Characterization and quantification of yolk proteins in the lobster, Homarus americanus

Yolk protein (vitellin, Vn) and its precursor (vitellogenin, Vg) were isolated and characterized in the ovary and hemolymph, respectively, of the adult female lobster, Homarus americanus. Vn had a molecular mass of 360 kDa when analyzed by gel filtration. When analyzed by SDS-PAGE, Vn had six bands (110, 105, 94, 90, 81, and 78 kDa). An anti-Vn antiserum was developed using purified Vn, and the antiserum was used to detect Vn and Vg by ELISA and western blot techniques. ELISA analysis of hemolymph proteins separated by gel filtration indicated that Vg was similar in mass to Vn (360 kDa). However, western blots of hemolymph proteins separated by SDS-PAGE indicated that Vg contained a pair of protein subunits, 194 kDa and 179 kDa. Furthermore, the elution profiles of Vn and Vg from anion exchange chromatography indicated that Vg had a more negative charge. Thus, Vg appears to be processed after its uptake by the ovary to form Vn. Vg was undetectable in hemolymph from adult males by either ELISA or by western blot analysis. However, hemolymph levels of Vg in adult females increased 40-fold during the reproductive cycle, rising from 18 microg/mL in ovarian stage II to 789 microg/mL at stage V. This increase correlates well with oocyte growth during the cycle. Hence, this method may be useful for studying the regulation of lobster vitellogenesis.     

Development and Application of an Effective Detection Method for Fish Plasma Vitellogenin Induced by Environmental Estrogens

Vitellogenin is a protein induced by estrogens, including environmental chemicals with estrogenic activity. To measure the effects of environmental estogens, we developed an effective and rapid one-step method of detecting and purifying fish plasma vitellogenin using a high-performance anion-exchange chromatography column, POROS-HQ. Vitellogenin in a plasma of estradiol-treated male fish (mummichog and red sea bream) was eluted as a single peak with a retention time of 10 minutes from the column, which gives an almost pure preparation as assessed by SDS-PAGE. The lowest detectable amount of vitellogenin was 2 μg per assay. The method was used to analyze the plasma vitellogenin level of aquacultured red sea breams caught in August, when the spawning season is over, and usually no vitellogenin is detected in either females or males, physiologically. However, the data showed that in addition to a few females, some male fish synthesized vitellogenin, suggesting that some chemicals or unknown factors with estrogenic activity have induced fish in the ocean to produce vitellogenin.     

Expression of Kaposi＇s Sarcoma-associated Herpesvirus ORFK8.1 and Its Preliminary Diagnostic Application

The ORFK8.1 of Kaposi's sarcoma associated-herpesvirus (KSHV) was expressed in a prokaryotic expression system. The expression of recombinant E.coli containing pQE-80L-orf K8.1 was induced by isopropyl-b-D-thiogalactopyranoside (IPTG). The fusion protein was purified by chromatyography. The expressed protein and its purified product were identified by sodium dodecyl sulfate-polyacrylamide gel eletrophoresis (SDS-PAGE). SDS-PAGE showed that a protein of 26 kDa was visualized as expected. A western blot assay was established to analyze the immunogenicity of purified recombinant 0RFK8.1 protein. The optimal condition of the recombinant ORFK8.1 ELISA assay was confirmed: the concentration of antigen was 5 ug/mL, the dilution of serum was 1:200. We used the ELISA method to investigate the recombinant ORF K8.1 protein's specificity, the data showed that the specificity of ORF K8.1 to detect KSHV was 100%. At the same time, 560 sera samples from Hubei province were detected by using ORFK8.1 ELISA to investigate KSHV seroprevalence in this region. The KSHV seroprevalence in Hubei province is shown to be 6.80%.     

Hybridization between Crocodylus acutus and Crocodylus moreletii in the Yucatan Peninsula: II. Evidence from microsatellites

Detecting and quantifying hybridization between endangered or threatened taxa can provide valuable information with regards to conservation and management strategies. Hybridization between members of the genus Crocodylus has been known to occur in captivity and in some wild populations. We tested for hybridization among wild populations of American crocodile (C. acutus) and Morelet's crocodile (C. moreletii) in the Yucatan Peninsula by comparing Bayesian assignment tests, based on microsatellite data, to mitochondrial and morphological assignments. Skin clips from 83 individuals were taken for genetic identification, and a total of 32 individuals (38.6%) exhibited some evidence of hybridization by combined morphological, mitochondrial and microsatellite analyses. The majority of hybrids were classified as F(2) hybrids and backcrosses to C. moreletii. Most of the introgression occurs in two national biosphere reserves located on the northern and eastern coasts of the Yucatan Peninsula. Preliminary tests did not find a significant decrease in hybridity across three life stages, thus far indicating a low level of selection against hybrids. Model-based analyses on multilocus genotypes of pure individuals returned little geographic partitioning in both C. acutus and C. moreletii.     

