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Cite this: DOI: 10.1039/c4an00720d
Competitive immunochromatographic assay for the detection of thiodiglycol sulfoxide, a degradation product of sulfur mustard† Manisha Sathe,* Shruti Srivastava, S. Merwyn, G. S. Agarwal and M. P. Kaushik An immunochromatographic assay (ICA) based on the competitive antigen-coated format using colloidal gold as the label was developed for the detection of thiodiglycol sulfoxide (TDGO), an important metabolite and degradation compound of sulphur mustard (SM). The ICA test strip consisted of a membrane with a detection zone, a sample pad and an absorbent pad. The membrane was separately coated with hapten–OVA conjugate (test line) and anti-rabbit mouse IgG (control line). The visual
Received 23rd April 2014 Accepted 24th July 2014
detection limit for TDGO by ICA detection was found to be 10 mg mL1. For validation, the ICA results obtained for spiked water samples were in good agreement with those obtained by indirect competitive inhibition enzyme-linked immunosorbent assay (ELISA) for TDGO. The assay time for detection was less
DOI: 10.1039/c4an00720d
than 10 min. The developed ICA has the potential to be a useful on-site screening tool for the
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retrospective detection of SM in environmental samples.
Introduction The development, production, stockpiling and use of chemical weapons are prohibited under the Chemical Weapons Convention (CWC).1 The most signicant chemical warfare agents (CWAs) in terms of military capacity and past use are the nerve and blister agents.2 For these reasons, the analysis of these compounds even at trace levels in environmental and biological matrices is of utmost importance. Sulphur mustard (SM) and its analogues such as sesquimustards belong to the blistering class of CWAs, which are classied as schedule 1A in the CWC.3 The blister agent SM remains one of the CW agents of greatest concern due to its advantageous physical properties and ease of production. In the environment, these compounds are susceptible to oxidation. Thiodiglycol (TDG) is the major hydrolytic breakdown product of SM (bis(2-chloroethyl)sulde), which oxidizes gradually in water to its sulfoxide analogue thiodiglycol sulfoxide (TDGO). In the presence of sunlight, further oxidation is then thought to occur, forming thiodiglycol sulfone (TDGO2; Fig. 1). Thus, TDGO and TDGO2 serve as important markers; their identication in any environmental matrix indicates the probable prior presence of SM, which is an important aspect in the verication analysis of CWC related chemicals.4 The conventional analytic methods for SM and its metabolites in biological and environmental samples are based
Process Technology Development Division, Discovery Centre, Defense R & D Establishment, Jhansi Road, Gwalior 474002, India. E-mail: drmanishasathe@drde. drdo.in; Fax: +917512340042; Tel: +917512390462 † Electronic supplementary 10.1039/c4an00720d
information
(ESI)
available.
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on chromatography and mass spectrometry.5–8 Recently, Neelam et al. reported a spectrophotometric technique for the detection of an SM analogue.9 These methods are used for off-site detection and involve high cost, sophistication, and time-consuming sample-preparation procedures. Immunochemical techniques have gained acceptance as a fast and cost-effective tools for separating and/ or detecting trace amounts of chemicals such as pesticides.10–12 However, the labour-intensive operations including repeated incubation and washing along with the enzyme reaction for the nal signal generation in enzyme-linked immunosorbent assay (ELISA) can be difficult to perform in non-specialized laboratories and in the eld. With the demand for speed and simplicity, a lateral-ow immunochromatographic assay (ICA) could be a more suitable alternative13,14 for the onsite detection of small molecular toxicants, their markers and other environmental contaminants. In the last few years, several research groups have demonstrated ICA for some pesticides15–21 and other environmental contaminants.22,23 The main feature of ICA using colloidal gold as the label is its single step assay procedure,
Fig. 1 Hydrolysis of sulfur mustard to produce thiodiglycol (TDG) and further oxidation to thiodiglycol sulfoxide (TDGO) and thiodiglycol sulfone (TDGO2).
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which is contrary to most prevalent types of immunoassay (for example, ELISA involves three to four steps). Another advantage of ICA is that it is easy and convenient to use because detection can be achieved with the naked eye. We are engaged in the development of methods for the retrospective detection of CWAs.24,25 Van der Schans et al. reported the detection of SM protein adducts and metabolites based on immunochemical methods.26 However, only one report in the literature has described the development of ICA for the detection of mustard gas; this study used a skin smear test developed by Securetec Detektions, Germany.27 To the best of our knowledge, ICA for the detection of TDGO has not been studied to date. Herein, in light of the CWC and the environmental concerns regarding abandoned CWAs, we report the development of ICA for the detection of TDGO using an in-housegenerated polyclonal antibody. The developed assay was further tested using spiked samples and validated by indirect competitive inhibition ELISA.
Experimental Reagents and instruments Unless stated otherwise, reagents including bovine serum albumin (crystalline, fraction V; BSA), chicken egg albumin (OVA), horseradish Peroxidase (HRP) and goat antirabbit IgG were obtained from Sigma-Aldrich (India). Solvents used for synthesis were of analytical grade. Reaction progress was monitored by thin-layer chromatography (TLC) using GF254 silica gel on glass plates with uorescent indicator. Chromatography was performed using silica gel (60–120 mesh). PD-10 gel ltration columns (Pharmacia, Sweden) were obtained from GE Healthcare, and 96-well, polystyrene Maxisorb plates were from Nunc (UK). The single beam scanning UV-visible spectrophotometer (Camspec M501) was from Camspec Analytical Instruments Ltd., Leeds UK. The ELISA plate reader used in this study was from Molecular Devices (USA). Electrospray ionization mass spectroscopy (ESI-MS) was performed on a Micromass Q-ToF high-resolution mass spectrometer with a Masslynx 4.0 data acquisition system. Matrix-assisted laser desorption ionization mass spectroscopy (MALDI-MS) data were recorded on an Applied Biosystems MALDI 4700 TOF. 1H-NMR spectra were collected in CDCl3 solutions on a Bruker AVANCE NMR spectrometer operating at 400 MHz (for 1H) using tetramethylsilane (TMS) as an internal standard. Chemical shis are reported in parts per million (ppm) downeld from TMS. Spin multiplicities are described as s (singlet), d (doublet), t (triplet), q (quartet) and m (multiplet). Coupling constants are reported in Hertz (Hz). Synthesis of hapten The haptens used for immunization and antigen coating were fabricated by a multistep synthesis,28 as shown in Scheme 1. Synthesis of TDGO (2)28a In a round-bottomed ask (50 mL) equipped with a magnetic stirrer, a solution of thiodiglycol (2 mmol, 0.244 g) in CH3OH
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Scheme 1 Synthetic scheme for the preparation of TDGO (5) from thiodiglycol.
(15 mL) was prepared. H2O2 (30%, 14 mmol, 1.4 mL) and ZrCl4 (4 mmol, 0.93 g) were added, and the mixture was stirred at room temperature for one hour. The progress of the reaction was monitored by TLC (eluent: 7/3 n-hexane/ethyl acetate). When the starting sulde had completely disappeared, the reaction mixture was quenched by adding water (15 mL) and extracted with chloroform (4 10 mL). The extract was then dried with anhydrous MgSO4. The ltrate was evaporated, and the corresponding thiodiglycol sulfoxide was obtained as a white solid in 65% yield. Synthesis of tert-butyl 2-(2-hydroxyethylsulnyl) ethyl carbonate (3)28b A mixture of 2 (0.8 g, 5.8 mmol), boc anhydride (1.92 g, 8.8 mmol), N,N-diisopropylethylamine (835 mg, 1.12 mL, 6.4 mmol), 4-dimethylaminopyridine (190 mg, 1.4 mmol) and dimethylformamide (DMF; 10 mL) was stirred at 40 C overnight. The DMF was removed in vacuo, and the residue was taken up in ethyl acetate (150 mL). The reaction was washed with brine (2 100 mL), saturated sodium bicarbonate (100 mL) and 0.01 M HCl (100 mL), followed by drying over sodium sulfate. Removal of the organics gave 3 (1.11 g) as an oily solid in 71% yield. Synthesis of 4-(2-(2-hydroxyethylsulnyl) ethoxy)-4oxobutanoic acid (4)28b A solution of 3 (1 g, 4.2 mmol) in pyridine (10 mL) was added dropwise to a stirred solution of succinic anhydride (420 mg, 4.2 mmol) dissolved in pyridine (10 mL). Aer stirring for 3 h at room temperature, 20 mL saturated K2CO3 was added to the reaction mixture and extracted with ethyl acetate. The water layer was acidied by adding 6 mol L1 HCl and extracted with ethyl acetate. The combined organic layer was dried over anhydrous sodium sulfate, ltered and evaporated under reduced pressure to afford 4 as a brown viscous liquid (1.1 g, 52%).
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Synthesis of 4-(2-(2-hydroxyethylsulnyl) ethoxy)-4oxobutanoic acid (TDGO 5)28b A solution of 4 (500 mg, 2 mmol) was dissolved in dichloromethane–triuoroacetic acid (DCM : TFA; 1 : 1; 15 mL) and stirred at 0 C for 2 h. The solvent was evaporated on a rotary evaporator, and diethyl ether was added into the resulting crude product, followed by extraction (2) with water. The aqueous solution was then basied by a saturated solution of sodium bicarbonate and extracted thrice with 15% isopropanol in chloroform, and the combined organic layer was dried over sodium sulphate. Aer the solvent was removed by rotary evaporation, TDGO 5 was afforded in 78% yield (320 mg).
