Production Of Polyclonal Antibodies Research Articles

Establishment of primary chicken embryo myoblast cell culture, antigenic epitopes prediction and production of anti activin receptor type IIB polyclonal antibody in chicken

The detection of activin receptor typeIIB (ACTRIIB) protein, a prominent negative muscle growth regulator has paramount value in augmenting growth traits through molecular breeding schemes in chicken. The study was formulated to establish primary chicken embryo myoblast culture (CEM) using 9th and 18th day chick embryos and to develop antibodies for immunodetection of ACTRIIB protein. The physicochemical and structural attributes of the ACTRIIB sequence were evaluated to identify substantial antigenic regions. The ACTRIIB sequence was transfected into CEM and expressed protein was injected subcutaneously into rats to produce hyperimmune serum. The average propensity of protein sequence for beta turns, surface accessibility, chain flexibility, antigenicity, hydrophilicity and linear epitopes was 0.978, 1.000, 0.991, 1.038, 1.258 and 0.512, respectively. The 9th day CEM exhibited confluency (80–90%) earlier than the 18th day. The expression of myogenic regulatory factors in 9th day myoblasts was higher than the 18th day by 7.28, 5.16, 6.28 and 6.93 folds for MYF5, MRF4, MYOG and MYOD, respectively. The ACTRIIB mRNA was downregulated by 2.54 folds on the 9th day compared to the 18th day myoblasts and protein varied significantly between 9th and 18th day myoblasts. The CEM culture can be harnessed unequivocally to investigate molecular mechanisms underlying muscle growth besides raising antibodies.

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Antibodies have important role in biological research and diagnosis. Polyclonal antibodies can be produced against more than one epitope of an antigen. Newcastle disease (ND) is one of the most devastating diseases that considerably effects the global poultry industry. Newcastle disease virus (NDV) possess Fusion (F) protein for the attachment and pathogenicity. In the present study TA plasmid having F gene was restricted by EcoR1 and it was ligated into pET30a expression vector. The F gene ligated pET30a was transformed in BL21DE3 expression strain. After the expression of NDV F protein, it was verified by the appearance of 67 kDa band on SDS-PAGE gel and western blot. The F protein was partially purified by the column of 70 kDa cut. The recombinant F protein was used for the generation of polyclonal antibodies. Two groups (experimental group and negative control group) of 3 months old rabbits were used. Two doses of recombinant F protein with interval of 15 days were injected subcutaneously for production of antibodies against the F protein. Antibodies were obtained from serum taken after scarifying the rabbits. Production of polyclonal antibodies was confirmed through Dot Blot Assay and ELISA. In this study maximum antibody titer was estimated to be 4.676 (OD at 450 nm) in case of using concentrated antibody. Produced polyclonal antibodies can be further purified in order to use as tools in biomedical and biochemical researches and diagnostic kits

PSVIII-2 Antibody yield for polyclonal egg yolk antibody (IgY) production in laying hens

Abstract Antibody production in egg yolks of immunized laying hens is an alternative to conventional mammalian production. Antibody yield peak and duration have not been described for immunoglobulin Y technology using Freund’s incomplete adjuvant (FIA) and C-phosphate-guanosine oligodeoxynucleotides (CpG-ODN) without the inclusion of Freund’s complete adjuvant for enhancing the immune response to an interleukin-10 (IL-10) peptide. This study sought to describe the antibody titer production for an 8 amino acid sequence from the surface of the bovine IL-10 protein (VMPQAENG) as the antigen emulsified with CpG-ODN and FIA in phosphate buffered saline (PBS). 60 hens were assigned to receive the complete vaccine (Peptide), 20 received the vaccine without the IL-10 peptide (Control), and 8 received a PBS injection (Blank). Hens were immunized with 0.25 mL in 4 locations, each breast and each thigh on days 1, 15 and 29. The complete vaccine delivered 0.6 mg IL-10 peptide, 8 µg CpG-ODN, and 0.33 mL FIA per hen on each vaccination day. Eggs were collected regularly until 175 days after the first immunization and the anti IL-10 peptide activities of the yolk were determined by ELISA. Egg titers by treatment were analyzed with a repeated measures ANOVA in SAS. The supplementation of FIA with CpG-ODN produced high titers, of over 100 µg of antibody per mL of yolk (µg Ab/mL yolk), around day 33 through day 76, with a slow decline through day 175 when average titers remained above 40 µg Ab/mL yolk. Peptide egg titers were significantly higher than Blank or Control titers from day 31 though day 175 (P < 0.0001). Titers recovered from Marcq et al. (2015) with similar methods were 1.5 to 7 times lower than these results over the same number of days.

