Higher Enzyme-linked Immunosorbent Assay Values Research Articles

The Role of GnRH Receptor Autoantibodies in Polycystic Ovary Syndrome.

ObjectiveIs polycystic ovary syndrome (PCOS) associated with activating autoantibodies (AAb) to the second extracellular loop (ECL2) of gonadotropin-releasing hormone receptor (GnRHR)?Design and MethodsWe retrospectively screened sera from 40 patients with PCOS and 14 normal controls (NCs) with regular menses using enzyme-linked immunosorbent assay (ELISA) for the presence of GnRHR-ECL2-AAb. We obtained similar data from 40 non-PCOS ovulatory but infertile patients as a control group (OIC) of interest. We analyzed GnRHR-ECL2-AAb activity in purified immunoglobulin (Ig)G using a cell-based GnRHR bioassay.ResultsThe mean ELISA value in the PCOS group was markedly higher than the NC (P = .000036) and the OIC (P = .0028) groups. IgG from a sample of 5 PCOS subjects, in contrast to a sample of 5 OIC subjects, demonstrated a dose-dependent increase in GnRHR-stimulating activity qualitatively similar to the acute action of the natural ligand GnRH and the synthetic agonist leuprolide. The GnRHR antagonist cetrorelix significantly suppressed (P < .01) the elevated GnRHR activity induced by IgG from 7 PCOS patients while the IgG activity level from 7 OIC subjects was unchanged. Five other OIC subjects had relatively high ELISA values at or above the 95% confidence limits. On further study, 3 had normal or low activity while 2 had elevated IgG-induced GnRHR activity. One suppressed with cetrorelix while the other did not. The copresence of PCOS IgG increased the responsiveness to GnRH and shifted the dosage response curve to the left (P < .01).ConclusionsGnRHR-ECL2-AAb are significantly elevated in patients with PCOS compared with NCs. Their presence raises important etiological, diagnostic, and therapeutic implications.

Comparison and evaluation of Renibacterium salmoninarum quantitative PCR diagnostic assays using field samples of Chinook and coho salmon

Renibacterium salmoninarum is a Gram-positive bacterium causing bacterial kidney disease (BKD) in susceptible salmonid fishes. Several quantitative PCR (qPCR) assays to measure R. salmoninarum infection intensity have been reported, but comparison and evaluation of these assays has been limited. Here, we compared 3 qPCR primer/probe sets for detection of R. salmoninarum in field samples of naturally exposed Chinook and coho salmon first identified as positive by nested PCR (nPCR). Additional samples from a hatchery population of Chinook salmon with BKD were included to serve as strong positive controls. The 3 qPCR assays targeted either the multiple copy major soluble antigen (msa) genes or the single copy abc gene. The msa/non-fluorescent quencher (NFQ) assay amplified R. salmoninarum DNA in 53.2% of the nPCR positive samples, whereas the abc/NFQ assay amplified 21.8% of the samples and the abc/TAMRA assay 18.2%. The enzyme-linked immunosorbent assay (ELISA) successfully quantified only 16.4% of the nPCR positive samples. Although the msa/NFQ assay amplified a greater proportion of nPCR positive samples, the abc/NFQ assay better amplified those samples with medium and high ELISA values. A comparison of the geometric mean quantity ratios highlighted limitations of the assays, and the abc/NFQ assay strongly amplified some samples that were negative in other tests, in contrast to its performance among the sample group as a whole. These data demonstrate that both the msa/NFQ and abc/NFQ qPCR assays are specific and effective at higher infection levels and outperform the ELISA. However, most pathogen studies will continue to require multiple assays to both detect and quantify R. salmoninarum infection.

