Goat Anti-rabbit IgG Research Articles

Chi-Ju-Di-Huang-Wan protects rats against retinal ischemia by downregulating matrix metalloproteinase-9 and inhibiting p38 mitogen-activated protein kinase.

BackgroundRetinal ischemia is a retinal disorder related to retinal vascular occlusion, glaucoma, diabetic retinopathy and age-related macular degeneration. The study aimed to evaluate the protective effects and underlying mechanisms of Chi-Ju-Di-Huang-Wan (CJDHW) against retinal ischemia in rats.MethodsHigh intraocular pressure (HIOP)-induced retinal ischemia was established in Wistar rats by raising their intraocular pressure to 120 mmHg for 60 min with in an eye whose anterior chamber was cannulated with a 30-guage needle adapted to a normal saline bottle through an intravenous line. This ischemic insult was followed by 1 or 7 days of reperfusion. The effects of CJDHW were studied by (i) electroretinogram (ERG); (ii) real-time polymerase chain reaction to determine the retinal mRNA levels of Thy-1 and matrix metalloproteinase-9 (MMP-9); (iii) Western blot analysis to determine the retinal protein levels of B cell lymphoma 2 (Bcl-2), heme oxygenase-1 (HO-1), phosphorylated-p38 mitogen-activated protein kinase (P-p38 MAPK) and MMP-9; (iv) hematoxylin and eosin (HE) staining; (v) fluorogold retrograde labeling; and (vi) terminal deoxynucleotidyl-transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) apoptosis assay. Moreover, after fixation with 4 % paraformaldehyde and 30 % sucrose, the isolated retinas were sectioned and immunolabeled with goat anti-choline acetyltransferase (ChAT) polyclonal antibody, mouse anti-vimentin monoclonal antibody and rabbit anti-glial fibrillary acidic protein (GFAP) polyclonal antibody. The retinal sections were then incubated with rhodamine-conjugated rabbit anti-goat antibody, fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG or FITC-conjugated goat anti-rabbit IgG. A daily oral intake of 3 mL of water (vehicle; Group 2) or CJDHW (2.8 or 4.2 g/kg/day; CJDHW2.8 or CJDHW4.2; Group 3 or 4) was given for 7 consecutive days either before (preischemic drug administration) or after HIOP-induced retinal ischemic injury (postischemic drug administration). In Group 5, an intravitreal injection of 4 μL of 0.5 mM SB203580 (p38 MAPK inhibitor) was performed on the ischemic eye 15 min before retinal ischemia. The control rats received a sham procedure (Group 1) where the saline reservoir was not raised.ResultsThe ischemia-induced changes (Group 2) were significantly modulated by pretreating the rats with 4.2 g/kg/day of CJDHW (Group 4; ERG: P < 0.001 on I/R day 7; HE stain: P < 0.001 on I/R day 7; TUNEL: P = 0.05 on I/R day 7; retrograde labeling: P = 0.007 on I/R day 7; Thy-1 mRNA: P = 0.02; MMP-9 mRNA: P < 0.001; Bcl-2 protein: P = 0.02; HO-1 protein: P = 0.03; P-p38 MAPK protein: P < 0.001; MMP-9 protein: P = 0.02). These modulations included the following features (Group 2 vs. 4), increased ERG b-wave amplitudes (0.38 ± 0.04 vs. 0.81 ± 0.03), increased inner retinal thickness (45.08 ± 2.85 vs. 67.98 ± 5.48 μm), increased ChAT immunolabeling, decreased vimentin/GFAP immunoreactivity, less numerous apoptotic cells in the ganglion cell layer (1.40 ± 0.55 vs. 0.60 ± 0.55), and more numerous retinal ganglion cells (887.73 ± 158.18 vs. 1389.02 ± 53.20). Moreover, increased Thy-1 (0.31 ± 0.15 vs. 0.78 ± 0.32) and decreased MMP-9 mRNA levels were found (4.44 ± 0.84 vs. 1.13 ± 0.34), respectively. Furthermore, the Bcl-2 protein level (0.78 ± 0.08 vs. 1.80 ± 0.34) was increased while the HO-1 (0.99 ± 0.20 vs. 4.15 ± 2.08), P-p38 MAPK (1.12 ± 0.18 vs. 0.57 ± 0.18) and MMP-9 levels were decreased (0.70 ± 0.23 vs. 0.39 ± 0.10). The ischemia-associated increases in P-p38 and MMP-9 protein levels were also attenuated by 0.5 mM SB203580 (P-p38 MAPK: 1.12 ± 0.18 vs. 0.18 ± 0.07, P < 0.001; MMP-9: 0.70 ± 0.23 vs. 0.21 ± 0.07, P = 0.002). This was also the case to the MMP_enzyme activity (Group 2 vs. 4: 5.03 ± 1.57 vs. 1.59 ± 0.47, P = 0.002; Group 2 vs. 5: 5.03 ± 1.57 vs. 1.35 ± 0.41, P = 0.001).ConclusionTreatment of the rats suffering from retinal ischemia with CJDHW inhibited apoptosis, increased antioxidative activity, downregulated MMP-9 and inhibited p38 MAPK.Electronic supplementary materialThe online version of this article (doi:10.1186/s13020-016-0109-6) contains supplementary material, which is available to authorized users.

