Direct Enzyme Immunoassay Research Articles

Validation of a 13,14-dihydro-15-keto-PGF 2α enzymeimmunoassay and its application for reproductive health monitoring in postpartum buffaloes

The objective of the present study was to validate a simple, sensitive and direct enzymeimmunoassay (EIA) procedure for 13,14-dihydro-15-keto-PGF 2α (PGFM) for use in buffaloes with postpartum reproductive disorders and determine the practicalities of using plasma concentrations of 13,14-dihydro-15-keto-PGF 2α for monitoring their reproductive health. The EIA was used for determination of the circulating levels of PGFM associated with the retention of fetal membranes, postpartum endometritis and variable postpartum intervals. The concentrations of PGFM with retention of fetal membranes in the periparturient period were lower as compared to buffaloes that had uneventful parturitions. Concentrations of PGFM associated with postpartum endometritis were elevated as compared to those in buffaloes free of reproductive tract infections. Buffaloes having higher plasma concentrations of PGFM in early postpartum period had shorter postpartum intervals, indicating the association between PGFM concentrations postpartum and uterine involution as well as the resumption of estrous cycle in this species. The study presents the possibility of using circulating PGFM concentrations for monitoring the postpartum reproductive health of buffaloes.

Rapid diagnosis of rabies in humans and animals by a dot blot enzyme immunoassay

The presently advocated tests for rapid diagnosis of rabies, such as the fluorescent antibody test (FAT) are expensive and require expertise to carry out and interpret the results. In this study, a simple direct dot blot enzyme immunoassay (DIA) has been developed and evaluated to detect the rabies antigen in brain specimens of animals and humans. The utility of this test in the ante-mortem diagnosis of human rabies has also been evaluated. Brain homogenates of suspected rabid animals (n = 250), humans (n = 16) and clinical samples like saliva (n = 12) and cerebrospinal fluid (CSF) (n = 12) were directly spotted on polyvinylidene difluoride membrane (PVDF) and the absorbed rabies nucleoprotein antigen was detected using biotinylated antinucleoprotein antibody followed by treatment with streptavidin peroxidase conjugate and color development with diamino benzedine (DAB). Rabies-infected and normal mouse brain homogenates were used as positive and negative controls, respectively. The results of this test were evaluated with fluorescent antibody technique (for brain samples) and mouse inoculation test (for saliva and CSF samples). A distinct dark brown color was seen in the positive control and all positive samples, while there was no color development with either the negative control or the negative samples. The concordance between the fluorescent antibody test (FAT) and dot immunoassay was 98.4% for brain samples, 83.3% for saliva and 91.6% for CSF samples. The specificity of the test was found to be 100%. The dot blot enzyme immunoassay (DIA) test described here is a sensitive, specific and rapid test for the post-mortem diagnosis of rabies in animals and humans. The utility of this test for the ante-mortem diagnosis of rabies needs to be further evaluated.

Development of Antibodies for the Detection of N-Acetyl-glufosinate

Glufosinate is a widely used herbicide, which is difficult to detect by conventional analytical techniques. For many other herbicides, suitable antibodies have been raised for immunoassay development. Unfortunately, glufosinate is a very small molecule and difficult to immunize with. Thus, a derivatization-assisted immunoassay (DAIA) using the target analyte N-acetyl-glufosinate (NAG) was constructed. The activated hapten was synthesized by a new approach, using a homobifunctional cross-linker suberic acid bis(N-hydroxysuccinimide ester). The preparation of a suitable conjugate, the immunization, and the characterization of polyclonal antibodies are shown. The determination of the conjugation density (hapten density) of the immunogens was performed by four different methods (high-performance liquid chromatography with a refractive index detector, total reflection X-ray fluorescence, inductively coupled plasma mass spectrometry, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry), which gave similar results. The limit of detection was 17 mug/L NAG in water for the direct competitive enzyme immunoassay. NAG is also a main metabolite of glufosinate in resistant transgenic plants. The antibodies might be useful for the selective detection of NAG in the presence of the parent compound glufosinate (cross-reactivity 0.13%) and other metabolites.

