Development and evaluation of a gp85 protein-based subgroup-specific indirect enzyme-linked immunosorbent assay for the detection of anti-subgroup J avian leukosis virus antibodies

Chang, Fangfang; Xing, Lixiao; Xing, Zhifeng; Yu, Mengmeng; Bao, Yuanling; Wang, Suyan; Farooque, Muhammad; Liu, Peng; Pan, Qing; Qi, Xiaole; Gao, Li; Li, Kai; Liu, Changjun; Zhang, Yanping; Cui, Hongyu; Wang, Xiaomei; Gao, Yulong

doi:10.1007/s00253-019-10320-4

Cited by 5 publications

(5 citation statements)

References 30 publications

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“…Nevertheless, the green fluorescence intensity varied among different cells, and in some cells even no green fluorescence was detected. The cell-to-cell variance in the intensity of fluorescent protein is also commonly reported in other cells stably transfected with fluorescent proteins [ 36 ]. The possible reasons include: (i) the copy number of the exogenous gene introduced into different cells is different; (ii) the activity of the promoters of the exogenous gene in different cells is different [ 37 ].…”

Section: Discussionmentioning

confidence: 98%

Deep learning-assisted high-content screening identifies isoliquiritigenin as an inhibitor of DNA double-strand breaks for preventing doxorubicin-induced cardiotoxicity

Chen,

Liu,

Zhao

et al. 2023

Biol Direct

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Background Anthracyclines including doxorubicin are essential components of many cancer chemotherapy regimens, but their cardiotoxicity severely limits their use. New strategies for treating anthracycline-induced cardiotoxicity (AIC) are still needed. Anthracycline-induced DNA double-strand break (DSB) is the major cause of its cardiotoxicity. However, DSB-based drug screening for AIC has not been performed possibly due to the limited throughput of common assays for detecting DSB. To discover new therapeutic candidates for AIC, here we established a method to rapidly visualize and accurately evaluate the intranuclear anthracycline-induced DSB, and performed a screening for DSB inhibitors. Results First, we constructed a cardiomyocyte cell line stably expressing EGFP-53BP1, in which the formation of EGFP-53BP1 foci faithfully marked the doxorubicin-induced DSB, providing a faster and visible approach to detecting DSB. To quantify the DSB, we used a deep learning-based image analysis method, which showed the better ability to distinguish different cell populations undergoing different treatments of doxorubicin or reference compounds, compared with the traditional threshold-based method. Subsequently, we applied the deep learning-assisted high-content screening method to 315 compounds and found three compounds (kaempferol, kaempferide, and isoliquiritigenin) that exert cardioprotective effects in vitro. Among them, the protective effect of isoliquiritigenin is accompanied by the up-regulation of HO-1, down-regulation of peroxynitrite and topo II, and the alleviation of doxorubicin-induced DSB and apoptosis. The results of animal experiments also showed that isoliquiritigenin maintained the myocardial tissue structure and cardiac function in vivo. Moreover, isoliquiritigenin did not affect the killing of HeLa and MDA-MB-436 cancer cells by doxorubicin and thus has the potential to be a lead compound to exert cardioprotective effects without affecting the antitumor effect of doxorubicin. Conclusions Our findings provided a new method for the drug discovery for AIC, which combines phenotypic screening with artificial intelligence. The results suggested that isoliquiritigenin as an inhibitor of DSB may be a promising drug candidate for AIC.

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Section: Discussionmentioning

confidence: 98%

Deep learning-assisted high-content screening identifies isoliquiritigenin as an inhibitor of DNA double-strand breaks for preventing doxorubicin-induced cardiotoxicity

Chen,

Liu,

Zhao

et al. 2023

Biol Direct

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“…However, this method was hampered by the professional requirements of testing personnel, laboratory environment, long turnaround time (over 7 days), high cost, and large reagent consumption. As to the complex epidemic of ALV, researchers have established many detection methods in different directions, such as virus isolation, reverse transcription–polymerase chain reaction (RT-PCR) (Garcia et al, 2003 ; Gao et al, 2014 ), quantitative reverse transcription–polymerase chain reaction (RT-qPCR) (Dai et al, 2015 ; Chen et al, 2018 ), reverse transcription–loop-mediated isothermal amplification (RT-LAMP) (Zhang et al, 2010 ; Peng et al, 2015 ) of molecular diagnostic, ELISA (De Boer and Osterhaus, 1985 ; Yun et al, 2013 ; Chang et al, 2020 ), and indirect immunofluorescence assays (IFA) (Pham et al, 1999 ; Zhang et al, 2021 ) of serological diagnostic. However, these methods have some weak points, for instance, RT-PCR and RT-qPCR require expensive and cumbersome instruments, and 2–3 pairs of primers for RT-LAMP tend to cause false positives because of nonspecific binding.…”

Section: Discussionmentioning

confidence: 99%

New rapid detection by using a constant temperature method for avian leukosis viruses

Chu

Zhang

et al. 2022

Front. Microbiol.

