Mean 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.

First Report of Tomato spotted wilt virus in Habanero and Tabasco Peppers in Florida.

In spring 2000, symptoms similar to thrips-vectored spotted wilt disease caused by Tomato spotted wilt virus (TSWV) were observed on habanero (Capsicum chinense) and tabasco (Capsicum frutescens) peppers in north Florida. Habanero peppers were from commercial fields grown for specialty markets and tabasco peppers were from research plots. Symptoms observed were leaf necrosis, fruit drop, necrotic stem lesions, and stunting. Fruit symptoms included chlorotic and necrotic spotting and distinct ring pattern and distortion. The incidence of symptomatic habanero peppers was 7 to 8% in one of the three production fields visited, and a lower incidence in two other fields (all in Jackson County). In tabasco pepper, TSWV was detected in spring and fall 1999, and spring 2000 seasons in 10 to 15% of the plants (Gadsden County). Adjacent tomato fields contained scattered plants exhibiting symptoms of TSWV. Diagnosis of TSWV from symptomatic stems, leaves, and fruit of habanero and tabasco peppers was confirmed by a double antibody sandwich enzyme linked immunosorbent assay (ELISA) using a commercially available kit (Agdia Inc., Elkhart, IN). ELISA values ranged from 1.57 to 1.95 for habanero pepper and 0.80 to 0.95 for tabasco pepper. The mean ELISA value of the negative controls was 0.001. To further verify TSWV infection, immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) was performed (1). The primer pair 5'-ATGTCTAAGGTTAAGCTC-3' and 5'-TTAAGCAAGTTCTGTGAG-3' represented the first and last 18 bases of the coding region of the nucleocapsid gene of TSWV, respectively, and produces approximately 800 bp PCR product (1). IC-RT-PCR gave a single DNA band of expected size in both habanero and tabasco samples, while no amplification was found in an uninfected pepper sample. This is the first report of TSWV on habanero and tabasco peppers in Florida. TSWV continues to be an economically important disease constraint to the production of tomato, pepper (C. annuum), peanut, and tobacco in the southeastern United States (observations from Georgia and Florida). Meanwhile, the known host range is expanding to include new species of cultivated vegetables.

Antibody response to toxic shock syndrome toxin-1 of Staphylococcus aureus in dairy cows

Antibody response to toxic shock syndrome toxin-1 (TSST-1) of Staphylococcus aureus in dairy cows was examined by enzyme linked immunosorbent assay (ELISA). Serum antibody to TSST-1 was not detected in 39 (76.5%) of 51 calves, which were 1–6 months of age. In contrast, TSST-1 antibody was demonstrated in 1728 (72.6%) of 2380 lactating cows housed on 36 dairy farms. The ELISA values of antibody ranged from 0.2 to 3.0 OD and presented a distribution with the peak at 1.6 OD. The mean ELISA value differed between farms, and it increased slightly along with parturient history. Somatic cell counts of milk from 174 lactating cows was compared with TSST-1 antibody and tst gene examined by polymerase chain reaction (PCR). The mean ELISA values in sera were approximately the same value (1.143–1.297 OD) in cows, with somatic cell counts in milk ranging from <100,000 to >1,000,000 cells per ml. The mean ELISA values in milk were lower than those of sera, but they rose as somatic cells increased. The tst gene of S. aureus was detected in 76.0–86.2% of the milk samples containing somatic cells >500,000 cells per ml, a level which indicates mastitis. The data suggests that many lactating cows may be infected by TSST-1- producing S. aureus.

Neutralizing antibodies to Escherichia coli Vero cytotoxin 1 and antibodies to O157 lipopolysaccharide in healthy farm family members and urban residents

