Comparison of the Ability of Three Radioimmunoassay to Detect Pregnancy-associated Glycoproteins in Bovine Plasma

Pregnancy‐associated glycoproteins (PAGs) constitute a large family of glycoproteins that are synthesized in the superficial layer of the ruminant placenta according to a spatial and temporal expression pattern. When PAGs are released in the maternal blood they can be used for pregnancy diagnosis, pregnancy follow‐up and for the monitoring of the trophoblastic function. Three different radioimmunoassay systems (RIA 1, RIA 2 and RIA 3) using antisera produced against PAG I₆₇ (RIA 1), PAG₅₅₊₆₂ (RIA 2) and PAG₅₅₊₅₉ (RIA 3) were used in this investigation in order to measure the PAG concentration in plasma samples withdrawn from pregnant cows and heifers during different periods following artificial insemination (AI). These systems were able to detect PAG molecules in the maternal blood as early as 21 days after AI in different concentrations (RIA 1: 0.43 ± 0.24 ng/ml, mean ± SD; RIA 2: 0.48 ± 0.24 ng/ml; RIA 3: 0.64 ± 0.37 ng/ml). On days 32 and 42 RIA 2 (4.30 ± 1.32 ng/ml and 5.56 ± 1.95 ng/ml) and RIA 3 (4.17 ± 1.15 ng/ml and 5.60 ± 1.89 ng/ml) presented significantly (p < 0.0001) higher PAG concentrations than those of RIA 1 (2.43 ± 0.81 ng/ml and 4.01 ± 1.48 ng/ml), respectively. After day 21, significant correlations (p < 0.0001; r ≥ 0.929) were determined between the three systems. Additionally the three individual PAG profiles presented in this study showed that PAG molecules secreted in the maternal blood between 21 and 50 days after AI were better recognized by the RIA 2 and RIA 3 systems. This study clearly indicated that the ability of a RIA test to recognize PAG molecules in the maternal blood can be improved by carefully selecting the antiserum.     

Correlation of five radioimmunoassay systems for measurement of bovine plasma pregnancy-associated glycoprotein concentrations at early pregnancy period

The measurement of serum or plasma PAG concentrations is currently used as a specific method for pregnancy diagnosis in cattle. In this study, the correlation between five radioimmunoassay systems (RIA-497, RIA-706, RIA-780, RIA-809 and RIA–Pool) developed for measurement of PAG concentrations in ruminant species was investigated in plasma from pregnant Friesian Holstein females. Plasma PAG concentrations (ng/mL) measured by different RIA systems were significantly correlated between them ( 0.81; P < 0.001). PAG concentrations increased significantly from Day 21 (n = 27) to 30 (n = 37) after AI by use of all PAG–RIA systems. From Day 30 to 80 after AI, lower PAG concentrations were observed when using the homologous system RIA-497. The addition of several proteinase inhibitors changed neither the non specific binding nor the B₀ binding to the tracer. Our results suggest that all tested PAG–RIA (RIA-497, RIA-706, RIA-780, RIA-809 and RIA–Pool) are highly correlated and can be useful to follow PAG concentrations in samples collected during the first trimester of gestation.     

Development and validation of a specific radioimmunoassay for equine osteocalcin

This study describes for the first time the development and validation of a sensitive and specific radioimmunoassay (RIA) for equine osteocalcin (OC) quantification using purified equine OC as standard, tracer, and immunogen for antibody formation in rabbits. The assay allowed to measure equine serum OC levels with a sensitivity of 0.2 ng/mL. Immunoreactive serum OC values of clinically normal, different-aged horses ranged from 3.68 to 127.31 ng/mL. Intra- and inter-assay coefficients of variation (CV) were 6.2 and 8.2%, respectively. Serial equine serum sample dilutions were linear. The recovery of equine OC from equine serum samples ranged from 93.88 to 107.9%. There was a tight correlation between OC values measured with the equine-specific OC RIA and two commercially available bovine-specific OC RIA kits. However, highest serum OC values were obtained with the equine-specific OC RIA. In conclusion, our equine-specific OC RIA is sensitive, linear, accurate, precise, and reproducible. The assay allowed to quantify OC in equine serum samples and might, therefore, be used to monitor equine osteoblast activity associated with bone diseases, exercise, therapy forms or diet.     

