Evaluation of Setaria cervi antigen in the diagnosis of human filariasis

Bovine filariid worm (Setaria cervi) antigen was evaluated for the immunodiagnosis of human filariasis. Patients with manifestations of filarial infection; [1) microfilaremia cases, (2) chronic clinical cases, (3) tropical pulmonary eosinophilia (TPE) cases) were taken for the investigations along with (4) normal endemic and non-endemic human subjects. CIEP, IFAT and ELISA tests were employed for detection of serum antibodies. There was a high degree of sensitivity shown by IFAT (1:64-1:256) and ELISA (1:200-1:20,800) in microfilaraemia cases and higher in chronic clinical cases with IFAT (1:128-1:1,024) and ELISA (1:12,800-1:102,400). Only one TPE case showed positive titre by IFAT (1:64) and ELISA (1:200) whereas sera from controls and patients with helminth infections did not show positive titres (1:100). In the case of CIEP, positive reaction was seen in only one case from group I.     

Immunodiagnostics of Toxocara canis in suspected ocular and visceral manifestations

Sera from patients with ocular and visceral manifestations of toxocariasis were tested by precipitin absorption (PAT), counter-immunoelectrophoresis (CIEP), indirect fluorescent antibody (IFAT) and ELISA. The ocular cases revealed a percentage of positivity of 36% by PAT, 0% by CIEP, 32% and 28% by IFAT with embryonated egg (EE) and frozen section antigens (FS) respectively and 40% by ELISA. The visceral cases revealed 44% positively by PAT, 24% by CIEP 52% and 48% by IFAT with EE and FS respectively and 52% by ELISA. Statistical evaluation was done to interpret the results.     

An antigen from Eimeria tenella merozoites for the indirect fluorescence antibody test.

The suitability of the antigen prepared from second generation merozoites of Eimeria tenella for the IFAT was confirmed. In comparison with other two antigens used until now in routine work, i. e., that prepared from histological sections of infected organs or that from in vitro released sporozoites, the merozoite antigen has the advantage of easy preparation in large quantities.     

Dynamics of antibody production in mice infected with Toxascaris leonina Linstow, 1909

Using counterimmunoelectrophoresis and ELISA tests the dynamics of antibody production in serum of mice experimentally infected with Toxascaris leonina was studied. The production of antibodies using both tests has already been detectable in serum of mice from 7 days post infection (DPI) and their level persisted till the end of the experiment, i.e. till 77 DPI. The most positive were reactions of sera with Antigens 1 and 3.     

Laboratory diagnosis of acute toxoplasmosis by immunosorbent agglutination assay (IgM ISA)

A group of 401 patients suspected for toxoplasmosis was examined by the indirect immunofluorescent antibody test (IFAT). All patients positive in IFAT (176) were examined by the immunosorbent agglutination assay (IgM ISA). In the IgM ISA 154 of them were negative, 10 temporarily and 12 high-positive. Some of high-positive patients were examined repeatedly; decrease of high levels of specific IgM antibodies occurred 2-9 months after the first examination. For the IgM ISA antigen prepared from peritoneal exudate of experimentally infected mice was used. The antigen was stable at 4 degrees C or in liquid nitrogen at least 1 year. The IgM ISA combined with IFAT and IgM IFAT was proved satisfactory for the diagnosis of acquired acute toxoplasmosis and can be recommended for laboratories with lower capacity.     

Development of a recombinant antibody ELISA test for the detection of Polymyxa betae and its use in resistance screening

An ELISA test was developed for the quantitative detection of the obligate parasite Polymyxa betae , the vector of Beet necrotic yellow vein virus (BNYVV), in infected sugarbeet roots. The test used monoclonal and polyclonal antibodies raised to a recombinantly expressed glutathione-S-transferase (GST) from P. betae . A close correlation was found between the number of P. betae zoospores in serially diluted suspensions and absorbance values in the ELISA test. Time-course studies of plants grown in naturally infested soils in controlled environment tests demonstrated the value of the ELISA test in screening for P. betae resistance. In preliminary tests, P. betae -resistant accessions of the wild sea beet ( Beta vulgaris ssp. maritima ), which might be used to restrict the transmission of BNYVV, were identified.     

Toxoplasmic antibodies in sera of HIV-infected persons.

