Microtiter Plate Enzyme-linked Immunosorbent Assay Research Articles

Feline Leukemia Virus p27 Antigen Concentration and Proviral DNA Load Are Associated with Survival in Naturally Infected Cats.

Longitudinal studies of cats naturally infected with feline leukemia virus (FeLV) are important for understanding disease outcomes. Levels of p27 antigen and copy numbers of proviral DNA have been associated with FeLV-infection courses. The purpose of this prospective study was to establish cutoff values for p27 antigen concentration and proviral DNA load that distinguished high positive from low positive groups of cats and to evaluate an association with survival. At enrollment, 254 cats were tested by point-of-care and microtiter plate enzyme-linked immunosorbent assays (ELISAs) for p27 antigen and real-time polymerase chain reaction (PCR) for proviral DNA. The 127 positive cats were retested monthly for six months and monitored for survival over the four-year study. A receiver operating characteristic-based analysis of samples with concordant or discordant qualitative results for p27 antigen and proviral DNA was used to establish cutoff values, and when applied to test results at enrollment for classifying cats as high positive or low positive, a significant difference in survival was observed. High positive cats had a median survival of 1.37 years (95% CI 0.83–2.02) from time of enrollment, while most low positive cats were still alive (93.1% survival). Quantitative results for p27 antigen concentration and proviral DNA load were highly correlated with survival times in FeLV-infected cats.

Total aflatoxin, aflatoxin B1 and ochratoxin a levels in Turkish wheat flour

This study was designed to assess the total aflatoxin (total AF), aflatoxin B1 (AFB1) and ochratoxin A (OTA) levels in 100 samples of wheat flour obtained from 7 different producing regions of Thrace by the microtitre plate enzyme-linked immunosorbent assay (ELISA). According to the EC Regulation and the Turkish Food Codex, the total AF, AFB1 and OTA levels were between minimum detection limit (0.05, <1.0, 0.025 μg/kg) and the maximum tolerable limit (4, 2, 3 μg/kg) in 43, 18 and 53 samples, respectively. Whilst 2, 2 and 28 samples had unacceptable contamination levels higher than the maximum tolerable limits, respectively. This is the first study in Thrace, Turkey on account of OTA, and the presence of OTA in 81% of analysed samples and taking part in South-Eastern Europe of sampling area would be indirectly related to Balkan Endemic Nephropathy and also be of risk to public healthy.

Evaluation of pap and sfa Genes Relative Frequency P and S Fimbriae Encoding of Uropathogenic Escherichia coli Isolated from Hospitals and Medical Laboratories; Yasuj City, Southwest Iran

Background: Urinary tract infections (UTI) are one of the common infections worldwide. Escherichia coli is the most common bacteria causing UTI. Objectives: The current study aimed at investigating the susceptibility pattern, biofilm formation, and the relationship between pap and sfa virulence genes and biofilm formation in E. coli strains isolated from patients with UTI from 2017 to 2018 in Yasuj City, Iran. Methods: In the current cross-sectional study, 130 E. coli strains were isolated from patients with UTI. Antimicrobial susceptibility testing was performed by disk diffusion method. The presence of pap and sfa genes was investigated by multiplex polymerase chain reaction (PCR). Biofilm formation was tested using microtiter plate assay and ELISA (enzyme-linked immunosorbent assay) reader for E. coli isolates. Results: Out of 130 E. coli strains isolated from UTI samples, 98 (75.38%) were positive for the biofilm formation, (14% strong, 31% moderate, and 53% weak). The pap and sfa genes were detected in 50% and 29% of the isolates, respectively. Majority of the isolates were resistant against ampicillin (80%) and susceptible to amikacin and meropenem (100%); the pap and sfa genes were detected in 92.85% and 85.71% of the biofilm-forming isolates, respectively. These genes in Uropathogenic E. coli (UPEC) have a significant relationship with strong biofilm formation (P < 0.0001). Conclusions: The obtained results indicated that the frequency of pap gene was higher than that of sfa in E. coli strains isolated from patients with UTI in Yasuj. The pap and sfa genes in E. coli strains had a significant relationship with strong biofilm formation. In addition, the frequency of biofilm formation in antibiotic-resistant E. coli was higher than that of antibiotic-sensitive strains. According to the results of the current study, it is recommended to administer Amikacin or Meropenem in Yasuj to treat UTI.

