Development of an assay to screen for inhibitors of tau phosphorylation by cdk5

A high-throughput assay for tau phosphorylation by cdk5/p25 is described. Full-length recombinant tau was used as a substrate in the presence of saturating adenosine triphosphate (ATP). Using PHF-1, an antibody directed specifically against 2 tau phosphorylation epitopes (serine 396 and serine 404), an enzyme-linked immunosorbent assay (ELISA)-based colorimetric assay was formatted in 384-well plates. The assay was validated by measuring kinetic parameters for cdk5/p25 catalysis and known inhibitors. Rate constants for the site-specific phosphorylations at the PHF-1 epitopes were determined and suggested preferential phosphorylation at these sites. The performance of this assay in a high-throughput format was demonstrated and used to identify inhibitors of tau phosphorylation at specific epitopes phosphorylated by cdk5/p25.     

Elisa for salivary and plasma estriol in pregnancy

A competitive, sensitive, and rapid enzyme-linked immunoadsorbent assay (ELISA) was developed for the determination of estriol in saliva and in plasma. Horseradish peroxidase (HRP) was used as the label enzyme; separation between free and bound steroid was carried out by insolubilized antibody prepared by adsorbing purified IgG of rabbit anti-6-oxoestriol-6-(0-carboxymethyl)oxime-BSA on polystyrene balls. The enzyme activity was measured by a colorimetric reaction using o-phenylenediamine dihydrochloride and hydrogen peroxide as substrate. The sensitivity of the assay was 12 pg/tube.In order to compare ELISA to RIA estriol estimations in different biological fluids, we selected six women during normal pregnancy, from the 30th to the 40th week of gestation. Salivary estriol was assayed by direct and extraction methods, while the corresponding plasma samples of the same subjects were analyzed only for unconjugated estriol by an extraction method.A good agreement was found between the results obtained by RIA and ELISA: r=0.897, p <0.001 between direct RIA and direct ELISA in saliva; r=0.909, p < 0.001 between extraction RIA and direct ELISA in saliva; and r=0.916, p < 0.001 between extraction RIA and extraction ELISA in plasma. A good correlation (r=0.793, p<0.001) was present between plasma samples by RIA and saliva samples by ELISA (direct method).These results indicate that: 1. ELISA is a reliable method for the determination of estriol in plasma and saliva. 2. Saliva samples can be used for the assay of estriol and therefore for the assessment of fetal conditions during pregnancy.     

Seasonal changes in serum leptin of the feral raccoon (Procyon lotor) determined by canine-leptin-specific ELISA

Several reports have been published on blood leptin concentrations in feral animals, including members of the Carnivora, using a commercially available multi-species radioimmunoassay (RIA) kit with anti-human leptin antibody. However, we observed weak immunoreactivity between recombinant canine leptin and anti-human leptin antibody, suggesting a limitation in the applicability of the RIA kit for leptin assays in Carnivora species. We tested the applicability of RIA and sandwich enzyme-linked immunosorbent assay (ELISA) with anti-canine leptin antibody to assay blood leptin in the dog (Canis familiaris) and the raccoon (Procyon lotor). When RIA was used for recombinant canine leptin and dog sera, values were much lower than those determined by ELISA at higher concentrations (>10 ng/ml), while rather higher at lower concentrations (<2 ng/ml). A similar discrepancy between the two methods was found for serum leptin concentrations in raccoons. Clear seasonal variations were observed by ELISA, but not by RIA, with high values in autumn (3.46+/-0.45 ng/ml) and low values in spring and summer (0.71+/-0.07 ng/ml). Serum leptin concentrations in raccoons correlated positively with their body weight (r=0.753) and body mass index (r=0.755), corroborating our previous findings of a strong positive correlation between serum leptin concentrations and body fat content in dogs. Thus, the canine leptin ELISA is useful for assays of dog and raccoon leptin, and blood leptin is a good marker of nutritional condition in the species of Carnivora assayed in this study.     

