Commercial Enzyme-linked Immunosorbent Assay Systems Research Articles

Line immunoassay for detection of IgG antibodies to hepatitis E virus (Hepeviridae, Orthohepevirus, Orthohepevirus A)

The diagnostic efficacy of methods for hepatitis E serodiagnostic varies over a wide range; therefore, the combined use of tests of various formats is recommended. The aim of the research was to develop a test system for the detection of IgG antibodies to hepatitis E virus (HEV) in human serum by linear immunoassay (LIA). Serum samples from patients with hepatitis and healthy individuals were tested using commercial enzyme-linked immunosorbent assay systems for the presence of IgG antibodies to viral agents causing hepatitis and other infections associated with liver pathology. Recombinant antigens ORF2 and ORF3 of HEV genotypes 1 and 3 were used. The "RecomLine HEV IgG/IgM" reagent kit (Mikrogen GmbH, Germany) was used as a comparison test system. The first Russian diagnostic kit "Blot-HEV", designed to detect IgG antibodies to individual HEV proteins in human serum using LIA, was developed. The antigenic base is represented by strips of a nitrocellulose membrane with immobilized recombinant antigens ORF2 (aa 406-660) and ORF3 (aa 1-113) of HEV genotypes 1 and 3, and control antigens in the form of discrete lines. The conjugate was mouse monoclonal antibodies to human class G immunoglobulins labeled with horseradish peroxidase. The chromogen solution contained the 3,3',5,5'-tetramethylbenzidine. A visual and digital recording of results was provided. The analytical sensitivity of the test kit was 0.625 IU/ml for ORF2 antigens and 2.5 IU/ml for ORF3 antigens. The absence of the influence of endogenous interfering substances on the results of the analysis and the absence of cross-reactions with antibodies to hepatitis pathogens of the other etiologies had been shown. The sensitivity of the test system compared to the "RecomLine HEV IgG/IgM" kit was 92%, specificity 97%. Shelf life in condition of storage was determined to be 12 months. The developed test can be used to confirm the results of ELISA in laboratory diagnosis of hepatitis E.

Serological and Molecular Investigation of Swine Hepatitis E Virus in Pigs Raised in Southern Italy

Hepatitis E virus (HEV) infection is a common acute hepatitis transmitted by the fecal-oral route. In developed countries, the virus has a zoonotic potential, and domestic pigs and wild boars are considered main reservoirs. To assess the prevalence of HEV-positive animals in the Calabria region (southern Italy) on a serological and molecular level, a total of 216 autochthonous healthy pigs (Apulo-Calabrese breed) were sampled. Both sera and feces were collected. Pigs were grouped based on age: 117 pigs <6 months and 99 pigs >6 months. By using a commercial enzyme-linked immunosorbent assay system, a total of 173 (80%) of the 216 pigs tested seropositive. In all sampled farms (n = 8), pigs with antibodies (immunoglobulin G) against HEV were detected at a level higher than 60%, with a significant difference among age groups (P < 0.0001). Moreover, 16 fattening pigs were found to be nested reverse transcription PCR positive and thus to shed viral genomes in their feces. These positive findings resulted in a prevalence of 48.4% on the farm level (16 of 35 pigs) and an overall prevalence of 7.4% at the animal level (16 of 216 pigs). Based on the present study, HEV seems to circulate among the autochthonous domestic pig population of southern Italy with a low sharing rate. Further studies exploring the origin of infection are needed to minimize the risk of human exposure and to reduce consequences for public health.

Assessing PCV2 antibodies in field pigs vaccinated with different porcine circovirus 2 vaccines using two commercial ELISA systems.

Porcine circovirus type 2 (PCV2) is the primary causative agent for post-weaning, multisystemic, wasting syndrome. Consequently, serologic detection of and vaccination against PCV2 are important for the swine industry. Among several serological tests, the enzyme-linked immunosorbent assay (ELISA) is commonly used to measure anti-PCV2 antibody levels. In the present study, we used two commercial ELISA systems to comparatively evaluate anti-PCV2 antibodies in field pigs treated with three different PCV2 vaccines. Among a total of 517 serum samples, the results of the two ELISAs were fully concordant for 365 positive and 42 negative samples, indicating 78.7% agreement. In addition, the Pearson coefficient (0.636) indicated a moderate correlation between data from the two ELISAs. Results from the farms with pigs vaccinated with the three different PCV2 vaccines demonstrated that most of the vaccinated animals underwent seroconversion. However, the increase and duration of antibody titers varied depending on the vaccine, the presence of maternal antibodies, and the vaccination program. PCV2 serologic status and anti-PCV2 antibody levels of herds from this study could be utilized to determine the best timing for vaccination and assessing vaccination compliance.