Interpreting dual ELISA and qPCR data for bacterial kidney disease of salmonids

Although there are a variety of methods available for the detection of Renibacterium salmoninarum, the causative agent of bacterial kidney disease in salmon and trout, the enzyme-linked immunosorbent assay (ELISA) is probably the most widely used method. However, ELISA measures bacterial antigen, which does not necessarily reflect the number of cells present. We hypothesized that dual analysis of kidney tissue by ELISA and a quantitative real-time polymerase chain reaction assay (qPCR) would provide complementary information about antigen level and the number of bacterial genomes. We found that DNA extracted from the insoluble fraction of the ELISA tissue preparation produced the same qPCR result as DNA extracted directly from frozen tissue, permitting true dual analysis of the same tissue sample. We examined kidney tissue in this manner from individual free-ranging juvenile Chinook salmon and antibiotic-treated captive subadult Chinook salmon and observed 3 different patterns of results. Among the majority of fish, there was a strong correlation between the ELISA value and the qPCR value. However, subsets of fish exhibited either low ELISA values with elevated qPCR values or higher ELISA values with very low qPCR values. These observations suggest a conceptual model that allows inferences about the state of infection of individual fish based on dual ELISA/qPCR results. Although this model requires further assessment through experimental infections and treatments, it may have utility in broodstock selection programs that currently apply egg-culling practices based on ELISA alone.

ELISA Analysis for Fusarium in Barley: Development of Methodology and Field Assessment

Evaluating Fusarium head blight (FHB) involves inoculating barley (Hordeum vulgare L.) with Fusarium graminearum Schwabe [teleomorph Gibberella zeae (Schwein.) Petch] followed by visual observation of disease and analysis for deoxynivalenol (DON). Disease symptoms and DON are not always correlated because both are affected by environmental variables. The objective of this study was to develop an enzyme‐linked immunosorbent assay (ELISA) for quantification of FHB in barley. Antibodies to F. graminearum were tested for reaction with other Fusarium spp. Antibodies from cell line IF8 reacted with Fusarium spp. tested, but not other Ascomycota. The ELISA method was developed using seed lots with no, low, and high levels of DON. Quantity of seed, volume of extraction buffer, and agitation time were tested and Fusarium quantified with ELISA. Five genotypes each for high, medium, and low ELISA values were selected from a field experiment using a doubled‐haploid mapping population in 2003. The lines were grown in 2004 and scored for FHB, DON, and ELISA. ELISA had lower error than FHB or DON. Lines selected for low, medium, and high ELISA in 2003 had low, medium, and high ELISA values in 2004. ELISA and DON were correlated in both field experiments (r = 0.51). ELISA and DON were correlated (r = 0.71) in samples selected from grain elevators in 1993 through 2003 indicating naturally occurring Fusarium spp. outside the B clade had no effect on the performance of the ELISA analysis.

Thrombosis and ELISA optical density values in hospitalized patients with heparin‐induced thrombocytopenia

The natural history of heparin-induced thrombocytopenia (HIT) in the absence of thrombosis was previously established using functional assays for confirmation of diagnosis (e.g. 14C serotonin release assay). An enzyme-linked immunosorbent assay (ELISA) that detects the presence of antibodies directed against the heparin-platelet factor-4 (PF4) complex has largely replaced functional assays in many medical centers. Although the ELISA is highly sensitive for detecting HIT antibodies, its usefulness for predicting thrombotic outcomes has not been clearly established. We performed a retrospective chart review of all hospitalized patients at a university hospital who tested seropositive for HIT by a commercial ELISA during 2001 and 2002. A total of 63 inpatients were identified as HIT positive by ELISA. Forty-eight patients had no apparent HIT-associated thrombosis at the time of HIT seropositivity (i.e. isolated HIT) and only one was treated prophylactically with a direct thrombin inhibitor. The 30-day thrombosis rate for patients with isolated HIT was 17% (eight of 48). Higher ELISA optical density (OD) measurements correlated significantly with thrombosis (1.41 +/- 0.87 vs. 0.79 +/- 0.46, P <0.001). Patients with isolated HIT and an OD measurement of > or = 1.0 demonstrated nearly a 6-fold increased risk of thrombosis compared with those with OD values between 0.4 and 0.99 (odds ratio 5.74, 95% confidence interval 1.73, 19.0; absolute rate of thrombosis, 36% vs. 9%, respectively, P=0.07). We conclude that in hospitalized patients with isolated HIT, the presence of heparin-PF4 antibodies detected by ELISA was associated with a significant risk of subsequent thrombosis and higher ELISA values were observed among patients suffering thrombotic events.