Production and application of polyclonal antibodies against SARS-CoV-2 viral spike protein. Development to rapid, highly sensitive diagnosis kit for early Corona viral detection among the population.

Pathogenic novel coronavirus SARS-CoV-2 is a recently rising infection that causes a high death rate in contaminated individuals. Many of the individuals who are infected are unaware of the infection because they may be asymptomatic carriers of the virus.To early analyze the viral disease, we proposed advancement of SARS-CoV-2 spike (S) proteins-explicit polyclonal antibodies-based kit described in this specific circumstance. The tests, if made accessible from variety of makers, can help smooth out early immunodetection of the crown viral infection. The survey Safe and Patient Friendly Healthcare suggests early diagnosis as the key to patients recovering from this deadly ailment.

A Simple and Efficient Genetic Immunization Protocol for the Production of Highly Specific Polyclonal and Monoclonal Antibodies against the Native Form of Mammalian Proteins.

We have generated polyclonal and monoclonal antibodies by genetic immunization over the last two decades. In this paper, we present our most successful methodology acquired over these years and present the animals in which we obtained the highest rates of success. The technique presented is convenient, easy, affordable, and generates antibodies against mammalian proteins in their native form. This protocol requires neither expensive equipment, such as a gene gun, nor sophisticated techniques such as the conjugation of gold microspheres, electroporation, or surgery to inject in lymph nodes. The protocol presented uses simply the purified plasmid expressing the protein of interest under a strong promoter, which is injected at intramuscular and intradermal sites. This technique was tested in five species. Guinea pigs were the animals of choice for the production of polyclonal antibodies. Monoclonal antibodies could be generated in mice by giving, as a last injection, a suspension of transfected cells. The antibodies detected their antigens in their native forms. They were highly specific with very low non-specific background levels, as assessed by immune-blots, immunocytochemistry, immunohistochemistry and flow cytometry. We present herein a detailed and simple procedure to successfully raise specific antibodies against native proteins.

Production and purification of polyclonal antibody against attenuated and wild type Leishmania infantum in dogs.

Antibodies are still widely used in several programs including early research, imaging, Targeting drug delivery system, Affinity chromatography, flowcytometry technic, diagnosis and treatment. Purification of antibody is a standard approach for detection of infection agent in different species. The reservoir hosts for Leishmania infantum are Dogs and they have active role in the transmission of leishmania to humans by the bite of a sand fly belonging to genus Phlebotomus and Lutzomiya. Consequently, elimination of dogs in endemic areas and vaccination of dogs contributes to reduction of the human and canine VL cases. Serological antibody tests such as IFAT (Indirect Fluorescent Antbody Test), DFAT (Direct Fluorescent Antbody Test), ELISA (Enzyme-Linked Immunosorbent Assay), PCR (Polymerase chain Reaction Assay) have been extensively used to investigate canine infection with L. infantum. In this study we produced and purified polyclonal antibody against attenuated and wild type leishmania infantum in dogs. Anti-leishmania in dog serums precipitated with ammonium sulphate. The IgG recovered from ammonium sulphate precipitation was subject to ion exchange chromatography (IEC) and the purity of IgG was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) under reduced condition. The purity of proteins were above 95% and then purified IgG was conjugated with FITC. We determined optimum titer of dog IgG by observation parasites under fluorescent microscope. The optimum dilution of prepared FITC conjugated dog IgG was 1: 400. This polyclonal antibody can be used for other applications in research, diagnosis and clinic.

Preparation and Characterization of Two Immunogens and Production of Polyclonal Antibody with High Affinity and Specificity for Darunavir.

