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Journal articles on the topic 'Cell-based-ELISA'

Author: Grafiati
Published: 1 April 2023

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1

Shankar, Gopi, and Donald A. Cohen. "Enhanced Cytokine Detection by a Novel Cell Culture-Based Elisa." Journal of Immunoassay 18, no. 4 (November 1997): 371–88. http://dx.doi.org/10.1080/01971529708005828.

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2

Ahmadzadeh, Maryam, Farzaneh Farshdari, Leila Nematollahi, Shayan Maleknia, and Elham Mohit. "Optimization of HER2-based and cell-based ELISA for detection of trastuzumab biosimilar." Koomesh journal 22, no. 2 (April 1, 2020): 341–50. http://dx.doi.org/10.29252/koomesh.22.2.341.

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3

FRIIS, TINA, BIRGITTE KJAER SORENSEN, ANNE-MARIE ENGEL, JORGEN RYGAARD, and GUNNAR HOUEN. "A quantitative ELISA-based co-culture angiogenesis and cell proliferation assay." APMIS 111, no. 6 (June 2003): 658–68. http://dx.doi.org/10.1034/j.1600-0463.2003.1110609.x.

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4

Bengleil, Mudafara, and Jeffrey R. Fry. "In situ cell-based ELISA for determination of multiple heat shock proteins." Toxicology 226, no. 1 (September 2006): 71. http://dx.doi.org/10.1016/j.tox.2006.05.095.

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5

Tizard, E. J., E. Baguley, G. R. Hughes, and M. J. Dillon. "Antiendothelial cell antibodies detected by a cellular based ELISA in Kawasaki disease." Archives of Disease in Childhood 66, no. 2 (February 1, 1991): 189–92. http://dx.doi.org/10.1136/adc.66.2.189.

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6

Franciotta, Diego, Gianvito Martino, Elena Brambilla, Elisabetta Zardini, Vera Locatelli, Alessandra Bergami, Carmine Tinelli, Gaetano Desina, and Vittorio Cosi. "TE671 Cell-based ELISA for Anti-Acetylcholine Receptor Antibody Determination in Myasthenia Gravis." Clinical Chemistry 45, no. 3 (March 1, 1999): 400–405. http://dx.doi.org/10.1093/clinchem/45.3.400.

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Abstract Background: Acetylcholine receptor (AChR) from human muscles is the antigen used currently in radioimmunoprecipitation assays (RIPAs) for the determination of anti-AChR antibodies in the diagnosis of myasthenia gravis (MG). Our aim was to develop and validate an ELISA using TE671 cells as the source of AChR. Methods: After TE671 cell homogenization, the crude AChR extract was used for plate coating. Anti-AChR antibodies were determined in 207 MG patients and in 77 controls. Results: The mean intra- and interassay CVs (for two samples with different anti-AChR antibody concentrations) were 9.7% and 15.7%, respectively. Test sensitivity and specificity, for generalized MG, were 79.5% (95% confidence interval, 72.8–85.0%) and 96.1% (89.0–99.1%). The detection limit was 2 nmol/L. Anti-AChR antibody concentrations from 53 MG patients, as tested with our ELISA, showed good agreement with an RIPA with a mean difference (SD) of 1.0 (5.6) nmol/L. Conclusion: Our ELISA is a simple screening test for the diagnosis of MG and enables rapid and inexpensive patient follow-up.
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Zarletti, Gianpaolo, Massimo Tiberi, Veronica De Molfetta, Maurizio Bossù, Elisa Toppi, Paola Bossù, and Giuseppe Scapigliati. "A Cell-Based ELISA to Improve the Serological Analysis of Anti-SARS-CoV-2 IgG." Viruses 12, no. 11 (November 8, 2020): 1274. http://dx.doi.org/10.3390/v12111274.

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Knowledge of the antibody-mediated immune response to SARS-CoV-2 is crucial to understand virus immunogenicity, establish seroprevalence, and determine whether subjects or recovered patients are at risk for infection/reinfection and would therefore benefit from vaccination. Here, we describe a novel and simple cell-ELISA specifically designed to measure viral spike S1-specific IgG produced in vitro by B cells in peripheral blood mononuclear cell (PBMC) cultures from a cohort of 45 asymptomatic (n = 24) and symptomatic (n = 21) individuals, with age ranging from 8 to 99 years. All subjects underwent ELISA serological screening twice, at the same time as the cell-ELISA (T2) as well as 35–60 days earlier (T1). Cryopreserved PBMCs of healthy donors obtained years before the COVID-19 pandemic were also included in the analysis. The preliminary results presented here show that out of 45 tested subjects, 16 individuals (35.5%) were positive to the cell-ELISA, 11 (24.5%) were concomitantly positive in the serological screening (T1 and/or T2), and only one person was exclusively positive in ELISA (T1) and negative in cell-ELISA, though values were close to the cutoff. Of note, five individuals (11.2%) tested negative in ELISA but positive in cell-ELISA and thus, they appear to have circulating B cells that produce antibodies against SARS-CoV-2, likely at levels that are undetectable in the serum, which challenges the negative results of the serological screening. The relative level of in vitro secreted IgG was measurable in positive subjects, ranging from 7 to 50 ng/well. Accordingly, all anti-SARS-CoV-2 antibody-positive subjects previously reported moderate to severe symptoms attributable to COVID-19, even though the RT-PCR data were rarely available to confirm viral infection. Overall, the described cell-ELISA might be an effective method for detecting subjects who encountered the virus in the past, and thus helpful to improve serological ELISA tests in the case of undetectable/equivocal circulating IgG levels, and a suitable and improved tool to better evaluate SARS-CoV-2-specific humoral immunity in the COVID-19 pandemic.
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8

Chuang, Kuo-Hsiang, Shey-Cherng Tzou, Ta-Chun Cheng, Chien-Han Kao, Wei-Lung Tseng, Jentaie Shiea, Kuang-Wen Liao, et al. "Measurement of Poly(ethylene glycol) by Cell-Based Anti-poly(ethylene glycol) ELISA." Analytical Chemistry 82, no. 6 (March 15, 2010): 2355–62. http://dx.doi.org/10.1021/ac902548m.

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9

Khodashenas, Shabanali, Saeed Khalili, and Mehdi Forouzandeh Moghadam. "A cell ELISA based method for exosome detection in diagnostic and therapeutic applications." Biotechnology Letters 41, no. 4-5 (April 8, 2019): 523–31. http://dx.doi.org/10.1007/s10529-019-02667-5.

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10

Stults, Cheryl L. M., and Bruce A. Macher. "Measurement of β-galactosyltransferase activity in cell extracts with an ELISA-based assay." Archives of Biochemistry and Biophysics 280, no. 1 (July 1990): 20–26. http://dx.doi.org/10.1016/0003-9861(90)90512-w.

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11

Hsu, Wei-Ting, Chia-Yu Chang, Chih-Hsuan Tsai, Sung-Chan Wei, Huei-Ru Lo, Robert John S. Lamis, Hui-Wen Chang, and Yu-Chan Chao. "PEDV Infection Generates Conformation-Specific Antibodies That Can Be Effectively Detected by a Cell-Based ELISA." Viruses 13, no. 2 (February 15, 2021): 303. http://dx.doi.org/10.3390/v13020303.

