Development of an ELISA for detection of an organophosphorus compound using monoclonal antibodies

This paper describes a specific and highly sensitive ELISA system using monoclonal antibodies in order to assay an organophosphorus compound. The soman derivative methyl phosphonic acid, p-aminophenyl 1,2,2,-trimethyl-propyl diester (MATP) served as model substance. In order to obtain antibody-producing hybridomas BALB/c mice were immunized with MATP linked onto human serum albumin (HSA). The spleen cells of immunized mice were fused with syngenic plasmacytomas of the non-producer-line X63Ag8.653 with the aid of polyethylene glycol. To eliminate undesirable cross-reaction, common screening procedures were modified by directly coating the ELISA plates with hapten. Five out of 15 positive cell-lines were cloned by limiting dilution and further propagated. The respective immunoglobulin class and subclass of the obtained monoclonal antibodies was determined. Four of which were identified as IgG₁, the other as IgG_2a. After enrichment of antibodies in ascites and their isolation by protein A-sepharose, the affinity of various monoclonal antibodies was estimated in competitive inhibition enzyme immunoassay (CIEIA) by measuring the IC₅₀ rates of free MATP. The rates were found to lie between 2.5 × 10^–6 mol/l and 4.3 × 10^–4 mol/l MATP. The IC₁₀ rate for detectable MATP concentration was 5.4 × 10^–7 mol/l MATP. Test duration was 280 min. The reactivity of the monoclonal antibodies with structurally related substances was used to check their specificity. Cross-reaction turned out to be negative. In order to develop a direct competitive ELISA, MATP was linked to horse radish peroxidase (HRPO) by adding a spacer. This helped to reduce total duration to 40 min. The detection level was further reduced to 1.3 × 10^–7 mol/l MATP (corresponding to 975 pg/25 l test-buffer) using the monoclonal antibody F71D7. Likewise, MATP was detected in goat serum, chicken serum, rabbit serum, milk and company's water in concentrations between 2.1 × 10^–7 mol/l (IC₁₀, company's water) and 4.9 × 10^–8 mol/l (IC₁₀, milk).     

血液灌流对硫线磷和硫酸阿托品的吸附作用

目的 定量研究血液灌流对有机磷农药硫线磷和其解毒药阿托品的吸附作用.方法 模拟临床血液灌流装置,对含硫线磷和硫酸阿托品的血样进行灌流吸附,分别用毛细管气相色谱法和高效液相色谱法测定硫线磷和硫酸阿托品的残留量.结果 吸附剂用量为0.5、1.0和1.5 g,包膜活性炭在灌流2.0 h后硫线磷的清除率均能达到90%以上,硫酸阿托品的清除率依次为61.9%、84.9%和88.9%;HA230树脂在灌流1.5 h后硫线磷清除率都达到90%以上,硫酸阿托品的清除率也依次高达88.0%、97.2%和98.4%;包膜活性炭灌流3.0h后,硫酸阿托品与硫线磷的比值最高为灌流前的10.1倍,而HA230树脂灌流后,此比值最高为灌流前的6.7倍.结论 包膜活性炭和HA230吸附树脂血液灌流1.5～2.0 h均能清除血中大部分硫线磷,而且均能增加血中硫酸阿托品和硫线磷浓度的比值.     

Detection of the organophosphorus nerve agent soman by an ELISA using monoclonal antibodies