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stirred for 30 min. The precipitated proteins (IgGs) were removed by centrifugation (Sigma 4K15 centrifuge) at 5000 rpm for 10 min at 4 C. The precipitated IgGs were washed twice with 1 : 1 (vol/vol) saturated ammonium sulfate to remove the remaining soluble proteins. The precipitate was then dissolved in 1 mL of 0.1 M PBS at pH 7.2 and desalted by dialysis. The IgGs from the dialysed product were affinity puried using a proteinA column (Ab purication kit-Sigma) as per their instruction manual. The antibody was then further puried by BSAsepharose column-4B to remove the anticarrier (BSA) antibodies. The produced polyclonal antibody was named antiTDGO pAb.
ELISA protocols Conjugation of TDGO 5 to proteins and characterization To conjugate TDGO 5 to carrier proteins, the carboxylic acid group of TDGO 5 was directly employed in the binding of the amino groups of the carrier proteins using the active ester method.29 TDGO 5 was conjugated to two types of protein, BSA and ovalbumin (OVA), in order to prepare TDGO 5–BSA and TDGO 5–OVA conjugates to be used as immunizing and coating antigens. Thus, TDGO 5 (15 mmol) together with N-hydroxysuccinimide (NHS; 1.5 mg, 15 mmol) and ethyl(dimethylaminopropyl) carbodiimide (EDC; 5.7 mg, 30 mmol) were dissolved in DMF (1 mL). The reaction mixture was incubated for 4 h at RT. The activated hapten was then added dropwise to a protein solution (10 mg mL1, 0.15 mM) made in 0.1 M borate buffer (pH 9) using a protein–hapten molar ratio of 1 : 40. The nal reaction volumes of the protein–hapten conjugates were kept constant at 1 mL for each preparation. The conjugates were incubated overnight at RT and then centrifuged for 5 min at 10 000 rpm to remove any precipitate. They were further puried by passing through a P10 gel ltration column (Pharmacia, Sweden). Fractions with the highest protein concentrations were determined by absorbance measurements at 280 nm using a molar extinction coefficient of 43 824 M1 cm1 on a UV spectrophotometer.30 The nal protein concentrations of the conjugates were determined using a Micro BCA™ protein assay kit (Pierce). Prepared conjugates were characterized by spectrophotometric analysis and MALDI-MS according to the reported method.31 Preparation of polyclonal antiserum and purication Two female New Zealand white rabbits weighing 1–1.5 kg were used to raise polyclonal antibodies (pAb) against conjugated haptens. The rabbits were initially immunized by the subcutaneous route using 500 mg of TDGO 5–BSA conjugate (one rabbit per immunogen) and Freund's complete adjuvant at four different sites. All the animals subsequently received a booster dose of 500 mg of TDGO 5–BSA conjugate along with Freund's incomplete adjuvant intramuscularly at 15 day intervals for 45 days. The animals were bled from the heart, and the sera were separated and stored at 20 C. For purication, 5 mL of saturated ammonium sulfate was slowly added dropwise to 5 mL of hyperimmune serum diluted 1 : 1, and the mixture was
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Indirect competitive inhibition ELISA. Checkerboard assay was performed using several dilutions of antibody sera and titrating against varying amounts of the coating antigens (i.e., TDGO 5–OVA conjugate). This assay gave a rough estimate of the appropriate antigen coating and antibody concentrations for competitive assays. The procedure for the checkerboard assays was the same as that for the competitive assays, except that the addition of thiodiglycol sulfoxide standard was omitted at the competition step. The inuences of pH (6.0, 6.5, 7.0 and 7.5) and buffer concentration (10, 20, 30 and 40 mM) were evaluated (see the ESI,† Table 1). The goat antirabbit IgG was conjugated with HRP according to the method given by
Table 1
Cross-reactivity studies by competitive inhibition ELISA
Entry Toxicant/pesticide IC50a (mg mL1) SD Cross- reactivityb (%)
1.
0.098 0.02
100
2.
0.13 0.14
75
3.
0.85 0.13
11
4.
0.54 0.24
18
5.
0.62 0.19
15
6.
0.58 0.23
16
a
Determined by competitive inhibition ELISA using antibody TDGO–pAb (1 : 2000). All reactions were performed in triplicate on microtitration plates. b Cross-reactivity (%) ¼ (IC50 of TDGO/IC50 of other compound) 100.
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M. Imagawa et al.32 All incubations except that for antigen coating were carried out at RT. In brief, microtiter plates were coated with 100 mL per well of a TDGO 5–OVA conjugate in carbonate–bicarbonate buffer (50 mM, pH 9.6) by overnight incubation at 4 C. The plates were washed ve times with phosphate buffered saline with Tween 20 (PBST, 10 mM PBS containing 0.05% Tween 20, pH 7.4) and blocked by incubation with 1% OVA in PBS (200 mL per well) for 1 h. Aer another washing step, 50 mL per well of TDGO dissolved in methanol– PBS and 50 mL per well of the antibody diluted with PBS were added. Aer incubation for 1 h, the plates were washed and 100 mL per well of a diluted (1 : 3000) goat anti-rabbit IgG labeled with HRP was added. The mixture was allowed to incubate for 1 h, and aer another washing step, 150 mL of substrate solution (1.25 mM 3,30 ,5,50 -tetramethylbenzidine and 1.6 mM hydrogen peroxide in acetate buffer, pH 5.0) was added to each well. The reaction was stopped aer an appropriate time period (typically 10 min) by adding 50 mL of 2 mol L1 H2SO4, and absorbance was recorded at 450 nm. Competitive curves were obtained by plotting the normalised signal (B/B0) against the logarithm of analyte concentration (Fig. 2). The inhibitory concentration (IC50) and limit of detection (LOD) were obtained from a fourparameter logistic equation33 of the sigmoidal curves.
Determination of cross-reactivities by indirect competitive inhibition ELISA Various simulants, biomarkers and degradation products of sulfur mustards were tested for cross-reactivity using the indirect competitive inhibition ELISA procedure described above. The cross-reactivity values were calculated as follows: (IC50 of TDGO/IC50 of compound) 100. The synthesized
Fig. 2 Inhibition curve for TDGO by inhibition ELISA using antiserum TDGO5–BSA diluted 1 : 2000 with TDGO5–OVA (0.2 mg per mL per well) as the coating antigen and 0.1% OVA as the blocking agent. %B/B0 ¼ (A – Aex/A0 Aex) 100, where A is absorbance, A0 is absorbance at zero dose of analyte and Aex is absorbance at an excess of analyte. Data represent the mean of three experiments. Vertical bars indicate SD about the mean. The LOD were calculated as the smallest concentration of analyte that produced a signal statistically different from the blank (Student's t-test).
