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Goat

Goats are a species of domesticated ruminant mammal belonging to the Bovidae family.
They are known for their hardy, adaptable nature and are raised for a variety of purposes, including meat, milk, fiber, and as pets.
Goats are found in diverse habitats around the world and are an important livestock animal in many agricultural systems.
Their unique foraging abilities and tolerance for marginal environments make them a valuable resource, particularly in developing regions.
Goat research is crucial for improving production, health, and welfare, as well as understanding the role of this species in sustainable food systems.
PubCompare.ai's AI-powered platform can help streamline Goat research by locating the best protocols and identifying the most reproducible and effective methods from the literature, preprints, and patents.

Most cited protocols related to «Goat»

For in situ hybridization analysis, cryostat sections were hybridized using digoxigenin-labeled probes [45 (link)] directed against mouse TrkA or TrkB, or rat TrkC (gift from L. F. Parada). Antibodies used in this study were as follows: rabbit anti-Er81 [14 (link)], rabbit anti-Pea3 [14 (link)], rabbit anti-PV [14 (link)], rabbit anti-eGFP (Molecular Probes, Eugene, Oregon, United States), rabbit anti-Calbindin, rabbit anti-Calretinin (Swant, Bellinzona, Switzerland), rabbit anti-CGRP (Chemicon, Temecula, California, United States), rabbit anti-vGlut1 (Synaptic Systems, Goettingen, Germany), rabbit anti-Brn3a (gift from E. Turner), rabbit anti-TrkA and -p75 (gift from L. F. Reichardt), rabbit anti-Runx3 (Kramer and Arber, unpublished reagent), rabbit anti-Rhodamine (Molecular Probes), mouse anti-neurofilament (American Type Culture Collection, Manassas, Virginia, United States), sheep anti-eGFP (Biogenesis, Poole, United Kingdom), goat anti-LacZ [14 (link)], goat anti-TrkC (gift from L. F. Reichardt), and guinea pig anti-Isl1 [14 (link)]. Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) to detect apoptotic cells in E13.5 DRG on cryostat sections was performed as described by the manufacturer (Roche, Basel, Switzerland). Quantitative analysis of TUNEL+ DRG cells was performed essentially as described [27 (link)]. BrdU pulse-chase experiments and LacZ wholemount stainings were performed as previously described [46 (link)]. For anterograde tracing experiments to visualize projections of sensory neurons, rhodamine-conjugated dextran (Molecular Probes) was injected into single lumbar (L3) DRG at E13.5 or applied to whole lumbar dorsal roots (L3) at postnatal day (P) 5 using glass capillaries. After injection, animals were incubated for 2–3 h (E13.5) or overnight (P5). Cryostat sections were processed for immunohistochemistry as described [14 (link)] using fluorophore-conjugated secondary antibodies (1:1,000, Molecular Probes). Images were collected on an Olympus (Tokyo, Japan) confocal microscope. Images from in situ hybridization experiments were collected with an RT-SPOT camera (Diagnostic Instruments, Sterling Heights, Michigan, United States), and Corel (Eden Prairie, Minnesota, United States) Photo Paint 10.0 was used for digital processing of images.
Publication 2005
Anabolism Animals Antibodies Apoptosis Bromodeoxyuridine Calbindins Calretinin Capillaries Cavia Cells Diagnosis Digoxigenin DNA Nucleotidylexotransferase Domestic Sheep Goat Immunohistochemistry In Situ Hybridization In Situ Nick-End Labeling LacZ Genes Lumbar Region Mice, House Microscopy, Confocal Molecular Probes Neurofilaments Neuron, Afferent Pulse Rate Rabbits Rhodamine rhodamine dextran Root, Dorsal Staining transcription factor PEA3 tropomyosin-related kinase-B, human

