Goat Polyclonal Antibody Research Articles

Fibronectin type III domain-containing protein 5 (FNDC5)-like immunoreactivity and mRNA abundance in domestic animal tissues

Irisin is a 112-amino acid peptide hormone that is cleaved from fibronectin type III domain-containing protein 5 (FNDC5), a type I transmembrane protein abundantly found in muscle tissue. Irisin is a putative mediator of the benefits of exercise, neuroprotection, bone growth, and cardiac health. However, few studies have focused on irisin in domestic animals. Further, whether processed irisin is detectable in domestic animal tissues remains uncertain. To address this, we determined FNDC5 mRNA and protein concentration in anatine (duck) and porcine (pig) skeletal muscle, and in equine (horse), swine, and anatine serum samples. RT-PCR analysis identified FNDC5 mRNA in all pig and duck skeletal muscle samples. An approximately 25 kDa band representing FNDC5 was detected in both pig and duck skeletal muscle. Fluorescence immunohistochemistry using a rabbit monoclonal FNDC5/irisin primary antibody and a goat polyclonal anti-rabbit secondary antibody localized FNDC5/irisin-like immunoreactivity in both the glandular and muscular regions of pig stomach. FNDC5/irisin-like immunoreactivity was also identified in horse, pig, and duck serum using a multispecies irisin ELISA. The average values of irisin-like immunoreactivity were 13.7 (duck), 15.4 (horse), and 7.0 (pig) ng/mL in samples tested. Our results support the presence of irisin precursor in several domestic animals. Processed irisin, however, was not detectable. Further studies are required to validate reliable tools to detect and quantify processed irisin in domestic animals.

A Polyclonal Antibody against a Burkholderia cenocepacia OmpA-like Protein Strongly Impairs Pseudomonas aeruginosa and B. multivorans Virulence.

Despite advances in therapies, bacterial chronic respiratory infections persist as life-threatening to patients suffering from cystic fibrosis (CF). Pseudomonas aeruginosa and bacteria of the Burkholderia cepacia complex are among the most difficult of these infections to treat, due to factors like their resistance to multiple antibiotics and ability to form biofilms. The lack of effective antimicrobial strategies prompted our search for alternative immunotherapies that can effectively control and reduce those infections among CF patients. Previous work from our group showed that the anti-BCAL2645 goat polyclonal antibody strongly inhibited Burkholderia cenocepacia to adhere and invade cultured epithelial cells. In this work, we showed that the polyclonal antibody anti-BCAL2645 also strongly inhibited the ability of P. aeruginosa to form biofilms, and to adhere and invade the human bronchial epithelial cell line CFBE41o-. The polyclonal antibody also inhibited, to a lesser extent, the ability of B. multivorans to adhere and invade the human bronchial epithelial cell line CFBE41o. We also show that the ability of B. cenocepacia, P. aeruginosa and B. multivorans to kill larvae of the Galleria mellonella model of infection was impaired when bacteria were incubated with the anti-BCAL2645 antibody prior to the infection. Our findings show that an antibody against BCAL2645 possesses a significant potential for the development of new immunotherapies against these three important bacterial species capable of causing devastating and often lethal infections among CF patients.

A-012 Novel Method for Determining the Concentration of Total N-terminal Pro-hormone BNP (NT-proBNP) in a Sample with Mass Spectrometric Detection

Abstract Background NT-proBNP, the N-terminal fragment of the hormone B-type natriuretic peptide (BNP), is a useful clinical biomarker for diagnostic and prognostic evaluation of heart failure (HF). Recent studies show that commercially available NT-proBNP assays underestimate concentrations of NT-proBNP due to the usage of antibodies targeting the glycosylated central region of the NT-proBNP molecule. With this, a clinical need arises to establish a robust and reliable assay method which can measure the total amount of NT-proBNP, independent of the glycosylation status of the molecule. Methods To determine the concentration of NT-proBNP in patient samples, a known quantity of labelled NT-pro-BNP was added to each sample as standard. The capture of the analyte (native and standard) was performed by Dynabeads™ M-270 Epoxy beads coated with polyclonal goat antibodies, targeting non-glycosylated sites of the NT-proBNP molecule. Magnetic beads were added to the sample and incubated overnight. The isolated analytes were trypsinated directly on the beads after treatment for denaturation by unfolding and oxidation/alkylation. The reaction was stopped by addition of formic acid and the tryptic peptides were cleaned on C18 SPE columns, dried by SpeedVac and resuspended in acetonitrile for separation and analysis on LC-MS/MS (Agilent 1200 Series LC/MS system coupled to an Agilent G6490 triple quadrupole mass spectrometer). Results The Gentian LC-MS/MS validation was conducted including five studies such as Linearity, LoQ, Trueness, Precision and Carry-Over. All studies passed our defined acceptance criteria. To elucidate how glycosylation influences measurements of NT-proBNP, 81 plasma and serum samples covering a concentration range from 100 ng/L to 25 000 ng/L were measured by the Gentian LC-MS/MS method and the Roche Elecsys® proBNP I assay. Within the clinically relevant range of 100–2000 ng/L, the results between both methods exhibit a linear correlation of R2 = 0.97 and show that the commercial assay consistently underestimates the concentration of NT-proBNP by an average factor of 4.3 (3.5–5.8). Furthermore, results reveal that the concentration measurements between the commercial assay and the LC-MS/MS method converge at higher NT-proBNP concentrations. Method comparison was repeated using the Siemens ADVIA Centaur® and the Siemens IMMULITE Immunoassay systems, confirming the results of underestimation of NT-proBNP in clinically relevant areas. For method robustness investigations, the LC-MS/MS method was set up and used at two geographically different sites by different operators, using two different mass spectrometry systems (Agilent 1200 with triple quadrupole and the nano-LC-ESI-Orbitrap from Thermo Fisher Scientific), resulting in CVs from 13% to 2% for NT-proBNP concentrations ranging from 220 ng/L to 9700 ng/L. Conclusion Gentian AS presents a robust and reliable mass spectrometry method, capable of detecting the total amount of NT-proBNP in a sample, independent of its degree of glycosylation. This method is suitable for serving either as a reference method for calibrating and ensuring the accuracy of existing methods, harmonization of existing methods, or as a novel method in clinical chemistry for accurately determining the concentration of total amount of NT-proBNP.

