进出口植物产品中对硫磷残留酶联免疫检测试剂盒的研制

目的采用间接竞争ELISA法对进出口植物产品中对硫磷残留量进行快速筛选。方法将对硫磷还原成氨基对硫磷,利用适当的方法将其与牛血清白蛋白(BSA)进行偶联,制备免疫原免疫家兔并获得特异性抗体。结果采用传统的盐析法提取特异性免疫球蛋白(IgG)并建立了对硫磷间接竞争ELISA快速检测方法。抑制试验表明其检测范围在(100.0±1.56)ng/mL之间,可用于进出口植物产品中对硫磷的定量检测。该方法与气相色谱检测符合率为96%。样品的平均回收率为103%。交叉反应显示该抗体仅与对硫磷、氨基对硫磷反应,而与甲基对硫磷、马拉硫磷、杀螟硫磷、乐果等结构类似物几乎无交叉反应。结论本试剂盒具有操作简便、快速、灵敏等特点。     

The development and application of a blocking ELISA kit for the diagnosis of infectious coryza

A blocking enzyme-linked immunosorbent assay (B-ELISA) kit for the diagnosis of infectious coryza was developed in this study. The kit was based on a recently described blocking ELISA that uses monoclonal antibodies to achieve specificity for antibodies to either Haemophilus paragallinarum serovar A or serovar C. The results showed that the B-ELISA kit detected 96 and 90%, respectively, of chickens vaccinated or challenged with H. paragallinarum serovar A. When used on chickens vaccinated or challenged with H. paragallinarum serovar C, the kit detected 77 and 40%, respectively, as positive. The majority of sera from vaccinated chickens were still positive on the serovars A and C ELISAs 4 months after vaccination. Based on pen trial data, the serovar A B-ELISA kit had a sensitivity of 95% and a specificity of 100%. The serovar C B-ELISA kit had a sensitivity of 73% and a specificity of 100%. A range of field sera was examined with the kit, generating results that correlated with the known vaccination/disease history of the flocks examined. As freeze drying the monoclonal antibodies and the conjugate had some effect on optimal working concentration, the kit used liquid solutions of these two reagents. The kit could be stored for 7 days at 37°C, 10 months at 4°C and more than 1 yearat -20°C. Our results suggest that the kit would be a useful aid in the diagnosis of infectious coryza in China and other countries where H. paragallinarum serovars A and C are the predominant or sole serovars.     

Study on the venoms of the principal venomous snakes from French Guiana and the neutralization

We studied some biochemical, toxic and immunological characteristics of the venoms of Bothrops atrox, Bothrops brazili and Lachesis muta, Viperidae responsible for most of the bites of venomous snakes in French Guiana. Chromatographic (HPLC) and electrophoretical profiles (SDS-PAGE), lethal, hemorrhagic, defibrinogenating, coagulant, thrombin like, proteolytic, fibrino(geno)lytic and phospholipase activities were studied. In addition, the neutralization of some toxic activities conferred by four antivenins was compared. The chromatographic and electrophoretic profiles were different for the three venoms, showing differences between Bothrops and L. muta venoms. In general, bothropic venoms showed the highest toxic and enzymatic activities, while the venom of L. muta showed the lowest lethal, hemorrhagic and coagulant activities. The enzymes of bothropic venoms responsible for gelatinolytic activity were around 50-90 kDa. All the venoms were able to hydrolyze a and beta chains of the fibrinogen, showing different patterns of degradation. Although all the antivenoms tested were effective to various degrees in neutralizing the venom of B. brazili and B. atrox, neutralization of L. muta venom was significantly better achieved using the antivenom including this venom in its immunogenic mixture. For the neutralization of L. muta venom, homologous or polyvalent antivenoms that include the "bushmaster" venom in their immunogenic mixture should be preferred.     

