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Microbiological Research Feb 2016Chlamydia psittaci (C. psittaci), an obligate intracellular agent of psittacosis, causes an atypical pneumonia in humans. The transmembrane head proteins (TMH) of C....
Chlamydia psittaci (C. psittaci), an obligate intracellular agent of psittacosis, causes an atypical pneumonia in humans. The transmembrane head proteins (TMH) of C. psittaci, putatively belong to the Inc family and presumably play similar roles. CPSIT_0844 and CPSIT_0846 were the putative TMH proteins of C. psittaci. To identify these two proteins, antisera were raised with fusion proteins which were prokaryotic expressed in Escherichia coli and purified. By immunofluorescence assay, CPSIT_0844 and CPSIT_0846 were localized in the inclusion membrane of C. psittaci-infected cells. By RT-PCR and western blot analysis to detect the temporal expression, CPSIT_0844 and CPSIT_0846 were detected as early as 12h post-infection (p.i.) and 6h p.i., separately; meanwhile, in secretions monitored with immunofluorescence assay, these proteins were observed in the inclusion membrane at 18h p.i. and remained in the inclusion membrane throughout the growth cycle. CPSIT_0844 and CPSIT_0846 could specifically be recognized by the antiserum of C. psittaci but failed to react with the antiserums of Chlamydia trachomatis and Chlamydia pneumoniae, which is consistent with the fact that they had no significant orthologs in C. trachomatis and C. pneumoniae. These results revealed that CPSIT_0844 and CPSIT_0846, the putative TMH family proteins, might be unique to C. psittaci and could be used to diagnose the infection caused by C. psittaci. Moreover, CPSIT_0844 and CPSIT_0846 could induce the expression of the inflammatory cytokines IL-1β, IL-6 and TNF-α in THP-1 cells, which might contribute to chlamydia-induced inflammatory pathologies.
Topics: Bacterial Outer Membrane Proteins; Bacterial Proteins; Chlamydia Infections; Chlamydophila psittaci; Cloning, Molecular; Cytokines; Escherichia coli; Fluorescent Antibody Technique; Genes, Bacterial; HeLa Cells; Humans; Immune Sera; Psittacosis; Recombinant Proteins; Tumor Necrosis Factor-alpha
PubMed: 26805615
DOI: 10.1016/j.micres.2015.11.005 -
Ukrainian Biochemical Journal 2015A previously unknown phenomenon of acquired polyreactivity for serum immunoglobulins, which were subjected either to solutions of KSCN (3.0-5.0 M), low/high pH (pH... (Review)
Review
A previously unknown phenomenon of acquired polyreactivity for serum immunoglobulins, which were subjected either to solutions of KSCN (3.0-5.0 M), low/high pH (pH 2.2-3.0), or heating to 58-60 degrees C, was described by us in 1990 year. Much later, eleven years after that, similar data were published by others, which completely confirmed our results concerning the influence of either chaotropic ions or the drastic shift of pH on immunoglobulins polyreactive properties. Our further investigations of polyreactive serum immunoglobulins (PRIG) properties have shown that the mechanism of non-specific interaction between PRIG and antigens much differs from the mechanism of interaction between specific antibodies and corresponding antigens. Later we have shown that the increasing of PRIG reactivity could be induced in vivo, and PRIG are one of serum components for human or animal sera. Then, it could be suggested that PRIG can perform certain biological functions. Studying of PRIG's effect on the phagocytosis of microbes by peritoneal cells or the tumor growth have shown that PRIG can play a certain role in protecting the body from infections and probably can influence on the development of various pathological processes. Recently we have also found that PRIG IgG contents significantly increases in aged people. These data demonstrate that further investigations of PRIG's immunochemical properties and studying of their biological role in organism protection from various diseases is very intriguing and important.
Topics: Aging; Animals; Antibody Specificity; Antigen-Antibody Reactions; Antigens; Enzyme-Linked Immunosorbent Assay; Humans; Hydrogen-Ion Concentration; Immune Sera; Immunoglobulins; Protein Binding; Thiocyanates
PubMed: 26502695
DOI: 10.15407/ubj87.03.005 -
Environmental Science & Technology Apr 2016A sensitive, competitive enzyme-linked immunosorbent assay (ELISA) for the detection of the antimicrobial triclosan (TCS; 2,4,4'-trichloro-2'-hydroxydiphenyl ether) was...
