Article
Recommended Immunological Assays to Screen for Ricin-Containing Samples Stéphanie Simon 1 , Sylvia Worbs 2 , Marc-André Avondet 3 , Dobryan M. Tracz 4 , Julie Dano 1 , Lisa Schmidt 4 , Hervé Volland 1 , Brigitte G. Dorner 2 and Cindi R. Corbett 4,5, * Received: 9 October 2015; Accepted: 4 November 2015; Published: 26 November 2015 Academic Editor: Andreas Rummel 1
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CEA Saclay, Institute of Biology and Technologies of Saclay, Laboratory for Immunoanalytical Researches, Gif-sur-Yvette 91191 cedex, France;
[email protected] (S.S.);
[email protected] (J.D.);
[email protected] (H.V.) Biological Toxins, Centre for Biological Threats and Special Pathogens, Robert Koch Institute, 13353 Berlin, Germany;
[email protected] (S.W.);
[email protected] (B.G.D.) Federal Department of Defence, Civil Protection and Sport—SPIEZ Laboratory, Spiez 3700, Switzerland;
[email protected] Bacteriology & Enteric Diseases Division, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, MB R3E 3R2, Canada;
[email protected] (D.M.T.);
[email protected] (L.S.) Department of Medical Microbiology and Infectious Diseases, University of Manitoba, Winnipeg, MB R3E 0J9, Canada Correspondence:
[email protected]; Tel.: +1-204-784-5929; Fax: +1-204-789-5009
Abstract: Ricin, a toxin from the plant Ricinus communis, is one of the most toxic biological agents known. Due to its availability, toxicity, ease of production and absence of curative treatments, ricin has been classified by the Centers for Disease Control and Prevention (CDC) as category B biological weapon and it is scheduled as a List 1 compound in the Chemical Weapons Convention. An international proficiency test (PT) was conducted to evaluate detection and quantification capabilities of 17 expert laboratories. In this exercise one goal was to analyse the laboratories’ capacity to detect and differentiate ricin and the less toxic, but highly homologuous protein R. communis agglutinin (RCA120). Six analytical strategies are presented in this paper based on immunological assays (four immunoenzymatic assays and two immunochromatographic tests). Using these immunological methods “dangerous” samples containing ricin and/or RCA120 were successfully identified. Based on different antibodies used the detection and quantification of ricin and RCA120 was successful. The ricin PT highlighted the performance of different immunological approaches that are exemplarily recommended for highly sensitive and precise quantification of ricin. Keywords: ricin; immunological detection; enzyme linked immunosorbent assay; lateral flow assay; proficiency test
1. Introduction Ricin is a highly toxic protein from the seeds of the castor oil plant (Ricinus communis) which has a worldwide distribution, growing naturally across tropical and subtropical regions and is often used as an ornamental plant. Castor beans are used for industrial production of castor oil for hydraulic fluids, paints, and other products [1]. Ricin protein accounts for approximately 1%–5% of an individual castor bean’s weight, which also contains a less toxic but highly homologous protein R. communis agglutinin abbreviated RCA120 [2]. Due to the wide availability of castor beans, the straight-forward
Toxins 2015, 7, 4967–4986; doi:10.3390/toxins7124858
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Toxins 2015, 7, 4967–4986
extraction process to obtain ricin protein, and its high toxicity for potential use as a bioweapon, ricin is classified by the CDC as a Category B select agent [3]. Furthermore, since the toxin has been explored for potential military use by different nations during World War II and later, ricin is the only proteotoxin listed by the Organization for the Prohibition of Chemical Weapons as a controlled chemical under Schedule 1 compounds [4], which prevents the unlawful production, possession, and transfer of ricin toxin. Historically, ricin has been used in previous criminal and bioterrorism attacks (reviewed by Bozza et al., 2015), most notably