Allergy Asthma Immunol Res.  2014 Jul;6(4):325-332. 10.4168/aair.2014.6.4.325.

Specific B-cell Epitope of Per a 1: A Major Allergen of American Cockroach (Periplaneta americana) and Anatomical Localization

• Sookrung N
• Khetsuphan T
• Chaisri U
• Indrawattana N
• Reamtong O
• Chaicumpa
• Tungtrongchitr A

• KMID: 2260213
• DOI: http://doi.org/10.4168/aair.2014.6.4.325

Abstract

PURPOSE
Cockroach (CR) is a common source of indoor allergens, and Per a 1 is a major American CR (Periplaneta americana) allergen; however, several attributes of this protein remain unknown. This study identifies a novel specific B cell epitope and anatomical locations of Per a 1.0105.
METHODS
Recombinant Per a 1.0105 (rPer a 1.0105) was used as BALB/c mouse immunogen for the production of monoclonal antibodies (MAb). The MAb specific B cell epitope was identified by determining phage mimotopic peptides and pair-wise alignment of the peptides with the rPer a 1.0105 amino acid sequence. Locations of the Per a 1.0105 in P. americana were investigated by immunohistochemical staining.
RESULTS
The rPer a 1.0105 (~13 kDa) had 100%, 98% and > or =90% identity to Per a 1.0105, Per a 1.0101, and Cr-PII, respectively. The B-cell epitope of the Per a 1.0105 specific-MAb was located at residues99 QDLLLQLRDKGV110 contained in all 5 Per a 1.01 isoforms and Per a 1.02. The epitope was analogous to the Bla g 1.02 epitope; however, this B-cell epitope was not an IgE inducer. Per a 1.0105 was found in the midgut and intestinal content of American CR but not in the other organs. The amount of the Per a 1 was ~544 degrees Cg per gram of feces.
CONCLUSIONS
The novel Per a 1 B-cell epitope described in this study is a useful target for allergen quantification in samples; however, the specific MAb can be used as an allergen detection reagent. The MAb based-affinity resin can be made for allergen purification, and the so-purified protein can serve as a standard and diagnostic allergen as well as a therapeutic vaccine component. The finding that the Per a 1 is contained in the midgut and feces is useful to increase yield and purity when preparing this allergen.

Keyword

American cockroach; B-cell epitope; hybridoma; Per a 1; Periplaneta americana, mimotope

MeSH Terms

Allergens
Amino Acid Sequence
Animals
Antibodies, Monoclonal
Bacteriophages
Cockroaches
Epitopes, B-Lymphocyte*
Feces
Gastrointestinal Contents
Hybridomas
Immunoglobulin E
Mice
Peptides
Periplaneta*
Protein Isoforms
Allergens
Antibodies, Monoclonal
Epitopes, B-Lymphocyte
Immunoglobulin E
Peptides
Protein Isoforms

Figure

• 

  Fig. 1 SDS-PAGE separated-rPer a 1.0105 stained by Coomassie Brillian blue G-250 dye (A) and probed with mouse monoclonal anti-6x-histidine antibody (B). Lanes M of both blocks, Protein molecular weight standards. Numbers at the left of both blocks are molecular masses of proteins in kDa.

• 

  Fig. 2 Western blot results for determining antigenic specificity of the produced MAb. Lanes 1 and 2, SDS-PAGE separated-rPer a 1.0105 blotted strips probed with culture supernatants of hybridoma clones ACR1 and ACR2, respectively. Lanes 3 and 4, SDS-PAGE separated E. coli BL21 (DE3) lysate blotted strips probed with culture supernatants of hybridoma clones ACR1 and ACR2, respectively (controls). Lane M, Protein molecular weight standards. Numbers at the left are protein molecular masses in kDa.

• 

  Fig. 3 Pairwise alignments of the phage mimotope peptide (MAbPer a 1.0105-1) with the Per a 1.0105 amino acid sequence for the identification of the Per a 1.0105 residues bound by the ACR1 MAb.

• 

  Fig. 4 Results of competitive ELISA for verification of the MAb epitope. The tested phages carrying the MAb mimotope (peptide that bound to the MAb) could inhibit the binding of 5 and 50 µg of MAb to native Per a 1.0105 compared with the irrelevant phages and diluent controls. # and *, different significantly from the inhibition by the irrelevant phages at 5 and 50 µg of MAb, respectively.

• 

  Fig. 5 Immunohistochemically (IHC) stained sections of P. americana midgut with Per a 1.0105-specific MAb (A) and diluent control (B) (400×magnification).

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