Identification, quantitation, and purification of cockroach allergens using monoclonal antibodies

“…Expressed rBla g 1 was purified from culture media by affinity chromatography over a 10A6 monoclonal antibody column as described previously [11] with modifications. Bound rBla g 1 was eluted with 0.05 M glycine in 50% ethylene glycol, pH 10.…”

Section: Purification and Sequencing Of Rbla Gmentioning

confidence: 99%

“…Purified rBla g 1 was measured in a quantitative twosite ELISA using mAb 10A6 for allergen capture and polyclonal anti-(rBla g 1) Ig for detection [11].…”

Section: Analysis and Quantification Of Expressed Rbla Gmentioning

confidence: 99%

“…Two consecutive amino-acid repeats (a duplex) comprise a distinct molecular unit of Bla g 1. Natural Bla g 1 was identified and purified as a protein with a molecular mass consistent with the presence of approximately two duplexes (molecular mass 42 kDa) [11,12]. Knowledge of the IgE binding epitopes of allergens is important for developing new hypoallergenic products for immunotherapy, as has been carried out for other indoor allergens such as Der p 2 [17].…”

mentioning

confidence: 99%

“…Several German cockroach allergens have been cloned including Bla g 1, Bla g 2, Bla g 4, Bla g 5 and Bla g 6 [6][7][8][9][10]. Bla g 1 is the only German cockroach allergen that shows antigenic cross-reactivity with an American cockroach (Periplaneta americana) allergen, Per a 1 [7,[11][12][13][14]. Bla g 1 and Per a 1 share 70% sequence identity, and the prevalence of IgE antibodies to the group I allergens in cockroach allergic patients is 30-50%.…”

mentioning

confidence: 99%

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IgE reactivity of tandem repeats derived from cockroach allergen, Bla g 1

Pomés

Vailes

Helm

et al. 2002

European Journal of Biochemistry

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Sensitization to cockroach allergens is associated with the development of asthma. Bla g 1 is a German cockroach allergen that shows allergenic cross‐reactivity with American cockroach allergen, Per a 1, and has a molecular structure composed of multiple tandem amino‐acid repeats. Two consecutive repeats are not identical but form a duplex that constitutes a basic molecular unit of Bla g 1. By molecular mass, purified natural Bla g 1 would contain approximately two duplexes. We investigated the pattern of IgE antibody binding to this repeated structure, and whether one or two duplexes are sufficient for IgE binding. Recombinant (r)Bla g 1 duplexes were expressed in Escherichia coli and in Pichia pastoris, and analyzed for monoclonal antibody and IgE antibody binding by ELISA and/or immunoblotting. Optimal rBla g 1 expression was obtained using methanol‐inducible P. pastoris (> 95% pure protein, yield ≈ 48 mg·L−1), and rBla g 1 was produced as multiple molecular forms of molecular mass 43, 32, 21 and 6 kDa, that were the result of proteolytic cleavage. There was an excellent correlation between IgE antibody binding to natural and recombinant Bla g 1 (r = 0.91, n = 29, P < 0.001), and immunoblot analysis showed that a single Bla g 1 duplex was sufficient for IgE antibody binding. The rBla g 1 is suitable for structural studies and a candidate for clinical use in diagnosis of cockroach allergy and development of new forms of immunotherapy.

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