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Publication 2008
1,2-dihexadecyl-sn-glycero-3-phosphocholine Alabaster austin Brain Stem Buffers Cells Cerebellum Chloroform Cholinergic Agents Cold Temperature Cycloheximide Deoxyribonucleases Digestion Dithiothreitol Endoribonucleases Ethanol G-substrate Goat HEPES inhibitors Isopropyl Alcohol Lipids Magnesium Chloride Mice, Laboratory Mice, Transgenic Motor Neurons Nonidet P-40 Polyribosomes Protease Inhibitors Purkinje Cells Ribosomal RNA RNA, Messenger Sodium Acetate Sodium Chloride Striatum, Corpus Teflon Tissues trizol
Seven millilitres of SARS-CoV-2 pseudoviruses with a titre of 1.86 × 105 TCID50/ml were pelleted through a 25% sucrose cushion by ultra-centrifugation at 100,000× g for 3 h. The layers of supernatant and sucrose were removed, and the resulting viral pellets were re-suspended in 100 μl PBS. Sixty microlitre prepared pseudoviruses were mixed with 15 μl 6× SDS-sample buffer. The mixture was heated for 5 min at 100°C. Fifteen microlitre samples were subjected to SDS-PAGE and immunoblotting. The VSV pseudotyped virus was prepared with the same procedure and used as the pseudovirus negative control, cell culture medium as negative control. The incorporation of the spike protein on the pseudovirus surface was confirmed using Western bolt with SARS-CoV-2 convalescent serum sample as the detection antibody with a 500-fold dilution. Goat anti-human IgG (Jackson ImmunoResearch, 109-035-0030) was used with a 1:8000 dilution as the secondary antibody.
Publication 2020
anti-IgG Buffers Cell Culture Techniques Cells Centrifugation Culture Media Goat Homo sapiens Immunoglobulins M protein, multiple myeloma Pellets, Drug SARS-CoV-2 SDS-PAGE Serum Sucrose Technique, Dilution Virus
A DNA panel composed of 96 U.S. goats from 6 breeds (35 Boer, 11 Kiko, 12 LaMancha, 15 Myotonic, 3 San Clemente, and 20 Spanish) was assembled to identify the most homozygous individual so as to minimize the number of scaffold conflicts due to heterozygous genomic regions57 (link). Genotypes were generated using Illumina’s Caprine53K SNP beadchip processed through Genome Studio (Illumina, Inc. San Diego, CA). The degrees of homozygosity of individuals were determined by raw counts of homozygous markers on the genotyping chip (Sayre, B.L., Sonstegard, T.S., Silverstein, J., Huson, H.J., Woodward-Greene, J., et al. Proceedings of the Brazilian Society of Animal Science, Campinas, SP, BR (2013)). Individuals were ranked by their counts of homozygous markers and the individual with the highest count was selected as the reference animal. An adult male of the San Clemente goat breed with 46.02% SNP-distance homozygosity (FROH) was selected from this survey as the reference animal.
Publication 2017
Adult Animals DNA, A-Form DNA Chips Genome Goat Heterozygote Hispanic or Latino Homozygote Males
The Cancer of the Prostate Strategic Urologic Research Endeavor (CaPSURE) is a national disease registry accruing men with biopsy-proven prostate adenocarcinoma, recruited from 40 urology practices, primarily community-based, across the United States. Informed consent is obtained from each patient under institutional review board supervision. Patients are treated according to their physicians’ usual practices, and are followed until time of death or withdrawal from the study. Additional details have been reported previously.13 (link), 14 (link) Eligibility for inclusion in the study was limited to men with prostate cancer diagnosed since 1992 who underwent prostatectomy as primary treatment and had at least six months of followup recorded in the registry. Those with clinically advanced disease (>cT3aN0M0) pre-operatively were ineligible, as were those had received neoadjuvant or adjuvant hormonal and/or radiation.
Detailed reporting of staging variables (ECE, SVI, SM) is variable among pathology reports accessioned to CaPSURE. In the main analysis, ECE, SVI, or SM reported as “unable to assess” were assumed to be negative; in a sensitivity analysis, cases without complete data for all variables were dropped. To examine whether cases with missing pathologic data (ECE, SVI, SM) differed from cases with complete data, we compared these groups with respect to their distributions of the original preoperative CAPRA score using a Wilcoxon rank-sum statistic. In all cases, patients with no lymphadenectomy performed were assumed to have negative LNI. Patients missing pathologic Gleason score and/or preoperative PSA were excluded.
The definition of biochemical recurrence was either 2 consecutive PSA values over 0.2 ng/ml15 (link) or any secondary treatment at least six months following surgery (treatment within six months was assumed to be adjuvant). Men not experiencing recurrence—including those dying of other causes—were censored at date of the last available PSA.
Publication 2011
Adenocarcinoma Biopsy Eligibility Determination Ethics Committees, Research Goat Hypersensitivity Lymph Node Excision Neoadjuvant Therapy Operative Surgical Procedures Patients Pharmaceutical Adjuvants Physicians Prostate Cancer Prostatectomy Prostatic Diseases Radiotherapy Recurrence Supervision

Most recents protocols related to «Goat»