The development of a specific and sensitive immunologic ELISA detection system for methylphosphonoflouridic acid. 1,2,2-trimethylpropylester (soman) by the use of monoclonal antibodies (MAbs) is described. The monoclonal antibodies F71D7, F71H10, F71B12 and F71H9 originally produced against the soman derivative methyl phosphonic acid,p-aminophenyl 1,2,2-trimethylpropyldiester (MATP) also reacted with soman in a previously developed, direct competitive ELISA. After optimizing the ELISA system by varying the reaction mixture and the solvents for the organophosphate, 5.0×10^–7 mol/l soman (80% purity), e.g. 2.5 ng or 2 ng pure soman per 25 l test buffer, could be detected after a total test duration of 40 min. A shortening of the incubation time to 10 min resulted in a drop of sensitivity to 1.8×10^–6 mol/l soman. Various alcohols which may be used as extraction media for soman from various materials (isopropanol, ethanol and methanol) were shown to inhibit peroxidase activity and thereby reduce the sensitivity of the test. However, the influence of alcohols decreased with the shortening of incubation time. All monoclonal antibodies showed little cross reactivity to sarin and no cross reactivity to tabun and VX. Judging on the reactivity of the MAbs with MATP and soman oxidazed by 1,2-dihydrobenzol, some reactivity with some other (non-toxic) soman analogues containing the same pinacolyl group can be expected. There was no evidence for stereoselectivity of the MAbs tested. Finally, soman could be detected in different biological samples like human serum, goat serum, rabbit serum, chicken serum, milk, and tap water in concentrations between 1.3×10^–6 and 2.0×10^–6 mol/l.     

胆碱酯酶监测在抢救有机磷农药中毒中的临床意义

目的 探讨胆碱酯酶活性在决定有机磷农药中毒抢救中氯磷定、阿托品的用量,停药时间及对患者预后判断的作用。方法 将口服有机磷农药中毒抢救传统治疗组列为对照组（３２例）,将口服有机磷农药中毒采用胆碱酯酶指导氯磷定、阿托品用量及停药列为观察组（５０例）。结果 观察组阿托品用量和氯磷定用量与对照组阿托品用量和氯磷定用量有显著差异（Ｐ〈０．０１）。观察组治愈５０例,治愈率１００％;对照组治愈２８例,治愈率８７     

Reactivating and protective effects of pyridinium compounds in human erythrocyte acetylcholinesterase inhibition by organophosphates in vitro

The reactivation of human erythrocyte acetylcholinesterase (AChE, EC 3.1.1.7) inhibited by O-ethyl-S-2-di-isopropylaminoethyl methylphosphonothioate (VX) and the protection against AChE inhibition by O-1,2,2-trimethylpropyl methylphosphonofluoridate (Soman) was studied with sixteen quaternized pyridinium compounds. TMB-4 which is known as a good reactivator of AChE inhibited by organophosphates proved to be the most effective reactivator. Of the tested newly synthetised compounds 3 were fairly good reactivators of methylethoxyphosphonylated AChE. These compounds have 2 pyridinium rings connected by a dimethylether link and a hydroxiiminomethyl group in position 2 of one pyridinium ring, while the radicals of the other pyridinium ring are benzoylcarbonyl, cyclohexylcarbonyl or amidocarbonyl residue.The rate of reactivation with these compounds followed a two-phase pattern, being fast at the beginning and then slowing down to an equilibrium. Kinetic treatment of the first-phase reaction course yielded the second-order rate constants of reactivation. All 3 compounds had similar reactivating efficiency (k _r values range from 0.8×10³ to 3.6×10³ M^–1 min^–1) and in effective concentrations (1 to 100 M) they also inhibited AChE (K _i(app) values range from 0.11 to 0.19 mM). Their reactivating properties were not better than those revealed by TMB-4 (k _r= 19.4×10³ M^–1 min^–1) which was tested as a reference compound.HGG-12, HGG-42 and HI-6 were also found to exert a good protective effect against AChE inhibition by Soman; no protection was obtained with TMB-4.     

经食道心脏起搏在救治口服有机磷农药中毒心跳骤停中的应用体会

目的探讨口服有机磷中毒患者心跳骤停时应用食道心脏调搏进行救治的疗效。方法15例心脏骤停患者,在常规治疗基础上应用食道心脏调搏方法抢救。结果15例中救治成功13例。结论经食道心脏起搏方法简便、操作速度快、无创、无需消毒、效果良好,适用于临床紧急起搏,尤其适用于基层医院。     