Chemiluminescence Immunoassay for S-Adenosylhomocysteine Detection and Its Application in DNA Methyltransferase Activity Evaluation and Inhibitors Screening

Aberrant methylation by DNA transferase is associated with cancer initiation and progression. For high-throughput screening of DNA methyltransferase (MTase) activity and its inhibitors, a novel chemiluminescence immunoassay (CLIA) was established to detect S-adenosylhomocysteine (SAH), the product of S-adenosylmethionine (SAM) transmethylation reactions. We synthesized two kinds of immunogens for SAH and characterized the polyclonal antibodies in each group. The antibody with higher titer was used to develop a competitive CLIA for SAH, in which SAH in samples would compete with SAH coated on microplate in binding with SAH antibodies. Successively, horseradish peroxidase labeled goat antirabbit IgG (HRP-IgG) was conjugated with SAH antibodies on the microplate. In substrate solution containing luminol and H2O2, HRP-IgG catalyzed luminol oxidation by H2O2, generating a high chemiluminescence signal. The method could detect as low as 9.8 ng mL(-1) SAH with little cross-reaction (3.8%) to SAM. Since higher DNA MTase activity leads to more production of SAH, a correlation between the chemiluminescence intensity and DNA MTase activity was obtained in the range from 0.1 to 8.0 U/mL of DNA MTase. The inhibition study showed that, in the presence of SAM as methyl donor, Lomeguatrib, 5-Azacytidine, and 5-Aza-2'-deoxycytidine could inhibit the DNA MTase activity with IC50 values of 40.57 nM, 2.26 μM, and 0.48 μM, respectively. These results are consistent with the published studies. The proposed assay does not depend on recognizing methylated cytosines in oligonucleotides (methyl acceptor) and showed the potential as an accessible platform for sensitive detection of DNA MTase activity and screening its inhibitors.

Aggregation-induced emissive nanoparticles for fluorescence signaling in a low cost paper-based immunoassay

Low cost paper based immunoassays are receiving interest due to their fast performance and small amounts of biomolecules needed for developing an immunoassay complex. In this work aggregation-induced emissive (AIE) nanoparticles, obtained from a diastereoisomeric mixture of 1,2-di-(4-hydroxyphenyl)-1,2-diphenylethene (TPEDH) in a one-step top-down method, are characterized through Dynamic Light Scattering (DLS), Scanning Electron Microscopy (SEM), and Zeta potential. By measuring the Zeta potential before and after labeling the nanoparticles with antibodies we demonstrate that the colloidal system is stable in a wide pH-range. The AIE-active nanoparticles are deposited on chitosan and glutaraldehyde modified paper pads overcoming the common aggregation-caused quenching (ACQ) effect. Analyte concentrations from 1000ng and below are applied in a model immunocomplex using Goat anti-Rabbit IgG and Rabbit IgG. In the range of 7.81ng–250ng, linear trends with a high R2 are observed, which leads to a strong increase of the blue fluorescence from the TPEDH nanoparticles.

Development of a lateral flow immunochromatographic assay for rapid detection of Mycoplasma pneumoniae-specific IgM in human serum specimens

Early diagnosis of Mycoplasma pneumoniae (MP) infection is crucial for prompt treatment and good patient outcome. However, serological tests to detect MP rapidly and conveniently are still lacking. This study aimed to use the fluorescent dye Alexa Fluor® 647 as the detection marker to develop a lateral flow immunochromatographic assay (LFIA) for detection of MP-specific IgM in serum specimen. Monoclonal mouse antibody against human IgM (μ-chain specific) and goat anti-rabbit IgG were labeled with Alexa Fluor® 647 (anti-IgM-AF647 and anti-IgG-AF647). A mixture of natural MP antigen and recombinant P1 antigen was coated as the test line (T line) and rabbit IgG was coated as the control line (C line) on a nitrocellulose (NC) membrane. The MP antigens captured IgM-anti-IgM-AF647 complex on the T line. Rabbit IgG captured anti-IgG-AF647 on the C line. The fluorescence intensity on the T line and C line was measured. Sartorius CN140 NC membrane showed higher sensitivity than CN95. The optimal reaction time for the LFIA was 10min. The area under the receiver operating characteristic curve based on 34 MP positive and 166 MP negative serum samples was 0.986 (p<0.001). The cutoff value of T/C area ratio was 0.3830. The LFIA strips did not react with serum from patients infected with non-MP pathogens including influenza viruses and bacteria causing respiratory tract infection. The intra-assay and inter-assay coefficients of variation were between 3.28% and 10.14%. The shelf life was calculated to be 2years at room temperature. The LFIA strips and the commercial EUROIMMUN kit showed consistent results on 372 serum specimens. The overall consistency rate was 96.37% with a Kappa value of 0.842 (p<0.001). The LFIA in the current study may be a sensitive and specific approach to detect early MP infection rapidly and conveniently.

