Abstract
This study presents data showing the performance of 6 commercial detection assays against ricin around concentrations specified as detection limits by the producers. A 2-fold dilution series of 20 ng/ml ricin was prepared and used for testing the lateral-flow kits: BADD, Pro Strips™, ENVI, RAID DX, Ricin BioThreat Alert, and IMASS™ device. Three of the 6 tested field assays (IMASS™ device, ENVI assay, and the BioThreat Alert assay) were able to detect ricin, although differences in the measured detection limits compared to the official detection limits and false-negative results were observed. We were not able to get the BADD, Pro Strips™, and RAID assays to function in our laboratory. We conclude that when purchasing a field responder assay, there is large variation in the specificity of the assays, and a number of in-house tests must be performed to ensure functionality.
This study presents data showing the performance of commercial detection assays against ricin around concentrations specified as detection limits by the producers. Three of the 6 tested field assays were able to detect ricin, although differences in the measured detection limits compared to the official detection limits and false-negative results were observed. The authors conclude that, when purchasing a field responder assay, there is large variation in the specificity of the assays, and a number of in-house tests must be performed to ensure functionality.
I
It has been proposed that national biological laboratories be set up to monitor an assay evaluation program in the field of bioterror agents, but this has not yet been implemented.1,3 Until an international quality system for biodetection technologies of first responder assays has been introduced, researchers dealing with bioterrorism will have to rely on specific scientific publications for evaluating various commercial assays.
In recent years, some commercial first responder kits and assays for use in toxin detection have been scientifically tested and evaluated.3-6 In a previous study by Slotved et al, 4 first responder assays were tested for their sensitivity for botulinum toxins. The study showed that several of the assays malfunctioned. It was therefore decided to test additional commercially available first line ricin toxin kits.
Ricin is a toxic protein consisting of 2 subunits called the A- and B-chains. 7 It is highly toxic to humans, and the LD50 is estimated to be 5-10 μg/kg body weight when inhaled and 1-20 mg/kg when ingested. 6 The toxin is a known bioterror threat, which is why these kits have the important function of either confirming or rejecting possible attacks with ricin. 8
Our goal in this study was to see whether commercial lateral flow tests were easy to use and able to detect pour ricin at the detection limit provided by the producers. Six commercial quick tests representing the majority of available test kits based on the lateral flow test system were selected. The study represents the first data showing the performance of 6 different commercial ricin detection assays using pure ricin as a test substance including data on detection limits and handling procedures.
Material and Methods
Reagents, Standard Ricin Solution
Ricinus communis (RCA 60), kindly provided by the Robert Koch Institut (stock solution 1.02 mg/ml), was diluted in phosphate-buffered saline (PBS: 82 mM NaCl, 43 mM Na2HPO4, 9.8 mM KH2PO4) to a concentration of 100 ng/ml.
A 2-fold dilution series of 20 ng/ml ricin to 0.04 ng/ml was prepared and used for testing the 6 lateral flow kits: BADD, Pro Strips™, ENVI, RAID DX, Ricin BioThreat Alert, and IMASS™ device. All the mentioned ricin concentrations used in this study constitute the ricin concentrations before starting the test procedure described in each of the 6 assay protocols.
Commercial Test Kits Evaluated
Six different test kits based on the lateral flow technique were evaluated. All of the tests are mentioned in the report by Ozanich et al. 2 The number of test performed with each assay was limited due to available tests purchased and the test results, in that this study only evaluated the test kits to see how they were to handle and perform against purified toxin.
The BADD Ricin Toxin test is a lateral flow test designed to detect ricin. Further information can be found on the company's homepage (BADD [Biowarfare Agent Detection Devices], Advnt Biotechnologies): www.advnt.org/products/biowarfare/baddbox/. Detection limit: 5 ng/ml.
The Pro Strips Rapid Screening System Multi-Agent BioWarfare Threat Detection Kit is described by the company instruction as a 1-device, 1-sample multiple threat–recognition kit. Pro Strips™ detects anthrax, ricin toxin, botulinum toxin, Y. pestis (plague), and Staphylococcal enterotoxin B (SEB). For further information, see the Pro Strips™ (Advnt Biotechnologies) website: www.advnt.org/products/biowarfare/prostrips/. Detection limit: 5 ng/ml.
