Enzyme-linked immunosorbent assay of changes in serum levels of growth hormone (cGH) in common carps (Cyprinus carpio)

The aim of the present study was to purify the common native carp growth hormone (ncGH), produce monoclonal antibodies (mAbs) to common native carp growth hormone (ncGH), and further enhance the sensitivity of enzyme-linked immunosorbent assays (ELISA) for ncGH. Additionally, we investigated changes in serum ncGH levels in carps raised in different environmental conditions. The recombinant grass carp (Ctenopharyngodon idella) growth hormone was purified and used as antigen to immunize the rabbit. The natural ncGH was isolated from the pituitaries of common carp. SDS-PAGE and Western blot utilizing the polyclonal anti-rgcGH antibody confirmed the purification of ncGH from pituitaries. Purified ncGH was then used as an immunogen in the B lymphocyte hybridoma technique. A total of 14 hybridoma cell lines (FMU-cGH 1-14) were established that were able to stably secrete mAbs against ncGH. Among them, eight clones (FMU-cGH1-6, 12 and 13) were successfully used for Western blot while nine clones (FMU-cGH 1-7, 9 and 10) were used in fluorescent staining and immunohistochemistry. Epitope mapping by competitive ELISA demonstrated that these mAbs recognized five different epitopes. A sensitive sandwich ELISA for detection of ncGH was developed using FMU-cGH12 as the coating mAb and FMU-cGH6 as the enzyme labeled mAb. This detection system was found to be highly stable and sensitive, with detection levels of 70 pg/mL. Additionally, we found that serum ncGH levels in restricted food group and in the net cage group increased 6.9-and 5.8-fold, respectively, when compared to controls, demonstrating differences in the GH stress response in common carp under different living conditions.

An enzyme-linked immunosorbent assay for rare minnow (Gobiocypris rarus) vitellogenin and comparison of vitellogenin responses in rare minnow and zebrafish (Danio rerio)

A competitive enzyme-linked immunosorbent assay (ELISA) was developed to determine vitellogenin (Vtg) in rare minnow (Gobiocypris rarus) based on the separation and purification of rare minnow Vtg (r-Vtg) as well as the production of polyclonal antibody against r-Vtg in rabbits. Three different ELISAs for measuring r-Vtg were then compared: (1) indirect ELISA with the antibody against carp (Cyprinus carpio) Vtg (c-Vtg) (IC-ELISA); (2) competitive ELISA with the antibody against c-Vtg, and using r-Vtg for coating the plates and preparing standard curve (CC-ELISA); (3) competitive ELISA with the antibody against r-Vtg, and using r-Vtg for coating the plates and preparing standard curve (CR-ELISA). The result showed that the homologous CR-ELISA was the most sensitive among the three assays for quantifying r-Vtg. The sensitivities to 17 alpha-ethinylestradiol (EE2) Of rare minnow and zebrafish (Danio rerio) were compared upon the establishment of homologous competitive ELISA. The lowest observed effect concentrations (LOECs) to induce Vtg were found to be 0.8 ng EE2 l(-1) for rare minnow and 4 ng EE2 l(-1) for zebrafish respectively. Afterwards, CR-ELISA was applied to measure Vtg concentration in whole body homogenate (WBH) of juvenile rare minnow fed by three diets (tubifex from wastewater treatment plant, Artemia nauplii and commercial pellet food), and the agreements between bioassay and GC-MS analysis demonstrated that rare minnow was a sensitive fish model for assessing estrogenic effects of endocrine disrupting compounds in aquatic environment. (c) 2006 Elsevier B.V. All rights reserved.

Rapid detection of virulent protease secreted by Vibrio anguillarum by dot enzyme-linked immunosorbent assay

Dot enzyme-linked immunosorbent assay (dot-ELISA), indirect ELISA and Western blot were performed to detect the virulent protease secreted by Vibrio anguillarum which was isolated from the diseased left-eyed flounder, Paralichthys olivaceous. Sensitivity results showed that dot-ELISA is a more sensitive, rapid and simple technique for the protease detection. The minimal detectable amount of protease is about 7 pg in the dot-ELISA test, while 7.8 ng in the indirect ELISA and 6.25 ng in the Western blot respectively. Protease could be detected 2 h after incubation of V. anguillarum in the 2216E liquid medium but enzyme activity was very low at that period. From 6 to 12 h, the amount and enzyme activity of protease increased markedly and reached maximum at stationary phase. Analysis of serum samples periodically collected from the infected flounders showed that after 2 h of infection by V. anguillarum, the pathogenic bacteria could be detected in the blood of the infected flounders but no protease was found. It was 5 similar to 6 h after infection that the protease was detected in blood and then the amount increased as infection advanced. Quantitative detection of protease either incubation in the medium or from the blood of infected flounders could be accomplished in virtue of positive controls of quantificational protease standards ("marker") so that the alterations of protease secretion both in vitro and in vivo could be understood generally. In addition, the indirect ELISA and dot-ELISA were also performed to detect V. anguillarum cells. Results indicated that the sensitivity of indirect ELISA to bacteria cells is higher than that of the dot-ELISA, and that the minimal detectable amount is approximately 10(4) cell/mL in the indirect ELISA, while 10(5) cell/mL in the dot-ELISA.