Aspartic Proteinase Members Secreted by the Ruminant Placenta: Specificity of Three Radioimmunoassay Systems for the Measurement of Pregnancy-associated Glycoproteins

Pregnancy‐associated glycoproteins (PAGs) isolated from the placenta of various ruminant species are enzymatically inactive members of the aspartic proteinase family. The measurement of these proteins in the maternal blood can be a good indicator of the presence of a live embryo. As certain aspartic proteinases are present in biological fluids in physiological and pathological conditions at various concentrations, it was necessary to determine the specificity of three radioimmunoassay (RIA) systems currently used for the detection of PAG molecules. Commercially available members of the aspartic proteinase family like pepsinogen, pepsin, chymosin, rennet, cathepsin D and renin were tested in a wide concentration range (10 ng/ml – 1 mg/ml). Pepsinogen cross‐reacted in RIA 1, RIA 2 and RIA 3 over 1 mg/ml, 50 μg/ml and 500 μg/ml concentrations, respectively. In the presence of pepsin, cross‐reaction was observed in RIA 1, RIA 2 and RIA 3 over 1 mg/ml, 500 μg/ml and 1 mg/ml concentrations, respectively. Chymosin and rennet could cross‐react in RIA 2 and RIA 3, while renin and cathepsin D did not decrease the binding of the tracer to antisera more, than that of the minimal detection limit. As the plasma/serum concentrations of the examined aspartic proteinases reported in the literature were outside the concentration range where cross‐reaction was observed, it can be concluded that these RIA systems were specific for the detection of PAGs in biological fluids.     

Assessment of Progesterone Concentration Using Enzymeimmunoassay, for Early Pregnancy Diagnosis in Sheep and Goats

The objective of this study was to determine a value of serum progesterone (P₄) concentration, assessed using an enzymeimmunoassay (EIA), for the early distinction between pregnant and non‐pregnant ewes and goats. Adult, non‐lactating ewes of Chios (n=53), Berrichon (n=30) and Sfakia (n=45) breeds were synchronized during the breeding season with progestagens and gonadotrophins and mated to fertile rams (Experiment I). Adult, lactating goats of Swiss breeds (Alpine and Saanen, n=104) and indigenous Greek breed (n=45) were synchronized during the transitional season with progestagens, PGF₂α and gonadotrophins. Cervical artificial insemination (AI) with fresh semen was applied once, 42–44 h after sponge removal (Experiment II). Jugular blood samples were collected on day 19 after sponge removal (ewes) or on day 21 after AI (goats) and serum P₄ concentration was determined by EIA. Progesterone concentrations ≥1.0, ≥1.5, ≥2.5 and ≥4.0 ng/ml were tested as indicative of pregnancy. Pregnancy diagnosis was verified on birth. In the case of sheep, using a discriminatory level of 2.5 ng/ml, overall accuracy of pregnancy diagnosis was 91.4% and predictive value of negative and positive diagnoses were 98.3 and 85.3%, respectively. In the case of goats, predictive value of negative diagnosis was 95.8 and 94.0% and predictive value of positive diagnosis 71.3 and 71.7%, for 1.5 and 2.5 ng/ml, respectively; overall accuracy was 79.2% using either level. The other discriminatory levels tested did not improve these results. A significant positive correlation was observed between P₄ concentration and the number of lambs or kids born, and further analysis indicated that this relationship is not a simple linear function. Based on the results of this study, P₄ concentrations of 2.5 ng/ml in the case of ewes and 1.5–2.5 ng/ml in the case of goats, determined with EIA, are proposed as discriminatory levels between pregnant and non‐pregnant animals, at an interval of one oestrous cycle after service.     