Adaptive Focused Acoustics (AFA) Improves the Performance of Microtiter Plate ELISAs

We investigated the use of Adaptive Focused Acoustics (AFA) technology to improve the performance of microtiter plate enzyme-linked immunosorbent assays (ELISAs). Experiments were performed with commercially available AFA instrumentation and off-the-shelf 96-well microtiter plate sandwich ELISAs. AFA was applied over a range of acoustic energies, temperatures, and durations to the antigen/antibody binding step of an ELISA for measuring HIV-1 p24 in tissue culture samples. AFA-mediated antigen/antibody binding was enhanced up to 2-fold over passive binding at comparable temperatures and was superior or comparable at low temperature (8–10 °C) to passive binding at 37 °C. Lower nonspecific binding (NSB), lower inter- and intra-assay coefficients of variation (CVs), higher Z′ factors, and lower limits of detection (LODs) were measured in AFA-mediated assays compared with conventional passive binding. In a more limited study, AFA enhancement of antigen/antibody binding and lower NSB was measured in an ELISA for measuring IGFBP-3 in human plasma. We conclude from this study that application of AFA to antigen/antibody binding steps in microtiter plate ELISAs can enhance key assay performance parameters, particularly Z′ factors and LODs. These features render AFA-mediated binding assays potentially more useful in applications such as high-throughput screening and in vitro diagnostics than assays processed with conventional passive antigen/antibody binding steps.

Power-free chip enzyme immunoassay for detection of prostate specific antigen (PSA) in serum

A power-free, portable “Chip EIA” was designed to render the popular Enzyme Linked Immunosorbent Assay (ELISA) more suitable for point-of-care testing. A number of microfluidic platforms have enabled miniaturization of the conventional microtitre plate ELISA, however, they require external pumping systems, valves, and electric power supply. The Chip EIA platform has eliminated the need for pumps and valves through utilizing a simple permanent magnet and magnetic nanoparticles. The magnetic nanoparticles act as solid support to capture the target and are then moved through chambers harboring different reagents necessary to perform a sandwich ELISA. The use of magnetic nanoparticles increases the volume-to-surface ratio reducing the assay time to 30min. Changing the color of horseradish peroxidase (HRP) substrate to green indicates a positive result. In addition, a quantitative read-out was obtained through the use of cellphone camera imaging and analyzing the images using Matlab®. Cell phones, including smart ones, are readily available almost everywhere. The Chip EIA device was used to assay total prostate specific antigen (tPSA) in 19 serum samples. The PSA Chip EIA was tested for accuracy, precision, repeatability, and the results were correlated to the commercial Beckman Colter, Hybritech immunoassay® for determination of tPSA in serum samples with a Pearson correlation coefficient (R2=0.96). The lower detection limit of the PSA Chip EIA was 3.2ng/mL. The assay has 88.9% recovery and good reproducibility (% CV of 6.5). We conclude that the developed Chip EIA can be used for detection of protein biomarkers in biological specimens.