抗磺胺嘧啶单克隆抗体的制备及其ELISA检测试剂盒的建立

目的 :制备抗磺胺嘧啶 (SD)的单克隆抗体 (mAb) ,并研制SD快速检测试剂盒。方法 :以SD BSA的偶联物作为抗原免疫BALB/c小鼠 ,采用杂交瘤技术制备抗SD的mAb。并用抗SDmAb和SD HRP酶标抗原 ,建立一种可检测样品中的游离SD分子的竞争法ELISA。结果 :获得 5株可分泌抗SDmAb的杂交瘤细胞 1A1、1B8、1E4、2C1和 3D9。其中 1A1、1B8和 1E4为IgG1,2C1和 3D9为IgG2。试剂盒的半数抑制率 (I5 0 )为 9.3μg/L ,理论最低检出量达到 0 .6μg/L ,对于不同样品中SD的回收率均高于 60 %。除与SD反应以外 ,该试剂盒对其他磺胺类药物检测的交叉反应均小于 3 %。结论 :成功地制备了抗SD的mAb ,并建立了一种可快速检测各种样品中SD的竞争ELISA试剂盒     

Enzyme-linked immunosorbent assay (ELISA) of changes in specific IgG antibodies to five venoms during venom immunotherapy

An enzyme-linked immunosorbent assay (ELISA) was developed that could measure titres of human IgG antibodies to five different venoms (honeybee, yellow jacket, yellow hornet, white-faced hornet, and wasp), and to honeybee phospholipase A. Changes in specific IgG anti-venom titres were measured in twenty patients that had systemic anaphylactic reactions to insect stings, and ten non-allergic controls. After being stung and prior to treatment all patients had anti-venom IgG titres greater than controls. Treatment with small doses of venom over 1–2 months resulted in prompt rises in anti-venom IgG titres that may represent secondary anemnestic responses primed by prior slings. All patients undergoing venom immunotherapy showed at least 2-fold increases in IgG antibody lo the venoms they were treated with by the time maintenance doses of 100 meg were achieved, with one exception. Significant cross-reactive increases in anti-vespid IgG antibodies to venoms not used for treatment occurred in nine of eighteen treated patients. Overall, ELISA of IgG antibodies lo five venoms allowed clear evaluation of the considerable variation of IgG responses among different patients. We conclude that serial determination of venom-specific IgG titres by ELISA offers an important adjunct to evaluating the results of venom immunotherapy.     

人N端前脑钠肽ELISA定量检测方法的建立及初步应用

建立一种检测人血清中N端前脑钠肽含量的双抗体夹心ELISA方法。采用配对人N端前脑钠肽抗体,通过对其包被条件与封闭条件、包被与酶标抗体工作浓度、反应模式、线性范围、批内批间精密性、回收率、干扰因子试验、临床标本检测等研究,建立人N端前脑钠肽ELISA定量检测方法并评价其性能。实验确定了最适包被抗体稀释用缓冲液为0.05mol/L pH9.6CB,温度及时间为4℃18h;最适封闭液为0.6%BSA,温度及时间为4℃18h;包被抗体最适浓度为4μg/ml,酶标抗体最佳浓度为1∶3000;最佳反应模式为37℃样本孵育30min,酶标二抗孵育30min,显色20min的两步法反应模式。性能评价显示建立的检测方法线性范围为50~500pg/ml,批内及批间精密度分别为2.3%和3.4%。高、中、低值回收率分别为101.2%、97.2%、93.7%。干扰因子试验结果显示黄疸、脂血及低浓度溶血对试验结果无较大影响,但标本随溶血程度的增加其影响程度也增大。共做了248份临床血清,200份用来评价试剂盒的临床使用性能,结果显示总体符合率为96.9%,说明可用于临床心衰患者的检测;同时与国外试剂盒对比检测48份临床血清,结果显示两者相关性较好,回归方程:Y=0.8017X+188.3,R2=0.9027。初步建立了一种检测人血清中N端前脑钠肽含量的ELISA方法,该方法具有较高的准确性和精密度。     

Allergens in Hymenoptera venoms: IV. Comparison of venom and venom sac extracts

Honeybee venom sac extract is compared with pure venom. All five known allergens of venom are present in venom sac extract. Enzyme analyses indicate that the sac extracts contain 11% to 16% venom. At least 10 additional components, several of which are proteins, are present in venom sac extract. Radioallergosorbent test (RAST) studies of yellow jacket venom and venom sac extract yielded a correlation of r = 0.94, with only some weakly reactive sera positive to only one preparation. A few sera were substantially more reactive with venom sac extract. Venom sac extracts appear to be suitable for in vitro diagnostic use, but the extraneous proteins and peptides may make them less suitable than pure venoms for use in immunotherapy.     