Application of methyl parathion hydrolase (MPH) as a labeling enzyme

Methyl parathion hydrolase (MPH) is an enzyme that catalyzes the degradation of methyl parathion, generating a yellow product with specific absorption at 405 nm. The application of MPH as a new labeling enzyme was illustrated in this study. The key advantages of using MPH as a labeling enzyme are as follows: (1) unlike alkaline phosphatase (AP), horseradish peroxidase (HRP), and glucose oxidase (GOD), MPH is rarely found in animal cells, and it therefore produces less background noise; (2) its active form in solution is the monomer, with a molecular weight of 37 kDa; (3) its turnover number is 114.70 +/- 13.19 s(-1), which is sufficiently high to yield a significant signal for sensitive detection; and (4) its 3D structure is known and its C-terminal that is exposed to the surface can be easily subjected to the construction of genetic engineering monocloning antibody-enzyme fusion for enzyme-linked immunosorbent assay (ELISA). To demonstrate its utility, MPH was ligated to an single-chain variable fragment (scFv), known as A1E, against a white spot syndrome virus (WSSV) with the insertion of a [-(Gly-Ser)(5)-] linker peptide. The resulting fusion protein MPH-A1E possessed both the binding specificity of the scFv segment and the catalytic activity of the MPH segment. When MPH-A1E was used as an ELISA reagent, 25 ng purified WSSV was detected; this was similar to the detection sensitivity obtained using A1E scFv and the HRP/Anti-E Tag Conjugate protocol. The fusion protein also recognized the WSSV in 1 mu L hemolymph from an infected shrimp and differentiated it from a healthy shrimp.

Selective, peroxidase substrate based "signal-on" colorimetric assay for the detection of chromium (VI)

Due to the potentially adverse effects of the chromium (VI) on the human health and also on the environment, the quantitative determination of Cr(VI) is of particular interest. This work herein reports a facile, selective and rapid colorimetric determination of Cr(VI) based on the peroxidase substrate-2,2'-azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid) diammonium salt (ABTS) as the color developing agent. ABTS, which was usually acted as peroxidase substrate for the enzyme linked immunosorbent assay, is used here for the first time to fabricate the "signal-on" colorimetric Assay for Cr(VI).

Detection of hepatitis B virus markers using a biosensor based on imaging ellipsometry

A biosensor based on imaging ellipsometry (BIE) has been developed and validated in 169 patients for detecting five markers of hepatitis B virus (HBV) infection. The methodology has been established to pave the way for clinical diagnosis, including ligand screening, determination of the sensitivity, set-up of cut-off values (CoVs) and comparison with other clinical methods. A matrix assay method was established for ligand screening. The CoVs of HBV markers were derived with the help of receiver operating characteristic curves. Enzyme-linked immunosorbent assay (ELISA) was the reference method. Ligands with high bioactivity were selected and sensitivities of 1 ng/mL and 1 IU/mL for hepatitis B surface antigen (HBsAg) and surface antibody (anti-HBs) were obtained respectively. The CoVs of HBsAg, anti-HBs, hepatitis B e antigen, hepatitis B e antibody and core antibody were as follows: 15%, 18%, 15%, 20% and 15%, respectively, which were the percentages over the values of corresponding ligand controls. BIE can simultaneously detect up to five markers within 1 h with results in acceptable agreement with ELISA, and thus shows a potential for diagnosing hepatitis B with high throughput.