Darunavir (DRV) is a potent antiviral drug used for treatment of infections with human immunodeficiency virus (HIV). Effective and safe treatment with DRV requires its therapeutic drug monitoring (TDM) in patient’s plasma during therapy. To support TDM of DRV, a specific antibody with high affinity is required in order to develop a sensitive immunoassay for the accurate determination of DRV in plasma. In this study, two new and different immunogens were prepared and characterized. These immunogens were the DRV conjugates with keyhole limpet hemocyanin (KLH) protein. The first immunogen (DRV-KLH) was prepared by zero-length direct linking of DRV via its aromatic amino group with the tyrosine amino acid residues of KLH by diazotization/coupling reaction. The second immunogen (G-DRV-KLH) was prepared by conjugation of the N-glutaryl derivative of DRV (G-DRV) with KLH. The 5-carbon atoms-spacing G-DRV hapten was synthesized by reaction of DRV via its aromatic amino group with glutaric anhydride. The reaction was monitored by HPLC and the chemical structure of G-DRV was confirmed by mass, 1H-NMR, and 13C-NMR spectroscopic techniques. The hapten (G-DRV) was linked to the KLH protein by water-soluble 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) coupling procedure. The pertinence of the coupling reactions of haptens to protein was confirmed, and the immunogens were characterized by ultraviolet (UV) spectrophotometry. Both DRV-KLH and G-DRV-KLH were used for the immunization of animals and the animal’s antiserum that showed the highest affinity was selected. The collected antiserum (polyclonal antibody) had very high affinity to DRV (IC50 value = 0.2 ng mL−1; defining IC50 as the DRV concentration that can inhibit antibody binding by 50% of its maximum binding) and high specificity to DRV among other drugs used in the combination therapy with DRV. Cumulative results from direct and competitive enzyme-linked immunosorbent assay (ELISA) using this polyclonal antibody proved that the immunogens were highly antigenic and elicited a specific polyclonal antibody. The produced polyclonal antibody is valuable for the development of highly sensitive and selective immunoassays for TDM of DRV.

In-silico characterization and RNA-binding protein based polyclonal antibodies production for detection of citrus tristeza virus

Citrus tristeza virus (CTV) is the etiologic agent of the destructive Tristeza disease, a massive impediment for the healthy citrus industry worldwide. Routine indexing of CTV is an essential component for disease surveys and citrus budwood certification for production of disease-free planting material. Therefore, the present study was carried out to develop an efficient serological assay for CTV detection based on the RNA binding protein (CTV-p23), which is translated from a subgenomic RNA (sgRNA) that accumulates at higher levels in CTV-infected plants. CTV-p23 gene was amplified, cloned and polyclonal antibodies were raised against recombinant CTV-p23 protein. The efficacy of the produced polyclonal antibodies was tested by Western blots and ELISA to develop a quick, sensitive and economically affordable CTV detection tool and was used for indexing of large number of plant samples. The evaluation results indicated that the developed CTV-p23 antibodies had an excellent diagnostic agreement with RT-PCR and would be effective for the detection of CTV in field samples. Furthermore, CTV-p23 gene specific primers designed in the present study were found 1000 times more sensitive than the reported coat protein (CTV-p25) gene specific primers for routine CTV diagnosis. In silico characterizations of CTV-p23 protein revealed the presence of key conserved amino acid residues that involved in the regulation of protein stability, suppressor activity and protein expression levels. This would provide precious ground information towards understanding the viral pathogenecity and protein level accumulation for early diagnosis of virus.

Kinetic analysis of effects of temperature and time on the regulation of venom expression in Bungarus multicinctus

Venom gland is a highly efficient venom production system to maintain their predatory arsenal. Venom toxins mRNA has been shown to increase abruptly in snake after venom expenditure, while the dynamics of venom accumulation during synthesis are poorly understood. Here, PacBio long-read sequencing, Illumina RNA sequencing (RNA-seq), and label-free proteome quantification were used to investigate the composition landscape and time- and temperature-dependent dynamics changes of the Bungarus multicinctus venom gland system. Transcriptome data (19.5223 Gb) from six adult B. multicinctus tissues were sequenced using PacBio RS II to generate a reference assembly, and average 7.28 Gb of clean RNA-seq data was obtained from venom glands by Illumina sequencing. Differentially expressed genes (DEGs) mainly were protein processing rather than venom toxins. Post-translational modifications provided the evidence of the significantly different proportions of toxins in the venom proteome with the changing of replenishment time and temperature, but constant of venom toxins mRNA in the venom gland transcriptome. Dynamic of toxins and genes involved in venom gland contraction suggesting the formation of the mature venom gland system would take at least 9 days. In addition, 59 toxin processing genes were identified, peptidylprolyl isomerase B of which underwent positive selection in Toxicofera. These results provide a reference for determining the extraction time of venom, production of polyclonal and monoclonal antibody for precise treatment plans of venomous snakebites, and construction of an in vitro synthesis system for snake venom protein.

