinchonine (LA40221) replicates per whole-blood sample were evaluated independently by 3 professionals, yielding 234 Cinchonine (LA40221) observations for the specificity determination. Randomized whole-blood samples were spiked with rVP40 antigen at the whole-blood LOD of 9.4 ng/test and at 2-fold serial dilutions up to 600 ng/test. A minimum of 6 random spiked samples at each level were evaluated by 3 professionals, for a minimum of 18 observations per level. Extra samples were spiked with rVP40 at (117 observations) and 2-fold above (90 observations) the whole-blood LOD to verify the reproducibility of the previously described analytical LOD. A total of 303 observations were made with spiked whole-blood samples. All samples were blinded and tested according to guidance in the package insert. After incubation for 15 minutes, each dipstick was evaluated independently Dig2 by 3 professionals. Sixteen donor serum samples were collected for ReEBOV RDT specificity and contrived sensitivity testing in a blinded, randomized design. Six replicates per serum sample were Cinchonine (LA40221) tested and evaluated independently by 3 professionals, yielding 249 observations for the specificity determination. Randomized serum samples were.