Secondary Ab Research Articles

Secondary ab interno stenting to manage early tube-related hypotony.

Trabeculectomy is the gold standard procedure to achieve control of intraocular pressure surgically. However, glaucoma drainage devices have their place in certain refractory glaucomas not amenable to control by the standard trabeculectomy. These devices come with their own set of complications, cognizance of which is critical to anticipate and manage appropriately. With the nonvalved devices, hypotony is one difficult problem to deal with. Several modifications have been described to manage refractory hypotony after tubes. We present here a simple, less invasive surgical technique of secondary ab interno suture stenting to manage tube-related early hypotony.

RNA Analysis Using Immunoassay Detection Format.

Oligonucleotide probe tagging and reverse transcriptase polymerase-chain reaction (RT-PCR) are the most widely used techniques currently used for detecting and analyzing RNA. RNA detection using labeled oligonucleotide probe-based approaches is suitable for point-of-care (POC) applications but lacks assay sensitivity, whereas RT-PCR requires complex instrumentation. As an alternative, immunoassay detection formats coupled with isothermal RNA amplification techniques have been proposed for handheld assay development. In this chapter, we describe a robust technique comprising of: (a) target RNA tagging with a complementary oligonucleotide probe labeled with a hapten moiety to form a DNA/RNA duplex hybrid; (b) complexing the DNA/RNA duplex with a pre-coated antibody (Ab) directed at the hapten moiety; (c) sandwich complex formation with an Ab that selectively recognizes the DNA/RNA structural motif; and (d) detection of the sandwich complex using a secondary Ab enzyme conjugate targeting the anti-DNA/RNA Ab followed by standard enzyme-linked immunosorbent assay (ELISA) visualization.

Hydrogenation behavior of a C14 Laves phase under ultra-high hydrogen pressure

For high pressure metal hydride (MH) compressor applications, it is important to have a thorough understanding of the hydrogenation properties of MH forming compounds under high pressure and temperature conditions, which are still little studied. Here, a Ti0.90V0.30Mn1.00Ni0.80 compound with MgZn2-type AB2 structure has been investigated by Sieverts’ method over a wide pressure and temperature range (up to 100 MPa and between −80 and 120 °C). This provided clear experimental evidence of the downward curvature of the van’t Hoff plot in the high-pressure region, which arises from the non-ideal behavior of hydrogen. In this paper, we also show that the high-sorption pressures can be well estimated by the van’t Hoff equation using low-pressure data. Ultimately, this study was combined with synchrotron radiation X-ray diffraction (SR-XRD) performed in an effort to monitor the structural evolution of the compound in situ under ultra-high hydrogen pressure. We found that even under extreme conditions of 2 GPa and 200 °C, both the main AB2 phase and the secondary CsCl-type AB phase were fully hydrogenated and their structures were conserved. The existence of a critical temperature for the AB2-H2 system was demonstrated from the evolution of the SR-XRD patterns recorded for several temperatures during the desorption process.

Highly sensitive sandwich-type immunosensor with enhanced electrocatalytic durian-shaped MoS2/AuPtPd nanoparticles for human growth differentiation factor-15 detection

Growth differentiation factor-15 (GDF-15) expression is a potential biomarker for the diagnosis, risk stratification, and prognosis of various cardiovascular diseases. A sandwich-type electrochemical immunosensor was constructed using amine-modified graphene-supported gold nanorods (NG/AuNPs) as a substrate platform and the durian-shaped MoS2/AuPtPd nanodendrite (NDs) as a label for secondary antibodies (Ab2) for the quantification of GDF-15. NG/AuNPs were used to increase the surface area of electrodes and immobilize primary antibodies (Ab1). The MoS2/AuPtPd NDs have a unique morphology with a high crystal surface index and good stability, ability to load secondary Ab (Ab2), and provide a larger active site for the catalysis of H2O2. In a clinical sample experiment, tissue sections were subjected to immunohistochemical analyses and western blotting. To demonstrate the feasibility of the immunosensor in realistic applications, we compared the performance of the immunosensor and a GDF-15 enzyme-linked immunosorbent assay (ELISA) kit by analyzing clinical human blood samples. As demonstrated by its remarkable performance throughout the study, the devised immunosensor may be a reliable and practical “liquid biopsy” tool for the serological detection of GDF-15. It has the potential to facilitate the early diagnosis and subsequent treatment of patients with cardiovascular disease.

