Evaluation of a False Positive Heartworm Test Using Mass Spectrometry Based Proteomics

Abstract

Zoetis' WITNESS HW Test is intended to detect the presence of canine heartworm antigens in a simple immunomigration test cassette. The WITNESS HW Test displays one or two colored lines to indicate negative or positive results, respectively, when testing blood, plasma, or serum. Within Zoetis, an indiscriminate number of tests were shown to be false positive by alternative means. Zoetis contracted the Southwest Michigan Innovation Center (SMIC) to use analytical tools including mass spectrometry to identify the false positive agent. In one previous study done by SMIC, the false positive colored line was analyzed by digesting with trypsin and analyzing the resulting peptides by mass spectrometry. The present study extended this previous study by using alternative digest conditions, making and using full-length antibody and Fc fragment coupled beads, and using a high abundant serum protein removal column in combination with the above procedures. Prior to the use of high abundant protein removal, the results were plagued with identification of only high concentration serum proteins regardless of sample type tested. Following separation of false positive serum using the high abundant removal column, the flow-through fraction and the eluted high abundant protein fraction were tested on the WITNESS HW Test. It was discovered that the false positive agent was in the flow-through fraction upon extended incubation with the WITNESS HW Test. Immunoprecipitation using Fc fragment coupled beads was found to be ineffective at false positive agent enrichment. Components identified in the false positive and absent in the negative sample will be considered as potential false positive agents. Additional experiments will be carried out to validate the causative agent and prevent false positive results on the WITNESS HW Test.

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