A Novel Immunoreagent for the Specific and Sensitive Detection of the Explosive Triacetone Triperoxide (TATP)

Triacetone triperoxide (TATP) is a primary explosive, which was used in various terrorist attacks in the past. For the development of biosensors, immunochemical µ-TAS, electronic noses, immunological test kits, or test strips, the availability of antibodies of high quality is crucial. Recently, we presented the successful immunization of mice, based on the design, synthesis, and conjugation of a novel TATP derivative. Here, the long-term immunization of rabbits is shown, which resulted in antibodies of extreme selectivity and more than 1,000 times better affinity in relation to the antibodies from mice. Detection limits below 10 ng L−1 (water) were achieved. The working range covers more than four decades, calculated from a precision profile. The cross-reactivity tests revealed an extraordinary selectivity of the antibodies—not a single compound could be identified as a relevant cross-reactant. The presented immunoreagent might be a major step for the development of highly sensitive and selective TATP detectors particularly for security applications.


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carbonate (DSC, tip of a spatula) was given to the stirring mixture to ensure a water-free solution [2]. After addition of N,N′-dicyclohexylcarbodiimide (DCC, 8.2 µmol, 1.71 mg in 22 µL anhydrous THF) the reaction batch stirred over night at room temperature and protected from light. This procedure was performed under argon atmosphere and the molar ratio of hapten, NHS and DCC was 5:6:6. In order to separate the solution with the activated NHS ester of the TATP hapten from the precipitate (mainly dicyclohexyl urea), the mixture was centrifuged at 15 °C and 16,400 rpm for 10 min. The supernatant was added dropwise to a sodium hydrogen carbonate buffered BSA solution (0.18 µmol, 12 mg in 899 µL 130 mM NaHCO 3 , pH 8) and stirred for 3.5 h at room temperature. The purification of the TATP immunogen was performed on a PD-10 desalting column (with Sephadex™ G-25, GE Healthcare) using diluted phosphate buffered saline (PBS, 1 mM sodium dihydrogen phosphate (NaH 2 PO 4 ·2 H 2 O), 7 mM sodium hydrogen phosphate (Na 2 HPO 4 · 2H 2 O), 14.5 mM sodium chloride (NaCl), pH 7.6) as the eluent. The conjugate was collected manually in a UV-transparent 96-well microtitration plate (UV-Star ® -96 Microplate, 96 Well, Greiner Bio-One, Frickenhausen, Germany) and measured at 280 nm in a MTP reader. A mean coupling ratio of 14 hapten molecules per BSA (molar ratio 38:1 in synthesis) was determined via MALDI-TOF-MS [3] (Figure S-1) and a protein concentration of 7.8 g L −1 was photometrically determined at 280 nm based on a BSA calibration.

Synthesis of HRP Conjugate
To perform an ELISA (enzyme-linked immunosorbent assay), an enzyme conjugate consisting of TATP hapten and horseradish peroxidase (HRP) is needed. The protocol is similar to the synthesis of the immunogen (see above). The hapten-NHS ester was prepared in anhydrous N,N′-dimethylformamide (DMF) without adding DSC. The molar ratio of TATP hapten (4.3 µmol, 1.37 mg in 80 µL DMF), NHS (8.5 µmol, 1.01 mg in 31.5 µL DMF), and DCC (8.5 µmol, 1.77 mg in 28.3 µL DMF) was set to 1:2:2. 62 µL of the centrifuged reaction mixture was dripped into a sodium bicarbonate buffered HRP solution (0.089 µmol, 3.9 mg in 630 µL 130 mM NaHCO 3 , approx. pH 8), which corresponds to a ratio of 21:1 of TATP hapten to protein in the reaction mixture. MALDI-TOF-MS [3] measurements showed a mean coupling ratio below 1 hapten molecule per HRP. The concentration of the purified HRP conjugate was determined to be 4.2 g L −1 , which finally was diluted 1:2 with Peroxidase Conjugate Stabilizer (Guardian™, Thermo Scientific) and stored at 4 °C .