A sensitive, competitive enzyme-linked immunosorbent assay (ELISA) for the detection of the antimicrobial triclosan (TCS; 2,4,4'-trichloro-2'-hydroxydiphenyl ether) was developed. Novel immunizing haptens were synthesized by derivatizing at the 4-Cl position of the TCS molecule. Compounds derived from substitutions at 4'-Cl and that replaced the 2'-OH with a Cl atom were designed as unique coating antigen haptens. Polyclonal rabbit antisera were screened against the coating antigen library to identify combinations of immunoreagents resulting in the most sensitive assays. The most sensitive assay identified was one utilizing antiserum no. 1155 and a heterologous competitive hapten, where the 2'-OH group was substituted with a Cl atom. An IC50 value and the detection range for TCS in assay buffer were 1.19 and 0.21-6.71 μg/L, respectively. The assay was selective for TCS, providing low cross-reactivity (<5%) to the major metabolites of TCS and to brominated diphenyl ether-47. A second assay utilizing a competitive hapten containing Br instead of Cl substitutions was broadly selective for both brominated and chlorinated diphenylethers. Using the most sensitive assay combination, we measured TCS concentrations in water samples following dilution. Biosolid samples were analyzed following the dilution of a simple solvent extract. The immunoassay results were similar to those determined by LC-MS/MS. This immunoassay can be used as a rapid and convenient tool to screen for human and environmental exposure.
Topics: Animals; Anti-Infective Agents; Cross Reactions; Enzyme-Linked Immunosorbent Assay; Female; Halogenated Diphenyl Ethers; Haptens; Immune Sera; Rabbits; Reproducibility of Results; Tandem Mass Spectrometry; Triclosan; Water Pollutants, Chemical
PubMed: 26937944
DOI: 10.1021/acs.est.5b05357 -
Infection, Genetics and Evolution :... Jul 2017In recent years, novel Bluetongue virus (BTV) serotypes have been isolated and/or sequenced by researchers within the field. During Bluetongue surveillance activities,...
In recent years, novel Bluetongue virus (BTV) serotypes have been isolated and/or sequenced by researchers within the field. During Bluetongue surveillance activities, we identified a putative novel BTV serotype in healthy goats from Sardinia, Italy. RNAs purified from blood and serum samples were positive for BTV by a generic real time RT-PCR and c-ELISA, respectively, whereas genotyping and serotyping were unsuccessful. By NGS, the whole genome sequence was obtained from two blood samples (BTV-X ITL2015 strains 34200 and 33531). Overall, Seg 2 of BTV-X ITL2015 shows the highest identity (75.3-75.5% nt/77.4-78.1% aa) with recently isolated BTV-27s from Corsica and with the last discovered BTV XJ1407 from China (75.9% nt /78.2% aa), whereas it is less related with BTV-25 from Switzerland (73.0% nt/75.0% aa) and BTV-26 from Kuwait (62.0% nt/60.5% aa). A specific RT-qPCR targeting Seg 2 of BTV-X ITL2015 was assessed in this study. Considering the Seg 2/VP2 identity of BTV-X ITL2015 with BTV-25, 26, 27s and BTV XJ1407 and that serum of BTV-X ITL2015 infected goats failed to neutralize all tested extant serotypes, we propose the existence of a novel BTV serotype circulating in goats in Sardinia. Isolation was so far unsuccessful thus hampering proper antigenic characterization.
Topics: Amino Acid Sequence; Animals; Asymptomatic Diseases; Bluetongue; Bluetongue virus; Enzyme-Linked Immunosorbent Assay; Epidemiological Monitoring; Genome, Viral; Goats; High-Throughput Nucleotide Sequencing; Immune Sera; Italy; Phylogeny; RNA, Viral; Reverse Transcriptase Polymerase Chain Reaction; Serogroup
PubMed: 28341545
DOI: 10.1016/j.meegid.2017.03.021 -
Turkish Journal of Medical Sciences Oct 2021SARS-CoV-2 disease was announced as a pandemic by The World Health Organization in early 2020. It is still threatening the world population. Here, we aimed to produce...
BACKGROUND/AIM/AIM
SARS-CoV-2 disease was announced as a pandemic by The World Health Organization in early 2020. It is still threatening the world population. Here, we aimed to produce hyperimmune sera that contain immunoglobulin G and F(ab')2 fragments sourced from horse antibodies as an urgent response to the pandemic.
