Generation and purification of monoclonal antibodies against Der f 2, a major allergen from &lt;i&gt;Dermatophagoides farinae &lt;/i&gt;

Chen, Huifang; Zhang, Kejun; Wang, Shan; Xu, Chenxia; Zou, Zehong; Tao, Ailin

doi:10.5582/ddt.2016.01029

Cited by 2 publications

(3 citation statements)

References 29 publications

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“…For example, mAbs against Der f 1 (a major allergen of the house dust mite Dermatophagoides farina) can be used for the detection of this allergen (41). In addition, Der f 2 is a major allergen from D. farina, and mAbs against Der f 2 can be used to create a precise quantitative method to identify allergen components in dust samples (35,42). Der f 7 is another major allergen of house dust mites, and mAbs against Der f 7 may be useful for environmental studies and for the standardization of mite allergen extracts (43,44).…”

Section: Discussionmentioning

confidence: 99%

“…Briefly, following intraperitoneal injection of hybridoma cells (5x10 5 ; stable cell lines secreting antibodies) in three mice, ascites fluid was produced in all three BALB/c mice within 7-14 days. Purified mAbs were obtained from the ascites fluid by affinity chromatography using protein A-agarose (Bio-Rad Laboratories GmbH, München, Germany), as previously described (35), and were analyzed by SDS-PAGE.…”

Section: Immunoreactivity Of Human Sera With Recombinant Pla Amentioning

confidence: 99%

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Expression and purification of a major allergen, Pla a 1, from Platanus acerifolia pollen and the preparation of its monoclonal antibody

Huang

et al. 2017

Molecular Medicine Reports

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Platanus acerifolia pollen is considered an important source of airborne allergens in numerous cities. Pla a 1 is a major allergen from P. acerifolia pollen. The present study aimed to express and purify Pla a 1, and to prepare its monoclonal antibody. In the present study, the Pla a 1 gene was subcloned into a pET‑28a vector and transformed into the ArcticExpress™ (DE3) RP Escherichia coli host strain. The purified Pla a 1 was then used to immunize BALB/c mice. When serum detection was positive, spleen cells were isolated from the mice and fused with SP2/0 myeloma cells at a ratio of 10:1. Hybridoma cells were screened by indirect ELISA and limiting dilution. Positive cells were used to induce the formation of antibody‑containing ascites fluid, and the antibodies were purified using protein A‑agarose. The results of the present study demonstrated that recombinant Pla a 1 was successfully expressed and purified, and exhibited positive immunoglobulin E‑binding to serum from patients allergic to P. acerifolia. A total of 11 hybridomas that steadily secreted anti‑Pla a 1 antibody were obtained and an immunoblotting analysis indicated that all of these monoclonal antibodies specifically recognized the Pla a 1 protein. These results suggested that specific anti‑Pla a 1 antibodies may be obtained, which can be used for the rapid detection of Pla a 1 allergens and in the preparation of vaccines against P. acerifolia pollen.

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Section: Discussionmentioning

confidence: 99%

Section: Immunoreactivity Of Human Sera With Recombinant Pla Amentioning

confidence: 99%

Expression and purification of a major allergen, Pla a 1, from Platanus acerifolia pollen and the preparation of its monoclonal antibody

Huang

et al. 2017

Molecular Medicine Reports

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“…Recombinant Ara h 2 (rAra h 2, referenced to GenBank accession number AAT00598.1) was expressed in E. coli and purified using a Strep-tag II column from inclusion bodies. Four 6–8-week-old Balb/C mice were immunized with purified rAra h 2 according to the procedure described previously [ 17 , 18 ]. In brief, mouse mAbs against rAra h 2 were produced by the fusion of the myeloma cells sp2/0 and the spleen cells from the mice immunized with the purified rAra h 2 for three times at 2-week intervals.…”

Section: Methodsmentioning

confidence: 99%

A Quantitative Method for Detecting Ara h 2 by Generation and Utilization of Monoclonal Antibodies

Chen

Zou

Tao

2018

Journal of Immunology Research

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Peanut (Arachis hypogaea) is one of the most common food allergens that can induce fatal anaphylaxis, and Ara h 2 is one of the major allergen components involved in peanut allergy. The aim of this study was to develop a quantitative method for detecting peanut allergen using monoclonal antibodies against Ara h 2. The splenocytes of immunized mice were fused with myeloma cells (SP2/0), and stable mAb-producing clones were obtained by limiting dilution. mAbs against Ara h 2 were isolated from mouse ascites, and specificity was confirmed by immunoblotting. Five mAbs with high purity and specific reactivity were obtained, which were referred to as 1-2E10, 2-1D5, 3-1C5, 4-1C2, and 5-1G4, respectively. After screening different mAb combinations for development of a sandwich ELISA, we selected 5-1G4 as the capture antibody and 1-2E10 as the detection antibody for the measurement of Ara h 2 from which an optimal correlation between the Ara h 2 concentration and the OD value was obtained. This sandwich ELISA could specifically detect Ara h 2 in peanut extract at concentrations as low as 5 ng/mL and up to 10 μg/mL. These mAbs can, therefore, serve as quantitative diagnostic reagents for peanut and peanut product risk assessment.

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Generation and purification of monoclonal antibodies against Der f 2, a major allergen from <i>Dermatophagoides farinae </i>

Cited by 2 publications

References 29 publications

Expression and purification of a major allergen, Pla a 1, from Platanus acerifolia pollen and the preparation of its monoclonal antibody

Expression and purification of a major allergen, Pla a 1, from Platanus acerifolia pollen and the preparation of its monoclonal antibody

A Quantitative Method for Detecting Ara h 2 by Generation and Utilization of Monoclonal Antibodies

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