Detection of pregnancy in sheep by radioimmunoassay of sera for pregnancy-specific protein B

A radioimmunoassay (RIA) for bovine pregnancy-specific protein B (bPSPB) has been shown to be a reliable test for pregnancy in cows. Pregnant ewes have a blood antigen that cross-reacts in this RIA. Two studies were conducted to determine the accuracy of detection of pregnancy in sheep using the bPSPB RIA. In Study 1, 33 ewe lambs were bred over a 70-d period in late fall. At 26, 56, and 83 d after the end of the breeding period, blood samples were collected for assay in the bPSPB RIA, and the Pregmatic 3 ultrasonic device was used to detect pregnancy. Pregmatic 3 detected pregnancy in 14, 27 and 28 ewes and nonpregnancy in 19, 6 and 3 ewes at Days 26, 56 and 83 past the breeding period, respectively. The bPSPB assay detected pregnancy in 32, 31 and 30 ewes and nonpregnancy in 1, 2 and 2 ewes at Days 26, 56 and 83 past breeding, respectively, Thirty ewes lambed and three did not. In Study 2, 180 multiparous ewes were bred over a 60-d period in summer. At 35 d after the end of the breeding period, blood samples were collected for assay in the RIA, and a real-time ultrasonic scan was done to detect pregnancy. Real-time ultrasonic testing detected pregnancy in 163 ewes and nonpregnancy in 17 ewes; whereas, the RIA detected pregnancy in 161 ewes and nonpregnancy in 19 ewes. One hundred fifty-nine ewes lambed and 21 did not. The bPSPB RIA detected pregnancy earlier and more accurately than the Pregmatic 3 ultrasonic device and was equally as accurate as the real-time scanning instrument. These studies demonstrate an accurate serological test for a pregnancy-specific antigen in sheep.     

Detection of staphylococcal enterotoxins by enzyme-linked immunosorbent assays and radioimmunoassays: comparison of monoclonal and polyclonal antibody systems

Murine monoclonal antibodies reactive with at least one of the serological types of staphylococcal enterotoxin were examined for use in assay systems for the detection of enterotoxin at the level of 1.0 ng of enterotoxin per ml. An antibody sandwich enzyme-linked immunosorbent assay was devised for each toxin type by identifying an effective antibody pair. One antibody (the coating antibody) was coated onto a polystyrene plate and removed the enterotoxin from the test solution; the second antibody (the probing antibody) was conjugated to horseradish peroxidase and detected the captured toxin. Enterotoxins A and E could be detected in the same system by the use of cross-reacting monoclonal antibodies. All subtypes of enterotoxin C could be detected in one assay system. Two effective systems were described for each of types B and D. Each of these systems, when compared with the homologous enterotoxin-specific polyclonal rabbit antibody systems, was found to compare favorably. The monoclonal enzyme-linked immunosorbent assay systems for the detection of enterotoxins A and C2 were examined for a variety of food extracts; no abnormal interference could be detected from these extracts. The monoclonal antibody systems were also compared with the homologous enterotoxin-specific polyclonal serum for the detection of enterotoxin by the competitive radioimmunoassay (RIA). Single monoclonal antibodies generally did not perform as well in the RIA as did the homologous toxin-specific polyclonal serum. However, pools of monoclonal antibodies were prepared that approached the sensitivity and precision of the polyclonal system for the detection of each toxin by the RIA.     

Pharmacokinetic study of a human recombinant interferon (Re-IFN-alpha A) in cynomolgus monkeys by 2'-5' oligoadenylate synthetase assay

We evaluated 2'-5'Oligoadenylate (2-5 A) synthetase assay for pharmacokinetic study of human interferon (IFN) in cynomolgus monkeys. The enzyme was induced in primary cultures of cynomolgus monkey kidney (PMK) cells as well as in FL cells in response to human recombinant IFN-alpha A treatment. The enzyme activity increased with IFN dose and, in parallel with the enzyme elevation, developed the antiviral state of the cells. The enzyme activity induced in the peripheral blood lymphocytes peaked at 6 to 12 hr after iv or im administration. The peak level of the enzyme activity depended on the IFN concentration of the blood and the activity rapidly decreased as serum IFN was cleared from the blood. These results indicate that human recombinant IFN-alpha A induces 2-5 A synthetase in monkey cells both in vitro and in vivo, and that the enzyme assay can be used to quantitatively monitor the host response after IFN administration.     