Referencing cross-reactivity of detection antibodies for protein array experiments.

Protein arrays are frequently used to profile antibody repertoires in humans and animals. High-throughput protein array characterisation of complex antibody repertoires requires a platform-dependent, lot-to-lot validation of secondary detection antibodies. This article details the validation of an affinity-isolated anti-chicken IgY antibody produced in rabbit and a goat anti-rabbit IgG antibody conjugated with alkaline phosphatase using protein arrays consisting of 7,390 distinct human proteins. Probing protein arrays with secondary antibodies in absence of chicken serum revealed non-specific binding to 61 distinct human proteins. The cross-reactivity of the tested secondary detection antibodies points towards the necessity of platform-specific antibody characterisation studies for all secondary immunoreagents. Secondary antibody characterisation using protein arrays enables generation of reference lists of cross-reactive proteins, which can be then excluded from analysis in follow-up experiments. Furthermore, making such cross-reactivity lists accessible to the wider research community may help to interpret data generated by the same antibodies in applications not related to protein arrays such as immunoprecipitation, Western blots or other immunoassays.

Identification of biofilms in post-tympanostomy tube otorrhea.

Otitis media is a common problem in the pediatric population. Despite antibiotic therapy, post-tympanostomy otorrhea can be difficult to treat. Biofilms have been shown to play a role in chronic and recurrent otitis media and are implicated in otorrhea. This study investigated both the microbial composition and the presence of biofilm fragments rich in extracellular DNA (eDNA) and the bacterial DNA-binding protein, integration host factor (IHF), in post-tympanostomy tube otorrhea. Clinical samples. Institutional review board approval was obtained, and samples were recovered from pediatric patients with tympanostomy tubes and persistent otorrhea for both microbial culture and biofilm analysis. For biofilm assessment, frozen samples were sectioned and then labeled using a rabbit anti-IHF, which was detected with goat anti-rabbit IgG conjugated to AlexaFluor 594. Samples were then counterstained with 4',6-diamidino-2-phenylindole (DAPI) to detect DNA, and images were captured by inverted light microscopy. Of 15 pediatric otorrhea samples analyzed, nine (60%) contained solids that were positive for labeling of IHF in association with a lattice of eDNA, and 75% yielded positive bacterial cultures. Bacterial culture results included H. influenzae, Methicillin-resistant Staphylococcus aureus, S. pneumoniae, M. catarrhalis, and P. aeruginosa. Positive labeling of otorrhea solids for eDNA and IHF, in combination with microbiological culture results, indicated that biofilms likely played a key role in chronic otorrhea. Moreover, as a known critical structural component of biofilms, these findings suggest that DNABII proteins in association with eDNA may serve as an important therapeutic target in post-tympanostomy tube otorrhea. NA. Laryngoscope, 126:1946-1951, 2016.

Rational design of target biomolecule-responsive hydrogels using biomolecular complex crosslinks