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Porcine epidemic diarrhea virus (PEDV) is a coronavirus that causes serious and highly contagious enteric disease in swine worldwide. In this study, we constructed a recombinant baculovirus (S-Bac) expressing full-length spike protein of the virulent epidemic genotype 2b (G2b) PEDV strain for serological studies of infected pigs. We found that most spike-specific antibodies produced upon PEDV infection in pigs are conformation-specific and they could be detected on S-Bac-infected insect cells by immunofluorescent assay, but they were insensitive to Western blot analysis, the typical method for antiserum analysis. These results indicated that spike conformation is crucial for serum recognition. Since it is difficult to purify trimeric spike membrane protein for conventional enzyme-linked immunosorbent assay (ELISA), we used S-Bac to generate a novel cell-based ELISA for convenient PEDV detection. We analyzed 100 pig serum samples, and our cell-based ELISA exhibited a sensitivity of 100%, a specificity of 97%, and almost perfect agreement [Cohen’s kappa coefficient value (κ) = 0.98] with immunocytochemical staining results. Our cell-based ELISA rapidly presented antigen for proper detection of conformation-specific antibodies, making PEDV detection more convenient, and it will be useful for detecting many viral diseases in the future.
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12

Wang, Xiaohong, Martin Sapp, Neil D. Christensen, and Joakim Dillner. "Heparin-based ELISA reduces background reactivity in virus-like particle-based papillomavirus serology." Journal of General Virology 86, no. 1 (January 1, 2005): 65–73. http://dx.doi.org/10.1099/vir.0.80472-0.

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The interaction between human papillomavirus (HPV) particles and cell surface heparan sulfate requires intact conformation of the HPV particles. Type-specific HPV serology is currently based on virus-like particles (VLPs) with intact conformation. Presence of incorrectly folded VLPs in VLP preparations is recognized as an important cause of cross-reactivity in HPV serology. Heparin-coated microtitre plates were evaluated for capturing conformationally correct VLPs and improving the type specificity of HPV serology. Hybrid VLPs between HPV16 and HPV11, which had been found to have significant reactivity with children's sera and a batch of HPV18 VLPs that had failed the quality control because of significant reactivity with sera from virginal women, were tested in parallel with heparin ELISA, ordinary ELISA and type-specific mAb capture ELISA. Control sera from children that had detectable reactivity with HPV16/11 hybrid VLPs in ordinary ELISA did not react in heparin-based ELISA, but some hybrid VLPs also had background reactivity in capture ELISAs. Control sera from virginal women that had some reactivity with a poor quality HPV18 VLP preparation in ordinary ELISA had no reactivity in heparin or capture ELISA, suggesting that certain VLP preparations expose cross-reactive epitopes that are not exposed on VLPs with heparin-binding ability. As the sensitivity was similar or only marginally affected by the use of heparin plates, use of heparin-coated plates may improve the type specificity of VLP-based ELISAs and reduce interassay variability attributable to variable quality of different VLP batches.
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Yin, Dehui, Qiongqiong Bai, Xiling Wu, Han Li, Jihong Shao, Mingjun Sun, Hai Jiang, and Jingpeng Zhang. "Paper-based ELISA diagnosis technology for human brucellosis based on a multiepitope fusion protein." PLOS Neglected Tropical Diseases 15, no. 8 (August 17, 2021): e0009695. http://dx.doi.org/10.1371/journal.pntd.0009695.

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Background Brucellosis, as a serious zoonotic infectious disease, has been recognized as a re-emerging disease in the developing countries worldwide. In china, the incidence of brucellosis is increasing each year, seriously threatening the health of humans as well as animal populations. Despite a quite number of diagnostic methods currently being used for brucellosis, innovative technologies are still needed for its rapid and accurate diagnosis, especially in area where traditional diagnostic is unavailable. Methodology/Principal findings In this study, a total of 22 B cell linear epitopes were predicted from five Brucella outer membrane proteins (OMPs) using an immunoinformatic approach. These epitopes were then chemically synthesized, and with the method of indirect ELISA (iELISA), each of them displayed a certain degree of capability in identifying human brucellosis positive sera. Subsequently, a fusion protein consisting of the 22 predicted epitopes was prokaryotically expressed and used as diagnostic antigen in a newly established brucellosis testing method, nano-ZnO modified paper-based ELISA (nano-p-ELISA). According to the verifying test using a collection of sera collected from brucellosis and non-brucellosis patients, the sensitivity and specificity of multiepitope based nano-p-ELISA were 92.38% and 98.35% respectively. The positive predictive value was 98.26% and the negative predictive value was 91.67%. The multiepitope based fusion protein also displayed significantly higher specificity than Brucella lipopolysaccharide (LPS) antigen. Conclusions B cell epitopes are important candidates for serologically testing brucellosis. Multiepitope fusion protein based nano-p-ELISA displayed significantly sensitivity and specificity compared to Brucella LPS antigen. The strategy applied in this study will be helpful to develop rapid and accurate diagnostic method for brucellosis in human as well as animal populations.
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Fu, Shengyu, and Qi Zhao. "Quantitation and Identification of Therapeutic Anti-CD22 Monoclonal Antibodies in a Cell-Based ELISA Method." Antibodies 11, no. 3 (August 16, 2022): 53. http://dx.doi.org/10.3390/antib11030053.

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Since they lack native soluble membrane antigens, the analysis and selection of antigen-specific antibodies are commonly performed on whole live cells. Here, we have developed a simple and convenient enzyme-linked immunosorbent assay (ELISA) based on cell membrane antigens. Soluble cell membrane proteins isolated from Raji cells were immobilized on the polystyrene microplate, which permitted the assessment of a therapeutic anti-CD22 monoclonal antibody. The experiments showed less variability in the intra-assay. Compared to the living cell ELISAs, the advantage of the assay is avoiding cell losses and high variation of optical density (OD) readings. We provide a quantitative and reproducible ELISA that can be potentially applied to the development of specific antibodies against cell surface antigens.
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Sauerbrei, Andreas, Anna Schäfler, Jörg Hofmann, Michael Schacke, Bernd Gruhn, and Peter Wutzler. "Evaluation of Three Commercial Varicella-Zoster Virus IgG Enzyme-Linked Immunosorbent Assays in Comparison to the Fluorescent-Antibody-to-Membrane-Antigen Test." Clinical and Vaccine Immunology 19, no. 8 (June 20, 2012): 1261–68. http://dx.doi.org/10.1128/cvi.00183-12.

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ABSTRACTCommercial serologic assays for varicella-zoster virus (VZV), which enable reliable determination of VZV immune status and are amenable to automation, are needed. The present study compares the automated performance of the VZV whole-cell enzyme-linked immunosorbent assay (ELISA) Enzygnost anti-VZV/IgG, the Euroimmun anti-VZV ELISA (IgG) based on highly purified viral proteins, and the VZV glycoprotein (gp)-based Serion ELISA Classic VZV IgG. The fluorescent-antibody-to-membrane-antibody (FAMA) test was used as a reference. A total of 638 serum samples from VZV-negative children, blood donors, varicella vaccinees, and bone marrow transplant recipients were included. The Enzygnost anti-VZV/IgG and the Serion ELISA Classic VZV IgG showed sensitivities of 99.6% and 99.2%, respectively, and the Euroimmun anti-VZV ELISA (IgG) had a significantly lower sensitivity of 90.5%. Specificity was calculated as 100% for both the Euroimmun anti-VZV ELISA (IgG) and for the Enzygnost anti-VZV/IgG, and the Serion ELISA Classic VZV IgG had a significantly lower specificity of 89.4%. Quantitative results of all ELISAs correlated well, but there was a poor quantitative correlation between the ELISAs and FAMA. In conclusion, this study does not show any superiority of a gp- and a protein-based ELISA compared to a whole-cell ELISA for the automated detection of VZV-specific IgG. The automated performance of the Enzygnost anti-VZV/IgG assay correlated best with the FAMA reference assay.
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16

Cruz, Taís F., Tatiana M. Kanashiro, Alessandra M. M. G. de Castro, Cintia M. Baldin, Leonardo J. Richtzenhain, and João P. Araujo Jr. "A double-antibody sandwich ELISA based on the porcine circovirus type 2 (PCV2) propagated in cell culture for antibody detection." Pesquisa Veterinária Brasileira 36, no. 12 (December 2016): 1171–77. http://dx.doi.org/10.1590/s0100-736x2016001200005.