Detection of Mu‐Opioid Receptors in SH‐SY5Y Human Neuroblastoma Cells by Flow Cytometry

PurposePharmacological actions of morphine are mediated through G‐protein‐coupled receptors (GPCRs), specifically mu‐opioid receptors (MORs). Morphine‐induced activation of GPCRs inhibits adenylyl cyclase and decreases 3’,5’‐cyclic adenosine monophosphate (cAMP). Neuroblastoma SH‐SY5Y cell lines endogenously express MORs. Our aim was to develop a flow cytometry (FC) assay for MORs on the surface of SH‐SY5Y cells. This assay will provide in‐depth understanding of opioid‐induced downstream cAMP signaling.MethodsSH‐SY5Y human neuroblastoma cells (ATCC® CRL2266™) were grown in DMEM/10% FBS and maintained in a humidified incubator at 37ºC/5%CO2. Cells were seeded in 96‐well white, opaque‐bottom plates (5×104cells/well). Transfection conditions were optimized using ViaFect™ (Promega™) and a stably transfected SH‐SY5Y cell line was established using GloSensor™ ‐23F cAMP plasmid (Promega™) containing hygromycin‐resistance gene. Cells were washed in FACS buffer (PBS + 2% FBS) and incubated for 1h at room temperature with monoclonal rabbit anti‐MOR antibody (Ab) (Novus Biologicals). Following the wash, cells were incubated with donkey anti‐rabbit secondary Ab (Abcam). We used monoclonal mouse anti‐muscarinic Ab (Novus Biologicals) and donkey anti‐mouse secondary Ab (Jackson Labs). Both secondary Abs are bound to a fluorophore (Alexa Fluor® 488) which emits light when excited by FITC channel frequency (excitation/emission: 495nm/519nm). A Cytoflex flow cytometer (Beckman Coulter) analyzes cells for FITC fluorescence emission. Multiple primary and secondary concentrations were tested; Human TruStain Fc blocker (BioLegend) was used to prevent nonspecific tagging.ResultsRelative fluorescence emission was measured as percent shift increase from the untagged control group. Because MOR Abs have been tested in a limited capacity, we tested for presence of muscarinic receptors (another endogenously expressed GPCR in these cells) to establish the FC assay. Initial trials with low primary and secondary MOR Ab concentrations showed limited shifts (~1‐2%), however, muscarinic receptor Abs bound to their receptors at lower concentrations compared to MOR Abs. Testing muscarinic receptors showed secondary Ab concentration‐dependent fluorescence shifts indicating that the technique was viable. Studies with increased concentration ratios of primary and secondary MOR Abs showed secondary Ab concentration‐dependent response with maximum % shifts reaching up to 16%. Use of Fc blocker eliminated nonspecific binding, increasing the percent shift.ConclusionCompared to untagged controls, antibody‐tagging on SH‐SY5Y cell surface produced greater immunofluorescence indicating presence of MORs in both non‐transfected and 23F cAMP plasmid transfected cells. Direct binding of receptor modulators leads to altered downstream cAMP signaling as shown in previous in vitro signaling studies and behavioral mouse models. Detection of the MOR is essential to ensure that cAMP signaling is regulated by MOR agonists. In future studies, we plan to investigate the expression of endothelin‐A receptor (ETAR) and explore potential interactions between MOR and ETAR in SH‐SY5Y cells.

Deciphering the role of the conjugate's phycoerythrin label in complement-mediated interference occurring in HLA single antigen Luminex bead assays.

Complement-mediated interference is a well described phenomenon in single antigen bead (SAB) Luminex assay that leads to falsely low or negative results for anti-HLA antibody (Ab). In a context of high amount of Ab, the enrichment of the Ab around the bead can lead to complement cascade activation and deposition, thereafter impairing Ab detection. EDTA is now routinely used to circumvent this interference. In this report, we attempted to decipher the role of the phycoerythrin (PE) label conjugated to the secondary Ab in this interference. Indeed, PE is a huge molecule (240 kDa) that could participate to limiting access of the conjugate to its Ab target on the bead. To this purpose, 22 sera displaying complement interference without pre-treatment with EDTA were compared on SAB assay with three detection strategies: the recommended PE-conjugated secondary Ab (IgGPE), an Alexa Fluor 532-conjugated Ab (IgGAF) bearing a tiny 724 Da fluorochrome, and a biotinylated Ab followed by PE-conjugated streptavidin (IgGBiot). Complement interference occurred with the three detection methods, but its depth, defined by the percentage of MFI loss with neat serum, was the highest for IgGPE. Our study highlighted the partial role of the PE fluorochrome in complement interference in SAB assays.