Example 1

a. Materials and Methods

i. Vector Construction

1. Virus-Like Particle

As most broadly neutralizing HPV antibodies are derived from the highly conserved N-terminal region of L2, amino acids 14-122 of HPV16 L2 were used to create HBc VLPs. L2 with flanking linker regions was inserted into the tip of the a-helical spike of an HBc gene copy which was fused to another copy of HBc lacking the L2 insert. This arrangement allows the formation of HBc dimers that contain only a single copy of L2, increasing VLP stability (Peyret et al. 2015). This heterodimer is referred to as HBche-L2. A dicot plant-optimized HPV16 L2 coding sequence was designed based upon the sequence of GenBank Accession No. CAC51368.1 and synthesized in vitro using synthetic oligonucleotides by the method described (Stemmer et al., 1995). The plant-optimized L2 nucleotide sequence encoding residues 1-473 is posted at GenBank Accession No. KC330735. PCR end-tailoring was used to insert Xbal and SpeI sites flanking the L2 aa 14-122 using primers L2-14-Xba-F (SEQ ID NO. 1: CGTCTAGAGTCCGCAACCCAACTTTACAAG) and L2-122-Spe-R (SEQ ID NO. 2: G GGACTAGTTGGGGCACCAGCATC). The SpeI site was fused to a sequence encoding a 6His tag, and the resulting fusion was cloned into a geminiviral replicon vector (Diamos, 2016) to produce pBYe3R2K2Mc-L2(14-122)6H.

The HBche heterodimer VLP system was adapted from Peyret et al (2015). Using the plant optimized HBc gene (Huang et al., 2009), inventors constructed a DNA sequence encoding a dimer comprising HBc aa 1-149, a linker (G2S)5G (SEQ ID NO. 39), HBc aa 1-77, a linker GT(G4S)2 (SEQ ID NO. 40), HPV-16 L2 aa 14-122, a linker (GGS)2GSSGGSGG (SEQ ID NO. 41), and HBc aa 78-176. The dimer sequence was generated using multiple PCR steps including overlap extensions and insertion of BamHI and SpeI restriction sites flanking the L2 aa 14-122, using primers L2-14-Bam-F (SEQ ID NO. 3: CAGGATCCGCAACC CAACTTTACAAGAC) and L2-122-Spe-R (SEQ ID NO. 2). The HBche-L2 coding sequence was inserted into a geminiviral replicon binary vector pBYR2eK2M (FIG. 3), which includes the following elements: CaMV 35S promoter with duplicated enhancer (Huang et al., 2009), 5′ UTR of N. benthamiana psaK2 gene (Diamos et al., 2016), intron-containing 3′ UTR and terminator of tobacco extensin (Rosenthal et al, 2018), CaMV 35S 3′ terminator (Rosenthal et al, 2018), and Rb7 matrix attachment region (Diamos et al., 2016).

2. Recombinant Immune Complex

The recombinant immune complex (RIC) vector was adapted from Kim et al., (2015). The HPV-16 L2 (aa 14-122) segment was inserted into the BamHI and SpeI sites of the gene encoding humanized mAb 6D8 heavy chain, resulting in 6D8 epitope-tagged L2. The heavy chain fusion was inserted into an expression cassette linked to a 6D8 kappa chain expression cassette, all inserted into a geminiviral replicon binary vector (FIG. 3, RIC vector). Both cassettes contain CaMV 35S promoter with duplicated enhancer (Huang et al., 2009), 5′ UTR of N. benthamiana psaK2 gene (Diamos et al., 2016), intron-containing 3′ UTR and terminator of tobacco extensin (Rosenthal et al, 2018), and Rb7 matrix attachment region (Diamos et al., 2016).

ii. Agroinfiltration of Nicotiana benthamiana Leaves

Binary vectors were separately introduced into Agrobacterium tumefaciens EHA105 by electroporation. The resulting strains were verified by restriction digestion or PCR, grown overnight at 30° C., and used to infiltrate leaves of 5- to 6-week-old N. benthamiana maintained at 23-25° C. Briefly, the bacteria were pelleted by centrifugation for 5 minutes at 5,000 g and then resuspended in infiltration buffer (10 mM 2-(N-morpholino)ethanesulfonic acid (MES), pH 5.5 and 10 mM MgSO4) to OD600=0.2, unless otherwise described. The resulting bacterial suspensions were injected by using a syringe without needle into leaves through a small puncture (Huang et al. 2004). Plant tissue was harvested after 5 DPI, or as stated for each experiment. Leaves producing GFP were photographed under UV illumination generated by a B-100AP lamp (UVP, Upland, CA).

iii. Protein Extraction

Total protein extract was obtained by homogenizing agroinfiltrated leaf samples with 1:5 (w:v) ice cold extraction buffer (25 mM sodium phosphate, pH 7.4, 100 mM NaCl, 1 mM EDTA, 0.1% Triton X-100, 10 mg/mL sodium ascorbate, 0.3 mg/mL PMSF) using a Bullet Blender machine (Next Advance, Averill Park, NY) following the manufacturer's instruction. To enhance solubility, homogenized tissue was rotated at room temperature or 4° C. for 30 minutes. The crude plant extract was clarified by centrifugation at 13,000 g for 10 minutes at 4° C. Necrotic leaf tissue has reduced water weight, which can lead to inaccurate measurements based on leaf mass. Therefore, extracts were normalized based on total protein content by Bradford protein assay kit (Bio-Rad) with bovine serum albumin as standard.