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ABSTRACT: Few studies have described enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies against porcine circovirus type 2 (PCV2) based on antigens produced in cell culture. Furthermore, few articles have described viral purification techniques for members of the family Circoviridae. This occurs because circoviruses are difficult to isolate, noncytopathogenic, and produce low viral titres in cell culture. Thus, for overcoming these difficulties in the cultivation of PCV2, this study aimed to develop a double-antibody sandwich ELISA based on the cell culture antigen PCV2b for the quantification of anti-PCV2 antibodies. A 20% and 50% discontinuous sucrose cushion was used for viral purification, which enabled the separation of cell culture proteins in the 20% sucrose cushion and a greater viral concentration in the 50% sucrose cushion. Following isopycnic centrifugation, PCV2 was concentrated in the band with density values from 1.330 to 1.395g/cm3. Viral purification was assessed using SDS-PAGE, indirect ELISA and electron microscopy. The standardised ELISA revealed a strong linear correlation (r= 0.826, p<0.001) when compared with a commercial ELISA kit. The assay exhibited low variability (inter-assay coefficient of variation of 4.24% and intra-assay of 1.80%) and excellent analytical specificity conferred by the capture antibody produced in rabbit. Thus, this ELISA is a rapid, specific and convenient method for the detection of antibodies against PCV2 in studies of experimental and natural infection, and in monitoring the response to vaccination on commercial farms.
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Soman, Gopalan, Xiaoyi Yang, Hengguang Jiang, Steve Giardina, and Gautam Mitra. "Comparison of GD2 binding capture ELISA assays for anti-GD2-antibodies using GD2-coated plates and a GD2-expressing cell-based ELISA." Journal of Immunological Methods 373, no. 1-2 (October 2011): 181–91. http://dx.doi.org/10.1016/j.jim.2011.08.016.

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18

CHEN, CHI H., HWIA C. DING, and TSUNG C. CHANG. "Rapid Identification of Bacillus cereus Based on the Detection of a 28.5-Kilodalton Cell Surface Antigen." Journal of Food Protection 64, no. 3 (March 1, 2001): 348–54. http://dx.doi.org/10.4315/0362-028x-64.3.348.

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Conventional procedures for the identification of suspect Bacillus cereus isolated on mannitol-egg yolk-polymyxin(MYP) agar may need several days. To facilitate the identification of the bacterium, an enzyme-linked immunosorbent assay (ELISA) was developed. The assay was based on the detection of a 28.5-kDa cell surface antigen of B. cereus. Bacterial colonies grown on MYP agar or nutrient agar were suspended in phosphate-buffered saline (pH 7.2) containing 0.1% Teepol. The cell suspensions were heated at 100°C for 5 min and added to the microtiter plates coated with antibodies against the 28.5-kDa antigen. After washing, the same antibodies labeled with horseradish peroxidase were used as secondary antibodies to reveal the signal of antigen-antibody reaction. For 38 strains of B. cereus and 127 strains of non-B. cereus bacteria (including 79 isolates of Bacillus spp.) tested, the sensitivity and specificity of the ELISA were 100 and 88.2%, respectively. Strains producing false-positive results were members of the B. cereus group (i.e., Bacillus anthracis, Bacillus mycoides, and Bacillus thuringiensis), which are genetically and biochemically similar to B. cereus. Similar ELISA results were obtained by using antibodies against another cell surface antigen with a molecular mass of 20 kDa. If members of the B. cereus group were recognized as a single species, the sensitivity and specificity of the ELISA were 100 and 99.1%, respectively. The ELISA could be used as a rapid method for presumptive identification of B. cereus grown on MYP agar.
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Liang, Huanbin, Heng Wang, Liangquan Zhang, Honglang Gu, and Guihong Zhang. "Development of a novel immunoperoxidase monolayer assay for detection of swine Hepatitis E virus antibodies based on stable cell lines expressing the ORF3 protein." Acta Veterinaria Hungarica 62, no. 2 (June 1, 2014): 243–56. http://dx.doi.org/10.1556/avet.2013.051.

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Hepatitis E virus (HEV) strains are classified into 4 genotypes by nucleotide sequencing. Genotypes 3 and 4 infect humans and animals via HEV-contaminated food or water. HEV RNA was detected by PCR and antibodies were detected by ELISA. Since human studies showed that HEV IgG antibodies in sera can persist for extended periods, diagnosis of HEV infection in swine or humans is mainly based on serological detection using commercial ELISA kits. However, there is no supplemental method to verify ELISA results. Hence, we developed a novel method used for mutual correction of these common processes. Here, a modified stable HepG2 cell line was transfected with pcDNA3.1-ORF3 to express the swine HEV ORF3 protein. Based on this cell line, a novel immunoperoxidase monolayer assay (IPMA) was developed to detect antibodies against HEV. The results show that this method has good specificity, sensitivity and repeatability. When used to investigate 141 porcine serum samples, the IPMA had a coincidence rate of 92.2% with a commercial ELISA kit. The established IPMA described herein is valuable as a supplemental method to ELISA and can differentiate infections by HEV and other viruses.
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Wang, Y., J. Guo, S. Qiao, Q. Li, J. Yang, Q. Jin, and G. Zhang. "GP5 Protein-based ELISA for the Detection of PRRSV Antibodies." Polish Journal of Veterinary Sciences 19, no. 3 (September 1, 2016): 495–501. http://dx.doi.org/10.1515/pjvs-2016-0062.

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AbstractPorcine reproductive and respiratory syndrome virus (PRRSV) is an important swine pathogen, causing huge economic losses each year worldwide. Immunization with vaccines containing the glycoprotein 5 (GP5) of PRRSV is the main measure to induce neutralizing antibodies and control the disease. Here, we developed a GP5 protein-based ELISA for detecting antibodies against PRRSV. The overall yield of purified GP5 inE. coliflask culture was more than 45 mg/L cell culture. Western blot and IFA indicated that the GP5 protein was highly immunogenic. After optimization and validation with IDEXX PRRS using 566 clinical sera, the DSN, DSP, and accuracy of GP5-ELISA were 81.39%, 75.96%, and 80.39%, respectively. Besides, GP5-ELISA is highly specific, showing no cross-reactions with sera against other important swine pathogens. Hence, GP5 is a good diagnostic antigen and the GP5 protein-based ELISA has the potential to be used in the field.
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Bhartiya, Nidhi M., Aliabbas A. Husain, Hatim F. Daginawala, Lokendra Singh, and Rajpal S. Kashyap. "Development of an Immunodiagnostic Test for Screening Human Brucellosis Cases Using the Whole-Cell Antigens of Brucella abortus." Malaysian Journal of Medical Sciences 27, no. 6 (December 29, 2020): 15–26. http://dx.doi.org/10.21315/mjms2020.27.6.3.

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Background: Human brucellosis is an important zoonotic disease of public health and often remains neglected owing to lack of sensitive and efficient diagnostic methods. This study evaluates diagnostic utility of in-house designed enzyme-linked immunosorbent assay (ELISA) using whole-cell antigens of Brucella abortus (B. abortus) S19 against the commercially available kits. Methods: A prospective cohort study involving different populations within the Vidarbha regions of Maharashtra, India was conducted through camps organised from May 2009 to October 2015. A total of 568 serum samples were collected from high-risk people recruited as study cohorts based on inclusion criteria, additional risk factors and clinical symptoms. Samples were evaluated by indirect ELISA using the whole-cell antigens of B. abortus. The results were compared with the commercially available IgG detection ELISA kit to ascertain the specificity and sensitivity of the developed test. Results: Fever, body ache, joint pain, lower back pain, loss of appetite and weight loss were major symptoms associated with the disease. With the cut-off of > 0.8, the positivity of brucellosis infection was at 12.32% (70/568) compared to 9.33% (53/568) as detected by the commercial kit. The in-house developed ELISA method yielded a sensitivity of 87.5% and specificity of 99.18% as compared to the commercial kits (sensitivity –80.30% and specificity –99.6%). Discussion: The B. abortus S19-derived whole-cell protein-based ELISA is rapid and cost- effective and can be used for screening brucellosis infection in lieu of the commercially available ELISA kits.
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Padige, Geetha, Ahmed T. Negmeldin, and Mary Kay H. Pflum. "Development of an ELISA-Based HDAC Activity Assay for Characterization of Isoform-Selective Inhibitors." Journal of Biomolecular Screening 20, no. 10 (July 31, 2015): 1277–85. http://dx.doi.org/10.1177/1087057115598118.