Enzyme-Linked Immunosorbent Assay (ELISA)
ELISA was performed at room temperature in transparent microtiter plates with a high binding surface (Thermo Scientific/Nunc Immuno 96 MicroWell™ Solid Plates MaxiSorp™, C96, No. 446612). Each well was coated with 200 µL of 0.9 mg L −1 anti-rabbit IgG in PBS (10 mM sodium dihydrogen phosphate (NaH 2 PO 4 · 2H 2 O), 70 mM sodium hydrogen phosphate (Na 2 HPO 4 ·2 H 2 O), 145 mM sodium chloride (NaCl), pH 7.6). The Parafilm ® -covered plates were shaken 18-24 h at 750 rpm followed by the first washing step. A 96-channel plate washer (ELx405 Select™, BioTek Instruments, Bad Friedrichshall, Germany) was used to wash the plates three times with washing buffer (washing buffer: 0.75 mM potassium dihydrogen phosphate (KH 2 PO 4 ), 6.25 mM dipotassium hydrogen phosphate (K 2 HPO 4 ), 0.025 mM sorbic acid potassium salt, 0.05% (v/v) Tween™20, pH 7.6). Next, 200 µL rabbit sera diluted in PBS containing 0.005% (w/v) BSA were pipetted in the wells and was incubated by shaking for 1 h. The required dilutions depended on the stage of the immunization and the type of experiment, varying from 1:5,000 to 1:600,000. Typically, serum of boost 7 was diluted 1:80,000 and serum after boost 11 was diluted 1:100,000. Thereafter, another washing step was performed and 200 µL of TATP standard solution was added in triplicate. Finally, 50 µL of diluted HRP conjugate was added. The standards (seven or 31, depending on the experiment) were prepared from a methanolic TATP stock solution of 10 g L −1 , by dilution in water, starting from 500 µg L −1 to 0.001 µg L −1 (seven calibrators) 2,700 µg L −1 to 0.0008 µg L −1 (31 calibrators). A blank of water was used as the first point in the calibration curve. The stabilized HRP conjugate (2.1 g L −1 ) was diluted from 1:20,000 to 1:600,000, depending on the experiment and the immunization stage. For boost 7, the HRP conjugate was diluted 1:100,000 and for boost 11 the dilution was 1:300,000, for example. After shaking standards and HRP conjugate for 30 min, the plate was washed again. Directly afterwards, 200 µL substrate solution was added to the wells and incubated on a plate shaker. The substrate solution for one plate was always freshly prepared with 21.6 mL citrate buffer (220 mM potassium dihydrogen citrate, 0.5 mM sorbic acid potassium salt, pH 4), 8.34 µL H 2 O 2 (30%) and 540 µl TMB solution (according to Frey et al. [4]: 40 mM 3,3′,5,5′-tetramethylbenzidine (TMB) and 8 mM tetrabutylammonium borohydride in N,N′-dimethylacetamide). The immobilized HRP conjugate and its substrates TMB and hydrogen peroxide cause the formation of a blue color, which turns to yellow, when 100 µL sulfuric acid (1 M H 2 SO 4 ) is added to stop the reaction after 10-45 min, depending on the ELISA conditions. A microplate spectrophotometer (SpectraMax Plus 384 , Molecular Devices) controlled by Softmax ® Pro 5.3 software measured the absorbance of each well at 450 nm using 620 nm as reference.
DADP (CAS No. 1073-91-2) was synthesized following the procedure of Dubnikova et al. [5]. Washed and dried TATP (0.47 mmol) was dissolved in anhydrous chloroform (500 µL) with a catalytic amount of p-toluenesulfonic acid (~0.002 mmol, <5 g L −1 ). The mixture was incubated for two weeks at room temperature and protected from light. Subsequently, the solvent was evaporated and the product was recrystallized from hot methanol. After the second recrystallization, the DADP crystals were kept in the methanolic mother liquor to avoid potential decomposition. A 10 g L −1 DADP stock solution in methanol was gravimetrically prepared without completely removing methanol from the crystals (Therefore, the concentration is only an estimated value.). The success of the DADP formation was proven via X-ray crystallography and NMR (data not shown).
HMTD (CAS No. 283-66-9) was synthesized according to Wierzbicki and Cioffi [6]. Hexamethylenetetramine (0.75 mmol) was dissolved in aqueous hydrogen peroxide (9.24 mmol, 30%). The mixture was cooled to 0 °C and after cautiously adding solid citric acid (0.92 mmol), the temperature was slowly increased to room temperature. Five days later, the formed HMTD crystals were thoroughly washed with water and ice-cold methanol and then dried carefully under reduced pressure. X-ray crystallography (data not shown) verified the resulting product in comparison to Schaefer et al. [7]. A 1 g L −1 stock solution was prepared gravimetrically in dimethyl sulfoxide (DMSO), because of the poor solubility of HMTD in numerous solvents.
Analogous to the production of TATP, three other cyclic triperoxides (raw isomer mixtures) were synthesized from butanone, 2-pentanone and 3-pentanone instead of acetone. As described before, 3 mmol of the corresponding ketone was mixed with hydrogen peroxide (1.5 mmol, 30%) and sulfuric acid (0.015 mmol, 2 M) at 0 °C. After 48 h, the stock solutions were prepared by dissolving the reaction mixtures completely in 10 ml methanol each. A TATP synthesis was carried out in parallel. Its yield of about 60% was determined with ELISA in comparison with the purified TATP. The concentrations of the other three triperoxides (tri-butanone triperoxide, tri-2-pentanone triperoxide, and tri-3-pentanone triperoxide) were assumed to be similar to the TATP synthesis. Because of the lack of crystals and difficulties to purify the desired triperoxide products, X-ray studies were unfeasible, but NMR measurements suggested the successful synthesis of the respective products (data not shown).
Stock methanol, estimated to be ~1 g L −1 , was diluted 1:100 in water to approximately 10 mg L −1 . This solution of TATP hapten was compared with a 10 mg L −1 TATP solution by HPLC (conditions specified in Walter et al. [8]) and thus, the concentration was calculated to be 8.88 mg L −1 . A series of six consecutive 1:10 dilutions in water was prepared to determine the cross-reactivity of the TATP hapten. Furthermore, all other stock solutions were also diluted sequentially 1:10 in water to have seven aqueous solutions of each substance. The dilution series of acetone, hydrogen peroxide, and 7-oxooctanoic acid started with 10 g L −1 as the highest concentration; DADP, 18-crown-6, 12-crown-4, TNT, RDX, HMTD, ammonium nitrate, and nitroguanidine with 100 mg L −1 , and the dilution series of PETN and HMX started with 50 mg L −1 and 10 mg L −1 , respectively. The three other cyclic triperoxides and the TATP control synthesis were diluted 1:200 and 1:2,000, respectively, independent of their concentration.