Development of Indirect Competitive ELISA and Visualized Multicolor ELISA Based on Gold Nanorods Growth for the Determination of Zearalenone.

In this study, a zearalenone (ZEN) hapten was designed and prepared against the mycotoxin ZEN, and the original coating ZEN-ovalbumin (ZEN-OVA) was prepared by conjugation with OVA. Based on the gold nanorods (AuNRs) of uniform size and stable properties synthesized by the seed-mediated method, the indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and the AuNRs growth-based multicolor ELISA for detecting ZEN toxin were further established. Under the optimal experimental conditions, the coating amount of ZEN-OVA: 0.025 μg/well, antibody (Ab) dilution factor: 32,000 times, blocking solution: 0.5% skimmed milk powder, enzyme-labeled secondary Ab diluted 10,000 times, and a pH of the PBS buffer at 7.4, the sensitivity (IC50) of the established ic-ELISA for ZEN detection reached 0.85 ± 0.04 μg/L, and the limit of detection (IC15) reached 0.22 ± 0.08 μg/L. In the multicolor ELISA based on the growth of AuNRs, as the content of ZEN increased, the mixed solution exhibited a significant color change from brownish red to colorless. ZEN concentration as low as 0.1 μg/L can be detected by the naked eye (brown red to dark gray). This study provided an effective analysis strategy for the rapid screening and accurate monitoring of the ZEN contaminant in foods.

Eclipsing Binaries in the Open Cluster Ruprecht 147. IV: The Active Triple System EPIC 219511354

Abstract We report follow-up spectroscopic observations of the 1.62 day, K-type, detached, active, near-circular, double-lined eclipsing binary EPIC 219511354 in the open cluster Ruprecht 147, identified previously on the basis of photometric observations from the Kepler/K2 mission. This is the fourth eclipsing system analyzed in this cluster. A combined analysis of the light curve and radial velocities yields accurate masses of M Aa = 0.912 ± 0.013 M ☉ and M Ab = 0.822 ± 0.010 M ☉ for the primary (star Aa) and secondary (Ab), along with radii of R Aa = 0.920 ±0.016 R ☉ and R Ab = 0.851 ± 0.016 R ☉, and effective temperatures of 5035 ± 150 and 4690 ± 130 K, respectively. Comparison with current models of stellar evolution for the known age and metallicity of the cluster reveals that both radii are larger (by 10%–14%) and both temperatures cooler (by ∼6%) than theoretically predicted, as is often seen in M dwarfs. This is likely caused by the significant stellar activity in the system, manifested here by 6% peak-to-peak out-of-eclipse variability, a filled-in Hα line, and its detection as an X-ray source. We also find EPIC 219511354 to be a hierarchical triple system, with a low-mass tertiary in an eccentric 220 day orbit.

MARKET INTEGRATION AND PRICE TRANSMISSION OF BEEF IN THE ARCHIPELAGIC STATE: THE CASE OF THE PROVINCES IN INDONESIA

The Archipelagic state condition brings challenges to market integration and price transmission on two provinces market pairs within and between the island. The duration of price correction is essential to ensure market integration. Therefore, this study aims to assess the integration of domestic markets for beef within an island and between the island and analyze the speed of adjustment (the correction) of the prices by two provinces within the island and between the islands. These aims, daily frequency (weekdays) of Beef Consumer Prices (BCP) of 4 types of beef (Primary Cut, Secondary Cut AB, Meat Manufacturing and Fancy and Variety Meat) from 01 January 2015 to 27 April 2018 containing 28,611 observations were obtained. The data were analyzed using the Vector Error Correction Model (VECM). This study reveals that the island's provinces have fewer integrated than the pairs provinces located between islands by comparing 31 pairs with 38 pairs. It can be concluded that from research, the price correction in the pairwise province within an island is stronger than the pairwise between the islands, with 10.33 % per day and 9.87 % per day, respectively. This study recommends the government to formulate policies that carry out equitable development, especially those that facilitate access to the central beef production in Indonesia to avoid market exploitation and spread price information along with provinces in Indonesia, especially in eastern Indonesia. Keywords: archipelagic state, market integration, Indonesia, beef, VECM

Prevalidation of an ELISA for Detection of a New Clinical Entity: Leishmania donovani-Induced Cutaneous Leishmaniasis.