TORCH-IgM捕获法ELISA试剂盒的研制和应用

目的 为了发展TORCH近期感染的IgM酶免疫诊断技术。方法 以抗人IgM McAb包板，加待检血清、TORCH抗原，接着加酶标记的抗TORCH—McAb，最后加3，3’，5，5’-四甲基联苯胺(TNB)显色。用这种自制的捕获法ELISA(C—ELISA)试剂盒和HOPE公司的间接法ELISA(I—ELISA)试剂盒平行检测1196份孕妇血清TORCH-IgM。结果C—ELISA试剂盒检出阳性血清67份，并经其他试验证实为阳性。其中4份用I—ELISA试剂盒检测为阴性。由乳胶凝集试验诊断为类风湿因子(RF)阳性的2份血清，经I—ELISA试剂盒检测为阳性，但经C—ELISA试剂盒检测为阴性。结论 在检测孕妇TORCH—IgM方面，C—ELISA比I-ELISA有更强的敏感性和更高的特异性。C—ELISA试剂盒是诊断TORCH近期感染的良好工具。     

人组织激肽释放酶单克隆抗体的制备及其ELISA检测试剂盒的建立

目的 制备人组织激肽释放酶(human tissue kallikrein,HK)单克隆抗体(McAb),并研制检测人尿中HK含量的ELISA试剂盒.方法 以一段HK特异多肽和血蓝蛋白(KLH)的耦联物作为抗原免疫BALB/c小鼠,并采用杂交瘤技术制备抗HK的单克隆抗体.然后建立快速检测HK含量的间接竞争ELISA试剂盒.结果 获得了8株可分泌抗HK的杂交瘤细胞株,其产生的抗体效价为1:25 600;经1:1600倍稀释后的竞争抑制率为0.88;经纯化后的纯度为100%,其标准曲线对线良好(r=0.990).结论 成功制备了高效价、高特异性和高纯度的抗HK的单克隆抗体.同时建立了一种可以快速检测人尿中HK含量的ELISA试剂盒.

Abstract：

Objective To prepare monoclonal antibody(McAb) against human tissue kallikrein (HK) and develop an ELISA kit allows for the in vitro quantitative determination of human tissue kallikrein in urine. Methods To generate a monoclonal antibody specific for TK, the synthetic TK peptide consisting of 12 amine acids(12P), was fused to keyhole limpet hemocyanin(KLH) and used for immunization. Using hybridoma screening, monoclonal secreting cell lines were identified and used to generate ascites in BALB/c mouse. Antibody was purified by affinity column chromatography. 12% SDS-PAGE and Western blot were used to visualize the purified antibody. This kit employs indirect competitive ELISA technique and BiotinAvidin System. 12P was fused to bovine serum albumin(BSA) and has been pre-coated onto a microplate at first. Standards and samples were added to the appropriate microplate wells with a biotin-conjugated McAb croplate well. A TMB substrate solution is added to each well. The enzyme-substrate reaction is terminating by the addition of a sulphuric acid solution and the color change is measured spectrotometrically at a wavelength of 450 nm. The concentration of tissue kallikrein in the samples is then determined by comparing the O.D. of the samples to the standard curve. Results 8 hybridoma cell lines secreting mAbs special to HK,SDS-PAGE and Western blot demonstrated successful preparing and purification of McAb( 100% ). The linearity of this ELISA kit is demonstrated(r =0. 990). The range of detection of the assay is 0.008 μg/ml to 0. 5 μg/ml. The assay remained stable, with no change in the values measured, over five cycles of freezing and thawing. Conclusion 8 McAbs against HK have been prepared successfully and possess high titer and specificity. The development of an ELISA kit for detecting HK can meet the needs of detection of HK in urine samples.     

