• IMMUNE NETWORK
• /
• 제14권3호
• /
• pp.164-170
• /
• 2014

JL1, a specific epitope on CD43, is a potential biomarker for the diagnosis of acute leukemia. Although qualitative assays for detecting leukemia-specific CD43 exist, there is a need to develop quantitative assays for the same. Here, we developed two novel monoclonal antibodies (mAbs), 2C8 and 8E10, recognizing different epitopes on CD43. These clones are capable of pairing with YG5, another mAb against JL1 epitope, because they were selectively obtained using sandwich ELISA. Antigens recognized by 2C8 and 8E10 were confirmed as CD43 by western blotting using the CD43-hFC recombinant protein. When expression on various leukemic cell lines was investigated, 2C8 and 8E10 displayed a disparity in the distribution of the epitope. Enzyme assays revealed that these mAbs recognized a sialic acid-dependent epitope on CD43. Using normal thymus and lymph node paraffin-embedded tissues, we confirmed a difference in the epitopes recognized by the two mAbs that was predicted based on the maturity of the cells in the tissue. In summary, we developed and characterized two mAbs, 2C8 and 8E10, which can be used with YG5 in a sandwich ELISA for detecting leukemia-specific CD43.

• 미생물과산업
• /
• 제16권2호
• /
• pp.2-9
• /
• 1990

This paper outlines research to study two aspects of Cryptooridium. First, specific antigenic determinants were identified and followed through the growth cycle of C. parvum to investigate antigenic sharing of molecular epitopes among the different life cycle stages. Secondly, the importance of passive immune protective mechanisms in cryptosporidial infection was assessed by following the course of infection in neonatal mice which have been subjected to treatments using either monoclonal antibodies (mAbs) or hyperimmune bovine colostrum.

• 한국양식학회지
• /
• 제16권2호
• /
• pp.124-127
• /
• 2003

Our previous studies of both microarray analysis in channel catfish muscle gene expression of 2 different ages and channel catfish muscle expressed sequence tag profiles demonstrated parvalbumin beta is one of the highly expressed muscle transcriptome. We have cloned and sequenced complementary DNA encoding the channel catfish parvalbumin which encode 109 amino acids. The deduced amino acid sequences of the catfish parvalbumin are highly conserved with those cloned from other teleosts. The availability of the catfish parvalbumin provides the opportunity of studying fish epitopes.

• Bulletin of the Korean Chemical Society
• /
• 제35권5호
• /
• pp.1279-1284
• /
• 2014

The binding of TATA-binding protein (TBP) to the TATA-box containing promoter region is aided by many other transcriptional factors including TFIIA and TFIIB. The mechanistic insight into the assembly of RNA polymerase II preinitation complex (PIC) has been gained by either directly altering a function of target protein or perturbing molecular interactions using drugs, RNAi, or aptamers. Aptamers have been found particularly useful for studying a role of a subset of PIC on transcription for their ability to inhibit specific molecular interactions. One major hurdle to the wide use of aptamers as specific inhibitors arises from the difficulty with traditional assays to validate and determine specificity, affinity, and binding epitopes for aptamers against targets. Here, using a technique called the bio-layer interferometry (BLI) designed for a label-free, real-time, and multiplexed detection of molecular interactions, we studied the assembly of a subset of PIC, TBP binding to TATA DNA, and two distinct classes of aptamers against TPB in regard to their ability to inhibit TBP binding to TFIIA or TATA DNA. Using BLI, we measured not only equilibrium binding constants ($K_D$), which were overall in close agreement with those obtained by electrophoretic mobility shift assay, but also kinetic constants of binding ($k_{on}$ and $k_{off}$), differentiating aptamers of comparable KDs by their difference in binding kinetics. The assay developed in this study can readily be adopted for high throughput validation of candidate aptamers for specificity, affinity, and epitopes, providing both equilibrium and kinetic information for aptamer interaction with targets.

• Asian Pacific Journal of Cancer Prevention
• /
• 제14권12호
• /
• pp.7335-7338
• /
• 2013

There is increasing evidence that natural killer (NK) cells play an important role in antitumor immunity following dendritic cell (DC) vaccination. Little is known, however, about the optimal stimulation of DCs by epitopes and NK interactions for cytotoxicity in tumors. In this study, DC cells activated by the HPV16E7.49-57 epitope and LPS were co-cultured with NK cells in vitro, and then used ot immunize mice to study CTL activity of TC-1, which constitutively expresses HPV16E6E7, with an LDH release assay. Cytotoxicity in mice immunized with DC loaded with epitope HPVE7.49-57 vaccine co-cultured with NK was enhanced significantly (p<0.01). In conclusion, talk-across between DC and NK cells enhances their functions, also improving cytotoxicity againsttumor cells, suggesting that activated DC-NK by epitopes has potential application for cancer-specific immuno-cellular therapy.

