Development of an ic-ELISA and immunochromatographic strip based on IgG antibody for detection of ω-conotoxin MVIIA

Wang, Rongzhi; Zhong, Yanfang; Wang, Juncheng; Yang, Hui; Yuan, Jun; Wang, Shihua

doi:10.1016/j.jhazmat.2019.03.129

Cited by 13 publications

(12 citation statements)

References 27 publications

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“…These fusion proteins were successfully expressed after IPTG induction and further purified by Ni 2+ -NTA affinity chromatography. The band sizes of the purified MBP-LK-αB-CTX4 and TRX-αB-CTX with high purity on the gel of SDS-PAGE were 60 kDa and 28 kDa, respectively, the same as the theoretical result [5]. Then, the purified MBP-LK-αB-CTX4 was used as the immunogen for animal immunization, and the titer of serum was tested by ELISA with the TRX-αB-CTX as the detection antigen (Figure 2D).…”

Section: Preparation Of αB-ctx and Animal Immunizationsupporting

confidence: 74%

“…Conotoxins, a diverse array of unique bioactive neurotoxins, are mainly secreted by different kinds of cone snails [ 3 , 4 ]. Conotoxins are often toxic to humans and animals and can cause convulsions and paralysis, even to death [ 5 ]. More than 80,000 natural conotoxins have been estimated to exist in various cone snails around the world [ 6 , 7 , 8 ], and all conotoxins can be divided into 26 gene superfamilies based on their conserved signal sequences and their characteristic cysteine framework [ 5 , 9 ].…”

Section: Introductionmentioning

confidence: 99%

“…Conotoxins are often toxic to humans and animals and can cause convulsions and paralysis, even to death [ 5 ]. More than 80,000 natural conotoxins have been estimated to exist in various cone snails around the world [ 6 , 7 , 8 ], and all conotoxins can be divided into 26 gene superfamilies based on their conserved signal sequences and their characteristic cysteine framework [ 5 , 9 ]. Among them, α-, µ- and ω-conotoxins are the most characterized families so far for their high specificity and affinity to ion channels [ 1 , 10 , 11 , 12 ].…”

Section: Introductionmentioning

confidence: 99%

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Detection of αB-Conotoxin VxXXIVA (αB-CTX) by ic-ELISA Based on an Epitope-Specific Monoclonal Antibody

Tang

Liu

Gao

et al. 2022

Toxins

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In view of the toxicological hazard and important applications in analgesics and cancer chemotherapeutics of αB-CTX, it is urgent to develop an accurate, effective and feasible immunoassay for the determination and analysis of αB-CTX in real samples. In this study, MBP-αB-CTX4 tandem fusion protein was used as an immunogen to elicit a strong immune response, and a hybridoma cell 5E4 secreting IgG2b against αB-CTX was successfully screened by hybridoma technology. The affinity of the purified 5E4 monoclonal antibody (mAb) was 1.02 × 108 L/mol, which showed high affinity and specificity to αB-CTX. Epitope 1 of αB-CTX is the major binding region for 5E4 mAb recongnization, and two amino acid residues (14L and 15F) in αB-CTX were critical sites for the interaction between αB-CTX and 5E4 mAb. Indirect competitive ELISA (ic-ELISA) based on 5E4 mAb was developed to detect and analyze αB-CTX in real samples, and the linear range of ic-ELISA to αB-CTX was 117–3798 ng/mL, with a limit of detection (LOD) of 81 ng/mL. All the above results indicated that the developed ic-ELISA had high accuracy and repeatability, and it could be applied for αB-CTX detection and drug analysis in real samples.

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Section: Preparation Of αB-ctx and Animal Immunizationsupporting

confidence: 74%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Detection of αB-Conotoxin VxXXIVA (αB-CTX) by ic-ELISA Based on an Epitope-Specific Monoclonal Antibody

Tang

Liu

Gao

et al. 2022

Toxins

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“…The results showed that the anti-NEK2 3A3 mAb could specifically recognize exogenous and endogenous NEK2 proteins, indicating that the antibody had good antigen specificity. Wang [ 24 ] prepared an anti-ω-CTX MVIIA monoclonal antibody with high affinity and specificity and applied it to the development of ELISA kits and immunocolloidal gold test strips. The detection limits were 0.14 μg/mL and 1 μg/mL, respectively, and the linear range of the ELISA was 0.20 ~ 7.22 μg/mL, indicating it can be used as a rapid detection method for screening actual ω-CTX MVIIA samples.…”

