Characterization Of Polyclonal Antibodies Research Articles

The Production and characterization of Polyclonal Antibodies Against Interferon Alpha in Mice

The polyclonal antibodies are used extensively for research purposes in many areas of biology, such as immunoprecipitation, histochemistry, enzyme linked immunosorbent assays (ELISA), diagnosis of disease and western blots. Typically, an animal’s immune system will generate a large group of antibodies that recognize several epitopes of a particular antigen. Interferon alpha plays an important role in immune response activation and therefore is of interest in studies related to autoimmune diseases. In this paper the production of antibodies against interferon was studied in order to quantify interferon production to analyze interferon levels in autoimmune disorders in the future. For the antibody production, one month old laboratory grade mice were injected with interferon alpha in combination with a Freund’s complete adjuvant for a course of five weeks after which the antibodies were obtained in mouse serum. Confirmation of the production of anti-interferon alpha antibodies was carried out by the Elisa, immune dot blot and western blot analysis. An interferon alpha of approximately 20.5-21.5 KDa was detected in immunedot blot test. These antibodies may be produced in these mouse models commercially and could be used in future for treatment of autoimmune diseases by managing the interferon levels in the patients.
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Development and characterization of polyclonal antibodies against subtype specific vitellogenin of the dojo loach, Misgurnus anguillicaudatus

Fish vitellogenin (Vtg) has become an important biomarker for assessing the estrogenic potency of chemicals and the exposure of animals to estrogenic contaminants present in aquatic environments. In the current study, the polyclonal antibodies against subtype-specific Vtgs of the dojo loach (Misgurnus anguillicaudatus) were developed and identified by gene recombination and expression. Immuno-biochemical analyses revealed that VtgAo1 protein appeared to be the major Vtg type in this species. Enhanced chemiluminescent Western blotting was developed using the antiserum against VtgAo1. Exposure of male loach to 17α-ethinylestradiol (EE2) and 17β-estradiol (E2) via water induced the VtgAo1 protein induction with a lowest-observed-effect concentration (LOEC) by 100 ng L−1 and 1000 ng L−1group at 7 day post-initiation (dpi), respectively. The results indicated that this method provided a valuable tool for detecting estrogenic activities in aquatic environments and excluded any difficulties expected in purification procedures to separate a highly pure Vtg subtype from circulating proteins, and the other Vtg subtypes.

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

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

Characterization of polyclonal antibodies to Herpes Simplex Virus types 1 and 2

ABSTRACTInfections with herpes simplex virus (HSV) types 1 and 2 have been linked to oral, facial, genital lesions, as well as some visceral organ changes in patients under immunosuppressed conditions. Immunohistochemistry (IHC) with HSV antibodies is used for identification of the viruses in tissue samples. In this study, two polyclonal antibodies, prepared separately with HSV-1 and HSV-2 immunogens, were characterized in comparison to a monoclonal antibody to HSV-1 (10A3). The polyclonal anti-HSV-1 and monoclonal antibody 10A3 were shown to be reactive to viral proteins of both HSV-1 and HSV-2 on Western blots, while the polyclonal anti-HSV-2 was reactive to HSV-2 proteins, but not to those of HSV-1. Cross-reactivity was not observed to proteins of six other frequently encountered herpes viruses. IHC characterization was performed on 29 cases of HSV-infected tissue samples, 61 samples infected with other herpes viruses and 35 samples without known infection. By IHC, the polyclonal anti-HSV-1 and a monoclonal antibody 10A3 exhibited a signal, mainly in a nuclear pattern, in all of the HSV-infected samples and not in other tissue types. A positive signal, mainly in the cytoplasm, was identified with the polyclonal anti-HSV-2 in 21 of the 29 HSV-infected samples. Genotyping analysis was successful in 14 of the HSV-infected samples, with IHC HSV-2 positivity correlative to the HSV-2 genotype. The results demonstrate that these antibodies are useful tools for identification of HSV-1 and HSV-2, and their combinatorial application may help to distinguish between these two types of infection.

Development and characterization of polyclonal antibody against human kappa light chain in rabbit.

