The use of enzyme-linked immunosorbent assay for the quantitation of Calloselasma rhodostoma (Malayan pit viper) venom and venom antibodies.

The specificity and sensitivity of an indirect and two (an ‘ordinary’ and a ‘rapid’) double sandwich enzyme-linked immunosorbent assay (ELISA) procedures for the quantitation of Calloselasma rhodostoma (Malayan pit viper) venom were examined. The three assays were equally sensitive and the accuracy of the assays was not substantially affected by individual variation in the venom composition. The specificity of the assays was examined against 26 venoms from snakes of the families Viperidae and Elapidae. While the double sandwich ELISA procedures were sufficiently specific to be used in the clinical immunodiagnosis of C. rhodostoma bite in Malaysia, the indirect ELISA procedure exhibited extensive cross-reactivity with other Malaysian pit viper venoms. Attempts were made to improve the specificity of the indirect ELISA procedure for the quantitation of C. rhodostoma venom. A ‘low ELISA cross-reactivity’ venom fraction (termed VF52) was isolated from C. rhodostoma venom by repeated Sephadex G-100 gel filtration chromatography. The indirect ELISA procedure using antibodies to VF52 as immunoreagent showed an improvement in specificity. The use of the indirect ELISA procedure for the detection of C. rhodostoma antibodies was also examined and the results show that the assay was sufficiently specific to be used for retrospective diagnosis of C. rhodostoma bite in Malaysia, in particular when VF52 was used as the coating antigen.     

Action of Calloselasma rhodostoma (Malayan pit viper) venom on human blood coagulation and fibrinolysis using computerized thromboelastography (CTEG)

The effects of Malayan pit viper (Calloselasma rhodostoma) venom on human blood coagulation and fibrinolysis were studied in vitro using computerized thromboelastography. At low concentrations the venom had a coagulant effect shown by faster onset of the coagulation process (shortened SP and R), faster progress of the clot (increased angle and shortened K), and increased coagulation (TEG) index. The maximum amplitude (MA) was not affected, suggesting that the venom had no apparent effect on platelet function; and clot lysis was similar to that in the controls, suggesting that there was no primary fibrinolytic activity. At higher concentrations the venom had anticoagulant effects, SP and R were progressively shortened, but there was poor/no progress in the clot formed, evident from prolonged or absent K, diminished MA and reduced angle. These results show that C. rhodostoma venom has both coagulant and anticoagulant actions. The coagulant action may be due to Factor X activator predominance at low concentrations, while the anticoagulant action could be due to ancrod action. TEG is able to demonstrate the dual effect of this venom, previously described as a paradox, and may be a useful tool in the diagnosis and monitoring of envenomation patients.     

α-Fibrinogenase from Agkistrodon rhodostoma (Malayan pit viper) snake venom

By means of DEAE-Sephadex A-50 column chromatography, Agkistrodon rhodostoma (Malayan pit viper) snake venom was separated into eleven fractions. Fraction II had fibrinogenolytic activity, and when further purified by gel filtration was homogeneous, as judged by sodium dodecylsulfate polyacrylamide gel electrophoresis. It had a single peptide chain with a molecular weight of 25,360 and an isoelectric point greater than 10. The fibrinogenolytic activity was completely destroyed after heating for 30 min at 60°C at pH 5.6, 7.4 or 8.8. This enzyme cleaved specifically the α(A) chain of monomeric fibrinogen, without cleaving the β(B) chain or γ chain. The specific fibrinogenolytic activity was 51 mg fibrinogen/min per mg protein. This enzyme showed proteolytic activities toward fibrinogen, fibrin and casein, but was devoid of phospholipase A and tosyl-l-arginine methylester esterase activities which are found in the crude venom. The fibrinogenolytic activity was inhibited by EDTA and cysteine, but not by ε-aminocaproic acid.     

