Identification of low molecular weight allergens of American cockroach and production of monoclonal antibodies.

BACKGROUND

Previous studies have identified two allergic fractions (Cr-PI and Cr-PII) from crude American cockroach extract, from which the 72- and 78-kilodalton (kD) components of Cr-PI have been identified as major allergens.

OBJECTIVE

The objective of this study was to identify important allergens in Cr-PII and produce monoclonal antibodies.

METHODS

Sera of cockroach-sensitive atopic patients were examined for IgE binding to Cr-PII proteins and monoclonal antibodies were generated by fusion between spleen cells of BALB/c mice immunized with Cr-PII and FO cells.

RESULTS

Immunoblotting revealed that eight allergenic compounds in Cr-PII are able to bind specific IgE. Allergens of 18, 28, 32 and 45 kD bound 16.7% (2/12), 100% (12/12), 83.3% (11/12), and 100% (12/12) of the atopic sera tested, respectively. Cell fusion resulted in three stable subclones secreting monoclonal antibodies, and none of the monoclonal antibodies recognized any epitopes of German cockroach and Cr-PI of American cockroach. One monoclonal antibody reacted strongly with the 28- and 32-kD allergens of Cr-PII, and two monoclonal antibodies were able to bind the 18- and 12-kD components of Cr-PII with similar epitope specificities. All the epitopes defined by monoclonal antibodies are recognized by human IgE as demonstrated by a fluoroallergosorbent test (FAST) inhibition assay. Five commercial cockroach extracts were compared for Cr-PII levels by monoclonal antibody-base ELISA, and all extracts were found to contain detectable Cr-PII.

CONCLUSION

The 28-, 32-, and 45-kD components of Cr-PII may be considered as additional important allergens of American cockroach and monoclonal antibodies can be used to identify and define American cockroach allergens.