The sera of 67 HIV-infected persons without clinical signs of Toxoplasma gondii infection and sera of 777 immunocompetent persons from three distinct regions of Czechoslovakia were examined for the presence of toxoplasmic antibodies using the complement-fixation test (CFT). Additionally Toxoplasma positive HIV+ individuals were re-examined for the presence of IgG and IgM toxoplasmic antibodies by ELISA methods. Results show that overall prevalence of toxoplasmic antibodies is not significantly greater in HIV-positive subjects (29.8%) than in the general population (26.1%). Similarities between these two tested groups were also documented by a close correlation of their geometrical means of titres (13.9 versus 14.5). All 20 HIV-infected patients who were positive in CFT were positive in ELISA IgG reaction, and none in ELISA IgM reaction. The detected antibody levels were suggestive of a latent Toxoplasma infection only. But because of the risk of the infection reactivation all of these patients should be attended to on a systematic basis.     

Correlation of the results of IFAT-DASS and ELISA tests in experimental cysticercosis of mice

The sera of mice experimentally infected with Taenia crassiceps were tested for the occurrence of the antibodies by the enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence using defined antigen substrate spheres (IFAT-DASS). Results of both tests were compared. From day 11 p.i. until the end of the experiment (day 108 p.i.) antibodies were detected by both tests. The maximal intensity of the ELISA and IFAT-DASS reactions was observed between days 63 and 94 and days 14 and 46 p.i., respectively. ELISA is an easy-to-do and objectively appraisable method, IFAT-DASS is more suitable for testing antigens weakly adsorbing to polystyrene plates.     

Evaluation of a polyclonal antibody immunoassay for detection and quantification of Macrophomina phaseolina

Detection and quantification of Macrophomina phaseolina , causal agent of charcoal/dry root rot disease in many crop plants, was carried out using the ELISA serological technique. Polyclonal antisera raised to water soluble extracts of mycelium, the residual water insoluble mycelial materials or ribosomal proteins were evaluated for specificity and cross-reactivity with 16 common soil fungi by ODD and DAS–ELISA. Soluble and cell wall antisera exhibited strong cross-reactivity with most of the fungal isolates. Ribosomal antibodies were less reactive to common soil fungi except Fusarium oxysporum f.sp. ciceri . Mycelial antigens of M. phaseolina on chickpea roots were detectable with DAS–ELISA at a minimum concentration of 10 ng g⁻¹ at 1 : 100 root : buffer dilution. Quantitative estimation of M. phaseolina on roots was evaluated by ELISA under different temperatures and moisture conditions, and in soil amended with a potential antagonist ( Trichoderma harzianum 25-92). A significant reduction in ELISA values was observed in T. harzianum -amended treatments. This method may be useful for detection and rapid screening of M. phaseolina under different environmental conditions.     

南芥菜花叶病毒单克隆抗体的研制及检测应用

将纯化的南芥菜花叶病毒(Arabis mosaic virus,ArMV)制剂免疫Balb/c小鼠,末次免疫后第3天取其脾细胞与SP2/0细胞融合,采用选择性培养基、有限稀释法克隆和间接ELISA方法进行筛选,成功获得了2株稳定分泌ArMV单克隆抗体的杂交瘤细胞株并分别命名为3F7,4G10。用间接ELISA方法对所获得的2个杂交瘤细胞株进行亚型鉴定分别为IgA(3F7)、IgG1(4G10)。间接ELISA效价测定结果:3F7为1:106,4G10为1:108。以单克隆抗体为包被抗体、多克隆抗体为检测抗体的TAS-ELISA检测试剂盒能检测感染ArMV的昆诺藜病汁液的灵敏度为1:1600。     

Humoral intestinal immunity against Encephalitozoon cuniculi (Microsporidia) infection in mice

Three strains of mice, BALB/c, IL-12 knock-out (KO) and INF-gamma knock-out, were chosen as an experimental model for the study of intestinal immunity induction against Encephalitozoon cuniculi Levaditi, Nicolau et Schoen, 1923 infection. Mice were infected perorally with 10(7) spores and re-infected with the same dose 70 days after the first infection. The anti-E. cuniculi IgA, IgG and IgM responses in sera and extracts of stool samples were determined by ELISA. Results have shown specific antibody production in the sera and intestinal secretions of all three strains of mice induced orally by E. ciniculi spores. BALB/c mice developed a stronger humoral immune response than IL-12 KO mice. The lowest antibody response developed in INF-gamma KO mice that succumbed to the infection within 28 days post infection.     