Enzymatic activities of Calloselasma rhodostoma (Malayan pit viper) venom

The enzyme contents of four venom samples of Calloselasma rhodostoma were analyzed. The venoms contained phosphodiesterase, alkaline phosphomonoesterase, 5'-nucleotidase, protease, phospholipase A, L-amino acid oxidase, hyaluronidase, arginine ester hydrolase, arginine amidase, fibrinogenase and coagulant enzyme activities. There is significant variation in the contents of coagulant enzyme, arginine ester hydrolase, hyaluronidase, protease, phosphodiesterase, alkaline phosphomonoesterase and L-amino acid oxidase. DEAE-Sephacel ion exchange chromatography of the venom resolved it into eight major protein fractions. The eight fractions were heterogeneous and exhibited more than one type of enzymatic activity. The 5'-nucleotidase, alkaline phosphomonoesterase, protease, coagulant enzyme, arginine ester hydrolase, arginine amidase and fibrinogenase exist in multiple forms.     

An enzyme-linked immunosorbent assay (ELISA) for the quantitation of urinary desmosine

An enzyme-linked immunosorbent assay (ELISA) method has been developed for the quantitation of the elastin cross-link desmosine in urine. The system employs Rabbit antisera directed to the conjugate of desmosine and bovine serum albumin which was conjugated using carbodiimide reagent. The ELISA was done in microtiter plates which were coated with a desmosine-gelatin conjugate. And the assay system is based on an inhibition immunoassay. With this assay system, desmosine could be detected in a range between 0.4-400 ng/ml. Recovery of desmosine (DES) added to urine was 90.6-117.0% as measured by this method. Anti-DES antisera obtained from rabbits, showed no cross-reaction to 19 standard amino acids, two elastines nor mouse acetone liver power (which contained degradated elastin). But iso-desmosine cross-reacted with the antisera 13-45% at the isodesmosine concentration range of 40-400 ng/ml. Column purification of the urinary desmosine with CF-1 cellulose will not be necessary for desmosine measurement in ELISA assay. This paper described the detail procedures for sample preparation and the desmosine measurement in urine. Desmosine measurement can be an effective marker for screening the lung elastin degradation caused by cigarette smoking and environmental pollution to human lung.     

Sandwich enzyme-linked immunosorbent assay for Taiwan cobra venom

Poisonous snake bite victims usually have difficulty identifying the species, and clinical manifestations alone are not reliable because of overlapping symptoms. Thus, it is important to develop a quick and reliable mean of identifying the snake responsible. We describe the development of a sandwich-ELISA method for detection of venom in biological samples and apply it to a case of snakebite to confirm the clinical diagnosis. The sandwich-ELISA takes 6 h to complete. Cobra venom antigen gave positive absorbance at about 500 pg/ml. Good linearity with R2 values over 0.99 were observed in dilution series of 1:100 ng/mL of cobra venom in calf serum and human urine. A snakebite initially thought to be Trimeresurus mucrosquamatus was proven cobra with a serum venom level up to 288 ngmL 3 h after envenoming. Sandwich-ELISA provides a rapid and accurate method for clinical identification and evaluation of toxic antigens circulating in individuals bitten by the Taiwan cobra snake.     

Detection of antibodies to myotoxin a and prairie rattlesnake (Crotalus viridis viridis) venom in three antisera using enzyme-linked immunosorbent assay and immunodiffusion

Immunodiffusion and enzyme-linked immunosorbent assay (ELISA) were used to compare three antisera for their content of antibodies against myotoxin a and C. v. virdis venom. No antibodies were detected in Wyeth's polyvalent (Crotalidae) antivenin against myotoxin a using immunodiffusion, whereas ELISA indicated a low titer of such antibodies. However, antimyotoxin a serum and anti-C. v. viridis venom both had higher titers than antivenin when tested against myotoxin a and crude venom in the ELISA. These results correlate well with previous data which indicated that antiserum to myotoxin a was more effective than antivenin in neutralizing the myotoxicity of C. v. viridis venom. The high content of antibodies to myotoxin a in anti-C. v. viridis venom supports the hypothesis that this monovalent antiserum might be effective in neutralizing rattlesnake (C. v. viridis) venom-induced myonecrosis.     