Comparison of biochemical and immunologic properties of venoms from four hornet species

Venoms of two American hornets, Vespula maculata (bald-faced hornet) and Vespula arenaria (yellow hornet), and two Old World hornets, Vespa crabro and Vespa orientalis, were investigated for biochemical and immunologic properties. They were similar with regard to enzymatic activities (phospholipase A, phospholipase B, and hyaluronidase) and molecular composition. Analysis of radioallergosorbent test (RAST) results obtained from 30 vespid-sensitive patients suggests extensive cross-reactivity between the venoms of V. arenaria, V. maculata, and V. crabro, but the venom of V. orientalis seems to lack at least one important allergen. Rabbit antisera to each of the four venoms contain precipitating antibodies to all four venoms. Strong cross-reactivity was found between the venoms of V. maculata, V. arenaria, and V. crabro and between V. crabro and V. orientalis. V. orientalis venom cross-reacts weakly with the venoms of both V. maculata and V. arenaria. It is concluded therefore that diagnosis and immunotherapy with commercially available venoms of V. maculata and V. arenaria should be adequate for most patients sensitive to V. maculata, V. arenaria, or V. crabro but probably not for a significant proportion of those individuals primarily sensitive to V. orientalis.     

Advantage of using a home-made ELISA kit for detection of Helicobacter pylori infection over commercially imported kits

PURPOSE: To evaluate a home-made ELISA kit for detection of Helicobacter pylori (Hp) infection and comparison of its immunologic criteria with those of foreign commercial kits. METHODS: A home-made IgG ELISA kit was developed using soluble antigenic fractions of Hp proteins. Confirmed sera were tested and serological criteria were evaluated through assessment of 199 serum samples. RESULTS: The accuracy, sensitivity and specificity values of home-made kit were 92, 92 and 90.4%, respectively. These immunologic criteria for Trinity kit were 95.2, 95.2 and 95% in comparison with IBL kit (91.3, 92.2 and 88.5%), BIOHIT kit (72.4, 41.6 and 94.1%) and HelicoBlot2.1 (94.2, 93.4 and 100%). Kappa agreement assessment demonstrated that two of the imported ELISA kits had fair to moderate agreement with the home-made kit while the other one had a poor agreement value. CONCLUSIONS: Apart from comparable values between the home-made kit and the most efficient imported kit (Trinity) there was significant cost benefit. Therefore, we recommend the home-made kit as a suitable substitution for detection of Hp infection in the Iranian population.     

Development and evaluation of an immunochromatographic kit for the detection of antibody to Plasmodium vivax infection in South Korea

Malaria is a major parasitic disease in tropical areas. Three to five hundred million people suffer from the disease and it kill a million people per year. Blood smear observation was developed for the diagnosis of malaria, but the examination needs skilled experts and exact diagnosis is time consuming. A kit based on immunochromatography can be a reliable and rapid method for clinical diagnosis, even in the hands of inexperienced personnel. However, all such currently developed kits can only diagnose P. falciparum malaria. In our previous report, the C-terminal region of P. vivax merozoite surface protein 1 (PvcMSP) was cloned and expressed in E. coli. In the present study, we developed an immunochromatographic kit using this PvcMSP for the diagnosis of specific antibody to P. vivax malaria in serum samples. The kit was used to examine sera from vivax malaria patients and non-malaria- infected person and the test showed 100% sensitivity (78/78) and 98.3% specificity (58/59). This result demonstrated that the immunochromatographic kit for P. vivax antibody detection is applicable for the rapid and precise diagnosis of P. vivax malaria.     