Comparison of 2 enzyme immunoassays and a radioimmunoassay for measurement of progesterone concentrations in bovine plasma, skim milk, and whole milk

The objective of this study was to compare 2 enzyme immunoassays (EIAs) with a radioimmunoassay (RIA) as to sensitivity and accuracy in the measurement of the progesterone (P4) concentration in bovine plasma, skim milk, and whole milk. The 72 samples from 24 lactating dairy cows expected to have either a high P4 concentration (cows in diestrus or pregnant) or a low P4 concentration (cows in estrus or anestrus) were analyzed by RIA, solid-phase EIA (SPEIA), which included a solvent extraction step, or direct EIA (DEIA) without solvent extraction. The overall mean concentrations of P4 did not differ (P < 0.4) among the assays. However, for the cows that were in diestrus or pregnant, the mean P4 concentrations (and standard error) were higher (P < 0.03), regardless of sample type, with RIA than with SPEIA, at 7.3 (0.7) and 6.1 (0.6) ng/mL, respectively. When only the high-P4 samples analyzed by RIA were compared, the mean P4 concentration was higher (P < 0.001) in whole milk than in skim milk, at 9.8 (1.0) and 4.1 (0.7) ng/mL, respectively. Although the mean P4 concentrations in the low-P4 samples did not differ (P < 0.80) among assays, the proportions of cows with a P4 concentration > or = 1 ng/mL were 3%, 14%, and 44% for RIA, SPEIA, and DEIA, respectively (P < 0.01; DEIA > SPEIA > RIA).     

Validation of a human progesterone enzyme immunoassay (EIA) kit for use on serum of cattle in Cameroon

A study aimed at validating a human progesterone enzyme immunoassay kit was carried out on cattle at Bambui, Cameroon. Progesterone ELISA Kits (EH-511) were obtained from Clinpro International. Forty-one cows were selected, of which 19 were pregnant and 22 within 14 days post partum. Blood samples were analysed and progesterone levels were deduced from a curve obtained from standard absorbance values (A ₄₅₀). Results show that 95.5% of postpartum cows had progesterone levels below 1 ng/ml, with the highest level being 0.75 ng/ml. The mean level was 0.5 ± 0.26 ng/ml. The cows in the ‘pregnant group’ had progesterone levels ranging from 3.5 to 17.5 ng/ml. This kit can be used for measuring progesterone levels in cattle. Levels of 1 ng/ml for two consecutive samples or one sample at or above 3 ng/ml are an indication of the presence of corpus luteum, while cows below 1 ng/ml will be in anoestrus.     

Direct radioimmunoassay of progesterone in bovine plasma using danazol (17-alpha-2,4-pregnadien-20-yno(2,3-d)isoxazol-17-ol) as a displacing agent.

The majority of progesterone in plasma is bound to cortisol-binding globulin and albumin carrier proteins. In the determination of plasma progesterone concentration by radioimmunoassay (RIA), it is necessary to remove these carrier proteins or displace the hormone from them. In the present study, we have examined the suitability of danazol (17-alpha-2,4-pregnadien-20-yno(2,3-d)isoxazol-17-ol), a synthetic steroid, to displace progesterone from plasma proteins in a direct RIA of bovine plasma. Accordingly, the progesterone content of bovine plasma samples was measured with a RIA using danazol as a displacing agent (direct RIA) and compared with results obtained with a RIA incorporating a preliminary solvent extraction step (extraction RIA). Danazol did not alter the standard curve for progesterone. Sensitivity (ED80) of the direct RIA (9.8 pg/tube) was comparable to that of the extraction RIA (10.3 pg/tube). Results for progesterone assayed in the direct RIA correlated well (r = 0.99) with the results obtained with the extraction RIA. The direct RIA was shown to be accurate; the mean recovery of known amounts of progesterone added to a sample of pooled bovine plasma was 98.5% +/- 3.29 (SEM). The direct RIA intra-assay coefficient of variation (CV) RIA for samples within the low concentration range (0.1-1.0 ng/mL), the medium concentration range (1.0-3.0 ng/mL) and the high concentration range (3.0-6.0 ng/mL) of progesterone were 8.1%, 8.3% and 7.73%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative determination of progesterone (P4) in canine blood serum using an enzyme-linked fluorescence assay