Human leishmaniasis which is considered a neglected tropical parasitic disease presents in three main clinical forms (i.e., cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL), and visceral leishmaniasis (VL)) that are mainly determined by its causative species. Leishmania donovani, the most virulent and visceralizing parasite, is increasingly reported to cause CL in many countries in the world. Although CL is generally not considered to evoke a humoral immune response except for a nonrobust and a variable response in minority of cases, VL is associated with a clear strong humoral response. However, humoral response in L. donovani-induced CL has not been well evaluated before. A suitable serology-based assay is an essential primary step in such a study. An indirect enzyme-linked immunosorbent assay (ELISA) based on Leishmania promastigote crude antigen (Ag) was designed and optimized in order to utilize in further serological studies on this new clinical entity. Optimization included quantification of crude Ag, checkerboard titration method for determination of optimal concentrations for coating Ag, human sera and secondary antibody (Ab) with suitable coating buffer, blocking buffer, and incubating temperatures. The selected coating buffer was 0.02 M phosphate buffer, pH 6.8, and the blocking buffer was 2% fetal bovine serum with 0.01 M phosphate-buffered saline. At least 1 μg of crude Ag was required for coating the ELISA plate, while 1 : 1000 serum was used as primary Ab. The optimized concentration of secondary Ab was 1 : 64000 which might be altered according to manufacturer recommendations. The assay specificity was pre-evaluated using sera (n = 20 from each category) from confirmed CL patients and controls (other skin diseases which mimic CL, other systemic diseases that mimic VL, nonendemic healthy controls, and endemic healthy controls). This procedure described an optimization procedure of an ELISA technique for detection of anti-Leishmania antibodies in patients with L. donovani caused CL.

Chrysanthemum Morifolium Extract And Ascorbic Acid-2-Glucoside (AA2G) Blend Inhibits UVA-Induced Delayed Cyclobutane Pyrimidine Dimer (CPD) Production In Melanocytes.

BackgroundSolar ultraviolet radiation (UV) induces DNA damages in skin via direct absorption of UVB or indirectly by photosensitization mediated through UVA. Recent findings have revealed that UVA induces cyclobutane pyrimidine dimer (CPD) generation via chemiexcitation in melanocytes hours after the exposure. This UVA-induced delayed CPD (dark CPD) constitutes the majority of CPD in melanocytes. These findings indicate that sun light can damage the skin hours after the exposure, suggesting the need for skin care products post sun exposure. The main objective of this study was to investigate whether a blend of Chrysanthemum Morifolium flower extract (Chrys) and vitamin C derivative, Ascorbic Acid-2-Glucoside (AA2G), can provide protective effects against reactive oxygen species, melanin formation and UVA-induced dark CPD.MethodsIntracellular ROS levels were measured in epidermal keratinocytes using DHR123 dye. Melanogenesis inhibition efficacy was determined using B16 cells. As for the dark CPD measurement, Melan-a cells were treated with or without actives for 6 days, then irradiated with UVA at various doses. Cells were exposed with anti-CPD mAb followed by secondary Ab. CPD levels were determined by measuring fluorescent intensity using a high content imaging analysis.ResultsChrys, AA2G and their blend at various concentrations demonstrated ROS scavenging activity. Though Chrys alone did not show significant melanogenesis inhibition in B16 assay, the blend of Chrys with AA2G demonstrated additive effects in comparison with AA2G alone. The blend of AA2G and Chrys at various concentrations exhibited enhanced efficacy for inhibiting dark CPD compared to AA2G alone.ConclusionThe results from this study indicate that the use of natural antioxidant, Chrys in combination with AA2G, provides protection against UVA-induced delayed CPD formation by enhancing ROS scavenging activity and melanogenesis inhibition. These findings could potentially be applied for formulating post-sun exposure skin care products, possibly extending to evening-after care products.

FliC's Hypervariable D3 Domain Is Required for Robust Anti-Flagellin Primary Antibody Responses.

Bacterial flagellin is a well-known agonist of the innate immune system that induces proinflammatory responses through the TLR5 and Naip5/6 recognition pathways. Several clinical trials investigating flagellin fusion proteins have demonstrated promising results for inducing protective immunity toward influenza virus, which has been largely attributed to flagellin's ability to activate TLR5. Our laboratory previously demonstrated that the Salmonella enterica serovar Typhimurium flagellin protein, FliC, induces Ab responses in mice through a third pathway that is independent of TLR5, Casp1/11, and MyD88. In this study, we further define the structural features of FliC that contribute to this unknown third pathway. By destroying the Naip5/6 and TLR5 recognition sites, we demonstrate that neither were required for the TLR5-, inflammasome- and MyD88-independent Ab responses toward FliC. In contrast, deletion of FliC's D3 or D0/D1 domains eliminated primary anti-flagellin Ab responses. For optimal primary and secondary anti-flagellin Ab responses we show that TLR5, inflammasome recognition, and the D3 domain of FliC are essential for flagellin's robust immunogenicity. Our data demonstrate that the D3 domain of FliC influences immunogenicity independent of the known innate recognition sites in the D0/D1 domains to augment Ab production. Our results suggest full-length FliC is critical for optimal immunogenicity and Ab responses in flagellin-based vaccines.