iv. SDS-PAGE and Western Blot

Clarified plant protein extract was mixed with sample buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 0.02% bromophenol blue) and separated on 4-15% polyacrylamide gels (Bio-Rad). For reducing conditions, 0.5M DTT was added, and the samples were boiled for 10 minutes prior to loading. Polyacrylamide gels were either transferred to a PVDF membrane or stained with Coomassie stain (Bio-Rad) following the manufacturer's instructions. For L2 detection, the protein transferred membranes were blocked with 5% dry milk in PBST (PBS with 0.05% tween-20) overnight at 4° C. and probed with polyclonal rabbit anti-L2 diluted 1:5000 in 1% PBSTM, followed by goat anti-rabbit horseradish peroxidase conjugate (Sigma). Bound antibody was detected with ECL reagent (Amersham).

v. Immunization of Mice and Sample Collection

All animals were handled in accordance to the Animal Welfare Act and Arizona State University IACUC. Female BALB/C mice, 6-8 weeks old, were immunized subcutaneously with purified plant-expressed L2 (14-122), HBche-L2 VLP, L2 RIC, or PBS mixed 1:1 with Imject® Alum (Thermo Scientific, Rockford, IL). In all treatment groups, the total weight of antigen was set to deliver an equivalent 5 μg of L2. Doses were given on days 0, 21, and 42. Serum collection was done as described (Santi et al. 2008) by submandibular bleed on days 0, 21, 42, and 63.

vi. Antibody Measurements

Mouse antibody titers were measured by ELISA. Bacterially-expressed L2 (amino acids 11-128) was bound to 96-well high-binding polystyrene plates (Corning), and the plates were blocked with 5% nonfat dry milk in PBST. After washing the wells with PBST (PBS with 0.05% Tween 20), the diluted mouse sera were added and incubated. Mouse antibodies were detected by incubation with polyclonal goat anti-mouse IgG-horseradish peroxidase conjugate (Sigma). The plate was developed with TMB substrate (Pierce) and the absorbance was read at 450 nm. Endpoint titers were taken as the reciprocal of the lowest dilution which produced an OD450 reading twice the background. IgG1 and IgG2a antibodies were measured with goat-anti mouse IgG1 or IgG2a horseradish peroxidase conjugate.

vii. Electron Microscopy

Purified samples of HBche or HBche-L2 were initially incubated on 75/300 mesh grids coated with formvar. Following incubation, samples were briefly washed twice with deionized water then negatively stained with 2% aqueous uranyl acetate. Transmission electron microscopy was performed with a Phillips CM-12 microscope, and images were acquired with a Gatan model 791 CCD camera.

viii. Statistical Analysis

The significance of vaccine treatments and virus neutralization was measured by non-parametric Mann-Whitney test using GraphPad prism software. Two stars (**) indicates p values <0.05. Three stars (***) indicates p values <0.001.

b. Design and Expression of HBc VLPs and RIC Displaying HPV16 L2

BeYDV plant expression vectors (FIG. 3) expressing either the target VLP HBche-L2, or L2 and HBche alone as controls, were agroinfiltrated into the leaves of N. benthamiana and analyzed for VLP production. After 4-5 days post infiltration (DPI), leaves displayed only minor signs of tissue necrosis, indicating that the VLP was well-tolerated by the plants (FIG. 4A). Leaf extracts analyzed by reducing SDS-PAGE showed an abundant band near the predicted size of 51 kDa for HBche-L2, just above the large subunit of rubisco (RbcL). HBche was detected around the predicted size of 38 kDa (FIG. 4B). Western blot probed with anti-L2 polyclonal serum detected a band for HBche-L2 at ˜51 kDa (FIG. 4B). These results indicate that this plant system is capable of producing high levels of L2-containing HBc VLP.

To express L2-containing MC, amino acids 14-122 of HPV16 L2 were fused with linker to the C-terminus of the 6D8 antibody heavy chain and tagged with the 6D8 epitope (Kim et al. 2015). A BeYDV vector (FIG. 3) expressing both the L2-fused 6D8 heavy chain and the light chain was agroinfiltrated into leaves of N. benthamiana and analyzed for RIC production. To create more homogenous human-type glycosylation, which has been shown to improve antibody Fc receptor binding in vivo, transgenic plants silenced for xylosyltransferase and fucosyltransferase were employed (Castilho and Steinkellner 2012). By western blot, high molecular weight bands >150 kDa suggestive of RIC formation were observed (FIG. 4C). Expression of soluble L2 RIC was lower than HBche-L2 due to relatively poor solubility of the RIC (FIG. 4C).