Progesterone (P4) measurement in the peripheral blood is an objective parameter for determination of reproductive functions in the bitch. This study evaluates an enzyme-linked fluorescence assay (ELFA) (Biomerieux, France) for the determination of progesterone validated for use in human. The ELFA is to be performed on the MiniVidas automated analyser which provides quantitative results within 45 min. Blood samples from a total of 27 female dogs of different breeds were used. To test the correctness of the ELFA 15 blood samples with a range of 0.3-40.0 ng/ml were compared to a radioimmunoassay (RIA) validated in the dog. The values obtained with the MiniVidas showed a high agreement (mean deviation 15%), deviations were in both directions and the correlation coefficient was 0.989. The coefficient of correlation according to Passing-Bablok test was 0.995. The intra-assay reproducibility in the MiniVidas system was tested on five samples (mean values 61.8, 6.8, 51.4, 43.7 and 1.1 ng/ml). The coefficients of variation (CV; 10-12 replicates) were 3.4%, 6.7%, 2.6%, 3.1% and 25.4%, respectively. Four serum samples (mean value 47.0, 15.1, 49.1 and 4.0 ng/ml) from different bitches were assayed singly in 10 separate series to test the inter-assay variability. The corresponding CV was 2.1%, 2.2%, 3.1% and 4.3% respectively. Samples from three dogs were used to test the accuracy of the assay. These samples were diluted (1/2, 1/4, 1/8 and 1/16) with charcoal-stripped human serum (Biomerieux, France) and tested in three runs. The expected values were met in a range of 60-75%. Measurement of progesterone for the detection of ovulation as well as prediction of parturition provided meaningful results. As a conclusion the use of the MiniVidas system for determination of P4 in peripheral blood of the bitch provides rapid and reliable results.     

用母猪血浆中硫酸雌酮浓度作早期妊娠诊断的研究

45头杂种母猪于配种后第28天采血,用放射免疫法测血浆中硫酸雌酮含量。以0.5ng/ml为界线作判断妊娠与否的标准。妊娠诊断的准确率是100％。妊娠母猪的血浆硫酸雌酮水平和胎儿数呈正相关(r=0.501,n=43,P<0.01)。     

Development of a canine trypsin-like immunoreactivity assay system using monoclonal antibodies

The radioimmunoassay (RIA) for trypsin-like immunoreactivity (TLI) is one of the most sensitive and specific tests for detecting exocrine pancreatic insufficiency (EPI). An abnormally low serum TLI concentration (<2.5 ng/ml) indicates end-stage EPI. Although RIA methods can be used to detect canine serum TLI, these procedures are beyond the capabilities of most veterinary clinics and general laboratories. Using monoclonal antibodies (mAbs), we developed an enzyme-linked immunosorbent assay (ELISA) for canine TLI and incorporated it into an immunochromatographic test (ICT) for the diagnosis of EPI. The ELISA was linear over TLI concentrations of 1-100 ng/ml. Levels of intra-assay coefficients of variance (CVs) were 1.8-6.1%, inter-assay CVs were 5.1-9.8%, and the recovery of TLI added to two samples of canine serum ranged from 89 to 111 and 93 to 108%, respectively. Good correlation (correlation coefficient, 0.974) occurred between the TLI values obtained by the ELISA method and those by RIA from 56 clinical samples. Serum TLI values in clinically healthy dogs ranged from 7.8 to 29.2 ng/ml by ELISA, and those from dogs with EPI were 0.0-0.6 ng/ml. The values were 0.0-287.4 ng/ml for dogs with pancreatitis, and those from dogs with gastrointestinal disease were 5.5-58.9 ng/ml. The only statistically significant difference (P<0.01) occurred between the TLI level of healthy dogs and those with EPI. The ICT kit showed high reproducibility, and the TLI values yielding negative results differed significantly (P<0.01) from those returning positive results. The ICT kit yielded negative results (indicating EPI) from clinical serum samples with TLI concentrations of 0.0-4.1 ng/ml by ELISA. Both the ELISA and ICT kit are useful tools in the diagnosis of canine EPI.     

