Quality control of Bacillus thuringiensis ssp. israelensis products based on toxin quantification with monoclonal antibodies

Abstract

Quality control of Bacillus thuringiensis ssp. israelensis (Bti) products is currently based on international toxic units (ITUs). The potency of products is related to the activity of a standard (IPS-82, Institute Pasteur, Paris) assessed in bioassays using Aedes aegypti as a target host. The procedure is time consuming and costly, often producing variable results. The activity of Bti is based on four different insecticidal crystal proteins (ICPs): Cry4Aa, Cry4Ba, Cry11Aa and Cyt1Aa. Monoclonal antibodies were produced using IPS-82 for immunisation and an enzyme-linked immunosorbent assay (ELISA) was developed. Antibodies were selected with specificity against Cry11A and Cyt1A. Cry4 specific antibodies could not distinguish between Cry4A and Cry4B. Within five replicate assessments of the three ICPs (in µg mg⁻¹ ICP protein), an error between 3 and 8% was recorded, whereas a 14% error was obtained comparing seven samples of the same production batch for ITUs mg⁻¹. The toxicity against A. aegypti expressed in ITUs correlated well with the results of the ELISA (correlation coefficient r Cyt 1=0.79; Cry 11=0.87; Cry 4=0.91) also when related to the sum of all ICPs (r=0.87). The ELISA can reduce efforts to determine Bti quality compared with the labour-intensive and variable ITU bioassay.

Keywords:

• Quality control
• Bacillus thuringiensis israelensis
• ELISA
• toxin quantification
• Cry4
• Cry11
• Cyt1
• bioassay
• international toxic unit (ITU)
• Aedes aegypti

Acknowledgments

We gratefully acknowledge the financial support of the Deutsche Bundesstiftung Umwelt (DBU Project 18965). Thanks are also due to Neil Crickmore for provision of recombinant strains and to Norbert Becker for assessing ITUs.