Polyclonal antibodies against kappa light chain are used to diagnose diseases producing free light chain. The kappa and lambda light chains are products of immunoglobulin synthesis and released into the circulation in minor amounts such as serum, cerebrospinal fluid, urine and synovial fluid in normal condition. The purpose of this study was the production and purification of polyclonal immunoglobulin G (IgG) against human kappa light chains. In this study, early human IgG was purified by ion-exchange chromatography, reduced with Dithiothreitol and heavy and light chains were separated with size-exclusion chromatography. Afterward, affinity chromatography with protein L Sepharose at pH 2.00 was displayed to be a dominant condition for the separation and purification of the kappa light chain of immunoglobulins from human serum. Eventually, the rabbit was immunized by human kappa light chains. The rabbit IgG was purified and labeled with horseradish peroxidase (HRP). Direct enzyme-linked immunosorbent assay was planned to determine the titer of HRP conjugated rabbit IgG against the human kappa light chain. The optimum titer of anti-kappa IgG was 1:16000. At the result, purified polyclonal anti-kappa is useful tool in biomedical and biochemical researches and diagnostic kits.

Production and characterization of polyclonal antibody against Bengal tiger (Panthera tigris tigris) immunoglobulin G

ABSTRACT The immunoglobulin G (IgG) antibody is important for detecting and evaluating immune responses. Despite increasing opportunities for tigers to be exposed to emerging diseases due to global changes coupled with increasing interaction with humans, there is no available antibody specific to tigers. Aims of this study were to produce a rabbit anti-Bengal tiger (Panthera tigris tigris) IgG polyclonal antibody and to determine its specificity. The molecular weight of the purified Bengal tiger IgG was approximately 170 kDa. The cross-reactivity of rabbit anti-Bengal tiger IgG to IgG of other wild felid species and other domestic animals was analyzed by indirect enzyme-linked immunosorbent assay. The cross reactivity to other related wild animals ranged from 53.69% to 75.63%. There was high cross-reactivity with domestic cats (63.58%) and pigs (38.45%) and low cross-reactivity with domestic cows (10.46%), goats (8.60%), sheep (7.25%), and chickens (6.91%). To confirm cross-reactivity with domestic animals’ IgG, western blotting was performed. These results indicated that the purified rabbit anti-Bengal tiger IgG polyclonal antibody strongly reacted with species in the family Felidae. The polyclonal antibody generated in this study has the potential to aid in the development of useful tools for further novel immunological investigations on tigers and related felid species.

Development and characterization of polyclonal antibodies against the linker region of the telomere-binding protein TRF2

Background: TRF2 (telomeric repeat binding factor 2) is an essential component of the telomere-binding protein complex shelterin. TRF2 induces the formation of a special structure of telomeric DNA and counteracts activation of DNA damage-response pathways telomeres. TRF2 has a poorly characterized linker region (udTRF2) between its homodimerization and DNA-binding domains. Some lines of evidence have shown that this region could be involved in TRF2 interaction with nuclear lamina. Results: In this study, the fragment of the TERF2 gene encoding udTRF2 domain of telomere-binding protein TRF2 was produced by PCR and cloned into the pET32a vector. The resulting plasmid pET32a-udTRF2 was used for the expression of the recombinant udTRF2 in E. coli RosettaBlue (DE3). The protein was isolated and purified using ammonium sulfate precipitation followed by ion-exchange chromatography. The purified recombinant protein udTRF2 was injected into guinea pigs to generate polyclonal antibodies. The ability of anti-udTRF2 antibodies to bind endogenous TRF2 in human skin fibroblasts was tested by western blotting and immunofluorescent staining. Conclusions: In this study, the recombinant protein udTRF2 and antibodies to it were generated. Both protein and antibodies will provide a useful tool for investigation of the functions of the udTRF2 domain and its role in the interaction between TRF2 and nuclear lamina.

Production and characterization of polyclonal antibody against Arabidopsis GIGANTEA, a circadian clock controlled flowering time regulator

Arabidopsis GIGANTEA (GI) is encoded by a single gene and highly conserved among vascular plants and its mutants display pleiotropic phenotypes involved in diverse biological processes such as light signaling, circadian clock, and sucrose metabolism as well as abiotic stress responses. However, molecular mechanisms of GI are largely unknown due to the lack of useful antibody. To date, the epitope tags have been widely used to detect GI in plants, but it needs to generate the transgenic plants which take a few months. Here, we produced polyclonal α-GI antibody using truncated variants of GI having amino-terminal (1–858 aa) and carboxyl-terminal (920–1173) regions as antigens. Both recombinant His-GI1-858 and His-GI920–1173 proteins were individually and successfully expressed in E. coli and immunized into rabbit. Anti-serum was purified by antigenspecific affinity purification method using both recombinant His-GI1–858 and His-GI920–1173 proteins. Purified polyclonal α-GI antibody not only detected endogenous GI proteins in wild-type Arabidopsis plants, but also reenacted its diel oscillations. Furthermore, the antibody showed cross-reactivity with the GI orthologs in other plants such as Chinese cabbage, rape and tomato. Our polyclonal GI antibody could help to determine the molecular mechanisms of GI involved in largely unknown pleiotropic responses in plants.