HETEROLOGOUS EXPRESSION OF TWO ACETYLCHOLINESTERASES OF COLORADO POTATO BEETLE LEPTINOTARSA DECEMLINEATA IN BACTERIA ESCHERICHIA COLI AND PRODUCTION OF FORM-SPECIFIC ANTIBODIES

The Colorado potato beetle Leptinotarsa decemlineata is a widespread pest of plants of the Solanaceae family. Huge economic loss caused by L. decemlineata around the world is multiplied by its ability to develop resistance to all major insecticide classes. Previously, such resistance was found to be associated with mutations in the target enzyme LdAChE2, orthologous to Drosophila melanogaster acetylcholinesterase. However, discovery of the second form of L. decemlineata acetylcholinesterase LdAChE1 has changed this view. In order to compare the role of two acetylcholinesterase forms in the Colorado potato beetle physiology and in pest resistance to insecticides, gene copies were cloned and their heterologous expression in bacteria E. coli was followed by production of polyclonal antibodies against the recombinant proteins. Immunoblotting with produced antibodies demonstrated the absence of cross-reactivity, a lower content of LdAChE1 in the tissues of L. decemlineata adults compared with the second form, and the association of LdAChE2 with membranes. Further immunoaffinity purification of natural enzymes from the beetle tissues as well as their heterologous expression in insect cell cultures should help to evaluate the role of each form in physiology of the pest and in its resistance to insecticides.

Rabbit anti-deer polyclonal antibody applied to the diagnosis of Mycobacterium avium subsp. paratuberculosis in red deer (Cervus elaphus)

Abstract The diagnosis of Johne’s Disease by isolating Mycobacterium avium subsp. paratuberculosis from feces requires 6 months and high-cost reagents, and therefore an indirect technique like ELISA, is often being used to avoid these obstacles. The aim of this work was the production of a rabbit anti-deer polyclonal antibody (anti-deer pAb) for use in an in-house ELISA, applying protoplasmic antigen paratuberculosis (PPA) as antigen. Two rabbits were immunized subcutaneously with three doses of deer semipurified gamma globulin emulsified with incomplete Freund's adjuvant. Rabbit serum was purified with protein A. Titers of anti-deer pAb against deer serum and cross-reaction against sera from other species were studied by ELISA and immunoblot. Samples of feces and sera from 16 deer were evaluated by fecal culture and the in-house PPA-ELISA. The results were analyzed statistically using ROC curves and the Kappa index to estimate sensitivity (Se), specificity (Sp) and the agreement level between both techniques. Subsequently, sera from three to five years old stags (n = 155) from a herd with reports of Johne’s Disease were evaluated using the in-house PPA-ELISA. Anti-deer pAb showed a specific titer of 2.56 × 105. Cross-reaction against cattle, sheep, llama and goat was detected by ELISA and immunoblot. The in-house PPA-ELISA showed a Se = 85.71 % and Sp = 88.89 %. The agreement level with fecal culture was substantial (κ = 0.62 ± 0.19) and the assay was able to identify 76.77 % of the deer in the suspected herd as positive. The anti-deer pAb obtained was not only useful in the development of the in-house PPA-ELISA, which allows us to reduce the costs of Johne’s Disease diagnosis, but it could also be used in other diagnosis tests in deer as well as other ruminant species.

