A new assay for the detection of Loxosceles species (brown recluse) spider venom

STUDY OBJECTIVE: Dermal lesions from unrelated arthropod species and medical causes appear similar to Loxosceles species (brown recluse spider) bites. This may result in delayed diagnosis and treatment. We developed a sensitive Loxosceles species venom enzyme-linked immunosorbent assay (ELISA) and characterized the specificity of the assay by evaluating antigenic cross-reactivity from a variety of North American arthropod venoms. METHODS: North American arthropod (14 spiders, 2 scorpions, and 1 bee) venoms were studied. Three venom amounts (diluted in 100 microL of ELISA buffer) were assayed: 16,000 ng, 2,000 ng, and 40 ng. The latter quantity was selected because this is the observed maximum amount of venom we detect when inoculating dermis with amounts likely to be deposited by a spider bite. The larger venom amounts are overwhelming quantities designed to test the limits of the assay for arthropod venom cross-reactivity. Similar amounts of Loxosceles species venom and bovine albumin served as positive and negative controls, respectively. RESULTS: At the lowest amount of venom tested (40 ng), the ELISA detected only the Loxosceles species positive control. When 2,000 ng was assayed, only Scytodes fusca and Kukulcania hibernalis arachnid venoms (in addition to Loxosceles species) cross-reacted to the assay. Finally, at 16,000 ng, the ELISA assay modestly detected Diguetia canities, Heteropoda venatoria, Tegenaria agrestis, Plectreurys tristes, Dolomedes tenebrosus, and Hadrurus arizonensis arachnid venoms. CONCLUSION: Cross-reactivity was observed in 8 of 17 North American arthropod venoms when large venom amounts were assayed with a Loxosceles species ELISA. By using a relevant quantity of venom, 40 ng, the assay was specific for Loxosceles species venom. The venom specificity of the ELISA may allow clinical application in Loxosceles species endemic regions of North America.     

Epidemiological studies of snake bite in French Guiana

The incidence of snake bite and the presence of venom antibody in previous snake bite victims was investigated in French Guiana. The incidence proved to be highest (600/100,000) in inhabitants of bush regions and lowest (45/100,000) in urban areas. Of 43 sera tested for specific venom antibody 22 (51%) were positive, and most of these individuals suffered severe or moderate poisoning. The main species involved, as assessed by detection of venom antibody by ELISA, were Lachesis muta, Bothrops brazili, B. bilineatus and B. atrox. The significance of these findings is discussed.     

Enzyme-linked immunosorbent assay for detection of anti-RNA polymerase III antibody: analytical accuracy and clinical associations in systemic sclerosis

OBJECTIVE: We have recently developed an enzyme-linked immunosorbent assay (ELISA) for detection of anti-RNA polymerase III (anti-RNAP III) antibody, using a recombinant fragment containing the immunodominant epitope as the antigen source. This study was conducted to assess the analytical accuracy and clinical associations of the anti-RNAP III ELISA in patients with systemic sclerosis (SSc). METHODS: To evaluate analytical sensitivity and specificity of the ELISA, both immunoprecipitation tests and ELISA were used to detect anti-RNAP III antibody in 534 SSc sera from patients at 3 medical centers. Sera from 522 SSc patients and 516 controls, including patients with other connective tissue diseases and blood bank donors, were also evaluated to assess the clinical sensitivity and specificity of the ELISA. Clinical findings in anti-RNAP III antibody-positive SSc patients were compared between patient groups stratified according to anti-RNAP III antibody levels determined by the ELISA. RESULTS: In SSc patients, our ELISA showed analytical sensitivity of 91% and analytical specificity of 99% compared with the immunoprecipitation assay (a gold standard for detection of anti-RNAP III antibody). The additional analysis using a large series of SSc and control sera showed that clinical sensitivity and specificity of the ELISA with respect to the diagnosis of SSc were 17% and 98%, respectively. A high level of anti-RNAP III antibody was associated with diffuse cutaneous SSc, higher maximum total skin score, and increased frequency of tendon friction rubs. CONCLUSION: The anti-RNAP III ELISA is analytically accurate and clinically specific. With this assay, testing for anti-RNAP III antibody can be made routinely available.     