• Journal of Microbiology and Biotechnology
• /
• 제4권4호
• /
• pp.290-295
• /
• 1994

30 monoclonal antibodies (mAbs) were produced against Bacillus thuringiensis subsp. canadensis. Out of the these, 6 mAbs were selected for further studies. SDS-PAGE analyses of sonicated antigens of 10 8. thuringiensis strains showed that they generally had both predominant protein antigens of molecular weights of 45 kilodalton (kd) except for shandogiensis and konkukian, and 37kd except for israelensis, tochigiensis, and shandogiensis, respectively. These results indicate that 4kd and 37kd may be important for demonstrating common antigens except for a few strains of B. thuringiensis. In comparing the result of the westem blot using mAbs with that of using polyclonal antibodies to canadensis, we found that immunoreactive proteins of 99 and 39 kd were identified as common antigens, which might act as antigenic determinants, and might be surface or flagella antigens. Reactivities of mAbs with 41 strains of 8. thuringiensis demonstrated that mAbs of C-1, C-3, C-4, C-S and C-6 except C-2 did not recognize epitopes of thuringiensis, but that all of the mAbs recognized epitopes of galleriae, kurstaki, dakota, tolrJokuensis, silo, toguchini, and leesis. The potential applications of the mAbs we produced would be useful tools for the clarification of taxonomy, investigation of antigenic relationship between B. thuringiensis strains, and localization of specific surface and flagella antigens.

• Journal of Periodontal and Implant Science
• /
• 제47권3호
• /
• pp.174-181
• /
• 2017

Purpose: Epitope spreading is a phenomenon in which distinct subdominant epitopes become major targets of the immune response. Heat shock protein (HSP) 60 from Porphyromonas gingivalis (PgHSP60) and peptide 19 from PgHSP60 (Pep19) are immunodominant epitopes in autoimmune disease patients, including those with periodontitis. It remains unclear whether Pep19 is a dominant epitope in subjects without periodontitis or autoimmune disease. The purpose of this study was to determine the epitope spreading pattern and verify Pep19 as an immunodominant epitope in healthy teenagers using dot immunoblot analysis. The patterns of epitope spreading in age-matched patients with type 1 diabetes mellitus (type 1 DM) and healthy 20- to 29-year old subjects were compared with those of healthy teenagers. Methods: Peptide from PgHSP60, Mycobacterium tuberculosis HSP60 (MtHSP60), and Chlamydia pneumoniae HSP60 (CpHSP60) was synthesized for comparative recognition by sera from healthy subjects and patients with autoimmune disease (type 1 DM). Dot immunoblot analysis against a panel of peptides of PgHSP60 and human HSP60 (HuHSP60) was performed to identify epitope spreading, and a densitometric image analysis was conducted. Results: Of the peptide from PgHSP60, MtHSP60, and CpHSP60, PgHSP60 was the predominant epitope and was most consistently recognized by the serum samples of healthy teenagers. Most sera from healthy subjects and patients with type 1 DM reacted more strongly with PgHSP60 and Pep19 than the other peptides. The relative intensity of antibody reactivity to Pep19 was higher in the type 1 DM group than in the healthy groups. Conclusions: Pep19 is an immunodominant epitope, not only in autoimmune disease patients, but also in healthy young subjects, as evidenced by their robust immunoreactivity. This result suggests that the Pep19-specific immune response may be an initiator that triggers autoimmune diseases.