Event Abstract Back to Event Rational design of target biomolecule-responsive hydrogels using biomolecular complex crosslinks Takashi Miyata1, 2*, Akifumi Kawamura1, 2* and Tadashi Uragami1, 2* 1 Kansai University, Department of Chemistry and Materials Engineering, Japan 2 Kansai University, Organization for Research and Development of Innovative Science and Technology, Japan Introduction: Stimuli-responsive hydrogels have attracted considerable attention as smart biomaterials because of their many future opportunities in biomedical fields. However, very few studies on biologically stimuli-responsive hydrogels that undergo changes in the volume in response to a target biomolecule, i.e., biomolecule-responsive hydrogels, have been undertaken in spite of their many potential applications to drug delivery systems and molecular diagnostics. We proposed a novel strategy for designing biomolecule-responsive hydrogels, which utilizes biomolecular complexes as dynamic crosslinks that dissociate and associate in response to a target biomolecule[1]. For example, the bioconjugated hydrogels with antigen-antibody and lectin-glycopolymer complex crosslinks exhibited unique biomolecule-responsive swelling/shrinking changes[2]-[5]. This paper describes target biomolecule-responsive hydrogels that undergo changes in the volume in response to target DNA or two antigens. Materials and Methods: Bioconjugated hydrogels with DNA duplex crosslinks (DNA-crosslinked hydrogels) were prepared by the copolymerization of the DNA duplexes with acryloyl groups, acrylamide (AAm) and N, N'-methylenebisacrylamide (MBAA) using redox initiator. Bioconjugated hydrogels with two antigen-antibody complex crosslinks were designed using rabbit γ-immunoglobulin G (IgG), bovine serum albumin (BSA) and their antibodies (goat anti-rabbit IgG, anti-BSA). As first networks, bioconjugated hydrogels with the IgG antigen-antibody complexes were prepared by copolymerization of the antigen-antibody complexes with acryloyl groups, AAm and MBAA using redox initiators. Then the second networks with BSA antigen-antibody complexes were formed by the similar copolymerization within the first networks to obtain bioconjugated hydrogels having an interpenetrating polymer network (IPN) structure with two antigen-antibody complex crosslinks (IgG-BSA IPN hydrogels). Results and Discussion: The swelling ratio of DNA-crosslinked hydrogels strongly depended on the base sequence of single-stranded (ss) DNA (Fig. 1). The investigations into the crosslinking density revealed that their DNA-responsive swelling was attributed to a decrease in their crosslinking density, which was caused by the dissociation of the DNA duplex crosslinks, in the presence of target ssDNA. The swelling ratio of IgG-BSA IPN hydrogels increased more highly in a buffer solution with both IgG and BSA than in a solution with either IgG or BSA alone (Fig. 2). The multiple antigens-responsive swelling of the IPN hydrogels was caused by dissociation of both IgG and BSA antigen-antibody complex crosslinks in the presence of both IgG and BSA. Furthermore, the IPN hydrogels regulated the permeation of a model drug in response to both IgG and BSA. Conclusion: Bioconjugated hydrogels that underwent changes in the volume in response to target DNA and two proteins were strategically designed using biomolecular complex crosslinks. Such biomolecule-responsive hydrogels have many potential applications as smart biomaterials for self-regulated DDS and biomolecular diagnosis.

Types and Sub Types of Von Willebrand Disease Classified on the Basis of Multimer Analysis

Introduction: Von Willebrand disease is a common genetically inherited bleeding disorder. Types of VWD classified on the basis of coagulation factor assays but it cannot characterize further subtypes or verdict the quality and quantity of VWF. Multimer analysis facilitates the classification and prerequisite parameter to accurate diagnosis of VWD.Objective: To characterize the type and subtypes of VWD type I and II on the basis of laboratory data and analysis of VWF multimer structure.Setting: NIBD Karachi, University of Bonn Germany, Children Hospital & Chughtai Lab Lahore, PAEC Islamabad and HMC Peshawar.Material And Method: VWF Ag, Ricof, FVIII, Glycoprotein Ib and multimer analysis of VWD plasma on 1.6% medium resolution SDS-agarose gel electrophoresis was carried out, and then VWF multimer were transferred on nitrocellulose membrane. Incubated nitrocellulose membrane with using polyclonal rabbit anti-human VWF antibody and Goat anti-rabbit IgG HRP conjugate then protein detected by immune reaction after evaluation of the membrane under luminescence.Results: We studied 57 index patients, 2(3.5%) type I, 4(7.01%) type II, 3(5.26%) unclassified and 48(15.78%) were type III vWD. In type I,II, unclassified and III mean vWF Ag was 52.28, 35.15, 54.5 , 5.7 IU/dl, vWF RCo: 19.5, 18, 38.8, 7.22 IU/dl, vWF Ag/ RCof: 0.655, 0.393, 0.74, 0.55, FVIII 69, 25.7, 61.7, 7.22 IU/dl and GpIb: 27.25, 19.8, 39.8, 4.1. Significant loss of large multimers in type II while multimers were subtle in 2 cases, normal pattern in type I and entirely absence of multimers pattern in case of type III observed.Conclusion: Types and subtypes of VWD classified on the basis of concentration and distribution of vWF multimer by functional and immunological assays but single test is not enough for appropriate diagnosis. We need molecular genetics for von Willebrand disease for further diagnosis. DisclosuresOldenburg:SOBI: Consultancy.

Platform Technologies for Directly Reconstructing 3D Living Biomaterials.

Bio-electrospraying and cell electrospinning is explored for reconstructing living biomaterials for regenerative biology and medicine. The investigations carried out in this study demonstrate these approaches as platform biotechnologies for tissue reconstruction for repair, replacement, and rejuvenation of damaged and/or ageing tissues and/or organs.

Actinin-4 Governs Dendritic Spine Dynamics and Promotes Their Remodeling by Metabotropic Glutamate Receptors

Dendritic spines are dynamic, actin-rich protrusions in neurons that undergo remodeling during neuronal development and activity-dependent plasticity within the central nervous system. Although group 1 metabotropic glutamate receptors (mGluRs) are critical for spine remodeling under physiopathological conditions, the molecular components linking receptor activity to structural plasticity remain unknown. Here we identify a Ca(2+)-sensitive actin-binding protein, α-actinin-4, as a novel group 1 mGluR-interacting partner that orchestrates spine dynamics and morphogenesis in primary neurons. Functional silencing of α-actinin-4 abolished spine elongation and turnover stimulated by group 1 mGluRs despite intact surface receptor expression and downstream ERK1/2 signaling. This function of α-actinin-4 in spine dynamics was underscored by gain-of-function phenotypes in untreated neurons. Here α-actinin-4 induced spine head enlargement, a morphological change requiring the C-terminal domain of α-actinin-4 that binds to CaMKII, an interaction we showed to be regulated by group 1 mGluR activation. Our data provide mechanistic insights into spine remodeling by metabotropic signaling and identify α-actinin-4 as a critical effector of structural plasticity within neurons.

