detection of the organophosphorus nerve agent sarin by a competitive inhibition enzyme immunoassay

Arch Toxicol (1995) 69:644-648 �9 Springer-Verlag 1995

Yong-xin Zhou �9 Qing-jin Yan - Yun-xiang Ci Zhen-quan Guo - Kang-Tai Rong �9 Wen-bao Chang Yu-fen Zhao

Detection of the organophosphorus nerve agent sarin by a competitive inhibition enzyme immunoassay

Received: 28 September 1994/Accepted: 2 February 1995

Abstract Two artificial antigens, N'N'-di(O, O-diisopropyl) phosphoryl L-lysine (DIP)- bovine serum albumin (BSA) conjugate (DIP-BSA) and DIP-KLH (keyhole limpet hemocyanin), were synthesized. Anti- bodies against sarin (O-isopropyl methylphosphonofluoridate) were obtained after immunization of rabbits with DIP-KLH conjugate. A competitive inhibition enzyme immunoassay (CIEIA) was developed to detect the organophosphorus nerve agent sarin. The antibody solutions could be inhibited by sarin as low as 10-6mol/l, and the standard curve was linear over 3 orders of magnitude. The coefficients of intraassay and interassay variation of this method were 5.4-6.2% (n = 11) and 8.0-9.5% (n = 6) at a sarin concentration range of 10-3-10-6 mol/1, respectively. The recovery of sarin in water samples at the concentration of 5• 10-5 mol/1 was in the range of 96.8 102.5%. The specificity of the antiserum was assessed by comparing the inhibition induced by sarin with soman, Vx, isopropyl alcohol and isopropyl methyl phosphonic acid. The results showed that less than 5 mmol/l soman, 2mmol/1 Vx, 16 mmol/l isopropyl alcohol and 8 mmol/l isopropyl methyl phosphonic acid did not influence the determination of sarin in water samples.

Key words Artificial antigen- Competitive inhibition enzyme immunoassay - Sarin �9 Detection

This work was supported by the National Nature Science Founda- tion and the State Education Commission of China

Y. Zhou �9 Y. Ci ( ~ ) �9 W. Chang Department of Chemistry, Peking University, Beijing, 100871, China

Q. Yah �9 Y. Zhao Department of Chemistry, Tsinghua University, Beijing, 100084, China

Z. Guo College of Life Science, Peking University, Beijing 100871, China

Introduction

In view of the widespread use of organophosphate, particularly in plant protection as insecticides, and as some of these substances may be used as chemical warfare agents, specific methods for detection are required. Physicochemical methods such as thin- layer chromatography, spectrometry (D'Agostino and Provos 1986) and gas-liquid chromatography (De Jong et al. 1987) exist to detect organo- phosphorous compounds. Though these methods are sensitive, they are time consuming and often difficult to interpret.

In recent years, numerous attempts have been made to produce hapten specific antibodies by conventional polyclonal techniques and the newest hybridoma tech- nology for generating monoclonal antibodies, with varying degrees of success. A competitive inhibition enzyme immunoassay for paraoxon (Hunter and Lenz 1981) and O-pinacolyl O-(p-amino) phenyl methylphos- phonate (MATP) (Schmidt et al. 1988) with polyclonal antibodies was developed. The detection of the organophosphorus nerve agent soman (O-pinacolyl methylphosphonofluoridate) by a competitive inhibition enzyme immunoassay (Hunter et al. 1982) and direct competitive ELISA using monoclonal antibody (Erhard et al. 1990) were also reported. The ability of anti-soman monoclonal antibodies (Lenz et al. 1984) and the anti-Vx antiserum (Rong and Zhang 1990) to compete with acetylcholinesterase (ACHE) for soman and Vx in vitro proved useful in a therapeutic or prophylactic mode for organophosphorus poisoning. The attempts to produce antibodies against sarin, the smallest molecule in the present organophosphorus nerve agent, have been made by a number of laboratories, but with no success (such as Dean 1982, 1987).