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compounds 3, 4 and TDGO 5 were also tested for cross-reactivity studies. Development of lateral ow assay Preparation of colloidal gold. Colloidal gold was prepared by the Frens method.34 In brief, in a 500 mL round-bottom ask, 250 mL of 0.01% AuCl4 in doubly distilled water was brought to boil with vigorous stirring. To this solution was added 3.75 mL of 1% trisodium citrate. The solution turned deep blue within 20 s, and the colour nally changed to wine red in 60 s. Boiling was pursued for additional 10 min, and the solution was stirred for another 15 min aer the removal of the heating source. This solution was stored in dark bottles at 4 C. Finally, the gold colloidal solution supplemented with 0.05% sodium azide (used as an antiseptic) was stored at 4 C for further study. The size of the colloidal gold was determined by Zeta analyzer. Optimization of assay conditions The performance of one-step ICA was very simple, requiring only the addition of the standard or sample solution to the well of the sample pad on the strip. However, various assay conditions such as blocking reagent, concentration of antibody and amount of coating antigen in the test region (test line) were optimized. Preparation of colloidal gold–antibody conjugate To determine the minimum amount of pAb needed to stabilize the gold nanoparticle solution, 250 mL of gold nanoparticle solution was added to series tubes, and different volumes (0 to 20 mL in PBS buffer) of anti-TDGO pAb (concentration 1 mg mL1) was added to each tube. Mixtures were then incubated for 15 min at RT, and 100 mL of 10% NaCl solution was added in all the vials. The colour of the samples changed from brilliant red to blue as the concentration of anti-TDGO pAb decreased. The optimum concentration of pAb for colloidal gold labeling was the lowest concentration of pAb solution that did result in a colour change (1 mg mL1); pAb solution (40 mL) at this optimum concentration of 1 mg mL1 was incubated with 1 mL of colloidal gold solution (pH 9.0) for 30 min at RT. Aer the addition of 625 mL of 10% BSA solution in 20 mmol L1 sodium borate (pH 9.0), the mixture was incubated at RT for another 10 min. Washing of the labeled pAb was performed by repeated centrifugation at 10 000 rpm and 4 C for 30 min using 20 mmol L1 sodium borate (pH 9.0) containing 1% BSA and 0.1% sodium azide. The precipitate was then resuspended in 500 mL of resuspension buffer and stored at 4 C for use. Following the same procedure, the antibody was conjugated with gold nanoparticles of different sizes (10, 20 and 40 nm). This antibodylabeled solution was used to prepare the conjugate pad. Preparation of immunochromatographic test strips An ICA test strip consisted of three pads (sample, conjugate release, and absorbent pads) and one nitrocellulose (NC) membrane with test and control zones. The test and control zones of the nitrocellulose membrane were dispersed with 4 mL This journal is © The Royal Society of Chemistry 2014
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of TDGO 5–OVA (2.8 mg mL1) conjugate and 0.5 mL of goat anti-rabbit IgG antibody (1 mg mL1), respectively. The treated NC membrane was dried for 10 min at RT. The anti-TDGO pAb–gold colloidal conjugate (5 mL per strip) was added to an untreated glass-ber membrane to be used as a conjugate release pad. The conjugate pad was air-dried for 5 min. The release pad was pasted on the plate by overcrossing 2 mm with the NC membrane. The sample pad was also pasted on the plate by overcrossing 2 mm with the release pad. The absorbent pad was pasted on the top of the membrane sheet. The whole assembled sheet was cut lengthwise and divided into strips (5 mm 75 mm). Strips prepared in this way were stored in sealed bags under dry conditions at laboratory temperature until use. Test procedure and principle The standard solution or the spiked sample of TDGO (40 mL) was added onto the sample pad, and the strip was eluted with the help of elution buffer to carry the sample towards the absorbent pad; a result could be seen aer 5 min. In the absence of TDGO in the sample solution, the antibody–gold nanoparticle conjugate was bound and trapped by the TDGO 5–OVA conjugate to form a visible line on the test zone. In contrast, if a sufficient concentration of TDGO is present in the sample solution, the free antigen occupied the antigen binding sites on the antibody–gold nanoparticle conjugates; consequently, the limited antibody–gold nanoparticle conjugates failed to bind with the TDGO 5–OVA conjugate on the test zone. The absence of a colour line on the test zone indicated a positive result. A control zone coated with goat anti-rabbit IgG was constructed to verify whether the assay has been performed properly; this control zone should show a red colour line under accurate operation regardless of the presence or absence of TDGO. A TDGO-free sample shows two red lines, whereas a positive sample with TDGO presents only one red line on the membrane. ICA optimization and selection of working solution The test strip made with unblocked NC membrane was further optimized in terms of the membrane pore sizes, capture antigen concentration, blocking agent, running buffer and percentage of Tween 20 in the running buffer. Aqueous samples containing a range of concentrations (1–100 mg mL1) of TDGO standard were prepared in 10% methanol in PBS (10 mM). These samples were tested by the ICA test strips. Visible changes were observed within 5 min, and the sensitivity of the ICA test strip was determined at different concentrations. The tested sample solution concentration with the best sensitivity over the ICA test strip was selected as the optimized working solution. Performance and cross-reactivity studies of ICA To determine the accuracy and the reproducibility of the developed ICA method, a standard solution of 1 mg mL1 TDGO was prepared in 10% methanol in PBS (10 mM). Tap water was spiked with appropriate aliquots of the standard solution to obtain nal concentrations of 3, 8, 17 and 45 mg mL1. The spiked and nonspiked water samples were tested three times by
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the ICA method and simultaneously analyzed by ELISA. Several compounds with structures similar to TDGO were tested for cross-reactivity using the developed ICA test strips. The crossreactivity was estimated visually on a positive/negative basis.
Results and discussion Synthesis and characterisation of the hapten–protein conjugates Hapten design and synthesis play a key role in the development of immunoassays. TDGO is a small molecule with a molecular weight of 138.04 g mol1. Because it is the important marker of the vesicant class of CWAs, their identication in samples submitted for analysis may imply the past contamination with mustards. In order to project 2,20 -sulnyl ethanol epitope outward to the protein, a four-carbon atom spacer arm and a terminal carboxylic group were introduced at one hydroxyl group. To prepare the immunogen and the coating antigen, TDGO was conjugated to a protein carrier molecule such as BSA and OVA. The hapten derivative of TDGO 5 was covalently attached through its carboxylic acid moiety to the lysine amino group of the carrier protein. The coupling utilized the active succinimidyl ester method, resulting in the formation of immunogen TDGO 5–BSA conjugate and TDGO 5–OVA as coating antigens. The free amino groups in the proteins before and aer conjugation were determined by MALDI-MS (refer to ESI†). The spectra were successfully obtained for both of the conjugates. The relative increase in the MW of the conjugates was manifested as a gradual shi in the mass peak as a function of hapten to protein ratio. This is interpreted as an increase in the conjugate hapten density; therefore, this method is able to determine the number of haptens per protein molecule. The observed value for BSA and OVA was used in the following equation to determine the hapten density: Number of haptens ¼ [(TDGO 5–BSA/TDGO 5–OVA conjugate MW–BSA/OVA Protein MW)]/TDGO 5 MW.
The hapten density in TDGO 5–BSA conjugate was found to be 28 molecules per BSA protein, while the hapten density of TDGO 5–OVA conjugate was 4–5 per OVA protein (refer to ESI†). Indirect competitive inhibition ELISA Maxisorp ELISA plates were used for all ELISA work. Antisera from the two rabbits injected with TDGO 5–BSA conjugate were collected one week aer the third booster injection. Because of high titer, the antisera from rabbit A was preferred for further purication steps. Antisera were rst puried by protein-A sepharose followed by BSA-sepharose-4B column to remove anti-carrier (BSA) antibodies. Anti-TDGO pAb displayed good reactivity against TDGO 5–OVA conjugate and demonstrated a signicantly lower reactivity with carrier proteins (BSA). The optimum dilution of anti-TDGO pAb was found to be 1 : 2000, and the selected concentration of coating antigen TDGO 5–OVA was 10 mg per mL per well. An optimization study was carried out in competitive format. Since methanol was used as the
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solvent to dissolve TDGO for competitive inhibition ELISA, it was desirable to assess the effect of methanol on ELISA performance. Consequently, the effect of methanol on the ELISA system was evaluated by preparing standard curves in buffers containing various amounts of methanol (10, 20, 30, and 40% in PBS). Increasing the concentration of methanol significantly affected the sensitivity of the assay (refer to ESI†); the sensitivity decreased with increasing methanol content. The optimum methanol concentration was 10%, which corresponded to the lowest IC50. The inuence of blocking reagent was investigated by using three blocking buffers (i.e., 5% skimmed milk powder, 1% BSA and 1% OVA); the highest sensitivity was observed when 1% OVA (in 10 mM PBS buffer) was used as the blocking buffer. The ELISA was more sensitive under neutral or slightly alkaline conditions; therefore, the optimum pH of the ELISA assay buffer was 7.5. The typical standard curve of the TDGO immunoassay is shown in Fig. 2. The IC50 and LOD values for TDGO were found to be 0.098 mg mL1 and 0.012 mg mL1, respectively. Cross-reactivity (CR) studies To determine the specicity of the optimized competitive inhibition ELISA, various compounds with structures similar to TDGO and other degradation products of SM were tested for cross-reactivity. Table 1 shows the cross-reactivity determined by the competitive inhibition ELISA. The highest interference was obtained for TDGO2, which showed a cross-reactivity of more than 75%. The cross-reactivity of the antibody for TDGO2 is understandable because it contains a sulfone group in its structure in place of the sulfoxide moiety. Other compounds, however, showed cross-reactivities of less than 20%, demonstrating the recognition of the 2-sulfonyl ethanol group by the generated polyclonal antibody. Synthesis and conjugation of gold nanoparticles (GNPs) to anti-TDGO pAb The synthesis of GNPs from gold chloride solution was conrmed by the absorption peak at 520 nm in the spectrum of the dark red colloidal gold solution. The direct absorption of pAb on the surfaces of GNPs by van der Waals forces and hydrophobic interactions was observed. Because the isoelectric point of the antibodies where they exist in zwitterion states is approximately pH 9.0, the positive amino terminals of the antiTDGO antibodies were targeted for conjugation with GNPs by adjusting the pH of the gold solution to 9.0 with 0.1M K2CO3. The size of the colloidal particles were found to be 40 nm. To attain a strong adsorption between the gold nanoparticles and pAb, a preliminary titration was performed by adding sodium chloride to gold solutions containing different amounts of antibody. Coating the gold particles with the optimal amount of antibody can prevent degradation of the gold surface and preserve the colour of the gold solution (wine red). The concentration of the GNPs–antibody conjugate was determined to be the key factor for competitive immunoassay. Therefore, the optimal immunoreagent (GNPs–antibody conjugate) concentration was determined and used to obtain a clear
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observable line on the NC membrane strips with the control and test samples. Results of preliminary titrations illustrated that 10 mg of anti TDGO–pAb was sufficient for stabilising the GNPs; this amount was selected for further experiments.