PP09.11 – 2460: Colocalization of HIF1 alpha and abnormal mitochondrial accumulations in muscle fibers in different neuromuscular diseases

Objective We perform the comparative study of immunohistochemical distribution of mitochondrial and hypoxia markers in abnormal mitochondrial clusters in striated muscle fibers of patients with myopathies. Such clusters, accordingly to recent data obtained by means of electron microscopy, play compensatory role in muscle tissue in different neuromuscular diseases. However, the regulatory impact's nature and the interaction with hypoxia are still unclear. Methods Fluorescence immunohistochemistry was performed. Primary mouse mitochondria monoclonal antibody, biotin conjugate (clone MTCO2) and Alexa Fluor 488 goat anti-mouse IgG as secondary antibody were used for mitochondria visualization. Visual image of HIF1 alpha was achieved by using primary rabbit polyclonal antibodies to HIF1 alpha and Alexa Fluor 555 goat anti-rabbit IgG secondary antibody. Paraffin embedded muscle tissue section slides obtained from 25 patients with mitochondrial myopathies, muscular dystrophies (DMD, merosinopathy, calpainopathy) and congenital myopathies (multicore, nemaline) were stained. Evaluation of the products is carried out by using The EVOS® FL Imaging System. Results Mitochondria and abnormal mitochondrial accumulations were visualized in subsarcolemmal zones of some muscle fibers using fluorescent immunohistochemistry. In the same muscle fibers we also observed increased expression HIF1 alpha; its localization matched with location of detected mitochondrial clusters, resulted in fluorescent lights overlapping and color change of subsarcolemmal zones luminescence. In the cases of muscular dystrophies it was observed that severity of pathological changes in muscles positively correlated with expression intensity of HIF1 alpha in the absence of mitochondrial marker expression. Conclusion The obtained results suggest that there is a colocalization of increased intracellular marker of hypoxia HIF1 alpha concentration and abnormal mitochondrial accumulations in different neuromuscular diseases. It can be explained by such clusters emerging in the areas of intracellular hypoxia. So hypoxia can be an important factor that stimulates mitochondrial proliferation, which may be important for tissue adaptation to different pathological conditions.

P161 – 2452: HIF1 alpha abnormal distribution in muscle fibers in the cases of nemaline myopathy

Objective The comparative study of immunohistochemical distribution of hypoxia marker HIF1 alpha and mitochondrial markers in striated muscle fibers of patients suffering from nemaline myopathy (NM) was performed for evaluation of tissue hypoxia's role in the pathogenesis of NM, characterized by rod-shaped structures, which originated from the Z-disks of the sarcomeres. Methods Fluorescence immunohistochemistry was performed. Primary mouse mitochondria monoclonal antibody, biotin conjugate (clone MTCO2) and Alexa Fluor 488 goat anti-mouse IgG as secondary antibody were used for mitochondria visualization. Visual image of HIF1 alpha was achieved by using primary rabbit polyclonal antibodies to HIF1 alpha and Alexa Fluor 555 goat anti-rabbit IgG as secondary antibody. Paraffin embedded muscle tissue section slides obtained from 3 NM patients were stained. Evaluation of the products is carried out by The EVOS® FL Imaging System. Results Mitochondria and abnormal mitochondrial accumulations were visualized in subsarcolemmal zones of some muscle fibers. In the same muscle fibers we observed increased expression of HIF1 alpha; its localization matched with location of detected mitochondrial clusters, resulted in fluorescent lights overlapping and color change of subsarcolemmal zones luminescence. Furthermore, the most intensive fluorescence and clearly delineated HIF1 alpha clusters were observed in the areas of rods accumulation, pointing pronounced tissue hypoxia in these regions. Conclusion While the diffuse subsarcolemmal zones of tissue hypoxia and increased number of mitochondrial clusters are often found in other myopathies, location of HIF1 alpha clusters in the rods areas is very unusual. Due to their brightness, HIF1 alpha clusters can be used in the quantification of muscular disorders severity at low magnification of fluorescence microscopy. No evidence whether these changes are secondary to rods accumulation or vice versa was found in the literature. In this way immunomorphological characterisation of energy muscle tissue metabolism of patients suffering from NM is of the current interest.