Interactions of Standard Antibody with Australia Antigens in Au Ag-Ab Radioimmunoassay

Human antisera against Australia (Au) antigen have been characterized by liquid-phase radioimmunoassay (RIA) for their precipitation of (125)I-labeled Au antigen. The end-point dilutions of sera (anti-Au) which precipitated 50% of (125)I-Au antigen by RIA correlated well with complement fixation titers but had a much wider range, indicating a greater precision and perhaps a better sensitivity of assay. Anti-Au serum diluted to precipitate 50% of (125)I-labeled Au antigen was used as standard antibody in RIA tests to detect either inhibition or enhancement of the reaction by preincubated mixtures of Au antigen and antibody specimens. Without free Au antigen or antibody in the resultant mixtures there was no inhibition or enhancement; the mixtures presumably contained immunoreactively equivalent proportions of Au antigen and antibody. RIA data for diagnostic specimens indicated an end-point sensitivity which was proportional to the dilution of the standard anti-Au sera used in the test. High concentrations of the standard antibody permitted detectable inhibition of (125)I-Au antigen precipitation at lower antigen specimen concentrations. Similarly, low concentrations of the standard antibody permitted detectable enhancement of (125)I-Au antigen precipitation at lower antibody specimen concentrations. Omitting the standard antibody altogether resulted in a more sensitive RIA for Au antibody in test sera.     

Detection of staphylococcal enterotoxins by enzyme-linked immunosorbent assays and radioimmunoassays: comparison of monoclonal and polyclonal antibody systems.

Murine monoclonal antibodies reactive with at least one of the serological types of staphylococcal enterotoxin were examined for use in assay systems for the detection of enterotoxin at the level of 1.0 ng of enterotoxin per ml. An antibody sandwich enzyme-linked immunosorbent assay was devised for each toxin type by identifying an effective antibody pair. One antibody (the coating antibody) was coated onto a polystyrene plate and removed the enterotoxin from the test solution; the second antibody (the probing antibody) was conjugated to horseradish peroxidase and detected the captured toxin. Enterotoxins A and E could be detected in the same system by the use of cross-reacting monoclonal antibodies. All subtypes of enterotoxin C could be detected in one assay system. Two effective systems were described for each of types B and D. Each of these systems, when compared with the homologous enterotoxin-specific polyclonal rabbit antibody systems, was found to compare favorably. The monoclonal enzyme-linked immunosorbent assay systems for the detection of enterotoxins A and C2 were examined for a variety of food extracts; no abnormal interference could be detected from these extracts. The monoclonal antibody systems were also compared with the homologous enterotoxin-specific polyclonal serum for the detection of enterotoxin by the competitive radioimmunoassay (RIA). Single monoclonal antibodies generally did not perform as well in the RIA as did the homologous toxin-specific polyclonal serum. However, pools of monoclonal antibodies were prepared that approached the sensitivity and precision of the polyclonal system for the detection of each toxin by the RIA.     

Development of a direct enzyme immunoassay of milk progesterone and its application to pregnancy diagnosis in cows

The purposes of this study were to develop an enzyme immunoassay (EIA) for determination of progesterone in unextracted whole milk and to apply it to pregnancy diagnosis. Paper fibers covalently coupled to antibody specifically and competitively bound ³H-progesterone and 11α-hydroxyprogesterone hemisucccinate-horseradish peroxidase and were stable for 9 mo at ?20°C. The sensitivity, recovery, precision, and cross reactivity of the EIA and a radioimmunoassay (RIA) of milk progesterone were evaluated, and showed that this EIA was comparable to the RIA. Milk samples were collected once daily for one estrous cycle from ten lactating Holstein cows and the progesterone levels were determined by RIA. Milk progesterone in 70 samples measured by EIA were highly correlated (r = 0.90) with the values of RIA for the same samples. Milk samples for pregnancy diagnosis by EIA of milk progesterone were obtained daily from days 20 to 24 after 115 artificial inseminations of 85 lactating Holstein cows. Pregnancy diagnosis by EIA was confirmed by rectal palpation at 30 to 60 days after insemination or return to estrus. The accuracy based on single, two, three, four, and five consecutive samples was from 67.2 to 80.0%, 77.3 to 84.0%, 79.2 to 87.5%, 82.0 to 85.4%, and 85.4%, respectively, for pregnancy diagnosis; and from 95.0 to 98.3% for nonpregnancy.     