Optimization and performance of the TDGO immunochromatographic strip A schematic description of the ICA test strip is shown in Fig. 3A. Aer the synthesis of the gold colloidal particle antibody probe, the suitability of the probe was tested on the strip and compared with the BSA–gold conjugate (refer to ESI†), which was used as a control (Fig. 3B). In the control test, there is no red line on the membrane aer testing with 10% MeOH/PBST. This result indicates that BSA used as a blocking reagent for gold particles does not generate a nonspecic binding to the coating antigen (TDGO 5–OVA conjugate) and goat anti-rabbit IgG immobilized on the test and control lines, respectively. Colour development was observed at the test and control lines when the ICA strip was assembled with the conjugate pad containing the gold–TDGO pAb probe and the test was performed with TDGO negative (0 mg mL1 in 10% MeOH/PBST). However, only one red line at the control line appeared for the application of TDGO positive (10 mg mL1 in 10% MeOH/PBST). This result indicates that the gold–TDGO pAb probe is specic to TDGO and can be used as a marker in the development of ICA. Among the various factors associated with the success of ICA, we found that gold colloidal particles with 40 nm sizes offered better visibility than 10 nm particles, likely due to their optimal size and the lower steric hindrance during the conjugation of the 40 nm particles and the antibody. In addition, NC membranes with various pore sizes (5, 8, 10 and 15 mm) were examined before assembly into the strip. On the membrane with 15 mm pore sizes, the GNPs demonstrated a more obvious visual effect and a faster migration. In the dipstick format, the major challenge is to minimize non-specic binding on the NC membrane, which is usually hydrophobic in nature. To do so, an array of blocking agents including skim milk, PEG, PVP, PVA and BSA were studied for their effectiveness in dipstick- based ICA. The blocking agent made by mixing 5% BSA, 5% sucrose and 5% PVP in phosphate buffer (20 mM, pH 7.4) for 20 min at RT yielded the best results. The presence of Tween 20 in the elution buffer also affected the colour development at the test line. Therefore, elution buffers with different percentages of Tween 20 (0.05%, 0.075% and 0.125%) were tested to determine the optimal buffer composition. Out of the three tested concentrations, the elution buffer containing 0.05% Tween 20 gave the optimal colour development at the test line. This can be attributed to the faster migration of antibody–gold conjugate at higher concentrations of Tween 20, leading to a shorter contact between the antibody–gold conjugate and the capture antigen. Therefore, a Tween 20 concentration of 0.05% was selected. The test strip was further optimised for the concentration of capture antigen at the test line. Images of ICA tests of TDGO standard solutions with concentrations ranging from 0.1 mg mL1 to 100 mg mL1 are presented in Fig. 3C. A negative control without TDGO gave a
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(A) Schematic illustrations of ICA test strip: C, control zone (anti-rabbit mouse IgG); T, test zone (TDGO–OVA). (B) Confirmation of TDGO pAb–gold conjugate: (1) the conjugate pad containing the BSA–gold conjugate; (2 and 3) the conjugate pads containing the TDGO pAb–gold conjugate. (C) Detection limit of TDGO by the ICA test strip; a concentration equal to or greater than 10 mg mL1 of TDGO was found to cause the red line at the test zone to disappear. Fig. 3
clear red colour at both the test line and the control line. Serial dilutions of TDGO 5–OVA with concentrations ranging from 0.1 mg mL1 to 1000 mg mL1 were prepared in borate buffer (0.5 mM, pH 9). When the concentration of TDGO 5–OVA was equal to or greater than 20 mg mL1, the test line exhibited a visible signal; however, at high concentrations (greater than 1.0 mg mL1) of TDGO 5–OVA, more free TDGO was needed in the sample to compete with TDGO 5–OVA conjugate. Finally 40 nm colloidal gold particles, a 15 mm NC membrane, a mixture of 5% BSA, 5% sucrose and 5% PVP as blocking agent, 0.05% Tween 20 in the elution buffer and greater than or equal to 1.0 mg mL1 of capture antigen were chosen for all ICA strips herein. The optimized conditions for the ICA test were chosen by visual evaluation of the strips in order to achieve balance between good line colour, good test sensitivity, minimum
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immunoreagent consumption and rapid test performance. With all the above optimal parameters, a detectable difference was observed in the intensity of colour development on NC membrane-containing strips. The maximum colour intensity was observed when no TDGO was present in the sample and the maximum amount of GNPs–antibody conjugate was bound to the immobilized conjugate. The colour intensity was less in the TDGO-containing samples as compared to the control because the binding sites for free TDGO of the GNPs–antibody conjugate were not available for binding with the immobilized conjugate on the NC membrane strip. As a result, a less intense red colour developed when the TDGO concentration in the sample was high, and a more intense colour developed when the concentration of TDGO in the sample was lower. The sensitivity of the ICA strip was determined by testing the TDGO standard samples in the concentration range from 1–100 mg mL1.
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Comparison of ELISA and ICA strip analyses of TDGO in spiked water samples
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Table 2
Samples
Spiked concentration (mg mL1)
ELISA results mg mL1 SD
ICAa results
1 2 3 4
3 8 17 45
3.54 0.45 9.56 0.15 16.50 0.32 46.70 0.24
— + +
a Visual assessment of the test line; () negative result; (+) positive result; () weakly positive result.
2
3 4 5 6
Fig. 3C demonstrates that the colour intensity of the test line for a 10 mg mL1 concentration of TDGO had a visuallydistinguishable difference when compared to the negative control. Thus, 10 mg mL1 of TDGO was considered to be the visual detection limit for the ICA test. At concentrations of TDGO equal to or higher than 10 mg mL1, the test line was invisible.
10
Analysis of spiked tap water samples
11
The accuracy of the proposed method was evaluated by spiked tap water sample experiments. Samples were spiked with four different concentrations (3, 8, 17 and 45 mg mL1) of TDGO and analyzed by the optimized ICA test strip protocol. The test and control lines were both observed at the concentrations of 3 and 8 mg mL1, while the test line disappeared at the 17 mg mL1 and 45 mg mL1 concentrations. The results of sample analysis obtained on a gold colloidal ICA test strip were in a good agreement with those obtained from competitive ELISA (Table 2). The strip can be directly dipped into a sample solution without further sample cleanup, and the assay can be completed within 10 min.
7 8 9
12 13 14 15 16
17
Conclusions The results presented in this paper demonstrate the successful application of the developed pAb against TDGO for the detection of TDGO in water samples using ICA. The visible detection limit was found to be 10 mg mL1 using the ICA method. The assay was very simple to perform, and the detection was completed within 10 min. In addition, the assay did not require intensive labor or expensive equipment for sample analysis. This technique possesses several advantages in terms of rapidity, specicity, and cost effectiveness, and it is well-suited for eld applications in on-site testing.
18
19 20 21 22 23
Acknowledgements
24
The authors thank the Director, DRDE, Gwalior for his keen interest and encouragement. 25
References 1 Convention on the Prohibition of the Development, Production, Stockpiling and Use of Chemical Weapons and
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on their Destruction, Technical Secretariat for the Organisation for the Prohibition of Chemical Weapons, The Hague, 1997. R. M. Black and R. W. Read, in: Chemical Weapons Convention Chemical Analysis, Sample Collection, Preparation and Analytical Methods, ed. M. Mesilaakso, Wiley, Chichester, 2005. L. Szinicz, Toxicology, 2005, 214, 167. R. M. Black, J. L. Hambrook, D. J. Howells and R. W. Read, J. Anal. Toxicol., 1992, 16, 79. J. Riches, W. R. Robert and R. M. Black, J. Chromatogr. B: Anal. Technol. Biomed. Life Sci., 2007, 845, 114. C. L. Young, D. Ash, W. J. Driskell, A. E. Boyer, R. A. Martinez, L. A. Silks and J. R. Barr, J. Anal. Toxicol., 2004, 28, 339. R. M. Black and R. W. Read, J. Anal. Toxicol., 2004, 28, 346. S. Popiel, J. Nawala, D. Dziedzic, M. Soderstrom and P. Vanninen, Anal. Chem., 2014, 86, 5865. Neelam, V. Singh and T. Gupta, Anal. Chim. Acta, 2014, 812, 222. V. S. Morozova, A. I. Levashova and S. A. Ermin, J. Anal. Chem., 2005, 60, 202. A. Y. Kolosova, J. H. Park and A. Sergei, Anal. Chim. Acta, 2004, 511, 323. Y. J. Kim, Y. A. Kim, Y. T. Lee and H. S. Lee, Anal. Chim. Acta, 2007, 591, 183. G. A. Posthuma-Trumpie, J. Korf and V. Amerongen, Anal. Bioanal. Chem., 2009, 393, 569. S. Gandhi, N. Caplash, P. Sharma and C. R. Suri, Biosens. Bioelectron., 2009, 25, 502. W. J. Gui, S. T. Wang, Y. R. Guo and G. N. Zhu, Anal. Biochem., 2008, 377, 202. J. Kaur, K. V. Singh, R. Boro, K. R. Thampi, M. Raje, G. C. Varshney and C. R. Suri, Environ. Sci. Technol., 2007, 41, 5028. C. Shi, S. Zhao, K. Zhang, G. Hong and Z. Zhu, J. Environ. Sci., 2008, 20, 1392. W. B. Shim, Z. Y. Yang, J. Y. Kim, J. G. Choi, J. H. Je, S. J. Kang, A. Y. Kolosova, S. A. Eremin and D. H. Chung, J. Agric. Food Chem., 2006, 54, 9728. S. Wang, C. Zhang, J. Wang and Y. Zhang, Anal. Chim. Acta, 2005, 546, 161. P. Zhou, Y. T. Lu, J. Zhu, J. B. Hong, B. Li, J. Zhou, D. Gong and A. Montoya, J. Agric. Food Chem., 2004, 52, 4355. J. Zhu, W. Chen, Y. Lu and G. Cheng, Environ. Pollut., 2008, 156, 136. D. W. Li, S. Wei, H. Yang, Y. Li and A. P. Deng, Biosens. Bioelectron., 2009, 24, 2277. Y. Zhou, F. G. Pan, Y. S. Li, Y. Y. Zhang, J. H. Zhang, S. Y. Lu, H. L. Ren and Z. S. Liu, Biosens. Bioelectron., 2009, 24, 2744. M. Sathe, S. R. Merwyn, R. Ghorpade, G. S. Agarwal, M. K. Rao, G. P. Rai and M. P. Kaushik, J. Hazard. Mater., 2011, 192, 1720. M. Sathe, R. Ghorpade, S. R. Merwyn, G. S. Agarwal and M. P. Kaushik, Analyst, 2012, 137, 406. G. P. Van der Schans, D. Noort, R. H. Mars-Groenendijk, A. Fidder, L.-F. Chau, L. P. A. De Jong and H. P. Benschop, Chem. Res. Toxicol., 2002, 15, 21.
