Diagnosis of pregnancy in moose using a bovine assay for pregnancy-specific protein B

Blood samples were collected from 26 moose (Alces alces ) and evaluated for the presence of an antigen that cross-reacted with antisera to bovine pregnancy-specific protein B (P-SPB). The objective of this study was to determine if the P-SPB radioimmunoassay (RIA) was a reliable indicator of pregnancy in these animals. In the first year of the study calf production the following summer was used as the index of previous pregnancy. In the second year all females were subjected to palpation per rectum after chemical immobilization. Seven of the 10 cows sampled in the first year were also sampled in the second year. All animals determined pregnant by rectal palpation were positive for P-SPB; however, P-SPB was not detected in males.     

Diagnosis of pregnancy by radioimmunoassay of a pregnancy-specific protein in the plasma of dairy cows

The accuracy and efficiency of progesterone (P4) and bovine pregnancy-specific protein B (bPSPB) radioimmunoassays (RIA) in detecting pregnant and nonpregnant dairy cows were compared at different stages of pregnancy. The study included 145 French Friesian heifers and cows from a single herd. A total of 175 artificial insemination (A.I.) and blood sampling procedures were performed. Animals were bled 24 d post AI for P4 RIA. They were bled at 24, 26, 30 to 35, and 70 +/- 9 after AI for bPSPB RIA. Females were declared nonpregnant when plasma P4 concentrations were lower than 1.5 ng/ml. With the bPSPB RIA, cows were nonpregnant when at least one of the B Bo x 100 replicates was higher than 95% in the RIA. When compared with palpations per rectum at 70 d, the accuracy of positive diagnoses (no. positive and pregnant/no. positive diagnoses) by P4 RIA at Day 24 was 67.2% (82 122 ). The accuracy of negative diagnoses was 98% (52 53 ). Accuracy of positive diagnoses by bPSPB RIA increased with gestation age (P<0.05) from 86.2% (50 58 ) on Day 24 to 98.8% (83 84 ) at time of palpation per rectum. Accuracy of negative diagnoses increased (P< 0.001) from Day 24 (71.8%; 84 117 ) to Days 30 to 35 (100%, 83 83 ). Efficiency in detecting nonpregnant females was much higher (P < 0.001) with the bPSPB RIA on Days 30 to 35 (90.2%; 83 92 ) than with the P4 RIA on Day 24 (56.5%, 52 92 ). It is concluded that 30 days after AI, the bPSPB RIA is an efficient test both for pregnancy prediction and detection of nonpregnant dairy cows.     

Immunochemical demonstration of a new pregnancy protein in the mare

An antiserum against the serum of a pregnant mare was absorbed with stallion serum. This antiserum then gave two precipitates in crossed immunoelectrophoresis with serum from pregnant mares as the antigen. The two precipitates exhibited beta-1 and alpha-2 electrophoretic mobility. Identity was demonstrated between the alpha-2 mobile protein and PMSG. The absorbed antiserum inhibited the biological action of the PMSG preparation when tested in mouse ovarian weight assays. The beta-1 mobile protein was not detected in the serum from non-pregnant mares, stallions or geldings and was detected earlier in pregnancy (Day 30) than was PMSG (Day 42).     

Diagnosis of pregnancy in wood bison using a bovine assay for pregnancy-specific protein B

Blood samples were collected from 51 wood bison (Bison bison athabascae ) and evaluated for the presence of an antigen that cross-reacted with antisera to pregnancy-specific protein B(PSPB). The objective of this study was to determine if the PSPB radioimmunoassay (RIA) was a reliable indicator of pregnancy in these animals. Pregnancy of mature females was determined either at autopsy (20 animals) or by palpation per rectum after chemical immobilization (18 animals). The antigen was not detected in either males or juvenile females. There was minor cross-reaction in sera of one of nine nonpregnant females that had been exposed to males. The antigen was found in the sera of 25 of 27 females that were confirmed pregnant. It was concluded that the PSPB RIA was a useful tool in the determination of pregnancy in wood bison.     

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.     

Comparison of ultrasonography, bovine pregnancy-specific protein B, and bovine pregnancy-associated glycoprotein 1 tests for pregnancy detection in dairy cows

At Days 26 to 58 after AI, 138 Holstein-Friesian dairy cows were repeatedly examined by ultrasonography, using a 7.5 MHz linear-array rectal transducer. The total calving rate was 37.6% (52/138), and late embryonic mortality occurred 8.6% of the cows (12/138). On the days of ultrasound scanning, blood samples were drawn from the jugular vein for measuring the concentration of bovine pregnancy-specific protein B (bPSPB) and bovine pregnancy-associated glycoprotein 1 (bPAG 1). When compared with calving results, there were no significant differences in accurate diagnosis of pregnant cows were found between the 3 methods. However, when recognition of an embryo proper with a beating heart was used as the criterion for positive ultrasonographic diagnosis significantly fewer (P < 0.001) pregnant cows were correctly identified than by the other 2 tests. When compared with the noncalving cows, significantly fewer (P < 0.001) false positive diagnoses were made by the 2 ultrasonographic tests than by the PSPB and bPAG 1 tests, while significantly fewer (P < 0.001) false positive diagnoses were made by the bPSPB test than by the bPAG 1 test. The accuracy of detecting nonpregnant animals by both protein tests was limited by the relatively long half-life of these proteins after calving and by early embryonic mortality.     