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Histone deacetylase (HDAC) proteins are promising targets for cancer treatment, with several HDAC inhibitors used clinically as anticancer drugs. Most HDAC inhibitors nonspecifically interact with all or many of the 11 HDAC isoforms. Isoform-selective HDAC inhibitors would be useful tools to dissect the individual functions of HDAC proteins in cancer formation, in addition to potentially displaying effective anticancer properties. We report here a robust HDAC activity assay for screening selective HDAC inhibitors, which is inspired by the traditional enzyme-linked immunosorbent assay (ELISA). The key feature of the ELISA-based HDAC activity assay is use of mammalian cell–derived HDAC isoforms instead of recombinant proteins. Importantly, the assay was validated with several known HDAC inhibitors. The ELISA-based HDAC activity assay will facilitate the characterization of isoform-selective HDAC inhibitors against mammalian cell–derived HDAC proteins, which will enhance HDAC-centered cancer research and provide a foundation for anticancer drug development.
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O'Sullivan, Catherine A., Patrick J. Joyce, Teresa Sloan, and Alan G. Shattock. "Capture immunoassay for the diagnosis of bovine mastitis using a monoclonal antibody to polymorphonuclear granulocytes." Journal of Dairy Research 59, no. 2 (May 1992): 123–33. http://dx.doi.org/10.1017/s0022029900030375.

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SummaryA direct capture enzyme-linked immunosorbent assay (ELISA) was developed to measure elevated polymorphonuclear granulocyte (PMN) antigens using horseradish peroxidase (EC 1.11.1.7) conjugated rabbit polyclonal anti-PMN antisera and a monoclonal antibody specific for PMN cells. Optical densities obtained in the ELISA were used to predict the cell counts of milk samples. Predicted counts were not significantly different from actual somatic cell counts (SCC). In a total of 156 bovine milk samples the correlation coefficient between somatic cell counting, taking > 500000 cells/ml as being indicative of mastitis, and the assay was 0·94, yielding an assay sensitivity of 95·2% and a specificity of 97·3%. In further trials the ELISA could detect elevated PMN antigens in milk with SCC as low as 100000 cells/ml. The results indicate that the monoclonal antibody based direct ELISA has excellent potential in the detection and determination of bovine mastitis.
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Holthoff, Hans-Peter, Stefan Zeibig, Valerie Jahns-Boivin, Johannes Bauer, Martin J. Lohse, Stefan Kääb, Sebastian Clauss, et al. "Detection of Anti–β1-AR Autoantibodies in Heart Failure by a Cell-Based Competition ELISA." Circulation Research 111, no. 6 (August 31, 2012): 675–84. http://dx.doi.org/10.1161/circresaha.112.272682.

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Jamieson, David, Nicola Cresti, Mark W. Verrill, and Alan V. Boddy. "Development and validation of cell-based ELISA for the quantification of trastuzumab in human plasma." Journal of Immunological Methods 345, no. 1-2 (June 2009): 106–11. http://dx.doi.org/10.1016/j.jim.2009.04.006.

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Collins, Ian, Paul Upton, and Gillian Wilson. "DEVELOPMENT OF AN ELISA PLATE-BASED METHOD FOR SCREENING POTENTIAL THERAPEUTICS WHICH INHIBIT CELL ADHESION." Shock 7, Supplement (March 1997): 122. http://dx.doi.org/10.1097/00024382-199703001-00494.

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27

Hong, Kyu, Leonard G. Presta, Yanmei Lu, Ashley Penn, Camellia Adams, Anan Chuntharapai, Jihong Yang, Wai Lee Wong, and Y. Gloria Meng. "Simple quantitative live cell and anti-idiotypic antibody based ELISA for humanized antibody directed to cell surface protein CD20." Journal of Immunological Methods 294, no. 1-2 (November 2004): 189–97. http://dx.doi.org/10.1016/j.jim.2004.09.003.

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28

Hoelzle, Katharina, Julia Grimm, Mathias Ritzmann, Karl Heinritzi, Paul Torgerson, Anja Hamburger, Max M. Wittenbrink, and Ludwig E. Hoelzle. "Use of Recombinant Antigens To Detect Antibodies against Mycoplasma suis, with Correlation of Serological Results to Hematological Findings." Clinical and Vaccine Immunology 14, no. 12 (October 17, 2007): 1616–22. http://dx.doi.org/10.1128/cvi.00345-07.

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ABSTRACT Porcine eperythrozoonosis is a disease with worldwide distribution caused by the unculturable hemotrophic bacterium Mycoplasma suis. Current serological testing utilizes crude M. suis antigens purified from the blood of experimentally infected pigs. These antigens show high variability and are restricted to specialized laboratories. We evaluated a novel serological assay based on two recombinant M. suis antigens (rMSG1 and rHspA1). Antigen specificity was proven by means of sera raised against nonhemotrophic mycoplasma and other relevant bacteria. Using experimental and convalescent-phase sera, rMSG1 and rHspA1 enzyme-linked immunosorbent assays (ELISAs) demonstrated sensitivities, specificities, and predictive values (94.0 to 100.0%) equal to or higher than those of the M. suis whole-cell ELISA. Field samples from 120 weaning piglets grouped by quantitative PCR results were used to evaluate the diagnostic capability of the new ELISA systems in comparison to that of the whole-cell ELISA. Assuming a 100.0% specificity of the PCR, the whole-cell ELISA, rHspA1 ELISA, and rMSG1 ELISA showed specificities of 84.8%, 83.8%, and 90.6% and sensitivities of 61.5%, 74.0% and 58.1%, respectively. Cohen's kappa coefficients comparing the recombinant ELISAs to the whole-cell ELISA indicate moderate to substantial agreement. The detection of anti-MSG1 and/or anti-HspA1 antibodies in pigs was significantly correlated with decreased hematocrit, erythrocyte numbers, and hemoglobin concentrations, indicating that a single seropositive result is connected with clinical and etiological significance. In conclusion, rMSG1 and rHspA1 are sensitive and specific serological and infection markers which are for the first time used independently of animal experiments. They are especially fit to be used in routine diagnosis, pathogenesis studies, and large-scale epidemiological investigations.
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Kroll, J. J., M. A. Eichmeyer, M. L. Schaeffer, S. McOrist, D. L. Harris, and M. B. Roof. "Lipopolysaccharide-Based Enzyme-Linked Immunosorbent Assay for Experimental Use in Detection of Antibodies to Lawsonia intracellularis in Pigs." Clinical Diagnostic Laboratory Immunology 12, no. 6 (June 2005): 693–99. http://dx.doi.org/10.1128/cdli.12.6.693-699.2005.

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ABSTRACT An enzyme-linked immunosorbent assay (ELISA) for Lawsonia intracellularis was developed and compared with a whole-cell antigen-based immunofluorescence antibody test (IFAT). The antigen-containing lipopolysaccharide (LPS) was derived from Percoll gradient purified cultures of L. intracellularis by using a modification of the Westphal hot phenol procedure. The antigen was bound directly to polystyrene 96-well microtiter plates, and the assay was performed in an indirect ELISA format. Specificity and sensitivity values based on 80 known positive and 80 known negative serum samples from controlled experimental trials were 93.7% and 88.7%, respectively. Serological results from a controlled L. intracellularis challenge exposure study confirmed the high specificity and sensitivity of this assay (100% and 99.5%, respectively). Comparisons between the LPS ELISA and the IFAT in detecting anti-Lawsonia antibodies in this controlled study revealed significantly more LPS ELISA-positive pigs than IFAT-positive pigs on days 21, 28, 35, and 42 (P = 0.003, 0.030, 0.002, and 0.006, respectively). This indirect ELISA (LPS ELISA) test is an improved method of detecting antibodies in pigs soon after exposure to L. intracellularis, regardless of isolate type (vaccine or wild type) in experimental studies. The LPS ELISA may be used as a tool to support future research trials on vaccine efficacy and to further understand the immune response induced by L. intracellularis.
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Hjelle, B., C. Wilson, S. Cyrus, P. Bradshaw, J. Lo, C. Schammel, T. Wiltbank, and S. Alexander. "Human T-cell leukemia virus type II infection frequently goes undetected in contemporary US blood donors." Blood 81, no. 6 (March 15, 1993): 1641–44. http://dx.doi.org/10.1182/blood.v81.6.1641.1641.