Visualisation of kupffer cells in the rat liver with poly- and monoclonal antibodies against microglial-specific protein Iba-1

BACKGROUND: A necessary attribute of any study, which is related to the cell biology of various structural components of the digestive tract, is the usage of modern immunohistochemical methods. One of the most difficult objects are resident liver macrophages, or Kupffer cells. This determines the high significance to develop a reliable approach for Kupffer cells visualization.
 AIM: The aim of the study was to develop a protocol for the immunohistochemical research of Kupffer cells in rat liver using two types of primary antibodies against Iba-1, and to analyze the advantages and disadvantages of this approach, taking into account the use of zinc-ethanol-formaldehyde as a fixative.
 MATERIALS AND METHODS: The study was carried out on liver samples of adult Wistar rats (n = 5). Goat polyclonal and rabbit monoclonal antibodies against calcium-binding protein Iba-1 were used for the ligh microscopy immunohistochemistry assay of resident liver macrophages.
 RESULTS: Using two types of antibodies it was shown by quantitative analysis that rabbit monoclonal antibodies most completely reveal Iba-1-immunopositive structures compared to goat polyclonal antibodies. Fixation with zinc-ethanol-formaldehyde made it possible to reveal Iba-1-immunopositive cells in all studied rat liver samples.
 CONCLUSIONS: Iba-1 immunohistochemistry using rabbit monoclonal antibodies was considered as the most optimal immunohistochemical approach. Fixation with zinc-ethanol-formaldehyde preserves the antigenic epitopes and allows the effective use of different antibodies.

The Study of Type I Collagen by Immunoblotting in Samples of Bone-Plastic Biomaterials

The type I collagen was studied in samples of two types of osteoplastic materials produced in the Biotech Research Institute of the Samara State Medical University using immunoblotting. The demineralized samples used in the work were compact bone powder and crushed material of human cancellous bone tissue. Collagen and its polypeptides were separated in a 5% polyacrylamide gel with 3.6 M urea according to the method of Hayashi and Nagai (1979). The advantage of the method is the separation under these conditions of type I and III collagen, as well as the α1(I) and α2(I) chains of type I collagen. Immunoblotting was carried out by diffusion method according to the method of Towbin et al. (1979) using nitrocellulose membranes (Santa Cruz, USA). Primary goat polyclonal antibodies to denatured collagen, 1:500 dilution (Millipore) were used. Peroxidase-conjugated secondary antibodies (mouse vs. goat), 1:80000 dilution (Sigma) were used also. It has been established that the bulk of the compact bone protein is localized between the α1- and α2-fractions of collagen. In samples of cancellous bone tissue, a molecular reduction of the protein is noted. Protein macromolecules with a gradually decreasing molecular weight and low molecular weight polypeptides migrating in the gel with a wide front up to the indicator line are detected. Due to the low specificity of osteoblast integrins in regenerating bone tissue, collagen polypeptides, as well as protein molecules retained in implants, can act as inducers of synthetic processes occurring in osteoblast nuclei. Protein fragmentation products in the implant can act as signaling molecules that trigger cascades of enzymatic reactions and intracellular signaling pathways.