Antihaemolytic and snake venom neutralizing effect of some Indian medicinal plants

ObjectiveTo validate traditional claims of usefulness of the Indian plants in management of poisonous snakebite and evaluate the antivenom properties displayed by the alcoholic extracts of Andrographis paniculata (A. paniculata), Crateva magna (C. magna), Gloriosa superba (G. superba) and Hydrocotyle javanica (H. javanica).MethodsThese plants were collected, identified and the extracts were prepared by using conventional Soxhlet ethanol extraction technique. The venom neutralization activity was accessed in mice (20-25g) and number of mortalities was observed against clinically important snake (Naja nigricollis) venom. Present study also deals with in vitro membrane stabilizing activity of these plants against hyposaline induced human red blood corpuscles (HRBC).ResultsExtracts of H. javanica and G. superba gave 80 % and 90 % protection to mice treated with minimum lethal dose of venom (LD₉₉). These two plants showed significant neutralization effect against the venoms of Naja nigricollis venom. H. javanica and G. superba (25-100 mg/mL) produced significant changes of membrane stabilization of human red blood cells (HRBC) exposed to hyposaline-induced haemolysis.ConclusionsWe conclude that probably due to presence of various phytochemicals plays an important role in the anti-venom potential of these Indian medicinal plants against Naja nigricollis venom. The above observations confirmed that A. paniculata, C. magna, G. superba and H. javanica plant extracts possess potent snake venom neutralizing capacity and could potentially be used as an adjuvants for antivenin therapy in case of snakebite envenomation, especially against the local effects of cobra venoms.     

A low-cost method to test cytotoxic effects of Crotalus vegrandis (Serpentes: Viperidae) venom on kidney cell cultures

The pathogenesis of the renal lesion upon envenomation by snakebite has been related to myolysis, hemolysis, hypotension and/or direct venom nephrotoxicity caused by the venom. Both primary and continuous cell culture systems provide an in vitro alternative for quantitative evaluation of the toxicity of snake venoms. Crude Crotalus vegrandis venom was fractionated by molecular exclusion chromatography. The toxicity of C. vegrandis crude venom, hemorrhagic, and neurotoxic fractions were evaluated on mouse primary renal cells and a continuous cell line of Vero cells maintained in vitro. Cells were isolated from murine renal cortex and were grown in 96 well plates with Dulbecco's Modified Essential Medium (DMEM) and challenged with crude and venom fractions. The murine renal cortex cells exhibited epithelial morphology and the majority showed smooth muscle actin determined by immune-staining. The cytotoxicity was evaluated by the tetrazolium colorimetric method. Cell viability was less for crude venom, followed by the hemorrhagic and neurotoxic fractions with a CT50 of 4.93, 18.41 and 50.22 microg/mL, respectively. The Vero cell cultures seemed to be more sensitive with a CT50 of 2.9 and 1.4 microg/mL for crude venom and the hemorrhagic peak, respectively. The results of this study show the potential of using cell culture system to evaluate venom toxicity.     

Enzyme‐linked immunosorbent assay for detection of Anti–RNA polymerase III antibody: Analytical accuracy and clinical associations in systemic sclerosis

Objective

We have recently developed an enzyme‐linked immunosorbent assay (ELISA) for detection of anti–RNA polymerase III (anti–RNAP III) antibody, using a recombinant fragment containing the immunodominant epitope as the antigen source. This study was conducted to assess the analytical accuracy and clinical associations of the anti–RNAP III ELISA in patients with systemic sclerosis (SSc).

Methods

To evaluate analytical sensitivity and specificity of the ELISA, both immunoprecipitation tests and ELISA were used to detect anti–RNAP III antibody in 534 SSc sera from patients at 3 medical centers. Sera from 522 SSc patients and 516 controls, including patients with other connective tissue diseases and blood bank donors, were also evaluated to assess the clinical sensitivity and specificity of the ELISA. Clinical findings in anti–RNAP III antibody–positive SSc patients were compared between patient groups stratified according to anti–RNAP III antibody levels determined by the ELISA.