An immunochemical method for detection and analysis of changes in methylome

DNA methylation is an important epigenetic modification involved in the ability of an organism to respond to stress and adaptation. It has been implicated in development, differentiation, oncogenesis, chromatin remodelling, nutrigenomics, and appears to play a pivotal role in many regulatory and adaptive functions. It is therefore important to analyze the status of DNA methylation and its changes under various developmental, carcinogenic, pharmacological, and environmental conditions. In this report we describe an immunochemical method for the detection of genome wide DNA methylation and its alterations under various conditions along with the analysis of DNA methyltransferase activity. The ability of this approach to detect and provide a map of methylomic changes in a genome facilitates assessment of various agents and conditions which can alter this important epigenetic signal. This experimental system permits rapid evaluation of potential target genes which would be modulated by DNA methylation changes and thus the gene networks that govern the processes.     

Physico-chemical characteristics of pregnancy-specific protein in the rat

Molecular weight of PSP, determined by gel-filtration on Sephadex G-200, is 130 000 +/- 4 000 D and, according to the Laemmli method with SDS, 85 000 +/- 2 000 D. The protein was detected in the zone of beta 1-globulins, its relative electrophoretic mobility is 0.45 +/- 0,2. PSP is heterogeneous by electric charge; at electrofocusing this protein was revealed in the following isoelectric points (pI); 5.85, 6.11, and 6.57. PSP was precipitated with ammonium sulfate (20-50% of saturation). Iron, carbohydrates, lipids were not found histochemically in PSP and it did not manifest the esterase and phosphatase activity.     

Detection of pregnancy by radioimmunoassay of a novel pregnancy-specific protein in serum of cows and a profile of serum concentrations during gestation

The development of a double antibody radioimmunoassay for a bovine pregnancy-specific protein (pregnancy-specific protein B; PSPB) is presented. By means of this assay, PSPB could be measured in serum of pregnant cows. Five dairy cows were bled throughout gestation to measure serum levels of PSPB. Serum concentrations (means +/- SE) exceeded 1 ng/ml by 30 days postbreeding and increased gradually through three months (9 +/- 0.6 ng/ml), six months (35 +/- 6 ng/ml), and nine months (150 +/- 75 ng/ml) of gestation. Maximum levels of PSPB (542 +/- 144 ng/ml) were reached two days before parturition and then steadily declined to less than 78 ng/ml by 21 days postpartum. In 21 cows bled daily from 15 through 30 days postbreeding, PSPB could be measured in a few cows before and in most cows by 24 days after breeding. In a commercial herd of 102 beef cows, the assay could detect pregnancy earlier and more accurately than the routine method of rectal palpation. This radioimmunoassay measures a unique antigen that, for the first time, provides a serological method for detecting pregnancy in cows.     

Purification of a ribonuclease from potato tubers and its use as an antigen in the immunochemical assay of this protein following tuber damage

Summary A method for the purification of a ribonuclease from potato tubers is described. The preparation was free from deoxyribonuclease and phosphodiesterase activities and possessed only slight phosphomonoesterase activity. Specific antibodies against the ribonuclease preparation were raised in rabbits. Two precipitin arcs were observed on Ouchterlony plates and three by the use of immunoelectrophoresis suggesting that the preparation contained three antigens. Development of one of the arcs on the diffusion plates could be prevented by prior absorption of the RNase preparation with an antiserum specific for phosphomonoesterase from potato tubers. Two of the arcs developing upon immunoelectrophoresis, one of which had low electrophoretic mobility and the other which migrated to the anode, corresponded in position to that of ribonuclease fractionated by agar gel electrophoresis. The remaining arc corresponded to the position of that arising when the RNase antigen was cross-reacted with specific antibodies against phosphomonoesterase from potato tubers. It was concluded that the anti-acid RNase antiserum may be useful for the immunochemical assay of RNase protein when used in conjunction with an anti-phosphomonoesterase antiserum and it was used for this purpose with homogenates derived from damaged and undamaged tuber tissue cv. Majestic. The observations suggested that RNase protein did not parallel the increase in ribonuclease activity following tissue damage and it was concluded that the enhanced RNase activity following mechanical damage may be due to activation of the pre-formed enzyme.     