MP11-03 FALSE POSITIVE PREGNANCY TESTS AMONG PEDIATRIC PATIENTS WITH AUGMENTATION ENTEROCYSTOPLASTIES

You have accessJournal of UrologyCME1 May 2022MP11-03 FALSE POSITIVE PREGNANCY TESTS AMONG PEDIATRIC PATIENTS WITH AUGMENTATION ENTEROCYSTOPLASTIES Zoë Baker, Arthi Hannallah, Hannah Dillon, Yuding Wang, and Evalynn Vasquez Zoë BakerZoë Baker More articles by this author , Arthi HannallahArthi Hannallah More articles by this author , Hannah DillonHannah Dillon More articles by this author , Yuding WangYuding Wang More articles by this author , and Evalynn VasquezEvalynn Vasquez More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002533.03AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: A high number of false positive pregnancy test results have been self-reported among patients with augmentation enterocystoplasties using over-the-counter (OTC) urine pregnancy tests. We sought to determine the accuracy of OTC pregnancy tests among pediatric patients who had previously undergone augmentation enterocystoplasties. METHODS: Patients at a tertiary care pediatric hospital who had previously undergone augmentation enterocystoplasties or Mitrofanoff appendicovesicostomies only were prospectively recruited. Urine samples were obtained and two OTC pregnancy tests, one point of care testing (POCT) urine pregnancy test, and urinalysis (UA) were completed. All pregnancy tests qualitatively assessed human chorionic gonadotropin (hCG). OTC Test A was a sandwich immunoassay employing monoclonal anti-β-hCG, goat anti-rabbit, anti-α-hCG, and rabbit Immunoglobulin G (IgG) antibodies and using chromatography to separate bound and free colored label. OTC Test B was a lateral flow immunoassay dipstick containing mouse monoclonal anti-β-hCG antibody colloidal gold conjugate pre-dried on a pad, mouse monocolonal anti-α-hCG antibodies, and goat anti-mouse IgG. The POCT test employed mouse monoclonal anti-hCG antibodies and goat polyclonal anti-hCG antibodies. RESULTS: Among 14 pediatric patients who had undergone augmentation cystoplasty, 12 (85.7%) had positive pregnancy test results on OTC Test A, including all 9 females and 60% of males (n = 3/5; Table 1). No patients had positive pregnancy test results on OTC Test B or the POCT pregnancy test (Table 1). No false positive OTC pregnancy test results occurred among 4 patients with Mitrofanoff appendicovesicostomies only. Among patients with enterocystoplasties, UA results indicated that the majority had at least trace levels of protein, blood, and leukocytes, most tested positive for nitrite, and mean urine pH was 6.7 (±0.5; Table 1). CONCLUSIONS: Preliminary findings indicate that certain OTC pregnancy tests qualitatively assessing hCG in urine may be inaccurate among patients who have previously undergone augmentation enterocystoplasties. Female patients of reproductive age should be counseled regarding appropriate pregnancy testing options to avoid unplanned pregnancy scares or concerns about ectopic pregnancies. Source of Funding: The Long Road Foundation © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e157 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Zoë Baker More articles by this author Arthi Hannallah More articles by this author Hannah Dillon More articles by this author Yuding Wang More articles by this author Evalynn Vasquez More articles by this author Expand All Advertisement PDF DownloadLoading ...

Development of a PTHrP chemiluminescent immunoassay to assess humoral hypercalcemia of malignancy

BackgroundParathyroid hormone related peptide (PTHrP) measurements are helpful in the evaluation and management of individuals suspected of humoral hypercalcemia of malignancy (HHM). AimTo develop a chemiluminescent assay for PTHrP quantitation, establish reference intervals, and evaluate its clinical performance. MethodPTHrP 1–86 was measured using a polyclonal rabbit antibody (capture) and an acridinium ester labeled goat polyclonal antibody for chemiluminescent detection. ResultsAssay imprecision was < 9% (intra-assay) and < 15% (inter-assay). The analytical measuring range was 0.16–50.5 pmol/L. No significant cross-reactivity was observed for PTH (1–84), PTHrP (107–139), and PTHrP (1–36); whereas PTHrP (38–94) showed 8.3% cross-reactivity. Comparison with the pre-existing Mayo assay showed a positive bias: new assay = 2.24 (pre-existing assay)-0.30 and r2 = 0.96. The reference interval was ≤ 0.7 pmol/L, however, a cut-off of ≤ 4.2 pmol/L yielded increased specificity (98%). Comparison of patients with HHM versus those without HHM resulted in an area under the ROC curve of 0.99. A significant inverse relationship between eGFR and PTHrP was observed (r = 0.738). PTHrP concentrations in patients with Chronic Kidney Disease (CKD) were ≤ 4.2 pmol/L. ConclusionThis assay is specific for PTHrP 1–86. A clinical decision limit of 4.2 pmol/L was sensitive and specific for patients with HHM.

Immunization With Recombinant Haemonchus contortus Y75B8A.8 Partially Protects Local Crossbred Female Goats From Haemonchus contortus Infection.