Evaluation of Human Anti Igg Polyclonal Antibody Production Conjugated with Peroxidase in Egg Yolk

Egg yolk is a rich and accessible source of yolk immunoglobulin (Y immunoglobulin). Presently, polyclonal antibodies from mammalian sources are used for diagnosis. Antibody production from egg yolk gives a higher yield and turnover than that from lab animals, and invasive methods such as phlebotomy and causing stress to the animals are not required. Due to the issues regarding mammalian antibodies, we aimed to evaluate the human anti-IgG polyclonal antibody production conjugated with peroxidase in egg yolk. Population of laying hens reared in Agriculture/Isfahan University of Technology were used in 2017. After immunizing hen against pure human IgG, specific IgY (yolk immunoglobulin) was purified from the yolk by sedimentation with polyethylene glycol (PEG6000). To assess the molecular weight and activity of the product, SDS-PAGE and ELISA-test were used, respectively. The complete molecular weight of IgY was 180 kDa and the molecular weight of its light and heavy chains were 27 and 67 kDa, respectively. Antihuman IgG IgY had a purity above 90%. The product of this study can be used to measure IgG class antibodies in order to diagnose different diseases.

Production of Polyclonal Antibodies against Pregnancy Associated Glycoprotein in Cattle

Pregnancy-associated glycoproteins (PAGs) are considered to be a large gene family in the trophoblasts of ruminants. PAGs determination precisely, PAG-1 in serum has been as the solid ground for pregnancy diagnosis in cow. Unluckily the PAG-1 and the antigenically related PAGs show a long half-life in serum less than 8 days and can be assessed 80 to 100 das postpartum, thereafter giving false results in cows bred within 60 days of calving. This study involves using novel polyclonal antibodies after immunizing rabbits and purification of the produced antibodies. The cattle are pregnant when the PAG hormone level is measured in a high level in a serum sample from the animal. It could give a precise method of detecting pregnancy at the early with few false positive results.

Synthetic Oligomers Mimicking Capsular Polysaccharide Diheteroglycan are Potential Vaccine Candidates against Encapsulated Enterococcal Infections.

Infections caused by Enterococcus spp. are a major concern in the clinical setting. In Enterococcus faecalis, the capsular polysaccharide diheteroglycan (DHG), composed of ß-d-galactofuranose-(1 → 3)-ß-d-glucopyranose repeats, has been described as an important virulence factor and as a potential vaccine candidate against encapsulated strains. Synthetic structures emulating immunogenic polysaccharides present many advantages over native polysaccharides for vaccine development. In this work, we described the synthesis of a library of DHG oligomers, differing in length and order of the monosaccharide constituents. Using suitably protected thioglycoside building blocks, oligosaccharides up to 8-mer in length built up from either Galf-Glcp or Glcp-Galf dimers were generated, and we evaluated their immunoreactivity with antibodies raised against DHG. After the screening, we selected two octasaccharides, having either a galactofuranose or glucopyranose terminus, which were conjugated to a carrier protein for the production of polyclonal antibodies. The resulting antibodies were specific toward the synthetic structures and mediated in vitro opsonophagocytic killing of different encapsulated E. feacalis strains. The evaluated oligosaccharides are the first synthetic structures described to elicit antibodies that target encapsulated E. faecalis strains and are, therefore, promising candidates for the development of a well-defined enterococcal glycoconjugate vaccine.

Purification of hyaluronidase from Naja melanoleuca venom and production of polyclonal antibodies

Purification of hyaluronidase from Naja melanoleuca venom and production of polyclonal antibodies

Polyclonal antibodies production from porcine gelatin and its preliminary study for immunosensor applications

Gelatin was extracted from porcine skin and used as an antigen to produce the polyclonal antibodies of porcine gelatin using New Zealand white rabbits for application in the development of an ELISA-based immunosensor for porcine gelatin recognition. The extraction of porcine gelatin from porcine skin was successfully performed with 20.52 % of yield. The characterization performed by using UV and IR spectroscopy indicated that the purified gelatin has a comparable quality against the commercial standard of porcine gelatin. Further analysis of the gelatin with SDS-PAGE method observed five separation lines with molecular weight bands of 57.06; 49.50; 42.94; 33.89; and 29.40 KDa, indicating that the gelatin can be used as an antigen to produce the polyclonal antibodies of porcine gelatin. After five injections of gelatin into the rabbit, ELISA characterization of the purified serum of the blood rabbit showed that the antibody with a concentration of 0.289 mg/mL could be produced.