Purification and characterization of a platelet aggregation inducer from Calloselasma rhodostoma (Malayan pit viper) snake venom

A potent platelet aggregation inducer was purified from Calloselasma rhodostoma snake venom by Sephadex G-75, CM-Sephadex C-50 and Sephacryl S-300 column chromatography. It was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a molecular weight estimated to be 28,160 +/- 1280. It was devoid of phospholipase A2, fibrino(geno)lytic and thrombin-like activities. The venom inducer elicited platelet aggregation and the serotonin release reaction in rabbit platelet-rich plasma and platelet suspension. Exogenous calcium was required for its platelet activation. Creatine phosphate/creatine phosphokinase and indomethacin did not inhibit the venom inducer-induced aggregation and release reaction. Mepacrine and verapamil preferentially inhibited aggregation, while PGE1 completely blocked both aggregation and release reaction. It is concluded that the venom inducer activates platelets through the activation of endogenous phospholipase A2 or C, leading to intracellular calcium mobilization, but independent of the ADP release reaction or thromboxane A2 formation.     

Caseinolytic and esteratic activities of Malayan pit viper venom and its proteolytic and thrombin-like fractions

Some enzymic activities of the venom of the Malayan pit viper (Agkistrodon rhodostoma) and its two major components were studied. The proteolytic component had strong caseinolytic activity but little esteratic activity using $\text{p-}\text{toluene sulphonyl-}\text{L}\text{-arginine methyl}$ ester (TAME) and N-benzoyl-L-arginine ethyl ester (BAEE) as substrates. On the other hand, the thrombin-like component had marked activity on TAME and BAEE but no caseinolytic activity.     

The anticoagulant effect of Bothrops castelnaudi snake venom (Castelnaud's pit viper)

An inhibitory effect of Bothrops castelnaudi venom was observed on the following systems: prothrombin time, activated partial thromboplastin time, thrombin time, thromboplastin generation time, activation of factor X by Russell's viper venom and Russell's viper venom activated factor X (factor Xa). This effect did not require previous incubation and was prevented by the addition of Bothrops-antivenom. The prolonged activated partial thromboplastin time was not shortened by increased phospholipid concentration (0.5-10 mg/ml), suggesting that the inhibitory effect is not due to an anti-phospholipid activity. No significant fibrinogenolytic activity was detected upon incubation of human fibrinogen with the venom, since physiological levels of thrombin-clottable material were still present. Compared to Bothrops jararaca venom, the proteolytic activity on casein and on azocoll was very low. Thrombin-induced clots of human plasma and fibrinogen were not lysed by the venom within 24 hr. The results indicate that the anticoagulant effect of Bothrops castelnaudi venom is exerted at least at two levels of the blood coagulation mechanism: (1) before prothrombin activation, by inhibiting factor X-activation and factor Xa activity; (2) by direct action on thrombin.     

Neutralization of lethality and proteolytic activities of Malayan pit viper (Calloselasma rhodostoma) venom with North American Virginia opossum (Didelphis virginiana) serum

Malayan pit viper (Calloselasma rhodostoma) envenomation is a major health problem in South East Asia. During envenomation, venom components mainly affect the hemostatic system. The sera from the North American Virginia opossums (Didelphis virginiana) were able to neutralize the venom of the Malayan pit viper. These natural inhibitors could be explored as potential therapeutics against envenomations of a variety of venomous snake species in different geographical habitats.     

