Monoclonal Antibodies to Proteins from Cocksfoot Grass <i>(Dactylis glomerata)</i> Pollen: Isolation and N-Terminal Sequence of a Major Allergen

Abstract

Monoclonal antibodies (Mabs) raised to an aqueous extract of cocksfoot grass (Dactylis glomerata) pollen have been characterised. Mab 1B9 was demonstrated by SDS-PAGE and Western blotting to recognise a major allergen of an approximate molecular weight of 28 kD in this extract, termed DG3, and a component with a molecular weight of between 35 and 40 kD in an extract of Secale cereale (cultivated rye) pollen. The 28 kD component of cocksfoot grass pollen isolated by affinity chromatography using Mab 1B9 was recognised by IgE antibodies in 80% (8 of 10) atopic sera, but only weakly by 25% (1 of 4) non-atopic sera tested. N-terminal sequencing of DG3 purified by affinity chromatography, 2 D electrophoresis and electroblotting to polyvinylidenedifluoride revealed significant homology with a group-V allergen (Phi p V) from timothy grass (Phleum pratense).

N·Terminal Amino Acid Sequence Homologies of Group V Grass Pollen Allergens

“…Furthermore, the reported se quence of a cDNA clone encoding the group IX isoaller gen from Poa pratensis [11], included in table 1, has a high degree of similarity to sequences obtained from the other grass species studied here. In a previous report [12], and in common with Matthiesen et al [13], we misassigned histidine residues to positions now shown to be occupied by hydroxyproline residues [5; this report] (table 1). This consistent misas-signment, resulting from the difficulty in detecting the characteristically small twin hydroxyproline peaks by HPLC following microscqucncing [5], was confirmed by repeated analyses using collagen as a control protein stan dard for hydroxyproline.…”

Identification and partial characterization of parathyroid hormone-related protein in human and bovine milk

“…MATERIALS and rabbit anti-[Tyr34]-hPTHrP(l-34) (SIR3DB) (Docherty, Dixon-Lewis, Milton et al 1989) were gifts from Dr H. M. Docherty, Department of Medicine, University of Birmingham, U.K. Ascitic fluid containing monoclonal antibody DG2B5 to a 28 000 Da protein extracted from Cocks¬ foot grass pollen (Walsh, Matthews, Denmeade et al 1990) was a gift from Dr D. J. Walsh, Wolfson Research Laboratories, Birmingham, West Midlands, U.K. The Elite ABC anti-mouse immunoglobulin (Ig)G kit was from Vector Laboratories, Peterborough, Cambs, U.K. All other chemicals were of the highest quality available.…”

Monoclonal antibodies against selected peanut allergens: Production and use as affinity agents

“…In examining the immunoblotting data, it was noteworthy that the antibodies bound to one major band and a number of minor bands in the SDS-PAGE-separated CPE; immunoblotting of the proteins with the monoclonal antibodies showed recognition of epitopes on bands at 12, 14, 23 and 25 kDa. Monoclonal antibodies usually bind to a single, unique site; however, reactivity of monoclonal antibodies to more than one protein band in an allergenic extract has been noted in other studies (Shen et al, 1988;Westphal et al, 1988;Jarolim et al, 1989;Walsh et al, 1990;Wu et al, 1990). Some of the bands could have been proteolysis products, or subunits of the other larger molecular weight proteins.…”