Characterization of polyclonal antibodies against nonstructural protein 9 from the porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syn- drome (PRRS) is considered to be one of the most important infectious diseases impacting the swine industry and is characterized by reproductive failure in late term gestation in sows and respiratory disease in pigs of all ages. The nonstructural protein 9 gene, Nsp9, encoding the RNA-dependent RNA polymerase, is generally regarded as fairly conserved when compared to other viral proteins. Antibodies againstNsp9 will be of great importance for the diagnosis and treatment of the causal agent, PRRS virus. A study was undertaken to generate polyclonal antibodies against the immunodominant Nsp9. For this purpose, the Nsp9 was expressed in Escherichia coli and subsequently used as an antigen to immunize New Zealand rabbits. Antiserum was identified via an indirect ELISA, and then verified based on the ability to react with both naturally and artificially expressed Nsp9. Results of virus neutrali- zation test showed that this antiserum could not neutralize the PRRSV. Nevertheless, this antiserum as a diagnostic core reagent should prove invaluable for further investiga- tions into the mechanism of PRRS pathogenesis.

Development of novel antibodies against non-structural proteins nsP1, nsP3 and nsP4 of chikungunya virus: potential use in basic research.

Chikungunya virus (CHIKV) has reemerged recently as an important pathogen, causing several large epidemics worldwide. This necessitates the development of better reagents to understand its biology and to establish effective and safe control measures. The present study describes the development and characterization of polyclonal antibodies (pAbs) against synthetic peptides of CHIKV non-structural proteins (nsPs; nsP1, nsP3 and nsP4). The reactivity of these pAbs was demonstrated by ELISA and Western blot. Additionally, in vitro infection studies in a mammalian system confirmed that these pAbs are highly sensitive and specific for CHIKV nsPs, as these proteins were detected very early during viral replication. Homology analysis of the selected epitope sequences revealed that they are conserved among all of the CHIKV strains of different genotypes, while comparison with other alphavirus sequences showed that none of them are 100% identical to the epitope sequences (except Onyong-nyong and Igbo Ora viruses, which show 100% identity to the nsP4 epitope). Interestingly, two different forms of CHIKV nsP1 and three different forms of nsP3 were detected in Western blot analysis during infection; however, further experimental investigations are required to confirm their identity. Also, the use of these antibodies demonstrated faster and enhanced expression profiles of all CHIKV nsPs in 2006 Indian outbreak strains when compared to the CHIKV prototype strain, suggesting the epidemic potential of the 2006 isolate. Accordingly, it can be suggested that the pAbs reported in this study can be used as sensitive and specific tools for experimental investigations of CHIKV replication and infection.

Preparation and characterization of polyclonal antibody against Kaposi's sarcoma-associated herpesvirus lytic gene encoding RTA.

Replication and transcription activator (RTA) is a critical lytic protein encoded by Kaposi's sarcoma-associated herpesvirus (KSHV). To prepare rabbit polyclonal antibody against RTA, three antigenic polypeptides of KSHV RTA were initially synthesized. The fragment of RTA was cloned into p3FlagBsd to construct the recombinant plasmid, pRTA-Flag. 293T and EA.hy926 cells were transfected with pRTA-Flag to obtain RTA-Flag fusion protein, which was detected using anti-Flag antibody. Next, New Zealand white rabbits were immunized with keyhole limpet hemocyanin-conjugated peptides to generate polyclonal antibodies against RTA. Enzyme-linked immunosorbent assays were performed to characterize the polyclonal antibodies, and the titers of the polyclonal antibodies against RTA were greater than 1:11,000. Western blotting and immunofluorescence assay revealed that the prepared antibody reacted specifically with the RTA-Flag fusion protein as well as the native viral protein in KSHV-infected primary effusion lymphoma cells. Collectively, our work successfully constructed the recombinant expression vector, pRTA-Flag, and prepared the polyclonal antibody against RTA, which was valuable for investigating the biochemical and biological functions of the critical KSHV lytic gene.

Preparation and Characterization of Polyclonal Antibody Against CD133 Derived from Camel

Preparation and Characterization of Polyclonal Antibody Against CD133 Derived from Camel

Generation and characterization of polyclonal antibody against part of immunoglobulin constant heavy υ chain of goose.