GG Tauri A: dark shadows on the ringworld

Context. With its high complexity, large size, and close distance, the ringworld around GG Tau A is an appealing case to study the formation and evolution of protoplanetary disks around multiple star systems. However, investigations with radiative transfer models usually neglect the influence of the circumstellar dust around the individual stars. Aims. We investigate how circumstellar disks around the stars of GG Tau A influence the emission that is scattered at the circumbinary disk and if constraints on these circumstellar disks can be derived. Methods. We performed radiative transfer simulations with the POLArized RadIation Simulator (POLARIS) to obtain spectral energy distributions and emission maps in the H-Band (near-infrared). Subsequently, we compared them with observations to achieve our aims. Results. We studied the ratio of polarized intensity at different locations in the circumbinary disk. We conclude that the observed scattered-light near-infrared emission is best reproduced if the circumbinary disk lies in the shadow of at least two coplanar circumstellar disks surrounding the central stars. This implies that the inner wall of the circumbinary disk is strongly obscured around the midplane, while the observed emission is actually dominated by the upper-most disk layers. In addition, the inclined dark lane (“gap”) on the western side of the circumbinary disk, which has been a stable, nonrotating, feature for approximately 20 yr, can only be explained by the self-shadowing of a misaligned circumstellar disk surrounding one of the two components of the secondary close-binary star GG Tau Ab.

Matrix Metalloproteinase‐9 Release from Porphyromonas gingivalis Lipopolysaccharide (LPS)‐treated Rat Brain Microglia In Vitro

BackgroundThe putative association between periodontal disease (PD), a chronic inflammatory disease and neuroinflammation, a condition which involves brain microglia, remains under investigation. We have reported that Porphyromonas gingivalis (Pg) lipopolysaccharide (LPS), a bacterium implicated in periodontitis, activates both classical (M1‐type) and alternative (M2‐type) activation of neonatal rat microglia (BMG) with concomitant release of superoxide anion, thromboxane B2, cytokines, and chemokines (Faseb Journal 32.1 Supplement: 702.2, 2018). We hypothesized that matrix metalloproteinase 9 (MMP‐9) release by Pg‐treated microglia might be detectable by both ELISA and visualized by confocal fluorescence imaging.MethodsP. gingivalis LPS (Pg LPS) 105 ng/mL from InvivoGen (San Diego, CA) and Escherichia coli LPS (Ec LPS) 026:B6 1 ng/mL from Difco Lab, Detroit, MI (positive control) were used to treat rat BMG for 18 hours at 35.9°C in vitro. MMP‐9 release was assessed with a rat MMP‐9 ELISA kit (R&D Systems). Confocal fluorescence imaging with a Nikon A1R laser confocal microscope was used to identify BMGs using a primary mouse anti‐rat CD11b/c antibody (Ab) (AbD SeroTec, Raleigh, NC), and secondary donkey anti‐mouse Ab (Thermo Fischer, Waltman, MA). MMP‐9 was visualized with a primary rabbit anti‐rat MMP‐9 polyclonal Ab, and secondary goat anti‐rabbit Ab, both from Abcam, Cambridge, MA.ResultsMMP‐9 presence in tissue culture supernates was detectable by ELISA in both Ec LPS and Pg LPS‐treated BMG. In contrast, MMP‐9 was observed by confocal fluorescence imaging only within Ec LPS‐treated BMG but not in Pg LPS‐treated BMG.ConclusionsBoth ELISA and confocal fluorescence imaging provide additional support for our working hypothesis, because Pg LPS stimulated BMGs to release MMP‐9. Thus, our current observations extend our studies on the effect of Pg LPS on rat brain microglia activation and contribute to further characterize the putative role of Pg LPS in both periodontitis, neuroinflammation and neurodegeneration.Support or Funding InformationSupport by College of Dental Medicine, College of Graduate Studies, and Chicago College of Osteopathic Medicine, Midwestern University are gratefully acknowledged.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The extreme O-type spectroscopic binary HD 93129A