李痘病毒单克隆抗体及TAS-ELISA试剂盒的研制

将纯化的李痘病毒(Plum pox virus,PPV)制剂免疫BALB/c小鼠,用SP2/0骨髓瘤细胞与经李痘病毒免疫的BALB/c小鼠的脾细胞融合,有限稀释法克隆和间接ELISA法筛选出2株稳定分泌李痘病毒单克隆抗体的杂交瘤细胞株3F1,7A8。用间接ELISA方法对所获得的2个杂交瘤细胞株进行亚型鉴定分别为IgG1、IgG3。间接ELISA方法测定腹水效价分别为3F1:1.0×106,7A8:1.0×105。以多克隆抗体为包被抗体、单克隆抗体为检测抗体的TAS-ELISA试剂盒与李痘病毒的D株系、M株系的病毒分离物均有反应,与同属的马铃薯A病毒、莴苣花叶病毒、西瓜花叶病毒2号、马铃薯Y病毒坏死株系不发生交叉反应。     

甲萘威酶联免疫吸附分析技术研究

合成了两种不同结构的甲萘威半抗原并与载体蛋白质共价偶联制备人工抗原。以人工抗原免疫动物获得对甲萘威具特异性的高效价抗体 ,建立并优化了痕量甲萘威的竞争性酶联免疫吸附测定法。该法测定甲萘威的线性浓度范围为 10 1～ 10 -4 μg/ml,检测限低于 0 .0 1ng/ml。其他结构类似的氨基甲酸酯类杀虫剂对甲萘威的分析无干扰。     

Monoclonal antibody for the detection and identification of a phytoplasma associated with rice yellow dwarf

One stable hybridoma clone, 247B11, secreting specific monoclonal antibody (MA) against the mycoplasmalike organism (MLO), newly be termed phytoplasma, associated with rice yellow dwarf (RYD) was produced by employing an immunization scheme for inducing the immunological tolerance of mice to rice antigens prior to the administration of RYD-phytoplasma immunogens. Neonatal BALB/c mice were first injected with nontarget rice antigens present in the immunogen preparation and were immunized intrasplenically with RYD-phytoplasmaenriched antigens prepared by Percoll density-gradient fraction 6 wk later. The MA was of the IgG1 class. With this MA, RYD-phytoplasma in diseased rice was specifically detected by indirect enzyme-linked immunosorbent assay (ELISA), immunofluorescent staining and tissue-blotting techniques. Antibody titer determined by indirect ELISA for hybridoma-culture supernatant was 5120. The antibody recognized two polypeptides, 16 kDa and 41 kDa of RYD-phytoplasma determined by western blotting. RYD-phytoplasma was differentiated serologically from the phytoplasmas associated with sweetpotato, peanut, loofah, paulownia, andIpomoea obscura witches' broom, aster yellows (NJ strain), elm yellows, and sugarcane white leaf both in indirect ELISA and immunofluorescent staining.     

Analysis of soluble Entamoeba histolytica antigen using enzyme-L inked electroimmunotransfer blots

Soluble antigens of ten strains of E. histolytica were studied by sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and enzyme-linked electroimmunotransfer blots (EITB). No relations of immune replicas to virulence, geographical origin and method of cultivation (xenic or axenic culture) were found. Antigens of all ten strains tested precipitated with anti-E. histolytica human serum in the area of 30-43 kD. Antigen of HK-9 strain created in this area a characteristic pattern with all sera containing the specific anti-E. histolytica antibodies and, therefore, EITB can be used for excluding false positive results in ELISA.     