In the present study, we have developed a competitive inhibition enzyme immunoassay for detection of the organophosphorus nerve agent sarin.

645

Materials and methods

Chemicals

Bovine serum albumin (BSA), goat anti-rabbit immunoglobulin- horseradish peroxidase conjugate (GAR-HRP) and tetramethyl benzidine (TMB) were purchased from Sigma, St Louis, USA. Keyhole limpet hemocyanin (KLH) was obtained from Professor K.T. Rong, Institute of Pharmacology and Toxicology, Beijing, China.

N~,N~-di(O,O-diisopropyl) phosphoryl L-lysine couplin 9 to BSA

Bovine serum albumin (BSA, 0.3 g) was dissolved in 16 ml mixed solvents (water:DMF:triethylamine=2:l:l), and added into a solution of the above lysine ester (0.8 g) in 5 ml DMF, then stirred slowly at 0~ for 16 h. The solution was dialyzed against distilled water (2 1 with three changes of water per day) at 4~ for 3 days and lyophilized; product DIP-BSA was obtained and stored at - 20~

3tp-NMR(H20, ppm) & 10.3, 8.2

N',N~-di(O,O-diisopropyl) phosphoryl L-lysine coupling to KLH

Instruments

31P-NMR and ~H-NMR spectra were recorded on a Brucker ACP- 200 spectrometer (USA); chemical shifts of IH-NMR are referenced to the internal tetramethyl silicane (TMS), and the 31p-NMR spectrum was referenced to an external solution of 85% HaPO 4. Posit- ive-ion FAB-MS data were obtained on a KYKY Zhp-5 double- focusing mass spectrometer (Scientific Instrument Factory, Beijing, China).

Preparation of DIP-protein conjugates

Synthesis of N~,N~-di(O,O-diisopropyl) phosphoryl L-lysine

L-lysine 20 mmot was dissolved in 20 ml mixed solvents (water: methyl alcohol: triethylamine = 1 : 1 : 2, 0~ added dropwise into solution of 40mmol diisopropyl phosphite (DIP) in 20 ml CCI4, then stirred at 0~ for 2 h. The organic solvents were removed under reduced pressure. The residue was extracted with ethyl acetate (20mix2), and then acidified to pH = 3 by 1 molfl hydrochloric acid. The water phase was extracted with ethyl acetate-tertbutyl alcohol (3:2 v/v, 20 mlx 3). The extract was washed with a saturated NaC1 solution (10mlxl ) and water (10 ml x 1), and dried using anhydrous MgSo 4. The solvents were evaporated and an oily product was obtained.

31P-NMR (EtOAc, ppm) 6: 8.26, 6.53 IH-NMR (CDCI3, ppm) & 1.0-t.5 (m, 30H, 4x (CH3)2C < and -(CH2)3-), 2.8-2.9 (brs, 2H, > N-CH2-, 3.0-3.3 (bs, 2H, 2 x -NH-), 3.6-3.8 (s, 1H, > N-CH-), 4.3--4.6 (m, 4H, 4 x > CH-O-), 11.5 (s, 1H, COOH) FAB-MS: m/z 475(MH +) for C18H40NzO8P2 Structure: ((CH3)2CHO)2PO-NH-CH2-CH2CH2CH2-CH(COOH)- NH-OP(OCH(CH3)2) 2

Synthesis of N',N'-di(O, O-diisopropyl) phosphoryl L-lysine N-hydroxylsuccinimide ester

N',N'-di(O,O-diisopropyl) phosphoryl L-lysine 3 mmol and 3.2 mmol N-hydroxylsuccinimide was dissolved in 20 ml ethyl acetate and added slowly into 4 ml triethylamine containing 3 mmol ethyl phosphodichloridate (EDCP) at 0~ and stirred for 5 h. The reaction solution was washed with saturated NaHCO 3 (10 ml x 5) as well as water (10 ml • 2) and then dried using anhydrous MgSo 4. The solvent was removed under vacuum and an oily product was obtained.