Use of estrone sulfate determination in goat blood for the detection of pregnancy and prediction of fetal number]

The study was carried out on 50 goats of Polish White breed. Blood samples were taken on day 60 after mating from V. jugularis. Pregnancy diagnosis was made using transcutan ultrasound scanning (Pie Medical Scanner 2000 with a 5 MHz curved array probe). The estrone sulphate level was determined by RIA method after extraction and hydrolysis with arylsulphatase/glucuronidase from Helix pomatia. As a threshold value for differentiation of pregnant from nonpregnant goats 1 ng/ml was assumed. In eight nonpregnant goats the estrone sulphate level was 0.61 +/- 0.21 ng/ml. In 42 pregnant goats the level was 6.1 +/- 3.5 ng/ml. The diagnosis was correct in all cases. The number of single, twin and triplet pregnancies was six, 34 and two, respectively. Estrone values in goats with twin pregnancies were significantly higher compared to those with single pregnancies (7.2 +/- 4.1 ng/ml vs. 4.5 +/- 1.8 ng/ml; p < or = 0.01). The results of this study indicate, that the determination of estrone sulphate in blood of goats may be useful for pregnancy diagnosis. The estrone sulphate level in goats with twins is higher, but due to high variation between animals the number of fetuses cannot be exactly predicted.     

Early Pregnancy Detection of Iraqi Riverine Buffalo (Bubalus bubalis) Using the BioPRYN Enzyme‐Linked Immunosorbent Assay for PSPB and the Progesterone Assay

This study was undertaken to detect pregnancy in Iraqi riverine buffalo (Bubalus bubalis) using three different methods (rectal palpation, plasma progesterone concentration and detection of the presence of pregnancy‐specific protein B (PSPB) with the BioPRYN^® enzyme‐linked immunosorbent assay (ELISA) test. The aim of the study was to identify the most sensitive, early and accurate method for detecting pregnancy. Twenty‐two female riverine buffalo that were 6.0 ± 0.93 years old were used. Four blood samples per buffalo were taken via jugular venipuncture at days 22–24, 32–34, 42–44 and 58–61 post‐mating (PM) to measure the progesterone concentration (ng/ml) and to detect the presence of plasma PSPB. The rectal palpation method was employed to evaluate all buffalo on days 42–44 and 58–61 PM. The BioPRYN^® test differed (p < 0.01) from the other tests with earlier accuracy for detecting pregnant and non‐pregnant buffalo. Eighty‐eight percent of pregnant and 76.9% of non‐pregnant buffalo were distinguished early (days 22–24 PM) using BioPRYN^® and plasma PSPB‐ELISA level (2.09 ± 0.12 ng/ml) in relation to 66.7% and 53.9% detected using the progesterone assay at similar days (4.30 ± 0.40 ng/ml). In conclusion, these results described, for the first time, the early and accurate pregnancy detection of water riverine buffalo using BioPRYN^® technology and provided the plasma levels of PSPB using an ELISA test. These findings will improve the reproductive and productive efficiency of Iraqi riverine buffalo by adapting the recent management and reproductive strategies in Iraq and in the world.     

Assessment of pregnancy‐associated glycoprotein (PAG) concentrations in swamp buffalo samples from fetal and maternal origins by using interspecies antisera