Context. HD 93129A was classified as the earliest O-type star in the Galaxy (O2 If*) and is considered as the prototype of its spectral class. However, interferometry shows that this object is a binary system, while recent observations even suggest a triple configuration. None of the previous spectral analyses of this object accounted for its multiplicity. With new high-resolution UV and optical spectra, we have the possibility to reanalyze this key object, taking its binary nature into account for the first time. Aims. We aim to derive the fundamental parameters and the evolutionary status of HD 93129A, identifying the contributions of both components to the composite spectrum Methods. We analyzed UV and optical observations acquired with the Hubble Space Telescope and ESO’s Very Large Telescope. A multiwavelength analysis of the system was performed using the latest version of the Potsdam Wolf-Rayet model atmosphere code. Results. Despite the similar spectral types of the two components, we are able to find signatures from each of the components in the combined spectrum, which allows us to estimate the parameters of both stars. We derive log(L∕L⊙) = 6.15, Teff = 52 kK, and log Ṁ = −4.7 [M⊙ yr−1] for the primary Aa, and log(L∕L⊙) = 5.58, Teff = 45 kK, and log Ṁ = −5.8 [M⊙yr−1] for the secondary Ab. Conclusions. Even when accounting for the binary nature, the primary of HD 93129A is found to be one of the hottest and most luminous O stars in our Galaxy. Based on the theoretical decomposition of the spectra, we assign spectral types O2 If* and O3 III(f*) to components Aa and Ab, respectively. While we achieve a good fit for a wide spectral range, specific spectral features are not fully reproduced. The data are not sufficient to identify contributions from a hypothetical third component in the system.

Abstract 5436: Novel single-domain antibody (sdAb), SBT-100, localizes in the cytoplasm to inhibit IL-6 mediated P-STAT3 nuclear translocation in cancer cells

Abstract BACKGROUND: Interleukin-6 (IL-6) is a multifunctional cytokine that is involved in immune defense and plays an important role in biologic activities of cells including tumor cells. These effects are mediated by several signaling pathways, including the signal transducer and transcription activator 3 (STAT3), which plays a critical role in the development, growth, and metastasis of cancer. Inside cancer cells within the tumor microenvironment IL-6 activates (phosphorylates) STAT3. The P-STAT3 dimers translocate into the nucleus where it turns on many genes (e.g., BCL-XL, MCL-1, Myc, Cyclin D1/D2, VEGF, MMP-2, etc.) that are necessary for tumorigenesis. Since there are no commercially available IL-6 or STAT3 inhibitors, blocking IL-6/STAT3 signaling is a potential therapeutic strategy for cancer. SBT-100 is a novel sdAb which binds P-STAT3 to inhibit its translocation into the nuclei of cancer cells, thereby suppressing the cancers growth. METHODS: Human cancer cell lines obtained from ATCC. The STAT3 reporter assay (Promega, WI). IHC staining with IL-6 stimulation, the primary antibody (Ab) was STAT3 (124H6) mouse mAb (Cell Signaling), secondary Ab was anti-mouse IgG (H&L), Alexa Fluor 488 (Cell Signaling), blocking Ab was SBT-100, and recombinant human IL-6 was from Peprotech. In vitro cancer cell suppression via MTT assay (3 day). Xenograft study, athymic nude mice were obtained from Envigo. RESULTS: The STAT3 reporter assay demonstrates that IL-6 activation of STAT3 is completely inhibited by 100ug/ml of SBT-100 (p<0.0012). SBT-100 shows significant (p<0.001) suppression of ten different human cancers in vitro which have constitutive expression of P-STAT3. Immunohistochemical (IHC) staining demonstrates that SBT-100 crosses the cell membrane and localizes within the cytoplasm of MDA-MB-231 cells. IHC staining of STAT3 shows IL-6 stimulation drives P-STAT3 into the nuclei of HEp-2 and PANC-1 cells and this translocation is inhibited by SBT-100. In vivo xenograft studies demonstrate that SBT-100 gives significant (p<0.001) tumor growth suppression of MDA-MB-231. CONCLUSION: SBT-100 inhibits IL-6 mediated function of P-STAT3 as demonstrated by two different assays. IHC staining demonstrates SBT-100 crosses the cell membrane to localize inside cancer cells. Based on its mechanism of action, SBT-100 can be a potential antibody targeted IL-6/STAT3 therapy for human cancer. Citation Format: Sunanda Singh, Genoveva Murillo, Avani Singh, Samara Singh, Meenakshi Parihar, Rajendra Mehta, Anjali Singh, Ashutosh Parihar. Novel single-domain antibody (sdAb), SBT-100, localizes in the cytoplasm to inhibit IL-6 mediated P-STAT3 nuclear translocation in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5436.