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27 Sufur mustard detector by skin smear test, Securetec detektions-system AG Germany, For more details please visit the website: http://www.securetec.net/en/products/ rapid-test-sulfur mustard. 28 (a) K. Bahrami, Tetrahedron Lett., 2006, 47, 2009; (b) M. Bodanszky and A. Bodanszky, The Practice of Peptide Synthesis, Springer- verlog Berlin Heidelberg, 1984. 29 C. N. Lieske, R. S. Klopcic, C. L. Gross, J. H. Clark, T. W. Dolzine, T. P. Logan and H. G. Meyer, Immunol. Lett., 1992, 31, 117.
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30 M. Sathe, M. Derveni, G. Broadbent, A. Bodlenner, K. Charlton, B. Ravi, M. Rohmer, M. R. Sims and D. C. Cullen, Anal. Chim. Acta, 2011, 708, 97. 31 K. V. Singh, J. Kaur, G. C. Varshney, M. Raje and C. R. Suri, Bioconjugate Chem., 2004, 15, 168. 32 M. Imagawa, S. Yoshitake, Y. Hamguchi, E. Ishikawa, Y. Nitsu, I. Urushizaki, R. Kanazawa, S. Tachibana, M. Nakazawa and H. Ogawa, J. Appl. Biochem., 1982, 4, 41. 33 G. M. Raab, Clin. Chem., 1983, 29, 1757. 34 G. Frens, Nat. Phys. Sci., 1973, 241, 20.
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Cite this: DOI: 10.1039/c4an00720d
Competitive immunochromatographic assay for the detection of thiodiglycol sulfoxide, a degradation product of sulfur mustard† Manisha Sathe,* Shruti Srivastava, S. Merwyn, G. S. Agarwal and M. P. Kaushik An immunochromatographic assay (ICA) based on the competitive antigen-coated format using colloidal gold as the label was developed for the detection of thiodiglycol sulfoxide (TDGO), an important metabolite and degradation compound of sulphur mustard (SM). The ICA test strip consisted of a membrane with a detection zone, a sample pad and an absorbent pad. The membrane was separately coated with hapten–OVA conjugate (test line) and anti-rabbit mouse IgG (control line). The visual
Received 23rd April 2014 Accepted 24th July 2014
detection limit for TDGO by ICA detection was found to be 10 mg mL1. For validation, the ICA results obtained for spiked water samples were in good agreement with those obtained by indirect competitive inhibition enzyme-linked immunosorbent assay (ELISA) for TDGO. The assay time for detection was less
DOI: 10.1039/c4an00720d
than 10 min. The developed ICA has the potential to be a useful on-site screening tool for the
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retrospective detection of SM in environmental samples.
Introduction The development, production, stockpiling and use of chemical weapons are prohibited under the Chemical Weapons Convention (CWC).1 The most signicant chemical warfare agents (CWAs) in terms of military capacity and past use are the nerve and blister agents.2 For these reasons, the analysis of these compounds even at trace levels in environmental and biological matrices is of utmost importance. Sulphur mustard (SM) and its analogues such as sesquimustards belong to the blistering class of CWAs, which are classied as schedule 1A in the CWC.3 The blister agent SM remains one of the CW agents of greatest concern due to its advantageous physical properties and ease of production. In the environment, these compounds are susceptible to oxidation. Thiodiglycol (TDG) is the major hydrolytic breakdown product of SM (bis(2-chloroethyl)sulde), which oxidizes gradually in water to its sulfoxide analogue thiodiglycol sulfoxide (TDGO). In the presence of sunlight, further oxidation is then thought to occur, forming thiodiglycol sulfone (TDGO2; Fig. 1). Thus, TDGO and TDGO2 serve as important markers; their identication in any environmental matrix indicates the probable prior presence of SM, which is an important aspect in the verication analysis of CWC related chemicals.4 The conventional analytic methods for SM and its metabolites in biological and environmental samples are based
Process Technology Development Division, Discovery Centre, Defense R & D Establishment, Jhansi Road, Gwalior 474002, India. E-mail: drmanishasathe@drde. drdo.in; Fax: +917512340042; Tel: +917512390462 † Electronic supplementary 10.1039/c4an00720d
information
(ESI)
available.
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See
DOI:
on chromatography and mass spectrometry.5–8 Recently, Neelam et al. reported a spectrophotometric technique for the detection of an SM analogue.9 These methods are used for off-site detection and involve high cost, sophistication, and time-consuming sample-preparation procedures. Immunochemical techniques have gained acceptance as a fast and cost-effective tools for separating and/ or detecting trace amounts of chemicals such as pesticides.10–12 However, the labour-intensive operations including repeated incubation and washing along with the enzyme reaction for the nal signal generation in enzyme-linked immunosorbent assay (ELISA) can be difficult to perform in non-specialized laboratories and in the eld. With the demand for speed and simplicity, a lateral-ow immunochromatographic assay (ICA) could be a more suitable alternative13,14 for the onsite detection of small molecular toxicants, their markers and other environmental contaminants. In the last few years, several research groups have demonstrated ICA for some pesticides15–21 and other environmental contaminants.22,23 The main feature of ICA using colloidal gold as the label is its single step assay procedure,
Fig. 1 Hydrolysis of sulfur mustard to produce thiodiglycol (TDG) and further oxidation to thiodiglycol sulfoxide (TDGO) and thiodiglycol sulfone (TDGO2).
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which is contrary to most prevalent types of immunoassay (for example, ELISA involves three to four steps). Another advantage of ICA is that it is easy and convenient to use because detection can be achieved with the naked eye. We are engaged in the development of methods for the retrospective detection of CWAs.24,25 Van der Schans et al. reported the detection of SM protein adducts and metabolites based on immunochemical methods.26 However, only one report in the literature has described the development of ICA for the detection of mustard gas; this study used a skin smear test developed by Securetec Detektions, Germany.27 To the best of our knowledge, ICA for the detection of TDGO has not been studied to date. Herein, in light of the CWC and the environmental concerns regarding abandoned CWAs, we report the development of ICA for the detection of TDGO using an in-housegenerated polyclonal antibody. The developed assay was further tested using spiked samples and validated by indirect competitive inhibition ELISA.
Experimental Reagents and instruments Unless stated otherwise, reagents including bovine serum albumin (crystalline, fraction V; BSA), chicken egg albumin (OVA), horseradish Peroxidase (HRP) and goat antirabbit IgG were obtained from Sigma-Aldrich (India). Solvents used for synthesis were of analytical grade. Reaction progress was monitored by thin-layer chromatography (TLC) using GF254 silica gel on glass plates with uorescent indicator. Chromatography was performed using silica gel (60–120 mesh). PD-10 gel ltration columns (Pharmacia, Sweden) were obtained from GE Healthcare, and 96-well, polystyrene Maxisorb plates were from Nunc (UK). The single beam scanning UV-visible spectrophotometer (Camspec M501) was from Camspec Analytical Instruments Ltd., Leeds UK. The ELISA plate reader used in this study was from Molecular Devices (USA). Electrospray ionization mass spectroscopy (ESI-MS) was performed on a Micromass Q-ToF high-resolution mass spectrometer with a Masslynx 4.0 data acquisition system. Matrix-assisted laser desorption ionization mass spectroscopy (MALDI-MS) data were recorded on an Applied Biosystems MALDI 4700 TOF. 1H-NMR spectra were collected in CDCl3 solutions on a Bruker AVANCE NMR spectrometer operating at 400 MHz (for 1H) using tetramethylsilane (TMS) as an internal standard. Chemical shis are reported in parts per million (ppm) downeld from TMS. Spin multiplicities are described as s (singlet), d (doublet), t (triplet), q (quartet) and m (multiplet). Coupling constants are reported in Hertz (Hz). Synthesis of hapten The haptens used for immunization and antigen coating were fabricated by a multistep synthesis,28 as shown in Scheme 1. Synthesis of TDGO (2)28a In a round-bottomed ask (50 mL) equipped with a magnetic stirrer, a solution of thiodiglycol (2 mmol, 0.244 g) in CH3OH
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Scheme 1 Synthetic scheme for the preparation of TDGO (5) from thiodiglycol.
