A Recombinant Positive Control for Serology Diagnostic Tests Supporting Elimination of Onchocerca volvulus.

Serological assays for human IgG4 to the Onchocerca volvulus antigen Ov16 have been used to confirm elimination of onchocerciasis in much of the Americas and parts of Africa. A standardized source of positive control antibody (human anti-Ov16 IgG4) will ensure the quality of surveillance data using these tests.A recombinant human IgG4 antibody to Ov16 was identified by screening against a synthetic human Fab phage display library and converted into human IgG4. This antibody was developed into different positive control formulations for enzyme-linked immunosorbent assay (ELISA) and rapid diagnostic test (RDT) platforms. Variation in ELISA results and utility as a positive control of the antibody were assessed from multiple laboratories. Temperature and humidity conditions were collected across seven surveillance activities from 2011-2014 to inform stability requirements for RDTs and positive controls. The feasibility of the dried positive control for RDT was evaluated during onchocerciasis surveillance activity in Togo, in 2014. When the anti-Ov16 IgG4 antibody was used as a standard dilution in horseradish peroxidase (HRP) and alkaline phosphatase (AP) ELISAs, the detection limits were approximately 1ng/mL by HRP ELISA and 10ng/mL by AP ELISA. Positive control dilutions and spiked dried blood spots (DBS) produced similar ELISA results. Used as a simple plate normalization control, the positive control antibody may improve ELISA data comparison in the context of inter-laboratory variation. The aggregate temperature and humidity monitor data informed temperature parameters under which the dried positive control was tested and are applicable inputs for testing of diagnostics tools intended for sub-Saharan Africa. As a packaged positive control for Ov16 RDTs, stability of the antibody was demonstrated for over six months at relevant temperatures in the laboratory and for over 15 weeks under field conditions.The recombinant human anti-Ov16 IgG4 antibody-based positive control will benefit inter-laboratory validation of ELISA assays and serve as quality control (QC) reagents for Ov16 RDTs at different points of the supply chain from manufacturer to field use

Lateral flow test strip scanned images showing test line (‘T’, left) and control line (‘C’, right) results after using as samples the dried anti-Ov16 IgG4-positive control subjected to elevated temperature stress over time.

<p>Time zero shown indicates intensity of the starting signal prior to storage at 25°C, 45°C, and variable cycling temperature (20°C to 40°C, daily) for up to 19 months.</p

Concentration-dependent absorbance values of the ELISA platform for the anti-Ov16 human IgG4 serological assay.

<p>(A) Absorbance units versus concentration of recombinant anti-Ov16 IgG4-positive control antibody. (B) HRP ELISA and AP ELISA absorbance values from both plasma/serum and DBS samples derived from identical sources: anti-Ov16 IgG4-positive control-spiked negative human sera, dilutions of an Ov-positive pool from microfilaria-positive patient plasmas and sera, and the average of nine negative plasmas and sera (from donors that have not traveled to an Ov-endemic area).</p

Environmental conditions during surveillance activities in Togo 2013 and 2014.

<p>A. Daily temperature profiles (dark grey lines) in degrees Celsius during surveillance activities. The mean temperature is plotted with the bold red line. B. Daily % relative humidity (dark grey lines) cycles during surveillance activities. The mean relative humidity is plotted with the bold green line. C. Frequency of recorded temperature and relative humidity pairs experienced by monitors attached to microscopes used during the surveillance activities. The color scale represents the frequency of observations represented by intense red (least frequent) to intense yellow (most frequent).</p