A gold standard method for the evaluation of antibody-based materials functionality: Approach to forced degradation studies

The scope of this paper is to present a gold standard method to evaluate functional activity of antibody (Ab)-based materials during the different phases of their development, after their exposure to forced degradations or even during routine quality control. Ab-based materials play a central role in the development of diagnostic devices, for example, for screening or therapeutic target characterization, in formulation development, and in novel micro(nano)technology approaches to develop immunosensors useful for the analysis of trace substances in pharmaceutical and food industries, clinical and environmental fields. A very important aspect in diagnostic device development is the construction of its biofunctional surfaces. These Ab surfaces require biocompatibility, homogeneity, stability, specificity and functionality.Thus, this work describes the validation and applications of a unique ligand binding assay to directly perform the quantitative measurement of functional Ab binding sites immobilized on the solid surfaces. The method called Antibody Anti-HorseRadish Peroxidase (A2HRP) method, uses a covalently coated anti-HRP antibody (anti-HRP Ab) and does not need for a secondary Ab during the detection step. The A2HRP method was validated and gave reliable results over a wide range of absorbance values. Analyzed validation criteria were fulfilled as requested by the food and drug administration (FDA) and European Medicines Agency (EMA) guidance for the validation of bioanalytical methods with 1) an accuracy mean value within +15% of the nominal value; 2) the within-assay precision less than 7.1%, and 3) the inter-day variability under 12.1%. With the A2HRP method, it is then possible to quantify from 0.04 × 1012 to 2.98 × 1012 functional Ab binding sites immobilized on the solid surfaces. A2HRP method was validated according to FDA and EMA guidance, allowing the creation of a gold standard method to evaluate Ab surfaces for their resistance under laboratory constraints. Stability testing was described through forced degradation studies after exposure of Ab-surfaces to storage, pH and aqueous-organic solvent mixture stresses.

176 Localization and Quantitative Expression of Phospholipase C Zeta in Equine Sperm Using Commercial Antibodies

Fertilization failure in vivo and in vitro (intracytoplasmic sperm injection, ICSI) can be caused by the inability of sperm to elicit intracellular calcium oscillations and to induce oocyte activation. Phospholipase C zeta (PLCz) is sperm-associated protein that can induce oocyte activation. Male infertility has been associated with PLCz deficiency in various species, although this has not been studied in the stallion. We hypothesised that the location and amount of PLCz on sperm varies among stallions. The aim of this study was to validate commercial antibodies (Ab) to detect PLCz on stallion sperm, and then to use these Ab to quantify the amount of PLCz, using flow cytometry, with the long-term goal of correlating PLCz on sperm with stallion fertility. Frozen-thawed sperm were analysed (20 stallions in 3 replicates) using 2 commercial Ab (anti-mouse PLCz M163 and anti-human PLCz H50, Santa Cruz Biotechnology, TX, USA). Western blot and immunofluorescence microscopy were used to validate Ab binding. For microscopy, sperm DNA was counterstained with 1 µg mL−1 Hoechst 33258. For flow cytometry, samples were incubated with Live Dead Fixable Far Red Stain Kit (Molecular Probes, Eugene, OR, USA), fixed, permeabilized, incubated overnight with primary Ab, and labelled with conjugated secondary Ab (anti-rabbit IgG Alexa Fluor 488, Molecular Probes). Green and far red mean fluorescence intensity (MFI) were measured for 20,000 cells per sample. Results are presented as mean ± SEM. Wilcoxon test, Spearman rank correlation, and linear regression were performed for analyses. Immunoblot analyses for both commercial Ab identified an immunoreactive band of ~70 kDa in sperm heads, tails, and whole sperm; β-tubulin was used as loading control and for normalization. Microscopy revealed PLCz in the acrosomal and post-acrosomal regions, connecting piece, midpiece, and tail. Post-acrosomal localization was the pattern most frequently observed (55%), followed by acrosomal plus post-acrosomal regions (25%). The PLCz labelling was observed on >85% of midpiece and tail regions, independent of Ab used. Flow cytometric evaluation revealed that percentage of live sperm was 47 ± 2%. Similar fluorescence intensity was exhibited for both Ab (M163 and H50) with a wide range of values among stallions [M163, mean 30.7 ± 1.9 × 103 (range, 8.8-82.2 × 103); H50: 25.5 ± 3.2 × 103 (7.3-55.0 × 103)]. The percentage of live sperm within a sample was not associated with Ab MFI. However, when samples were gated for live/dead cells, live sperm exhibited higher (P < 0.001) MFI than dead sperm for M163 (42.6 ± 6.0 v. 30.6 ± 3.9 × 103) and H50 (38.4 ± 4.7 v. 25.6 ± 3.7 × 103). There was a strong and positive correlation between M163 and H50 MFI for total sperm and live sperm (total: r = 0.81, P < 0.001; live: r = 0.71; P < 0.001). In conclusion, 2 anti-PLCz commercial antibodies detected equine PLCz, and the PLCz was localised on the sperm as described. Flow cytometric evaluation showed that stallions have different quantities of PLCz on their sperm, and this may provide a mean to determine if PLCz on stallion sperm is associated with fertility.