(15 mL) was prepared. H2O2 (30%, 14 mmol, 1.4 mL) and ZrCl4 (4 mmol, 0.93 g) were added, and the mixture was stirred at room temperature for one hour. The progress of the reaction was monitored by TLC (eluent: 7/3 n-hexane/ethyl acetate). When the starting sulde had completely disappeared, the reaction mixture was quenched by adding water (15 mL) and extracted with chloroform (4 10 mL). The extract was then dried with anhydrous MgSO4. The ltrate was evaporated, and the corresponding thiodiglycol sulfoxide was obtained as a white solid in 65% yield. Synthesis of tert-butyl 2-(2-hydroxyethylsulnyl) ethyl carbonate (3)28b A mixture of 2 (0.8 g, 5.8 mmol), boc anhydride (1.92 g, 8.8 mmol), N,N-diisopropylethylamine (835 mg, 1.12 mL, 6.4 mmol), 4-dimethylaminopyridine (190 mg, 1.4 mmol) and dimethylformamide (DMF; 10 mL) was stirred at 40 C overnight. The DMF was removed in vacuo, and the residue was taken up in ethyl acetate (150 mL). The reaction was washed with brine (2 100 mL), saturated sodium bicarbonate (100 mL) and 0.01 M HCl (100 mL), followed by drying over sodium sulfate. Removal of the organics gave 3 (1.11 g) as an oily solid in 71% yield. Synthesis of 4-(2-(2-hydroxyethylsulnyl) ethoxy)-4oxobutanoic acid (4)28b A solution of 3 (1 g, 4.2 mmol) in pyridine (10 mL) was added dropwise to a stirred solution of succinic anhydride (420 mg, 4.2 mmol) dissolved in pyridine (10 mL). Aer stirring for 3 h at room temperature, 20 mL saturated K2CO3 was added to the reaction mixture and extracted with ethyl acetate. The water layer was acidied by adding 6 mol L1 HCl and extracted with ethyl acetate. The combined organic layer was dried over anhydrous sodium sulfate, ltered and evaporated under reduced pressure to afford 4 as a brown viscous liquid (1.1 g, 52%).
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Synthesis of 4-(2-(2-hydroxyethylsulnyl) ethoxy)-4oxobutanoic acid (TDGO 5)28b A solution of 4 (500 mg, 2 mmol) was dissolved in dichloromethane–triuoroacetic acid (DCM : TFA; 1 : 1; 15 mL) and stirred at 0 C for 2 h. The solvent was evaporated on a rotary evaporator, and diethyl ether was added into the resulting crude product, followed by extraction (2) with water. The aqueous solution was then basied by a saturated solution of sodium bicarbonate and extracted thrice with 15% isopropanol in chloroform, and the combined organic layer was dried over sodium sulphate. Aer the solvent was removed by rotary evaporation, TDGO 5 was afforded in 78% yield (320 mg).
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stirred for 30 min. The precipitated proteins (IgGs) were removed by centrifugation (Sigma 4K15 centrifuge) at 5000 rpm for 10 min at 4 C. The precipitated IgGs were washed twice with 1 : 1 (vol/vol) saturated ammonium sulfate to remove the remaining soluble proteins. The precipitate was then dissolved in 1 mL of 0.1 M PBS at pH 7.2 and desalted by dialysis. The IgGs from the dialysed product were affinity puried using a proteinA column (Ab purication kit-Sigma) as per their instruction manual. The antibody was then further puried by BSAsepharose column-4B to remove the anticarrier (BSA) antibodies. The produced polyclonal antibody was named antiTDGO pAb.
ELISA protocols Conjugation of TDGO 5 to proteins and characterization To conjugate TDGO 5 to carrier proteins, the carboxylic acid group of TDGO 5 was directly employed in the binding of the amino groups of the carrier proteins using the active ester method.29 TDGO 5 was conjugated to two types of protein, BSA and ovalbumin (OVA), in order to prepare TDGO 5–BSA and TDGO 5–OVA conjugates to be used as immunizing and coating antigens. Thus, TDGO 5 (15 mmol) together with N-hydroxysuccinimide (NHS; 1.5 mg, 15 mmol) and ethyl(dimethylaminopropyl) carbodiimide (EDC; 5.7 mg, 30 mmol) were dissolved in DMF (1 mL). The reaction mixture was incubated for 4 h at RT. The activated hapten was then added dropwise to a protein solution (10 mg mL1, 0.15 mM) made in 0.1 M borate buffer (pH 9) using a protein–hapten molar ratio of 1 : 40. The nal reaction volumes of the protein–hapten conjugates were kept constant at 1 mL for each preparation. The conjugates were incubated overnight at RT and then centrifuged for 5 min at 10 000 rpm to remove any precipitate. They were further puried by passing through a P10 gel ltration column (Pharmacia, Sweden). Fractions with the highest protein concentrations were determined by absorbance measurements at 280 nm using a molar extinction coefficient of 43 824 M1 cm1 on a UV spectrophotometer.30 The nal protein concentrations of the conjugates were determined using a Micro BCA™ protein assay kit (Pierce). Prepared conjugates were characterized by spectrophotometric analysis and MALDI-MS according to the reported method.31 Preparation of polyclonal antiserum and purication Two female New Zealand white rabbits weighing 1–1.5 kg were used to raise polyclonal antibodies (pAb) against conjugated haptens. The rabbits were initially immunized by the subcutaneous route using 500 mg of TDGO 5–BSA conjugate (one rabbit per immunogen) and Freund's complete adjuvant at four different sites. All the animals subsequently received a booster dose of 500 mg of TDGO 5–BSA conjugate along with Freund's incomplete adjuvant intramuscularly at 15 day intervals for 45 days. The animals were bled from the heart, and the sera were separated and stored at 20 C. For purication, 5 mL of saturated ammonium sulfate was slowly added dropwise to 5 mL of hyperimmune serum diluted 1 : 1, and the mixture was
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Indirect competitive inhibition ELISA. Checkerboard assay was performed using several dilutions of antibody sera and titrating against varying amounts of the coating antigens (i.e., TDGO 5–OVA conjugate). This assay gave a rough estimate of the appropriate antigen coating and antibody concentrations for competitive assays. The procedure for the checkerboard assays was the same as that for the competitive assays, except that the addition of thiodiglycol sulfoxide standard was omitted at the competition step. The inuences of pH (6.0, 6.5, 7.0 and 7.5) and buffer concentration (10, 20, 30 and 40 mM) were evaluated (see the ESI,† Table 1). The goat antirabbit IgG was conjugated with HRP according to the method given by
Table 1
Cross-reactivity studies by competitive inhibition ELISA
Entry Toxicant/pesticide IC50a (mg mL1) SD Cross- reactivityb (%)
1.
0.098 0.02
100
2.
0.13 0.14
75
3.
0.85 0.13
11
4.
0.54 0.24
18
5.
0.62 0.19
15
6.
0.58 0.23
16
a
Determined by competitive inhibition ELISA using antibody TDGO–pAb (1 : 2000). All reactions were performed in triplicate on microtitration plates. b Cross-reactivity (%) ¼ (IC50 of TDGO/IC50 of other compound) 100.
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M. Imagawa et al.32 All incubations except that for antigen coating were carried out at RT. In brief, microtiter plates were coated with 100 mL per well of a TDGO 5–OVA conjugate in carbonate–bicarbonate buffer (50 mM, pH 9.6) by overnight incubation at 4 C. The plates were washed ve times with phosphate buffered saline with Tween 20 (PBST, 10 mM PBS containing 0.05% Tween 20, pH 7.4) and blocked by incubation with 1% OVA in PBS (200 mL per well) for 1 h. Aer another washing step, 50 mL per well of TDGO dissolved in methanol– PBS and 50 mL per well of the antibody diluted with PBS were added. Aer incubation for 1 h, the plates were washed and 100 mL per well of a diluted (1 : 3000) goat anti-rabbit IgG labeled with HRP was added. The mixture was allowed to incubate for 1 h, and aer another washing step, 150 mL of substrate solution (1.25 mM 3,30 ,5,50 -tetramethylbenzidine and 1.6 mM hydrogen peroxide in acetate buffer, pH 5.0) was added to each well. The reaction was stopped aer an appropriate time period (typically 10 min) by adding 50 mL of 2 mol L1 H2SO4, and absorbance was recorded at 450 nm. Competitive curves were obtained by plotting the normalised signal (B/B0) against the logarithm of analyte concentration (Fig. 2). The inhibitory concentration (IC50) and limit of detection (LOD) were obtained from a fourparameter logistic equation33 of the sigmoidal curves.
Determination of cross-reactivities by indirect competitive inhibition ELISA Various simulants, biomarkers and degradation products of sulfur mustards were tested for cross-reactivity using the indirect competitive inhibition ELISA procedure described above. The cross-reactivity values were calculated as follows: (IC50 of TDGO/IC50 of compound) 100. The synthesized
Fig. 2 Inhibition curve for TDGO by inhibition ELISA using antiserum TDGO5–BSA diluted 1 : 2000 with TDGO5–OVA (0.2 mg per mL per well) as the coating antigen and 0.1% OVA as the blocking agent. %B/B0 ¼ (A – Aex/A0 Aex) 100, where A is absorbance, A0 is absorbance at zero dose of analyte and Aex is absorbance at an excess of analyte. Data represent the mean of three experiments. Vertical bars indicate SD about the mean. The LOD were calculated as the smallest concentration of analyte that produced a signal statistically different from the blank (Student's t-test).
