γ-Hydroxyphosphonate inducing single-chain antibodies capable of catalyzing soman hydrolysis

Abstract

A γ-hydroxyphosphonate P 6 ( O 1 -methyl- O 2 -(1, 2, 2-trimethylpropyl)-2-hydroxy-5- nitrophenyl methylphosphonic acid) which is proposed to be an analog of the transition state in hydrolysis of soman was synthesized. Artificial antigens were obtained by conjugating P 6 to the carrier proteins BSA (bovine serum albumin) and LPH ( Limulus polyphenus hemocyanin). Mice were immunized with P 6 -LPH and recombinant single-chain antibody phage display library was constructed. After 4 rounds of panning against P 6 -BSA and competitive inhibition enzyme immunoassay, more than 70 strains of phage antibodies capable of binding soman were obtained and 11 of them can accelerate the hydrolysis reaction of soman. One of them (EP 6 ) was studied further. Soluble single-chain antibody was prepared and purification was performed by gel filtration and ion exchange chromatography. The kinetic experiment was carried out showing that the turnover number k cat = 198 min -1 and the rate enhancement k cat / k uncat = 122 419. When 0.16 mg mL -1 EP 6 was preincubated in vitro with 0.132 mmol L -1 (220 mg kg -1 = 1.1×LD 95 ) of soman prior to the administration to mice by subcutaneous route, all animals (19 mice) survived whereas all the control mice (14) treated with PBS and soman died within 30 min. Furthermore, EP 6 could prolong the latent time of spasm and death when mice were passively immunized with EP 6 intravenously 15 min before 1×LD 95 of soman challenge. These results demonstrate that EP 6 is able to increase the rate of soman degradation and protect against soman's toxicity, especially in vitro.