Direct enzyme immunoassay of progesterone in bovine plasma

ABSTRACTThe present study was undertaken to develop a novel, practical and simple procedure for enzyme immunoassay (EIA) of plasma progesterone in cows. Diluted plasma was heated for 70°C for 30 min and applied directly to wells of a microtitre plate without extraction. Then plasma was incubated with antiprogesterone antibody and horseradish peroxidase‐labeled progesterone. The sensitivity of the assay was estimated as 4.4 pg/mL (0.11 pg/well). The intra‐assay and interassay coefficients of variation were 5.7–19.1% and 6.6–19.3%, respectively. When 0.3, 1 and 3 ng of progesterone were added to plasma, the recovery rates ranged between 79.9 and 108.4%. Only 4 h were needed to complete an assay to measure progesterone concentration. To apply the present direct EIA, progesterone concentration in plasma was assayed in crossbred cows used for the embryo transfer program. During insertion of controlled‐internal drug release (CIDR), progesterone concentrations were kept at a high level, although the removal of CIDR with treatment of dinoprost trometamine reduced progesterone concentration drastically. These results suggest that the present direct EIA is a practical and suitable method for measuring the plasma concentration of progesterone.

Improved enzyme immunoassay for group-specific determination of penicillins in milk

Polyclonal antibodies against penicillin antibiotics were raised in rabbits after immunization with an ampicillin-bovine serum albumine conjugate. The specificity and sensitivity of these antibodies were tested in a competitive direct enzyme immunoassay (EIA), using an ampicillin-horseradish peroxidase conjugate as the labelled antigen, and penicillin G as the reference antibiotic. The 50% binding inhibition concentration of penicillin G standard curves was at 2–4 ng ml−1, with a detection limit (20% binding inhibition) at 0.5–1 ng ml−1. The antiserum showed strong relative cross-reactivities with all penicillin-type betalactam antibiotics tested, including those regulated by maximum residue limits (MRLs) within the European Union (ampicillin 64%, amoxicillin 24%, oxacillin 100%, cloxacillin 29%, dicloxacillin 27%, and nafcillin 120%). After alkaline hydrolysis of the betalactam ring, no cross-reactivity of the respective penicillins was observed anymore. Cephalosporin-type betalactam antibiotics did not react in this test system. The recovery rates for penicillin G in artificially contaminated milk samples (2–32 ng ml−1) were at 89–97%. Penicillin G, ampicillin, amoxicillin, oxacillin, cloxacillin, dicloxacillin and nafcillin were all detectable in spiked milk samples at or below the respective MRL level. Comparison analysis of the EIA and a microbiological inhibition assay using violative milk samples obtained from routine milk testing scheme in Bavaria showed excellent agreement between both methods.

Validation of a sensitive enzymeimmunoassay for 13,14-dihydro-15-keto-PGF 2α in buffalo plasma and its application for reproductive health status monitoring

A simple, sensitive and direct enzymeimmunoassay (EIA) procedure on microtitre plates using the second antibody coating technique was standardized and validated for the determination of 13,14-dihydro-15-keto PGF 2α (PGFM) in unextracted buffalo plasma. The assay was carried out directly in 20 μl of buffalo plasma. PGFM standards prepared in charcoal stripped hormone-free plasma were used. The sensitivity of the assay was 0.4 pg/well, which corresponded to 20 pg/ml plasma. Plasma volumes for the assay ranging from 10 to 50 μl did not influence the PGFM standard curve; however, a slight drop in the OD 450 was observed with higher plasma volumes. Biological validation of the assay was carried out in buffalo plasma samples obtained during physiological states of cyclicity, peri-estrus, post-insemination, reproductive tract infection and persistent corpus luteum conditions. A pulsatile pattern of plasma PGFM release was observed prior to estrus when PGFM was determined in blood samples collected at hourly intervals of time. The PGFM pulsatility was not observed when blood sampling frequency of either 4 or 12 h was considered. The PGFM levels stayed high in peripheral circulation of buffaloes with reproductive tract infections and remained low throughout the sampling period in buffaloes having persistent corpus luteum. After an initial increase post-insemination, the plasma PGFM levels showed minor fluctuations. The assay was found to be sufficiently reliable and specific for estimation of PGFM levels in buffaloes. The standardization and validation of PGFM assay in buffalo opens the prospects of using PGFM levels as an indicator for reproductive health status monitoring in this species.