Development of an on-site plum pox virus detection kit based on immunochromatography

Sharka disease, caused by plum pox virus (PPV), is the most serious viral disease of stone fruit trees. Among the eight known strains of the virus, PPV-D is the most important due to its recent global spread. Although enzyme-linked immunosorbent assay (ELISA) is the most common approach for diagnosing sharka, it involves time-consuming steps and requires expensive equipment and trained technicians. In this study, an on-site PPV detection kit based on immunochromatography was developed using polyclonal antibodies against the coat protein (CP) of a PPV-D isolate. The immunochromatographic (IC) assay kit was as sensitive as a commercial ELISA system for detecting Japanese PPV-D isolates. Moreover, it was easy to use (a one-step procedure), and results could be obtained on-site within 15 min without special laboratory equipment. The IC assay kit detected the virus from every aerial part of symptomatic Japanese apricot trees. In a detailed study of viral localization in leaves, the most suitable plant parts for use in the IC assay were symptomatic mesophyll tissues and the region from the petiole to the main vein. A positive reaction was also observed using the CP of other major (PPV-M and PPV-Rec) and minor (PPV-EA, PPV-W, and PPV-T) strains.

Food allergenic proteins in lactose as the pharmaceutical excipient (P6222)

Abstract Most drugs contain pharmaceutical excipients which are pharmacologically inactive substances used as vehicles to deliver the active ingredients of a medication. Some of these pharmaceutical excipients are produced from foods containing allergenic proteins (e.g., milk, egg, peanut, soybean, and sesame) and removing these completely from the excipients is difficult. Therefore, if individuals with food allergies consume drugs containing allergenic food-derived excipients, they are at risk for developing specific allergic symptoms. We determined the levels of total protein in lactose found in pharmaceutical excipients and ethical drugs known to contain lactose by spectrophotometric analyses. The level of total protein in lactose was approximately 1 mg/g. We also determined the levels of allergenic proteins in lactose using commercial enzyme-linked immunosorbent assay systems. The milk proteins in lactose were detected in the range of 1.4-13.1 μg/g. Furthermore, we performed a luciferase-based in vitro elicitation test (EXiLE test) in addition to western-blot analysis using the sera from patients with milk allergies in order to examine the allergenicity of protein residues in lactose. The result of the EXiLE test indicated that the protein residues in lactose have the ability to crosslink human FcϵRI on mast cells.

Analyses of the results of different test systems in the 2005 global proficiency testing schemes for infectious bursal disease virus and Newcastle disease virus antibody detection in chicken serum

The results of global proficiency testing schemes (PTS) for serological tests to detect antibodies against infectious bursal disease virus (IBDV) and Newcastle disease virus (NDV) in chicken serum, in which 125 and 120 laboratories, respectively, participated from Africa, Asia, Europe, Central and South America, were used to analyse the performances of different antibody test systems such as virus neutralization tests, haemagglutination inhibition tests, enzyme-linked immunosorbent assays and agar gel precipitation tests. All laboratories were asked to carry out their routine diagnostic tests for the detection of IBDV and NDV antibodies as usual. This global ring trial provided a large amount of data on variation within and between laboratories and test systems used worldwide. The data showed that the variation between the quantitative test results of different laboratories (R between) using the IBDV virus neutralization tests and the NDV haemagglutination inhibition test was higher (about double) compared with the variation within commercial enzyme-linked immunosorbent assay systems. Although both tests are often referred to and used as the “gold standard” in experimental and scientific studies, official procedures and for the validation of tests, this study shows that there is an urgent need for a global implementation of recommended test procedures and/or the inclusion of international reference sera in these studies.