Immunodiagnosis of bunchy top viruses in abaca with polyclonal antibodies against their recombinant coat proteins

Polyclonal antibodies against abaca bunchy top virus (ABTV) and banana bunchy top virus (BBTV) proteins are necessary for immuno-based detection of these two viruses in abaca (Musa textilis Nee). In this study, recombinant bunchy top viral coat proteins fused with a 6xHis tag at the N-terminus were expressed in E. coli BL21StarTM(DE3)pLysS strain and purified under denaturing conditions. Purified recombinant ABTV and BBTV coat proteins were used as antigens for the production of rabbit polyclonal antibodies. IgG was purified and evaluated by Direct Antigen Coating (DAC)-ELISA and further optimized by testing primary to secondary antibody dilution combinations. Analysis of ABTV and BBTV-infected abaca samples using the optimized DAC-ELISA assay showed that the anti-ABTV CP IgG can react to BBTV and that anti-BBTV CP IgG can react to ABTV, hence, a cross-reaction. The study demonstrates the advantage of using recombinant DNA technology for mass production of antigens for antibody production. Although specificity of the polyclonal antibodies may have been compromised when renatured recombinant proteins were used as immunogens, the ability of the purified IgGs to detect positive abaca samples reveals that the DAC-ELISA can be routinely used for screening disease-free abaca planting materials.

Production and Characterization of Polyclonal Antibodies for Diagnosis of Pregnancy in Cattle

Production and Characterization of Polyclonal Antibodies for Diagnosis of Pregnancy in Cattle

Immunogenicity and antitumor efficacy of a novel human PD-1 B-cell vaccine (PD1-Vaxx) and combination immunotherapy with dual trastuzumab/pertuzumab-like HER-2 B-cell epitope vaccines (B-Vaxx) in a syngeneic mouse model

ABSTRACT Therapeutic blockade of PD-1/PD-L1 signaling with monoclonal antibodies (mAbs) has shown clinical success and activity across a broad set of cancer subtypes. However, monotherapy with PD-1/PD-L1 inhibitors are only effective in a subset of patients and ongoing studies show efficacy of treatment depends on a combinatorial approach. Contrary to mAbs chimeric B-cell cancer vaccines incorporating a “promiscuous” T-cell epitope have the advantage of producing a polyclonal B-cell antibody that can potentially induce memory B- and T-cell responses, while reducing immune evasion and suppression. Here, we describe a novel PD-1 B-cell peptide epitope vaccine (amino acid 92–110; PD1-Vaxx) linked to a measles virus fusion peptide (MVF) amino acid 288–302 via a four amino acid residue (GPSL) emulsified in Montanide ISA 720VG that aims to induce the production of polyclonal antibodies that block PD-1 signaling and thus trigger anticancer effects similar to nivolumab. In preclinical studies, the PD1-Vaxx outperformed the standard anti-mouse PD-1 antibody (mAb 29F.1A12) in a mouse model of human HER-2 expressing colon carcinoma. Furthermore, the combination of PD1-Vaxx with combo HER-2 peptide vaccine (B-Vaxx) showed enhanced inhibition of tumor growth in colon carcinoma BALB/c model challenged with CT26/HER-2 cells. The PD-1 or combined vaccines were safe with no evidence of toxicity or autoimmunity.

Production of polyclonal antibodies to the coat protein gene of Indian isolate of Apple stem grooving virus expressed through heterologous expression and its use in immunodiagnosis

Apple stem grooving virus (ASGV) belonging to the genus Capillovirus under family Betaflexiviridae is one of the widely distributed latent viruses mainly on pome fruit trees. It infects apple causing considerable economic losses that is a threat to the apple industry. To study the occurrence and incidence of ASGV disease, sensitive antisera based diagnostic tool has been developed, which would be helpful in large scale indexing, certification and quarantine programmes. Coat protein gene of ASGV was cloned into pET-32a(+) and pHIS-Parallel expression vectors. Expression characteristics of the proteins expressed from both the systems were compared and taken for raising the antisera. Sensitivity and specificity of the antiserum against virus infection was compared by Double Antibody Sandwich (DAS-ELISA). It was observed that the antisera raised from the protein expressed from pHIS-Parallel expression system (smaller ~ 3 kDa His tag) was more sensitive as compared to the antisera raised from the protein expressed from pET-32a(+) expression system (~ 18 kDa His tag) and the commercially available antisera. The antisera could also be successfully used for western blotting of infected samples and in DAS-ELISA. Thus, this study presents the first report on production of polyclonal antiserum against recombinant coat protein of ASGV from India, for reliable detection of the virus.