3~p-NMR(EtOAc, ppm) 6: 8.91, 6.24 XH-NMR(CDC13, ppm) 6:1.1-1.3 (m, 30H), 2.0(s, 4H, -(CH2)2-CO ), 2.8-2.9 (brs, 2H), 4.0-4.1 (m, 1H), 4.4-4,6(m, 4H) FAB-MS: m/z 572(MH +) for C22Ha3N3OIoP2

The same procedure as above was used to couple with KLH.

31P-NMR(HzO, ppm) 6: 10.0, 8.2

Immunization schedule

Equal volumes of artificial antigen DIP-KLH conjugate in sterilized 0.9% NaCI and Freund's complete adjuvant (FCA) or incomplete adjuvant (FIA) were mixed and emulsified. FIA was prepared with wool grease and liquid paraffin (1:4, w/w). The concentration of Bacillus Calmette-Guerin in the emulsions was 35 mg/ml for FCA emulsion. New Zealand White rabbits of both sexes, weighing between 2.5 and 3.0 kg, were housed one per cage and supplied with food and water ab libitum. They were immunized five times. For the initial immunization, 20mg/kg body weight DIP-KLH in FCA emulsion on day 0 (day of first immunization) were injected intrader- mally into the pads of two hind legs of each rabbit. DIP-KLH 0.2 mg in 0.4 ml FIA emulsions were injected into the popliteal lymph node on day 10. Then, for the two booster injections, 500 lag DIP-KLH in 1 ml FIA emulsions were injected into multiple sites on the animal's back on day 40 and 70, respectively. After bleeding through the ear vein and titer determination, the solution of 20 lag DIP-KLH into 0.2 ml 0.9% NaCI was injected into the ear vein as the last booster injection. Seven days after the last injection, the rabbits were bled. The red cells were removed by centrifugation, and sera were collected and frozen for subsequent use.

Purification of antibody

Rabbit immunoglobulin was prepared by two precipitations of rabbit serum with 33% saturated (NH4)2SO4. Following exhaustive dialysis against 0.01 mol/l phosphate buffer, pH 7.2, an IgG fraction was prepared using a Protein G-Sepharose 4 Fast Flow Column (MAb Trap TMG, Pharmacia LKB Biotech., Uppsala, Sweden). The eluted antibodies were dialysed against distilled water, sterilized by passage through a 0.22 tam membrane fitter (Millipore, Bedford, Mass., USA), lyophilized and stored at - 20~

Enzyme immunoassay

DIP-BSA was diluted to a concentration of 10.0 lag/ml in 0.05 mol/1 carbonate buffer, pH 9.6, and aliquot of 200 lal were added into each well of 96-well polystyrene microliter plates (Flow Laboratories, Vienna, Va., USA). The plates were incubated at 4~ overnight and subsequently washed three times with PBS-Tween (10 mmol/l PBS, 0.05% Tween-20, pH 7.2).

An initial experiment was performed to determine the antiserum titer. A 100 lal aliquot of serum samples of serial dilution was added into each well coated with DIP-BSA and incubated at 37~ for lh followed by three washings with PBS-Tween. Thirty minutes after adding 100lal of 1/1000 dilution of GAR-HRP to each well, the

646

plates were washed and 100 H1 TMB substrate solution (100 gl 6 mg/ml TMB and 15 gl 30% of H 2 0 2 in 10 ml phosphate buffer, 0.1 mol/1, pH 6.0) was added and reacted at room temperature in the dark for 30 min and then 50 p.l 2 mol/l HzSO 4 was added. The absorbance of each well was measured at 450 nm by Minireader II (Dynatech, Alexandria, Va., USA). Each point was a mean of the absorbance values in duplicate at least. Antibody titer was charac- terized by serum dilution, corresponding to an absorbance of 50%, the highest absorbance, and used for the competitive inhibition enzyme immunoassay.