呼吸机联合阿托品与氯解磷定治疗重度有机磷农药中毒的临床研究

目的观察呼吸机联合不同剂量阿托品和氯解磷定治疗濒死重度有机磷农药中毒的临床效果。方法117例濒死重度有机磷农药中毒患者,根据是否用使用呼吸机分为呼吸机联合阿托品氯解磷定组（治疗组）,仅使用阿托品氯解磷定、未用呼吸机组（对照组）。治疗组根据人院后最大阿托品用量分为两亚组,适量组：最大用量≤100mg／h;大剂量组：最大用量〉100mg／h。结果治疗组痊愈率高于对照组,差异有统计学意义（P〈0．01）。两亚组比较,适量组死亡率和阿托品中毒率低于大剂量组,阿托品总用量少于大剂量组（P〈0．01）。两亚组间机械通气时间、ICU住院时间、总住院时间差异无统计学意义（P〉0．05）。结论抢救濒死重度有机磷农药中毒,应在阿托品联合复能剂的基础上应用机械通气,机械通气要及时,阿托品应该足量、个体化使用,尽量避免大剂量应用,用药过程中严密观察患者的反应。     

Effects of organophosphorus pesticides and their ozonation byproducts on gap junctional intercellular communication in rat liver cell line.

The effects of organophosphorus pesticides (OPs), oxons and their ozonation byproducts on gap junctional intercellular communication (GJIC) on cultured BRL cell line were investigated using scrape loading and dye transfer (SL/DT) technique. The neutral red uptake assay was used to identify the non-cytotoxic levels of diazinon, parathion and methyl-parathion applied to GJIC assay. The concentration-dependent inhibition of GJIC was observed over a range of 50-350 mg/l diazinon, parathion and methyl-parathion after 90 min incubation compared with the vehicle control. However, oxons and ozonation byproducts of OPs had no inhibition effect on GJIC at any of the concentrations tested. The inhibition of GJIC by OPs was reversible after removal of the tested pesticides followed by incubation with fresh medium. The present study suggested that the ozonation treatment could be used for the detoxification of drinking water and food crops contaminated with diazinon, parathion and methyl-parathion without formation of GJIC toxicity.     

急性有机磷中毒患者心肌酶和心电图的变化及临床意义

目的　探讨急性有机磷中毒 (AOPP)患者血清肌酸激酶 (CK)、肌酸激酶同工酶 (CK -MB)、乳酸脱氢酶 (LDH)和心电图的变化规律及其临床意义。方法　 6 1例AOPP患者 (中毒组 )在中毒后第 1～ 10天分别检测血清心肌酶和心电图 ,同时选择 41例正常人作对照。结果　 (1)与对照组比较中毒组的CK、CK -MB、LDH水平在第 1～ 10天均明显增高 ,差异有显著性 (P <0 0 5～ 0 0 1) ,在第 2～ 4天有较高的峰值平台 ;(2 )轻、中、重度中毒组和对照组间血清中CK、CK -MB、LDH水平的两两比较差异均有显著性 (P <0 0 1) ;(3)心电图异常改变构成比随中毒程度加重而增大 ,重度中毒组心电图异常改变占严重心律失常的主要比例。结论　在中毒的第 2～ 4天组织损害越严重 ,血清中各酶水平增加越明显 ,则组织损害更加严重 ;中毒程度越深 ,心电图异常改变越多 ,严重的心律失常也更多 ,对重度中毒患者应进行心电监护 ,以避免心源性猝死     

经鼻气管插管机械通气在急性有机磷农药中毒伴呼吸衰竭中的临床应用

目的:探讨经鼻气管插管机械通气抢救急性有机磷中毒伴呼吸衰竭的临床应用价值。方法:32例急性有机磷农药中毒伴呼吸衰竭患者,随机分成2组,实验组(16例)给予经鼻气管插管机械通气;对照组(16例)行经口气管插管机械通气治疗。机械通气模式先后A/C,SIMV PSV PEEP或PSV PEEP。结果:实验组16例患者中,15例治愈,1例死亡,平均住院时间8.6d。对照组中16例,15例治愈,1例死亡,平均住院时间12.5d。治愈率两者差异无统计学意义(P>0.05),但平均住院时间两者差异有显著统计学意义(P<0.01)。结论:及时经鼻气管插管使用机械通气抢救急性有机磷农药中毒伴呼吸衰竭是有效的,且能有效缩短住院时间。