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Abstract Serologic screening for human T-cell leukemia virus type I (HTLV-I) infection was begun in US blood banks with the licensure of enzyme- linked immunosorbent assays (ELISA) in December 1988. We examined the donation histories of the first 60 Western blot (WB)-confirmed HTLV- I/II positive donors to one blood center and found 8 had made 16 previous donations that scored negative on the screening ELISA. All 16 donations had ELISA absorbance below the cutoff for a positive assay, but still well above that of the average donation (17.6% +/- 5.7% of the cutoff). In a more extensive study, 17 donations from a total of 61,752 at six blood centers were both ELISA-positive and WB-positive for HTLV-I (4) or HTLV-II (13), and 218 samples had ELISA absorbance greater than 50% of the ELISA cutoff. One hundred seventy-eight of the 218 were tested further by WB and 11 were found positive. All 11 positives were confirmed by polymerase chain reaction; 10 had HTLV-II and 1 had HTLV-I. Thus, the HTLV-I-based screening ELISA missed at least 10 of 23, or 43% (95% confidence interval, 23% to 66%), of HTLV- II infections, compared with 1 of 5, or 20%, of HTLV-I infections.
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Hjelle, B., C. Wilson, S. Cyrus, P. Bradshaw, J. Lo, C. Schammel, T. Wiltbank, and S. Alexander. "Human T-cell leukemia virus type II infection frequently goes undetected in contemporary US blood donors." Blood 81, no. 6 (March 15, 1993): 1641–44. http://dx.doi.org/10.1182/blood.v81.6.1641.bloodjournal8161641.

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Serologic screening for human T-cell leukemia virus type I (HTLV-I) infection was begun in US blood banks with the licensure of enzyme- linked immunosorbent assays (ELISA) in December 1988. We examined the donation histories of the first 60 Western blot (WB)-confirmed HTLV- I/II positive donors to one blood center and found 8 had made 16 previous donations that scored negative on the screening ELISA. All 16 donations had ELISA absorbance below the cutoff for a positive assay, but still well above that of the average donation (17.6% +/- 5.7% of the cutoff). In a more extensive study, 17 donations from a total of 61,752 at six blood centers were both ELISA-positive and WB-positive for HTLV-I (4) or HTLV-II (13), and 218 samples had ELISA absorbance greater than 50% of the ELISA cutoff. One hundred seventy-eight of the 218 were tested further by WB and 11 were found positive. All 11 positives were confirmed by polymerase chain reaction; 10 had HTLV-II and 1 had HTLV-I. Thus, the HTLV-I-based screening ELISA missed at least 10 of 23, or 43% (95% confidence interval, 23% to 66%), of HTLV- II infections, compared with 1 of 5, or 20%, of HTLV-I infections.
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Cromwell, Evan, Byungrae Cho, Ori Hoxha, Neil Bristol, Cathy Olsen, and Oksana Sirenko. "Multi-parametric Cell-Based Inflammation Assays for Cytokines and Cell Surface Antigens." Journal of Immunology 202, no. 1_Supplement (May 1, 2019): 51.20. http://dx.doi.org/10.4049/jimmunol.202.supp.51.20.

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Abstract Macrophages and vascular cells upregulate expression of cytokines and adhesion molecules in response to inflammation stimuli. Monitoring these molecules can provide a physiological read-out for inflammation. We present results from a multiparametric assays that used a combination of low-volume ELISA for secreted cytokines and automated cell imaging for cell surface markers to evaluate the effect of different mediators on inflammatory response with two cell models. First, human monocytic THP-1 cells were differentiated to macrophages and activated by LPS. Phenotypic changes in cell morphology were quantified by cell area and number of adherent cells using TL imaging. Secretion of IL-8, IL-1β and TNF-α was quantified by an automated low-volume ELISA (PuMA System). Second, primary human umbilical vein endothelial cells (HUVEC) were stimulated with inflammation cytokines (TNF-α, IFN-γ, and IL-1β), then PuMA System was used to quantify amount of MCP-1, IL-8, and IL-6 in cell supernatants. Expression of adhesion molecules VCAM and HLADR was quantified by measurement of total fluorescence from antibody-stained cells using an ImageXpress Pico system. Concentration dependent inhibition of inflammation by the compounds AG126 and MG132, SB202190 and PDTC were measured and EC50s calculated. Clear differences in cytokine expression were seen between the compounds consistent with reported mechanism of actions of the kinase inhibitors. The combination of imaging and microfluidic-based assays provides an efficient multiparametric assay system that can test the efficacy of anti-inflammatory compounds versus toxicity and provide insight into their MoA by selective inhibition of markers triggered by different signaling pathways.
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Lee, Brian W., Russell F. Bey, Mary J. Baarsch, and Randy R. Simonson. "ELISA Method for Detection of Influenza A Infection in Swine." Journal of Veterinary Diagnostic Investigation 5, no. 4 (October 1993): 510–15. http://dx.doi.org/10.1177/104063879300500402.

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An antigen-capture enzyme-linked immunosorbent assay (ELISA) was developed to monitor virus shedding associated with experimental infection with a field strain of swine influenza in pigs. The assay consisted of a monoclonal anti-nucleoprotein capture antibody and a biotinylated rabbit anti-influenza A (H1N1) sandwich antibody. The antigen-capture system was capable of detecting as little as 1 ng/ml purified virus. The ELISA system surpassed egg cultivation procedures in the detection of low levels of shedding virus. Egg cultivation procedures indicated that most viral shedding had ceased by day 10 postinfection. In contrast, antigen-capture ELISA still showed an ongoing presence of viral antigen. A virus-capture ELISA, using this capture-sandwich antibody system, is equivalent in sensitivity to conventional egg inoculation procedures for the detection of the early phases of virus shedding. The automative potential of an ELISA-based system coupled with a substantially reduced assay time requirement give this virus-capture ELISA a distinct advantage over other cell culture or egg-based diagnostic techniques.
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Amigot, Jose Antonio, Montserrat Torremorell, and Carlos Pijoan. "Evaluation of Techniques for the Detection of Toxigenic Pasteurella Multocida Strains from Pigs." Journal of Veterinary Diagnostic Investigation 10, no. 2 (April 1998): 169–73. http://dx.doi.org/10.1177/104063879801000209.