Amoebiasis in foreign students

A total of 2,883 foreign students at the age of 18-30 years were examined for amoebiasis after their arrival to Czechoslovakia. Stool examinations revealed the presence of Entamoeba histolytica in 112 of them (3.9%). Students from 38 countries were found to be infected with this parasite. In a set of 2,064 students from these countries E. histolytica prevalence in stool was 5.4%. There were greater differences in the prevalence between individual countries inside a geographical region than between individual geographical regions. The highest E. histolytica prevalence in stool was found in students from tropical and southern Africa (6.7% of 745 examined) and the lowest in students from South-eastern Asia (3.1% of 321 examined). In a simple cross-section study, antibodies against E. histolytica were detected by enzyme-linked immunosorbent assay (ELISA) in the sera of 1,001 persons. Antibodies were detected in 7.9% of students at the following titres: 1:200 in 4.5%, 1:600 in 1.5%, 1:1,800 in 1.9%. Antibodies occurred more frequently in students carrying E. histolytica cysts (X2 = 14.9). Titre of ELISA antibodies in patients with confirmed liver abscess was higher than 1:1,800. counterimmunoelectrophoresis (CIEP) test was used for serum examinations of patients who had been demonstrated by ELISA to be seropositive and of those carrying E. histolytica cysts. In a set of 170 patients CIEP antibodies were also more frequent in those carrying E. histolytica cysts (X2 = 26.95). A comparison of the results of ELISA and CIEP tests in the same patients revealed that CIEP antibodies were more dependent on the actual parasitization with E. histolytica than ELISA antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)     

An investigation of the reliability of ELISA as a practical test for detecting potato leaf roll virus and potato virus Y in tubers

Enzyme-linked immunosorbent assay (ELISA), involving mechanical sampling of tubers, was compared with a growing-on test in which virus was assessed visually, and with ELISA on leaves. The percentage infection of potato leaf roll virus and potato virus Y on samples of potential seed tubers sent for advisory test during the winter, was determined by each method. The tuber ELISA underestimated the incidence of both viruses, and was less accurate than the growing-on or leaf ELISA. The effect on the reliability of tuber ELISA of the amount and distribution of virus in the tuber, the compromises made to make the test fast and simple, and the quality of anti-serum and composition of the extraction buffer are discussed.     

Detection of tobacco rattle virus by ELISA and test plants in main sprouts of tulip bulbs during storage at different temperatures

The detectability of tobacco rattle virus (TRV) in the main sprouts of primarily and secondarily infected tulip bulbs of cv. Apeldoorn stored at different temperatures from the lifting in July up to February is described. Detection by ELISA was not affected by the size of the main sprout, nor by the size of the bulbs. The rates of TRV-infected bulbs found by ELISA were highest during storage at 13, 9 or 5°C continuously, and when temperatures were lowered from 20 or 17°C to 5°C in October. The percentages detected via test plants, but undetectable by ELISA were also lowest at these temperatures. The unfavourable effect of continuous storage at 20, 17, or 2°C as expressed in low ELISA absorbances not significantly different from the mean value of healthy bulbs, was largely overcome during long storage by the change of temperature down to 5°C from 20 and 17°C or upwards from 2°C. The reverse from 5°C upwards to 17 and 20°C affected the detectability by ELISA unfavourably. The rate of detection via test plants in the main sprout and in the small sprouts from different positions in bulbs was only possible at low percentages.The effect of some factors, like different temperatures during storage, detectability of different TRV serotypes, interference of irregular occurrence of TRV in the removed scale and basal-plate tissue with the main sprout, and variable recurrence of TRV in progeny bulbs, is discussed in view of its impact on routine testing of bulbs during storage.     

稻瘟病菌一假定几丁质酶多克隆抗体的制备及其检测

 为检测几丁质酶的表达,制备稻瘟病菌的一假定几丁质酶多克隆抗体,探索其可能运用。将稻瘟病菌(Magnaporthe oryzae) 几丁质酶基因克隆入融合表达载体pET\|32a,并在大肠杆菌Escherichia coli BL21(DE3)中进行诱导表达。表达菌株经0.5 mmol/L IPTG诱导4～6 h后,用Ni²⁺\|NTA亲和柱纯化蛋白,得到可溶的几丁质酶\|His融合蛋白。以该融合蛋白免疫新西兰雄兔,制备多克隆抗体。ELISA分析表明该抗体效价达1∶204 800,特异性良好。用该抗体ELISA检测了稻瘟病菌4个不同田间菌株中,不同的菌株表达量不同。对稻瘟病菌侵染水稻3、5和7d后的病叶进行抗原Western验证,结果表明,稻瘟病菌不同的侵染时间其几丁质酶表达量有明显差异。 几丁质酶表达的差异是否与菌株间致病型等生物学和生理学差异有关,目前正在进一步研究。