Grb10 characterization in bovine cumulus oocyte complexes from different follicle sizes

The objective of this study was to investigate the mRNA expression and protein localization of Grb10 gene in bovine cumulus-oocyte complexes (COCs) from different follicle sizes. Firstly, it was investigated the mRNA expression to correlate with maturation rates. COCs from follicles at 1-3, 4-6, 6-8 and &amp;gt;8mm were used to evaluate Grb10 gene expression by qRT-PCR assay and nuclear maturation rates. It was observed that more competent oocytes (from follicles at 6-8 and &amp;gt;8mm; P&amp;gt;0.05), had lower Grb10 mRNA expression levels when compared to the oocytes from follicles at 1-3 and 4-6mm (P&amp;gt;0.05). After it was performed an immunofluorescence analysis in COCs from different follicle sizes (1-3, 4-6, 6-8 and &amp;gt;8mm) to investigate Grb10 protein localization. Samples were incubated with primary antibody: Polyclonal rabbit anti-Grb10 (1:100). Primary antibody was detected using goat anti-rabbit IgG antibody conjugated with Alexa Fluor 488 (1:500). Positive fluorescence signal was detected in all analyzed samples but less evident in COCs from largest follicles. These results characterized Grb10 gene in bovine COC and provide evidences for its involvement during oocyte molecular maturation.

Antibody conjugates bispecific for intercellular adhesion molecule 1 and allergen prevent migration of allergens through respiratory epithelial cell layers

Antibody conjugates bispecific for intercellular adhesion molecule 1 and allergen prevent migration of allergens through respiratory epithelial cell layers

An in-house indirect immunofluorescence test for ensuring that the cell bank is contamination free

Background: Cross-contamination between cells is a usual mistake at cell culture laboratories and cell banks worldwide. MRC5 diploid cell and Rk-13, Vero and Hela continuous cell lines are used in different stages of human viral vaccines propagation at Razi Vaccine and Serum Research Institute of Iran. However diploid and continuous cells are propagated at separated cell culture laboratory and continuous cells can contaminate MRC5 diploid cells. Therefore, a sensitive test is needed. World Health Organization recommends few molecular and cellular techniques to cell characterization. Materials and Methods: The present study was therefore designed to set up an indirect immunofluorescence (IIF) test as follows: Polyclonal anti-MRC5 cell and anti-rabbit antibodies were prepared in rabbit and goat, respectability. Anti-rabbit IgG was purified using protein G affinity chromatography, conjugated to fluorescein isothiocyanate (FITC), and then further purified to remove unbound FITC using Sephadex G 25 chromatography. Using double immunodiffusion assay, purification of homemade anti-rabbit IgG was asssayed by observation of a single arch. Results: The titer of homemade FITC conjugated goat anti rabbit IgG was measured 1/16 vs 1/8 of commercial type. Fluorescein/protein molar ratio of local made fluorescein goat anti-rabbit IgG was measured 3.44 and its protein concentration and FITC concentration were determined 1.198 mg/ml and 0.01 mg/ml, respectively. Conclusion: Moreover, homemade IIF test showed 100% intra-laboratory reproducibility. Purity of three batches of MRC5 working seed cell was verified using in- house IIF test and no contamination to continuous cell lines was found .

AMPK Subcellular Localisation in &amp;lt;i&amp;gt;Dictyostelium discoideum&amp;lt;/i&amp;gt;

The Dictyostelium discoideum AMP-activated protein kinase (AMPK) snfA subcellular localization was studied in AX2 and stable HPF strains by use of AMPK antipeptide antibody and goat anti-rabbit Alexa-Flour 488-conjugated IgG antibody. The AMPK exhibited cytosolic localization patterns and uniform focalised concentrations in wild type and the strains alike. Constitutive activation and attenuation of the α subunit expression did not affect subcellular distribution of AMPK. However, snfA expression was more intense in strains in which AMPK was constitutively active compared with the AX2 but lesser in attenuation strains. The localisation of the snfA reinforced the putative standing that it had a plethora of cytoplasmic functions. Moreover, the oxidative cellular function would require a ubiquitous system and might coordinately regulate responses to metabolic requirements. Furthermore, the developmental phases of the life cycle would support the cytosolic localization; and since organelles were potentially reorganized or removed entirely during the transition from vegetative living to fruiting body morphology. This study provided insight into the subcellular distribution of AMPK in Dictyostelium discoideum. We demonstrated that AMPK localization was steady in AX2 and derived strains whether constitutively active or anti-sense inhibited depicting extreme genetic states.