Induction of vitellogenesis by estradiol-17beta and development of enzyme-linked immunosorbant assays to quantify plasma vitellogenin levels in green turtles (Chelonia mydas)

Treatment of juvenile green turtles (Chelonia mydas) with estradiol-17beta resulted in the induction of a 200 kDa plasma protein, consistent with vitellogenin (Vtg). The N-terminal 15 amino acids of the anion exchange purified protein shared sequence homologies with vitellogenins of several vertebrate species. Rabbit antiserum raised against purified Vtg recognized the plasma protein as well as several yolk proteins. Monoclonal antibody (Mab) HL1248, produced by inoculating mice with turtle yolk granules, showed specificity for plasma Vtg as well as a set of yolk proteins 120, 82, 43 and 32 kDa in size. The N-terminal 22 amino acids of the 43 kDa yolk protein was similar to the lipovitellin I subunit of Vtg of several vertebrate species. The peptide mass map of the 82 kDa yolk protein shared enough ions with that of purified plasma Vtg to support the conclusion that this protein was derived from plasma Vtg. Taken together, these results validate the specificity of Mab HL1248 for Vtg. Using purified Vtg concentration standards, competition and antigen capture enzyme-linked immunosorbant assays (ELISAs) were shown to quantitatively detect Vtg in green turtle plasma. Pre-induced plasma of juvenile turtles had Vtg levels of 2-4 micrograms/ml whereas post-estradiol exposure samples had 38-40 mg/ml. The plasma Vtg concentration of a nesting female turtle was 4.6 mg/ml, approximately 20-fold higher than that of a non-nesting adult female. The antigen capture ELISA will be useful in population studies of this endangered species, to detect vitellogenesis in females that will nest in a given year and to detect inappropriate Vtg levels in turtles exposed to xenoestrogens.     

Development and validation of a homologous zebrafish (Danio rerio Hamilton–Buchanan) vitellogenin enzyme-linked immunosorbent assay (ELISA) and its application for studies on estrogenic chemicals

Vitellogenin (VTG) was isolated by anion exchange chromatography from plasma of female zebrafish (Danio rerio) induced with 17α-ethinylestradiol (EE2). The purity of the VTG isolate was confirmed by polyacrylamide gel electrophoresis (SDS-PAGE). Purified VTG was used to raise polyclonal antibodies in rabbits and the specificity of the antisera for VTG confirmed by Western blot analysis of plasma proteins separated by SDS-PAGE. The antibodies cross-reacted with two proteins in the plasma of female zebrafish, with molecular masses of approximately 142 and 171 kDa. No cross-reactivity was observed with any other plasma proteins. A competitive enzyme-linked immunosorbent assay (ELISA) was developed using the polyclonal zebrafish VTG (z-VTG) antibodies and purified z-VTG as ligand and standard, respectively. The z-VTG ELISA was sensitive with a detection limit of between 2.0 and 3.0 ng purified VTG/ml, and a working range between 3 and 500 ng/ml (30–85% binding). The ELISA demonstrated precision, with inter- and intra-assay variations of 7.5±2.7 and 4.9±1.4%, respectively. Plasma from adult zebrafish and whole body homogenates from juvenile zebrafish diluted parallel with the z-VTG standard in the ELISA, validating the assay for quantifying z-VTG in both of these tissues. Exposure of adult male zebrafish to EE2 via water induced a concentration-dependent induction of VTG with a lowest observed effect concentration (LOEC) ≤1.67 ng EE2/l (for a 21-day exposure). The homologous z-VTG ELISA provides a valuable tool for the study of environmental estrogens in zebrafish.     

