Establishment of Reverse Direct ELISA and Its Application in Screening High-Affinity Monoclonal Antibodies

Zhang, Chunmei; Li, Rende; Li, Yongming; Song, Chaojun; Liu, Zhijia; Zhang, Yun; Xu, Zhuwei; Zhuang, Ran; Yi, Jun; Yang, Angang; Yang, Kun; Jin, Boquan

doi:10.1089/hyb.2012.0010

Cited by 4 publications

(4 citation statements)

References 10 publications

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“…We used an indirect ELISA to titer five McAbs. Figure 1 A shows that the titer of 1A1, 3A9, 1H10, and 4H1 was 1:64,000, and the titer of 1D8 was 1:16,000, according to the principle of P/N ≥ 2.0 [ 45 ]. Antibodies with a higher dilution had a higher titer, which meant they had a strong ability to bind antigen.…”

Section: Resultsmentioning

confidence: 99%

Enzyme-Antibody-Modified Gold Nanoparticle Probes for the Ultrasensitive Detection of Nucleocapsid Protein in SFTSV

Yuqin

Zhao

et al. 2020

IJERPH

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As humans and climate change continue to alter the landscape, novel disease risk scenarios have emerged. Sever fever with thrombocytopenia syndrome (SFTS), an emerging tick-borne infectious disease first discovered in rural areas of central China in 2009, is caused by a novel bunyavirus (SFTSV). The potential for SFTS to spread to other countries in combination with its high fatality rate, possible human-to-human transmission, and extensive prevalence among residents and domesticated animals in endemic regions make the disease a severe threat to public health. Because of the lack of preventive vaccines or useful antiviral drugs, diagnosis of SFTS is the key to prevention and control of the SFTSV infection. The development of serological detection methods will greatly improve our understanding of SFTSV ecology and host tropism. We describe a highly sensitive protein detection method based on gold nanoparticles (AuNPs) and enzyme-linked immunosorbent assay (ELISA)—AuNP-based ELISA. The optical sensitivity enhancement of this method is due to the high loading efficiency of AuNPs to McAb. This enhances the concentration of the HRP enzyme in each immune sandwich structure. The detection limit of this method to the nucleocapsid protein (NP) of SFTSV was 0.9 pg mL−1 with good specificity and reproducibility. The sensitivity of AuNP-based ELISA was higher than that of traditional ELISA and was comparable to real-time quantitative polymerase chain reaction (qRT-PCR). The probes are stable for 120 days at 4 °C. This can be applied to diagnosis and hopefully can be developed into a commercial ELISA kit. The ultrasensitive detection of SFTSV will increase our understanding of the distribution and spread of SFTSV, thus helping to monitor the changes in tick-borne pathogen SFTSV risk in the environment.

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Section: Resultsmentioning

confidence: 99%

Enzyme-Antibody-Modified Gold Nanoparticle Probes for the Ultrasensitive Detection of Nucleocapsid Protein in SFTSV

Yuqin

Zhao

et al. 2020

IJERPH

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“…We used lipopolysaccharides (LPS, 100 ng/ml, Sigma-Aldrich) as a positive control, cells were tested following incubation for 3 h after treatments. HMGB1, histone 3 and HSP70 levels were measured by direct ELISA using a self-coating protocol, as previously described ( Zhang et al, 2012 ). All samples were measured on a single 96-well plate for each cytokine, the cultured medium was centrifuged at 800 g for 5 min at 4°C and the supernatant was used as the sample.…”

Section: Methodsmentioning

confidence: 99%

Loss of Ca2+ entry via Orai–TRPC1 induces ER stress, initiating immune activation in macrophages

Conceicao

Sun

Zboril

et al. 2019

Journal of Cell Science

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Activation of cellular stresses is associated with inflammation; however, the mechanisms are not well identified. Here, we provide evidence that loss of Ca 2+ influx induces endoplasmic reticulum (ER) stress in primary macrophages and in murine macrophage cell line Raw 264.7, in which the unfolded protein response is initiated to modulate cytokine production, thereby activating the immune response. Stressors that initiate the ER stress response block storedependent Ca 2+ entry in macrophages prior to the activation of the unfolded protein response. The endogenous Ca 2+ entry channel is dependent on the Orai1-TRPC1-STIM1 complex, and the presence of ER stressors decreased expression of TRPC1, Orai1 and STIM1. Additionally, blocking Ca 2+ entry with SKF96365 also induced ER stress, promoted cytokine production, activation of autophagy, increased caspase activation and induced apoptosis. Furthermore, ER stress inducers inhibited cell cycle progression, promoted the inflammatory M1 phenotype, and increased phagocytosis. Mechanistically, restoration of Orai1-STIM1 expression inhibited the ER stress-mediated loss of Ca 2+ entry that prevents ER stress and inhibits cytokine production, and thus induced cell survival. These results suggest an unequivocal role of Ca 2+ entry in modulating ER stress and in the induction of inflammation.

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“…Finally, the dissociation constant (K D ) was evaluated by measuring the slope of the linear dependence [ 27 ].…”

Section: Methodsmentioning

confidence: 99%

Distinct B-Cell Specific Transcriptional Contexts of the BCL2 Oncogene Impact Pre-Malignant Development in Mouse Models

Zawil

Marchiol

Brauge

et al. 2022

Cancers

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Upregulated expression of the anti-apoptotic BCL2 oncogene is a common feature of various types of B-cell malignancies, from lymphoma to leukemia or myeloma. It is currently unclear how the various patterns of deregulation observed in pathology eventually impact the phenotype of malignant B cells and their microenvironment. Follicular lymphoma (FL) is the most common non-Hodgkin lymphoma arising from malignant germinal center (GC) B-cells, and its major hallmark is the t(14:18) translocation occurring in B cell progenitors and placing the BCL2 gene under the control of the immunoglobulin heavy chain locus regulatory region (IgH 3′RR), thus exposing it to constitutive expression and hypermutation. Translocation of BCL2 onto Ig light chain genes, BCL2 gene amplification, and other mechanisms yielding BCL2 over-expression are, in contrast, rare in FL and rather promote other types of B-cell lymphoma, leukemia, or multiple myeloma. In order to assess the impact of distinct BCL2 deregulation patterns on B-cell fate, two mouse models were designed that associated BCL2 and its full P1-P2 promoter region to either the IgH 3′RR, within a “3′RR-BCL2” transgene mimicking the situation seen in FL, or an Ig light chain locus context, through knock-in insertion at the Igκ locus (“Igκ-BCL2” model). While linkage to the IgH 3′ RR mostly yielded expression in GC B-cells, the Igκ-driven up-regulation culminated in plasmablasts and plasma cells, boosting the plasma cell in-flow and the accumulation of long-lived plasma cells. These data demonstrate that the timing and level of BCL2 deregulation are crucial for the behavior of B cells inside GC, an observation that could strongly impact the lymphomagenesis process triggered by secondary genetic hits.

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Establishment of Reverse Direct ELISA and Its Application in Screening High-Affinity Monoclonal Antibodies

Cited by 4 publications

References 10 publications

Enzyme-Antibody-Modified Gold Nanoparticle Probes for the Ultrasensitive Detection of Nucleocapsid Protein in SFTSV

Enzyme-Antibody-Modified Gold Nanoparticle Probes for the Ultrasensitive Detection of Nucleocapsid Protein in SFTSV

Loss of Ca2+ entry via Orai–TRPC1 induces ER stress, initiating immune activation in macrophages

Distinct B-Cell Specific Transcriptional Contexts of the BCL2 Oncogene Impact Pre-Malignant Development in Mouse Models

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