Concurrent hyperadrenocorticism in a minature schnauzer with severe Trichophyton mentagrophytes infection

A 4‐year‐old intact female schnauzer dog was presented with a history greater than 1 year of alopecia, thick crusting and secondary seborrhea with mild pruritus. The dog was diagnosed pituitary‐dependant hyperadrenocorticism (PDH) concurrent with severe Trichophyton mentagrophytes infection. The dog showed generalized alopecia, enlarged pendulous abdomen, mild polydipsia, polyuria and polyphagia. Liver enzymes alanine aminotransferase (ALT) and alkaline phosphatase (ALKP) were normal. ACTH stimulation and low‐dose dexamethasone suppression tests were performed prior to therapy. The concentrations of serum cortisol were determined by radioimmunoassay (RIA) and direct enzyme immunoassay (EIA) methods. The results of both assay systems indicated PDH. Fungal cultures showed T. mentagrophytes infection in 4 days. Histological examination of a skin biopsy specimen revealed severe irregular acanthosis accompanied by compact hyperkeratosis, and numerous dermatophytic spores and hyphae heavily colonized in the keratin layer. After 3 months of oral terbinafine (25 mg kg−1, once daily) and mitotane therapy, the hair regrew and the result of an ACTH stimulation test were normal. Three sets of biweekly fungal cultures were negative after 6 months. In this case, terbinafine was given concurrently with mitotane without detectable adverse effects (no abnormal clinical signs were observed and liver enzymes remained within normal ranges). Thus, the combination of terbinafine and mitotane appears to be a safe therapeutic protocol in treatment of PDH with dermatophytosis. This study was supported by The Association of Veterinary Dermatology, Taipei.

Development and Application of an Enzyme Immunoassay Based on a Monoclonal Antibody against Gonyautoxin Components of Paralytic Shellfish Poisoning Toxins

With a gonyautoxin 2/3 (GTX2/3)–specific monoclonal antibody (designated GT-13A) and a saxitoxin-horseradish peroxidase conjugate (STX-HRP), a direct competitive enzyme immunoassay (GTX-EIA) was established and its sensitivity to various toxin components was investigated. The concentrations resulting in 50% inhibition of the binding of STX-HRP to the solid-phase GT-13A antibody for GTX2/3, decarbamoyl-GTX2/3 (dc-GTX2/3), N-sulfocarbamoyl-GTX2/3 (C1/2), GTX1/4, STX, and neosaxitoxin (neoSTX) in GTX-EIA were found to be 0.28, 0.41, 0.52, 3.46, 4.06, and 89.37 ng/ml, respectively. When the minimum detection limit was assumed to be at a toxin concentration causing 30% inhibition of the binding of STX-HRP to the solid-phase GT-13A antibody, the detection limits for GTX2/3, dc-GTX2/3, C1/2, GTX1/4, STX, and neoSTX were found to be 0.15, 0.18, 0.19, 1.09, 1.50, and 22.93 ng/ml, respectively. These results indicate that all of the GTX components examined and STX are detectable at concentrations lower than the regulatory limit of 80 μg/100 g of shellfish tissue, even when a minimum dilution factor of 100 is applied to tissue extracts with the extraction procedure of the Association of Official Analytical Chemists. Therefore, GTX-EIA is thought to be a useful qualitative screening method for GTX components and STX in the mass monitoring of toxin-contaminated shellfish.

Evaluation of canine ACTH stimulation test with direct enzyme immunoassay for serum cortisol in Taiwan

In this study, we evaluated cortisol concentrations from 53 healthy dogs in response to ACTH stimulation test. The concentrations of serum cortisol were directly determined by a competitive enzyme immunoassay (EIA) with a 104‐fold diluted self‐made monoclonal antibody. The sensitivity of EIA was 0.1 ng mL−1 with intra‐assay and interassay coefficients of variation of 8.28 ± 0.09 and 14.60 ± 0.28%, respectively. The recovery rates for 1, 5 and 10 ng mL−1 of cortisol in dog serum were 77.8–128.7% with direct detection. The parallelism test revealed its reliability to detect directly the cortisol concentration in canine serum without extraction. In general, this EIA showed low crossreactivities with a variety of steroids. In the ACTH stimulation tests, we gave each dog 250 µg ACTH (Cortrosyn® Organon) intramuscularly and collected blood samples before and 1 h after administration of ACTH. The cortisol concentrations from 53 healthy dogs were 0.85–14.63 ng mL−1 pre‐ and 3.06–37.22 ng mL−1 post‐test, respectively. Sex and age did not significantly affect the cortisol concentrations in response to ACTH stimulation, but the large breeds (body weight > 25 kg; most of them were German shepherd dogs) showed lower cortisol responses than the smaller breeds. Thus, the results of this test suggested that body weight and breed might play an important role in adrenal dynamic tests.