Reproductive dynamics of a tropical freshwater crocodilian: Morelet's crocodile in northern Belize

Morelet's crocodile Crocodylus moreletii has not been well-studied and many aspects of its life history are unknown. In particular there is a notable paucity of information on nesting and reproductive ecology. We studied the nesting ecology of Morelet's crocodile in northern Belize from 1992 through 1995. Nesting occurs at the onset of the wet season in mid-June and continues through mid-July (mean oviposition date =1 July±10 days). Eggs hatch from mid-August through mid- to late September. Nesting effort at our primary study site remained relatively constant during 1992, 1993 and 1995, but nearly doubled in 1994; this appeared to reflect a regional trend. Natural and man-made islands are heavily used as nesting sites. Nesting success in 1993 and 1994 was consistently higher on natural islands when compared with man-made islands or shoreline sites. Nest losses were primarily due to flooding and raccoon Procyon lotor predation. Losses from predation were greatest in 1994 when unseasonably low water levels facilitated predator access to nests. Females probably reach sexual maturity in 7–8 years after attaining a total length of 150 cm. Mean clutch size (25.0±7.6; range=9–42; n =73) did not differ among years. Mean clutch size, egg width (EW), egg length, egg mass (EM) and clutch mass were positively correlated with female snout–vent length (SVL). Mean EW was the best predictor of female SVL. A partial correlation analysis of egg and clutch attributes found that independent of female SVL, EM increases with increasing clutch size.     

Oral and cloacal microflora of wild crocodiles Crocodylus acutus and C. moreletii in the Mexican Caribbean

Bacterial cultures and chemical analyses were performed from cloacal and oral swabs taken from 43 American crocodiles Crocodylus acutus and 28 Morelet's crocodiles C. moreletii captured in Quintana Roo State, Mexico. We recovered 47 bacterial species (28 genera and 14 families) from all samples with 51.1% of these belonging to the family Enterobacteriaceae. Fourteen species (29.8%) were detected in both crocodile species and 18 (38.3%) and 15 (31.9%) species were only detected in American and Morelet's crocodiles, respectively. We recovered 35 bacterial species from all oral samples, of which 9 (25.8%) were detected in both crocodile species. From all cloacal samples, we recovered 21 bacterial species, of which 8 (38.1%) were detected in both crocodile species. The most commonly isolated bacteria in cloacal samples were Aeromonas hydrophila and Escherichia coli, whereas in oral samples the most common bacteria were A. hydrophila and Arcanobacterium pyogenes. The bacteria isolated represent a potential threat to crocodile health during conditions of stress and a threat to human health through crocodile bites, crocodile meat consumption or carrying out activities in crocodile habitat. We especially warn about the presence of Salmonella arizonae and S. typhi, which cause enteritis and septicemia in crocodiles and salmonellosis and typhoid fever in humans. The risk of bacterial contamination from crocodiles to humans could increase in the future because of the accelerated destruction of crocodile habitat, which could lead to an augmentation of human-crocodile interactions. Information on bacterial diversity reported here could help in the choice of antibacterial products in case of infections that are of crocodile origin.     

Hybridization between Crocodylus acutus and Crocodylus moreletii in the Yucatan Peninsula: I. Evidence from mitochondrial DNA and morphology

The American crocodile (Crocodylus acutus) and the Morelet's crocodile (C. moreletii) are broadly sympatric in Belize and Mexico. The presence of morphologically anomalous individuals in the overlapping range area suggests possible hybridization between these species. Analysis of 477 base pairs of the mitochondrial tRNA(Pro)-tRNA(Phe)-Dloop region revealed the presence of pure C. acutus (N=43) and C. moreletii (N=56), as well as a high proportion of interspecific hybrids (N=17, 14.6%) in the Yucatan Peninsula, Mexico. Although all individuals could be assigned to one species or other based on phenotypic characters, some had been characterized as potential hybrids in the field by anomalous scale counts. The hybridization zone lies along the area of sympatry between C. acutus and C. moreletii investigated in this study, but extends further inland if hybrid localities from Belize are included. Hybridization in the Yucatan Peninsula is bidirectional, which indicates considerably more genetic contact between these species than previously recognized, and is probably more detrimental to the genetic integrity of smaller C. acutus populations. A more intensive study of the pattern of hybridization is warranted and supports continued classification of C. acutus as a critically threatened species in the Yucatan Peninsula.     

Ticks from a Morelet's crocodile in Belize.