A simple, rapid and large capacity ELISA for biologically active native and recombinant human IFN gamma

A rapid and sensitive enzyme-immunoassay for native and recombinant human interferon gamma is described. The test is performed in one step at room temperature and is based on the sandwich principle. The IFN gamma preparation is distributed with horse radish peroxidase-labeled monoclonal antibody to IFN gamma in microtiter plates previously coated with a second mab against IFN gamma. The amount of the IFN gamma mab sandwich fixed in the microtiter plate wells is proportional to the color developed after the addition of peroxidase-specific substrate. The two mab's used in the test neutralize IFN gamma and are directed against the same epitope. For this reason they can only detect the biologically active dimeric form of IFN gamma. The IFN gamma-ELISA works in phosphate buffer as well as in tissue culture medium or human serum. As the assay is routinely performed in 2 hours, the limit of detection is 3 U/ml of IFN gamma (0.3 ng/ml). If the assay is performed in 16 hours, the limit of detection decreases to 0.5 U/ml IFN gamma (0.06 ng/ml). The conditions to preserve the activity of IFN gamma preparation as standard are discussed.     

A preliminary report on the effect of dietary energy on prostaglandin F2 alpha production in vitro, interferon-tau synthesis by the conceptus, endometrial progesterone concentration on days 9 and 15 of pregnancy and associated rates of embryo wastage in ewes

Two groups of ewes were fed to provide 1.70 x (high energy group; n = 15) or 0.56 x (low energy group; n = 15) energy requirements for maintenance of liveweight from 14 d before a synchronized mating in November until slaughter at 9 or 15 d after mating. We investigated the effects on interferon-tau (IFN tau) secretion by the conceptuses, prostaglandin F2 alpha (PG) production in vitro by endometrial tissue, and associated rates of embryo mortality, endometrial progesterone content and progesterone production by luteal tissue. No differences between groups in pregnancy rate were detected on Day 9 between the 2 groups. Proportionately (6/6 vs 2/5), there were more pregnant ewes in the high energy group on Day 15, although this difference did not reach significance (P = 0.06). The proportion of corpora lutea represented by embryos was significantly lower in undernourished ewes (P < 0.05). Secretion in vitro of PG was lower in the 2 pregnant ewes of the low energy group on Day 15, and it was accompanied by higher IFN tau secretion by conceptuses recovered from these ewes. However, the limited number of pregnant ewes recorded on Day 15 prevented any statistical comparison. Neither mean endometrial content of progesterone nor ovarian venous progesterone concentrations and production of progesterone by luteal were affected by nutrition. The provisional results of the present experiment indicate that undernutrition may induce a reduction in the rate of secretion of IFN tau and can therefore increase production of PG from the endometrium. This could initiate luteolysis. The lower pregnancy rates observed in underfed ewes could be mediated through this alteration in the signal of maternal recognition of pregnancy. However, these findings remain to be shown in further experiments including a larger number of animals, as they only represent data from 2 undernourished animals.     

Development of an enzyme‐linked immunosorbent assay (ELISA) for Luteinizing Hormone (LH) in the elephant (Loxodonta africana and Elephas maximus)

A simple, rapid enzyme‐linked immunosorbent assay (ELISA) for the measurement of LH in plasma and serum of elephants (Loxodonta africana and Elephas maximus) has been developed, validated, and used for comparative studies. Purified elephant LH (eleLH) diluted in elephant plasma was used as standards (0.78–50 ng/ml). A monoclonal antibody against the β‐subunit of bovine LH (518B₇) was used as the capture antibody. The second antibody (a polyclonal rabbit anti‐human LH antibody), conjugated to horseradish peroxidase, cleaved a substrate (tetramethyl benzidine), resulting in a color change. The total assay time was approximately 2½ hr, with incubations at room temperature. Sensitivity was found to be 1.56 ng/ml. Cross‐reactivities to elephant FSH and TSH were low: 0.9% and 0.15%, respectively. The accuracy of the assay was demonstrated by comparing the ELISA with a validated eleLH radioimmunoassay (RIA), progesterone data, and ultrasound observations. Blood samples from 18 Asian and African elephant cows were analyzed with the ELISA and RIA, and an additional 11 cows were used to describe endocrine parameters for LH and progesterone using only RIA. No difference was found in LH peak concentrations between the ELISA and RIA. The time from the progesterone decline to the first LH peak, and the time between the two peaks were similar between species. Asian cows had higher LH peaks than African cows. Ultrasound confirmed the time of ovulation occurring with the second LH peak. Three cows were inseminated and confirmed to be pregnant using this ELISA as a timing device. Instrumentation is not always required, as LH peaks approximating 3 ng/ml can be visually observed. In conclusion, this ELISA can be used as a field test to determine time of ovulation for artificial insemination (AI) or natural breeding of both species of elephants, and thus is an important tool for the preservation of captive populations worldwide. Zoo Biol 23:65–78, 2004. © 2004 Wiley‐Liss, Inc.     