Haemonchus contortus Y75B8A.8 (Hc8) derived from H. contortus excretory–secretory (ES) products was identified as a functional inhibitor of goat interleukin 2 (IL-2). It may act as a vaccine candidate for the development of therapeutic strategies against H. contortus infection. In this research, recombinant Hc8 (rHc8) and goat anti-rHc8 polyclonal antibodies were employed to evaluate the protective capacities of Hc8 antigen against H. contortus infections via active and passive immunization trials, respectively. In both trials, local crossbred female goats aged 9–12 months old were randomly divided into three groups, five in each group, respectively. Parasitological examinations, including fecal egg counts (FEC), cumulative FEC (cFEC), and worm burdens, were performed. In addition, antibody levels in mucosal homogenate (MH) samples and hematological and immunological parameters were detected. In the passive trial, goats were intravenously immunized with 5 mg total IgG containing anti-rHc8 goat polyclonal antibodies. After twice immunization, compared with the challenged control group, cFEC was reduced by 39%. In addition, there was a 46% reduction of worm burdens compared with the challenged controls. In the active immunization trials, 500 μg of recombinant Hc8 protein was given subcutaneously twice to 9–12-month-old local crossbred female goats with a 2-week interval, resulting in the generation of high levels of antigen-specific circulating antibodies. Besides, cFEC and abomasal worm burden were reduced by 70 and 55%, respectively, compared with the challenged control group. In addition, immunized goats had higher mucosal homogenate IgA and hemoglobin levels than the challenged controls in both passive and active immunization trials. These preliminary results demonstrated the immunoprophylactic effects of Hc8 antigen and will inform new studies on ES proteins in developing subunit recombinant vaccines against H. contortus.

Zika Virus Non-Structural Protein 1 Antigen-Capture Immunoassay.

Infection with Zika virus (ZIKV), a member of the Flavivirus genus of the Flaviviridae family, typically results in mild self-limited illness, but severe neurological disease occurs in a limited subset of patients. In contrast, serious outcomes commonly occur in pregnancy that affect the developing fetus, including microcephaly and other major birth defects. The genetic similarity of ZIKV to other widespread flaviviruses, such as dengue virus (DENV), presents a challenge to the development of specific ZIKV diagnostic assays. Nonstructural protein 1 (NS1) is established for use in immunodiagnostic assays for flaviviruses. To address the cross-reactivity of ZIKV NS1 with proteins from other flaviviruses we used site-directed mutagenesis to modify putative epitopes. Goat polyclonal antibodies to variant ZIKV NS1 were affinity-purified to remove antibodies binding to the closely related NS1 protein of DENV. An antigen-capture ELISA configured with the affinity-purified polyclonal antibody showed a linear dynamic range between approximately 500 and 30 ng/mL, with a limit of detection of between 1.95 and 7.8 ng/mL. NS1 proteins from DENV, yellow fever virus, St. Louis encephalitis virus and West Nile virus showed significantly reduced reactivity in the ZIKV antigen-capture ELISA. Refinement of approaches similar to those employed here could lead to development of ZIKV-specific immunoassays suitable for use in areas where infections with related flaviviruses are common.

Establishment and evaluation of an indirect ELISA for detection of antibodies to goat Klebsiella pneumonia

BackgroundKlebsiella pneumonia, a Gram-negative bacterium belonging to the genus Enterobacter, causes many human and livestock diseases. Notably, infected goats may develop pneumonia, septicemia, which can lead to occasional death, resulting in great economic losses in goat-farming industry. However, there are little systematic methods for detection of goat Klebsiella pneumoniae in livestock production.ResultsIn this study, we developed a Klebsiella pneumoniae goat polyclonal antibody and established an indirect ELISA method to detect the Klebsiella pneumoniae. After screening and optimizing the conditions for detection, we determined the optimal working dilutions of the coated-bacterial antigen, the polyclonal antibody, and the enzyme-labeled secondary antibody that were 1:800 (2.99 × 107 CFU/ml), 1:6400, and 1:5000, respectively. The optimal condition of coating and blocking were both 4 °C for 12 h. The optimal dilution buffers of bacterial antigen, the antibodies, and the blocking buffer were 0.05 mol/L carbonate buffer, 1% BSA phosphate buffer, and 1.5% BSA carbonate buffer, respectively. The cut-off value was determined to be 0.28, and the analytical sensitivity was 1:800 (dilution of a positive sample). Furthermore, there was no cross-reaction between the coated antigen and goat serum positive for antibodies against other bacteria, indicating that indirect ELISA could detect Klebsiella pneumoniae specifically in most cases. The average coefficients of variation of intra-assay and inter-assay were 4.37 and 5.17% indicating favorable reproducibility of indirect ELISA. In the detection of clinical veterinary samples, the positive rate of indirect ELISA was 6.74%, higher than that of conventional agglutination assays.ConclusionsTaken together, we successfully established an indirect ELISA method for detecting antibodies against Klebsiella pneumoniae in goats, which can be applied in production.