Pregnancy‐associated glycoproteins (PAG) constitute a large family of glycoproteins found in the outer placental epithelial cell layer of the placenta in Eutherian species. In ruminants, they are noted to be structurally closely related among the different species. This study was designed to determine PAG concentrations in maternal and fetal plasma, allantoic and amniotic fluids in buffalo species. Antisera (AS) generated in rabbits against distinct PAG molecules were used in three radioimmunoassay (RIA)‐PAG systems: RIA‐1 (antiserum raised against bovine PAG67kDa; AS#497), RIA‐2 (antiserum raised against caprine PAG55 + 62 kDa; AS#706) or RIA‐3 (antiserum raised against buffalo PAG; AS#859). Samples were collected at a slaughterhouse (n = 67). PAG concentrations determined by RIA‐2 gave significantly higher results in both allantoic and amniotic fluids (12.7 ± 2.1 ng/mL and 24.0 ± 7.3 ng/mL, respectively). Regarding maternal and fetal plasma, PAG concentrations obtained by RIA‐2 (21.8 ± 2.4 ng/mL and 20.2 ± 2.5 ng/mL, respectively) and RIA‐3 (25.0 ± 2.2 ng/mL and 21.9 ± 3.2 ng/mL, respectively) were higher than those obtained by RIA‐1 (15.5 ± 1.4 ng/mL and 16.1 ± 1.8 ng/mL, respectively). The correlation among the three systems was very high. The study clearly reveals the ability of different PAG‐RIA systems to measure PAG concentration in swamp buffalo samples.     

Technical note: time-resolved immunofluorometric assay for growth hormone in ruminants

A noncompetitive, time-resolved immunofluorometric assay (TRIFMA) was developed using a selected pair of monoclonal antibodies (mab) raised against recombinant bovine GH, with the catching mab immobilized on microtiter plate wells and the detection mab labeled with Eu3+ as a tracer, arranged as a sandwich. Plates were coated with mab1.15 (680 ng/well) using a phosphate buffer (pH 4.9), and then blocked with assay buffer containing 1% (wt/vol) BSA. The assay procedure involved incubation of 50 microL of sample (plasma or serum) and 200 microL of assay buffer containing 25 ng of mab1.2-Eu3+ conjugate for 4 h at 25 degrees C. Plates were then washed six times, incubated for 5 to 10 min with 250 microL of enhancement solution, and fluorescence read with a time-resolved fluorometer. The sensitivity of the assay was 0.1 ng/mL, and the working range was 0.2 to 200 ng/mL. Recovery of quantitative amounts of bovine GH added to plasma samples was close to 100%. Cross-reactivity with other bovine pituitary hormones or with GH from nonbovidae or cervidae species was not significant. Intra- and interassay CV during routine operation was 4.4 and 10.7%, respectively (mean = 3.54 ng/mL). Plasma concentrations of bovine GH determined by TRIFMA correlated closely (r2 > or = 0.93) with RIA results, with a conversion ratio of 0.62 when the higher specificity of the monoclonal antibodies was taken into account. The TRIFMA is a reliable alternative to RIA methods because the assay employs no radiolabeled or hazardous chemicals, delivers results rapidly, and has little risk of down periods.     

二氟沙星残留检测的Ci-ELISA方法建立

以合成的二氟沙星（DIF）人工抗原为基础，制备出了高效价抗DIF血清（1∶29×104），并建立了DIF间接竞争酶联免疫吸附测定法(Ci-ELISA法)。标准曲线的线性回归方程为y=-0.223x+0.9631(r=0.9929)，中值（I50）=119.21 ng/ml，最低检测限（LOD）=1.14 ng/ml，线性范围为1.14~10000 ng/ml，方法的平均批内变异系数（CV）为4.83%,平均批间CV为10.69%。DIF以浓度15~10000 ng/ml在鸡肉中添加时，回收率为81.24%~90.64%，变异系数为8.18%~11.75%。     