Development of single-chain variable fragment (scFv) antibodies against hapten benzo[a]pyrene: a binding study

Due to its highly carcinogenic and mutagenic effect on humans, a maximum tolerable limit of 10 ng/L of benzo[a]pyrene (B[a]P) in drinking water was set by the European Commission (Council Directive 98/83/EC). Although several polyclonal and monoclonal antibodies (mAb) for the detection of B[a]P and other polycyclic aromatic hydrocarbons (PAH) have been developed by others, a traditional enzyme-linked immunosorbent assay (ELISA) with a limit of quantification of 10 ng/L for monitoring B[a]P has not been developed. With this in mind, several single-chain variable fragment (scFv) antibodies were created using existing mAbs against the extremely hydrophobic hapten B[a]P, and their heavy and light chains recombined to make unique variable light (V(L)) and heavy (V(H)) chain combinations. Their binding behaviour was investigated using microtiter plate ELISA and surface plasmon resonance techniques. Specifically, the coding sequences for V(L) and V(H) chains of 10 murine anti-B[a]P antibody producing hybridoma cell lines were isolated by degenerate oligonucleotide primer sets, cloned in phagemid pIT2 and transferred into Escherichia coli HB2151. To systematically investigate the interaction of the V(L) and V(H) domains, three high-affinity B[a]P-specific and one nonspecific clone were selected and recombined to build a set of 16 different V(L) and V(H) combinations. On the basis of our data, it was shown that the V(H) plays the major role for specific binding of B[a]P, whilst the V(L) can, in some cases, increase the final sensitivity of the assay by one order of magnitude. Furthermore, the sequence analysis of scFvs indicates that the complementarity determining region H3 plays a major role in affinity, whilst cross-reactivity to seven other PAHs demonstrates the importance of the V(L) in providing cross-reactivity.

Screening and characterization of new monoclonal anti-benzo[a]pyrene antibodies using automated flow-through microarray technology

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants, which can cause cancer in humans. The maximum tolerable limit of benzo[a]pyrene (B[a]P) in drinking water was set to 10ng/L by the European Commission (Council Directive 98/83/EC), because of its highly carcinogenic and mutagenic effect on humans. In the present investigation, mice were immunized with B[a]P-bovine serum albumin conjugates and 110 generated hybridoma cell lines screened by different techniques to identify clones that produce anti-B[a]P antibodies. Subsequently, a new automated flow-through biochip noncompetitive direct chemiluminescence immunoassay (CLEIA) was compared with conventional indirect and direct enzyme-linked immunosorbent assays (ELISAs). It was demonstrated that the microchip-based screening method compared to ELISA was fast and very sensitive with use of only nanoliter volumes of supernatant. Forty clones could be evaluated in less than 5min. Six high affinity monoclonal antibodies with different cross-reactivities (CR) for individual PAHs were identified by the chip-based assay and indirect microtiter plate ELISA. In comparison, the direct ELISA in the microtiter plate failed to identify three of these clones. The four antibodies with the highest affinity had half maximum inhibitory concentrations (IC50 values) between 0.31 and 0.92μg/L for B[a]P. Affinity constants of these four antibodies were determined by surface plasmon resonance using a water soluble B[a]P-peptide. The observed CR pattern of the four monoclonal antibodies for 16 tested PAHs was quite different. Only one specific antibody for B[a]P was observed, while others were more suitable for class-specific PAH determination.

Serological markers for Leishmania donovani infection in Nepal: agreement between direct agglutination test and rK39 ELISA

Visceral leishmaniasis (VL) is an important vector-borne disease caused by Leishmania donovani in the Indian subcontinent. The actual incidence and role of asymptomatic infections in the region are not wellknown. We used the direct agglutination test (DAT) and the rK39 ELISA as L. donovani infection markers in 10 VL endemic villages in Nepal. DAT titre distribution showed two subgroups in the population (infected and non-infected individuals), while rK39 did not. The agreement between both tests was moderate (j = 0.53; 95% CI 0.49–0.57). More research is needed to develop validated markers for Leishmania infection.