Detection of Epithelial Cell Adhesion Molecule in Feline Normal and Tumor Cell Lines and Tissues With Selected Commercial Anti-human EpCAM Antibodies.

Epithelial cell adhesion molecule (EpCAM) is a transmembrane protein expressed at intercellular junctions in epithelial cells. As an epithelial biomarker, it used for immunologic-based capture of epithelial-derived circulating tumor cells (CTCs) in human patients with different carcinomas. EpCAM expression has not been described in normal or neoplastic epithelial tissues in cats. Our goal was to find a commercial antibody that recognizes surface EpCAM expression for CTC detection. We tested two anti-human EpCAM antibodies, designated for use with flow cytometry, for detection of surface EpCAM expression on feline cell lines derived from normal mammary and renal epithelia and mammary and oropharyngeal squamous cell carcinomas in cats. Only one of the antibodies, a goat polyclonal antibody, labeled normal and neoplastic feline mammary epithelial cells and oropharyngeal squamous cell carcinoma cells; no labeling was observed for normal feline kidney epithelial cells. At low dilution, this antibody immunohistochemically stained the intercellular junctions of normal pancreatic, intestinal and mammary epithelium, as well as neoplastic mammary epithelium in feline tissues; however, oral mucosa, skin, and an oropharyngeal squamous cell carcinoma showed no positive immunostaining. The antibody only weakly bound feline squamous cell carcinoma cell lines under static adhesion. Our results indicate that EpCAM is expressed in specific epithelia in cats but is variably expressed in feline mammary tumors and oropharyngeal squamous cell carcinoma. A higher avidity cross-reactive or feline-specific antibody will be required to further investigate EpCAM expression in normal and neoplastic feline tissue or for detecting CTCs in the blood of tumor-bearing cats.

Endothelial eNAMPT amplifies pre-clinical acute lung injury: efficacy of an eNAMPT-neutralising monoclonal antibody.

RationaleThe severe acute respiratory syndrome coronavirus 2/coronavirus disease 2019 pandemic has highlighted the serious unmet need for effective therapies that reduce acute respiratory distress syndrome (ARDS) mortality. We explored whether extracellular nicotinamide phosphoribosyltransferase (eNAMPT), a ligand for Toll-like receptor (TLR)4 and a master regulator of innate immunity and inflammation, is a potential ARDS therapeutic target.MethodsWild-type C57BL/6J or endothelial cell (EC)-cNAMPT−/− knockout mice (targeted EC NAMPT deletion) were exposed to either a lipopolysaccharide (LPS)-induced (“one-hit”) or a combined LPS/ventilator (“two-hit”)-induced acute inflammatory lung injury model. A NAMPT-specific monoclonal antibody (mAb) imaging probe (99mTc-ProNamptor) was used to detect NAMPT expression in lung tissues. Either an eNAMPT-neutralising goat polyclonal antibody (pAb) or a humanised monoclonal antibody (ALT-100 mAb) were used in vitro and in vivo.ResultsImmunohistochemical, biochemical and imaging studies validated time-dependent increases in NAMPT lung tissue expression in both pre-clinical ARDS models. Intravenous delivery of either eNAMPT-neutralising pAb or mAb significantly attenuated inflammatory lung injury (haematoxylin and eosin staining, bronchoalveolar lavage (BAL) protein, BAL polymorphonuclear cells, plasma interleukin-6) in both pre-clinical models. In vitro human lung EC studies demonstrated eNAMPT-neutralising antibodies (pAb, mAb) to strongly abrogate eNAMPT-induced TLR4 pathway activation and EC barrier disruption. In vivo studies in wild-type and EC-cNAMPT−/− mice confirmed a highly significant contribution of EC-derived NAMPT to the severity of inflammatory lung injury in both pre-clinical ARDS models.ConclusionsThese findings highlight both the role of EC-derived eNAMPT and the potential for biologic targeting of the eNAMPT/TLR4 inflammatory pathway. In combination with predictive eNAMPT biomarker and NAMPT genotyping assays, this offers the opportunity to identify high-risk ARDS subjects for delivery of personalised medicine.

Platelet-Associated Antibodies By Flow Cytometry in Adult Patients with Immune Thrombocytopenia before and after Splenectomy