Expression and characterization of a recombinant fibrinogenolytic serine protease from green pit viper (Trimeresurus albolabris) venom

Viper venom serine proteases (SPs) display several effects on hemostatic system. Molecular cloning showed that Trimeresurus albolabris venom comprised a mixture of five SPs with thrombin-like (2), fibrinogenase (2) and plasminogen-activating (1) activities. Because only few fibrinogenolytic SP sequences were reported, we decided to express albofibrase, a novel fibrinogenase from T. albolabris using Pichia pastoris system. The recombinant active form of enzyme was 30 kDa including 2.2 kDa of glycosylation. Albofibrase showed an fibrinogenase activity. In addition, a plasminogen activating and clotting effect were detectable. Albofibrase prolonged APTT and PT in a time-dependent manner. The effect was neutralized by pre-incubation with equine antivenom to T. albolabris. Therefore, the protein is potentially useful as a new anticoagulant as the antidote is clinically available. Sequence analysis compared with other snake venom fibrinogenases and SPs could not find any unique residues responsible for their various effects. Structure–function relationship should be further studied using mutagenesis in order to explore the mechanisms of venom protease functional diversity.     

A sensitive enzyme-linked immunosorbent assay for evaluating the concentration of bee venom in rat plasma

A simple and reproducible enzyme-linked immunosorbent assay (ELISA) was developed to determine the concentration of bee venom in rat plasma. The intra- and inter-assay coefficients of variation for the ELISA were less then 3% between 0.1 and 1,000 ng mL(-1) venom, and the sensitivity of the detection was 0.1 ng mL(-1). Total recovery of the bee venom added to rat plasma was determined. Using this ELISA, serum levels of bee venom were easily determined. The rats were administered a single intravenous injection or oral dose of bee venom (1 mg kg(-1) of body weight). The bioavailability of the bee venom under the two administrations was compared using pharmacokinetic parameters. Results showed that intravenous administration of bee venom produced high plasma concentrations with a short half-life. The area under the curve for oral administration was 10 times lower than for intravenous administration. This loss of bee venom may be due to the degradation that occurs in the enzymatic and acidic environment of the gastrointestinal tract.     

The application of immunoassay techniques,including enzyme-linked immunosorbent assay (ELISA), to snake venom research

R. D. G. Theakston. The application of immunoassay techniques, including enzyme-linked immunosorbent assay (ELISA), to snake venom research, Toxicon21, 341–352, 1983. — The development and application of immunoassay techniques in relation to snake venom research is reviewed. Enzyme linked immunosorbent assay (ELISA) is compared with radioimmunoassay, immunodiffusion, immunofluorescence, haemagglutination and immunoelectrophoresis. It is concluded that ELISA is the most versatile immunoassay technique so far applied to the field of venom research, its main advantages over other methods including relatively high levels of sensitivity and specificity, reproducibility, simplicity and ease of sample collection. It can also be readily modified into kit form and is easily adapted for use in large scale epidemiological studies and for accurate retrospective diagnosis of snake bite. None of the other assay systems considered fulfil these criteria to the same extent.ELISA is helping to advance epidemiological knowledge of snake bite, in exploring the role of active immunisation and in the compilation of accurate clinical patterns of envenoming. Other applications of the test include its use for potency screening of both new and developed commercially available antivenoms and for the detection of monoclonal antibodies which should eventually result in increased specificity of the assay system by eliminating cross reactions between venoms and antibodies of closely related species.     

Fractionation and enzymatic activities of common viper (Vipera berus berus) venom

By means of Sephadex G-100 column chromatography, Vipera berus berus venom has been separated into ten fractions which have been tested for the following enzyme activities: phosphomonoesterase, phosphodiesterase, 5′-nucleotidase, protease, arginine ester hydrolase, hyaluronidase, ribonuclease and phospholipase. Isoelectric focusing over a pH range of 3–10 revealed fractions with the following isoelectric points: arginine ester hydrolase, 4·0 and 4·5; phosphodiesterase, 4·0 and 6·3; proteases, 4·5 and 6·1; 5′-nucleotidase, 5·6; phospholipase, 5·1; 6·7 and 9·3. Phosphomonoesterase and hyaluronidase lost their activities during the electrofocusing procedure. Organophosphorus inhibitors and ethylenediaminetetra-acetic acid have been used to classify the proteases and the arginine ester hydrolase. The protease with the higher mol. wt and with a pI of 6·1 was a metalloenzyme while the protease with the lower mol. wt and with a pI of 4·5 as well as arginine ester hydrolase were serine esterases.     