Results

In SSc patients, our ELISA showed analytical sensitivity of 91% and analytical specificity of 99% compared with the immunoprecipitation assay (a gold standard for detection of anti–RNAP III antibody). The additional analysis using a large series of SSc and control sera showed that clinical sensitivity and specificity of the ELISA with respect to the diagnosis of SSc were 17% and 98%, respectively. A high level of anti–RNAP III antibody was associated with diffuse cutaneous SSc, higher maximum total skin score, and increased frequency of tendon friction rubs.

Conclusion

The anti–RNAP III ELISA is analytically accurate and clinically specific. With this assay, testing for anti–RNAP III antibody can be made routinely available.

     

Characterization of a monoclonal antibody that neutralizes the hyaluronidase activity of Russell's viper venom

Three monoclonal antibodies (WPN1, WPN2 and WPN3) raised against a partially purified fraction of Russell's viper venom (RVV) were characterized. All three monoclonal antibodies reacted with crude RVV when tested by ELISA, but only two (WPN1, WPN2) neutralized its hyaluronidase activity. WPN1 was the more potent and was effective at an antigen: antibody ratio of 1:3. Furthermore, WPN1 was shown to recognize only the 14,000 MW component of crude RVV. This has been identified in a previous study to be hyaluronidase. This antibody was also found to recognize some components of Calloselasma rhodostoma venom which also possesses potent hyaluronidase activity. The potential therapeutic role of antibodies that neutralize the hyaluronidase component of snake venoms should be investigated further.     

视神经脊髓炎特异性抗体三种检测方法比较

目的 评价组织间接免疫荧光检测(IIF)、细胞免疫荧光(CBA)及ELISA 3种不同检测视神经脊髓炎(NMO)特异性抗水通道蛋白4(AQP4)抗体的方法。方法 NMO 29例、多发性硬化(MS)23例、健康志愿者50例,分别采用IIF、CBA及ELISA方法检测血清中的抗AQP4抗体,比较各种方法诊断NMO的敏感性和特异性以及阳性结果的一致性。结果 3种方法检测的抗AQP4抗体诊断NMO的敏感性CBA法(72.4％)＞IIF法(62.1％)＞ELISA法(51.7％);特异性CBA法(100％)＞ ELISA法(98.6％) ＞IIF法(97.3％)。采用Kappa检验评估各种检测方法一致性,CBA、IIF、ELISA 法均具有较好的一致性,其中CBA法与ELISA法检测一致性最好(P＜0.01)。结论 检测抗AQP4 抗体以CBA法敏感性和特异性最佳,CBA与ELISA法阳性结果具有较好的一致性。     

Krait bite requiring high dose antivenom: a case report

Anti snake venom (ASV) is the most specific therapy available for treatment of snakebite envenomation. The ASV available in Nepal are polyvalent ASV produced in India and are effective against envenomation by cobra and krait, the two most common species found in Eastern Nepal. Neurotoxic signs respond slowly and unconvincingly and continuous absorption of venom may cause recurrent neurotoxicity. Therefore, close observation and continuous administration of ASV is essential to save the victim. We report a case of neurotoxic envenomation due to bite by common krait (Bangarus caeruleus). The victim required very high dose of polyvalent ASV for reversal of neurological manifestations.     

Protein C activators from snake venoms and their diagnostic use

Proteinases converting the zymogen protein C (PC) of vertebrates into activated PC have been detected in several snake venoms. Most PC activators have been purified from venom of snake species belonging to the genera of the Agkistrodon complex. Unlike the physiological, thrombin-catalyzed PC activation reaction which requires thrombomodulin as a cofactor, most snake venom activators directly convert the zymogen PC into the catalytically active form which can easily be determined by means of coagulation or chromogenic substrate techniques. Due to this feature, the fast-acting PC activator Protac from Agkistrodon contortrix contortrix (southern copperhead snake) venom has found a broad application in diagnostic practice for the determination of disorders in the PC pathway. Recently, screening assays for the PC pathway have been introduced, based on the observation that the PC pathway is probably the most important physiological barrier against thrombosis.     