Introduction Immune thrombocytopenic purpura (ITP), or primary immune thrombocytopenia is an autoimmune disease characterized by isolated thrombocytopenia (number of platelets in peripheral blood is less than 100×109/L) and splenic production of antibodies against platelet glycoprotein complexes and megakaryocytes, resulting in hemorrhagic syndrome. Circulating platelets attached by autoantibodies that leads to accelerated removal of these cells by spleen macrophages. The therapy for patients with newly diagnosed ITP with hemorrhagic syndrome and/or severe thrombocytopenia (number of platelets in peripheral blood &amp;lt;10-20×109/L) includes corticosteroids and characterized by a low rate of remission (just around 20-30%). For cases of ITP resistant to corticosteroid therapy recommends one of the second-line method of treatment - splenectomy (SE). Approximately 60-80% of the patients achieve complete remission after splenectomy. There is a technique for assessment of platelet-associated antibodies (PAA) classes IgG, IgM and IgA on platelets by flow cytometry. This method is a commodious, easy, quick, and relatively cheap and applied to estimate autoimmunity status of patients with thrombocytopenia. However, this method characterized by low specificity. Aim The aim of the study was to determine the correlation between level of PAA of IgG, IgM, and IgA classes in the peripheral blood of adults with ITP before SE, 5-7 days and 3 months after SE. Patients and methods The study included 21 patients with ITP (4 cases of persistent ITP and 17 cases with chronic form). Median age was 36.9 years, M:F ratio was 1: 4.25 (men was older than women - 46.0 years old versus 34.7). All patients underwent from 1 to 3 lines of therapy and were recommended for SE due to resistance to treatment. The PAA level was measured at three time points (before SE, 5-7 days, and three months after SE) by flow cytometry (Becton Dickinson FACS Canto II). Goat polyclonal antibodies against human IgG, IgM, IgA labeled with fluorescein isothiocyanate (FITC) (Cedarlane) were used to determine antibodies of various classes. Anti-CD41a labeled with phycoerethrin (Becton Dickinson) was used to determined platelets. PAA level was assessed based on the mean of fluorescence intensity (MFI) of the FITC-channel. Statistic analysis was carried out using GraphPad Prism 6.01. Wilcoxon signed-rank test had been used for pair comparison. The value of 0.05 had been taken as reliable. Results MFI levels of PAA IgA (391 vs 198, p = 0.005) and IgM (275 vs 142, p&amp;lt;0.0001) significantly decreased in patients after SE compared with the initial level (level before SE). Level of MFI PAA IgM also remained reduced (275 vs 138, p=0.0084) three months after SE (Fig. 1). MFI levels of PAA IgG did not change. Conclusions Using of flow cytometry to determinate platelet-associated immunoglobulins for diagnostic of ITP remains controversial. Despite the fact, this test can be recommended for monitoring of PAA from patients with ITP after SE. In addition, the results confirm the fact that most cells producing antiplatelet antibodies seems to be residing in a spleen. Disclosures No relevant conflicts of interest to declare.

Aging Process in Dermal Fibroblast Cell Culture of Green Turtle (Chelonia mydas)

Senescence in green turtle is an interesting process to study because until now no clear explanation has been established about senescence at cellular or molecular level in this species. One of common markers used for detecting senescence is telomere shortening. Reduced telomerase activity may also cause senescence. The aims of this research are to establish and identify dermal fibroblast cell culture from green turtle and also to compare telomere length and telomerase activity from the cells subculture 5 and 14. Cells were identified with Rabbit Anti-Vimentin Polyclonal Antibody and Goat Polyclonal Antibody using confocal microscope. Telomere length was obtained using TeloTAGGG Telomere Length Assay (Roche), while telomerase activity was obtained using TeloTAGGG Telomerase PCR ElisaPlus. Primary cell culture from green turtle skin showed fibroblastic morphology and immunocytochemistry results using vimentin antibody proved that the culture was fibroblast cell. Measurement of telomere length and telomerase activity showed that telomere length and telomerase activity of subculture 14 was greater than subculture 5. However, based on morphology, green turtle fibroblast skin cell culture showed senescent morphology. Therefore, possible aging mechanism that the green turtle fibroblast skin cell culture underwent did not go through both telomere shortening and reduced telomerase activity.

A universal multiplex assay format for ultra-sensitive cytokine quantification in human serum and plasma

Abstract Quantifying prospective biomarkers of immune signaling in biological samples requires high assay sensitivity. The Simoa Planar Array provides greater sensitivity than ELISA, but has a finite assay menu. This study aimed to develop a universal multiplex ‘homebrew’ planar immunoassay format for development of high sensitivity assays for any analyte. Anchor antibodies specific for a peptide tag were microprinted in microwell plates. The peptide tags were conjugated to analyte-specific capture antibodies via maleimide chemistry, enabling high affinity binding of capture to anchor antibody. Bound sample antigen was sandwiched between the peptide-tagged capture and biotinylated detector antibodies; chemiluminescent detection was used to quantify signal. This ‘homebrew’ approach successfully detected sub picomolar levels of cytokines and chemokines in human serum and plasma in a multiplex assay measuring IL-5, IL-6, IL-10, IL-22, TNFa. When compared to commercially available planar assays the universal planar assay format provided comparable LLOQ and LOD with sample correlation (R2) &amp;gt;0.97 for all analytes tested. The universal planar assay was used to successfully screen multiple antibody pairs for human and mouse biomarkers and to develop robust assays using mouse, rabbit or goat monoclonal and polyclonal antibodies. This universal multiplex planar homebrew assay format combines the accuracy, reproducibility and high sensitivity of Simoa Planar Array technology with total analyte flexibility, and provides assay developers a simple and versatile method to rapidly develop robust high-sensitivity multiplex assays for any analyte of their choice.