in the assassination of Bulgarian dissident Georgi Markov in 1978 and mail letter attacks in the United States in 2003 and 2013 [5]. Ricin is an A-B toxin (~60 kDa), consisting of A and B subunits linked by a single disulfide bond. While the B-chain is responsible for binding to different oligosaccharide residues on the cell surface, the ricin A-chain acts as a type 2 ribosome-inactivating protein, depurinating a single adenosine residue in the 28S ribosomal RNA. This adenosine prevents binding of the elongation factor and inhibits protein synthesis, resulting in cell death [6,7]. In contrast to ricin, RCA120 consists of a dimer of two associated ricin-like molecules, each of which contains A- and B-chains (~32 kDa and ~36 kDa) resulting in a ~120 kDa protein [8]; in one publication, a disulphide bond between the two A-chains of RCA120 has been shown by X-ray crystallography [9]. Detection of ricin is complicated by the fact that the A- and B-chains or ricin and RCA120 show a high degree of homology of 93% and 84% on amino acid levels, respectively [10]. Following human exposure via ingestion, injection, or inhalation, the toxic effects of ricin may include nausea, vomiting, dehydration, respiratory failure, and circulatory collapse [1]. Clinical cases of ricin poisoning via ingestion of castor beans occur accidentally or in suicide attempts [11–13]. Studies on the toxicity of ricin have been previously reviewed [1,11], and measurements can vary greatly depending on the type of toxicological assessment. The least toxic route of ricin poisoning is via ingestion, with an estimated the lethal dose (LD50 ) of 1 to 20 mg/kg of human body weight [2]. There are cases of self-inflicted ricin toxin injection [14], and the LD50 from animal models is 5–10 µg/kg in mice [15] and 3–5 µg/kg in rats [16]. Inhalational ricin toxic effects have an estimated LD50 of 3–5 µg/kg [17]. The detection of the ricin protein depends on immunological, mass spectrometry, or functional activity assays [18–21]. There are many advantages to using immunological-based diagnostic tests for ricin, which have previously demonstrated specificity and sensitivity for ricin toxin. For example, conventional enzyme-linked immunosorbent assay (ELISA)-based methods for detecting ricin toxin have previously been reported with sensitive lower limits of detection (LOD), including: Griffiths et al., (1986), LOD = 20 ng/mL; Leith et al., (1988), LOD = 0.2 ng/mL; Poli et al., (1994), LOD = 0.1 ng/mL; Alderton and Paddle (1997), LOD = 0.01 ng/mL; Shyu et al., (2002a), LOD = 1 ng/mL; and Pauly et al., (2009), LOD = 0.002 ng/mL [22–27]. Field-deployable diagnostic tests, such as hand-held lateral flow assay devices (LFA) are another effective immunoassay option for ricin detection, with reported lower limits of detection of 10 ng/mL [28] and 14 ng/mL [29]. Alternative immunoassays for ricin detection include liquid microsphere-based arrays [27], colloidal gold particles [30] and electrochemiluminescence [31]. While the above mentioned immunological methods successfully distinguish ricin from other lectins, the high sequence homology between ricin and RCA120 poses a challenge to immunological methods. Herein, we describe the immunological detection of ricin and/or RCA120 within four international laboratories during the 2013 EQuATox ricin proficiency panel. For background information on the EQuATox project please see Worbs et al. [32]. At the outset of the EQuATox program there was a lack of an internationally accepted ricin toxin standard for analytical detection and identification of ricin within unknown samples. This study represents an important milestone for the acceptance of an international standard material for ricin toxin, as it would enable direct comparison of multiple laboratory approaches for ricin detection and identification. The EQuATox 2013 international proficiency panel consisted of nine blinded samples, containing 1.2 mL liquid samples (S1–S8) which were spiked with ricin toxin and/or RCA120 at various concentrations.
