Preparation and Identification of Monoclonal Antibody against Abrin-a

Li, Xiaobing; Yang, Wei; Zhang, Yi; Zhang, Zhigang; Kong, Tao; Li, Dongna; Tang, Junhua; Liu, Zhaoxi; Liu, Guowen; Wang, Zhe

doi:10.1021/jf202534y

Cited by 19 publications

(12 citation statements)

References 15 publications

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“…Both assays were highly sensitive, but they were not designed for exclusive detection of intact or active abrin due to the nature of antibodies used. A llama-derived single domain antibody based ELISA reported in 2011 by Goldman et al [ 22 ] detected Abrus agglutinin, exclusively, leaving all abrin isoforms undetected. Assays that fail to detect major abrin isoforms could yield misleading diagnostic results, with a consequent risk to human health.…”

Section: Discussionmentioning

confidence: 99%

Detection of Abrin Holotoxin Using Novel Monoclonal Antibodies

Patfield

Cheng

et al. 2017

Toxins

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Abrin, a member of the ribosome-inactivating protein family, is produced by the Abrus precatorius plant. Having the potential to pose a severe threat to both human and animal health, abrin is classified as a Select Agent by the U.S. Department of Health and Human Services. However, an immunoassay that is specific for intact abrin holotoxin has not yet been reported. In this study, seven new monoclonal antibodies (mAbs), designated as Abrin-1 through Abrin-7 have been developed. Isotyping analyses indicate these mAbs have IgG1, IgG2a, or IgG2b heavy-chains and kappa light-chains. Western blot analyses identified two abrin A-chain specific mAbs, Abrin-1 and Abrin-2, and four B-chain specific mAbs (Abrin-3, -5, -6, and -7). A sandwich enzyme-linked immunosorbent assay (ELISA), capable of detecting a mixture of abrin isoforms and agglutinins was developed using B-chain specific Abrin-3 for capture and A-chain specific Abrin-2 as detector. The ELISA is highly sensitive and detects 1 ng/mL of the abrin holotoxin in phosphate-buffered saline, nonfat milk, and whole milk, significantly below concentrations that would pose a health concern for consumers. This ELISA also detects native abrin in plant extracts with a very low background signal. The new abrin mAbs and ELISA should be useful for detecting this potent toxin in the milk supply chain and other complex matrices.

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Section: Discussionmentioning

confidence: 99%

Detection of Abrin Holotoxin Using Novel Monoclonal Antibodies

Patfield

Cheng

et al. 2017

Toxins

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“…The abrin-UPT-LFA had detection limits as low as 0.1 ng/mL. A monoclonal capture ELISA with an LOD of 7.8 ng/mL [ 27 ] and use of aptamers for abrin detection in a colorimetric assay with an LOD of 0.05 nM (3.1 ng/mL) [ 28 ] also have been reported. Immunoassay performance fundamentally depends on the performance of the antibodies used as well the assay format.…”

Section: Discussionmentioning

confidence: 99%

A Monoclonal–Monoclonal Antibody Based Capture ELISA for Abrin

Tam

Cheng

et al. 2017

Toxins

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Abrin, one of the most highly potent toxins in the world, is derived from the plant, Abrus precatorius. Because of its high toxicity, it poses potential bioterror risks. Therefore, a need exists for new reagents and technologies that would be able to rapidly detect abrin contamination as well as lead to new therapeutics. We report here a group of abrin-specific monoclonal antibodies (mAbs) that recognize abrin A-chain, intact A–B chain toxin, and agglutinin by Western blot. Additionally, these mAbs were evaluated for their ability to serve as capture antibodies for a sandwich (capture) ELISA. All possible capture–detector pairs were evaluated and the best antibody pair identified and optimized for a capture ELISA. The capture ELISA based on this capture–detector mAb pair had a limit of detection (L.O.D) of ≈1 ng/mL measured using three independent experiments. The assay did not reveal any false positives with extracts containing other potential ribosome-inactivating proteins (RIPs). Thus, this new capture ELISA uses mAbs for both capture and detection; has no cross-reactivity against other plant RIPs; and has a sensitivity comparable to other reported capture ELISAs using polyclonal antibodies as either capture or detector.

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“…(13,14) The spleen was aseptically removed and the spleen cells were fused with the SP2/0 myeloma cells at a rate of 5 to 10: 1 in the presence of polythylene glycol (50%, w/v). After fusion, the cells were cultured in RPMI-1640 medium containing 15-20% fetal calf serum and HAT for 8-14 days.…”

Section: Cell Fusion and Hybridoma Clone Screeningmentioning

confidence: 99%

Preparation and Identification of Monoclonal Antibodies Against ω-Conotoxin MVIIA

Ma²,

Li³

et al. 2014

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy

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o-Conotoxins MVIIA (o-CTX MVIIA) is a peptide with 25 amino acid residues. It is a selective and reversible N-type voltage-gated calcium channel blocker, which could be used as an analgesic for pain. To date, there are no monoclonal antibodies (MAb) for immunoassay against o-conotoxin MVIIA. In this study, an MAb against o-conotoxin MVIIA was prepared. The conotoxin-coding DNA sequence was chemically synthesized and cloned into expression vector pGEX-6p-1 and pET32a ( + ), respectively. The fusion protein GST-CTX was expressed and purified, and was used to immunize BALB/c mice for preparing the anti-CTX antibody. The spleen cells were fused with SP2/0 myeloma cells after the titer of antiserum was detected and qualified. After being screened by indirect ELISA and cloned by limiting dilution, a hybridoma named 4A12, which produces monoclonal antibody specifically against o-CTX MVIIA, was successfully obtained. It was found that there are 102 chromosomes in the 4A12 cell, and the subclass for the MAb is IgM. The MAb affinity against o-CTX MVIIA was 7.33 · 10 9 L/mol, and the cross-reaction test showed that the MAb specifically bound o-CTX MVIIA. The MAb could be used as a specific antagonist for o-CTX MVIIA in the physiological study on the CaV channels in the nervous system.

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Preparation and Identification of Monoclonal Antibody against Abrin-a

Cited by 19 publications

References 15 publications

Detection of Abrin Holotoxin Using Novel Monoclonal Antibodies

Detection of Abrin Holotoxin Using Novel Monoclonal Antibodies

A Monoclonal–Monoclonal Antibody Based Capture ELISA for Abrin

Preparation and Identification of Monoclonal Antibodies Against ω-Conotoxin MVIIA

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