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Recently acquired field isolates and archived isolates from our collection of Pasteurella multocida were analyzed for production of dermonecrotic toxin. Detection of the toxin was carried out using a fetal lung feline (FLF) cell line and a commercial enzyme-linked immunosorbent assay (ELISA) kit. The dermonecrotic toxin gene ( ToxA) was also detected using a polymerase chain reaction (PCR) technique. Results from the 3 methods were compared. Field isolates (group 1) came from a commercial herd that had clinical signs of atrophic rhinitis. Fifty-six (17.9%) strains were isolated from 312 nasal swabs. Thirty-five of these strains belonged to serotype A and the rest (21/56), although probably serotype D, were not characterized further. All of these strains were toxin negative based on both the ELISA and FLF cell culture results. Five isolates gave faint bands in the PCR reaction, and the rest (51/56) were PCR negative. PCR and ELISA were also performed from the initial swab cultures (mixed cultures); 7 samples gave faint PCR bands, but ELISA results were all negative. Archived strains (group 2) had been isolated from clinical cases of atrophic rhinitis and from cases of pulmonary pasteurellosis. A total of 76 strains were analyzed; 46 were serotype A, and the rest (30) were serotype D. ELISA and FLF cell culture tests were negative for all serotype A strains; however, 3 strains showed faint bands in the PCR reaction. Fourteen serotype D strains showed positive results in both the ELISA and the FLF cell culture tests. PCR from these samples also gave positive results showing a strong band in the gel. However, 4 strains that were ELISA and FLF cell culture negative showed a faint band in the PCR reaction. The 3 methods gave similar results in the detection of the P. multocida dermonecrotic toxin. However, complete agreement among the tests was achieved only when strong PCR bands were considered positive. This is the first report that demostrates the use of FLF cell line for the detection of toxigenic P. multocida.
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SCAPIGLIATI, GIUSEPPE, Gianpaolo Zarletti, Massimo Tiberi, Veronica De Molfetta, Elisa Toppi, and Paola Bossù. "Development of a Cell-based ELISA assay to assess memory IgG produced in vitro by B cells specific for spike protein of SARS-CoV-2." Journal of Immunology 208, no. 1_Supplement (May 1, 2022): 65.23. http://dx.doi.org/10.4049/jimmunol.208.supp.65.23.

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Abstract Knowledge of antibody-mediated immune response to SARS-CoV-2 is crucial to understand the virus immunogenicity, establish seroprevalence and determine possible risks for infection/reinfection. An antibody presence, indicative of an humoral immune response, can be evidenced at early/intermediated stages after infection and antibodies are mainly detected by ELISA-based platforms. However, a major drawback of ELISA systems is a decrease of serum antibody titers during time. On the other hand, circulating antigen-specific memory B cells (MBC) may be present for long time after immunization/infection. The spike S1 protein has been shown to be a serological marker for Covid-19, and we describe a novel, simple and robust Cell-ELISA assay specifically designed to measure viral spike S1 protein by detecting specific IgG produced in vitro by MBC in PBMC (doi:10.3390/v12111274). By applying the Cell-ELISA assay to a cohort of 150 asympto/symptomatic individuals, age ranging from 8–100 years, we detected individuals resulted negative in ELISA but positive in Cell-ELISA, thus showing the presence of MBC that produce in vitro antibodies against SARS-CoV-2 at levels that are undetectable in the serum. These data may challenge the negative results obtained from the serological screening and indicate a previous antigen exposure in the possible absence of serum IgG. We also detected a presence of MBC specific for S1 protein for &gt;10 months after viral infection (doi:10.3390/v13091704), suggesting a potent antigenicity, and evaluated the relative amount of in vitro secreted IgG in positive subjects. The proposed assay could be a candidate tool to monitor the presence of an effective IgG antibody memory after SARS-CoV-2 infection and vaccination.
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Yu, Fuxun, Mai Quynh Le, Shingo Inoue, Hong Thi Cam Thai, Futoshi Hasebe, Maria del Carmen Parquet, and Kouichi Morita. "Evaluation of Inapparent Nosocomial Severe Acute Respiratory Syndrome Coronavirus Infection in Vietnam by Use of Highly Specific Recombinant Truncated Nucleocapsid Protein-Based Enzyme-Linked Immunosorbent Assay." Clinical Diagnostic Laboratory Immunology 12, no. 7 (July 2005): 848–54. http://dx.doi.org/10.1128/cdli.12.7.848-854.2005.

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ABSTRACT Severe acute respiratory syndrome (SARS) is a recently emerged human disease associated with pneumonia. Inapparent infection with SARS coronavirus (CoV) is not well characterized. To develop a safe, simple, and reliable screening method for SARS diagnosis and epidemiological study, two recombinant SARS-CoV nucleocapsid proteins (N′ protein and NΔ121 protein) were expressed in Escherichia coli, purified by affinity chromatography, and used as antigens for indirect, immunoglobulin G enzyme-linked immunosorbent assays (ELISA). Serum samples collected from healthy volunteers and SARS patients in Vietnam were used to evaluate the newly developed methods. The N′ protein-based ELISA showed a highly nonspecific reaction. The NΔ121 protein-based ELISA, with a nonspecific reaction drastically reduced compared to that of the nearly-whole-length N′ protein-based ELISA, resulted in higher rates of positive reactions, higher titers, and earlier detection than the SARS-CoV-infected cell lysate-based ELISA. These results indicate that our newly developed SARS-CoV NΔ121 protein-based ELISA is not only safe but also a more specific and more sensitive method to diagnose SARS-CoV infection and hence a useful tool for large-scale epidemiological studies. To identify inapparent SARS-CoV infections, serum samples collected from health care workers (HCWs) in Vietnam were screened by the NΔ121 protein-based ELISA, and positive samples were confirmed by a virus neutralization test. Four out of 149 HCWs were identified to have inapparent SARS-CoV infection in Vietnam, indicating that subclinical SARS-CoV infection in Vietnam is rare but does exist.
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Spurgers, Kevin B., Clarence R. Hurt, Jeffrey W. Cohen, Lori T. Eccelston, Cathleen M. Lind, Vishwanath R. Lingappa, and Pamela J. Glass. "Validation of a cell-based ELISA as a screening tool identifying anti-alphavirus small-molecule inhibitors." Journal of Virological Methods 193, no. 1 (October 2013): 226–31. http://dx.doi.org/10.1016/j.jviromet.2013.06.007.

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LIU, ZONGLIN L., and HANS P. BLASCHEK. "Monoclonal Antibody-Based ELISA for Detection of Clostridium perfringens Alpha-Toxin." Journal of Food Protection 59, no. 6 (June 1, 1996): 621–25. http://dx.doi.org/10.4315/0362-028x-59.6.621.

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A monoclonal antibody-based ELISA was developed to detect alpha-toxin present in Clostridium perfringens bacterial cell lysates and cell-free culture supernatants, Monoclonal antibodies against C. perfringens alpha-toxin were produced in hybridoma tissue culture supernatants and in BALB/c mice ascites fluid, The monoclonal antibodies obtained from hybridoma culture supernatant and ascites fluid showed identical antigen specificity, but the latter showed a higher titer, with a 50% endpoint at 1/4,000. The monoclonal antibodies were specific for phospholipase C produced by C. perfringens, but not by Bacillus cereus. The lower limit of phospholipase C detection was 16 ng/ml. The dose-dependent relationship between absorption at 490 nm and concentration of phospholipase C diluted in HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid) or Trypticase glucose yeast broth fit a four-parameter and a quadratic model, respectively. The monoclonal antibody-based ELISA developed is a rapid, sensitive and specific detection method and can be used for quantitative characterization of C. perfringens alpha-toxin.
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Frankfurt, Oskar S., and Awtar Krishan. "Microplate Screening for Apoptosis with Antibody to Single-Stranded DNA Distinguishes Anticancer Drugs from Toxic Chemicals." Journal of Biomolecular Screening 8, no. 2 (April 2003): 185–90. http://dx.doi.org/10.1177/1087057103253326.