The ENVI Assay System is an immunochromatographic rapid test. As described in the company instruction, the kit contains a disposable rapid test for screening environmental samples for anthrax, ricin toxin, SEB, smallpox, and botulinum toxin. For further information, see ENVI Assay System and ChemPro Reader Module (Environics Oy): www.swanenviron.com/securitytec.html. Detection limit: 5 ng/ml.
The RAID DX kit contains a RAID 8 test and a RAID TOX test. The RAID 8 test detects anthrax, ricin toxin, botulinum toxin, SEB, plague, brucella, tularemia, and orthopox. The RAID TOX test detects ricin toxin, botulinum toxin, and SEB and has a positive and a negative control. The assay is used as a follow-up test to evaluate whether there is a “hook effect” due to oversampling. For further information, see Alexeter Technologies, LLC©: www.alexeter.com/biow/index.asp. Detection limit: 6 ng/ml.
Ricin BioThreat Alert® Kit TC-8008-025 is a lateral flow test that detects ricin (can be read visually) using a BioThreat Alert® Reader. For further information, see Tetracore, Inc.: http://www.tetracore.com/bio-warfare/. Detection limit: 2-5 ng/ml.
Biothreat identification IMASS™ device is a lateral flow test that detects anthrax, ricin toxin, botulinum toxin A/B, Francisella tularensis, plague, Brucella spp., Burkholderia mallei (glanders), and SEB. For further information, see BBI Detection: http://www.bbidetection.com/products/biothreat-detection-imass-device/. Detection limit: 1 ng/ml.
Results
Results of tests of the first-responder lateral-flow kits are presented in Table 1.
Six Lateral Flow Tests for the Detection of Ricin
One test reacted positively after 30 minutes. However, according to the manufacturer, this has to be considered a false-positive reaction.
Positive after direct application and negative with the swab application provided by the producer.
Two RAID 8 and 2 RAID TOX tests were tested.
The BADD ricin kit was tested 3 times with a 20-ng/ml ricin solution with negative results. Because of the lack of positive responses, despite an alleged detection limit of 5 ng/ml ricin, it was decided not to perform further tests with this assay.
The Pro Strips™ test was tested 5 times with ricin (20 ng/ml) with negative results within 15 minutes after sample application. After 30 minutes, however, 1 test showed a positive reaction. Three of the 5 tests showed malfunction in that adding drops to the sample wells did not result in a sample flow that was able to reach all test windows. The detection limit of Pro Strips™ is stated to be 5 ng/ml for ricin, and according to the manufacturer, the kits are to be read at 15 minutes and are not supposed to be read after 30 minutes It was decided not to perform further tests.
The ENVI Assay System was tested using 6 kits with ricin concentrations at 20 ng/ml, 10 ng/ml, and 5 ng/ml. The tested kits (2 kits) showed positive reactions against 20 ng/ml ricin when adding the suspension directly and by using the attached swab (provided by the producer). At 10 ng/ml, the kit was positive after direct application, while the second kit with the swab application was negative. At 5 ng/ml, both kits showed negative signals. The detection limit of the ENVI Assay System is stated to be 5 ng/ml.
Two RAID 8 and 2 RAID TOX tests were each tested with ricin (20 ng/ml) with a negative response. In 1 of the RAID 8 tests, a false-positive reaction for orthopox was observed when testing with ricin. The detection limit of the RAID 8 test is stated to be 6 ng/ml. It was decided not to perform further tests.
Ricin BioThreat Alert was tested against ricin with 5 tests per concentration (20 ng/ml, 10 ng/ml, 5 ng/ml, 2.5 ng/ml, and 1.25 ng/ml). The tests are meant to be read in an optical reader, but in this study the kits were read both visually and by using the recommended reader. The kit reacted with positive results both visually and with the reader for 20 ng/ml for all 5 strips tested, although in 1 the reader first showed a negative response. Because the visual response was positive, the strip was retested in the reader, and this time a positive response appeared. When testing 5 kits with 10 ng/ml, 3 were found positive with the reader, and of these 3, 2 were found positive under visual inspection. Three kits were tested with 5 ng/ml ricin, and all 3 tested positive with the reader, while visual inspection was negative. Kits tested with 2.5 ng/ml and 1.25 ng/ml all showed negative results. The detection limit is 2-5 ng/ml of ricin according to the manufacturer.