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Additionally, one solid sample (S9) was distributed that contained a naturally contaminated organic fertilizer containing R. communis shred. The task was to analyse the nine samples qualitatively and/or quantitatively with respect to the content of ricin, RCA120 or both related lectins (see Worbs et al. [32], this issue of Toxins). Both potential analytes were announced at the beginning of the exercise and the choice of methodology to work on this task was free. Additionally, the participants were asked to provide the results of two independent measurements. Considering the restricted volume of sample provided, the laboratories had to carefully plan their analysis strategy. In this report, we assess the capacity of four out of seventeen participant laboratories to detect, differentiate, and quantify ricin and RCA120 in these proficiency panel samples with immunological assays. The overall proficiency panel test results are discussed in Worbs et al. [32], in this issue of Toxins. 2. Results and Discussion 2.1. Overview of Individual Participant Laboratory Assay Validation Studies Before the PT, each of the immunological approaches applied by the four laboratories underwent Toxins 2015, 7 an independent, critical validation within the different laboratories addressing similar,4 but not identical topics. The scope of validation and procedures varied including the use of different ricin topics. The standard scope of validation and procedures varied the use validation of different ricin (and/or (and/or RCA120) materials. The results of including the in-house studies are briefly RCA120) standard materials. The results of the in-house validation studies are briefly summarized in summarized in Figure 1. Please note the numbering of ELISA- and LFA-formats follows the Figure 1. Please note the numbering of ELISA- and LFA-formats follows the numbering in Worbs et al., numbering in Worbs et al., (2015) on ricin PT results this issue of Toxins [32]. (2015) on ricin PT results this issue of Toxins [32].
Figure 1.1.Cont. Figure Cont.
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1. Overview of individual participant laboratory validationstudies studies for Figure 1. Figure Overview of individual participant laboratory validation for different different ELISAs detecting ricin and/or (a) totypical (d) display typical curves standardofcurves four detecting ELISAs ricin and/or RCA120 (a) toRCA120 (d) display standard four of different ELISA different ELISA usedparticipating in the ricin PTinstitutions by four participating based on different used in the ricin PT by four based oninstitutions different antibodies as described in antibodiesSection; as described in the Experimental Section. (e) and (f) display curves the Experimental (e) and (f) display standard curves of two ELISAstandard developed for RCA120 of two ELISA developed for RCA120 detection. The absorption is plotted against concentration detection. The absorption is plotted against concentration of the lectin. Indicated below the pictures Indicated below the pictures are resultsbyofthe thefour individual validation are resultsofofthe thelectin. individual validation studies performed institutions priorstudies to the ricin PT. performed by the four institutions prior to the ricin PT. LOD = limit of LOD = limit of detection; ULOQ = upper limit of detection; LLOQ = lower limit detection; of detection; SD = ULOQ = upper of detection; LLOQ = lower limit of detection; SD = standard deviation; standard deviation; n = limit sample size. n = sample size.
2.1.1. ELISA 2.1.1.3 ELISA 3 ELISA ELISA 3, developed at Public Health Agency of Canada—National Microbiology Laboratory 3, developed at Public Health Agency of Canada—National Microbiology Laboratory (Canada),(Canada), is an antigen-capture ELISA using monoclonal antibodies (RAC18 and is an antigen-capture sandwich-type sandwich-type ELISA using monoclonal antibodies (RAC18 and RBC11) RBC11) that detectboth both AB and B chains of ricin (Figure 1a). This ELISA validated to that detect A and chains of ricin toxin [33] toxin (Figure[33] 1a). This ELISA is validated to ISOis17025 ISO 17025standards standards an accredited test in the NationalLaboratory Microbiology Laboratory as an as accredited test in the National Microbiology (NML, Canada) quality(NML, system. Canada) quality system. A validation thoroughstudy validation study assessedcharacteristics assay performance A thorough assessed assay performance by testing characteristics accuracy, inter- andby testing assay precision, linearity, limits of detection and quantitation, diagnostic and sensitivity