An enzyme-linked immunosorbent assay (ELISA) to detect antibodies to Escherichia coli O157 lipopolysaccharide (LPS) was developed with sera from 63 children with confirmed recent E. coli O157 infection and from 256 age-stratified urban controls. The median ELISA values for control and case sera were 0.05 (interquartile range, 0 to 0.20; mean +/- standard deviation [SD], 0.15 +/- 0.22) and 1.41 (interquartile range, 1.11 to 1.59; mean +/- SD, 1.41 +/- 0.53), respectively (P < 0.001). With a breakpoint of 0.59 (mean ELISA value of the control sera + 2 SDs), the assay had a sensitivity, specificity, and positive and negative predictive values of 95, 94, 80, and 98%, respectively, for recent E. coli O157 infection. The O157 LPS assay and Vero cytotoxin (VT) 1-neutralizing-antibody (NAb) assay were used to compare the relative frequencies of O157 LPS antibodies and VT1-NAbs in an age-stratified urban population from Toronto, Ontario, Canada, and in 216 healthy family members from dairy farm in southern Ontario. The frequency of O157 LPS antibodies was about threefold higher in dairy farm residents (12.5%) than in urban residents (4.7%) (P < 0.01). Similarly, the frequency of VT1-NAbs was about sixfold higher in dairy farm residents (42.0%) than in urban residents (7.7%) (P < 0.001). These findings are consistent with a greater level of exposure of dairy farm residents to VT-producing E. coli (VTEC) strains. The high rate of seropositivity to VT1 in farm residents probably reflects the booster effect of repeated VTEC exposures and argues against a sustained generalized immunosuppressive effect of VT1. Seroepidemiological studies may help in assessing the level of exposure of different populations to VTEC strains.

Patterns of Antibody Levels to the 96 tR Recombinant Protein of Plasmodium falciparum in Children over a Six-Month Period

Several Plasmodium falciparum asexual stage antigens have been reported as possible candidate antigens for vaccines against malaria. One such is the 96-kDa antigen. We used the 96 tR recombinant protein to determine the levels of antibodies to this protein over a 6-mo period in children 4 mo to 15 yr old, who make up the population at risk in Cameroon. The mean enzyme-linked immunosorbent assay (ELISA) on 83 children at the start of the study in June was 0.270, with 13% of these children having ELISA values considered to be positive (> or = 0.36). In the follow-up study in early December, the mean ELISA was 0.320 with 20% of the children having positive values. In the period between June and December, 85% of the children who returned for follow-up had had 1 or more attacks of malaria. The mean ELISA values of this latter group had increased from 0.235 in June to 0.318 in December, with 6% of the children in June having positive values compared with 18% in December. These findings reinforce our evidence from a previous study that reactivity to the 96-kDa antigen is related to adequate exposure or repeated infection.

Enzyme-linked immunosorbent assay for detection of immunoglobulin G antibodies to Escherichia coli Vero cytotoxin 1

The frequency of Vero cytotoxin 1 (VT1)-neutralizing antibody (NAb) in serum specimens from 790 age-stratified (0 to 70 years) control individuals from Toronto was 61 of 790 (7.7%), with a peak of 19% in the 20- to 30-year-old age group and a second peak of 16.7% in the 60- to 70-year-old age group. A total of 568 serum specimens, including 538 from the 790 Toronto control subjects, 21 from patients from three outbreaks of VT-producing Escherichia coli (VTEC) infection, and 9 known VT1-NAb-positive serum specimens from patients with hemolytic-uremic syndrome (HUS), were then tested for the presence of anti-VT1 immunoglobulin G (IgG) by an enzyme-linked immunosorbent assay (ELISA). The mean ELISA values of 522 VT1-NAb-negative serum specimens and 46 VT1-NAb-positive serum specimens were 0.09 +/- 0.06 (range, 0 to 0.56) and 0.78 +/- 0.66 (range, 0.16 to 2.91), respectively (P < 0.001; Student's t test). With a breakpoint of 0.21 (mean ELISA value of the VT1-NAb-negative sera + 2 standard deviations), the sensitivity, specificity, positive predictive value, and negative predictive value of the VT1 IgG ELISA compared with those of the VT1-NAb assay were, respectively, 95.7, 98.7, 86.3, and 99.6%. There were nine discrepant serum specimens, of which seven were anti-VT1 IgG positive and VT1-NAb negative and two were anti-VT1 IgG negative and VT1-NAb positive. The ELISA was also used for testing 238 control serum specimens from The Netherlands, Japan, and India and acute- and convalescent-phase serum specimens from 42 Toronto patients with HUS. The frequencies of anti-VT1 IgG (with VT1-NAb frequencies in parantheses) in control sera from the Netherlands, Japan, and India were 6% (3%), 1.1% (0%), and 12% (10%), respectively, with no age clustering. The frequencies of anti-VT1 IgG seropositivity in HUS patients were 5 of 14 (35.7%) in patients with unknown toxin exposure, 2 of 22 (9.1%) in individuals with known exposure to VT1 plus VT2 or VT1 alone, and 0 of 6 (0%) in patients exposed to only VT2. Development of serum anti-VT1 IgG response appears to be the exception rather than the rule in sporadic HUS patients infected with VTEC expressing VT1. However, in two family outbreaks associated with VTEC strains expressing VT1 alone and VT1 plus VT2, respectively, the presence of anti-VT1 IgG in virtually all exposed individuals who remained symptom free suggests that the presence of antibody was associated with protection.