Detection of a novel HLA-DQ specificity: serological and immunochemical analyses by a monoclonal antibody

A monoclonal antibody (mAb) with a novel human B-cell allospecificity was produced by immunizing a C3H/He mouse with the human B lymphoblastoid cell line EBV-Wa (HLA-DR4/Dw15/DQblank homozygous). The mAb, termed HU-46, reacted with B cells from not only DR4/Dw15-positive individuals but also certain DRw8/Dw8-positive ones whose DQ phenotypes had not yet been defined. Two-dimensional gel analyses indicated that the mAb recognized class II antigens which were encoded by the HLA-DQ locus. Furthermore, in genetic analysis, the gene encoding the class II antigen detected by HU-46 met the Hardy-Weinberg condition as a fourth allele of the DQ locus. We provisionally labeled this novel DQ specificity DQWa.     

Effects of embryo size at transfer (whole versus demi) and early pregnancy progesterone supplementation on embryo growth and pregnancy-specific protein bovine concentrations in recipient dairy heifers

The objectives of this study were to evaluate embryonic size and survival, plasma progesterone (P4) and pregnancy-specific protein bovine (PSPB) concentrations in early pregnancies (n = 99) following the transfer of one whole (n = 66) or one demi (n = 33) embryo to recipient virgin dairy heifers. The experiment was designed to evaluate the fixed effects of embryo size at transfer (whole or demi embryo) on Day 7 of the estrous cycle (Day 0 = estrus) and P4 supplementation between Days 7 to 19 through an intravaginal device (yes or no) on plasma P4 and PSPB concentrations and on embryo measurements. Plasma P4 concentrations were measured by RIA on Days 0, 7, 14, 19, 21, 25, 35, 42, 49, 56 and 63 of pregnancy and, PSPB concentrations were measured by ELISA on Days 7, 21, 25, 35, 42, 49, 56 and 63. The presence of an embryonic vesicle was detected on Day 25, embryonic/fetal movements and heartbeat were evaluated on Days 42 and 63 and embryo measurements [crown-rump length (CRL) and width at mid body] were obtained on Day 42 through ultrasonography.In non-supplemented pregnancies, Day 42 whole embryos had higher (P < 0.05) CRL and width than demi embryos, but the difference averaged only 1 to 2 mm. In P4 supplemented pregnancies, whole and demi embryos attained a similar size on Day 42 of pregnancy. Embryo size at transfer, early exogenous P4 supplementation and their interactions had no effects (P > 0.05) on plasma P4 concentrations. However, the post-hoc LSD evaluation showed that plasma P4 concentrations on Day 25 were higher (P < 0.001) in whole than in demi embryo derived pregnancies and, that exogenous P4 supplementation increased (P < 0.05) plasma P4 concentrations on Day 19 of pregnancy. The plasma PSPB detection rate on Days 7 to 63 of pregnancy was similar in pregnancies resulting from the transfer of whole and demi embryos. From a total of 93 recipients remaining pregnant until Day 63, plasma PSPB was constantly undetectable on Day 7, was detected in 4% of Day 21 samples, 41% of Day 25, 95% of Day 35, 96% of Day 42, 99% of Day 49 and in 100% of samples of Days 56 and 63. Concentrations of PSPB increased (P < 0.05) from Days 21 to 42 and from Days 56 to 63, with a plateau between Days 42 to 56. Demi embryo pregnancies had higher (P < 0.05) plasma PSPB concentrations on Days 35 and 42 than whole embryo pregnancies. Progesterone supplementation had a positive effect (P < 0.01) on PSPB concentrations from Days 35 to 63. Concentrations of PSPB were similar in non-supplemented whole and demi embryo pregnancies from Days 7 to Day 63. In contrast, in supplemented recipients, demi embryo pregnancies had higher (P < 0.05) PSPB concentrations on Days 25 to 42 than whole embryo pregnancies. No significant correlation was found between P4 and PSPB concentrations or between the concentrations of these hormones and embryonic measurements on Day 42. In conclusion, demi embryos experienced a compensatory growth until Day 42 of pregnancy, attaining a similar size to that of whole embryos and originating conceptuses producing similar plasma PSPB concentrations to those of whole embryo derived conceptuses. Embryonic growth and conceptus secretion of PSPB were positively stimulated by early pregnancy exogenous P4 treatment.     