accuracy,intrainterand intra- dilutional assay precision, dilutional linearity, limits ofand detection quantitation, and specificity [34]. Accuracy and precision wereAccuracy assessed byand testing internal quality (IQC) and diagnostic sensitivity and specificity [34]. precision were control assessed by testing standards prepared at known ricin toxin concentrations (using ricin toxin standard prepared in 2009 at internal quality control (IQC) standards prepared at known ricin toxin concentrations (using ricin RKI, Germany). Datainfrom 23 individual ELISA plates were final data set, which was toxin standard prepared 2009 at RKI, Germany). Dataincluded from in 23theindividual ELISA plates were performed by multiple operators on separate days (see Methods for full description of validation study included in the final data set, which was performed by multiple operators on separate days (see methodology). The lower limit of detection (LOD) is 0.284 ng/mL of ricin toxin, the empirical lower Methods for full description of validation study methodology). The lower limit of detection (LOD) is limit of quantitation is 0.464 ng/mL, and the upper limit of quantitation is 9.5 ng/mL (n = 252) [34]. 0.284 ng/mL of ricin toxin, the empirical lower limit of quantitation is 0.464 ng/mL, and the upper During the validation study, ELISA performance characteristics also demonstrated acceptable levels of limit of quantitation is 9.5 (CV% ng/mL (n =n252) [34]. During the error, validation study, ELISA performance intra-assay precision ≤ 25%, = 105), accuracy (≤25% n = 105), dilutional linearity characteristics alsoslope demonstrated acceptable precision (CV% ď 25%, n = 105), (r2 = 0.936, = 1.062, intercept = −0.09, levels n = 168),ofasintra-assay well as diagnostic sensitivity and specificity 2 = 0.936, slope = 1.062, intercept = ´0.09, accuracy (100%) (ď25%forerror, n = 105), dilutional linearity (r selected plant seed samples, including rosary peas and other seeds. n = 168), as well as diagnostic sensitivity and specificity (100%) for selected plant seed samples, including rosary peas and other seeds. 2.1.2. ELISA 6 ELISA 6 developed at CEA Saclay (France) is a sandwich-type ELISA using monoclonal antibodies (RB37 and RA36) against ricin toxin [35] (Figure 1b). The lower limit of detection of this immunoassay is 12 pg/mL and the cross reactivity with RCA120 is 0.1% (calculated as the percentage of ricin concentration/RCA120 concentration giving the same absorbance in the linear range of the standard curve, 0.5 AU). No cross reaction were observed with other lectins (24 lectins have been tested). The intra-assay coefficients of variation were determined by assaying five times on the same day five different ricin concentrations (30, 50, 100, 300 and 1500 pg/mL). Inter-assay coefficients were
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determined by repeating this experiment on six different days. Intra-assay coefficients of variation (CV%) were lower than 10% and the inter-assay coefficients variation were below 15% for all the concentrations. As both CVs were below 20% for 30 pg/mL we considered this concentration as the limit of quantification. The reference material used for these experiments was an in-house purified ricin calibrated using its absorbance. 2.1.3. LFA 4 Additionally, CEA Saclay developed an LFA test which is available commercially by NBC-sys (Saint Chamond, France). The test has a limit of detection of 1 ng/mL (using an in-house reference material), with a cross-reactivity for RCA120 of 0.1% (calculated as the percentage of LOD for ricin/LOD for RCA120) and an assay running time of 15 min. 2.1.4. ELISA 4 The Robert Koch Institute, Germany, has developed two different sandwich-ELISA, one preferentially detecting ricin with only little cross-reactivity to RCA120 (Figure 1c) and the other preferentially detecting RCA120 with low cross-reactivity to ricin (Figure 1e; for graphical representation of cross-reactivities please refer to Worbs et al. [32]. The ricin-specific ELISA is based on a combination of two monoclonal antibodies and detects both chains of ricin (anti-ricin B chain: mAb R109 and anti-ricin A chain: mAb R18; [27]), while the RCA120-specific ELISA combines a monoclonal with a polyclonal chicken antibody (mAb ARK4 and chicken IgY RC22) [36,37]. In a validation study for the ricin-specific ELISA, the half maximal effective concentration (EC50 ) of the ELISA as point of highest precision with respect to quantification was determined at 115 ˘ 32 pg/mL with a limit of detection at 2 pg/mL. The working range of the ricin-specific ELISA as the range in which obtained results have a precision of