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.

Field evaluation of the use of an ELISA to detect chloroquine and its metabolites in blood, urine and breast-milk

The evaluation of an enzyme-linked immunosorbent assay (ELISA) for chloroquine and its metabolites in blood, urine and breast-milk is reported. ELISA blood levels, following standard treatment with chloroquine of pregnant and non-pregnant women, showed mean values comparable to other analytical methods. Blood chloroquine concentrations were estimated at day 0, 350–400 ng/ml; day 2, 1000–1500 ng/ml; day 14, 350–400 ng/ml; day 28, 180–350 ng/ml. In a separate sample a significant association was observed between history of chloroquine use in the previous 2 weeks and blood ELISA values ( P < 0·01). Mean ELISA values in breast-milk were higher than in corresponding whole blood samples. High concentrations of chloroquine in urine were observed. There was a weak association of the ELISA of urine and blood samples collected at the same time ( P = 0·076). This study confirms the low sensitivity (<55%) of the Dill-Glazko test in urine, which is 100–1000 times less sensitive than the ELISA; the latter can detect 10–20 ng chloroquine per ml. A cut-off value for blood positivity 2 weeks following therapeutic drug ingestion was determined (40% ELISA inhibition), which can be used to make decisions about subject selection in drug sensitivity tests or population surveys. There are several advantages with the ELISA under field conditions. These include direct measurement of whole blood concentration; avoidance of problems of urine collection; suitability of finger-prick samples, especially with young children; application to population surveys to monitor drug use; and selection of subjects for drug sensitivity tests and monitoring of blood levels during in vivo tests.

Rapid diagnosis of acute Epstein-Barr virus infection by an indirect enzyme-linked immunosorbent assay for specific immunoglobulin M (IgM) antibody without rheumatoid factor and specific IgG interference

An indirect enzyme-linked immunosorbent assay (ELISA) for detection of Epstein-Barr virus-specific immunoglobulin M (IgM) antibody was developed with commercial reagents. Sera containing rheumatoid factor (RF) (as little as 0.5 IU/ml) coupled with specific IgG resulted in false-positives in the ELISA. This interference was eliminated by the use of anti-human IgG antibodies to remove RF and IgG. Thus, pathogen-specific IgG complexes to which IgM-RF could be bound during the subsequent test were inhibited, and competition between specific IgG and IgM was also prevented. Of the 1,672 serum specimens tested, 353 were found to be Epstein-Barr virus IgM antibody positive by indirect immunofluorescence (IF). Compared with the IF test, the ELISA showed 96.6% sensitivity, 99.7% specificity, and 99% accuracy. Further evidence indicated that most of the 12 ELISA false-negatives were IF false-positives. There was a linear correlation between mean ELISA values and increasing IF titers (r = 0.96). However, the IF test has the disadvantages that it lacks automated reading and requires considerable technical expertise, both of which restrict the range of laboratories performing the test. The indirect ELISA has the advantages that it is simple and rapid and can be automated. All the reagents used in this assay are commercially available, have been prestandardized, and are stable.