Haemonchus contortus transthyretin domain - containing protein (HcTTR): A promising vaccine candidate against Haemonchus contortus infection

Haemonchus contortus transthyretin domain-containing protein (HcTTR) with 136 amino acids belongs to a transthyretin-like (TTL) family member. In our previous study, it was reported that HcTTR was a novel antagonist of the goat cytokine Interleukin 4 (IL-4), and was involved in the regulation of host immune responses, implying that it might be applied for therapeutic strategies or vaccine development against the infection of H. contortus. Thus, the protective capacities of HcTTR against H. contortus infections via active and passive immunization trials were examined. For the passive protection trials, H. contortus-infected goats were intravenously immunized twice with 5 mg of total IgG containing anti-rHcTTR goat polyclonal antibodies. The results showed that the significant rates of reduction in egg shedding and worm burden was 58.12% and 64.61%, respectively, as compared with the positive control group. For the active protection trials, local goats were vaccinated twice with 500 μg of recombinant HcTTR to generate antigen-specific circulating antibodies, resulting in 63.7% reduction in eggs shedding and 66.4% reduction in worm burdens after H. contortus challenge. In both passive and active trials, the immunized goats displayed higher mucosal IgA levels and less anaemic compared to the challenged positive controls. Pen trials indicated that HcTTR generated partial immune protective effects against H. contortus challenge and it could be a promising vaccine candidate for development of effective strategy to control H. contortus.

Prostate-specific antigen levels in patients with risk factors for prostate carcinoma disorders

ABSTRACT Objective: To verify the association between the prostate-specific antigen (PSA) serum concentration and the presence of risk factors for prostate diseases in adult patients with urogenital infections. Materials and methods: Analytical cross-sectional study of PSA in 60 patients aged 40 to 65 years, from January to December 2013. PSA quantification was performed in serum by solid-phase chemiluminescence in two label cycles: 1. mouse monoclonal antibody (mAb); and 2. goat polyclonal antibody (pAbs). After each cycle the free antigen was removed. The readings were taken on the INMULITE ONE Siemens® automated equipment. From the PSA, 0.0-2.5 ng/ml was used to calculate the antigen distribution trend, and a bivariate statistical analysis of PSA was as opposed to previous exams, and endogenous and environmental risk factors detected. Results: Patients between 50-60 years of age, with a family history of urogenital and sexually transmitted infections, toxic habits, and exposure to occupational risk prevailed. Sixty-two percent of patients presented normal PSA levels, and the remaining presented slightly elevated and very high PSA levels. In the statistical analysis, a significant association (p < 0.001) of PSA was found as opposed to previous tests. A significant association (p = 0.001) was also found between PSA versus age, family history, personal pathological history, toxic habits, and risk exposure. Conclusion: The high association rate found between PSA versus age and other risk factors could be used as a predictive value for prostate cancer (Pca) or other prostate disorders.

High Expression of Trophoblast Cell Surface Antigen 2 in Triple-negative Breast Cancer Identifies a Novel Therapeutic Target

Abstract Objectives Patients with triple-negative breast cancer (TNBC) respond poorly to current therapeutic modalities. The newly FDA-approved drug conjugate, Sacituzumab Govitecan, targeting antitrophoblastic cell surface antigen 2 (Trop-2), offers a new modality to treat these subtypes of breast carcinoma (BC). Our study examines expression of Trop-2 by immunohistochemistry (IHC) in TNBC. Methods Forty cases of TNBC were selected from our files (34 ductal, 4 pleomorphic lobular, 1 metaplastic, and 1 mixed ductal and lobular carcinoma). A control group included 11 luminal A-like, 11 luminal B-like, and 8 HER2-like BC. Immunostaining for Trop-2 was performed on 4-micron-thick tissue sections using goat polyclonal antibody (R&amp;D Systems). Three pathologists individually scored the % and intensity of membranous staining. The % of staining was defined as &lt;10%, 10%-50%, and &gt;50%. The intensity of staining was scored as 3+ (crisp circumferential membranous staining), 2+ (medium membranous staining), 1+ (patchy weak membranous staining), and 0 (no staining). A Kruskal-Wallis H-test and pairwise comparison with Bonferroni correction was performed to compare % and intensity of staining in TNBC to control group. Results TNBC exhibited stronger Trop-2 staining in both intensity and extent when compared to the control. The result was significantly different, χ2 (4) = 17.427, P = .001. A pairwise comparison with Bonferroni correction found significant difference between TNBC (42.55) and luminal A (16.65) (P &lt; .001). There was significant agreement (P &lt; .001) among 3 independent pathologists for assessing % and intensity of staining. Conclusion The extent and intensity of staining for Trop-2 is higher in TNBC than in other molecular subtypes of BC. These findings suggest that patients with TNBC may potentially benefit from this targeted therapy. Furthermore, identification of Trop-2 in other molecular subtypes may expand the therapeutic potential of this new drug. Further studies are needed to determine the efficacy of this marker.