Optimizing a protein-specific ELISA for the detection of protein-marked insects

A series of tests were conducted to determine if the sensitivity and efficiency of an established rabbit immunoglobulin (IgG)-specific enzyme-linked immunosorbent assay (ELISA) could be improved for detecting a protein mark on insects. Five variations of ELISA were examined for their ability to detect a rabbit IgG mark on the convergent lady beetle, Hippodamia convergens Guérin-Méneville. The conventional sandwich ELISA was the most sensitive immunoassay based on the strength of the ELISA reaction and the proportion of individuals scoring positive for rabbit IgG. ELISAs were also conducted on rabbit IgG-marked beetles that were either homogenized or soaked in sample buffer prior to the assay. Results showed that homogenized beetles yielded higher ELISA values in the conventional ELISA than soaked beetles, but the qualitative response (i.e., percentage scoring positive for the mark) was about the same for up to 18 days after marking. Therefore, in some instances, simply soaking an individual beetle might be a viable alternative to the labour-intensive homogenization of the sample. Sandwich ELISAs with immunoreagent incubation intervals held constant at 5, 10, 20, or 60 min were also examined for their ability to detect rabbit-IgG-marked beetles. Results showed that the ELISA with immunoreagent incubations of 60 min yielded a significantly higher ELISA reading than the shorter intervals. However, all the marked beetles examined scored positive for the presence of rabbit IgG, regardless of the incubation interval. Finally, a test was conducted to determine if the conventional ELISA could also detect the presence of relatively inexpensive normal rabbit serum on marked beetles. Beetles marked with rabbit serum diluted one part rabbit serum to one, four, or eight parts water yielded statistically similar ELISA values to those beetles marked with the conventional rabbit IgG mark.

Seroepidemiological study of Epstein-Barr virus infection in Bangladesh.

A seroepidemiological study was carried out on 502 sera to determine the prevalence of EBV infection in a group of Bangladeshi people (age range: 15 days-90 years). All sera were tested for IgG antibody to the EBV viral capsid antigen (VCA) by a commercially available enzyme linked immunosorbent assay (ELISA) and the negative sera were checked subsequently by indirect immunofluorescence (IF) methods. The prevalence of EBV infection in the study group was 81.27%. 42.37% of infants had antibodies to EBV by the age of 1 year. A significant rise in the percentage of seropositives between 0-1- and 1-2-year-old children was demonstrated, indicating a high rate of primary infection at these ages. The prevalence of IgG antibody to VCA was 87.93% in the 2-10 years age group and was sustained at over 85% thereafter. Higher ELISA values were more common both in the 0-2- and > 25-year age groups, the latter being statistically significant (P < 0.025). Similar higher values were also observed in females as compared to males (P = 0.05). Eighteen out of 109 negative sera and two equivocal sera by ELISA were found to be positive by indirect IF, indicating a negative predictive value of 82% for ELISA. The concordance between the two methods was 97% with ELISA proving to be less sensitive than indirect IF. It is concluded that the prevalence of EBV infection in Bangladeshi population is similar to that observed in other developing countries and that ELISA can be used for seroepidemiological surveys; however, the sera negative by ELISA should be checked routinely by indirect IF.

Definition of Populations at Risk for Plasmodium falciparum Infection in Three Endemic Areas of Cameroon

Blood samples were collected from 285 individuals attending hospitals in 1 of 3 different regions of Cameroon. Of these, 89 had Plasmodium falciparum parasitemia. Prevalence in the Douala region was drastically reduced above the age of 19. In the Njinikom and Bamenda regions, on the other hand, an appreciable decline in prevalence was not observed until over the age of 49. Enzyme-linked immunosorbent assay (ELISA) values indicate that in Douala, high antibody titers to P. falciparum were present in all age groups tested. In Njinikom and Bamenda, an age dependence was seen in the response, with sera from individuals above 20 having significantly higher ELISA values compared with those below age 20. Generally, individuals with high antibody titers had low or no parasitemia. Results suggest that future malaria control and treatment measures might target high risk populations such as those defined in this study.