The IMASS™ device was tested against ricin (20 ng/ml, 10 ng/ml, 5 ng/ml, 2.5 ng/ml, 1.25 ng/ml, and 0.625 ng/ml). Two test kits showed positive responses for 20 ng/ml, and another 2 showed positive responses for 10 ng/ml. Testing 5 ng/ml, 2.5 ng/ml, 1.25 ng/ml, and 0.625 ng/ml showed negative responses. The detection limit is 1 ng/ml of ricin according to the manufacturer.
Discussion
There are several options for identifying first responder assays for detection of toxins, such as directly searching the internet or using public reports, 2 which have listed biodetection technologies for first responders. However, published studies presenting data on the performance of first responder assays are scarce, and reports only present the assays and refer to available scientific studies, but do not evaluate the assays.
In the present study, 6 commercially available first responder assays based on the lateral flow technique were tested for their possible use in the detection of ricin. According to Ozanich et al 2 (except for the previous study by the authors 4 ) and a literature search performed by the authors, it is possible to find peer-reviewed articles testing and evaluating only 2 of the 6 commercial lateral flow techniques. Only the BADD (botulinum toxin) 9 and the BioThreat Alert assays (ricin,6,10 botulinum toxin, 10 SEB, 10 plague, 10 anthrax, 10 and abrin 11 ) have previously been evaluated in studies. In the study by Slotved et al, 4 the BADD, Pro Strips™, the ENVI Assay System, and RAID 8 and 2 RAID TOX tests were tested for botulinum toxin detection and found not applicable for first responder use because of several observed functional failures and lack of positive responses against toxins above the stated detection limits.
Of the 6 assays, only 1—the BioThreat Alert assay—has previously been evaluated for ricin detection.6,10 Both studies found this assay to be reliable as a screening assay in the field and laboratory. In our test (Table 1) and evaluation of the 6 assays, we found that all of the assays showed some failures in ricin detection. With 3 of the assays (BADD, Pro Strips™, and RAID assay), we were not able to achieve a reliable response for ricin with the tested concentration. With the IMASS™ device and the ENVI assay, we were able to achieve a response although the detection limit was found to be higher than stated by the producers. With the BioThreat Alert assay and reader, we were able to detect ricin at the detection limit stated by the producer; however, false-negative tests were also observed. In humans, the lethal ricin concentration (LD50) has been estimated to be 5-10 μg/kg body weight via inhalation and 1-20 mg/kg via ingestion. 6 This may roughly be translated into a concentration of 5-10 ng/ml by inhalation and 1,000-20,000 ng/ml by ingestion. Based on these numbers, this study finds that the IMASS™ device, the ENVI assay, and the BioThreat Alert assay all may be used as possible first responder assays in the field for ricin detection.
The assays are described by the companies as easy to use; however, the experience from this study was that, for all the assays, in-house test training and evaluations have to be performed because of the differences between the company specifications and what is observed in the laboratory, particularly since the stated detection level for the field assays provided by the companies is often not a level that can be achieved in the laboratory (Table 1). Therefore, an additional cost for in-house testing has to be taken into consideration when evaluating these field responder assays. However, it also has to be mentioned that if peer-reviewed articles are available, as is the case with the extensive data on the BioThreat Alert assay,6,10 the number of in-house tests with, for example, spiked samples can be reduced.
A limitation of this study is that its only purpose was to perform a first-time evaluation of the test kits to see how easy they were to handle and how they performed against purified toxin. The authors were, therefore, well aware that further tests are needed for in-house adjustment, before the assays can be used in routine procedures.
In conclusion, this study found that only 3 of the 6 tested field assays (IMASS™ device, ENVI assay, and the BioThreat Alert assay) were able to detect ricin at levels close to the detection limit, although differences in detection limits and the presence of some false-negative results were observed. We were not able to get the BADD, Pro Strips™, and RAID assays to function in our laboratory. Consequently, when purchasing a field responder assay, it has to be expected that in-house evaluation tests will have to be performed before its introduction to the first responder toolbox. We further recommend that duplicate or triplicate test standard procedures always be performed under field conditions when using a field responder assay because of possible failures of the assays.