The accuracy of enzyme-linked immunosorbent assay and latex agglutination progesterone test for the validation of estrus and early pregnancy diagnosis in dairy cattle

The accuracy of the enzyme-linked immunosorbent assay (ELISA) and the latex agglutination (LA) on-farm progesterone kit for detecting estrus and diagnosing early pregnancy was investigated in this study. Italian Friesian dairy cows (n=82) from 6 dairy herds were used for the collection of foremilk samples at the time of breeding and at 19, 21, and 23 days post insemination. Pregnancy status was ascertained by uterine palpation per rectum 40 to 60 days post insemination. Progesterone levels were affected by herd, percentage of milk fat, and the day of testing x diagnosis interaction. Validation of estrus by qualitative on-farm tests was 74.6% (LA) and 100.0% (ELISA) accurate using 0.5 ng/ml of progesterone as the RIA estimate for estrus. The accuracy rate for early pregnancy diagnosis by RIA was 68.4 to 83.8% for day 19 and day 21, respectively, while the detection rate for nonpregnancy was 84.6 to 100% on day 19 and day 21, respectively, as compared with uterine palpation per rectum. The average accuracy rate for early pregnancy diagnosis ranged from 84.7 to 92.3% for the LA and ELISA tests, respectively; the nonpregnancy rate was correctly predicted 93.9% to 68.2% for the LA and ELISA tests, respectively.     

Detection of thyroid microsomal and thyroglobulin antibodies by new sensitive radioimmunoassay in Hashimoto's disease; comparison with conventional hemagglutination assay

We evaluated clinical usefulness of thyroid microsomal antibody (MCAb) and thyroglobulin antibody (TGAb) measured by new sensitive radioimmunoassays (RIA). These assays are simple and reproducible; the intra- and inter-assay coefficients of variation were 3.6-6.8% and 6.6-13.2% in the MCAb assay, and 3.2-7.7% and 7.6-12.3% in the TGAb assay, respectively. In 126 patients with Hashimoto's disease, the antibody activity determined by this RIA correlated with that determined by the hemagglutination assay (HA) (r = 0.848 for MCAb, r = 0.686 for TGAb, p less than 0.001). MCAb was detected by RIA in all of 115 HA-positive and 4 of 11 HA-negative patients, and TGAb by RIA in all of 84 HA-positive and 29 of 42 HA-negative patients: the prevalence of MCAb was 94% and that of TGAb was 90% in the disease. Moreover, some showed high antibody activity only in RIA. In another group of 14 patients with biopsy-proved Hashimoto's disease with no antibody activity by routine HA tests, serum MCAb was detected in 3 (21%), TGAb in 11 (79%), and both activities in 2 (14%). Our results indicate that (1) the RIA tests are more sensitive than the conventional HA test, and that (2) the present RIA test for TGAb is more sensitive than that for MCAb in detecting autoimmune abnormalities, especially in patients with biopsy-proved Hashimoto's disease who give negative results in the HA test.     

Degradation of tau protein by puromycin-sensitive aminopeptidase in vitro

Tau, a microtubule associated protein, aggregates into intracellular paired helical filaments (PHFs) by an unknown mechanism in Alzheimer's disease (AD) and other tauopathies. A contributing factor may be a failure to metabolize free cytosolic tau within the neuron. The buildup of tau may then drive the aggregation process through mass action. Therefore, proteases that normally degrade tau are of great interest. A recent genetic screen identified puromycin-sensitive aminopeptidase (PSA) as a potent modifier of tau-induced pathology and suggested PSA as a possible tau-degrading enzyme. Here we have extended these observations using human recombinant PSA purified from Escherichia coli. The enzymatic activity and characteristics of the purified PSA were verified using chromogenic substrates, metal ions, and several specific and nonspecific protease inhibitors, including puromycin. PSA was shown to digest recombinant human full-length tau in vitro, and this activity was hindered by puromycin. The mechanism of amino terminal degradation of tau was confirmed using a novel N-terminal cleavage-specific tau antibody (Tau-C6g, specific for cleavage between residues 13-14) and a C-terminal cleavage-specific tau antibody (Tau-C3). Additionally, PSA was able to digest soluble tau purified from normal human brain to a greater extent than either soluble or PHF tau purified from AD brain, indicating that post-translational modifications and/or polymerization of tau may affect its digestion by PSA. These results are consistent with observations that PSA modulates tau levels in vivo and suggest that this enzyme may be involved in tau degradation in human brain.     