Rhodococcus equi: Equine neutrophil chemiluminescent and bactericidal responses to opsonizing antibody

The opsonic capacity of serum containing R. equi-specific antibody was compared with antibody-deficient sera using luminol-dependent chemilumenscence (LDCL) and bactericidal assays. These assays incorporated peripheral blood polymorphonuclear neutrophilic leukocytes (PMNL) exposed to R. equi opsonized with neonatal equine pre-colostral serum (control) or serum from foals with R. equi infections (principal). All sera were complement inactivated at 56°C for 30 min. Bacteria were obtained from the lung of a foal with R. equi pneumonia. Neutrophils were obtained from one adult horse for LDCL and another for bactericidal assays. Chemiluminescence of PMNL exposed to R. equi opsonized with control or principal sera was measured in a liquid scintillation counter. Mean peak LDCL within 1 h was significantly ( P<0.01) higher with principal sera (2.4 × 10 5 cpm) than with control sera (0.018 × 10 5 cpm). A radioisotope bactericidal assay was used to determine the effect of control or principal sera on PMNL capacity to kill R. equi. Mean peak percent kill of R. equi by PMNL within 2 h was significantly ( P<0.01) higher with principal sera (95.2%) than with control sera (54.6%). Enzyme-linked immunosorbent assay (ELISA) values for R. equi-specific antibody were determined on all sera. Mean ELISA values were significantly ( P<0.01) higher for principal sera (71.8) than for controls (0.0). This investigation documents the presence and biological effectiveness of opsonic activity in complement-inactivated sera from foals with R. equi infections and R. equi-specific antibody.

Assessment of an enzyme linked immunosorbent assay for the detection of cattle infected with Mycobacterium bovis.

An indirect enzyme linked immunosorbent assay (ELISA) using an unpurified antigen was assessed for its accuracy in detecting Mycobacterium bovis infected cattle. The ELISA test recorded sensitivities of 88.7% and 63%, respectively, for infected cattle tuberculin tested positive and for infected cattle never tuberculin tested. Specificity was determined at 52.6% for cattle from confirmed free herds which had never been tuberculin tested. Significant differences in the mean ELISA values were recorded between the 3 groups. No evidence was found for long term effects of tuberculin testing upon the titre of antibodies detected by the ELISA in unaffected cattle. The indirect ELISA using the unpurified antigen of this assay was considered to be unsuitable as an alternative to tuberculin testing for the detection of M. bovis infected animals.

Evaluation of ELISA for the diagnosis of paragonimiasis westermani

Possible applicability of an enzyme-linked immunosorbent assay (ELISA) for the diagnosis of paragonimiasis westermani was examined using crude extract of adult Paragonimus westermani (Pw) as the test antigen. The mean ELISA values of the sera from Pw egg-positive cases and from clinically suspected cases were more than 22 times higher than that of the healthy control. No, or only marginal, cross-reactivity was observed against Pw antigen using sera from patients with various parasitic diseases other than paragonimiasis, except that from schistosomiasis and clonorchiasis cases. When serum samples from the endemic area were tested by ELISA and double diffusion (DD), sera from skin test (ST)-positive and DD-positive cases were all positive with ELISA, whereas approximately 19% of ST-positive but DD-negative cases were positive for ELISA. Diagnosis by ELISA values correlated well with, and seemed to be more sensitive than, that by DD test. In some definite paragonimiasis cases, the ELISA value was markedly reduced after drug-treatment with bithionol. These results suggest that ELISA is useful not only in mass screening but also in the evaluation of the efficacy of drug treatment in paragonimiasis.

Circulating IgA- and IgG-class antigliadin antibodies in dermatitis herpetiformis detected by enzyme-linked immunosorbent assay.

A sensitive and technically simple enzyme-linked immunosorbent assay (ELISA) was developed to demonstrate circulating IgA- and IgG-class antibodies to gliadin, a component of wheat gluten. Serum samples from 24 patients with dermatitis herpetiformis (DH), 5 with coeliac disease (CD) and 75 normal controls were analysed. Antigliadin antibodies (AGA) of the IgA class were detected in 71% of DH patients, all of the CD patients and 19% of the controls. IgG-AGA was found in over 90% of DH patients and controls and in all of the CD patients. The mean ELISA values of both IgA- and IgG-class AGA were significantly higher in DH patients than in the controls. The occurrence of circulating IgA-class AGA is compatible with the hypothesis that these antibodies can be deposited in the skin, e.g. as immune complexes, or due to cross-reactivity of gliadin and dermal reticulin.