An enzyme-linked immunosorbent assay (ELISA) for methylphenidate (Ritalin ) in urine

A direct enyzme-linked immunosorbent assay (ELISA) for urinary immunoreactive methylphenidate (Ritalin), in which a standard 96-well microtiter plate is used, is described. For this ELISA, a methylphenidate-thyroglobulin conjugate is immobilized to the microtiter plate and competes with methylphenidate in the standard or urine sample for antibody-binding sites. After washing, the sheep methylphenidate antibody bound to immobilized methylphenidate is detected with peroxidase-labelled goat antisheep IgG. Following a further wash, tetramethylbenzidine is added, color is developed, and the plate is read at 450 nm on an ELISA plate reader. This method is unaffected by drugs of abuse and is suitable for routine use in the toxicology laboratory.     

Development of a rapid and sensitive enzyme-linked immunosorbent assay (ELISA) for measuring venom antigens after an experimental snake bite

, , and . Development of a rapid and sensititve enzyme-linked immunosorbent assay (ELISA) for measuring venom antigens after an experimental snake bite. Toxicon 26, 1157–1167, 1988.—We describe a new ELISA which allows the measurement of the concentration of venom antigens in whole blood. The assay can be performed in less than 20 min and requires a 200 μl sample of blood. It allows the accurate evaluation of concentrations of Vipera ammodytes venom in quantities smaller than 1 ng/ml of blood. Using this ELISA, we were able to follow in rabbits the kinetics of experimental envenomation with non-lethal doses of venom. This ELISA was also used to measure post mortem the level of venom antigens in various tissues such as liver, kidney, muscles and abdominal serosity of a rabbit. The method, which might be adapted to measure envenomation by other snake species, seems to be sufficiently rapid and sensitive to allow routine evaluation of the gravity of a snake bite in humans and to estimate the efficacy of immunotherapy.     

Partial purification of acetylcholinesterase from the venom of the shore pit viper (Trimeresurus purpureomaculatus)

Trimeresurus purpureomaculatus venom acetylcholinesterase has been partially purified by Sephadex G-200 gel filtration chromatography and DEAE Sephacel ion exchange chromatography. The enzyme has a mol. wt of 58,600. It was strongly inhibited by physostigmine salicylate and edrophonium chloride and exhibited substrate inhibition at high substrate concentration. The content of acetylcholinesterase in Trimeresurus purpureomaculatus venom was estimated to be much less than 0.3%.     

牛乳中抗幽门螺杆菌特异性抗体的间接ELISA法的建立

目的 建立抗幽门螺杆菌(Hp)特异性抗体的ELISA检测方法,用于牛乳中抗Hp特异性抗体的检测.方法 超声Hp全菌抗原接种奶牛,按多克隆抗体制备技术制备牛抗Hp抗血清.用纯化牛IgG免疫家兔.制备兔抗牛IgG多克隆抗体.硫酸铵盐析,DEAE-32柱层析分别纯化牛与兔IgG.兔抗牛IgG以辣根过氧化物酶标记.用Hp全菌抗原包被反应板,建立间接ELISA法用于牛乳抗Hp抗体的检测.结果 建立的抗Hp间接ELISA法的最佳包被抗原量为1.47μg/孔,HRP-兔抗牛IgG 1∶600稀释,标准曲线方程为A=0.0124 0.3988 lnC,相关系数r=0.999 8,线性范围为1.8～145μg/ml,回收宰在86.4%～92.8%,变异系数为9.4%～12.3%.结论 初步建立牛乳中抗Hp抗体的间接ELISA测定法,可用于Hp免疫牛乳中抗Hp特异性IgG的检测.