Progesterone profile in buffaloes during various stages of oestrous cycle using radioimmunoassy technique

Investigation were carried out to study the norms of progesterone concentration in the blood serum of buffaloes during various phases of oestrous cycle. Twenty four animals (12 heifers and 12 cows) were used. The blood serum samples were stored at -20 degrees C until processed for progesterone assay. The progesterone concentrations were measured by the radioimmunoassay technique. The progesterone levels were 0.360 +/- 0.062 and 0.334 +/- 0.066 ng/ml on the day of oestrus in buffalo-heifers and buffalo-cows, respectively. The values were around 1 ng/ml till day 6, followed by a gradual increase to a peak average value of 4.888 +/- 0.399 and 5.119 +/- 0.415 ng/ml on day 15 of the cycle in heifers and cows, respectively. Thereafter, the progesterone concentration fell abruptly to a level similar to that at oestrus. The mean progesterone value a day before oestrus was 0.488 +/- 0.067 and 0.577 +/- 0.053 ng/ml in buffalo-heifers and buffalo-cows, respectively. The mean progesterone concentration of different days of the cycle (except day 16) did not differ significantly (P / -0.01) between heifers and cows.     

Use of milk progesterone enzyme immunoassay for early pregnancy diagnosis in cows

An enzyme immunoassay using beta-galactosidase as the tracer was applied to determine milk progesterone level in cows. The novel method was reliable and practicable and required only a spectrophotometer and a centrifuge as major equipment. The milk progesterone enzyme immunoassay successfully diagnosed early pregnancy in cows. Milk samples were collected from 268 Holstein-Friesian cows in commercial dairy herds on 20, 21 or 22 days(usually 21 days) after insemination. Progesterone level in skim milk higher than 1.0 ng/ml indi-cated pregnancy. Pregnancy was confirmed by rectal palpation on 60 to 120 days after insemination. The accuracy of the milk progesterone test was 60.0 % (132 220 ) for a positive diagnosis and 100 % (48 48 ) for a negative result. A high incidence of embryonic death, 27.9 % (51 183 ), may have reduced accuracy for a positive test result. The enzyme immunoassay may be an alternative to radioimmunoassay in milk progesterone analysis for pregnancy diagnosis.     

Enzyme immunoassay of progesterone in the feces from beef cattle to monitor the ovarian cycle

The present study was undertaken to measure fecal progesterone concentration of beef cattle using antibody against authentic progesterone and to examine whether this method can monitor the ovarian cycle in beef cattle. Rectal fecal samples collected from 14 beef cattle were mixed with 6 ml of 100% methanol and shaken for 15 min. After centrifugation, supernatant was extracted with petroleum ether followed by an enzyme immunoassay (EIA) for progesterone. Specificity of the assay was examined by HPLC separation of fecal solution followed by the EIA in each fraction. The present assay identified only progesterone but not other metabolites in the feces sample that was extracted with petroleum ether. Sensitivity of the assay was estimated to be 0.0055 ng/ml (0.11 ng/g). Coefficient variations of intra- and inter-assay were 9.6-10.9% and 10.8-16.6%, respectively. Recovery rates ranged between 73 and 84%. Patterns in the fecal progesterone concentrations during the ovarian cycle were almost parallel to the plasma concentrations. A significant positive correlation was established between the fecal and plasma progesterone concentrations in individual animal (r=0.59-0.84, P<0.001, n=10) as well as pooled data (r=0.70, P<0.001, n=65). Fecal progesterone concentrations of day 0 (showing the nadir of concentration) of the ovarian cycle were less than 50 ng/g, which increased significantly toward day 9 (P<0.01). From days 14 to 18, there was significant reduction of fecal progesterone concentration (P<0.01). Ovarian cycles had at least 48 ng/g (mean=74 ng/g) of difference between minimum and maximum fecal progesterone concentrations. All cattle at days 9, 11 and 14 had higher fecal progesterone concentrations by more than 20 ng/g compared with day 0. These results suggest that the present EIA is suitable to measure the progesterone in cattle feces and can monitor ovarian cycle.     