Urinary leukotriene levels are increased during exacerbation of atopic eczema/dermatitis syndrome. Relation to clinical status.

Leukotrienes are potent mediators of allergic inflammation and their role in the pathogenesis of allergic disorders, particularly asthma, is well established. Their importance in the pathogenesis of atopic eczema/dermatitis syndrome (AEDS) is still unclear. We aimed to compare urinary cysteinyl leukotriene (Cys-LT) levels during exacerbation and remission of AEDS in relation to clinical status, IgE levels, and eosinophil counts. Urinary Cys-LTs were measured by direct enzyme immunoassay in 17 adult patients with AEDS and in 17 healthy controls in whom atopy had been excluded. Cys-LTs were compared during exacerbation and remission of AEDS in relation to the clinical status measured by SCORAD. Total IgE levels were measured by enzyme-linked immunoassay (ELISA). Mean clinical score during the exacerbation was 64.3 +/- 3.1 and during remission 22.4 +/- 4 (P < 0.01). Cys-LTs levels were significantly higher during the exacerbation of AEDS than in the control group (230.9 +/- 20.8 vs 123.2 +/- 9.9 pg/mg creatinine; P < 0.005). During the remission, the difference between AEDS patients and the control group was not significant (96.3 +/- 8.7 vs 123.2 +/- 9.9 pg/mg creatinine; P = 0.8). During AEDS exacerbation Cys-LTs levels were significantly correlated with the clinical status (rS = 0.73, P < 0.01) and with eosinophil counts (r = 0.86; P < 0.01) but not with the duration of the disease, age of patients, or IgE levels. Our results point to enhanced biosynthesis of Cys-LTs during the AEDS exacerbations. Inflammatory cells, e.g. eosinophils are the most probable source of Cys-LTs. A strong correlation between Cys-LT levels and clinical status may in part explain preliminary clinical observations of efficacy of leukotriene antagonists in alleviating symptoms of AEDS.

IgE-reactive proteins from Stachybotrys chartarum

Stachybotrys chartarum has been associated with idiopathic pulmonary hemorrhage in infants. This is thought to be mycotoxin-related. There are increasing numbers of reports linking this fungus to the indoor environment of patients with other pulmonary problems, including allergies and asthma. Given the potential significance of this fungus as a pulmonary pathogen, this work evaluates the antigenic proteins of S. chartarum as to their molecular size and the prevalence of immunoglobulin (Ig)E and IgG directed against them in the general population. S. chartarum was isolated from a local home. S. chartarum for extract production was grown on minimum salts and glucose. Plasma from 132 healthy individuals was evaluated for IgE and IgG directed against S. chartarum using direct and inhibition enzyme immunoassay. The number and molecular size of those proteins that were bound by IgE from pooled sera known to contain IgE to S. chartarum were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblotting. Enzyme immunoassay indicated 65 of 132 (49.2%) sera tested contained IgG against S. chartarum and 13 of 139 (9.4%) sera tested contained IgE against S. chartarum. Pooled sera identified two IgE-binding proteins from extracts of S. chartarum spores and mycelia. These proteins are 34 and 52 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblot. We conclude sensitivity to S. chartarum is potentially much more widespread than previously appreciated. This fungus may impact the asthmatic and allergic population through both immunologic and toxic mechanisms. Its significance in the milieu of allergenic fungi may need to be re-evaluated.

Production and application of new monoclonal antibodies specific for a fecal Helicobacter pylori antigen.