Development of rapid, specific and sensitive detection of Cucumber mosaic virus

Cucumber mosaic virus (CMV) causes major losses to thousand agricultural and horticultural crops around the world. Unlike other plant pathogens, there are no direct methods available yet to control viruses and, consequently, the current measures rely on indirect tactics to manage the viral diseases. Hence, methods for detection and identification of viruses, both in plants and vectors, play a critical role in virus disease management. A rapid assay for diagnosis of CMV, which can be employed in both laboratory and field, is essential. Therefore, this study was undertaken to develop a procedure for detection of the CMV in infected plants using a monoclonal and polyclonal antibodies. Dotimmunobinding assays (DIBA) are useful alternatives to microtitre plate enzyme-linked immunosorbent assay (ELISA). Nine monoclonal antibodies were readily used for detected CMV by TAS-ELISA and DIBA of infected plants. DIBA has about the same sensitivity as ELISA in microtiter plates, but it has the additional advantages of simplicity, quick completion in the field or office on large numbers of samples, economy, and can be quantified using densitometry.

Development of rapid, specific and sensitive detection of Cucumber mosaic virus

Cucumber mosaic virus (CMV) causes major losses to thousand agricultural and horticultural crops around the world. Unlike other plant pathogens, there are no direct methods available yet to control viruses and, consequently, the current measures rely on indirect tactics to manage the viral diseases. Hence, methods for detection and identification of viruses, both in plants and vectors, play a critical role in virus disease management. A rapid assay for diagnosis of CMV, which can be employed in both laboratory and field, is essential. Therefore, this study was undertaken to develop a procedure for detection of the CMV in infected plants using a monoclonal and polyclonal antibodies. Dotimmunobinding assays (DIBA) are useful alternatives to microtitre plate enzyme-linked immunosorbent assay (ELISA). Nine monoclonal antibodies were readily used for detected CMV by TAS-ELISA and DIBA of infected plants. DIBA has about the same sensitivity as ELISA in microtiter plates, but it has the additional advantages of simplicity, quick completion in the field or office on large numbers of samples, economy, and can be quantified using densitometry.

Development and analytical performance evaluation of an automated chemiluminescent immunoassay for pro-gastrin releasing peptide (ProGRP)

Pro-gastrin releasing peptide (ProGRP) concentrations in blood play an important role in the diagnosis and treatment of patients with small cell lung cancer (SCLC). The automated quantitative ARCHITECT ProGRP assay was developed to aid in the differential diagnosis and in the management of SCLC. The purpose of this study was to evaluate the analytical performance of this chemiluminescent microparticle immunoassay at multiple sites. ARCHITECT ProGRP measures ProGRP using a two-step sandwich using monoclonal anti-ProGRP antibodies coated on paramagnetic microparticles and labeled with acridinium. Analytical performance of the assay was evaluated at four sites: Abbott Japan, Denka Seiken, the Johns Hopkins University, and the University of Munich. Total precision (%CV) for nine analyte concentrations was between 2.2 and 5.7. The analytical sensitivity of the assay was between 0.20 pg/mL and 0.88 pg/mL. The functional sensitivity at 20% CV was between 0.66 pg/mL and 1.73 pg/mL. The assay was linear up to 50,000 pg/mL using a 1:10 autodilution protocol. The calibration curve was stable for 30 days. Comparison with the Fujirebio microtiter plate enzyme-linked immunosorbent assay (EIA) ProGRP assay gave a slope of 0.93 and a correlation coefficient (r) of 0.99. These results demonstrate that the ARCHITECT ProGRP assay has excellent sensitivity, precision, and correlation to a reference method. This assay provides a convenient automated method for ProGRP measurement in serum and plasma in hospitals and clinical laboratories.

Development of a microtiter plate ELISA and a dipstick ELISA for the determination of the organophosphorus insecticide fenthion.