High prevalence of HIV-1 and hepatitis C virus coinfection among injection drug users in the southeastern region of Yunnan, China

The southeastern region of Yunnan province is a key site for drug trafficking and HIV-1 infection spread from the west of Yunnan and Laos to southeastern China. To investigate the prevalence of HIV-1 infection and hepatitis C virus (HCV) coinfection among injection drug users (IDUs) in southeastern Yunnan, three cohorts of 285 addicts, including 242 IDUs and 43 oral drug users, living in the cities of Gejiu and Kaiyuan and the county of Yanshan were studied. HIV-1 and HCV infections were detected by enzyme-linked immunosorbent assay and/or polymerase chain reaction. Data on the age, sex, risk behavior, drug use history, employment, ethnic background, and marriage status were obtained by interview. The overall prevalence of HIV-1 infection was 71.9%. The rate of HCV coinfection among 138 HIV-1-infected IDUs was 99.3%. Most HIV-infected IDUs were 20 to 35 years old (86.7%) and were ethnic Han (75.9%), suggesting that the epidemic in Yunnan is no longer confined to non-Han ethnic minorities, HIV prevalence in female IDUs (81.2%) was significantly higher than in male IDUs (68.2%) (p <.05). The prevalence of HIV infection reached 68.4% after 1 year of injection drug use. Needle/syringe sharing is the major high risk factor for the spread of HIV-1 and HCV infections. Large-scale educational campaigns are urgently needed to reduce the spread of HIV and HCV infection in these regions.

Detection of microcystins in environmental samples using surface plasmon resonance biosensor

An indirect inhibitive surface plasmon resonance (SPR) immunoassay was developed for the microcystins (MCs) detection. The bioconjugate of MC-LR and bovine serum albumin (BSA) was immobilized on a CM5 sensor chip. A serial premixture of MC-LR standards (or samples) and monoclonal antibody (mAb) were injected over the functional sensor surface, and the subsequent specific immunoreaction was monitored on the BIAcore 3000 biosensor and generated a signal with an increasing intensity in response to the decreasing MCs concentration. The developed SPR immunoassay has a wide quantitative range in 1-100 mu g L-1. Although not as sensitive as conventional enzyme-linked immunosorbent assay (ELISA), the SPR biosensor offered unique advantages: (I) the sensor chip could be reusable without any significant loss in its binding activity after 50 assay-regeneration cycles, (2) one single assay could be accomplished in 50 min (including 30-min preincubation and 20-min BIAcore analysis), and (3) this method did not require multiple steps. The SPR biosensor was also used to detect MCs in environmental samples, and the results compared well with those obtained by ELISA. We conclude that the SPR biosensor offers outstanding advantages for the MCs detection and may be further developed as a field-portable sensor for real-time monitoring of MCs on site in the near future. (C) 2009 Published by Elsevier B.V.

Effects of adrenergic agents on the expression of zebrafish (Danio rerio) vitellogenin Ao1

Teleost vitellogenins (VTGs) are large multidomain apolipoproteins, traditionally considered to be estrogen-responsive precursors of the major egg yolk proteins, expressed and synthesized mainly in hepatic tissue. The inducibility of VTGs has made them one of the most frequently used in vivo and in vitro biomarkers of exposure to estrogen-active substances. A significant level of zebrafish vtgAo1, a major estrogen responsive form, has been unexpectedly found in heart tissue in our present studies. Our studies on zebrafish cardiomyopathy, caused by adrenergic agonist treatment, suggest a similar protective function of the cardiac expressed vtgAo1. We hypothesize that its function is to unload surplus intracellular lipids in cardiomyocytes for "reverse triglyceride transportation" similar to that found in lipid transport proteins in mammals. Our results also demonstrated that zebrafish vtgAo1 mRNA expression in heart can be suppressed by both (x-adrenergic agonist, phenylephrine (PE) and beta-adrenergic agonist, isoproterenol (ISO). Furthermore, the strong stimulation of zebrafish vtgAo1 expression in plasma induced by the beta-adrenergic antagonist, MOXIsylyl, was detected by Enzyme-Linked ImmunoSorbent Assay (ELISA). Such stimulation cannot be suppressed by taMOXIfen, an antagonist to estrogen receptors. Thus, Our present data indicate that the production of teleost VTG in vivo can be regulated not only by estrogenic agents, but by adrenergic signals as well. (c) 2009 Elsevier Inc. All rights reserved.