MATERIALS AND METHODS
SARS-CoV-2 was produced and inactivated with three different methods [formaldehyde (FA), formaldehyde, and binary ethylene amine (FA + BEI), and heat treatment]. After in vitro inactivation control, immunogens were mixed with Freund’s adjuvant, thereafter horses (n: 2 for FA, 4 for FA + BEI, 2 for heat inactivation) and New Zealand rabbits (n: 6 for FA, 6 fo r FA + BEI, 6 for heat inactivation) were immunized four times. Neutralizing antibody levels of the sera were measured at the 4th, 6th, and 8th weeks. When the antibodies were detected at the peak level, plasma was collected from horses and hyperimmune sera procured after the purification process.
RESULTS
Horses and rabbits produced highly neutralizing antibodies against the SARS-CoV-2 in FA and FA + BEI inactivation groups, foreign proteins were removed effectively after purification.
CONCLUSION
This study presents a profitable practice to develop specific antisera in horses against SARS-CoV-2 for emergency and low-cost response. In further studies, new purification methods can be used to increase the efficiency of the final product.
Topics: Animals; Horses; Immune Sera; Immunologic Factors; Rabbits; SARS-CoV-2; COVID-19 Drug Treatment
PubMed: 34092050
DOI: 10.3906/sag-2101-304 -
Free Radical Biology & Medicine Feb 2019The only general technique that allows the unambiguous detection of free radicals is electron spin resonance (ESR). However, ESR spin trapping has severe limitations... (Review)
Review
The only general technique that allows the unambiguous detection of free radicals is electron spin resonance (ESR). However, ESR spin trapping has severe limitations especially in biological systems. The greatest limitation of ESR is poor sensitivity relative to the low steady-state concentration of free radical adducts, which in cells and in vivo is much lower than the best sensitivity of ESR. Limitations of ESR have led to an almost desperate search for alternatives to investigate free radicals in biological systems. Here we explore the use of the immuno-spin trapping technique, which combine the specificity of the spin trapping to the high sensitivity and universal use of immunological techniques. All of the immunological techniques based on antibody binding have become available for free radical detection in a wide variety of biological systems.
Topics: Animals; Antibodies, Monoclonal; Chickens; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Enzyme-Linked Immunosorbent Assay; Free Radicals; Haptens; Immune Sera; Limit of Detection; Nitrogen Oxides; Pyrroles; Rabbits; Spin Labels; Spin Trapping; Vaccination
PubMed: 30552998
DOI: 10.1016/j.freeradbiomed.2018.11.009 -
Endocrinology Jul 2016Estrogens regulate normal sexual and reproductive development in females. Their actions are mediated mainly by estrogen receptor (ER)α and ERβ. Understanding the...
Estrogens regulate normal sexual and reproductive development in females. Their actions are mediated mainly by estrogen receptor (ER)α and ERβ. Understanding the function of ERs necessitates knowing their cellular location and protein partners, which, in turn, requires reliable and specific antibodies. Several antibodies are available for ERα; however, discrepancies in immunoreactivity have been reported for ERβ. Here, we have developed antisera for mouse ERβ (mERβ) using a specific C-terminal 18-amino acid peptide conjugated to mariculture keyhole limpet hemocyanin. Sprague Dawley rats were immunized, and the resulting antisera were characterized by Western blot analysis of nuclear extracts from tissues of wild-type (WT) mice, and mice genetically modified to lack either ERα (CERαKO) or ERβ (CERβKO). An approximately 56-kDa protein was detected in the hypothalamus, uterus, ovary, mammary gland, testes, and epididymis of WT mice, consistent with the predicted molecular size of ERβ. In addition, the same protein band was identified in in vitro synthesized mERβ protein and in the mammary glands of CERαKO mice. The approximately 56-kDa protein was not observed in in vitro synthesized mERα protein or in any tissue examined in the CERβKO mice. Immunohistochemistry using the antisera revealed ERβ staining in the granulosa cells of WT ovaries and in the mediobasal hypothalamus, paraventricular nucleus, and cerebral cortex in the WT adult mouse brain. These data suggest that the novel rat anti-mERβ sera are specific to ERβ to allow investigators to explore to cellular and physiological role of ERβ in the brain and other mouse tissues.