After rigorous genetic optimization, the N. benthamiana system is capable of producing very high levels of recombinant protein, up to 30-50% of the total soluble plant protein, in 4-5 days (Diamos et al. 2016). Using this system, we produced and purified milligram quantities of fully assembled and potently immunogenic HBc VLPs displaying HPV L2 through a simple one-step purification process (FIGS. 4A-4C and 6).

c. Purification and Characterization of HBche-L2 and L2 RIC

To assess the assembly of HBc-L2 VLP, clarified plant extracts containing either HBche-L2 or HBche were analyzed by sucrose gradient sedimentation. HBche-L2 sedimented largely with HBche, which is known to form VLP, though a small increase in density was observed with HBche-L2, perhaps due to the incorporation of L2 into the virus particle (FIG. 5A). To demonstrate particle formation, sucrose fractions were examined by electron microscopy. Both HBche and HBche-L2 formed ˜30 nm particles, although the appearance of HBche-L2 VLP suggested slightly larger, fuller particles (FIGS. 5C and 5D). As most plant proteins do not sediment with VLP, pooling peak sucrose fractions resulted in >95% pure HBche-L2 (FIG. 5B), yielding sufficient antigen (>3 mg) for vaccination from a single plant leaf.

L2 RIC was purified from plant tissue by protein G affinity chromatography. By SDS-PAGE, an appropriately sized band was visible >150 kDa that was highly pure (FIG. 5B). Western blot confirmed the presence of L2 in this band, indicating proper RIC formation (FIG. 5B). L2 RIC bound to human complement C1q receptor with substantially higher affinity compared to free human IgG standard, suggesting proper immune complex formation (FIG. 5E).

d. Mouse Immunization with HBche-L2 and L2 RIC

Groups of Balb/c mice (n=8) were immunized, using alum as adjuvant, with three doses each of 5 μg L2 delivered as either L2 alone, HBche-L2 VLP, L2 RIC, or a combination of half VLP and half RIC. VLP and RIC, alone or combined, greatly enhanced antibody titers compared to L2 alone by more than an order of magnitude at all time points tested (FIG. 6). After one or two doses, the combined VLP/RIC treatment group outperformed both the VLP or RIC groups, reaching mean endpoint titers of >200,000, which represent a 700-fold increase over immunization with L2 alone (FIG. 6). After the third dose, both the VLP and combined VLP/RIC groups reached endpoint titers >1,300,000, a 2-fold increase over the RIC alone group. To determine the antibody subtypes produced by each treatment group, sera were assayed for L2-binding IgG1 and IgG2a. All four groups produced predominately IgG1 (FIG. 7, note dilutions). However, RIC and especially VLP-containing groups had an elevated ratio of IgG2a:IgG1 (>3-fold) compared to L2 alone (FIG. 7).

In vitro neutralization of HPV16 pseudovirions showed that the VLP and RIC groups greatly enhanced neutralization compared to L2 alone (FIG. 5, p<0.001). Additionally, VLP and RIC combined further enhanced neutralization activity ($5-fold, p<0.05) compared to either antigen alone, supporting the strong synergistic effect of delivering L2 by both platforms simultaneously.

In this study, by displaying amino acids 11-128 on the surface of plant-produced HBc VLPs, L2 antibody titers as high as those seen with L1 vaccines were generated (FIG. 6). Mice immunized with L2 alone had highly variable antibody titers, with titers spanning two orders of magnitude. By contrast, the other groups had much more homogenous antibody responses, especially the VLP-containing groups, which had no animals below an endpoint titer of 1:1,000,000 (FIG. 6). These results underscore the potential of HBc VLP and RIC to provide consistently potent immune responses against L2. Moreover, significant synergy of VLP and RIC systems was observed when the systems were delivered together, after one or two doses (FIG. 6). Since equivalent amounts of L2 were delivered with each dose, the enhanced antibody titer did not result from higher L2 doses. Rather, these data suggest that higher L2-specific antibody production may be due to augmented stimulation of L2-specific B cells by T-helper cells that were primed by RIC-induced antigen presenting cells. Although treatment with VLP and RIC alone reached similar endpoint titers as the combined VLP/RIC group after 3 doses, virus neutralization was substantially higher (>5-fold) in the combined group (FIG. 8). Together, these data indicate unique synergy exists when VLP and RIC are delivered together. Inventors have observed similarly significant synergistic enhancement of immunogenicity for a variety of other antigens.

Mice immunized with L2 alone had highly variable antibody titers, with titers spanning two orders of magnitude. By contrast, the VLP and VLP/RIC groups had much more homogenous antibody responses, with no animals below an endpoint titer of 1:1,000,000 (FIG. 6). These results underscore the potential of HBc VLP and RIC to provide consistently potent immune responses against L2.