Comparison of the allergenicity and antigenicity of Polistes venom and other vespid venoms

The crossantigenicity of Polistes venom with other vespid venoms was examined with rabbit and human antisera. Venom preparations from various Polistes species were obtained by electrical stimulation of individual insects and venom sac dissection. Rabbit antibodies were raised to the venom (P. apachus) and venom sac extract (P. exclamans). Human antisera were obtained from patients allergic to Polistes and other vespid venoms. The venom appeared to be more potent than the venom sac preparations in reactions with rabbit IgG and human IgE antibodies. Among the Polistes species, P. exclamans, P. instablis, and P. apachus venoms showed several lines of precipitation with rabbit antisera, and P. annularis and P. fuscatus venoms only one line, suggesting quantitative or qualitative antigenic differences. In RAST analysis, most sera reacted equally to all Polistes species but occasional exceptions were noted, again suggesting differences in venom allergens. P. exclamans-coupled discs gave the most consistent results. In gel diffusion experiments, there was no crossreactivity between Polistes and yellow jacket venoms and only limited crossreactivity between Polistes and hornet venoms. Patients sensitive to Polistes venom showed varying degrees of reactivity to yellow jacket and hornet venoms in RAST analysis. Patients sensitive to other vespid venoms also showed varying degrees os sensitivity to Polistes venom. Polistes venom appears to contain a genus-unique antigen (allergen). In addition, there appear to be some crossreacting antigens in Polistes and other vespid venoms but to a much lesser degree than found previously in the analysis of the relationship of yellow jacket and hornet venoms.     

进出口植物产品中甲霜灵残留量酶联免疫检测试剂盒的研究

目的本研究采用直接竞争ELISA法对进出口植物产品中甲霜灵残留量进行快速筛选.方法将甲霜灵转化成甲霜灵酸,利用适当的方法将其与辣根过氧化物酶(HRP)进行偶联,制备酶标半抗原.采用传统的盐析法提取IgG并建立了甲霜灵直接竞争ELISA快速检测方法.结果抑制试验表明其检测范围在(0.78～50.0)ng/mL之间,可用于进出口植物产品中甲霜灵的定量检测.该方法与气相色谱检测符合率为94%.样品的平均回收率为94%.交叉反应显示该抗体仅与甲霜灵、甲霜灵酸起反应,不与结构相似的毒草胺、甲草胺、异丙甲草胺、新燕灵和己酰甲草胺起交叉反应.结论本试剂盒具有操作简便、快速、灵敏等特点.     

Improved ELISA for the detection of HBsAg

A sandwich ELISA for hepatitis B virus surface antigen (HBsAg) was developed using monoclonal anti-HBs for the solid phase and horse-radish peroxidase labelled sheep anti-HBs.

The sensitivity was approx. 0.3 U/ml HBsAg, in the standard test procedure,comprising two incubation steps of 1 h at 37°C, or in a shortened procedure comprising two incubation steps of 30 min at 50°C. A slightly reduced sensitivity (approx. 0.5 U/ml) was obtained when the two incubations were combined in a one-step incubation for 1 h at 37°C. All three procedures were completed by an incubation for 30 min at room temperature with peroxide and tetra-methylbenzidine.

The number of false positives obtained when donor sera were screened was below 0.5%. False positive reactions occurred more frequently, but still to a limited extent, when previously selected sera containing rheumatoid factor or other possibly interfering factors were tested with the standard procedure. Most sera containing factors that interfere with a commercial ELISA for HBsAg using sheep anti-HBs coated plates, were negative. Rheumatoid factor positive sera seldom gave false positive results.

The lower detection limit became approx. 0.1 U/ml when the cut-off was reduced, while the number of false positives in a donor population only increased to 1.5%. The results obtained with reagents from four different batches indicate a stability of up to 4 wk at 37°C and for at least 26 wk at 4°C.     

