SummaryBackground Parietaria pollen is one of the most important causes of pollinosis in Mediterranean countries. Parietaria judaica pollen extract presents two major allergens, Par j 1 and Par j 2, that belong to the lipid transfer protein family.Objective To develop an ELISA for quantification of both major allergens of P. judaica pollen extracts, and to assert correlation of these allergens content with the allergenic activity of extracts.Methods Natural Par j 1–Par j 2 allergens were purified by gel filtration, ion exchange, and affinity chromatography and identified by mass spectrometry. Rabbit antisera were obtained using this protein preparation as antigen and used for immunoaffinity purification of nPar j 1–Par j 2. BALB/c mice were immunized with the immunopurified nPar j 1–Par j 2 and after fusion and screening by direct ELISA, 5D4 monoclonal antibody was selected as capture antibody to develop a quantitative two‐site ELISA. Bound proteins were detected by a biotinylated Par j 1–Par j 2‐specific polyclonal antibody.Results The optimized ELISA was developed from 25 to 8000 pg/mL of purified Par j 1–Par j 2, and a linear portion of 200–1000 pg/mL. The intraassay and interassay coefficients of variation were lower than 7% and 14% respectively. The assay was very sensitive and specific as it had a detection limit of 25 pg/mL and did not detect reactivity with the same family plants, as Urtica. Par j 1–Par j 2 allergens content was measured in 14 P. judaica and two P. officinalis pollen extracts showing a significant correlation with their allergenic activity measured by enzyme allergosorbent test inhibition.Conclusions The results proved the usefulness of the two‐sandwich ELISA for the standardization of Parietaria pollen extracts intended for clinical use, because of its good correlation with allergenic potency.

01 Oct 1990·Journal of NeurochemistryQ2 · MEDICINE

Oligosaccharide‐Related Epitope Specific for a Brain‐Specific Glycoprotein, 1D4 Antigen

Q2 · MEDICINE

Article

Author: Hachiro Nakagawa ; Shin-ichiro Sano ; Shunji Ito ; Masahiko Nakamura

Abstract: The characteristics of glycosylation of a brain‐specific glycoprotein, 1D4 antigen, and the epitope recognized by its monoclonal antibody were studied. Removal of high‐mannose and hybrid types of N‐linked oligosaccharides by treatment with endoglycosidase H converted the molecular mass of the 1D4 antigen from 89 kDa to 78 kDa, but did not affect its reactivity with the 1D4 monoclonal antibody. Removal of all types of N‐linked oligosaccharides by treatment with glycopeptidase F or removal of both N‐ and O‐linked oligosaccharides by chemical treatment caused both reduction of the molecular mass of the antigen to 63 kDa and loss of its reactivity with the monoclonal antibody. These results suggest that the 1D4 monoclonal antibody recognizes a complex‐type oligosaccharide‐related epitope specific for the 1D4 antigen. Results also showed that N‐linked giycosy lation was not responsible for the charge heterogeneity of the 1D4 antigen. The oligosaccharide chain‐related epitope was detected in rat brain but not in mouse, rabbit, or bovine brain, but the 1D4 antigen was recognized in rat and mouse brains with antiserum (polyclonal antibodies). These findings indicate that the oligosaccharide‐related epitope is species specific. Furthermore, results with neuraminidase‐treated 1D4 antigen indicated that sialic acids were not involved in the oligosaccharide‐related epitope. These findings suggest that the 1D4 antigen may have the oligosaccharide structure specific for rat brain and itself.

100 Deals associated with Monoclonal antibody BW494(Universitätsklinikum Köln)

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