A mathematical model of pregnancy recognition in mammals

In this paper we develop a mathematical model of the luteal phase of the reproductive cycle in mammals with the aim to generate a systems understanding of pregnancy recognition. Pregnancy recognition is initiated by the production of interferon tau (IFNτ) by the growing conceptus. This ensures that the maternal corpus luteum (CL) remains viable to secrete progesterone, which is critical for providing a uterine microenvironment suitable for embryonic growth. Our mathematical model describes the interactions among the CL, the reproductive hormones and the hormone receptors in the uterus. It also characterises the complex interactions amongst the uterine oestrogen, progesterone and oxytocin receptors that control the sensitivity of the uterus to oestrogen, progesterone and oxytocin, respectively. The model is represented by a dynamical system and exhibits qualitative features consistent with the known experimental results in sheep. A key factor identified was a time-dependent threshold for the IFNτ signal below which the presence of the embryo might not be recognised and thus pregnancy would likely fail. Furthermore, the model indicated that if the IFNτ signal is later than around day 13 of the cycle, then pregnancy will not be recognised irrespective of the IFNτ concentration. The thresholds in the concentration and time of the IFNτ signal is a screening mechanism whereby only embryos of sufficient quality are able to prevent luteolysis (i.e. regression of the CL). The effect of progesterone secretion rate from the CL on pregnancy recognition was investigated. The model suggests that if the secretion rate is low then the initiation of the IFNτ signal is delayed, which in turn compromises the likelihood of a pregnancy being recognised by the CL. Furthermore, pregnancy recognition does not occur below a critical threshold in the progesterone secretion rate. In summary, the model can be used to identify the most favourable conditions for pregnancy recognition.     

Effect of interferons on the replication of alcelaphine herpesvirus-1 of malignant catarrhal fever

Four isolates of alcelaphine herpesvirus-1 of malignant catarrhal fever (MCF) were tested for their inducibility of and sensitivity to various interferons. Viral isolates from an Indian gaur (Bos gaurus), a greater kudu (Tragelaphus strepsiceros) and two wildebeest (Connochaetes gnou) calves did not induce measurable interferon (IFN) in bovine fetal kidney cells. However, these low passages of each virus were all highly cell-associated and viral replication was inhibited at these passages by IFN at 14 IFN units/0.05 ml recovered from NDV-infected MDBK cells and at 7.6 IFN units/0.05 ml of IFN from NDV-infected bovine macrophages. The herpesvirus from the Indian gaur and greater kudu and high passages (greater than 50) of the cell-free WC-11 strain of alcelaphine herpesvirus-1 also were inhibited in their replication by recombinant IFN of bovine and human origins as determined by a fluorescent focus unit (FFU) reduction assay. The concentrations of IFN required to produce a 50% reduction in herpesvirus-produced FFU ranged between 6.4 and 480 IFN units. These findings promote the use of IFN as part of the regimens of treatment of captive endangered ruminant species with clinical MCF.     

Urinary eCG patterns in the mare during pregnancy

Blood and urine samples collected from 12 mares at frequent intervals from 25 to 210 d of pregnancy were analyzed for equine chorionic gonadotropin (eCG). Blood and urine samples were collected daily through two consecutive ovulatory periods from five cyclic mares for comparative purposes. Separate radioimmunoassays (RIA) were developed to detect eCG in the urine and plasma. A simple and quick commercial dipstick enzyme-linked immunospecific assay (ELISA), developed for eCG in the blood, was also utilized in this study to detect eCG in the urine. In the 12 pregnant mares, eCG concentrations in both the plasma and urine as detected by RIA rose significantly on Day 40, peaked by Day 60 and slowly dropped to low levels by Day 200. The dipstick ELISA appeared more reliable for eCG in the plasma than in the urine of the five pregnant mares tested. However, on peak days (50 to 60), both the plasma and urine tested positive in all five mares. Similar eCG profiles were observed when urine samples from seven of the mares were assayed in the dipstick ELISA and RIA. The highest percentage of mares (86%) were positive for eCG by ELISA between Days 65 and 85. The highest concentration of eCG in the urine as detected by RIA was observed between Days 55 and 90. ECG-like immunoactivity was not detected by the ELISA in the urine of cyclic mares, but the RIA showed variable patterns with increases in immunoactivity that could not be correlated with physiological events. In summary, eCG in urine follows a similar profile as the eCG in plasma of mares during their first trimester of pregnancy.