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compounds 3, 4 and TDGO 5 were also tested for cross-reactivity studies. Development of lateral ow assay Preparation of colloidal gold. Colloidal gold was prepared by the Frens method.34 In brief, in a 500 mL round-bottom ask, 250 mL of 0.01% AuCl4 in doubly distilled water was brought to boil with vigorous stirring. To this solution was added 3.75 mL of 1% trisodium citrate. The solution turned deep blue within 20 s, and the colour nally changed to wine red in 60 s. Boiling was pursued for additional 10 min, and the solution was stirred for another 15 min aer the removal of the heating source. This solution was stored in dark bottles at 4 C. Finally, the gold colloidal solution supplemented with 0.05% sodium azide (used as an antiseptic) was stored at 4 C for further study. The size of the colloidal gold was determined by Zeta analyzer. Optimization of assay conditions The performance of one-step ICA was very simple, requiring only the addition of the standard or sample solution to the well of the sample pad on the strip. However, various assay conditions such as blocking reagent, concentration of antibody and amount of coating antigen in the test region (test line) were optimized. Preparation of colloidal gold–antibody conjugate To determine the minimum amount of pAb needed to stabilize the gold nanoparticle solution, 250 mL of gold nanoparticle solution was added to series tubes, and different volumes (0 to 20 mL in PBS buffer) of anti-TDGO pAb (concentration 1 mg mL1) was added to each tube. Mixtures were then incubated for 15 min at RT, and 100 mL of 10% NaCl solution was added in all the vials. The colour of the samples changed from brilliant red to blue as the concentration of anti-TDGO pAb decreased. The optimum concentration of pAb for colloidal gold labeling was the lowest concentration of pAb solution that did result in a colour change (1 mg mL1); pAb solution (40 mL) at this optimum concentration of 1 mg mL1 was incubated with 1 mL of colloidal gold solution (pH 9.0) for 30 min at RT. Aer the addition of 625 mL of 10% BSA solution in 20 mmol L1 sodium borate (pH 9.0), the mixture was incubated at RT for another 10 min. Washing of the labeled pAb was performed by repeated centrifugation at 10 000 rpm and 4 C for 30 min using 20 mmol L1 sodium borate (pH 9.0) containing 1% BSA and 0.1% sodium azide. The precipitate was then resuspended in 500 mL of resuspension buffer and stored at 4 C for use. Following the same procedure, the antibody was conjugated with gold nanoparticles of different sizes (10, 20 and 40 nm). This antibodylabeled solution was used to prepare the conjugate pad. Preparation of immunochromatographic test strips An ICA test strip consisted of three pads (sample, conjugate release, and absorbent pads) and one nitrocellulose (NC) membrane with test and control zones. The test and control zones of the nitrocellulose membrane were dispersed with 4 mL This journal is © The Royal Society of Chemistry 2014
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of TDGO 5–OVA (2.8 mg mL1) conjugate and 0.5 mL of goat anti-rabbit IgG antibody (1 mg mL1), respectively. The treated NC membrane was dried for 10 min at RT. The anti-TDGO pAb–gold colloidal conjugate (5 mL per strip) was added to an untreated glass-ber membrane to be used as a conjugate release pad. The conjugate pad was air-dried for 5 min. The release pad was pasted on the plate by overcrossing 2 mm with the NC membrane. The sample pad was also pasted on the plate by overcrossing 2 mm with the release pad. The absorbent pad was pasted on the top of the membrane sheet. The whole assembled sheet was cut lengthwise and divided into strips (5 mm 75 mm). Strips prepared in this way were stored in sealed bags under dry conditions at laboratory temperature until use. Test procedure and principle The standard solution or the spiked sample of TDGO (40 mL) was added onto the sample pad, and the strip was eluted with the help of elution buffer to carry the sample towards the absorbent pad; a result could be seen aer 5 min. In the absence of TDGO in the sample solution, the antibody–gold nanoparticle conjugate was bound and trapped by the TDGO 5–OVA conjugate to form a visible line on the test zone. In contrast, if a sufficient concentration of TDGO is present in the sample solution, the free antigen occupied the antigen binding sites on the antibody–gold nanoparticle conjugates; consequently, the limited antibody–gold nanoparticle conjugates failed to bind with the TDGO 5–OVA conjugate on the test zone. The absence of a colour line on the test zone indicated a positive result. A control zone coated with goat anti-rabbit IgG was constructed to verify whether the assay has been performed properly; this control zone should show a red colour line under accurate operation regardless of the presence or absence of TDGO. A TDGO-free sample shows two red lines, whereas a positive sample with TDGO presents only one red line on the membrane. ICA optimization and selection of working solution The test strip made with unblocked NC membrane was further optimized in terms of the membrane pore sizes, capture antigen concentration, blocking agent, running buffer and percentage of Tween 20 in the running buffer. Aqueous samples containing a range of concentrations (1–100 mg mL1) of TDGO standard were prepared in 10% methanol in PBS (10 mM). These samples were tested by the ICA test strips. Visible changes were observed within 5 min, and the sensitivity of the ICA test strip was determined at different concentrations. The tested sample solution concentration with the best sensitivity over the ICA test strip was selected as the optimized working solution. Performance and cross-reactivity studies of ICA To determine the accuracy and the reproducibility of the developed ICA method, a standard solution of 1 mg mL1 TDGO was prepared in 10% methanol in PBS (10 mM). Tap water was spiked with appropriate aliquots of the standard solution to obtain nal concentrations of 3, 8, 17 and 45 mg mL1. The spiked and nonspiked water samples were tested three times by
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the ICA method and simultaneously analyzed by ELISA. Several compounds with structures similar to TDGO were tested for cross-reactivity using the developed ICA test strips. The crossreactivity was estimated visually on a positive/negative basis.
Results and discussion Synthesis and characterisation of the hapten–protein conjugates Hapten design and synthesis play a key role in the development of immunoassays. TDGO is a small molecule with a molecular weight of 138.04 g mol1. Because it is the important marker of the vesicant class of CWAs, their identication in samples submitted for analysis may imply the past contamination with mustards. In order to project 2,20 -sulnyl ethanol epitope outward to the protein, a four-carbon atom spacer arm and a terminal carboxylic group were introduced at one hydroxyl group. To prepare the immunogen and the coating antigen, TDGO was conjugated to a protein carrier molecule such as BSA and OVA. The hapten derivative of TDGO 5 was covalently attached through its carboxylic acid moiety to the lysine amino group of the carrier protein. The coupling utilized the active succinimidyl ester method, resulting in the formation of immunogen TDGO 5–BSA conjugate and TDGO 5–OVA as coating antigens. The free amino groups in the proteins before and aer conjugation were determined by MALDI-MS (refer to ESI†). The spectra were successfully obtained for both of the conjugates. The relative increase in the MW of the conjugates was manifested as a gradual shi in the mass peak as a function of hapten to protein ratio. This is interpreted as an increase in the conjugate hapten density; therefore, this method is able to determine the number of haptens per protein molecule. The observed value for BSA and OVA was used in the following equation to determine the hapten density: Number of haptens ¼ [(TDGO 5–BSA/TDGO 5–OVA conjugate MW–BSA/OVA Protein MW)]/TDGO 5 MW.