ELISA confirmation of acute and past envenoming by the monocellate Thai cobra (Naja kaouthia)

The monocellate Thai cobra (Naja kaouthia) is a major cause of snake bite mortality and morbidity throughout Thailand, but neither the local nor the systemic effects of its venom are diagnostic. Species diagnosis is important because only monospecific antivenoms are available for treatment in Thailand. We tested the ability of the ELISA technique to detect venom antigen in the sera of 58 acute snake bite cases including 4 fatalities, and venom antibody in 51 patients bitten between 1 month and 19 years previously. N. kaouthia venom antigen was found in 8 of 33 patients with only local envenoming and in 14 of 20 with local plus systemic (neurotoxic) envenoming, but the mean venom concentration was 33 times greater in the latter group. The serum of 1 fatal case contained banded krait (Bungarus fasciatus) but no cobra venom antigen. N. kaouthia venom antibody was present in sera of patients bitten between 1 month and 7 years previously. Antibody was found in 6 of 8 patients who had had local envenoming alone but in only 19 of 41 who had had systemic envenoming treated by antivenom. The titer of antibody declined with an approximate half time of 2-3 years. One patient had a significant titer of B. fasciatus venom antibody. This study confirms the value of ELISA-immunodiagnosis and the predominance of N. kaouthia as a cause of neurotoxic envenoming in the Bang Phli area. However, the attribution of 1 fatal case to B. fasciatus bite suggests that patients with neurotoxic signs should be given B. fasciatus antivenom if they fail to respond to cobra antivenom.     

Detection of Mycobacterium tuberculosis in clinical samples using insertion sequences IS6110 and IS990

To detect Mycobacterium tuberculosis in clinical samples, we used the M. tuberculosis-complex specific insertion sequence IS990 as the target in a simple DIG-PCR ELISA assay, as this element is present as a single copy in all strains of M. tuberculosis we have examined to date. The IS990 test was compared with a similar PCR that utilizes IS6110 as target. For detection of PCR product, digoxigenin-11-dUTP (DIG-dUTP) was incorporated into the product. After amplification, the PCR product was hybridized with biotinylated capture probe, which was complementary to the inner part of the amplicon. The hybrid was captured onto streptavidin-coated microtiter plate and DIG-labeled PCR product was detected using a peroxidase-conjugated antibody to DIG. We evaluated DIG-PCR ELISA for the detection of M. tuberculosis DNA in 265 respiratory and non-respiratory specimens taken from patients with known and suspected tuberculosis disease or from controls. The sensitivity and specificity of both IS990-based test and IS6110-based test was 96.5% and 95.3% respectively, comparable to the sensitivity and specificity of the IS6110-based test. The results demonstrate that the IS990 PCR ELISA test is a rapid and sensitive tool for the detection and identification of M. tuberculosis in clinical samples, and may have advantages to the more widely used IS6110-based tests, particularly in areas where IS6110-negative strains are found.     

人工合成gp210多肽抗原在原发性胆汁性肝硬化中的应用

目的 尝试用gp210抗原的羧基末端氨基酸序列合成多肽做抗原,探索一种简单实用的抗gp210抗体的检测方法.方法 采用棋盘试验法建立抗gp210抗体的酶联免疫吸附试验(ELISA)检测方法,分别用ELISA方法与免疫印迹法检测原发性胆汁性肝硬化(PBC)组与对照组的抗gp210抗体.结果 gp210抗原的工作浓度为5 μg/ml.血清抗gp210抗体的吸光度(A)值>0.61(x+3s)为阳性.两种方法在检测抗gp210抗体之间差异无统计学意义(p=0.617),二者之间有很强的相关性(r=0.868,P=0.000).结论 人工合成gp210多肽抗原与天然纯化抗原敏感性和特异性基本一致,可以用于临床检测抗gp210抗体.     