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The effect of anticancer drugs and toxic compounds on cultures of human leukemic cells was evaluated by an enzyme-linked immunosorbent assay (Apoptosis ELISA) that uses a monoclonal antibody against single-stranded DNA to quantitate the apoptotic cells. The concentrations of 13 anticancer drugs, which increased Apoptosis ELISA absorbance, were close to the cytotoxic concentrations determined by the long-term cell survival assay. Short-term tetrazolium-based microculture tetrazolium (MTT) assay was significantly less sensitive than the Apoptosis ELISA and the cell survival assay for all anticancer drugs. For 6 drugs, cytotoxic concentrations measured by the MTT assay were at least 1 log higher than the concentrations inducing apoptosis. Importantly, in contrast to the anticancer drugs, 14 toxic chemicals did not increase the Apoptosis ELISA absorbance at cytotoxic concentrations. The difference in apoptosis induction by the anticancer drugs and the toxic chemicals was especially large in cultures treated with drug concentrations 2-fold higher than the IC50 dose. Although all of the anticancer drugs tested induced intense ELISA reaction (mean absorbance 2.0), all toxic chemicals tested did not induce apoptosis. The Apoptosis ELISA assay could have useful applications in drug development as it can distinguish between clinically useful anticancer drugs and toxic compounds, has sensitivity similar to that of the long-term cell survival assay, and provides insight into the mechanism of drug cytotoxicity by differentiating between compounds killing cells by apoptosis and necrosis. ( Journal of Biomolecular Screening 2003:185-190)
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Pilely, Katrine, Martin Rask Johansen, Rikke Raaen Lund, Thomas Kofoed, Thomas Kjærsgaard Jørgensen, Lars Skriver, and Ejvind Mørtz. "Monitoring process-related impurities in biologics–host cell protein analysis." Analytical and Bioanalytical Chemistry 414, no. 2 (October 1, 2021): 747–58. http://dx.doi.org/10.1007/s00216-021-03648-2.

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AbstractDuring biologics development, manufacturers must demonstrate clearance of host cell impurities and contaminants to ensure drug purity, manufacturing process consistency, and patient safety. Host cell proteins (HCPs) are a major class of process-related impurities and require monitoring and documentation of their presence through development and manufacturing. Even in residual amounts, they are known to affect product quality and efficacy as well as patient safety. HCP analysis using enzyme-linked immunosorbent assay (HCP-ELISA) is the standard technique, due to its simple handling, short analysis time, and high sensitivity for protein impurities. Liquid chromatography mass spectrometry (LC–MS) is an orthogonal method for HCP analysis and is increasingly included in regulatory documentation. LC–MS offers advantages where HCP-ELISA has drawbacks, e.g., the ability to identify and quantify individual HCPs. This article summarizes the available knowledge about monitoring HCPs in biologics and presents the newest trends in HCP analysis with current state-of-the-art HCP measurement tools. Through case studies, we present examples of HCP control strategies that have been used in regulatory license applications, using an MS-based coverage analysis and HCP-ELISA and LC–MS for HCP quantification. This provides novel insight into the rapid evolving strategy of HCP analysis. Improvements in technologies to evaluate HCP-ELISA suitability and the implementation of orthogonal LC–MS methods for HCP analysis are important to rationally manipulate, engineer, and select suitable cell lines and downstream processing steps to limit problematic HCPs.
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AlGhounaim, Mohammad, Yves Longtin, Milagros Gonzales, Joanna Merckx, Nicholas Winters, and Caroline Quach. "Clostridium difficile Infections in Children: Impact of the Diagnostic Method on Infection Rates." Infection Control & Hospital Epidemiology 37, no. 9 (June 6, 2016): 1087–93. http://dx.doi.org/10.1017/ice.2016.123.

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BACKGROUNDPolymerase chain reaction (PCR) assays based on the detection of the toxin B gene are replacing enzyme-linked immunosorbent assay (ELISA)–based toxin production detection or cell cytotoxicity assay in most laboratories.OBJECTIVETo determine the proportion of pediatric patients diagnosed withClostridium difficile infection by PCR who would have also been diagnosed by ELISA and to compare the clinical characteristics of PCR+/ELISA+ vs PCR+/ELISA− patients.METHODSUsing the microbiology laboratory information system, stool samples positive for C. difficile by PCR between October 2010 and July 2014 were identified. Using frozen stool specimens, an ELISA for toxin A and B was performed. A retrospective medical chart review was conducted to obtain demographic and clinical data. Duplicate samples were excluded.RESULTSA total of 136 PCR-positive samples underwent ELISA testing: 54 (40%) were positive for toxin A or B. The mean (SD) age of the entire cohort was 8.5 (6.2) years. There was no difference in age, gender, clinical manifestation, previous medical problems, and management between patients positive or negative by ELISA. However, patients positive by ELISA were more likely to have had a recent exposure to antibiotics (67.9% vs 50%; crude odds ratio, 2.1 [95% CI, 1.03–4.28]).CONCLUSIONIn our pediatric population, 60% of patients with C. difficile diagnosed by PCR had no toxin detectable by ELISA. ELISA-negative patients were less likely to have received an antibiotic recently compared with ELISA-positive patients. These results highlight the need to standardize laboratory criteria for the diagnosis of C. difficile infections in children.Infect Control Hosp Epidemiol 2016;37:1087–1093
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Flood, Veronica H., Patricia A. Morateck, Pamela A. Christopherson, Kenneth D. Friedman, Joan Cox Gill, and Robert R. Montgomery. "Unique ELISA-Based Assays for VWF-GPIb Interactions and the Impact of Racial Differences on VWF Testing." Blood 112, no. 11 (November 16, 2008): 423. http://dx.doi.org/10.1182/blood.v112.11.423.423.

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Abstract Diagnosis of von Willebrand disease (VWD) relies primarily on assays of von Willebrand factor (VWF) function (VWF:RCo) and concentration of VWF protein (VWF:Ag). VWF:RCo is a surrogate measure of VWF activity for VWF interaction with the GPIb receptor on platelets. For VWD screening purposes, some have advocated that VWF:RCo is the most appropriate single test, but variability and reproducibility of VWF:RCo assays between laboratories has been problematic. Alternative assays have therefore been sought. Type 2 VWD variants, 2A, 2B, and 2M, are characterized by a discrepancy between the VWF:Ag and VWF:RCo, yet numerous factors can affect this ratio. Previously, our laboratory has reported a cell based assay to measure VWF interaction with the GPIb complex by flow cytometry, but such assay instrumentation is not available in hemostasis testing laboratories. Plasma samples were collected from 75 healthy donors enrolled in the TS Zimmerman Program for the Molecular and Clinical Biology of VWD, including 44 African American (AA) and 31 Caucasian subjects. VWF:Ag and VWF:RCo levels were performed in a central laboratory, as were collagen binding (VWF:CB) and propeptide (VWFpp) testing. Two ELISA-based assays were developed to measure VWF interaction with GPIb using recombinant GPIbα – one using normal GPIb with ristocetin (VWF:RCo ELISA) and the other a mutant form of GPIbα containing the platelet-type mutations D235Y and M239V that does not require ristocetin (VWF:IbCo ELISA). A monoclonal antibody is used to capture the rGPIb and to orient the GPIb for subsequent interaction with VWF. Serially diluted plasma samples were incubated for 1 hour with or without added ristocetin and monoclonal antibodies to VWF were used to detect the presence of VWF. Both assays utilized a 10 minute agitation at the end of the plasma incubation step. For all subjects, the mean VWF:Ag was 142, the mean VWF:RCo was 124, and the mean VWF:RCo/VWF:Ag ratio was 0.90. The two new assays yielded similar activity results when all the control subjects were analyzed, with a mean of 104 for the VWF:RCo ELISA and a mean of 108 for the VWF:IbCo ELISA. Both assays correlated well with each other and with the VWF:RCo. R squared values as determined by linear regression were 0.79 for the comparison of the VWF:RCo ELISA with the VWF:IbCo ELISA and 0.80 for comparison of either with the regular VWF:RCo. When the results were analyzed by race, however, a significant difference was seen for the two ristocetin-containing assays. The mean VWF:RCo/VWF:Ag ratio for the AA controls was 0.85, compared to 0.95 for the Caucasian controls (p&lt;0.025). For the ristocetin ELISA, the mean was 0.54 for the AA controls and 0.79 for the Caucasian controls (p&lt;0.001). However, no significant racial difference was seen for the VWF:IbCo ELISA with mean of 0.72 for the AA controls and 0.81 for the Caucasian controls (p=NS). Other VWF ratios have been proposed to be used to classify VWD – VWF:CB/VWF:Ag, FVIII/VWF:Ag, and VWFpp/VWF:Ag, but none were significantly different by race. The use of ELISA-based assays to determine VWF function is therefore feasible and may alleviate some of the problems inherent in the traditional VWF:RCo assay, including reproducibility and the technical demands of the assay. The VWF:IbCo assay may also eliminate racial differences in the VWF activity to antigen ratio, thus preventing the potential for erroneous diagnosis of VWD. Furthermore, the ELISA-based assays can be performed using standard hemostasis laboratory instrumentation.
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Zhang, Yuan, Gang Xu, Lu Zhang, Jiakai Zhao, Pinpin Ji, Yaning Li, Baoyuan Liu, et al. "Development of a double monoclonal antibody–based sandwich enzyme-linked immunosorbent assay for detecting canine distemper virus." Applied Microbiology and Biotechnology 104, no. 24 (November 7, 2020): 10725–35. http://dx.doi.org/10.1007/s00253-020-10997-y.