Fc gamma receptors are present on immune cells and strongly impact antibody effector functions such as antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity (Jefferis 2009). In mice, these interactions are controlled in part by IgG subtypes. IgG1 is associated with a Th2 response and has limited effector functions. By contrast, IgG2a is associated with a Th1 response and more strongly binds complement components (Neuberger and Raj ewsky 1981) and Fc receptors (Radaev 2002), enhancing effector functions and opsonophagocytosis by macrophages (Takai et al. 1994). Immunization with L2 alone was found to produce low levels of IgG2a, however immunization with RIC and VLP produced significant increases in IgG2a titers. VLP-containing groups in particular showed a 3-fold increase in the ratio of IgG2a to IgG1 antibodies (FIG. 7). Importantly, production of IgG2a is associated with successful clearance of a plethora of viral pathogens (Coutelier et al. 1988; Gerhard et al. 1997; Wilson et al. 2000; Markine-Goriaynoff and Coutelier 2002).

The glycosylation state of the Fc receptor also plays an important role in antibody function. Advances in glycoengineering have led to the development of transgenic plants with silenced fucosyl- and xylosyl-transferase genes capable of producing recombinant proteins with authentic human N-glycosylation (Strasser et al. 2008). Antibodies produced in this manner have more homogenous glycoforms, resulting in improved interaction with Fc gamma and complement receptors compared to the otherwise identical antibodies produced in mammalian cell culture systems (Zeitlin et al. 2011; Hiatt et al. 2014; Strasser et al. 2014; Marusic et al. 2017). As the known mechanisms by which RIC vaccines increase immunogenicity of an antigen depend in part on Fc and complement receptor binding, HPV L2 RIC were produced in transgenic plants with silenced fucosyl- and xylosyl-transferase. Consistent with these data, we found that L2 RIC strongly enhanced the immunogenicity of L2 (FIG. 6). However, yield suffered from insolubility of the RIC (FIG. 4C). We found that the 11-128 segment of L2 expresses very poorly on its own in plants and may be a contributing factor to poor L2 RIC yield. Importantly, we have produced very high yields of RIC with different antigen fusions. Thus, in some aspects, antibody fusion with a shorter segment of L2 could substantially improve the yield of L2 RIC.

e. Neutralization of HPV Pseudovirions

Neutralization of papilloma pseudoviruses (HPV 16, 18, and 58) with sera from mice immunized IP with HBc-L2 VLP and L2(11-128) showed neutralization of HPV 16 at titers of 400-1600 and 200-800, respectively (Table 1). More mice IP-immunized with HBc-L2 VLP had antisera that cross-neutralized HPV 18 and HPV 58 pseudoviruses, compared with mice immunized with L2(11-128). Anti-HBc-L2 VLP sera neutralized HPV 18 at titers of 400 and HPV 58 at titers ranging from 400-800 (Table 1), while anti-L2(11-128) sera neutralized HPV 18 at a titer of 200 and HPV 58 at a titer of 400 (Table 1). None of the sera from intranasal-immunized mice demonstrated neutralizing activity, consistent with lower anti-L2 titers for intranasal than for intraperitoneal immunized mice.

TABLE 1
L2-specific serum IgG and pseudovirus neutralization
titers from IP immunized mice
Neutralization of Pseudoviruses
ImmunogenSerum IgGHPV 16HPV 18HPV 58
HBc-L2>50,000 400
~70,0001600400400
>80,0001600400800
L2 (11-128)~8000 200
~12,000 400
~50,000 800200400

Patent 2024
3' Untranslated Regions 5' Untranslated Regions AA 149 Agrobacterium tumefaciens aluminum potassium sulfate aluminum sulfate Amino Acids Animals Animals, Transgenic Antibodies Antibody Formation Antigen-Presenting Cells Antigens B-Lymphocytes Bacteria Bromphenol Blue Buffers Cell Culture Techniques Cells Centrifugation Chromatography, Affinity Cloning Vectors Cold Temperature Combined Modality Therapy complement 1q receptor Complement Receptor Complex, Immune Complex Extracts Cytotoxicities, Antibody-Dependent Cell Cytotoxin Digestion DNA, A-Form DNA Sequence Edetic Acid Electron Microscopy Electroporation Enzyme-Linked Immunosorbent Assay Epitopes ethane sulfonate Fc Receptor Females Formvar Fucosyltransferase G-substrate Gamma Rays Genes Genes, vif Glycerin Goat Helix (Snails) Helper-Inducer T-Lymphocyte Homo sapiens Homozygote Horseradish Peroxidase Human papillomavirus 16 Human papillomavirus 18 Human Papilloma Virus Vaccine IGG-horseradish peroxidase IgG1 IgG2A Immune Sera Immunoglobulin Heavy Chains Immunoglobulins Immunologic Factors Institutional Animal Care and Use Committees Introns Inventors L2 protein, Human papillomavirus type 16 Light Macrophage Mammals Matrix Attachment Regions Mice, Inbred BALB C Microscopy Milk, Cow's Morpholinos Mus Necrosis Needles Nicotiana Oligonucleotide Primers Oligonucleotides Open Reading Frames Opsonophagocytosis Papilloma Pathogenicity Plant Development Plant Extracts Plant Leaves Plant Proteins Plants Plants, Transgenic polyacrylamide gels Polystyrenes polyvinylidene fluoride prisma Protein Glycosylation Proteins Punctures Rabbits Receptors, IgG Recombinant Proteins Replicon Reproduction Response, Immune Ribulose-Bisphosphate Carboxylase Large Subunit Satellite Viruses SDS-PAGE Serum Serum Albumin, Bovine Sodium Ascorbate Sodium Chloride sodium phosphate Specimen Collection Stars, Celestial Strains Sucrose Sulfate, Magnesium Syringes System, Immune Technique, Dilution Tissue, Membrane Tissues Transferase Transmission Electron Microscopy Triton X-100 Tromethamine Tween 20 Ultraviolet Rays uranyl acetate Vaccination Vaccines Vaccines, Recombinant Virion Viroids Virus Vision Western Blotting xylosyltransferase
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Example 4