The predictive value, sensitivity and specificity of palpation per rectum and transrectal ultrasonography for the determination of bovine luteal status

Luteal status (classifications 1 and 3 = high progesterone levels, 2 and 4 = low progesterone levels) was diagnosed in randomly cyclic Holstein cows, using palpation per rectum and B-mode transrectal ultrasonography, respectively. The results were compared in 2x2 contingency tables with radioimmunoassay (RIA) classifications. Cows with milk progesterone levels of 5.0 ng/ml or higher were classified high and cows with levels below 5.0 ng/ml of progesterone were low. The predictive value, sensitivity and specificity results were 1) 88.2, 68.2 and 88.9% for Classification 1; 69.6, 88.9 and 68.2 for Classification 2; 90.9, 83.3 and 87.5 for Classification 3; and 77.8, 87.5 and 83.3 for Classification 4. Within each test, the diagnosis of high progesterone status was more accurate than of low status. The types of errors are discussed. A correlation coefficient of 0.68 resulted between RIA progesterone concentration and ultrasonographic luteal diameter. Ultrasonography was a better diagnostic test than palpation per rectum.     

Competitive enzyme immunoassay for human chorionic somatomammotropin using the avidin-biotin system

Human chorionic somatomammotropin (HCS) is determined by an enzyme immunoassay where HCS competes with biotin-labeled HCS for insolubilized anti-HCS antibodies. Enzymelabeled avidin is then used to reveal the amount of bound HCS. The system proves to be sensitive (1 ng/ml of HCS can be detected) and results agree with radioimmunoassay determinations (correlation coefficient = 0.979). Kinetics of the avidin-biotin reaction and coating of polystyrene wells are also investigated.     

Enzyme-linked immunosorbent assay for determination of cyclosporin a in the whole blood

A test-system based on enzyme-linked immunosorbent assay (ELISA) for quantitative determination of cyclosporin A (CSA) in human whole blood has been developed. The detection limit of the method was 25 ng/ml, the linearity of the method in the concentration range of 60–1400 ng/ml varied from 94 to 105%, the variation coefficient did not exceed 8%. The novel method exhibited good correlation with radioimmunoassay and polarization fluoroimmunoassay methods; the linear regression coefficients were 0.965 and 0.984, respectively. The developed test system is stable for at least 9 months when stored at 4°C and can be used in clinical practice.     

Simple and sensitive progesterone detection in human serum using a CdSe/ZnS quantum dot-based direct binding assay

In this study, we developed a CdSe/ZnS quantum dot (QD)-based immunoassay for use in determining the presence of progesterone (P₄) in human serum. Hydrophilic QDs were conjugated to anti-progesterone antibody (P₄Ab) via ethyl-3-(dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) as coupling reagents. After purification, the P₄Ab–QD conjugates were immobilized onto the wells of a 96-well microtiter plate, and a direct-binding immunoassay based on the binding of P₄ to immobilized P₄Ab–QD conjugates had a detection limit of 0.21 ng/ml and a sensitivity of 1.37 ng/ml, with a linear range of 0.385 to 4.55 ng/ml. The proposed immunoassay was successfully used to determine the P₄ concentration in real human serum, and the results showed a good correlation with the accredited radioimmunoassay (RIA).     

Development of quantitative immunoenzyme and radioimmunologic analysis of lambda-free immunoglobulin light chains

New quantitative techniques of radioimmunoassay (RIA) and enzyme immunoassay (EIA) were developed for determination of free light lambda-chains of immunoglobulins (immunometric sandwich-like variants) in biological fluids using two types of monoclonal specific antibodies (MAB-1 and MAB-2) to different epitopes of the antigen molecule: MAB-1 were immobilized on a solid phase (polystyrene beads), whereas MAB-2 were labeled with iodine or with the enzyme. The test systems prepared can be used for determination of concentrations from 25 to 1000 ng/ml, are very sensitive (25 ng/ml), and the analysis time is 5 h. The two methods were compared, and their clinical and diagnostic validity was evaluated in patients with various diseases associated with disorders in the antibody synthesis by the immune system.     

An electrochemical immunosensor for milk progesterone using a continuous flow system

An electrochemical biosensor for cow's milk progesterone has been developed and used in a competitive immunoassay under thin-layer, continuous-flow conditions. Single-use biosensors were fabricated by depositing anti-progesterone monoclonal antibody (mAb) onto screen-printed carbon electrodes (SPCEs). Three operational steps could be identified: (1) Competitive binding of sample/conjugate (alkaline-phosphatase-labelled progesterone, AP-prog) mixture, (2) establishment of a steady-state amperometric baseline current and (3), measurement of an amperometric signal in the presence of enzyme substrate (1-naphthyl phosphate, 1-NP). In the thin-layer cell, the enzyme product, 1-naphthol, showed electrochemical behaviour consistent with bulk conditions and gave a linear amperometric response under continuous-flow conditions (E_app=+0.3 V vs. Ag/AgCl) over the range 0.1–1.0 μg/ml. After pre-incubating biosensors with progesterone standards, signal generation within the cell (substrate CONCENTRATION=5 mM) was recorded amperometrically as rate (nA/s) or maximum current (i_max, nA). Response values for milk standards were approximately 50% of those prepared in buffer. In both cases, calibration plots over the range 0–50 ng/ml progesterone were obtained. By conducting sample binding under flowing conditions, only 7% of the previous response was obtained, even at a substrate concentration of 50 mM, resulting in low signal:noise ratio. Using a stop-flow arrangement (i.e. quiescent sample binding, followed by continuous flow), low-noise amperograms were obtained at [1-NP]=5 mM. Calibration plots were obtained over the range 0–25 ng/ml, with a coefficient of variation of 12.5% for five replicate real milk samples.     