Effects of gibberellin A(3) on growth and microcystin production in Microcystis aeruginosa (cyanophyta)

Environmental. factors that affect the growth and microcystin production of microcystis have received worldwide attention because of the hazards microcystin poses to environmental safety and public health. Nevertheless, the effects of organic anthropogenic pollution on microcystis are rarely discussed. Gibberellin A(3) (GA(3)) is a vegetable hormone widely used in agriculture and horticulture that can contaminate water as an anthropogenic pollutant. Because of its common occurrence, we studied the effects of GA3 on growth and microcystin production of Microcystis aeruginosa (M. aeruginosa) PCC7806 with different concentrations (0.001-25mg/L) in batch culture. The control was obtained without gibberellin under the same culture conditions. Growth, estimated by dry weight and cell number, increased after the GA3 treatment. GA3 increased the amounts of chlorophyll a, phycocyanin and cellular-soluble protein in the cells of M. aeruginosa PCC7806, but decreased the accumulation of water-soluble carbohydrates. In addition, GA3 was observed to affect nitrogen absorption of the test algae, but to have no effect on the absorption of phosphorus. The amount of microcystin measured by enzyme-Linked immunosorbent assay (ELISA) increased in GA3 treatment groups, but the stimulatory effects were different in different culture phases. It is suggested that GA3 increases M. aeruginosa growth by stimulating its absorbance of nitrogen and increasing its ability to use carbohydrates, accordingly increasing cellular pigments and thus finally inducing accumulation of protein and microcystin. (C) 2007 Elsevier GmbH. All rights reserved.

Detection of cutaneous antibodies in excised skin explants from grass carp, Ctenopharyngodon idella (Valenciennes), immune to Scophthalmus maximus rhabdovirus

This study determined whether cutaneous antibodies were present in excised skin explants of grass carp, Ctenopharyngodon idella, immune to Scophthalmus maximus rhabdovirus (SMRV). Culture fluid from immune skin explants were assayed by indirect enzyme-linked immunosorbent assay (iELISA), Western blot, indirect immunofluorescent assay (IFA) and flow cytometry (FCM). iELISA showed that cutaneous antibody titres were much lower (1:12) than antiserum titres (1:1458) from intraperitoneally immunized grass carp. The phosphoprotein and matrix protein antigens of purified SMRV proteins were recognized by cutaneous antibodies from skin culture fluid using Western blot. The skin culture fluid produced staining signals in viral assembly sites and cytoplasm of SMRV-infected epithelioma papulosum cyprini (EPC) cells by IFA. FCM showed that 4.39% SMRV-infected EPC cells were detected, while non-specific reaction was seen in 2% of control cells. This is the first description of cutaneous antibodies against SMRV in grass carp.

Generation and characterization of monoclonal antibodies against the flounder Paralichthys olivaceus rhabdovirus

Two MAbs (3C7 and 3C9) against flounder Paralichthys olivaceus rhabdovirus (PORV) were generated with hybridoma cell fusion technology and characterized by an indirect enzyme-linked immunosorbent assay, isotype test, Western blot and immunodot analysis and immunofluorescence assay. Isotyping tests demonstrated that both of the two MAbs belonged to IgM subclass. Western blot analysis showed the MAbs reacted with 42, 30, and 22 kDa viral proteins, which were localized within the cytoplasm of PORV-infected grass carp ovary (GCO) cells analyzed by indirect immunofluorescences tests. The MAb 3C7 was also selected at random for detecting virus antigens in the inoculated grass carp tissues by immunohistochemistry assay. Flow cytometry tests showed that at the 36 h postinfection (0.25 PFU/cell), the 23% PORV-infected GCO cells could be distinguished from the uninfected cells with the MAb 3C7. Such MAbs could be useful for diagnosis and potential treatment of viral infection. (C) 2007 Elsevier B.V. All rights reserved.

A novel chemiluminescent immunoassay for microcystin (MC) detection based on gold nanoparticles label and its application to MC analysis in aquatic environmental samples

A novel chemiluminescent immunoassay method based on gold nanoparticles was developed for the detection of microcystins (MCs). The immunoassay included three main steps: indirect competitive immunoreaction, oxidative dissolution of gold nanoparticles, and indirect determination for MCs with Au3+-catalysed luminol chemiluminesent system. The method has a wide working range (0.05-10 mu g L-1, r(2) = 0.9914), the limit of detection was determined to be 0.024 mu g L-1, which is much lower than the World Health Organization's proposed guidelines (1 mu g L-1) for drinking-water. The proposed method was applied to MC analysis in natural water and fish tissue samples, and most results in the proposed method were in agreement with the conventional indirect competitive enzyme-linked immunosorbent assay method, which indicated that the new chemiluminescent immunoassay was sensitive, reliable, and suitable for MC analysis in natural water and fish tissue samples.