• 한국동물위생학회지
• /
• 제44권4호
• /
• pp.185-194
• /
• 2021

Polyglutamine extension in the coding sequence of mutant huntingtin causes neuronal degeneration associated with the formation of insoluble polyglutamine aggregates in Huntington's disease (HD). Mutant huntingtin can form aggregates within the nucleus and processes of neurons possibly due to misfolding of the proteins. To better understand the mechanism by which an elongated polyglutamine causes aggregates, we have developed an in vitro binding assay system of polyglutamine tract from truncated huntingtin. We made GST-HD exon1 fusion proteins which have expanded polyglutamine epitopes (e.g., 17, 23, 32, 46, 60, 78, 81, and 94 CAG repeats). In the present emergence of new study adjusted nanotechnology on protein chip such as surface plasmon resonance strategy which used to determine the substance which protein binds in drug discovery platform is worth to understand better neurodegenerative diseases (i.e., Alzheimer disease, Parkinson disease and Huntington disease) and its pathogenesis along with development of therapeutic measures. Hence, we used strengths of surface plasmon resonance (SPR) technology which is enabled to examine binding specificity and explore targeted molecular epitope using its electron charged wave pattern in HD pathogenesis utilize conjugated mutant epitope of HD protein and its interaction whether wild type GST-HD interacts with mutant GST-HD with maximum binding affinity at pH 6.85. We found that the maximum binding affinity of GST-HD17 with GST-HD81 was higher than the binding affinities of GST-HD17 with other mutant GST-HD constructs. Furthermore, our finding illustrated that the mutant form of GST-HD60 showed a stronger binding to GST-HD23 or GST-HD17 than GST-HD60 or GST-HD81. These results indicate that the binding affinity of mutant huntingtin does not correlate with the length of polyglutamine. It suggests that the aggregation of an expanded polyglutamine might have easily occurred in the presence of wild type form of huntingtin.

• Clinical and Experimental Vaccine Research
• /
• 제12권1호
• /
• pp.47-59
• /
• 2023

Purpose: The development and study of hepatitis C virus (HCV) vaccine candidates' individualized responses are of great importance. Here we report on an HCV DNA vaccine candidate based on selected envelope (E1/E2) epitopes. Besides, we assessed its expression and processing in human peripheral blood mononuclear cells (PBMCs) and in vivo cellular response in mice. Materials and Methods: HCV E1/E2 DNA construct (EC) was designed. The antigen expression of EC was assayed in PBMCs of five HCV-uninfected donors via a real-time quantitative polymerase chain reaction. Serum samples from 20 HCV antibody-positive patients were used to detect each individual PBMCs expressed antigens via enzyme-linked immunosorbent assay. Two groups, five Swiss albino mice each, were immunized with the EC or a control construct. The absolute count of lymph nodes' CD4⁺ and CD8⁺ T-lymphocytes was assessed. Results: Donors' PBMCs showed different levels of EC expression, ranging between 0.83-2.61-fold in four donors, while donor-3 showed 34.53-fold expression. The antigens expressed in PBMCs were significantly reactive to the 20 HCV antibody repertoire (all p=0.0001). All showed comparable reactivity except for donor-3 showing the lowest reactivity level. The absolute count % of the CD4⁺ T-cell significantly increased in four of the five EC-immunized mice compared to the control group (p=0.03). No significant difference in CD8⁺ T-cells % was observed (p=0.89). Conclusion: The inter-individual variation in antigen expression and processing dominance was evident, showing independence in individuals' antigen expression and reactivity levels to antibodies. The described vaccine candidate might result in a promising natural immune response with a possibility of CD4⁺ T-cell early priming.

• IMMUNE NETWORK
• /
• 제21권6호
• /
• pp.39.1-39.18
• /
• 2021

The high virulent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus that emerged in China at the end of 2019 has generated novel coronavirus disease, coronavirus disease 2019 (COVID-19), causing a pandemic worldwide. Every country has made great efforts to struggle against SARS-CoV-2 infection, including massive vaccination, immunological patients' surveillance, and the utilization of convalescence plasma for COVID-19 therapy. These efforts are associated with the attempts to increase the titers of SARS-CoV-2 neutralizing Abs (nAbs) generated either after infection or vaccination that represent the body's immune status. As there is no standard therapy for COVID-19 yet, virus eradication will mainly depend on these nAbs contents in the body. Therefore, serological nAbs neutralization assays become a requirement for researchers and clinicians to measure nAbs titers. Different platforms have been developed to evaluate nAbs titers utilizing various epitopes sources, including neutralization assays based on the live virus, pseudovirus, and neutralization assays utilizing recombinant SARS-CoV-2 S glycoprotein receptor binding site, receptor-binding domain. As a standard neutralization assay, the plaque reduction neutralization test (PRNT) requires isolation and propagation of live pathogenic SARS-CoV-2 virus conducted in a BSL-3 containment. Hence, other surrogate neutralization assays relevant to the PRNT play important alternatives that offer better safety besides facilitating high throughput analyses. This review discusses the current neutralization assay platforms used to evaluate nAbs, their techniques, advantages, and limitations.