The hapten density in TDGO 5–BSA conjugate was found to be 28 molecules per BSA protein, while the hapten density of TDGO 5–OVA conjugate was 4–5 per OVA protein (refer to ESI†). Indirect competitive inhibition ELISA Maxisorp ELISA plates were used for all ELISA work. Antisera from the two rabbits injected with TDGO 5–BSA conjugate were collected one week aer the third booster injection. Because of high titer, the antisera from rabbit A was preferred for further purication steps. Antisera were rst puried by protein-A sepharose followed by BSA-sepharose-4B column to remove anti-carrier (BSA) antibodies. Anti-TDGO pAb displayed good reactivity against TDGO 5–OVA conjugate and demonstrated a signicantly lower reactivity with carrier proteins (BSA). The optimum dilution of anti-TDGO pAb was found to be 1 : 2000, and the selected concentration of coating antigen TDGO 5–OVA was 10 mg per mL per well. An optimization study was carried out in competitive format. Since methanol was used as the
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solvent to dissolve TDGO for competitive inhibition ELISA, it was desirable to assess the effect of methanol on ELISA performance. Consequently, the effect of methanol on the ELISA system was evaluated by preparing standard curves in buffers containing various amounts of methanol (10, 20, 30, and 40% in PBS). Increasing the concentration of methanol significantly affected the sensitivity of the assay (refer to ESI†); the sensitivity decreased with increasing methanol content. The optimum methanol concentration was 10%, which corresponded to the lowest IC50. The inuence of blocking reagent was investigated by using three blocking buffers (i.e., 5% skimmed milk powder, 1% BSA and 1% OVA); the highest sensitivity was observed when 1% OVA (in 10 mM PBS buffer) was used as the blocking buffer. The ELISA was more sensitive under neutral or slightly alkaline conditions; therefore, the optimum pH of the ELISA assay buffer was 7.5. The typical standard curve of the TDGO immunoassay is shown in Fig. 2. The IC50 and LOD values for TDGO were found to be 0.098 mg mL1 and 0.012 mg mL1, respectively. Cross-reactivity (CR) studies To determine the specicity of the optimized competitive inhibition ELISA, various compounds with structures similar to TDGO and other degradation products of SM were tested for cross-reactivity. Table 1 shows the cross-reactivity determined by the competitive inhibition ELISA. The highest interference was obtained for TDGO2, which showed a cross-reactivity of more than 75%. The cross-reactivity of the antibody for TDGO2 is understandable because it contains a sulfone group in its structure in place of the sulfoxide moiety. Other compounds, however, showed cross-reactivities of less than 20%, demonstrating the recognition of the 2-sulfonyl ethanol group by the generated polyclonal antibody. Synthesis and conjugation of gold nanoparticles (GNPs) to anti-TDGO pAb The synthesis of GNPs from gold chloride solution was conrmed by the absorption peak at 520 nm in the spectrum of the dark red colloidal gold solution. The direct absorption of pAb on the surfaces of GNPs by van der Waals forces and hydrophobic interactions was observed. Because the isoelectric point of the antibodies where they exist in zwitterion states is approximately pH 9.0, the positive amino terminals of the antiTDGO antibodies were targeted for conjugation with GNPs by adjusting the pH of the gold solution to 9.0 with 0.1M K2CO3. The size of the colloidal particles were found to be 40 nm. To attain a strong adsorption between the gold nanoparticles and pAb, a preliminary titration was performed by adding sodium chloride to gold solutions containing different amounts of antibody. Coating the gold particles with the optimal amount of antibody can prevent degradation of the gold surface and preserve the colour of the gold solution (wine red). The concentration of the GNPs–antibody conjugate was determined to be the key factor for competitive immunoassay. Therefore, the optimal immunoreagent (GNPs–antibody conjugate) concentration was determined and used to obtain a clear
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observable line on the NC membrane strips with the control and test samples. Results of preliminary titrations illustrated that 10 mg of anti TDGO–pAb was sufficient for stabilising the GNPs; this amount was selected for further experiments.
Optimization and performance of the TDGO immunochromatographic strip A schematic description of the ICA test strip is shown in Fig. 3A. Aer the synthesis of the gold colloidal particle antibody probe, the suitability of the probe was tested on the strip and compared with the BSA–gold conjugate (refer to ESI†), which was used as a control (Fig. 3B). In the control test, there is no red line on the membrane aer testing with 10% MeOH/PBST. This result indicates that BSA used as a blocking reagent for gold particles does not generate a nonspecic binding to the coating antigen (TDGO 5–OVA conjugate) and goat anti-rabbit IgG immobilized on the test and control lines, respectively. Colour development was observed at the test and control lines when the ICA strip was assembled with the conjugate pad containing the gold–TDGO pAb probe and the test was performed with TDGO negative (0 mg mL1 in 10% MeOH/PBST). However, only one red line at the control line appeared for the application of TDGO positive (10 mg mL1 in 10% MeOH/PBST). This result indicates that the gold–TDGO pAb probe is specic to TDGO and can be used as a marker in the development of ICA. Among the various factors associated with the success of ICA, we found that gold colloidal particles with 40 nm sizes offered better visibility than 10 nm particles, likely due to their optimal size and the lower steric hindrance during the conjugation of the 40 nm particles and the antibody. In addition, NC membranes with various pore sizes (5, 8, 10 and 15 mm) were examined before assembly into the strip. On the membrane with 15 mm pore sizes, the GNPs demonstrated a more obvious visual effect and a faster migration. In the dipstick format, the major challenge is to minimize non-specic binding on the NC membrane, which is usually hydrophobic in nature. To do so, an array of blocking agents including skim milk, PEG, PVP, PVA and BSA were studied for their effectiveness in dipstick- based ICA. The blocking agent made by mixing 5% BSA, 5% sucrose and 5% PVP in phosphate buffer (20 mM, pH 7.4) for 20 min at RT yielded the best results. The presence of Tween 20 in the elution buffer also affected the colour development at the test line. Therefore, elution buffers with different percentages of Tween 20 (0.05%, 0.075% and 0.125%) were tested to determine the optimal buffer composition. Out of the three tested concentrations, the elution buffer containing 0.05% Tween 20 gave the optimal colour development at the test line. This can be attributed to the faster migration of antibody–gold conjugate at higher concentrations of Tween 20, leading to a shorter contact between the antibody–gold conjugate and the capture antigen. Therefore, a Tween 20 concentration of 0.05% was selected. The test strip was further optimised for the concentration of capture antigen at the test line. Images of ICA tests of TDGO standard solutions with concentrations ranging from 0.1 mg mL1 to 100 mg mL1 are presented in Fig. 3C. A negative control without TDGO gave a
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(A) Schematic illustrations of ICA test strip: C, control zone (anti-rabbit mouse IgG); T, test zone (TDGO–OVA). (B) Confirmation of TDGO pAb–gold conjugate: (1) the conjugate pad containing the BSA–gold conjugate; (2 and 3) the conjugate pads containing the TDGO pAb–gold conjugate. (C) Detection limit of TDGO by the ICA test strip; a concentration equal to or greater than 10 mg mL1 of TDGO was found to cause the red line at the test zone to disappear. Fig. 3
clear red colour at both the test line and the control line. Serial dilutions of TDGO 5–OVA with concentrations ranging from 0.1 mg mL1 to 1000 mg mL1 were prepared in borate buffer (0.5 mM, pH 9). When the concentration of TDGO 5–OVA was equal to or greater than 20 mg mL1, the test line exhibited a visible signal; however, at high concentrations (greater than 1.0 mg mL1) of TDGO 5–OVA, more free TDGO was needed in the sample to compete with TDGO 5–OVA conjugate. Finally 40 nm colloidal gold particles, a 15 mm NC membrane, a mixture of 5% BSA, 5% sucrose and 5% PVP as blocking agent, 0.05% Tween 20 in the elution buffer and greater than or equal to 1.0 mg mL1 of capture antigen were chosen for all ICA strips herein. The optimized conditions for the ICA test were chosen by visual evaluation of the strips in order to achieve balance between good line colour, good test sensitivity, minimum
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immunoreagent consumption and rapid test performance. With all the above optimal parameters, a detectable difference was observed in the intensity of colour development on NC membrane-containing strips. The maximum colour intensity was observed when no TDGO was present in the sample and the maximum amount of GNPs–antibody conjugate was bound to the immobilized conjugate. The colour intensity was less in the TDGO-containing samples as compared to the control because the binding sites for free TDGO of the GNPs–antibody conjugate were not available for binding with the immobilized conjugate on the NC membrane strip. As a result, a less intense red colour developed when the TDGO concentration in the sample was high, and a more intense colour developed when the concentration of TDGO in the sample was lower. The sensitivity of the ICA strip was determined by testing the TDGO standard samples in the concentration range from 1–100 mg mL1.
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Comparison of ELISA and ICA strip analyses of TDGO in spiked water samples
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Samples
Spiked concentration (mg mL1)
ELISA results mg mL1 SD
ICAa results
1 2 3 4
3 8 17 45
3.54 0.45 9.56 0.15 16.50 0.32 46.70 0.24
— + +
a Visual assessment of the test line; () negative result; (+) positive result; () weakly positive result.
2
3 4 5 6
Fig. 3C demonstrates that the colour intensity of the test line for a 10 mg mL1 concentration of TDGO had a visuallydistinguishable difference when compared to the negative control. Thus, 10 mg mL1 of TDGO was considered to be the visual detection limit for the ICA test. At concentrations of TDGO equal to or higher than 10 mg mL1, the test line was invisible.
10
Analysis of spiked tap water samples
11
The accuracy of the proposed method was evaluated by spiked tap water sample experiments. Samples were spiked with four different concentrations (3, 8, 17 and 45 mg mL1) of TDGO and analyzed by the optimized ICA test strip protocol. The test and control lines were both observed at the concentrations of 3 and 8 mg mL1, while the test line disappeared at the 17 mg mL1 and 45 mg mL1 concentrations. The results of sample analysis obtained on a gold colloidal ICA test strip were in a good agreement with those obtained from competitive ELISA (Table 2). The strip can be directly dipped into a sample solution without further sample cleanup, and the assay can be completed within 10 min.
7 8 9
12 13 14 15 16
17
Conclusions The results presented in this paper demonstrate the successful application of the developed pAb against TDGO for the detection of TDGO in water samples using ICA. The visible detection limit was found to be 10 mg mL1 using the ICA method. The assay was very simple to perform, and the detection was completed within 10 min. In addition, the assay did not require intensive labor or expensive equipment for sample analysis. This technique possesses several advantages in terms of rapidity, specicity, and cost effectiveness, and it is well-suited for eld applications in on-site testing.
18
19 20 21 22 23
Acknowledgements
24
The authors thank the Director, DRDE, Gwalior for his keen interest and encouragement. 25
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