Evaluation of estrogenic activities and mechanism of action of perfluorinated chemicals determined by vitellogenin induction in primary cultured tilapia hepatocytes

Perfluorochemicals (PFCs) are emerging persistent organic pollutants (POPs) and are widely present in the environment, wildlife and humans. Recently, reports have suggested that PFCs may have endocrine-disrupting activities. In the present study, we have developed a non-competitive enzyme-linked immunosorbent assay (ELISA) method to investigate estrogenic activities of selected PFCs using vitellogenin (VTG) induction in primary cultured hepatocytes of freshwater male tilapia (Oreochromis niloticus). Cultured hepatocytes were exposed to various concentrations of perfluorooctanyl sulfonate (PFOS), pentadecafluorooctanoic acid (PFOA), 1H, 1H, 2H, 2H-nonafluoro-1-hexanol (4:2 FTOH), 1H, 1H, 2H, 2H-perfluorooctanol (6:2 FTOH) and 1H, 1H, 2H, 2H-perfluoro-1-decanol (8:2 FTOH) for 48h, while 17 beta-estradiol (E2) and 4-nonylphenol (4-NP) were used as positive controls. A dose-dependent induction of VTG was observed in E2-, 4-NP-, PFOS-, PFOA- and 6:2 FrOH-treated cells, whereas VTG levels remained unchanged in the 4:2 FTOH and 8:2 FTOH exposure groups at the concentrations tested. The estimated 48-h EC50 values for E2,4-NP, PFOS, PFOA and 6:2 FTOH were 4.7 x 10(-7), 7.1 x 10(-6), 1.5 x 10(-5), 2.9 x 10(-5) and 2.8 x 10(-5) M, respectively. In the time-course study, significant VTG induction took place at 24 h (E2), 6 It (4-NP), 48 It (PFOS), 48 It (PFOA), 72 It (4:2 FTOH), 12 h (6:2 FTOH), 72 h (8:2 FTOH), and increased further after 96 It of exposure. Co-exposure to binary mixtures of individual PFCs and E2 for 48 It significantly inhibited E2-induced hepatocellular VTG production in a dose-dependent manner except for 4:2 FTOH. The estimated 48-h IC50 (concentration of a compound that elicits 50% inhibition of maximally E2-induced VTG) values for PFOS, PFOA, 6:2 FTOH and 8:2 FTOH were 3.1 x 10(-7), 5.1 X 10(-7), 1.1 X 10(-6) and 7.5 x 10(-7) M, respectively. In order to further investigate the estrogenic mechanism of PFCs, the hepatocytes were co-exposed to binary mixtures of individual chemicals (E2,4-NP, PFOS, PFOA and 6:2 FTOH) and the known estrogen receptor inhibitor tamoxifen for 48 h; tamoxifen significantly inhibited the ability of these chemicals to stimulate vitellogenesis. The overall results demonstrated that PFOS, PFOA and FTOHs have estrogenic activities and that exposure to a combination of E2 and PFCs produced anti-estrogenic effects. The results of the estrogen receptor inhibition assay further suggested that the estrogenic effect of PFCs may be mediated by the estrogen receptor pathway in primary cultured tilapia hepatocytes. (c) 2007 Elsevier B.V. All rights reserved.

Characterization and application of monoclonal antibodies against turbot (Scophthalmus maximus) rhabdovirus

Five monoclonal antibodies (mAbs), 1G8, 1H9, 2D2, 2D3, and 2F5, against Scophthalmus maximus rhabdovirus (SMRV) were prepared. Characterization of the mAbs included indirect enzyme-linked immunosorbent assay, isotyping, viral inhibition assay, immunofluorescence staining of virus-infected cell cultures, and Western blot analysis. Isotyping revealed that 1G8 and 1H9 were of the IgG2b subclass and that the other three were IgM. 2D2, 2D3, and 2F5 partially inhibited SMRV infection in epithelioma. papulosum cyprinid (EPC) cell culture. Western blotting showed that all five mAbs could react with two SMRV proteins with molecular masses of approximately 30 kDa (P) and 26 kDa (M). These two proteins were localized within the cytoplasm of SMRV-infected EPC cells by immunofluorescence assay. Also, progressive foci of viral replication in cell cultures were monitored from 6 to 24 h, using mAb 2D3 as the primary antibody. A flow cytometry procedure was used to detect and quantify SMRV-infected (0.01 PFU/cell) EPC cells with mAb 2D3, and 10.8% of cells could be distinguished as infected 36 h postinfection. Moreover, mAb 2D3 was successfully applied for the detection of viral antigen in cryosections from flounder tissues by immunohistochemistry tests.