Enzyme-linked immunosorbent assay using recombinant antigens expressed in mammalian cells for serodiagnosis of tick-borne encephalitis

A recombinant plasmid that expresses the tick-borne encephalitis (TBE) virus premembrane (prM) and envelope (E) proteins in mammalian cells was constructed. Recombinant proteins retained antigenic and conformational structures similar to those of native virus proteins, and transfected cells released virus-like particles (VLPs), which were 1.13–1.14 g/ml in density and 20–30 nm in diameter, into the culture medium. Recombinant E proteins were used for the development of an enzyme-linked immunosorbent assay (ELISA) to detect TBE virus-specific IgM and IgG antibodies in serum. The results of this ELISA correlated well with the results of commercial ELISA, when tested with 95 serum samples from clinically TBE-suspected patients. In addition, ELISA using recombinant antigens showed no cross-reactivity against serum from Japanese encephalitis (JE) patients, despite the cross-reactivity shown by commercial ELISA systems. These observations indicated that this newly developed ELISA system could distinguish tick-borne encephalitis from Japanese encephalitis infection, and that it constitutes a useful and safe alternative to conventional ELISA systems.

The ability of the enzyme-linked immunosorbent assay to detect antibody against Staphylococcus aureus in milk following experimental intramammary infection.

Changes in the milk antibody levels against Staphylococcus aureus were measured at the start of an experimental intramammary instillation of either S. aureus (Study I) or Staphylococcus hyicus (Study II). A commercial enzyme-linked immunosorbent assay system was used. Twenty-one Holstein cows were enrolled in Study I and 15 Holstein cows were used in Study II. Pathogen instillation began 21 days before the start of the non-lactating period. Cows received intramammary antibiotic treatment in all quarters immediately after the last milking, the start of the non-lactating period. Lacteal secretions were collected before the start of the non-lactating period, and during the immediate postpartum period in both studies, and during the non-lactating period in Study I. Milk was cultured for mastitis pathogens and S. aureus antibody levels and somatic cell counts were determined from all samples. There was an approximate 2-week delay in the elevation in antibody levels in response to the instillation of S. aureus. Antibody levels remained elevated in cows with S. aureus intramammary infections postpartum, but were below threshold in cows where intramammary infections were cured during the non-lactating period. Antibody levels were elevated by S. hyicus intramammary infections, remained elevated for the first 12 days postpartum, but were below threshold by day 21 postpartum. Cows with incipient intramammary S. aureus infections might be misclassified as false negatives by the antibody test. However, results suggest that cows with S. hyicus intramammary infections that were not cured would not be misclassified if milk is withheld from test for the first 30 days postpartum, as recommended by the manufacturer of the test.

Determination of Aflatoxins in Foods Using HPLC and a Commercial ELISA System

A commercially available enzyme linked immunosorbent assay (ELISA) system and a high performance liquid chromatography (HPLC) method were used simultaneously to analyze 178 samples of foodstuffs for total aflatoxins. High correlation coefficient values between results of the two methodologies were obtained (>0.96) with nuts, nut products, peanuts, and peanut butter. However, poor correlation was obtained from results of cereals and grain samples. The ELISA system demonstrated a high degree of reproducibility between wells (p<0.01). Of the 35 samples of nut and nut products examined, aflatoxins were detected in five samples at levels ranging from 40 to 276 μg/kg. However, of 73 peanut and peanut butter samples only one peanut sample contained 61 μg/kg, and of 70 samples of processed cereal and grain, three samples contained 6–10 μg/kg total aflatoxins. Both methods were also applied to six peanut meal samples, with known aflatoxin concentrations, provided in an interlaboratory collaborative trial. The results obtained by the HPLC and ELISA methods were in close agreement with the mean results obtained by the participating laboratories.

Technical improvements of a commercial ELISA to detect antibodies against bovine herpesvirus 1

Prevention and control of the spread of IBR/IPV virus in cattle presumes exact diagnosis even with large sample collections. The requirements are fulfilled firstly by the application of the most sensitive testing methods for virus specific antibodies, such as the enzyme-linked immunosorbent assay (ELISA), and secondly by the introduction of bovine milk as a specimen for sampling. Data are presented demonstrating the application of a commercial ELISA system for evaluation of large sample collections and its optimization for this purpose.