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The avian leukemia virus causes avian leukemia (AL), a severe immunosuppressive disease in chickens (ALV). Since the 1990s, the diversity of ALV subpopulations caused by ALV genome variation and recombination, and the complexity of the infection and transmission, with currently no effective commercial vaccine and therapeutic for ALV, has resulted in severe economic losses to the chicken business in various parts of the world. Therefore, as a key means of prevention and control, an effective, rapid, and accurate detection method is imperative. A new real-time reverse transcription recombinase-aided amplification (RT-RAA) assay for ALV with rapid, highly specific, low-cost, and simple operational characteristics have been developed in this study. Based on the amplification of 114 base pairs from the ALV P12 gene, real-time RT-RAA primers and a probe were designed for this study. The lowest detection line was 10 copies of ALV RNA molecules per response, which could be carried out at 39°C in as fastest as 5 min and completed in 30 min, with no cross-reactivity with Marek's disease virus, avian reticuloendothelial virus, Newcastle disease virus, infectious bronchitis virus, infectious bursal disease virus, infectious laryngotracheitis virus, and avian influenza virus. Furthermore, the kappa value of 0.91 (>0.81) was compared with reverse transcription–polymerase chain reaction (RT-PCR) for 44 clinical samples, and the coefficients of variation were within 5.18% of the repeated assays with three low-level concentration gradients. These results indicate that using a real-time RT-RAA assay to detect ALV could be a valuable method.

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“…According to the characteristics of host range, viral envelope protein, and cross-neutralization patterns, ALVs can be classified as endogenous or exogenous viruses. Exogenous ALVs can be classified into subgroups (A, B, C, D, J and K) in the chicken, which can cause different pathological lesions in chickens( Liang et al., 2019 ; Chang et al., 2020 ). Compared with other subgroups of ALVs, ALV-J mainly causes hematopoietic malignancy with myeloid leukemia and hemangioma in the chicken ( Cheng et al., 2010 ).…”

Section: Introductionmentioning

confidence: 99%

“…The Gp85 of ALV-J located on the viral surface mediates viral binding to cellular receptor on host cell membranes ( Venugopal et al., 1998 ) and can determine the specificity of different subgroups and the host range. Moreover, the sequence of ALV-J gp85 has a low homology rate with that of other exogenous subgroups ( Chang et al., 2020 ),thereby subgroups of ALV can be distinguished according to the sequence and antigenicity of gp85 .…”

Section: Introductionmentioning

confidence: 99%

“…Monoclonal antibody ( MAb ) has been widely developed and effectively applied in the detection of pathogenic microbes ( Garcia-Lunar et al., 2019 ; Chaudhari et al., 2020 ). MAbs applied against ALV-J have been successfully prepared ( Qin et al., 2001 ; Sun et al., 2012 ; Li et al., 2015 ; Chang et al., 2020 ) and used to establish some rapid and specific methods for the detection of ALV-J ( Liu et al., 2018 ). Although previous research reported that MAbs against Gp85 of ALV-J that could be used for identifying the antigen, there is still no commercial testing kits for ALV-J appeared in the clinic.…”

Section: Introductionmentioning

confidence: 99%

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Preparation of a novel monoclonal antibody against Avian leukosis virus subgroup J Gp85 protein and identification of its epitope

Wang

Chen²,

Zhu³

et al. 2021

Poultry Science

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Avian leukosis virus subgroup J ( ALV-J ) is an avian oncogenic retrovirus that has caused huge economic losses in the poultry industry due to its great pathogenicity and transmission ability. However, the continuous emergence of new strains would bring challenges to diagnosis and control of ALV-J. .This study focuses on preparing the monoclonal antibody ( MAb ) against ALV-J Gp85 and identifying its epitope. The truncated ALV-J gp85 gene fragment was amplified and then cloned into expression vectors. Purified GST-Gp85 was used to immune mice and His-Gp85 was used to screen MAb. Finally, a hybridoma cell line named J16 that produced specific MAb against the ALV-J. Immunofluorescence assay showed that MAb J16 specifically recognized ALV-J rather than ALV-A or ALV-K infected DF-1 cells. To identify the epitope recognized by MAb J16, fourteen partially overlapping ALV-J Gp85 fragments were prepared and tested by Western blot. The results indicated that peptide 150-LIRPYVNQ-157 was the minimal epitope of ALV-J Gp85 recognized by MAb J16. Alignment analysis of Gp85 from different ALV subgroups showed that the epitope keep high conservation among 36 ALV-J strains, but significant different from that of ALV subgroup A, B, C, D, E and K. Overall, we prepared a MAb specific against ALV-J and identified peptide 150-LIRPYVNQ-157 as a novel specific epitope of ALV-J Gp85, which may assist in laying the foundation for specific ALV-J detection methods.

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Development and evaluation of a gp85 protein-based subgroup-specific indirect enzyme-linked immunosorbent assay for the detection of anti-subgroup J avian leukosis virus antibodies

Cited by 5 publications

References 30 publications

Deep learning-assisted high-content screening identifies isoliquiritigenin as an inhibitor of DNA double-strand breaks for preventing doxorubicin-induced cardiotoxicity

Deep learning-assisted high-content screening identifies isoliquiritigenin as an inhibitor of DNA double-strand breaks for preventing doxorubicin-induced cardiotoxicity

New rapid detection by using a constant temperature method for avian leukosis viruses

Preparation of a novel monoclonal antibody against Avian leukosis virus subgroup J Gp85 protein and identification of its epitope

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