Recording and interpretation of the results obtained by using the ELISA Sevatest commercial immunoenzyme kit for detecting Toxoplasma gondii antibodies

The results of the repeated tests of 295 serum samples from patients, previously examined by means of enzyme immunoassay screening and traditional serological tests (complement fixation, indirect immunofluorescence, and indirect hemagglutination) and 115 serum samples from healthy donors, studied by enzyme immunoassay techniques with the use of the commercial kits Sevatest ELISA, have been analyzed. The methodological approach permitting the determination of the final titer of the serum under study, taken in a dilution of 1:800, by its optical density has been used. The mean geometric titer for the control group of donors, determined in the enzyme immunoassay, has been 1:800. This fact suggests that titers exceeding 1:1,600 should be considered diagnostically significant.     

Development of an immunoenzyme test system for detecting antibodies to commercial strains of feed yeasts

ELISA as a specific and highly sensitive test system was used for the examination of large groups of workers employed in the production of fodder protein to detect antibodies to the production strains of fodder yeast. The results yielded by ELISA correlated well with those obtained by the serological luminescent techniques, but antibody titers determined by means of ELISA are 10-20 times higher.     

Comparison of the sensitivity of the serological latex and ELISA tests

A sensitivity of the serological latex and ELISA tests were compared in carnation mottle virus diagnosis. For the latex test carnation mottle virus (CaMV) antiserum was sensibilized with latex suspension for RF-test. Sensibilized antiserum was used in 1: 200 dilution, as compared with fresh antiserum. For ELISA the γ-globuline fraction of antiserum was conjugated with alkaline phosphatase. The optimal dilution in both, CaMV fraction of antisera for coating of plates and γ-globuline-enzyme conjugate were in the ratio of 1: 500, 2 μg of antibodies in 1 ml. The dilution end point of carnation mottle virus in sap from carnation leaves was 1.6 × 10^?4 to 1.25 × l0^?5 and 1 × 10^?4 to 1.25 × l0^?5, when serological latex and ELISA tests were used. As indicated, ELISA as compared to the latex test was found to be more sensitive for carnation mottle diagnosis. As the latex test is considered to be simpler and cheaper, and in addition, showing the same assurance as the biological test onChenopodium amaranticolor, the latex test is recommended for carnation mottle virus detection.     

Comparative evaluation of four commercial tests for presumptive identification of Candida albicans

Four commercially available tests (Albicans ID2, Chromalbicans Agar, CHROMagar Candida, and BactiCard Candida) and the germ tube (GT) test for presumptive identification of Candida albicans were evaluated using clinical isolates of C. albicans (n=89) and of non-albicans yeasts (n=107). Sensitivities and specificities of all tests regarding the identification of C. albicans were greater than 92%, except for Chromalbicans Agar plates (88.7% after 48 h) and their specificity was 86%. Overall, the four commercial systems were easy to use and are good systems for the routine identification of C. albicans.     

Diagnostic validation of selected serological tests for detecting scrub typhus

Clinical diagnosis of scrub typhus is often difficult because the symptoms are very similar to those of other febrile illness such as dengue, leptospirosis, malaria and other viral hemorrhagic fevers. Though better diagnostic tests are available for rickettsial diseases and scrub typhus elsewhere, the Weil–Felix test is still commonly used in India, mainly because microimmunofluorescence assays (M‐IFA) were not available in India till recently and relevant staff had insufficient training. The present study was performed to investigate the performance of M‐IFA, IgM ELISA, and Weil–Felix test on 546 non‐repeated serum samples from subjects suspected of having scrub typhus. One hundred and forty‐three of these 546 samples were positive by M‐IFA; these cases were also confirmed clinically to have scrub typhus based on their dramatic responses to doxycycline therapy. IgM ELISA was positive in 122 of the 143 M‐IFA positive cases and the Weil–Felix test in 96. Though the Weil–Felix test is a heterophile agglutination test, it was found in this study to have good specificity but far too little sensitivity to use as a routine diagnostic test. IgM ELISA can be a good substitute for M‐IFA. Incorporation of multiple prototype antigens on M‐IFA slides is likely one of the reasons for its superior performance. As newer and better diagnostic assays become available for scrub typhus diagnosis in developed countries, it will be imperative to also use such tests in other endemic countries to prevent over‐ or under‐diagnosis of scrub typhus.     

