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A sensitive monoclonal antibody-based enzyme-linked immunosorbent assay to quantify Parietaria judaica major allergens, Par j 1 and Par j 2.
Clin Exp Allergy. 2006 Jan; 36(1):87-93.CE

Abstract

BACKGROUND

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.

Authors+Show Affiliations

Research and Development Department, Bial-Arístegui, Bilbao, Spain.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

16393270

Citation

Arilla, M C., et al. "A Sensitive Monoclonal Antibody-based Enzyme-linked Immunosorbent Assay to Quantify Parietaria Judaica Major Allergens, Par J 1 and Par J 2." Clinical and Experimental Allergy : Journal of the British Society for Allergy and Clinical Immunology, vol. 36, no. 1, 2006, pp. 87-93.
Arilla MC, González-Rioja R, Ibarrola I, et al. A sensitive monoclonal antibody-based enzyme-linked immunosorbent assay to quantify Parietaria judaica major allergens, Par j 1 and Par j 2. Clin Exp Allergy. 2006;36(1):87-93.
Arilla, M. C., González-Rioja, R., Ibarrola, I., Mir, A., Monteseirín, J., Conde, J., Martínez, A., & Asturias, J. A. (2006). A sensitive monoclonal antibody-based enzyme-linked immunosorbent assay to quantify Parietaria judaica major allergens, Par j 1 and Par j 2. Clinical and Experimental Allergy : Journal of the British Society for Allergy and Clinical Immunology, 36(1), 87-93.
Arilla MC, et al. A Sensitive Monoclonal Antibody-based Enzyme-linked Immunosorbent Assay to Quantify Parietaria Judaica Major Allergens, Par J 1 and Par J 2. Clin Exp Allergy. 2006;36(1):87-93. PubMed PMID: 16393270.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - A sensitive monoclonal antibody-based enzyme-linked immunosorbent assay to quantify Parietaria judaica major allergens, Par j 1 and Par j 2. AU - Arilla,M C, AU - González-Rioja,R, AU - Ibarrola,I, AU - Mir,A, AU - Monteseirín,J, AU - Conde,J, AU - Martínez,A, AU - Asturias,J A, PY - 2006/1/6/pubmed PY - 2006/5/4/medline PY - 2006/1/6/entrez SP - 87 EP - 93 JF - Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology JO - Clin Exp Allergy VL - 36 IS - 1 N2 - BACKGROUND: 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. SN - 0954-7894 UR - https://www.unboundmedicine.com/medline/citation/16393270/A_sensitive_monoclonal_antibody_based_enzyme_linked_immunosorbent_assay_to_quantify_Parietaria_judaica_major_allergens_Par_j_1_and_Par_j_2_ L2 - https://doi.org/10.1111/j.1365-2222.2006.02406.x DB - PRIME DP - Unbound Medicine ER -