Aim

The aim of the study was to evaluate the ability of selected CD40 and CEACAM5 targeting RUBY™ bsAbs to bind both their targets simultaneously as well as their potential cross-reactivity with additional members of the CEA protein family was evaluated by ELISA.

Materials and Methods

96-well plates were coated with 0.5 μg/mL antigen, hCEACAM-1 (2244-CM-050, R&D Systems), hCEACAM-5 (4128-CM-050, R&D Systems), hCEACAM-6 (3934-CM-050, R&D Systems) or CEACAM-8 (9639-CM-050, R&D Systems) in PBS over night at 4° C. After washing in PBS/0.05% Tween 20 (PBST), the plates were blocked with PBST, 2% BSA for at least 30 minutes at room temperature before a second round of washing. RUBY bsAbs, diluted in PBST, 0.5% BSA, were then added and allowed to bind for at least 1 hour at room temperature. After washing, plates were incubated with either 50 μl detection antibody (0.5 μg/ml HRP conjugated goat anti human-kappa light chain, #STAR127P, AbD Serotec) for analysis of binding to CEACAM protein family proteins or 0.5 μg/ml biotinylated hCD40-muIg (504-030, Ancell) followed by HRP conjugated streptavidin (21126, Pierce) for confirmation of dual antigen binding. Finally, a final round of washing was performed and bound complexes detected using SuperSignal Pico Luminescent as substrate and luminescence signals were measured using Fluostar Optima.

Results and Conclusions

All evaluated RUBY™ bsAbs was indeed able to bind to both CD40 and human CEACAM5 simultaneously (FIG. 2), although with varying potency. In general, bsAbs carrying 1132 as CD40 binding antibody (Multi46-Multi49) displayed lower potency in the dual target ELISA, as compared to bsAbs carrying G12_mut. Also, Multi38 displayed reduced dual target binding compared to other G12_mut based bsAbs, likely due to lower CEACAM5 binding of Fab6 than other evaluated CEACAM5 binding antibodies.

As can be seen in FIG. 3, a majority of the evaluated CD40 and CEACAM5 targeting RUBY™ bsAbs did not cross react with any of the other CEA family members evaluated. However, a limited number of the assayed bsAb did show significant cross-reactivity with CEACAM1 (Multi38, Multi39, Multi45 and Multi 49) or CEACAM6 (Multi40).

All in all, it can be concluded that all evaluated RUBY™ bsAbs have the ability to bind CD40 and CEACAM5 simultaneously and a majority of the set was specific for CEACAM5, with no or little detectable binding to other evaluated members of the CEA protein family.

Patent 2024
Antibodies Antigens biliary glycoprotein I Binding Proteins Carcinoembryonic Antigen carcinoembryonic antigen-related cell adhesion molecule 6, human Cross Reactions Enzyme-Linked Immunosorbent Assay Family Member Gene Products, Protein Goat Homo sapiens Immunoglobulin kappa-Chains Immunoglobulins Luminescence Streptavidin Tween 20 Vision

Example 2

Bovine serum albumin (BSA), erbB2 extracellular domain (HER2) and streptavidin (100 μl of 2 μg/ml) were separately coated on Maxisorp 96 well plates. After blocking with 0.5% Tween-20 (in PBS), biotinylated and non-biotinylated hu4D5Fabv8-ThioFab-Phage (2×1010 phage particles) were incubated for 1 hour at room temperature followed by incubation with horseradish peroxidase (HRP) labeled secondary antibody (anti-M13 phage coat protein, pVIII protein antibody). FIG. 8 illustrates the PHESELECTOR Assay by a schematic representation depicting the binding of Fab or ThioFab to HER2 (top) and biotinylated ThioFab to streptavidin (bottom).