Immunoglobulin Y (abbreviated as IgY) is a type of immunoglobulin that is the major antibody in bird, reptile, and lungfish blood. IgY consists of two light (λ) and two heavy (υ) chains. In the present study, polyclonal antibody against IgYFc was generated and evaluated. rIgYCυ3/Cυ4 was expressed in Escherichia coli, purified and utilized to raise polyclonal antibody in rabbit. High affinity antisera were obtained, which successfully detected the antigen at a dilution of 1:204,800 for ELISA assay. The antibody can specifically recognize both rIgYCυ3/Cυ4 and native IgY by Western bolt analysis. Furthermore, the serum of Grus japonensis or immunoglobulin of chicken, duck, turkey, and silkie samples and dynamic changes of serum GoIgY after immunogenicity with GPV-VP3-virus-like particles (GPV-VP3-VLPs) can be detected with the anti-GoIgYFc polyclonal antibody. These results suggested that the antibody is valuable for the investigation of biochemical properties and biological functions of GoIgY.

Production and characterization of polyclonal antibody against a synthetic peptide from β-actin protein.

Antibodies against actin, as one of the most widely studied structural and multifunctional housekeeping proteins in eukaryotic cells, are used as internal loading controls in western blot analyses. The aim of this study was to produce polyclonal antibody against a synthetic peptide derived from N-terminal region of β-actin protein to be used as a protein loading control in western blot and other assay systems. A synthetic peptide derived from β-actin protein was designed and conjugated to Keyhole limpet hemocyanin (KLH) and used to immunize a white New Zealand rabbit. The antibody was purified from serum by affinity chromatography column. The purity of the antibody was determined by SDS-PAGE and its ability to recognize the immunizing peptide was measured by ELISA. The reactivity of the antibody with β-actin protein in a panel of different cell lysates was then evaluated by western blot. In addition, the reactivity of the antibody with the corresponding protein was also evaluated by Immunocytochemistry and Immunohistochemistry in different samples. The antibody could recognize the immunizing peptide in ELISA. It could also recognize β-actin protein in western blot as well as in immunocytochemistry and immunohistochemistry. Our data suggest that this antibody may be used as an internal control in western blot analyses as well as in other immunological applications such as ELISA, immunocytochemistry and immunohistochemistry.

Generation and Characterization of Polyclonal Antibodies Against Mouse T-cell Immunoglobulin and Immunoreceptor Tyrosine-based Inhibitory Domain by DNA-based Immunization

Mouse T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory domain (TIGIT) is a newly identified surface protein expressed in regulatory, memory, natural killer (NK), and activated T cells. Several studies indicate that mouse TIGIT is a vital immunomodulator that can control the activities of both NK and T cells and plays an important role in transplantation tolerance. In this study, we designed a vector, TIGIT-pcDNA3.1 (+), that encodes the complete coding sequence of mouse TIGIT. The vector was intramuscularly injected into rats, and then the specific antisera were harvested and purified using a protein A/G PLUS-agarose affinity column. Western blot and immunohistochemistry analyses revealed that the antibodies generated by DNA immunization can bind with the mouse TIGIT. Using these antibodies in immunoblots, TIGIT was detected in lysates of mouse organs, T cells from mouse lymph nodes, and recombinant mouse fusion protein of TIGIT and Fc fragment. Immunohistochemistry analysis of normal mouse kidney showed that immunoreactivity was located on endothelial cells of glomerular capillary loops and peritubular capillaries. Our results demonstrated that the DNA immunization of rats through intramuscular injection was a simple and easily available method of producing polyclonal antibodies that can be used to detect and analyze mouse TIGIT expression in mouse systems.

High yield expression of duck hepatitis A virus VP1 protein in Escherichia coli, and production and characterization of polyclonal antibody

VP1 protein, the capsid protein of duck hepatitis A virus (DHAV), contains critical epitopes for inducing a protective immune response. Due to its low-level expression in Escherichia coli (E. coli), the function of this protein is poorly characterized. In this study, a codon-optimized VP1 gene was chemically synthesized in terms of the codon usage bias in E. coli and subcloned into pET32a (+) to increase its expression. The recombinant VP1 fusion protein was purified from inclusion body by Ni2+ affinity chromatography His-Bind Resin and used to raise the rabbit anti-DHAV-VP1 polyclonal antibody. The expression of the codon-optimized VP1 gene in E. coli was significantly increased when compared to the wild-type VP1 gene, having an at least 17-fold increase. Western blot analysis showed that the recombinant protein was recognized by the rabbit anti-DHAV polyclonal antibody. Western blot also demonstrated that the rabbit anti-DHAV-VP1 polyclonal antibody could recognize the purified VP1 fusion protein specifically, and in the indirect immunofluorescent assays (IFA), the antibody was able to probe the VP1 protein in DHAV-1 infected cells. In conclusion, codon optimization increased dramatically DHAV VP1 expression in E. coli and the His-tagged VP1 fusion protein showed good antigenicity and immunogenicity.