伏马菌素B1免疫学检测方法的研究

目的制备抗伏马菌素B1(Fumonisin B1,FB1)单克隆抗体,在此基础上建立一种可用于定量检测玉米中FB1含量的间接竞争ELISA方法。方法人工合成免疫原BSA-FB1和包被抗原OVA-FB1,免疫BALB/c小鼠,采用杂交瘤细胞融合技术制备抗FB1的单抗,并对其特性进行鉴定。以OVA-FB1作为包被抗原,FB1为竞争抗原,两者与一定量的抗FB1单抗竞争反应,建立检测FB1的间接竞争ELISA方法,对玉米样品进行初步检测应用。结果获得了1株稳定分泌抗FB1单克隆抗体的杂交瘤细胞(命名为5H12-C6-C2)。对间接竞争ELISA方法进行反应条件优化后,建立了检测FB1的标准曲线,回归方程为y=1.113-0.313x,相关系数R2为-0.990,最适可测范围为10ng/ml~1000ng/ml,灵敏度为90.88ng/nl,最低检测限为7.83ng/ml,方法的批内和批间平均变异系数分别为3.24%和2.45%,样品添加平均回收率为94.32%。结论成功地制备了抗FB1特异性单抗,建立了FB1的间接竞争ELISA检测方法,为进一步研制FB1检测试剂盒奠定了基础。     

Cross neutralization of common Southeast Asian viperid venoms by a Thai polyvalent snake antivenom (Hemato Polyvalent Snake Antivenom)

Snake envenomation is a serious public health threat in many rural areas of Asia and Africa. Antivenom has hitherto been the definite treatment for snake envenomation. Owing to a lack of local production of specific antivenom, most countries in these regions fully depend on foreign supplies of antivenoms. Often, the effectiveness of the imported antivenoms against local medically important species has not been validated. This study aimed to assess cross-neutralizing capacity of a recently developed polyvalent antivenom, Hemato Polyvalent Snake Antivenom (HPAV), against venoms of a common viper and some pit vipers from Southeast Asia. Neutralisation assays showed that HPAV was able to effectively neutralize lethality of the common Southeast Asian viperid venoms examined (Calloselasma, Crytelytrops, Popeia, and Daboia sp.) except for Tropidolaemus wagleri venom. HPAV also effectively neutralized the procoagulant and hemorrhagic activities of all the venoms examined, corroboratively supporting the capability of HPAV in neutralizing viperid venoms which are principally hematoxic. The study also indicated that HPAV fully prevented the occurrence of hematuria and proteinuria in mice envenomed with Thai Daboia siamensis venom but was only partially effective against venoms of Myanmar D. siamensis. Thus, HPAV appears to be useful against its homologous venoms and venoms from Southeast Asian viperids including several medically important pit vipers belonging to the Trimeresurus complex. Nevertheless, the effectiveness of HPAV as a paraspecific antivenom for treatment of viperid envenomation in Southeast Asian region requires further assessment from future clinical trials     

Direct nephrotoxicity of Russell's viper venom demonstrated in the isolated perfused rat kidney

Envenoming by Russell's Viper (Vipera russelli) is an important cause of acute renal failure. The mechanism of renal damage is unresolved. It is difficult to obtain evidence of a direct nephrotoxic action because of the coincidental disturbance to the systemic circulation. We studied the action of Russell's Viper venom on the function of the isolated perfused rat kidney. Direct nephrotoxic action was indicated by a dose dependent decrease in inulin clearance and an increase in fractional excretion of sodium seen at venom concentrations down to 50 ng/ml, a concentration likely to be achieved in the human circulation after envenoming. The isolated perfused kidney was also used to assess the efficiency of antivenom and for a comparison with snake venoms from the Thai cobra (Naja kauothia) and the Nigerian Saw-Scaled Viper (Echis ocellatus).     

ELISA法检测血清包虫IgG抗体试剂盒的应用评价

目的对ELISA(酶联免疫吸附试验)法检测血清包虫IgG抗体试剂盒进行评价,为其应用提供参考。方法收集2012-01-12就诊的1242位可疑包虫病患者的血清标本,采用ELISA检测血清棘球蚴IgG抗体。结果1242例中就诊患者中对包虫感染有明确诊断(确诊或排除包虫病)的有1130例,其中棘球蚴IgG抗体阳性159例(14.07%),棘球蚴IgG抗体阴性971例(85.93%);以临床诊断为标准得到灵敏度87.06%,特异性91.87%,Youden指数为0.78。结论ELISA法检测棘球蚴IgG抗体的灵敏度和特异性较高,可用于包虫病患者的流行病学调查;并结合B超等影像学检查用于临床包虫病的辅助诊断。