Detection of Toxoplasma gondii-specific antibodies in pigs using an oral fluid-based commercial ELISA: Advantages and limitations

Toxoplasma gondii is a major food-borne parasite and undercooked meat of infected pigs represents an important source of infection for humans. Since infections in pigs are mostly subclinical, adequate diagnostic tests for use at the farm level are pursued. Oral fluid (OF) was shown to be a promising matrix for direct and indirect detection of infections with various pathogens in pigs. The objective of this study was to assess whether T. gondii infections in pigs could be diagnosed using an indirect ELISA kit adapted for OF samples (OF-ELISA). Routine serology and OF-immunoblot (IB) were used as standards for the comparison. For this, serial OF samples from sows (n = 8) and fatteners (n = 3) experimentally inoculated with T. gondii oocysts, individual field samples from potentially exposed sows (n = 9) and pooled OF samples from potentially exposed group-housed fatteners (n = 195 pig groups, including 2,248 animals) were analysed for antibodies against T. gondii by ELISA. For individual animals, OF-ELISA exhibited a relative diagnostic specificity of 97.3% and a relative diagnostic sensitivity of 78.8%. In experimentally infected animals, positive OF-ELISA results were observed from 1.5 weeks post inoculation (pi) until the end of the experimental setup (8 to 30 weeks pi); however, values below the estimated cut-off were occasionally observed in some animals despite constant seropositivity. In potentially exposed individual animals, OF- and serum-ELISA results showed 100% agreement. In group-housed fatteners, antibodies against T. gondii could be reliably detected by OF-ELISA in groups in which at least 25% of the animals were seropositive. This OF-ELISA, based on a commercially available serum-ELISA, may represent an interesting non-invasive screening tool for detecting pig groups with a high exposure to T. gondii at the farm level. The OF-ELISA may need further adjustments to consistently detect individual infected pigs, probably due to variations in OF antibody concentration over time.     

Use of specific Bordetella pertussis antibodies in immunoenzyme analysis for detecting the pertussis antigen

The competitive EIA technique with the use of peroxidase-labeled B. pertussis antigen has been developed. The data obtained in our investigations suggest the possibility of using this technique for the detection of B. pertussis antigen in faucial smears obtained from patients.     

Evaluation of a rapid ELISA technique for detection of circulating antigens of Toxoplasma gondii

To evaluate a modified rapid ELISA method for detecting CAg during Toxoplasma gondii infection, we analyzed the specificity and sensitivity of the ELISA method by using experimental Toxoplasma infection in rabbits and also tested this method in human samples including 5428 serum, 548 cerebrospinal fluid and two breast milk samples. We prepared PcAb, and used it for rapid one-step sandwich ELISA testing in which an incubation time in the regular ELISA procedure was omitted. This method detected CAg at the concentration of 31.2 ng/mL, and no cross-reaction was found with antigens of protozoa (Cryptosporidium parvum, Plasmodium falciparum), trematode (Schistosoma japonicum, Paragonimus sp.) and nematode (Brugia malayi, Ancylostoma duodenale, Ascaris lumbricoides and Trichinella spiralis). CAg was detected in rabbit serum 3 days after infection, and optical density values reached a peak 9-13 days after infection, then declined gradually. Among human serum samples, the positive rate of CAg was 2.11% in cerebral paralysis patients, whereas it was 0.22% or 0.71% in patients without neurological symptoms or in uncomplicated pregnant women. The difference among these three groups was statistically significant (P < 0.05). The positive rate of cerebrospinal fluid samples from cerebral paralysis patients was 10.58%. There is a statistically significant difference between the positive rates of meat-packing workers and blood donors (P < 0.01). In the retrospective analysis, CAg was detected in accordance with the onset of clinical symptoms, suggesting that CAg could reflect the clinical course in humans. Together with these results, CAg detected in the modified rapid sandwich ELISA could be a sensitive marker for acute and active infection of T. gondii.     

Rapid serological screening of human sera for the presence of measles virus antibodies in solid-phase immunoenzyme analysis

A single dilution technique has been used for the determination of antimeasles antibody titer. The method involved the plotting of the calibration curve and the characterization of the serum by arbitrary "evaluation units" in comparison with the specially selected positive serum whose titer was taken to be equal to 100 "evaluation units". By means of this method 57 sera obtained from children immunized against measles and 118 sera from non-vaccinated adults aged 18-22 years were examined. The values of the calculated titers were similar to those determined experimentally. This recommends this method for seroepidemiological investigations aimed at determining the level of herd immunity to measles.     