The aim of the present study was to establish monoclonal antibodies that could be used to produce a diagnostic test composed of one kind of monoclonal antibody recognizing a fecal Helicobacter pylori antigen. The need to develop such a test arose from disadvantages of the diagnostic test that uses a polyclonal antibody or plural kinds of monoclonal antibodies, such as the lower specificity for H. pylori antigen and the difficulty of reproduction with consistent quality. Mice were immunized with sonicated cells of the coccoid form of H. pylori, and fecal samples from H. pylori-positive subjects were screened by a direct sandwich enzyme immunoassay (EIA) for antibody production from 32 hybridoma clones. The three stable clones produced antibodies (21G2, 41A5, and 82B9) that reacted with the same soluble antigen. Gel filtration chromatography showed that the molecular masses of the cellular antigen and the fecal antigen were the same, 260 kDa. The antigen was labile in response to sodium dodecyl sulfate and heat treatments. A single-step direct sandwich EIA using a single monoclonal antibody, 21G2, was developed. The EIA could detect the antigen in 41 H. pylori clinical isolates and in fecal samples from seven H. pylori-positive subjects. Several kinds of Helicobacter species (Helicobacter felis, Helicobacter hepaticus, Helicobacter mustelae, and Helicobacter cinaedi) except H. pylori, major bacteria in feces (Campylobacter jejuni, Bacteroides vulgatus, Bifidobacterium breve, Bifidobacterium infantis, and Escherichia coli), and fecal samples from six H. pylori-negative subjects showed negative results. These results indicate that the new monoclonal antibodies and the new specific EIA would be useful as a noninvasive method of diagnosis of H. pylori infection.

Direct Enzyme Immunoassay of Estrone Sulfate in the Plasma of Cattle.

The present study was undertaken to develop a novel, practical and simple procedure for enzyme immunoassay of plasma estrone sulfate (E1S) in cows. Diluted plasma was applied directly to wells without extraction and incubated with anti-estrone antibody and horseradish peroxidase-labeled estrone. The sensitivity of the assay was estimated as 40 pg/ml. The intra-assay and inter-assay coefficients of variation were 7.8-9.6% and 7.2-14.4%, respectively. When 2, 5, 10 and 20 ng of E1S were added to 1 ml of plasma, the recovery rates ranged between 84.6 and 98.5%. Only 3 hr were needed to complete an assay to measure E1S concentration. Plasma concentrations of E1S were measured in six crossbred cows after the first trimester of pregnancy. A dramatic increase in the plasma concentrations of E1S was observed from days 240 to 284 of gestation (7.40-13.00 ng/ml to 18.00-36.00 ng/ml). These results suggest that the present direct enzyme immunoassay is a practical and suitable method for measuring the plasma concentration of E1S.

Comparing tandem repeats and multiple antigenic peptides as the antigens to detect antibodies by enzyme immunoassay

Short synthetic peptides are useful alternatives to whole lysate or recombinant proteins as the antigens used for serodiagnosis of bacterial or viral infections. However, certain known antigenic peptides displayed low seroreactivities in direct enzyme immunoassay. This was believed to be due to the low coating efficiency, a constrained orientation, or loss of flexibility required for optimal antibody binding. Using a model peptide system derived from the V3-loop of HIV-1 gp120, we demonstrated that low antigenicity could be overcome by using either tandem repeats (TR) or multiple antigenic peptides (MAPs) which contained the same amino acid sequence as the monomeric peptide. In our model system, a four-branch MAP was a better choice compared to the tandem repeats because of the MAP's slightly higher sensitivity and lower cost of production.

Laboratory diagnosis of Clostridium difficile disease

The laboratory diagnosis of Clostridium difficile-associated disease (CDAD) is based on culture and toxin detection in fecal specimens. Culture is performed on a commercially available selective media. C. difficile colony morphology is typical when viewed under a dissecting microscope. Definitive identification is best obtained by gas liquid chromatography. Culture is very sensitive but, when used alone without toxin testing, it leads to low specificity and misdiagnosis of CDAD when high rates of asymptomatic carriage exist. Toxin detection by a tissue culture cytotoxin assay followed by neutralisation with specific antiserum is often considered the standard. However, this approach lacks sensitivity and has not detected up to 30% of patients with confirmed CDAD. Multiple enzyme immunoassays (EIAs) have been introduced by various manufacturers for the detection of toxin A alone or for both toxins A and B. Some of these are designed to give results in less than 1 h. Comparative studies of EIA kits reported that the sensitivity and specificity are slightly lower than cytotoxin assays. Toxigenic culture tests C. difficile isolates for toxin production: colonies isolated on selective media are tested for in-vitro toxin production either by a cytotoxicity assay or by direct EIA. It has higher sensitivity than the cytotoxicity assay and equivalent specificity. In the routine laboratory, culture and toxin detection should be performed on every specimen and, in culture-positive and fecal toxin-negative cases, toxigenic cultures should be performed on isolated colonies.