Topics: Animals; Epididymis; Estrogen Receptor beta; Female; Hypothalamus; Immune Sera; Male; Mammary Glands, Animal; Mice; Ovary; Rats; Rats, Sprague-Dawley; Testis; Uterus
PubMed: 27105387
DOI: 10.1210/en.2016-1122 -
Cold Spring Harbor Protocols Sep 2021If antibodies against a particular antigen are available, that antigen can be purified and used for further immunizations, and antigens thus purified can show enhanced...
If antibodies against a particular antigen are available, that antigen can be purified and used for further immunizations, and antigens thus purified can show enhanced immunogenicity. Purified immune complexes can be injected directly, or while coupled to beads; the presence of antibodies and/or beads stimulates phagocytosis and usually will not influence the response. This method provides a useful means of antigen enrichment for a variety of applications, such as using antibodies raised against a denatured antigen to harvest a native protein for further immunizations, or when using a monoclonal antibody as an intermediate to the preparation of polyclonal antisera. Injecting antibody-coated antigens has also been used to mask a particularly immunodominant epitope on an antigen, and thereby develop a response against other epitopes. The amount of antigen needed to elicit a strong response using immune complexes will vary from one compound to another. Doses as low as 50 ng of antigen have been used successfully when delivered this way.
Topics: Antibodies, Monoclonal; Antigen-Antibody Complex; Antigens; Epitopes; Immune Sera
PubMed: 34470860
DOI: 10.1101/pdb.prot099978 -
JCI Insight Sep 2020Preexisting humoral immunity to recombinant adeno-associated virus (AAV) vectors restricts the treatable patient population and efficacy of human gene therapies....
Preexisting humoral immunity to recombinant adeno-associated virus (AAV) vectors restricts the treatable patient population and efficacy of human gene therapies. Approaches to clear neutralizing antibodies (NAbs), such as plasmapheresis and immunosuppression, are either ineffective or cause undesirable side effects. Here, we describe a clinically relevant strategy to rapidly and transiently degrade NAbs before AAV administration using an IgG-degrading enzyme (IdeZ). We demonstrate that recombinant IdeZ efficiently cleaved IgG in dog, monkey, and human antisera. Prophylactically administered IdeZ cleaved circulating human IgG in mice and prevented AAV neutralization in vivo. In macaques, a single intravenous dose of IdeZ rescued AAV transduction by transiently reversing seropositivity. Importantly, IdeZ efficiently cleaved NAbs and rescued AAV transduction in mice passively immunized with individual human donor sera representing a diverse population. Our antibody clearance approach presents a potentially new paradigm for expanding the prospective patient cohort and improving efficacy of AAV gene therapy.
Topics: Animals; Antibodies, Neutralizing; Antibodies, Viral; Dependovirus; Dogs; Genetic Therapy; Humans; Immune Sera; Immunity, Innate; Immunization, Passive; Immunoglobulin G; Macaca; Mice; Proteolysis; Recombinant Proteins; Transduction, Genetic
PubMed: 32941184
DOI: 10.1172/jci.insight.139881 -
Methods in Molecular Biology (Clifton,... 2021Antibodies against Streptococcus pneumoniae (pneumococcus) following vaccination are crucial for host protection against invasive pneumococcal infections. The antibodies...
Antibodies against Streptococcus pneumoniae (pneumococcus) following vaccination are crucial for host protection against invasive pneumococcal infections. The antibodies induced by pneumococcal vaccines act as opsonins to mediate bacterial uptake and killing by host phagocytic cells, especially polymorphonuclear leukocytes (PMNs) also called neutrophils. Therefore, it is important to measure not only the levels of antibodies induced by a pneumococcal vaccine candidate but their actual functional capacity in mediating bacterial opsonization and killing by PMNs. Here, we describe a protocol to demonstrate effective deposition of vaccine-induced antibodies on the surface of S. pneumoniae by flow cytometry and subsequent opsonophagocytic killing (OPH) by murine bone-marrow derived PMNs.
Topics: Animals; Antibodies, Bacterial; Biomarkers; Bone Marrow Cells; Flow Cytometry; Immune Sera; Mice; Neutrophils; Phagocytes; Pneumococcal Infections; Pneumococcal Vaccines; Streptococcus pneumoniae
PubMed: 32959268
DOI: 10.1007/978-1-0716-0795-4_33