应用单克隆抗体测定人弓形虫IgM抗体的研究

为了检测人血清弓形虫ＩｇＭ抗体,采用抗人ＩｇＭ单克隆抗体和特异性抗弓形虫单克隆抗体建立捕获ＥＬＩＳＡ法,并与ＰＣＲ方法进行了比较。结果检测１０６５份献血员血清,检出阳性３例,用ＰＣＲ方法检测呈阳性结果;检测２３例类风湿病人血清及２份弓形虫ＩｇＧ抗体阳性血清均为阴性反应。说明该方法不受类风湿因子（ＲＦ）和特异性ＩｇＧ抗体的干扰,同时也表明捕获ＥＬＩＳＡ检测人血清中弓形虫ＩｇＭ抗体特异性,敏感性良好。     

基于猪德尔塔冠状病毒重组核衣壳蛋白的ELISA抗体检测方法的建立与评价

【目的】猪德尔塔冠状病毒(Porcine deltacoronavirus,PDCoV)是近年来新发现的一种猪肠道冠状病毒,2014年首次暴发于美国,随后亚洲多个国家也相继报道,对养猪业构成了巨大威胁。以大肠杆菌表达纯化的PDCoV重组核衣壳(N)蛋白为包被抗原,建立检测PDCoV抗体的间接ELISA方法,为PDCoV的血清抗体检测和流行病学调查提供工具。【方法】以PDCoV CHN-HN-2014株的基因组RNA为模板,通过RT-PCR扩增PDCoV核衣壳蛋白(N)基因的全长cDNA,将其插入原核表达载体pET-30a中,构建原核表达质粒p ET30a-N,转化大肠杆菌Rosetta(DE3),经异丙基-β-D-硫代半乳糖苷(Isopropyl-beta-D-thiogalactopyranoside,IPTG)诱导表达,以纯化的重组N蛋白为包被抗原,建立PDCoV N-ELISA抗体检测方法,评估其特异性、敏感性、稳定性,并用于临床血清的检测。【结果】SDS-PAGE电泳检测证实表达的重组N蛋白主要以可溶性形式存在,Western blotting证实表达的重组蛋白具有反应活性。用纯化的重组蛋白建立的N-ELISA具有良好的特异性、敏感性、稳定性。与中和试验同时检测148份免疫猪血清和102份临床血清,两种方法的阳性符合率为88.99%,阴性符合率为92.90%,总符合率为91.20%。用建立的ELISA方法检测267份临床血清,PDCoV抗体阳性血清的比率为66.67%。【结论】建立的猪德尔塔冠状病毒N-ELISA抗体检测方法与中和试验的符合率高,可用于PDCoV血清抗体检测和流行病学调查。     

Sporulation and survival of Toxoplasma gondii oocysts in different types of commercial cat litter

Toxoplasma gondii oocysts are environmentally resistant and can survive outdoors for many months in dry and cold climates. In the present study, sporulation and survival of T. gondii oocysts was studied in different types of cat litters commercially available in the United States. Oocysts sporulated within 2-3 days in all types of cat litters and occasionally remained viable for 14 days. Results indicate that cat litter should be changed daily to prevent sporulation and infectivity to people.     

Ultrastructural and biochemical studies on the immunohistochemistry of Toxoplasma gondii antigens using monoclonal antibodies

To determine the cellular distribution of Toxoplasma antigens, RH strain tachyzoites were incubated with either one of three monoclonal antibodies (FMC 19, FMC 20, FMC 22) to T. gondii, or one of two controls (the murine myeloma protein MOPC 21, or phosphate buffered saline), and then incubated with peroxidase-labelled goat-antimouse IgG. Diaminobenzidine was added as substrate and electron microscopy was used to localize the reaction. All three antibodies bound to the entire periphery of the tachyzoite surface membrane. To ascertain the chemical composition of the antigens against which seven monoclonal antibodies (FMC 18, FMC 19, FMC 20, FMC 22, FMC 23, 2G11, 3E6) to T. gondii reacted, untreated, pronase-treated, or periodate-treated tachyzoites were incubated with the antibodies or MOPC 21, and then with [125I]-Protein A. The pronase-treated tachyzoites showed reduced binding for six of the antibodies, compared with the reduction in binding of MOPC 21 with the pronase-treated parasites. The periodate-treated tachyzoites had reduced binding for FMC 18 only. The results of these experiments confirm that most Toxoplasma surface antigens are protein in nature, and are consistent with the hypothesis that at least one cytoplasmic antigen is secreted onto the parasite cell surface.     