Allergens in Hymenoptera venom XIII: Isolation and purification of protein components from three species of vespid venoms

Pure venoms were collected from individual insects of the species Dolichovespula maculata, white-faced hornet, Vespula squamosa, southern yellow jacket, and Polistes exclamans, paper wasp (one species). The venoms were first fractionated by high-resolution gel filtration on a 1.6 m column of Sephadex G-75 superfine, and the components were then purified by high-performance, ion-exchange chromatography on a Mono-S cation exchange column followed by a further gel filtration step. The isolated components were evaluated for purity by sodium dodecyl sulfate polyacrylamide gel electrophoresis by use of two different types of silver stains, by assays for enzyme activities, and by immunodiffusion with the use of rabbit antisera. The protein components were isolated in highly purified states by these techniques. Only three significant proteins were found in V. squamous venom: phospholipase (PL) A and B, hyaluronidase (HYAL), and antigen 5 (Ag 5). D. maculata venom contained HYAL, Ag 5, two isozymes of PL A and B, a high-molecular-weight protein, and several trace proteins. No significant amounts of proteases were found in D. maculata venom. P. exclamans venom contained HYAL, PL A and B, Ag 5, a high-molecular-weight protein, and several minor proteins. In all three venoms the PL A and B activities were found to be in the same molecule and did not separate. Trace components with apparent PL A activity were observed in the venoms. The venoms were screened for a variety of esterases, proteases, peptidases, glucosidases, and phosphatases, and none were detected in more than trace amounts. Vespid venoms do not appear to contain significant amounts of acid phosphatases as bee venoms do.     

肠道病毒71抗原ELISA检测方法构建及其应用

目的 建立肠道病毒71型(EV71)抗原的ELISA检测方法.用于EV71的抗原定量、TCID50试验及EV71免疫原性与免疫保护性的关系分析.方法 以高亲和力的EV71中和单抗K8G2和Y8H2-HRP构建检测EV71抗原的双抗体夹心ELISA方法.比较本方法与显微镜观察法检测的EV71 TCID50.分析EV71抗原的特异比活与免疫血清中和抗体水平的关系.结果 本试剂定量检测抗原的线性范围为0.125 ～4.0 U/ml,R2 =0.9911,试剂不与EV71之外的受试物反应,试剂的回收率为0.89 ～ 1.16,变异系数小于15％,37℃9 d的热回收率大于85％.本法与显微镜观察法检测的EV71 TCID50的相关系数r=0.990.21株EV71抗原的特异比活与免疫血清中和抗体水平的相关系数r=0.930.结论 构建了EV71抗原ELISA检测试剂,可用于EV71的抗原含量检测、TCID50分析,为特异比活与免疫血清中和性关系研究提供了一些有价值的信息.     

Fluorometric ELISA for the detection and quantitation of metallothionein

The development of a heterogeneous fluorometric ELISA for the detection and quantitation of metallothionein (MT) is described. A radioimmunoassay (RIA) previously developed in our laboratory is used as a reference assay to characterize the performance of the ELISA. The standard curves (logit-log regressions) that are typical of either assay have similar ranges (customarily from 20 000 to 100 pg of competing antigen); both assays are capable of quantitating MT in unknowns with 5-10% accuracy. Aspects of MT measurement in cytosols and physiological fluids are discussed.     

Evaluation of the Phadebact CSF test for detection of the four most common causes of bacterial meningitis

A five-center collaborative study was undertaken to determine the suitability of the Phadebact CSF test kit and the Phadebact group B Streptococcus reagent for routine use by clinical laboratories to detect antigens of common organisms causing bacterial meningitis. The kits employ staphylococcal protein A coagglutination to detect the antigens of Haemophilus influenzae types a, b, c, d, e, and f, Neisseria meningitidis groups A, B, C, Y, and W135, Streptococcus pneumoniae (83 serotypes), and group B Streptococcus. A total of 2,817 individual tests were performed on 577 cerebrospinal fluid specimens. The percent positive specimens detected by coagglutination was as follows: overall, 84%; H. influenzae, 97%; group B Streptococcus, 75%; S. pneumoniae, 71%; and N. meningitidis, 58%. Eighty-five of the specimens were also tested by counterimmunoelectrophoresis. Coagglutination was more sensitive than counterimmunoelectrophoresis because it detected 74% of the positive specimens, whereas counterimmunoelectrophoresis detected only 65%. No false-positive results were obtained with coagglutination. The Phadebact CSF test kit is recommended for routine use in screening cerebrospinal fluid samples for antigens of the common organisms causing bacterial meningitis along with the Gram stain and culture for delayed confirmation of the rapid results.