The methods for the determination of satin and DIP were essentially the same as the antiserum titer determination except that a dilution of 1:106 antiserum (or 8gg/ml purified antibody solution) was mixed for 30 rain at room temperature with an equal volume of sarin (or DIP) of serial log dilution and then added into wells coated with DIP-BSA. DIP-BSA coated wells incubated with antiserum only served as the noninhibited positive controls, while wells coated with unconjugated BSA incubated with antiserum controlled for nonspecific binding of antiserum.

Results

Preparation of antiserum

0.80

0,64

0.4e

0.32

0 , t 6

o, oo 1,oo

&

, J " \ ~ ~ 4 0 5.80 B,20 10.60 13,00

-LOC~Antlsarum DllutionSl

Rmbl01t 1 - I v . Rabbit 2 ---~-" Rel:~it 3

Fig. 2 Anti-DIP antiserum titer. Each point is the mean of three replicates

-- LOG 1 2 3 4 5 6 7 RI 0.73 0.72 0.71 0.68 0.66 0.60 0.49

OD R2 0.74 0.73 0.72 0.71 0.64 0.58 0.34 R3 0.72 0.72 0.72 0.71 0.70 0.68 0.57

- L O G 8 9 10 i1 12 13 R1 0.23 0.13 0.09 0.06 0.04 0.03

OD R2 0.15 0.12 0.07 0.03 0.01 0 R3 0.34 0.28 0.19 0.11 0.06 0.03

Representative results for the production of antibody by three rabbits over a period of 12 weeks are given in Fig. 1. Antibody titer was detected as early as week 3 after initial injection. The most drmnatic in- crease in antibody titer was observed after two subsequent booster injections of DIP-KLH. Due to the novel artificial antigen and the special immunization schedule, the antisera obtained from all three experimental rabbits were adequate for immunoassay studies (Fig. 2).

I e+O~ 1 e+08

le+07

1000000

100000

1oooo t--

lOOO lOO lO

1

0.1 0

/ / . . - . n / ~

,6/-., ""

20 50 80 lO0 ImmuNzstlo~ Time (DAY~

i Flsbbl~ 1 ' - & " Flsl~lt 2 - - e - - Rabbtt 3

Fig. 1 Anti-DIP antiserum titer of rabbits after immunization with DIP-KLH. Each point is the mean of double replicates. ~ booster injection time Days 0 20 50 80 100

R1 0 95 322 9.87 x 105 3.98 x 107 Titer R2 0 60 180 3.81x105 7.94x106

R3 0 143 434 6.63x106 1.25x10 s

I - -

100 .

6O 40 ~" 2O

0 I I I - ~ ,a,'~-.-~ 0 2 4 6 8

�9 -t.CX741nhINt orl wml/L

�9 DiP - A... ~ I I N

lO

Fig. 3 CIEIA for detection of sarin and DIP inhibition of rabbit 3 antiserum by various satin and DIP concentrations. All results are the average of four analysis sets (SD was always lower than 6.2%)

-- Log [DIP] 0.301 1.000 1.30i 2.301 3.301 4.301 Inhibi t ion% 97.8 95.6 93.5 91.3 58.7 37.0

- Log [DIP] 6.301 7.301 8.301 9.301 Inhibi t ion% 6.50 2.17 0 0

- Log sarin 0.427 1.427 2.427 3.427 4.427 5.427 Inhibi t ion% 98.1 96.3 63.0 48.1 35.2 18.5

- Log sarin 6.427 7.427 8.427 Inhibi t ion% 3.7 0 0

5.301 23.9

CIEIA for detection of diisopropyl phosphite and sarin

In CIEIA the antibody solutions were inhibited by various DIP concentrations. A positive correlation was seen between DIP concentration and inhibition of antibody activity (Fig. 3). The CIEIA detected DIP concentrations as low as 10-7 mol/1 and the standard curve

generated was nearly linear between 10 - 3 and 10 . 6 mol/1 DIP. Figure 3 also showed that the antibody solutions could be inhibited by sarin concentrations as low a s 10 - 6 tool/l, and the standard curve was linear over 3 orders of magnitude. This allows for the preparation of dilutions of test samples over a wide concentration range, with the assurance that one or more values will fall on the standard curve.