A Model System for Concurrent Detection of Antigen and Antibody Based on Immunological Fluorescent Method

This paper describes a combined antigen/antibody immunoassay implemented in a 96-well plate using fluorescent spectroscopic method. First, goat anti-human IgG was used to capture human IgG (model antigen); goat anti-human IgG (Cy3 or FITC) was used to detect the model antigen; a saturating level of model antigen was then added followed by unlabelled goat anti-human IgG (model antibody); finally, Cy3 labelled rabbit anti-goat IgG was used to detect the model antibody. Two approaches were applied to the concomitant assay to analyze the feasibility. The first approach applied FITC and Cy3 when both targets were present at the same time, resulting in 50 ng/mL of the antibody detection limit and 10 ng/mL of antigen detection limit in the quantitative measurements of target concentration, taking the consideration of FRET efficiency of 68% between donor and acceptor. The sequential approach tended to lower the signal/noise (S/N) ratio and the detection of the model antigen (lower than 1 ng/mL) had better sensitivity than the model antibody (lower than 50 ng/mL). This combined antigen/antibody method might be useful for combined detection of antigens and antibodies. It will be helpful to screen for both antigen and antibody particularly in the situations of the multiserotype and high-frequency mutant virus infections.

171 EXPRESSION OF THE OVULATION-INDUCING FACTOR/NERVE GROWTH FACTOR HIGH AFFINITY RECEPTOR IN THE OVARIES OF COWS DURING THE PERIOVULATORY PERIOD

Ovulation-inducing factor/nerve growth factor (OIF/NGF) influences ovulation and corpus luteum (CL) size and function in camelids and, remarkably, cows. The ovulation effect in induced ovulators is mediated at the hypothalamo-pituitary-axis, but the site and mechanism of action of the luteotrophic effect is unknown. This mechanism may be an important aspect of OIF/NGF in spontaneous-ovulators. The objective of this experiment was to detect changes in expression of the high affinity OIF/NGF receptor (TrkA) in the ovary during the periovulatory period in cattle. Cows (n = 14) were examined daily by transrectal ultrasonography to determine the day of ovulation (Day 0), and were assigned randomly to be unilaterally ovariectomized on Day 2, 4, 6, or in the preovulatory period before or after the LH surge. Cows assigned to preovulatory groups were given a luteolytic dose of prostaglandin when the dominant follicle of the second follicular wave was ≥10 mm and growing. Cows assigned to the pre-LH group were ovariectomized 24 h after prostaglandin treatment. Cows assigned to the post-LH group were given LH 24 h after prostaglandin treatment and were ovariectomized 18–20 h later. Cows were allowed to rest for one complete interovulatory interval and re-assigned to a different day-group on which the remaining ovary was removed (n = 4 to 5 ovaries/day-group). Ovaries were fixed in paraformaldehyde, and 5-μm sections of ovarian tissue representing the dominant follicle, largest subordinate follicle, and the CL were treated for enzymatic antigen retrieval and blocked in 1% BSA for 1 h. Slides were incubated overnight with the primary antibody (rabbit anti-human TrkA) and for 2 h with the secondary antibody (goat anti-rabbit IgG). Slides were evaluated by fluorescence microscopy. Images of 3 arbitrarily chosen fields (40×) were captured for each structure for each day-group, and the average proportion of immunoreactive cells in the theca layer and luteal tissue was estimated using ImageJ software (National Institutes of Health, Bethesda, MD, USA). The intensity of individual cell immunofluorescence was also scored as no reaction (0), faint (1), weak to moderate (2), or strong (3). Data were compared among groups by two-way ANOVA. The proportion of immunoreactive cells was higher in the dominant follicle than in the subordinate follicle or the CL (P &lt; 0.001); no effect of day-group or interaction was detected. The intensity score increased from faint on Day 2 to strong on Day 6, and remained strong in the pre- and post-LH groups. A discernible pattern of change in intensity score was not evident for the subordinate follicle or the CL. The greater proportion of TrkA positive cells and greater immunoreactive intensity in the maturing dominant follicle support the hypothesis that the luteotrophic effect of OIF/NGF in cattle is a result of a local increase in the expression of TrkA in the theca layer of the dominant follicle. Research was supported by the Natural Sciences and Engineering Research Council of Canada.

182 IMMUNOHISTOCHEMISTRY LOCALIZATION OF GROWTH DIFFERENTIATION FACTOR 9 (GDF-9) AND BONE MORPHOGENETIC PROTEIN 15 (BMP-15) IN CANINE FOLLICLES THROUGHOUT ESTRUS CYCLE