In previous studies, polyclonal antibodies against the organophosphorus insecticide fenthion were obtained and an indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed for this pesticide. In this study, using these antibodies and an enzyme tracer, direct competitive ELISAs for fenthion in microtiter plate and dipstick formats were developed. The microtiter plate ELISA showed an IC(50) value of 1.2 microg/L with a detection limit of 0.1 microg/L. The antibodies showed negligible cross-reactivity with other organophosphorus pesticides. The use of the dipstick format using Immunodyne as a support membrane allowed the quick visual detection of fenthion in concentrations >10 microg/L. The IC(50) value of the dipstick format using reflectance detection was 15 microg/L with a detection limit of 0.5 microg/L. The recoveries of fenthion from spiked vegetable samples using the two formats without any prior enrichment or cleanup steps were 87-116%.

Evaluation and validation of commercially available enzyme-linked immunosorbent assays (ELISAs) specific for atrazine, chlorpyrifos, and diazinon in aqueous phase

The performance of commercially available microtiter plate enzyme-linked immunosorbent assays (ELISA) kits specific for the determination of triazines (atrazine), chlorpyrifos, and diazinon was evaluated for sensitivity, intra-assay repeatability, and accuracy using samples of known concentration in aqueous solution. Mean percent recovery values were not significantly different among concentrations for diazinon (One-way parametric ANOVA, P=0.46, n=72). However, mean percent recovery values were significantly different among concentrations for both atrazine and chlorpyrifos analyses (One-way parametric ANOVA, P<0.0001, n=36 for both analyses), and were highly dependent on concentrations for chlorpyrifos (% recovery=−0.155 (concentration)+238.448, r 2=0.91, P<0.0001, n=36). All methods demonstrated a high degree of statistical separation between readings at various concentrations (One-way parametric ANOVA followed by Student–Neuman–Keuls (SNK) multiple range test, P<0.0001 for all analyses) and a close correlation between known concentrations and concentrations derived from ELISA for all three analytes (diazinon, r=0.985, P<0.0001, n=72; atrazine r=0.967, P<0.0001, n=36; chlorpyrifos r=0.947, P<0.0001, n=36). Statistical comparisons between known concentrations and concentrations derived from ELISAs showed that diazinon values were significantly ( P<0.01, n=12 per concentration level) overestimated for all concentration levels. Chlorpyrifos concentrations were significantly ( P<0.01, n=6 per concentration level) overestimated at lower concentrations and significantly ( P<0.01, n=6 per concentration level) underestimated at higher concentrations. ELISA-derived atrazine concentrations were statistically similar to known concentrations for most concentration levels ( P>0.05, n=6 per concentration level). Results indicate that ELISA kits are excellent for screening purposes, although consistent overestimation of ELISA for diazinon at all concentration levels and chlorpyrifos at lower concentrations levels must be resolved before the kits can be used routinely for regulatory compliance monitoring.

Steroidogenic Pathway to Estradiol-17β Synthesis in the Ovarian Follicles of the Protogynous Wrasse, Pseudolabrus sieboldi

The bambooleaf wrasse, Pseudolabrus sieboldi, is a diandric protogynous fish with a diurnal rhythm of ovarian development, and females spawn daily during the spawning season. This study investigated the steroidogenic pathway for estradiol-17β (E2) biosynthesis in vitellogenic ovarian follicles of the bambooleaf wrasse. We incubated follicles in vitro with radioactively labeled steroid precursors, and measured serum steroid levels using microtiter plate enzyme-linked immunosorbent assays (ELISAs). ELISAs for estrone (E1) and testosterone (T) were developed. The experiments showed that E2 was synthesized from pregnenolone via 17-hydroxypregnenolone, dehydroepiandrosterone, androstenedione, and E1. T was not produced from any radiolabeled precursors, and exogenous T was not converted to E2. During the spawning season, serum levels of E2 and E1 showed similar patterns, with a diurnal rhythm of high levels at 03:00 hr associated with active ovarian vitellogenic follicles. In contrast, serum T levels were constant and relatively low compared to levels of E2 and E1. These results indicate that E2 is synthesized via E1, rather than T, in the ovarian follicles, and suggest that T detected in the blood is likely derived from extra-follicular tissues.