Affinity-purified antibodies of defined specificity for use in a solid-phase microplate radioimmunoassay of human Tamm-Horsfall glycoprotein in urine.

Rabbit antibodies to human Tamm-Horsfall glycoprotein (prepared by salt precipitation from normal urine) were purified by affinity chromatography using columns containing Tamm-Horsfall glycoprotein linked to CNBr-activated Sepharose 4B. The specificity of these antibodies was determined by analysis of their binding characteristics on Western blots of Tamm-Horsfall protein from sodium dodecyl sulphate/polyacrylamide gradient gels and comparison with the reactivity of monoclonal antibodies to this glycoprotein. Optimal conditions of adsorption to poly(vinyl chloride) microtitre plates were established such that these purified antibodies could be used in a solid-phase radioimmunoassay for the determination of urinary Tamm-Horsfall-glycoprotein concentration. The specificity of the immunoassay was confirmed by competitive inhibition of the urinary Tamm-Horsfall glycoprotein by purified freeze-dried material in solution. A standard curve obtained with this material showed the radioimmunoassay to have a sensitivity of at least 5 ng/ml, with linearity between 30 and 600 ng/ml. The mean coefficient of variation over the linear section of the curve was 11.3 +/- 2.2% (n = 13). The effects of dialysis and freezing of urine samples before determination of Tamm-Horsfall-glycoprotein concentrations were investigated and the mean 24 h urinary excretion rate in 60 normal donors was shown to be 84.9 +/- 44.1 mg.     

Plasma progesterone levels during pregnancy in the little brown bat Myotis lucifugus (Vespertilionidae)

Plasma progesterone was measured by radioimmunoassay in individual female Myotis lucifugus throughout pregnancy and lactation. Progesterone levels, which averaged 6.7 +/- 0.7 ng/ml in late hibernation, rose to a mean of 18.9 +/- 6.7 ng/ml in unimplanted bats collected in the first two weeks after arrival at a maternity roost. Analysis of progesterone levels in bats in which the developmental stage of the embryo was known revealed two sharp, transient increases in plasma progesterone during the preimplantation period. The first, with values of 30-45 ng/ml, occurred at ovulation. The second, with values of 20-30 ng/ml, coincided with blastocyst formation. Progesterone levels increased exponentially from a mean of 7.4 +/- 1.0 ng/ml during early implantation to peak values of 100-200 ng/ml (means = 136.2 +/- 15.6) in the last two weeks of pregnancy, and showed no evidence of either a midpregnancy or prepartum decline. Despite involution of the corpus luteum at the end of pregnancy, progesterone levels averaged 9.0 +/- 1.0 ng/ml during lactation and did not decline until the end of lactation. In bats undergoing abortion, mean levels of plasma progesterone were already less than 6 ng/ml, equivalent to levels in nonbreeding females. The results indicate that the progesterone profile of pregnant M. lucifugus, though generally resembling those of other bats, exhibits several distinctive features. The sharp rise in plasma progesterone coinciding with blastocyst formation has not been reported in other mammals and suggests a possible role of progesterone in the cavitation process. In addition, peak values of plasma progesterone in late pregnancy were conspicuously higher than levels reported in other verpertilionid bats. The levels did not appear to fall before parturition, although such falls have been reported in other bats.     