Parasitism of crocodilians by ticks has rarely been reported, and to our knowledge only seven published accounts exist. On 3 July 1999, we collected four ticks from a subadult Morelet's crocodile (Crocodylus moreletii) captured in northern Belize. These were identified as Amblyomma dissimile (one female), and Amblyomma sp. (two nymphs, one larva). The crocodile was captured on land approximately 100 m from water, and all four ticks were attached to loose skin on the lateral surface of the tail. Crocodilians are most susceptible to terrestrial ectoparasites, including ticks, during overland movements. However, most such movements occur in response to drought, when tick questing activity is suppressed, which likely accounts for the small numbers of tick specimens recorded from crocodilians and the absence of any noticeable impact of parasitism on host fitness.     

Effects of estradiol-17beta treatment on in vitro and in vivo synthesis of two distinct vitellogenins in tilapia.

Two distinct vitellogenins (VTG) were purified from the blood of estradiol-17beta (E(2))-injected tilapia, Oreochromis mossambicus. Enzyme-linked immunosorbent assays (ELISA) of each VTG were developed to examine effects of E(2) treatment on induction of VTG synthesis in the primarily cultured tilapia hepatocytes. Two VTG molecules (VTG210 and VTG140) had apparent molecular masses of 370 and 220 kDa by gel filtration and 210 and 140 kDa by SDS-PAGE, respectively. Western blot analyses showed that antibodies raised against the purified VTG210 and VTG140 reacted only with each protein band. Furthermore, ELISA for each VTG was specific for target VTG. When E(2) was added into the media of primarily cultured tilapia hepatocytes, VTG210 and VTG140 were both detected from E(2) concentrations of 1x10(-7) M and 5x10(-7) M, respectively. Time course experiments showed that there was a difference in the detection time of VTG210 and VTG140 after the hormone treatment. Although the injection of different E(2) doses induced both VTGs in the plasma of male tilapia, the concentration of VTG210 was nearly five to eight times higher than that of VTG140. These results suggest that E(2) is a direct inducer of both VTGs in the tilapia hepatocytes in vitro and in vivo, and that there is difference in the hormone response in inducing the VTGs in the tilapia hepatocytes.     

Development and application of a monoclonal antibody-based sandwich ELISA for quantification of Japanese medaka (Oryzias latipes) vitellogenin

Vitellogenin (Vtg) was purified from ascitic fluid of a 17beta-estradiol (E2)-treated female Japanese medaka by anion-exchange chromatography. The molecular mass of medaka Vtg by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), corresponding to the Vtg monomer, was 200 kDa. BALB/c mice were immunized with purified-Vtg and two hybridoma clones producing specific antibodies against medaka Vtg were selected. The specificity of these monoclonal antibodies (mAbs) was evaluated by Western blot analysis of the plasma proteins separated on SDS-PAGE, and no cross-reactivity was observed with plasma proteins from control males. A sandwich enzyme-linked immunosorbent assay (ELISA) for quantification of medaka Vtg was developed using these mAbs. The assay range was between 1 and 100 ng/ml, and the intra- and inter-assay variations determined from plasma samples were within 7.7 and 8.5%, respectively. Recovery of medaka Vtg added to plasma was 92-111%. In a plasma dilution test, plots of Vtg concentration gave a straight line. After exposure of male medaka to E2 (10 ng/l), Vtg appeared in liver and plasma on the first day and reached a maximum on the 3rd to 5th day. The sandwich ELISA could be useful for the detection of estrogenic properties, and the medaka Vtg bioassay could be a very sensitive and good tool for screening of endocrine disrupting compounds.     

Bluegill (Lepomis macrochirus) vitellogenin: purification and enzyme-linked immunosorbent assay for detection of endocrine disruption by papermill effluent

Vitellogenin (VTG) is a highly specific marker of exposure to environmental estrogens and has been used extensively in field and laboratory studies of estrogenic endocrine disruption in fishes. The purpose of this study was to develop and validate a sensitive, competitive, enzyme-linked immunosorbent assay (ELISA) specific for bluegill (Lepomis macrochirus) vitellogenin. Bluegill VTG was purified by anion exchange chromatography on DEAE-agarose. The polypeptide had an apparent mass of 170 kDa and was specifically recognized by the rabbit antiserum raised against bluegill female-specific plasma protein. Plasma samples from vitellogenic females diluted in parallel with the purified VTG standard curve in the ELISA. The detection limit of the assay was 29 ng/ml and the working range extended to 2700 ng/ml. Recovery of purified VTG was 85.8+/-9.5%, intra-assay variation was 6.4% and interassay variation was 12.3%. We used this ELISA to analyze the seasonal cycle of vitellogenesis in female bluegill and to evaluate potential disruption of this process by exposure to bleached kraft mill effluent (BKME). Captive female bluegill stocked in outdoor experimental streams in New Bern, NC had the lowest levels of VTG, estradiol-17beta (E2), and testosterone (T) and the smallest oocyte diameters in January, but these variables increased in March and remained elevated through August, suggesting an extended spawning season. Plasma VTG, E2, T and oocyte diameter were unaffected by exposure to BKME concentrations as high as 30%. Development of the VTG ELISA allowed rapid and convenient analysis of plasma samples to evaluate exposure to potential endocrine disrupting compounds.     