Barley Yellow Dwarf Virus Multiplication and Host Plant Tolerance in Durum Wheat

AbstractA Canadian PAV‐like isolate of barley yellow dwarf virus (BYDV) was used to infect durum wheat (Triticum durum) cultivars previously identified in field trials involving artificial inoculation as highly sensitive (12 IDSN74), slightly tolerant (La Dulce), and relatively tolerant (Boohai and 12 IDSN227) to BYDV. The cultivars were inoculated in the greenhouse as seedlings, and indexed for virus accumulation by enzyme‐linked immunosorbent assay (ELISA) at various intervals between 3 and 60 days thereafter. Mean ELISA values were somewhat consistent with tolerance levels for 4 durum wheat cultivars, but the use of ELISA to screen for BYDV resistance in durum wheat is not practical. The magnitude of the difference between sensitive and tolerant cultivars for the mean ELISA value is not high enough, and it may be necessary to average readings between 3 and 60 days after inoculation to obtain somewhat meaningful ELISA data. The effect of vector aphid numbers on virus titre and aerial biomass in the sensitive durum wheat cv. Karim was also evaluated. There was no significant effect on virus content in a preliminary trial, but a second trial revealed that more viruliferous aphids per plant resulted in higher ELISA values. Infestation with 32 or 50 viruliferous Rhopalosiphum padi per plant depressed biomass yield below the level observed with 1–10 aphids per plant.

Detection of a Picorna-like Virus, Himetobi P Virus, in Organs and Tissues of Laodelphax striatellus by Immunogold Labeling and Enzyme-Linked Immunosorbent Assay

Himetobi P virus (HiPV)-infected planthoppers ( Laodelphax striatellus) were dissected and HiPV antigen present in salivary gland, heads, midguts, hindguts (plus Malpighian tubules), and sexual organs were examined by enzyme-linked immunosorbent assay (ELISA). Extremely high ELISA values were obtained in midguts. HiPV antigen was present in low amounts in hindgut samples, but was not detected in salivary glands. Electron microscopy showed that epithelial cells of midguts were heavily infected with HiPV. Infected cells were vacuolated and collapsed. Spherical viruses, which were specifically labeled with the HiPV-immunogold conjugate, were observed dispersed or aggregated in cytoplasms and vacuoles and occasionally in crystalline arrays. Large numbers of virus particles were found in lumens of midgut, hindgut, and Malpighian tubules. The feces of planthoppers contained extremely high concentrations of HiPV particles. Contaminations of host plant leaf surfaces with HiPV seem to be mainly by feces.

Screening of durum wheats for pasta-making quality with monoclonal antibodies for gliadin 45

Monoclonal antibodies specific for gliadin band 45 (gli 45) of common wheat cultivar 'Marquis' were treated against durum wheat gliadins using an enzyme-linked immunosorbent assay (ELISA). In a test of 15 durum cultivars, high ELISA values were associated with gli 45, high gluten strength, and high sodium dodecyl sulfate (SDS) sedimentation volume; low ELISA values were associated with gli 42, low gluten strength, and low SDS-sedimentation volume. Analysis of 120 F5 lines from the backcrosses Ward/Vic//Vic and Ward/Vic//Ward confirmed that a high reaction to ELISA was statistically correlated with the presence of gli 45, with high gluten strength, and with high SDS-sedimentation volume. It was concluded that monoclonal antibodies specific for gli 45 have potential as a test for rapid screening of durum wheat breeding populations for desirable pasta-making quality.Key words: monoclonal antibodies, gliadin, enzyme-linked immunosorbent assay, durum wheat.

Electron Microscopical and Serological Detection of Virus‐like Particles Associated with Lettuce Big Vein Disease

AbstractVery labile rod‐shaped particles measuring 324 and 152 nm × 18 nm were isolated only from lettuce plants affected with lettuce big vein (LBV) but not from healthy ones. An antiserum to these particles was prepared which enabled us to diagnose LBV‐affected plants, using immunosorbent electron microscopy (ISEM) and enzyme‐linked immunosorbent assay (ELISA). Clearly positive reactions were obtained in ISEM and ELISA even when symptoms of LBV‐affected plants raised in soil from various locations were indistinct. Higher ELISA values were obtained with extracts from leaves than with those from roots. In ISEM high numbers of particles were trapped from extracts of LBV‐affected plants with antiserum to tobacco stunt virus.