Studies on serum progesterone levels in relation to occurrence of uterine torsion in buffaloes (Bubalus bubalis )

Serum samples from 32 buffaloes suffering from uterine torsion at approximately the completion of the gestation period were analyzed for progesterone concentrations by radioimmunoassay. Fifteen buffaloes had progesterone concentrations of more than 1.0 ng/ml (1.65 +/- 0.13 ng/ml; 1.1 to 2.8 ng/ml), while the serum progesterone concentrations of the remaining 17 buffaloes were below 1.0 ng/ml (599 +/- 59 pg/ml; 200 to 900 pg/ml). The occurrence of uterine torsion was associated with labour and/or abdominal pain around the expected time of parturition at the completion of the gestation period. High progesterone concentrations at labour in cases of torsion in almost half of the buffaloes may suggest that there were instances of premature labour resulting from an impaired hormonal milieu. Disturbances in the onset of labour owing to hormonal imbalance as evidenced by high progesterone levels may also contribute to the causation of uterine torsion. Cases of uterine torsion having low progesterone values are also discussed in this study.     

Chemiluminescence enzyme immunoassay (CLEIA) for the determination of chloramphenicol residues in aquatic tissues.

A competitive indirect chemiluminescence enzyme immunoassay (ic-CLEIA) for chloramphenicol (CAP) residues in shrimp has been developed. After optimization (incubation time, concentration of Tween-20, concentration of PBS and pH), the method gave a limit of detection of 0.01 ng/mL and a detection range of 0.03-23.7 ng/mL, with an ED(50) of 0.47 ng/mL. The method has been validated on spiked shrimp samples in terms of precision (intra- and interassay coefficient variations of less than 10% and 15%, respectively) and accuracy (mean recovery 95-123%). The assay performance is better than the ELISA method which is widely used to detect chloramphenicol and indicates that the CLEIA method can be used to test aquatic samples instead of ELISA.     

Evaluation of an on-farm blood progesterone test for predicting the day of parturition in cattle

An on-farm blood progesterone enzymeimmunoassay (EIA) was evaluated as a diagnostic test to predict the time of calving within a 24-hour period in near-term dairy cows. Blood samples were taken daily from 45 cows beginning 5 days prior to their expected due dates until calving, and plasma was stored at -20 degrees C until all cows had calved. The EIA test was performed on frozen-thawed plasma samples, and progesterone concentrations were determined to be low (positive test for calving within 24 hours) or high (negative test for calving within 24 hours). Sensitivity, specificity and predictive value of the EIA to accurately determine parturition within 24 hours were 86.7, 90.8 and 75.0%, respectively. The EIA correctly predicted the day of parturition in 168 of 187 (89.8%) plasma samples. Ten additional cows were similarly monitored except the EIA was performed on whole blood immediately after collection, and the sensitivity, specificity and predictive value of the test were 80.0, 97.6 and 88.9%, respectively. The day of parturition was correctly predicted in 49 of 52 (94.2%) whole blood samples. More than 95% of the cows calved within 24 hours when their plasma progesterone reached < 1.3 ng/ml. When results of the EIA were compared with those of a radioimmunoassay (RIA), the EIA findings were used to correctly classify 190 of 232 (81.9%) plasma samples as having low (< 2.0 ng/ml) or high (>/= 2.0 ng/ml) concentrations of progesterone. The EIA test was found to be a quick, practical means of estimating progesterone concentrations in bovine plasma or whole blood and was a useful test for predicting the day of parturition in cows.     

A simple enzymeimmunoassay of milk progesterone for monitoring fertility in dairy buffaloes

A simple, sensitive, direct (without extraction) enzymeimmunoassay (EIA) was usec to determine progesterone levels in whole milk samples of 400 Nili-Ravi dairy buffaloes. The mean progesterone values 22 d after A.I. were significantly higher in pregnant (16.6 +/- 9.2 ng/ml) than nonpregnant (below 5 ng/ml) animals. The mean progesterone values were below 0.34 +/- 0.12 (the detection limit) both at estrus and in cases of clinically diagnosed inactive ovaries, 3.18 +/- 1.9 at proestrus, 2.25 +/- 1.2 postestrus and 13.22 +/- 6.74 at Day 10 of the estrous cycle. Twenty buffaloes confirmed pregnant for 2 to 3 mo, had a mean value of 20.3 +/- 4.5 ng/ml. The EIA test is very reliable in the selection of nonpregnant buffaloes (100 %) and the confirmation of inactive ovaries and of estrus. Differential diagnosis of inactive or active ovaries can be made by analyzing two milk samples at a 7-d interval.     