Ectromelia in Morelet's crocodile from Belize

Two Morelet's crocodiles (Crocodylus moreletii) captured on 21 March 1997 and 20 April 1998 in the New River system, Belize exhibited ectromelia of one forelimb. External and radiograph examination appears to indicate limb agenesis of unknown etiology, as there is no apparent scarring or skeletal trauma. These two individuals represent the only cases of missing limbs from 642 individuals captured in this study and to our knowledge, the first reported cases in Morelet's crocodile. Several factors including age and diet of the reproducing female, extremes in nest conditions (egg incubation temperature and humidity), and exposure to environmental contaminants can cause developmental abnormalities in crocodilians and may have contributed to the condition observed in these animals. Survival rates for hatchling crocodilians are generally low, and embryonic malformations such as ectromelia may constitute an added disadvantage to survival. However, both individuals examined in this study were vigorous and appeared in good condition.     

Component proteins in crude extract of adult Paragonimus westermani purified by immunoaffinity chromatography using monoclonal antibodies.

The nature of 2 component proteins in crude saline extract of adult Paragonimus westermani was investigated. By immunoaffinity chromatography using monoclonal antibodies (MAb) as ligands, the proteins were purified from the crude extract. Band 1 protein in disc-polyacrylamide gel electrophoresis (PAGE) was purified by PFCK-136 MAb. The protein, known to have molecular mass of 440 kDa, was composed of 23, 46 and 92 kDa subunits when observed by reducing SDS-PAGE and SDS-PAGE/immunoblot. This protein was originated from eggs of the worm as revealed by immunohistochemical staining with PFCK-136 Mab. Another affinity purified protein utilizing PFCK-44 MAb was the band 4 protein of 17 kDa in disc-PAGE. This was a monomer protein in reducing SDS-PAGE and SDS-PAGE/immunoblot. The protein was produced at intestinal epithelium of the worm.     

Vitellogenin-derived yolk proteins of white perch,Morone americana: purification,characterization, and vitellogenin-receptor binding

The objectives of this study were to 1) purify and characterize vitellogenin-derived yolk proteins of white perch (Morone americana), 2) develop a nonisotopic receptor binding assay for vitellogenin, and 3) identify the yolk protein domains of vitellogenin recognized by the ovarian vitellogenin receptor. Four yolk proteins derived from vitellogenin (YP1, YP2 monomer [YP2m] and dimer [YP2d], and YP3) were isolated from ovaries of vitellogenic perch by selective precipitation, ion exchange chromatography, and gel filtration. The apparent molecular masses of purified YP1, YP2m, and YP2d after gel filtration were 310 kDa, 17 kDa, and 27 kDa, respectively. YP3 appeared in SDS-PAGE as a approximately 20-kDa band plus some diffuse smaller bands that could be visualized by staining for phosphoprotein with Coomassie Brilliant Blue complexed with aluminum nitrate. Immunological and biochemical characteristics of YP1, YP2s, and YP3 identified them as white perch lipovitellin, beta'-components, and phosvitin, respectively. A novel receptor-binding assay for vitellogenin was developed based on digoxigenin (DIG)-labeled vitellogenin tracer binding to ovarian membrane proteins immobilized in 96-well plates. Lipovitellin from white perch and vitellogenin from perch and other teleosts effectively displaced specifically bound DIG-vitellogenin in the assay, but phosvitin and the beta'-component could not, demonstrating for the first time that the lipovitellin domain of teleost vitellogenin mediates its binding to the oocyte receptor. Lipovitellin was less effective than vitellogenin in this regard, suggesting that the remaining yolk protein domains of vitellogenin may interact with its lipovitellin domain to facilitate binding of vitellogenin to its receptor.     