A universal planar assay format for high sensitivity cytokine quantification in human serum and plasma

Abstract Quantifying prospective biomarkers of immune signaling in biological samples requires high assay sensitivity. The Simoa Planar Array provides greater sensitivity than ELISA, but has a finite assay menu. This study aimed to develop a universal ‘homebrew’ planar immunoassay format for development of high sensitivity assays for any analyte, by using an anchor antibody/peptide tag system to develop a universal capture plate. Anchor antibody specific for a peptide tag was microprinted in microwell plates. The peptide tag was conjugated to analyte-specific capture antibodies via maleimide chemistry, enabling high affinity binding of capture to anchor antibody. Bound sample antigen was sandwiched between the peptide-tagged capture and biotinylated detector antibodies; chemiluminescent detection was used to quantify signal. This ‘homebrew’ approach successfully detected sub picomolar levels of cytokines and chemokines in human or mouse serum and plasma, including IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-22, ITAC, MIP1a and MIP3b. When compared to commercially available planar assays the universal planar assay format provided comparable LLOQ and LOD with sample correlation (R2) &amp;gt;0.97 for all analytes tested. The universal planar assay was used to successfully screen multiple antibody pairs for human and mouse biomarkers and to develop robust assays using mouse, rabbit or goat monoclonal and polyclonal antibodies. This universal planar homebrew assay format combines the accuracy, reproducibility and high sensitivity of Simoa Planar Array technology with total analyte flexibility, and provides assay developers a simple and versatile method to rapidly develop robust high-sensitivity assays for any analyte of their choice.

SUN-LB059 A Rare Case Of Hypoglycemia Secondary To Proinsulin-Intermediate Secreting Tumor

IntroductionPancreatic neuroendocrine tumors (PNETs) that solely produce proinsulin (PI) manifesting with hypoglycemia and normal or low serum insulin (IN) levels are rare. Des 64-65 is a PI intermediate. We report a novel case of hypoglycemia secondary to a possible PI intermediate proinsulinoma. Case descriptionA 60-year-old diabetes educator presented with two years of double vision, light-headedness, and paresthesias aggravated by stress and alleviated with meals. She had a history of hypothyroidism treated with levothyroxine. Fingerstick glucose with symptoms was 46 (60-140 mg/dl). Physical examination was unremarkable. A fasting hypoglycemia test confirmed serum glucose of 47 mg/dl after 16 hours. Serum IN was 2.2 (3.0-25.0uIU/ml), C-peptide 1.1 (0.8-3.0ng/ml), and PI 262.4 (≤ 18.8 pmol/L). PI was measured by radioimmunoassay (RIA) using a polyclonal goat antibody (AB) with specificity for the B-C junction which does not distinguish between PI and its intermediate forms. IN autoantibodies were negative. β-hydroxybutyrate was ≤ 0.28 (0.32 mmol/L). IN secretagogue panel was negative. Endoscopic ultrasound revealed a 14 x 8 mm isoechoic mass in the pancreatic tail. Fine needle aspirate was positive for synaptophysin. The Ki-67 index was 3%. Immunohistochemistry (IHC) showed low IN and PI intensity. GS-9A8, a monoclonal AB from the Developmental Studies Hybridoma Bank at the University of Iowa, was used in the IHC. This antibody binds to the B-C junction of human and monkey proinsulin and stains only intact, non-processed PI found in the immature secretory granules of islet beta-cells. It does not cross-react to insulin or C-peptide. Positron emission tomography did not reveal hypermetabolic activity or distant metastases. Robotic distal pancreatectomy confirmed a well-differentiated PNET that was low intensity for IN and PI on IHC. Post-operatively, her glucose was 117 mg/dl, PI 8.8 pmol/L, IN 3 uIU/ml, C-peptide 0.8 ng/ml. She remains asymptomatic at 6 month follow up. DiscussionHypoglycemia in the setting of a pancreatic mass is usually due to IN producing PNETs. PI secreting tumors are a rare entity. PI acts on the IN receptor demonstrating a weak hypoglycemic effect. Our case showed low intensity IHC for IN and PI, with low serum IN and elevated serum PI. The low intensity IN and PI IHC using a monoclonal AB that stains only intact, non-processed PI, associated with an elevated serum PI level using an RIA that employs a polyclonal goat AB with specificity for the B-C junction and which does not distinguish between PI and its intermediate forms, suggests that the tumor was possibly producing a PI intermediate with an intact B-C junction, likely des 64-65 PI. Low intensity IN and PI IHC would suggest unregulated release with decreased intracellular storage of PI. PI or PI intermediate secreting PNET must be considered when evaluating hypoglycemia. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. s presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.