Influenza Pneumonia: A Descriptive Study

To describe the clinical features and complications of patients hospitalized with influenza during the 1999-2000 influenza season. We reviewed all cases of patients with influenza admitted to a large metropolitan referral hospital during the 1999-2000 season. Thirty-five adult patients (15 men and 20 women) tested positive for influenza A by direct enzyme immunoassay. A fourfold to sevenfold increase in the number of influenza cases was observed over previous years. Most patients had serious comorbid illnesses (88%), such as diabetes and chronic respiratory and heart disease. Seventeen patients developed pneumonia; these patients tended to be older (mean +/- SD, 63 +/- 13 years vs 51 +/- 19 years, respectively; p = 0.04) and had a higher incidence of chronic lung disease (41% vs 6%, respectively; p = 0.02) than the patients without pneumonia. Shortness of breath was the only symptom that distinguished patients with pneumonia from those with an upper respiratory tract illness alone. Antiviral treatment was begun 4 +/- 3 days from initiation of symptoms in patients with pneumonia and consisted of oseltamivir (35.2%), rimantadine (5.8%), or a combination of both (17.6%). Respiratory and/or blood culture results were positive in five patients (29%), Staphylococcus aureus was isolated in five patients, and Streptococcus pneumoniae was isolated in one patient. Ten of the patients with pneumonia (58.8%) were admitted to the ICU, and 5 patients (29%) died. The length of ICU stay and mechanical ventilation were 28 +/- 26 days and 21.5 +/- 20.5 days, respectively. Death in most pneumonia patients was attributed to respiratory failure. The recognized number of hospital admissions for influenza increased fourfold to sevenfold over previous years, most likely due to the implementation of rapid diagnostic tests for influenza. Patients with signs and symptoms of influenza and shortness of breath should undergo chest radiography. Hospitalization of patients with influenza pneumonia occurred in both previously healthy and immunocompromised patients and had a high mortality. S aureus was the most common bacterial isolate in patients with influenza pneumonia. Empiric antibiotics with staphylococcal activity should be used pending culture results in patients with influenza pneumonia. The effectiveness of oseltamivir and rimantadine in treating patients with influenza pneumonia remains to be determined.

Changes in rates of salivary estriol increases before parturition at term

Objective: The aim of this study was to characterize the increases of salivary estriol concentrations before the onset of labor at term. Study Design: Salivary estriol concentrations were measured in weekly patient-collected samples by means of a sensitive (mean ± SD threshold, 0.025 ± 0.001 ng/mL; coefficient of variation, 3.8%) direct enzyme immunoassay in a microtiter plate format. The salivary estriol concentrations in 16 healthy pregnant women were characterized from 30 weeks' gestation until the time of parturition and delivery. Samples were stored frozen at collection and analyzed in batches after delivery. Results: The median salivary estriol concentration profile revealed a nonlinear rise beginning from 30 weeks' gestation (0.89 ng/mL) until term (2.70 ng/mL, an increase of 201%). At 35 weeks' gestation the salivary estriol concentration median value increased sharply (positive inflection point, 50%-93% increase) at a demarcation between a slower increase during early pregnancy and a more rapid increase during late pregnancy. This positive inflection point associated with a late pregnancy increase characterized subgroups of pregnancies according to the lengths of gestation as follows: early term (delivered at <38 weeks 1 day's gestation), middle term (delivered at 38 weeks 1 day–40 weeks' gestation), and late term (delivered at >40 weeks' gestation). Five weeks before delivery the mean (±SEM) rate of rise in salivary estriol concentration was 0.50 ± 0.13 ng/mL per week to 0.84 ± 0.26 ng/mL per week in the early term group. The increase in rate for the middle term group was 0.32 ± 0.06 ng/mL per week to 0.37 ± 0.26 ng/mL per week, whereas in the late term group the rate of salivary estriol concentration rise was 0.37 ± 0.03 ng/mL per week to –0.03 ± 0.25 ng/mL per week. Conclusion: These data demonstrate in normal pregnancies (1) that a direct, nonradiometric measure of salivary estriol concentration can be used to monitor the late pregnancy increase in estriol production, (2) that 35 weeks' gestation marks a positive inflection point of the onset of increased estriol production, and (3) that the late pregnancy rise in salivary estriol concentration shows distinct patterns that tend to be characteristic of the length of pregnancy. These data support the concept that the rate of increase of estriol production is related to the timing of the onset of labor. (Am J Obstet Gynecol 2001;184:123-30.)