Nature and Clonality of the Fluoresceinated Secondary Antibody in Luminex Multiplex Bead Assays Are Critical Factors for Reliable Monitoring of Serum HLA Antibody Levels in Patients for Donor Organ Selection, Desensitization Therapy, and Assessment of the Risk for Graft Loss.

Luminex multiplex immunoassays enable simultaneous monitoring of Abs against multiple Ags in autoimmune, inflammatory, and infectious diseases. The assays are used extensively to monitor anti-HLA Abs in transplant patients for donor organ selection, desensitization, and assessing the risk for graft rejection. To monitor IgG Abs, fluoresceinated IgG constant H chain-binding polyclonal F(ab')2 (IgHPolyFab) is used as the fluoresceinated secondary Ab (2nd-Ab), whereas IgG subclasses are monitored with Fc-specific monoclonal whole IgG (FcMonoIgG). The fluorescent signal from the 2nd-Ab is measured as mean florescence intensity (MFI). When IgHPolyFab is used, the signal is amplified as a result of the binding of multiple polyclonal Fabs to the C region of primary IgH. The reliability of such amplification for Ab measurements was not validated, nor were MFIs compared with 1:1 binding of FcMonoIgG to primary Abs. Comparing the MFIs of anti-HLA Abs obtained with IgHPolyFab and FcMonoIgG against normal human sera, IVIg, and allograft recipients' sera, it was observed that the number of HLA-Abs was notably higher with IgHPolyFab than with FcMonoIgG The MFIs of anti-HLA Abs also remained higher with IgHPolyFab in the normal sera and in IVIg, but the reverse was true when the autologous and allogeneic IgG concentrations were augmented in allograft recipients. Indeed, MFIs of the de novo allo-HLA Abs were markedly higher with FcMonoIgG than with IgHPolyFab. Serum titration established the superiority of FcMonoIgG for monitoring MFIs of de novo allo-HLA Abs in allograft recipients. Avoiding false amplifications of the number and MFIs of anti-HLA IgG with FcMonoIgG may minimize immunosuppressive therapies, maximize the number of donors for patients waiting for allografts, and enable better prediction of graft rejection.

Generation and characterization of a human-mouse chimeric high-affinity antibody that detects the DYKDDDDK FLAG peptide

DYKDDDDK peptide (FLAG) is a useful tool for investigating the function and localization of proteins whose antibodies (Abs) are not available. We recently established a high-affinity monoclonal antibody (mAb) for FLAG (clone 2H8). The 2H8 Ab is highly sensitive for detecting FLAG-tagged proteins by flowcytometry and immunoprecipitation, but it can yield nonspecific signals in immunohistochemistry of mouse tissues because it is of mouse origin. In this study, we reduced nonspecific signals by generating a chimeric 2H8 Ab with Fc fragments derived from human immunoglobulin. We fused a 5′ terminal cDNA fragments for the Fab region of 2H8 mAb with 3′ terminal cDNA fragments for Fc region of human IgG1. We transfected both chimeric plasmids and purified the resulting human-mouse chimeric 2H8. The chimeric 2H8 Ab successfully detected FLAG-tagged proteins in flowcytometry with anti-human IgG secondary Ab with comparable sensitivity to 2H8 mAb. Importantly, chimeric 2H8 detected specific FLAG peptide signals without nonspecific signals in immunohistochemical analysis with mouse tissues. This human-mouse chimeric high-affinity anti-FLAG Ab will prove useful for future immunohistochemical analysis of mouse tissues.