Standard HRP reaction was carried out and the absorbance was measured at 450 nm. Thiol reactivity was measured by calculating the ratio between OD450 for streptavidin/OD450 for HER2. A thiol reactivity value of 1 indicates complete biotinylation of the cysteine thiol. In the case of Fab protein binding measurements, hu4D5Fabv8 (2-20 ng) was used followed by incubation with HRP labeled goat polyclonal anti-Fab antibodies.

Patent 2024
Anti-Antibodies Bacteriophage M13 Bacteriophages Biological Assay Biotinylation Cardiac Arrest Cysteine ERBB2 protein, human Goat herstatin protein, human Horseradish Peroxidase Immunoglobulins Proteins Serum Albumin, Bovine Streptavidin Sulfhydryl Compounds Tween 20
Not available on PMC !

EXAMPLE 8

In order to determine whether Nanobodies could inhibit the interaction of native CD80 and CD86 with CD28-Ig or CTLA4-Ig, Raji cells were incubated with serial dilutions of purified protein from confirmed clones or an irrelevant Nanobody. Next, either HuCD28-HuIgG1 or HuCTLA4-HuIgG1 was added to the cells/Nanobody suspension without washing the cells in between. After a wash step, cell-bound CD28- or CTLA4-HuIg was revealed using a phycoerythrin-conjugated F(ab′)2 derived from affinity purified goat-anti-human IgG1 antiserum (bovine serum protein crossabsorbed). Percentage inhibition was determined based on MFI values of controls having received an irrelevant specificity Nanobody (high control) or no CD28- or CTLA4-Ig fusion protein at all (low control).

Example FACS profiles of representative inhibitory and non-inhibitory Nanobodies are shown in FIG. 7.

Results from both ELISA and FACS based assays are summarized in Table C-6.

Patent 2024
Biological Assay Bos taurus Cardiac Arrest Cells Clone Cells CTLA-4-Ig CTLA4 protein, human Enzyme-Linked Immunosorbent Assay Goat Homo sapiens IgG1 Immune Sera Phycoerythrin Proteins Psychological Inhibition Serum Proteins Technique, Dilution VHH Immunoglobulin Fragments

Example 6

To assess the binding of the chimeric anti-FOLR1 mAbs to cells that are known to express FOLR1, SK-OV-3 (ATCC® HTB-77™) cells were plated at 80,000 cells per well on a 96-well non-binding U-bottom plate (Greiner Bio-One, Cat #: 650901). In some experiments, 90,000 cells per well were used. The cells were incubated in 50 μL FACS buffer (HBSS with 0.1% BSA and 0.05% sodium azide) containing mAbs at different concentrations on ice for 15 minutes. After wash, cells were incubated in 50 μL FACS buffer containing 3.5 μg/mL F(ab′)2-Goat anti-Human IgG Fc conjugated to Alexa Fluor® 488 (Invitrogen, Cat #: H10120) on ice for 15 minutes in dark and washed again. Cells were analyzed using an Attune NxT flow cytometer. The results for the binding of the chimeric anti-FOLR1 mAbs to SK-OV-3 cells are shown in FIGS. 3A-3D. MFI, mean fluorescence intensity.

Patent 2024
alexa fluor 488 Buffers Cells Chimera Figs Fluorescence FOLR1 protein, human Goat Hemoglobin, Sickle Homo sapiens Immunoglobulin G Monoclonal Antibodies Sodium Azide

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More about "Goat"

Goats are a versatile species of ruminant mammals belonging to the Bovidae family.
These hardy, adaptable animals are raised for a variety of purposes, including meat, milk, fiber, and even as beloved pets.
Goats can be found in diverse habitats around the world and are an essential livestock in many agricultural systems, particularly in developing regions.
Goat research is crucial for improving production, health, and welfare, as well as understanding the role of this species in sustainable food systems.
PubCompare.ai's AI-powered platform can help streamline your goat-related studies by locating the best protocols and identifying the most reproducible and effective methods from the literature, preprints, and patents.
Optimizing your goat research with PubCompare.ai's innovative solution can involve leveraging various techniques and materials, such as PVDF membranes for protein analysis, DAPI for nuclear staining, Triton X-100 for cell permeabilization, Alexa Fluor 488 for fluorescent labeling, Bovine serum albumin for blocking, Hoechst 33342 for live-cell imaging, RIPA lysis buffer for protein extraction, and FBS for cell culture.
With the Odyssey Infrared Imaging System, you can even visualize and quantify your experimental results with high precision.
By incorporating these tools and techniques, you can enhance your goat research and gain valuable insights that contribute to the understanding and advancement of this important livestock species.
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