Uterine and oviducal protein secretion during early pregnancy in the mouse

Changes in the protein composition of the embryo's environment during early development were studied by analysis of proteins synthesized and secreted by oviducal and uterine explants on Days 1-6 of pregnancy. Although secretions from ampullar and isthmic oviduct and uterus contained many proteins in common, each area also produced its own characteristic proteins. In the uterus, changes in the secretion pattern were found during the peri-implantation period, including both increases and decreases in particular proteins which appear to be dependent on the presence of embryos. Embryo-induced effects on uterine secretion began between 09:00 h of Day 4 and 09:00 h of Day 5. Oviducal secretions exhibited many of the embryo-dependent proteins found in the uterus, but the expression of these proteins did not appear to be influenced by the presence of embryos on Day 1 or Day 3. The characteristic pattern of secreted protein expression by each portion of the reproductive tract may reflect the specialization of each area for certain developmental events.     

Validation of a new pregnancy-associated glycoprotein radioimmunoassay method for the detection of early pregnancy in ewes

The aim of the current study was to describe the use of a pool of different antisera raised against pregnancy-associated glycoproteins (PAGs; purified from both ovine and caprine placentas) for early pregnancy diagnosis in ovine species. Sixty-three pluriparous Sarda ewes (Ovis aries) were synchronized. Blood samples were withdrawn on Days 18, 24, 26, 28, 30, and 50 after mating. These samples were assayed for progesterone (radioimmunoassay [RIA] including an extraction step) and for pregnancy-associated glycoproteins (RIA-706 and RIA-srPool). Progesterone concentrations were under 1.0 ng/mL in all nonpregnant Sarda ewes. In pregnant ewes, mean progesterone concentrations ranged from 2.4 ng/mL (Day 24, single pregnancies) to 4.4 ng/mL (Day 28, multiple pregnancies). During all periods of examination, PAGs remained lower than 0.8 ng/mL in nonpregnant ewes. On Day 18 of pregnancy, PAG concentrations could be detected in 26 of 43 (60.5%) and in 41 of 43 (95.3%) pregnant ewes using the RIA-706 and RIA-srPool methods, respectively. From Day 24 to Day 50, using both RIA methods, PAGs could be detected in all pregnant ewes. On Day 24, the best threshold for pregnancy diagnosis was obtained by use of RIA-srPool, maximal concentration in nonpregnant ewes being 0.3 ng/mL and minimal concentration in pregnant ewes being 4.8 ng/mL. In general, progesterone and PAG concentrations were higher in multiple pregnancies than in single pregnancies. However, because of large individual variations, single pregnancies could not be differentiated from multiple pregnancies.     

First purification of the antiquitin protein and demonstration of its enzymatic activity

Antiquitin is an evolutionarily conserved protein believed to play a role in the regulation of cellular turgor. Based on sequence analysis, this protein is classified as a member of the aldehyde dehydrogenase superfamily. All previous studies on antiquitin have been confined to the nucleotide level, and the protein has never been purified and characterized. In the present investigation, the antiquitin protein was purified for the first time. An acetaldehyde-oxidizing protein was isolated from the liver of black seabream (Mylio macrocephalus) by chromatographies on alpha-cyanocinnamate Sepharose and Affi-gel Blue agarose, followed by ammonium sulfate precipitation. The purified protein was identified as antiquitin by the first 18 N-terminal amino acid residues which showed 83.3% identity with the deduced sequence of human antiquitin. Electrophoretic mobility studies indicated that black seabream antiquitin is a tetramer with a subunit molecular mass of 57.5 kDa. Kinetic analysis of the purified protein indicated that it catalyzes the oxidation of acetaldehyde with K(m) and V(max) values of 2.0 mM and 1.3 U/mg, respectively. The longer aliphatic propionaldehyde and the aromatic benzaldehyde are also substrates of the purified enzyme. The enzyme is highly specific towards NAD+ as the coenzyme and is totally inactive towards NADP+. Maximal enzymatic activity was found at about pH 9-10.     

Comparative immunochemical characteristics and serological activity of the antigens of Cysticercus tenuicollis and Taenia hydatigena

Data are given on immunochemical analysis and serological activity of different antigens from larval and imaginal forms of Taenia hydatigena. Considerable heterogeneity and close antigenic affinity of the parasite's extracts under study both between each other and with the host's proteins, excluding the antigens from T. hydatigena, which has no common components with the latter, are established. In the homologous system in each extract under study there were recognised no less than 5 to 9 antigenic components. It is shown, however, by the method of adsorption of heterologous antibodies that the number of specific antigens in each of them does not exceed 1 or 2. All antigens happened to be serologically active, but the highest diagnostic efficiency was shown by extracts from scolices of C. tenuicollis and T. hydatigena. Antiparasitic antibodies were followed by these antigens in the sera of experimentally infected sucking pigs from the 10th day of the infection. They reached their maximum level on the 24th day and then was observed a gradual fall of the titre of specific antibodies, the level of which by the 115th day did not actually differ from initial values. The highest sensitivity and specificity in the immunoenzyme reaction under experimental conditions was displayed by the extracts from scolices of C. tenuicollis.