The Latex Allergen Hev B 5 Is an Antigen with Repetitive Murine B-Cell Epitopes

Details

• Personal Author:
  Beezhold, Donald H. ; Hickey VL ; O'Hehir RE ; Sutherland MF
• Description:
  Hev b 5 is a potent latex allergen. In this study, we characterize the linear B-cell epitopes for three monoclonal antibodies (mAbs) to Hev b 5. The mAbs included 2 IgG1 (6A10, 3G3) and 1 IgG2b (6F6) isotypes. We used SPOTscan analysis with overlapping octapeptides to identify the binding regions for the antibodies and then methionine substitution analysis to further define the critical amino acids (aa) in each epitope. Site-directed mutagenesis was used to selectively eliminate the IgG binding for each epitope and single and multiple mutations were expressed as recombinant GST fusion proteins. Antibody recognition of the mutant proteins was determined by inhibition ELISA. All three mAbs recognized the same aa sequence by SPOTs analysis with slight variations, and this epitope was repeated 3 times in the Hev b 5 sequence; APETEK (63-68), PAEGEK (120-125), and PAEEEK (126-131). Sequential methionine substitution by SPOTsalogue identified K68, E122, and K131 as critical aa in each epitope to change by site-directed mutagenesis. Inhibition ELISA with the mutant proteins indicated that epitope 126-131 was the dominant epitope, but mutation of epitope 120-125 was also required to eliminate mAb reactivity to Hev b 5. The antibodies did not appear to recognize the epitope 63-68 in the recombinant fusion protein. We identified an immunodominant B-cell epitope in Hev b 5 that is repeated 3 times within the sequence, making Hev b 5 multivalent. Well-characterized monoclonals recognizing repeated epitopes would be a good choice for immunodetection of Hev b 5 in glove extracts where individual epitopes could get altered by the manufacturing process. [Description provided by NIOSH]
• Subjects:
  Amino Acids Antibodies Antigens Biological Assay Hypersensitivity Mutagens Occupational Health Safety
• Keywords:
  Allergens Amino-acids Antibody-response Bioassays Mutagenesis Proteins
• ISSN:
  1018-2438
• Document Type:
  Text
• Genre:
  Journal Article
• Place as Subject:
  OSHA Region 3 ; Pennsylvania ; West Virginia
• CIO:
  National Institute for Occupational Safety and Health (NIOSH)
• Division:
  HELD - Health Effects Laboratory Division
• Topic:
  Workplace Safety & Health
• Location:
  North America
• Pages in Document:
  334-340
• Volume:
  134
• Issue:
  4
• NIOSHTIC Number:
  nn:20025318
• Citation:
  Int Arch Allergy Immunol 2004 Aug; 134(4):334-340
• Contact Point Address:
  Dr. Donald Beezhold, NIOSH, 1095 Willowdale Road, Morgantown, WV 26505
• Email:
  zec1@cdc.gov
• Federal Fiscal Year:
  2004
• NORA Priority Area:
  Disease and Injury: Asthma and Chronic Obstructive Pulmonary Disease
• Peer Reviewed:
  True
• Source Full Name:
  International Archives of Allergy and Immunology
• Collection(s):
  National Institute for Occupational Safety and Health
• Main Document Checksum:
  urn:sha-512:804ef4ab7cdd0a1336dfea88f7f10bebb663b7a4124bb18f6e1df71066011f9c5d272d92dd8112ac8ad21df50efbf84b0b3995f5586e496dd02668c9611f53d5
• Download URL:
  https://stacks.cdc.gov/view/cdc/196754/cdc_196754_DS1.pdf
• File Type:
  [PDF - 124.59 KB ]