An enzyme immunoassay for plasma betamethasone

A sensitive enzyme immunoassay for plasma betamethasone was developed using betamethasone-3-(O-carboxymethyl)oxime-β-D-galactosidase conjugate as a labelled antigen and 4-methylumbelliferyl-β-D-galactoside as a fluorescence substrate. The performances of the enzyme immunoassay were compared with that of a radioimmunoassay using ³H-betamethasone and the same antiserum. The minimal detectable level for the enzyme immunoassay was 0.15 pg/tube or 0.15 ng/ml of plasma, which was remarkably more sensitive than the radioimmunoassay level of 10 pg/tube or 2 ng/ml of plasma. The specificity was sufficient, in particular, the cross reactivity of cortisol was 0.008%. However, the precision of the enzyme immunoassay was inferior to that of the radioimmunoassay.     

Development of a quantum dot-based fluorescent immunoassay for progesterone determination in bovine milk

The use of semiconductor quantum dots (QDs) as fluorescent labels to develop a competitive immunoassay for sensitive detection and quantification of progesterone in cow's milk is described. Colloidal water-soluble CdSe/ZnS QDs are conjugated to an antigen derivative (progesterone-BSA conjugate) and a simple methodology is optimised to determine the antigen concentration in the final bioconjugate. The obtained QD-linked antigens were then employed together with unlabelled anti-progesterone monoclonal antibodies, as the biological recognition elements, in the development of the quantitative QDs-based fluorescent immunoassay for progesterone in bovine milk. After optimization, the developed immunoassay proved to cover a progesterone concentration range from 0.3 to 14.5 ng/mL in cow milk. Milk samples were just diluted 10-fold with deionised water and directly analysed with the proposed immunoassay, without additional sample pre-treatment or analyte extraction. The minimum detectable level (IC(10)) of the developed immunoassay turned out to be 0.1 ng/mL of progesterone in bovine milk. The sensitivity (IC(50)) achieved was 2.2 ng/mL with a reproducibility of 3.5% RSD as obtained from the results of the analysis of the triplicate of same samples but in three different days. Applicability of the proposed methodology was evaluated by analyzing cow's milk samples enriched with known concentrations of progesterone and recoveries better than 90% were achieved.     

Seasonal variation in serum levels of steroid hormones and their relation with seminal quality and libido in buffalo bulls

Blood samples from 15 breeding male Murrah buffaloes were collected during the winter, summer and monsoon seasons. Seminal characteristics and sexual behaviour were also studied. Serum samples were analysed for testosterone, progesterone and estradiol-17beta levels by radioimmunoassay. The studies showed significantly lower values for testosterone during winter (0.53 +/- 0.06 ng/ml) than during summer (1.22 +/- 0.19 ng/ml) and monsoon (1.06 +/- 0.12 ng/ml). The progesterone level was lowest during monsoon (84 +/- 9 pg/ml), intermediate during winter (115 +/- 14 pg/ml) and highest during summer (224 +/- 24 pg/ml). The mean level of estradiol-17beta was almost double (9 +/- 0.7 pg/ml) during monsoon as compared to winter (5 +/- 0.1 pg/ml). The correlations between hormone levels, seminal characteristics and sexual behaviour were of low magnitude.     

Development of Quantitative Enzyme Immunoassay and Radioimmunoassay of λ-Free Light Chains of Immunoglobulins

New quantitative techniques of radioimmunoassay (RIA) and enzyme immunoassay (EIA) were developed for determination of free light -chains of immunoglobulins (immunometric sandwich-like variants) in biological fluids using two types of monoclonal specific antibodies (MAB-1 and MAB-2) to different epitopes of the antigen molecule: MAB-1 were immobilized on a solid phase (polystyrene beads), whereas MAB-2 were labeled with iodine or with the enzyme. The test systems prepared can be used for determination of concentrations from 25 to 1000 ng/ml, are very sensitive (25 ng/ml), and the analysis time is 5 h. The two methods were compared, and their clinical and diagnostic validity was evaluated in patients with various diseases associated with disorders in the antibody synthesis by the immune system.     

An enzyme immunoassay for secretin

A sensitive and specific enzyme immunoassay for secretin was developed with the use of enzyme-labeled antigens. Synthetic porcine secretin and its carboxy-terminal fragments (residues 11-27 and 18-27) were conjugated with beta-D-galactosidase for use in the immunoassay, and the assay method with the latter fragment (residues 18-27) linked to beta-D-galactosidase was found to be the most sensitive. The minimum amount of secretin detectable by this method was 1-2.5 pg/assay. Serum levels of secretin after intravenous injection of the peptide in rats were determined by both the enzyme immunoassay and a commercial radioimmunoassay kit. The correlation coefficient between the levels measured by the two methods was 0.984. The enzyme immunoassay could detect immunoreactive secretin levels in normal human sera, giving a value of 16.9 +/- 2.2 pg/ml (mean +/- SE of six human subjects).