Section: Discussionmentioning

confidence: 99%

Preparation of an anti-NEK2 monoclonal antibody and its application in liver cancer

Chen

Yang

et al. 2021

BMC Biotechnol

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Background Never in mitosis gene-A (NIMA)-related expressed kinase 2 (NEK2) is a serine/threonine protein kinase regulated by the cell cycle. The purpose of this study was to obtain NEK2 protein to prepare an anti-NEK2 monoclonal antibody (mAb) and explore the application of the anti-NEK2 mAb of therapeutic and diagnostic in hepatocellular carcinoma (HCC). Results The NEK2 gene sequence was cloned from the normal liver cell line HL7702, and the full-length NEK2 gene sequence was cloned into the prokaryotic expression vector pET30a and transformed into Escherichia coli BL21 (DE3) cells. The recombinant fusion protein was obtained under optimized conditions and injected in BALB/c mice to prepare an anti-NEK2 mAb. By screening, we obtained a stable hybridoma cell line named 3A3 that could stably secrete anti-NEK2 mAb. Anti-NEK2 3A3 mAb was purified from ascites fluid. The isotype was IgG1, and the affinity constant (Kaff) was 6.0 × 108 L/mol. Western blot, indirect enzyme-linked immunosorbent assay (iELISA), immunofluorescence and immunocytochemical analyses showed that the mAb could specifically recognize the NEK2 protein. MTT assays showed that the mAb 3A3 could inhibit the proliferation of HCC cells. KEGG pathway analysis showed that NEK2 might affected pathways of the cell cycle. Moreover, NEK2-related genes were mainly enriched in the S and G2 phases and might act as tumor-promoting genes by regulating the S/G2 phase transition of HCC cells. Conclusions An anti-NEK2 mAb with high potency, high affinity and high specificity was prepared by prokaryotic expression system in this study and may be used in the establishment of ELISA detection kits and targeted treatment of liver cancer.

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“…To evaluate the accuracy of the strip test, five different kinds of actual snail or shellfish samples were purchased randomly from the Fuzhou markets in China. The collected samples were mashed and centrifuged at 10,000 r/min for 5 min [ 31 ], and the supernatant was used for evaluating the αB-CTX residue in real samples by the developed AuNPs-/AuNFs-based strip. All the steps of detection were the same as described above.…”

Section: Methodsmentioning

confidence: 99%

Development of Immunochromatographic Strip for Detection of αB-VxXXIVA-Conotoxin Based on 5E4 Monoclonal Antibody

Tang

Liu

Chen

et al. 2022

Toxins

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Given the application of αB-VxXXIVA-conotoxin (αB-CTX) in analgesics and cancer chemotherapeutics, and its threat to humans, it is urgent to develop a rapid, effective and accurate method for the analysis and detection of αB-CTX in real shellfish and medicine drug samples. In the present study, two different immunochromatographic strips were established for αB-CTX detection, based on the monoclonal antibody 5E4 against αB-CTX, and the visual limits of detection (vLOD) for the colloidal gold nanoparticles-based strip (AuNPs-based strip) and nanoflowers-based strip (AuNFs-based strip) were 4 μg/mL and 1.5 μg/mL, respectively. The developed AuNPs-/AuNFs-based strips have good specificity and accuracy, and the detection results were analyzed in less than 10 min, without using an instrument. In view of the excellent repeatability and usability, the established methods could be applied to detect and analyze the content of αB-CTX in real samples.

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Development of an ic-ELISA and immunochromatographic strip based on IgG antibody for detection of ω-conotoxin MVIIA

Cited by 13 publications

References 27 publications

Detection of αB-Conotoxin VxXXIVA (αB-CTX) by ic-ELISA Based on an Epitope-Specific Monoclonal Antibody

Detection of αB-Conotoxin VxXXIVA (αB-CTX) by ic-ELISA Based on an Epitope-Specific Monoclonal Antibody

Preparation of an anti-NEK2 monoclonal antibody and its application in liver cancer

Development of Immunochromatographic Strip for Detection of αB-VxXXIVA-Conotoxin Based on 5E4 Monoclonal Antibody

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