The coefficients of intraassay and interassay variation of this method were 5.4%-6.2% (n = 11) and 8.0-9.5% (n = 6) at a sarin concentration range of 10-3-10-6 mol/1, respectively. The recovery of sarin in water samples at a Vx of 5 x 10 -5 tool/1 was in the range of 96.8-102.5%. The specificity of the antiserum was assessed by comparing the inhibition induced by sarin with soman, Vx, isopropyl alcohol and isopropyl methyl phosphonic acid. The results showed that less than 5 mmol/1 soman, 2mmol/1 Vx, 16 mmol/1 isopropyl alcohol and 8 mmol/l isopropyl methyl phosphonic acid did not influence the determination of sarin in water samples. DIP-protein conjugates appear to be quite stable and entirely nontoxic, so the assay is particularly well suited for detecting the nerve agent sarin.

Discussion

It is well known that organophosphorus compounds may be used as pesticides and warfare agents. Although rapid progress on the development of new physicochemical methods for their detection and on some specific antidotes for their therapeutics or pro- phylaxis has been made during recent years, methods for analysis and prophylactics of organophosphorus have not been improved dramatically. It has been sug- gested that the immunoassay and immunoprophylactic treatment would be a desirable approach for organophosphorus compounds, and what is more, development of abzymes (catalytic antibodies) is a new important way to study reactions related to organophosphorus compounds.

To obtain the antibodies, chemical vaccines and abzymes, it is necessary to design artificial antigens of organophosphorus compounds. Most organophosphorus compounds have no reactive group for the coupling reaction with protein carriers, and it is necessary to introduce a ."bridge" group into the organophosphorus molecule. If the bridge group is more immunogenic than the organophosphorus molecule, antibodies directed against the bridge group occur with some regularity. In a typical experiment (Hunter et al. 1982), p-aminophenyl group and diazo bond were used for the bridge. Mice were immunized with soman deri- vation MATP linked onto BSA (MATP-BSA). Only one in six mouse spleen cells produced antibodies re- cognizing the hapten soman, but the cells producing antibodies to the bridge (or part of the hapten-bridge)

647

o II

H3C O H3C H7C310- P-OIC3H7 \ // H c~CHO, ~/~ OIC, Hr NH! Oil

H3C~cHO" PNF I~3c~CHO, rNH O =p-NH--(CH2)4-CH-C-NH--proteln c I H3 H3C OiC3H z

a b c

Fig. 4 The structure of sarin, DIP and DIP-protein conjugate. a sarin, b DIP, e DIP-protein

were about 93.75%. The polyclonal antibodies of rabbits immunized with MATP-KLH did not erossreact with soma, but were inhibited by the p-nitrophenyl ester analog of soman (Lenz et al. 1984). These facts further proved that when the bridge is larger and more complex than the hapten in structure (e.g. containing more ring-like structure), the antibodies are mainly against the bridge or/and the bridge-hapten but not the hapten. We chose L-lysine, which is contained in proteins carriers (such as BSA and KLH), as the bridge of the conjugating hapten (here, an organophosphorus compound) and protein carriers, thus avoiding the influence of anti-bridge antibodies.