Skim milk progesterone in pregnant cows from insemination throughout lactation

The skim milk progesterone profile was assessed by radioimmunoassay, without extraction, from the day of insemination (day 0) until the cows were dried off on day 225 of gestation. A total of 418 samples were collected from 154 pregnant Holstein cows. The daily variation in skim milk progesterone was recorded from day 1 until day 45 of pregnancy to detect the commencement of progesterone secretion from the corpus luteum after insemination. Subsequent determinations were made every 2 weeks from day 46 until lactation ceased. On the day of artificial insemination and for the first 2 days after insemination, all the cows had a basal progesterone concentration <0.1 ng/ml. A rise in progesterone (0.2±0.1 ng/ml) was first detected on the third day after insemination. The progesterone values then increased significantly (p<0.001) until day 15.The values then remained nearly constant (2.5–3.5 ng/ml) until day 106 of pregnancy, when they began to decline. Between days 120 and 180 of gestation, progesterone was significantly decreased (2.2–2.9 ng/ml) before it rose again to the previous plateau (3.5–3.9 ng/ml) around day 180. The progesterone concentration then remained at the higher level until the animals were dried off.Abbreviations AI artificial insemination - RIA radioimmunoassay     

Enzyme Immunoassays for the Determination of Ovine LH and FSH

The development of competitive enzyme immunoassays for ovine plasma LH (oLH) and FSH (oFSH) is described. Standards and plasma samples were preincubated with diluted antiserum to oLH or oFSH and the reacted solution (100 μl per well) was transferred to plates previously coated with oLH or oFSH, respectively. The second antibody used was anti‐rabbit IgG horseradish peroxidase. The measuring range was 0.39–50 ng/ml for each hormone and the 50% relative binding sensitivity was 9 ng/ml for oLH. The respective value for oFSH was 3.5 or 34 ng/ml with different hormone and antibody preparations used for the assay. The enzyme immunoassays were used to determine oLH and oFSH levels in plasma from ewes of two breeds during the oestrous cycle. The assays detected the first FSH surge coincident with the LH surge, the second FSH surge about 24 h later and the periodic fluctuations of FSH concentrations during the luteal phase of the oestrous cycle. These enzyme immunoassays are an efficient and economic alternative to the established radioimmunoassays (RIA) for oLH and oFSH.     

A chicken leptin-specific radioimmunoassay

Recombinant chicken leptin was used to produce an antiserum in order to develop a specific and sensitive radioimmunoassay (RIA) for chicken leptin in plasma and serum. We have used either murine or chicken leptin as tracer and competition curves were performed using recombinant chicken leptin. Variations in leptin plasma levels in different chicken strains and various nutritional states were correlated with the physiological status. Leptin plasma concentrations were regulated by the nutritional state with higher levels in the fed state as compared to the fasted state (3.36 +/- 0. 13 versus 2.78 +/- 0.11 ng/ml) and being dependent upon the age. Higher leptin levels were found in 22 week-old as compared to 15 week-old layer chickens (2.709 +/- 0.172 versus 1.478 +/- 0.102 ng/ml). We have also shown that the multispecies leptin RIA kit (LINCO Inc.) underestimated leptinemia compared to the chicken leptin- specific RIA reported here. In conclusion the RIA developed in the present study is specific to the chicken and thus may be considered as powerful tool for investigating the physiological significance of leptin in chickens.     

Development and evaluation of a rapid enzyme-immunoassay system for measurement of the urinary concentration of estrone-3-glucuronide in a female giant panda (Ailuropoda melanoleuca)

To detect estrus for reproductive management, and to determine the relationship between urinary estrogen and estrous behavior, in a female giant panda, we developed and evaluated a rapid enzyme immunoassay (EIA) system for urinary Estrone-3-glucuronide (E1G) using commercial reagents. The developed EIA system took only around 3 hours, including all procedures to obtain a result. It indicated good reproducibility (intra-assay CV of 5.16%, interassay CV of 15.4%) and sensitivity (lowest standard concentration was 0.0156 ng/ml) for measurement of the urinary concentrations of E1G in the giant panda. There was a positive correlation (r=0.934) with the data for estrone (E1) in the same samples, as measured by radioimmunoassay (RIA) performed in a commercial laboratory. The changes in the E1G concentrations were almost synchronous with the changes in E1 assayed by RIA in urine collected during 4 consecutive estrous seasons. The dynamics of urinary E1G measured by this system highly correlated with the occurrence of the presenting estrous behavior in the giant panda. The above results indicate that this assay system may be normally, rapidly and practically used for measurement of the urinary concentration of E1G in the giant panda.