Determination of Polycyclic Aromatic Hydrocarbons in Contaminated Water and Soil Samples by Immunological and Chromatographic Methods

An immunoassay was developed that can be used for the detection of polycyclic aromatic hydrocarbons (PAHs) in water, landfill leachate, and soil. As test format an indirect competitive microtiter plate ELISA (enzyme-linked immunosorbent assay) was applied. While groundwater samples from a former manufactured gas plant site could be analyzed directly, soil and landfill leachate had to be extracted and required at least a 100-fold dilution prior to immunochemical measurement. PAHs could be recovered from fortified reference soils as well as aged field samples with high yield using 1-h ultrasonication with acetonitrile. Extraction efficiency was comparable to Soxhlet extraction and ultrasonication with tetrahydrofurane. Recovery was lower with agitation but would still be acceptable for use in an on-site field test to provide rapid, semiquantitative, and reliable test results for making environmental decisions such as identifying “hot spots”, site mapping, monitoring of remediation processes, and selecting site samples for laboratory analysis. Classification of ELISA data showed that it was possible to estimate the PAH contamination in soils with about 5% false positive and 5% false negative results that may have arisen from heterogeneity of samples, cross-reactivity of compounds with a similar structure, humic acids, or unknown interferences.

Use of D-dimer assays in the diagnosis of venous thrombosis.

In the course of fibrin formation, the D-domains of adjacent fibrin molecules within the fibrin polymer are covalently linked by factor XIIIa, leading to the formation of a D-domain dimer. Proteolysis of this cross-linked fibrin generates fibrin fragments D-dimer and E as terminal products. Fragment D-dimer therefore is an indicator for the proteolysis of cross-linked fibrin, whereas the monomeric fragment D can stem from fibrinogen and non-cross-linked fibrin. Various monoclonal antibodies have been prepared that distinguish between fragments D-dimer and D and allow the detection of fibrin derivatives in the presence of fibrinogen. These anti-D-dimer-antibodies have been shown to react with fragment D-dimer, but also detect dimeric D-domains within larger fibrin compounds, including cross-linked fibrin complexes generated in an early phase of coagulation activation. Assay systems for D-dimer antigen therefore may uncover intravascular clot formation early, by detection of fibrin complexes, and after completion of clot formation, by the detection of proteolytic fragments released from the particulate clot. Various trials have shown that low concentrations of D-dimer antigen in the blood exclude recent venous thrombosis or pulmonary embolism. Elevated levels may be caused by venous thrombotic disease, but also by a variety of other conditions, leading to intra- or extravascular fibrin formation. Assay systems include manual immunoagglutination assays, immunofiltration assays, microtiter plate enzyme-linked immunosorbent assay (ELISA) systems, automated ELISA systems, and latex-enhanced photometric immunoassays. According to clinical studies, D-dimer assays may be the "first line" of technical screening in symptomatic outpatients with suspected venous thrombosis or pulmonary embolism, but further prospective management trials, and improved standardization of assay systems, are needed for the validation of this approach.

A stable and highly sensitive 3,3′,5,5′-tetramethylbenzidine-based substrate reagent for enzyme-linked immunosorbent assays

3,3′,5,5′-Tetramethylbenzidine (TMB) is a widely used chromogen for horseradish peroxidase-based detection systems because it yields reaction products with high absorption coefficients and lacks carcinogenicity. Unfortunately, TMB is labile and poorly soluble in aqueous buffers and such solutions must be freshly prepared before each experiment. Moreover, substrate depletion can occur under assay conditions. To overcome these problems we have developed a two-component TMB substrate system which has a lower detection limit and is more sensitive than many commercially available TMB reagents when compared in microtiter plate enzyme-linked immunosorbent assays. Both components of the substrate system are stable for at least 1 year at 4°C and the usual discoloration of TMB stock solutions is prevented by the addition of a stabilizer that decomposes upon mixing of the two components.