鸭疫里氏杆菌多种血清型间接ELISA检测方法的建立

【目的】鸭疫里氏杆菌(Riemerella anatipestifer,RA)是一种重要的禽病病原,分为21个血清型。但一直缺乏一种针对多种血清型广泛适用的抗体检测方法。前期的研究表明,外膜蛋白A (Outer membrane protein A,OmpA)广泛存在于多种血清型的RA菌株中,是一种重要的免疫原性蛋白,并且其基因序列在RA血清型之间具有高度的保守性,提示其可以作为RA感染血清抗体检测的靶点分子。以重组蛋白OmpA建立间接酶联免疫吸附试验检测RA的抗体。【方法】通过诱导表达条件的摸索及蛋白纯化,获得适用于ELISA包被的重组OmpA抗原。通过Western-blot证明重组蛋白OmpA是否与RA多种血清型发生免疫学反应。进行方阵试验以确定ELISA抗原的最佳包被浓度、被检测血清的反应浓度。重复性、特异性和敏感性试验检查该方法的实用性。【结果】实验证实加入1%乙醇的诱导培养基有利于重组蛋白的可溶性表达。Western-blot结果表明,重组蛋白OmpA可以与1、2、6、10、11、13、14和17型多种RA主要流行血清型有良好的免疫反应性。经方阵试验确定抗原的最佳包被浓度为8 mg/L,待检血清的最佳稀释度为1:160。所建立检测方法具有良好的重复性、特异性和敏感性。【结论】实验建立的鸭疫里氏杆菌多种血清型间接ELISA检测方法可以用于免疫后抗体消长以及感染性抗体的检测。     

Monoclonal antibodies against nucleoside triphosphate hydrolase-II can reduce the replication of Toxoplasma gondii

Nucleoside triphosphate hydrolase (NTPase) is an abundant protein secreted by the obligate protozoan parasite Toxoplasma gondii, which has a wide specificity toward NTP. In the present study, two monoclonal antibodies (mAbs, MNT1 and MNT2) against recombinant T. gondii NTPase-II (rTgNTPase-II) were developed. Western blot analysis displayed that these two mAbs can recognize specifically rTgNTPase-II as well as a 63 kDa molecule in tachyzoites soluble antigens that corresponded to native NTPase-II. T. gondii tachyzoites pretreated with two mAbs were observed under Confocal Laser Microscope and a specific reaction was displayed on tachyzoites after indirect fluorescence antibody test (IFAT). When COS-7 cells were co-cultured with tachyzoites pretreated with two mAbs, the number of intracellular parasites per infected cell was significantly decreased compared with the control. Furthermore, incubation of T. gondii tachyzoites with two mAbs can inhibit NTPase activity in the presence of dithiothreitol, which hinted that the reduction of tachyzoite replication might be owing to the inhibition of NTPase-II by the mAbs. The passive immunization test indicated that the transferred mAbs can significantly prolong the survival time of challenge infected mice. Taken together, we concluded that the mAbs against NTPase-II can reduce the replication of T. gondii and have a crucial effect on the protection of host from T. gondii infection.     

Seroprevalence of Toxoplasma gondii antibodies in wild dolphins from the Spanish Mediterranean coast

Although Toxoplasma gondii infection has been found occasionally in cetaceans, little is known of the prevalence of antibodies to T. gondii in wild dolphins. Antibodies to T. gondii were determined in serum samples from 58 dolphins stranded in the Spanish Mediterranean coast. Modified agglutination test was used to determine T. gondii antibodies, and a titer of 1:25 was considered indicative of T. gondii infection. Antibodies to T. gondii were found in 4 of 36 striped dolphins (Stenella coeruleoalba), in 2 of 4 common dolphins (Delphinus delphis), in 4 of 7 bottlenose dolphins (Tursiops truncatus), and in 1 harbour porpoise (Phocoena phocoena). Antibodies were not found in 9 Risso's dolphins (Grampus griseus) and in 1 long-finned pilot whale (Globicephala melas) surveyed. The results indicate that T. gondii infection is frequent in at least 3 dolphin species from the Mediterranean Sea.     

Production and characterization of a panel of monoclonal antibodies to Toxoplasma gondii antigens

A representative collection was obtained containing 68 monoclonal antibodies (MAB) to Toxoplasma gondii antigens, which was characterized by the binding with the below fractions of tochizoites in the immune-enzyme assay (IEA) and immunoblotting (IB): membrane (MEM), somatic (water-soluble, SOM) and excretory-secretory (ES). Most of MABs were produced to MEM antigens (43), 6 MABs reacted with the somatic fraction, and 3 MABs reacted with both fractions. Two MABs to ES antigen were detected in the latter group. An analysis of MABs in concurrent IEA and IB revealed the immune-dominant proteins of the MEM and SOM fractions of antibodies to T. gondii tochizoites (p30 and p27, respectively). The presence of 2 non-overlapping antigenic determinants was shown for p30. Further research would detect MABs that could be used in the diagnosis of toxoplasmosis.