Among different developmental proteins, growth differentiation factor-9 (GDF-9) and bone morphogenic protein 15 (BMP-15), members of the transforming growth factor β (TGFb) superfamily, have been described in many species as key mediators for folliculogenesis. The aim of this study was to analyse in canines the presence and localization of GDF-9 and BMP-15 proteins in follicle cells and oocytes during follicle development. To determine the immunolocalization of GDF-9 and BMP-15, sections of 4% paraformaldehyde in PBS ovaries obtained from 15 bitches at different stage of oestrous cycle (proestrus-oestrus n = 5; diestrus n = 5; anestrus n = 5) were dehydrated (xylene–ethanol solutions) and embedded into paraffin blocks. Individual paraffin sections (5 µm) were inactivated with methanol/H2O2. Sections were blocked at 37°C in the peroxidase reagent and incubated with rabbit anti-human GDF-9 (1 : 200) or goat anti-human BMP-15 (1 : 150) antibodies overnight at 4°C and with secondary antibodies goat anti-rabbit IgG and donkey anti-goat IgG, both HRP conjugated. Previously negative control sections received rabbit serum. Sections were stained with 1 mg mL–1 3,3-diaminobenzidine (DAB) and counterstained with haematoxylin. All sections were examined at magnification × 200 using an IX71 inverted microscope (Olympus, Center Valley, PA, USA), with an IX2-RFA lamp and a ProgRes CapturePro camera (Jenoptik AG, Jena, Germany). The same exposure time was applied to all samples and digital photos were taken and subsequently analysed. The intensity of immunostaining in both oocyte and follicular cells of each follicle was evaluated with Image J version 1.45s software (National Institutes of Health, Bethesda, MD, USA). The images were transformed to grey scale and the corrected total cell mark was calculated by subtracting the mean staining of background reading to the integrated density of each sample. The results showed that samples of all slices contained primordial, primary, secondary and pre-antral follicles as well as different sizes of antral follicles showed positive GDF-9 and BMP-15 immunoreactions in the oocyte cytoplasm and in the follicles cells. No such labelling was found in the negative controls. Follicle sizes differed in GDF-9 and BMP-15 immunoreactions according to oestrous cycle. The pattern of GDF-9 immunostaining in proestrus, oestrus, and diestrus was more intense within preantral rather than antral follicles/oocytes, whereas BMP-15 immunoreaction was prominent mainly in proestrus stage increasing to antral stage. These preliminary results indicated temporal pattern of GDF-9 and BMP-15 in canine ovarian follicles which might play important roles in follicular/oocyte development and ovulation during oestrous cycle. Research was funded by FONDECYT Grant 1140658.

263 ADDING RESVERATROL TO THE EXTENDER AFFECTS PROTEIN TYROSINE PHOSPHORYLATION IN BUFFALO SPERM

Cryopreservation induces remarkable capacitation- like changes in buffalo sperm (Kadirvel et al. 2011 Theriogenology 75, 1630–1639; Elkhawagah et al. 2014 J. Buffalo Sci. 3, 3–11). The aim of this study was to evaluate the effect of resveratrol, a natural phytoalexin with antioxidant properties, on capacitation status of frozen-thawed buffalo sperm, assessed by protein tyrosine phosphorylation assay. Three ejaculates from four bulls were used for the trial. Each ejaculate was split into two equal aliquots and diluted at 37°C with BioXcell extender containing no supplement (control) or 50 µM resveratrol, to a final concentration of 30 × 106 spermatozoa per mL. After 4 h at 4°C, straws were frozen in an automated system. Immediately after thawing, sperm motility was evaluated by phase-contrast microscopy, sperm viability by Trypan Blue/Giemsa staining and localization of phosphotyrosine proteins by indirect immunofluorescence, as described Kadirvel et al. (2011 Theriogenology 75, 1630–1639). Briefly, after thawing, semen was centrifuged (300 × g, 10 min), fixed in 2% formaldehyde for 1 h at 4°C, and sperm pellets were incubated overnight at 4°C in modified phosphate buffer saline containing 2% BSA. After centrifugation, sperm pellets were resuspended, diluted 1 : 10 in mPBS, smeared onto slides, air-dried, and permeabilized with absolute ethanol for 5 min. Then, spermatozoa were incubated with rabbit anti-phosphotyrosine primary antibody for 1 h at room temperature in a humid chamber. Slides were incubated with secondary antibody, FITC conjugated goat anti-rabbit IgG, for 1 h in a dark humid chamber at room temperature and mounted with 90% glycerol. A total of 100 spermatozoa were screened per slide and classified as described (Luño et al. 2013 Reproduction 146, 315–324): pattern A: uniform fluorescence over the entire acrosome (low capacitation level); pattern E: signal in the equatorial segment (medium capacitation level); and pattern EA: fluorescence at both equatorial and acrosomal areas (high capacitation level). Data were analysed by chi-square. There were no significant differences between control and treated groups for sperm motility (50.0 and 55.0%, respectively) or viability (77.4 and 72.9%). The percentage of sperm cells that did not exhibit fluorescence was very low (2.4 and 4.3% in the control and resveratrol groups, respectively). In resveratrol-treated group, pattern E was higher than the control (4.9 and 2.0%; P &lt; 0.01). More interestingly, in the resveratrol-treated group, an increased percentage of sperm with pattern A (79.6 and 52.5%) and a decreased percentage of sperm with pattern EA (12.2 and 43.1%) were recorded. Based on decreased sperm with a high capacitation level (EA pattern) and increased sperm with low capacitation level (A pattern) at thawing, we concluded that adding resveratrol to semen extender before cryopreservation of buffalo was beneficial.