Enzyme immunoassay method for total urinary follicle-stimulating hormone (FSH) beta subunit and its application for measurement of total urinary FSH

Objective: To develop and validate an enzyme-linked immunosorbent assay (ELISA) for total urinary follicle-stimulating hormone (FSH) that can be used to evaluate frozen-thawed urine samples. Design: A method was developed for dissociating urinary FSH into subunits by boiling. An ELISA for the free beta subunit of FSH was used to measure total FSH in urine samples collected from male and female volunteers. The urinary FSH values were compared with serum FSH concentrations in paired samples. Setting: A clinical research laboratory. Patient(s): Serum and urine samples from normal male and female volunteers and from men with infertility were used to develop and validate an assay for total urinary FSH. Intervention(s): None. Result(s): Urinary FSH concentrations in frozen-thawed urine were highly correlated to matched serum FSH concentrations. Conclusion(s): A microtiter plate ELISA for FSH beta subunit provides an economical method for evaluating FSH secretion in men and women. This method has practical applications for population-based epidemiologic studies in which urine samples are collected in the held and stored frozen before analysis.

Circulating levels and in vitro production of two maturation-inducing hormones in teleost: 17α,20β-dihydroxy-4-pregnen-3-one and 17α,20β,21-trihydroxy-4-pregnen-3-one, in a daily spawning wrasse, Pseudolabrus japonicus

The female bambooleaf wrasse, Pseudolabrus japonicus, spawns daily during the spawning season, and exhibits a diurnal rhythm of ovarian development. In the present study, we have investigated: (1) circulating levels of 17αa, 20β-dihydroxy-4-pregnen- 17,20β-P) and 17α,20β,21-trihydroxy-4-pregnen-3-one (20β-S) in females sampled at different times of the day during spawning season in captivity, and (2) in vitro production of 17,20β-P and 20β-S by follicle-enclosed oocytes at seven different develo tal stages. In addition, we developed a microtiter plate enzyme-linked immunosorbent assay (ELISA) for 17,20β-P. Serum levels of 17,20β-P and 20β-S showed similar diurnal changes; substantial increases in these levels occurred around the time of germinal vesicle breakdown (GVBD). In vitro experiments showed that massive production of 17,20β-P and 20β-S occurred in follicles collected just before or during GVBD. Further, acute decreases in 17,20β-P and 20β-S production were found in the ovarian follicles just prior to ovulation, suggesting inactivation of the maturation-inducing hormone (MIH). These results, taken together with our previous data on the occurrence of GVBD in vitro, suggest a role for both 17,20β-P and 20b-S as MIHs in the bambooleaf wrasse.

Evaluation of Microtiter-Plate Enzyme-Linked Immunosorbent Assay for the Analysis of Triazine and Chloroacetanilide Herbicides in Rainfall

Triazine and chloroacetanilide concentrations in rainfall samples collected from a 23-state region of the United States were analyzed with microtiter-plate enzyme-linked immunosorbent assay (ELISA). Thirty-six percent of rainfall samples (2072 out of 5691) were confirmed using gas chro matography/mass spectrometry (GC/MS) to evaluate the operating performance of ELISA as a screening test. Comparison of ELISA to GC/MS results showed that the two ELISA methods accurately reported GC/MS results (m = 1), but with more variability evident with the triazine than with the chloroacetanilide ELISA. Bayes's rule, a standardized method to report the results of screening tests, indicated that the two ELISA methods yielded comparable predictive values (80%), but the triazine ELISA yielded a false-positive rate of 11.8% and the chloroacetanilide ELISA yielded a false-negative rate of 23.1%. The false-positive rate for the triazine ELISA may arise from cross reactivity with an unknown triazine or metabolite. The false-negative rate of the chloroacetanilide ELISA probably resulted from a combination of low sensitivity at the reporting limit of 0.15 μg/L and a distribution characterized by 75% of the samples at or below the reporting limit of 0.15 μg/L.