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Abstract Canine distemper virus (CDV) infection causes mass mortality in diverse carnivore species. For effective virus surveillance, rapid and sensitive assays are needed to detect CDV in field samples. In this study, after BABL/c mice were immunized with recombinant CDV-fusion (F) protein, monoclonal antibodies (mAbs) against recombinant CDV-F protein (designated 1A5, 1A6, and 7D5) were produced using traditional hybridoma cell technology. Next, capture antibody (1A6, 800 ng/well) and horseradish peroxidase (HRP)–conjugated detection antibody (HRP-7D5, 1:100, 500 ng/well) were used in a double monoclonal antibody–based sandwich enzyme-linked immunosorbent assay (ELISA) for CDV detection after optimization of both mAb amounts per well using a checkerboard titration test. Based on sandwich ELISA test results for 120 known CDV-negative samples, the cutoff value for a positive result was set to an OD450 nm value ≥ 0.196. As compared with test results obtained from commercial immune colloidal gold test strips, the low limits of detection for the two assays were revealed to be 100 TCID50 per 100 μL. In addition, the sandwich ELISA agreed 100% and 96.4% with commercial immune colloidal gold test strips when testing serum and stool samples. The sandwich ELISA assay provided statistically similar CDV detection. Thus, the sandwich ELISA developed here to detect CDV in fecal and serum samples provided good sensitivity, high specificity, and good reproducibility and should serve as an ideal method for large-scale surveillance of CDV infections in carnivores. Key points • Three CDV mAbs that recognized different epitopes and bound to virion were generated. • The sandwich ELISA based mAbs to detect CDV in fecal and serum samples was developed. • The sandwich ELISA is an ideal method for detecting CDV infections in the field.
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López, Lissett, Angel Venteo, Marga García, Ana Camuñas, Ana Ranz, Julia García, Javier Sarraseca, Carmen Anaya, and Paloma Rueda. "Antigen-Capture Blocking Enzyme-Linked Immunosorbent Assay Based on a Baculovirus Recombinant Antigen to Differentiate Transmissible Gastroenteritis Virus from Porcine Respiratory Coronavirus Antibodies." Journal of Veterinary Diagnostic Investigation 21, no. 5 (September 2009): 598–608. http://dx.doi.org/10.1177/104063870902100503.

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A new commercially available antigen-capture, blocking enzyme-linked immunosorbent assay (antigen-capture b-ELISA), based on baculovirus truncated-S recombinant protein of Transmissible gastroenteritis virus (TGEV) and 3 specific monoclonal antibodies, was developed and evaluated by examining a panel of 453 positive Porcine respiratory coronavirus (PRCoV), 31 positive TGEV, and 126 negative field sera by using another commercially available differential coronavirus b-ELISA as the reference technique to differentiate TGEV- from PRCoV-induced antibodies. The recombinant S protein-based ELISA appeared to be 100% sensitive for TGEV and PRCoV detection and highly specific for TGEV and PRCoV detection (100% and 92.06%, respectively), when qualitative results (positive or negative) were compared with those of the reference technique. In variability experiments, the ELISA gave consistent results when the same serum was evaluated on different wells and different plates. These results indicated that truncated recombinant S protein is a suitable alternative to the complete virus as antigen in ELISA assays. The use of recombinant S protein as antigen offers great advantages because it is an easy-to-produce, easy-to-standardize, noninfectious antigen that does not require further purification or concentration. Those advantages represent an important improvement for antigen preparation, in comparison with other assays in which an inactivated virus from mammalian cell cultures is used.
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Foster, Camila N., Ursula A. Rossi, M. Raquel Castaño Zubieta, Victor Vanzini, and Carlos A. Rossetti. "Evaluation of B. melitensis whole-cell lysate antigen-based indirect ELISA for the serodiagnosis of caprine brucellosis." Research in Veterinary Science 147 (October 2022): 1–6. http://dx.doi.org/10.1016/j.rvsc.2022.03.015.

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Pi-Estopiñan, Franciscary, María Teresa Pérez, Anitza Fraga, Gretchen Bergado, Geidy D. Díaz, Ivette Orosa, Marianniz Díaz, et al. "A cell-based ELISA as surrogate of virus neutralization assay for RBD SARS-CoV-2 specific antibodies." Vaccine 40, no. 13 (March 2022): 1958–67. http://dx.doi.org/10.1016/j.vaccine.2022.02.044.

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Yanamandra, Mahesh, Labanyamoy Kole, Archana Giri, and Sayan Mitra. "Development of highly sensitive cell-based AKT kinase ELISA for monitoring PI3K beta activity and compound efficacy." Journal of Immunoassay and Immunochemistry 38, no. 6 (November 2, 2017): 663–74. http://dx.doi.org/10.1080/15321819.2017.1385027.

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Pellett, Sabine, William H. Tepp, Eric A. Johnson, and Dorothea Sesardic. "Assessment of ELISA as endpoint in neuronal cell-based assay for BoNT detection using hiPSC derived neurons." Journal of Pharmacological and Toxicological Methods 88 (November 2017): 1–6. http://dx.doi.org/10.1016/j.vascn.2017.04.013.

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Craigo, Jodi K., Corin Ezzelarab, and Ronald C. Montelaro. "Development of a high throughput, semi-automated, infectious center cell-based ELISA for equine infectious anemia virus." Journal of Virological Methods 185, no. 2 (November 2012): 221–27. http://dx.doi.org/10.1016/j.jviromet.2012.07.007.

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Begum, Taslima, Humayun Sattar, Md Ruhul Amin Miah, and AFM Shahidur Rahman. "Evaluation of indirect immunofluroscence on HEP-2 cell and enzyme immunoassay methods for detection on antinuclear antibodies." Bangladesh Journal of Medical Microbiology 7, no. 1 (January 1, 2013): 11–14. http://dx.doi.org/10.3329/bjmm.v7i1.19315.

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Abstract:
The suspicion of autoimmune disease primarily starts with clinical symptoms. The ELISA and IIF (indirect immunofluorescence) on HEp-2 cell methods are comparable for detecting ANA in patient with auto immune diseases (AID). 102 patients attending Rheumatology out patient department as new or old cases with provisional diagnosis of SLE, RA, MCTD and other Connective Tissue disorder (CTD) screened for specific autoantibody by ELISA (anti CCP, ANA and anti dsDNA) were subjected to indirect immunofluorescence on HEp-2 cell line. Diagnostic accuracy based on ELISA and indirect immunoflurescence on HEp-2 cell line showed the results of sensitivity, specificity, PPV, NPV, accuracy of ANA were found to be 78.9%, 33.3%, 78.9%, 33.3%. 68% respectively. Sensitivity, specificity, PPV, NPV and the accuracy of anti-dsDNA was 80%, 40%, 80%.40% and 70% respectively. Sensitivity, specificity, PPV, NPV, and the accuracy of anti CCP was found to be 64.3%, 72.2%, 64.3%, 72.2% and 68.8% respectively. The results indicates there is a need for screening all suspected autoimmune patient by indirect immunofluorescence on HEp-2 cell line before going to specific test.DOI: http://dx.doi.org/10.3329/bjmm.v7i1.19315 Bangladesh J Med Microbiol 2013; 07(01): 11-14
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