The main purpose of this paper was to develop an immunoassay for the organophosphorus nerve agent sarin. Because sarin has no reactive group for coupling with proteins, and its characteristic group, unlike other organophosphorus nerve agents (such as soman and Vx), is on O-isopropyl phosphoryl phosphoryl group, we considered that DIP, which has two O-isopropyl groups, was suitable to conjugate its L-lysine derivative to a protein carrier as the artificial antigen for the introduction of anti-sarin antibody in animals (Fig. 4). DIP derivatives were easily obtained through the reaction of DIP with amino acids or peptides (Zhao et al. 1988), and successfully conjugated to protein carriers by means of activated ester (Zhao and Ma 1991). This preparation has been shown to be adequate for antibody production. Anti-sarin antibody, as we report here, is the antibody to the smallest organophosphorus molecule so far.

For so small an organophosphorus molecule, the present CIEIA for sarin was considered a sensitive immunological method. As there is a need to detect sarin concentrations of below 1 p.mol/1 in drinking water, a more sensitive assay, such as time-resolved fluorescence immunoassay using the antibodies obtained, should be developed.

The implications of the antibodies in the immunop- ropylactic and immunotherapeutic treatment will be studied in mice in a passive immunization regimen. The antibodies can be injected into the mice by the intra- venous route immediately before sarin challenge or by the intraperitoneal route at different times before sarin challenge, and their survival and the dose-effect rela- tionship can be examined.

648

In conclusion, two artificial antigen, DIP-BSA and DIP-KLH, were prepared through proteins coupling with N-phosphoryl L-lysine, and then polyclonal antiserum was obtained after immunization of rabbits with DIP-KLH. A competitive inhibition enzyme immunoassay was used to detect DIP and sarin concentrations as low as I0-7 and 10-6 mol/1, respectively.

Acknowledgements The authors would like especially to thank Pro- fessor Rong K.T. from the institute of Pharmacology and Toxicol- ogy for his help.

References

D'Agostino PA, Provost LR (1986) Capillary column ammonia chemical ionization mass spectrometry of organophosphorus chemical warfare agents and stimulants. Biomed Environ Mass Spectrum 13:231-236

Dean RG (1982) Ultrasensitive detection of chemical substance. Defence Advanced Research Projects Agency, R & D Status Report, USA. AD-A122300

Dean RG (1987) Ultrasensitive detection of chemical substance. Defence Advanced Research Projects Agency, R & D Status Report, USA. AD-A188149

De Jong LPA, Bijleveld EC, Van Dijk C, Benschop HP (1987) Assay of the chiral organophosphate soman in biological samples. Int J Environ Anal Chem 29:179-197

Erhard MH, Kuhlmann R, Szinicz L, Losch U (1990) Detection of the organophosphorus nerve agent soman by an ELISA using monoclonal antibodies. Arch Toxicol 64:580-585

Hunter Jr KW, Lenz DE (1981) Detection and Quantification of the organophosphate insecticide paraoxan by competitive inhibition enzyme immunoassay. Life Sci 30:355-361

Hunter Jr KW, Lenz DE, Brimfield AA, Naylor IA (1982) Quantifi- cation of the organophosphorus nerve agent soman by competitive inhibition enzyme immunoassay using monoclonal antibody. FEBS Lett 149:147-154

Lenz DE, Hunter Jr KW, Benschop HP, DeJong LPA, Van Dijk C, Clow TR (1984) Studies using a monoclonal antibody against soman. Fundam. Appl Toxicol 4:S156-$164

Rong KT, Zhang LJ (1990) Immunologic protection against VX intoxication in experimental animals. Pharmacol Toxicol 67: 255-259

Schmidt P, Kuhlmann R, Losch U (1988) A competitive enzyme immunoassay for detection and quantification of organophosphorus compound. Z Naturforsch 43c: 167 172

Zhao YF, Ma XB (1991) Synthesis of N-phosphoryl di(or tri)-peptides through the active ester method. Phosphorus Sulfur Silicon 61:9-18

Zhao YF, Ji G J, Zeng JN, Xue CB (1988) Synthesis of N-O, O- diisophosphoryl amino acids and dipeptides. Synthesis 6: 444-448

detection of the organophosphorus nerve agent sarin by a competitive inhibition enzyme immunoassay

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