Development of male-incubated ovaries in the gypsy moth,Lymantria dispar

Ovaries from Lymantria dispar females were transplanted into an environment lacking vitellogenin, the male milieu, in order to determine how the presence of vitellogenin in the hemolymph affects the process of protein uptake by gypsy moth oocytes. When undeveloped ovaries from newly ecdysed last instar females were transplanted into males of the same stage, follicles detached from the germarium and increased in size, but the growth of oocytes proceeded more slowly than those from female controls. Although chorion fromation was delayed in male-grown ovaries, scanning electron microscopy of chorionated eggs recovered from adult males showed that a chorion with normal surface architecture was formed by the adult stage. SDS-PAGE analysis of the male-grown ovaries and hemolymph from males receiving ovaries showed that vitellogenin production was not stimulated by the organ transplant and only male hemolymph proteins were internalized by the male-incubated ovaries. Thus, in the absence of vitellogenin, endocytosis of male hemolymph proteins occurred, but the rate of oocyte growth was slowed.     

血管性血友病因子裂解蛋白酶的稳定表达及其活性检测

本研究旨在得到重组的血管性血友病因子裂解蛋白酶(ADAMTS13),进一步研究其在血栓止血中的作用。利用脂质体将编码ADAMTS13全长序列的重组质粒pSecTag-ADAMTS13转染Hela细胞,用潮霉素(Hygromycin-B)筛选得到阳性克隆细胞株,并扩大培养,收集上清。利用Ni-NTA琼脂糖柱、梯度咪唑淋洗法纯化蛋白,SDS-PAGE和Westernblotting鉴定纯化产品纯度和免疫学活性,采用GST-His双抗夹心法测定蛋白剪切活性。结果显示,成功获得一株能恒定分泌重组ADAMTS13蛋白的细胞株ADAMTS2-4,每1L培养上清可纯化得到5.8mg重组蛋白。Western blotting结果显示,ADAMTS13多抗能与重组蛋白在190kDa处显单一条带,并且蛋白具有6.4U/mL的剪切活性(每毫升正常人混合血浆中ADAMTS13活性为1U)。重组蛋白具有较好的免疫原活性和酶活性,为进一步研究ADAMTS13作用机理和运用奠定了良好的基础。     

Presence of oxidized protein hydrolase in human cell lines, rat tissues, and human/rat plasma

Oxidized protein hydrolase (OPH), an 80 kDa serine protease whose activity is inhibited by diisopropyl fluorophosphate (DFP), has been isolated from human erythrocytes [Fujino, T. et al. (1998) J. Biochem. 124, 1077-1085]. The presence of OPH in various biological samples was examined by enzyme-linked immunosorbent assay (ELISA) and immunoblotting using an anti-OPH antibody raised against OPH purified from human erythrocytes, and by [(3)H]DFP-labeling and successive SDS-PAGE/fluorography. Solubilized samples of human cell lines including K-562 cells, THP-1 cells and Jurkat cells, and rat tissues including brain, heart, liver, kidney, and testis, inhibited the anti-OPH antibody binding to OPH in ELISA. Immunoblotting of lysates of K-562 cells, THP-1 cells and Jurkat cells showed four immunoreactive protein bands including an 80 kDa protein. Immunoprecipitation of the [(3)H]DFP-labeled K-562 cell lysate and successive SDS-PAGE/fluorography showed the presence of only the 80 kDa DFP-reactive protein with OPH antigenic activity. The level of the 80 kDa immunoreactive protein in K-562 cells rose as the cells differentiated toward erythrocytes. Immunoblotting of human and rat plasma showed two immunoreactive protein bands, including the 80 kDa protein, and SDS-PAGE/fluorography of [(3)H]DFP-labeled rat and human plasma showed the presence of only the 80 kDa DFP-reactive protein. The results indicate that OPH is present in a wide variety of biological samples.     

Monoclonal antibody-based immunoassay of vitellogenin in the blood of male channel catfish (Ictalurus punctatus).

1. A monoclonal antibody to vitellogenin of channel catfish (Ictalurus punctatus) was made, and its specificity was demonstrated using Western blots of serum from female fish, estradiol-treated male fish, untreated male fish, vitellogenin purified by three different methods and egg extracts. 2. An enzyme-linked immunosorbent assay (ELISA), using this monoclonal antibody, detected vitellogenin in the plasma of 59 out of 60 untreated 17-24-month-old male channel catfish with a mean concentration of 338 micrograms/ml and a maximum concentration of 4240 micrograms/ml. 3. Vitellogenin levels in male channel catfish were unrelated to testicular stage, gonadosomatic index and month.