IgE antibodies to food allergens detected by ELISA, RAST and monkey PCA

IgE antibody to twelve common food and inhalant allergens was measured by enzyme-linked immunosorbent assay (ELISA) in the sera of thirteen atopic patients with one or more allergic disorders (asthma occurring in eleven; rhinitis in ten; eczema in six; urticaria in four; mouth and gastro-intestinal symptoms in six), of twelve non-atopic patients with various clinical symptoms (asthma in four; rhinitis in four; eczema in one; urticaria in two; mouth and gastro-intestinal symptoms in four) and sixteen cord blood sera. The atopic patients had significantly higher IgE ELISA values to the twelve allergens tested than non-atopic persons (P less than 0.01) and cord blood sera (P less than 0.001). Further investigation of IgE antibody to egg white in a group of twenty-two maternal-infant-paired sera using RAST and ELISA techniques showed two cord blood sera that had reproducible RAST values significantly greater than the mean of the maternal group. This was confirmed by RAST inhibition studies but not by monkey PCA tests. High IgE ELISA values to egg white extract did not correlate with RAST results.

Evaluation of an enzyme immunoassay for serodiagnosis of infections with Theileria parva and T annulata

An enzyme linked immunosorbent assay (ELISA) was used to determine antibody levels in cattle infected with Theileria parva and T annulata, using antigens prepared from the intra-erythrocytic piroplasm stage of the parasites. Antibody levels in calves infected with T parva increased from the 16th day after infection to reach peak values at days 28 to 35 and then declined rapidly, but in calves infected with T annulata antibody levels rose steadily up to day 40. Similar patterns of antibody production were shown by indirect fluorescent antibody tests. Sera from animals infected with T parva gave higher ELISA values with the antigen prepared from the homologous parasite species than with the antigen prepared from T annulata, but sera from cattle infected with T annulata gave similar high ELISA values with antigens prepared from both T parva and T annulata. Sera from animals infected with T mutans, T sergenti, T velifera, Babesia divergens, B major and B bovis gave only slight or no cross reactions with the piroplasm antigens, but serum from a calf infected with B bigemina cross reacted at a significant level with both piroplasm antigens.

Detection of Neisseria meningitidis cell envelope antigen by enzyme-linked immunosorbent assay in patients with meningococcal disease

Sera from various populations and cerebrospinal fluid (CSF) from meningitis patients were tested for Neisseria meningitidis cell envelope antigen by an enzyme-linked immunosorbent assay (ELISA) sandwich system. The minimum optical density (OD) for antigen detection in CSF was defined as the mean value obtained with specimens from a group of tuberculous meningitis patients plus two standard deviations. By this criterion, antigen was detected by ELISA in four of five CSF specimens from group A meningococcal meningitis patients. Very high ELISA values were obtained with sera from fulminating cases but control sera and sera obtained from meningococcal meningitis nonfulminating cases could not be clearly differentiated by this technique. The possible reasons for this are discussed. The results of this study show that as little as 15 ng/ml (protein) of cell envelope antigen can be detected by the ELISA sandwich test and suggest that the technique may be of value in clinical diagnosis of meningococcal disease.

Diagnosis of Reaginic Allergy with House Dust, Animal Dander and Pollen Allergens in Adult Patients

An enzyme-linked immunosorbent assay (ELISA) employing allergen adsorbed onto the internal surface of a plastic tube, and alkaline phosphatase conjugated anti-IgE was used for the determination of specific IgE antibodies to various inhalant allergens in serum samples from 255 individuals with asthma and/or allergic rhinitis. A total of 541 analyses were carried out and the results were compared with those of provocation tests, skin tests and the RAST. It was found that negative ELISA values with high probability indicated nonallergy and very high ELISA values (class greater than or equal to 5) indicated allergy. However, the proportion of positive ELISA values which did not correspond to clinical allergy was high, as were the number of intermediate, inconclusive values (class 1-2). Although significant correlations between the ELISA values and provocation test, skin test or the RAST were obtained with some allergens, it was concluded that our version of the ELISA has not advantages over currently used methods for allergy diagnosis.