Derivation and Properties of Recombinant Fab Antibodies to Coplanar Polychlorinated Biphenyls

Recombinant Fab antibodies (rFabs) specific for coplanar polychlorinated biphenyls (PCBs) were derived from a hybridoma cell line (Chiu et al. Anal. Chem. 1995, 67, 3829-3839). Immunoglobulin V(H)-C(H1) and V(L)-C(L) sequences from S2B1 messenger RNA were amplified by PCR and cloned into the M13 phagemid vector pComb3H. Phage displaying rFab were enriched by panning on a PCB hapten conjugate and expressed as soluble rFabs in Escherichia coli XL-1 Blue. Two rFab clones competitively bound PCBs 77 and 126 with half-maximal inhibition (I(50)) of 10-13 ppb in indirect and direct enzyme immunoassays (EIAs), with selectivity nearly identical to that of whole S2B1 IgG and its Fab fragments prepared by papain digestion. These results, and comparison of N-terminal amino acid sequences of MAb S2B1 and the rFab, indicated that rFab S2B1 is a functional copy of the MAb. The rFab S2B1 sequences have 75-89% sequence identity with antibodies that bind nitrophenyl haptens and are being used to construct a three-dimensional computational model of the PCB binding site.

Monoclonal Antibodies Showing Surface Reactivity with Lactobacillus and Pediococcus Beer Spoilage Bacteria

Five monoclonal antibodies (Mabs) were isolated based upon reactivity with both a Lactobacillus and a Pediococcus beer spoilage isolate. Immunoblot analysis and Proteinase K treatment both suggested that none of the antigens recognized by the Mabs were proteins. Since the Mabs were known to bind surface-accessible antigenic sites on Gram-positive bacteria, it was proposed that lipoteichoic acid (LTA) was the antigen recognized. Each of the Mabs was purified and conjugated with horseradish peroxidase. Isolation of LTA, followed by direct antibody binding and antibody competition enzyme immunoassays, confirmed this. To determine whether the five Mabs were binding the same site on the LTA, the ability of each Mab to inhibit the binding of each of the enzyme-conjugated Mabs was determined. The data obtained allow us to propose that the five Mabs recognize three spatially distinct epitopes on the bacterial LTA. As the Mabs were found to bind to most beer spoilage Lactobacillus and Pediococcus isolates tested, these Mabs could prove useful in rapid detection of these Gram-positive beer-spoilage bacteria.

Development and evaluation of a direct enzyme immunoassay for oestrone sulphate in urine as a tool for diagnosis of early pregnancy in swine.

A competitive inhibition-type enzymeimmunoassay (EIA) has been developed using 3-hemisuccinate-oestrone-peroxidase as conjugate for direct measurement of the hormone in swine urine. The method has a minimum detection level at 0.3 ng ml(-1) and satisfactory specificity, recovery and reproducibility. In a field trial with a group of 387 sows (7 in oestrus, 16 non-pregnant and 364 pregnant sows at several stages post service), it was shown that the assay is potentially an accurate pregnancy test in assessing the viability of the fetoplacental unit from day 23 up to day 30 post service. The assay is well suited for routine testing, particularly as a swine early pregnancy diagnosis test since urine sampling is easier and does not disturb the animal, while in the present assay there is no restriction in the time of sampling and the sample storage conditions.