Preparation and characterization of polyclonal antibody against resting spores of Olpidium virulentus, fungal vector of lettuce big-vein disease

Research on Olpidium virulentus, the vector of the serious lettuce big-vein disease, is difficult because its resting spores persist in soil and retain virus for years and the fungus is not culturable. We developed protocols to obtain purified, viable resting spores from infected roots using an enzymatic treatment and two-step density gradient centrifugation and to generate a polyclonal antibody against these spores. The antibody was species-specific in a direct immunostaining assay and in western blots produced two specific bands (ca. 30.5, 29.0 kDa) against resting spores. The detection limit for resting spores in an indirect ELISA was 200 spores/100 μL.

Production and characterization of antibodies against crosslinked gelatin nanoparticles and first steps toward developing an ELISA screening kit

Nanotechnologies are finding a growing range of applications in the food sector. Nanoparticles are used notably to add vitamins and other nutrients to foods and beverages without affecting taste and color. They are also used to develop new tastes, preserve food texture, control the release of flavors, improve the bioavailability of compounds such as antioxidants and vitamins, and monitor freshness with nanosensors. Crosslinked gelatin nanoparticles are a component of nano-sized carriers for nutrient and supplement delivery in foods and related products. This paper describes the production and characterization of polyclonal antibodies against gelatin nanoparticles. Two immunization schemes were investigated: subcutaneous injection with and without a first intravenous injection. Two enzyme-linked immunosorbent assay formats were used to characterize the antibodies: an inhibition format with an antigen-coated plate for detection of the immune response and a sandwich format for development of the method. The antibodies showed good sensitivity with an IC50 equal to 0.11 ng mL(-1) using indirect ELISA format and a good specificity for the nanomaterials, without significant cross-reactivity against native gelatin. The limit of detection was determined-0.42, 0.27, 0.26, and 0.24 μg mL(-1) for apple, orange juice, milk, and soft drink matrices, respectively. ELISA technology offers rapid, low-cost assays for screening foods, feeds, and beverages. We have studied a prototype ELISA for detection of gelatin-based nanocarrier systems. Fruit juices, milk, and a soft drink were the matrices selected for assay development.

Production and characterization of polyclonal antibodies against CD38 (159.3)

Abstract CD38 is a 42 kDa transmembrane glycoprotein widely expressed in hematopoietic cells. Anti-CD38 agonistic antibodies induce proliferation, migration, and apoptosis of B lymphocytes. Some of the available monoclonal antibodies do not recognize CD38 by Western blot. Then, we decide to prepare antisera that can be equally useful in biochemical and functional assays. To reach this goal, we used the extracellular domain of murine CD38 fused with a V5 epitope and a Histidin tag or fused with GST as antigens. These proteins were characterized by western blot, enzymatic activity and by reactivity with the mAB NIM-R5, which recognizes a conformational epitope. We immunized New Zealand rabbits and a female goat with the CD38-V5-Histidin tag fusion protein. Antibodies from both species stained cells from C57BL/6 but not from CD38 deficient mice. In addition, the polyclonal antibodies recognized in extracts from splenic cells a band of the expected weight. Finally, these antibodies induced B lymphocyte proliferation of mouse splenic cells. These results demonstrated the production of functional reagents that can be used in the analysis of CD38 in B cells.

Preparation and Characterization of Polyclonal Antibody Against Severe Acute Respiratory Syndrome-associated Coronavirus Spike Protein

A truncated gene (designated S1) encoding the receptor-binding domain (RBD) in the spike (S) protein of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) was amplified by PCR. The gene was cloned into prokaryotic expression vector pGEX-6P-1, resulting in a recombinant plasmid pGEX-SARS-S1. Subsequently, pGEX-SARS-S1 was transformed into host cells BL21(DE3)pLysS, and the expression of the S1 protein was induced by isopropyl β-D-thiogalactoside (IPTG). Polyclonal antibody against SARS-CoV S1 protein was generated in a rabbit immunized with the purified S1 protein. The reactivity of the antibody to the SARS-CoV S1 protein was confirmed by Western blot analysis. ELISA indicated that the antibody against SARS-CoV S1 protein had no cross reaction with S1 proteins of transmissible gastroenteritis virus, a porcine coronavirus, and infectious bronchitis virus, an avian coronavirus. The SARS-CoV S1 protein and its antibody are valuable reagents for related studies.