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Keywords:

  • allergen;
  • cockroach;
  • ELISA;
  • potency;
  • standardization

Summary

Rationale The competition ELISA assay is used to determine the potency of US standardized allergen extracts. We have been concerned that the competition ELISA is not sensitive to changes in individual allergen levels. This study was designed to determine the sensitivity of the competition ELISA to detect the specific loss of Bla g 1 and Bla g 2 in cockroach extracts.

Methods German cockroach extract E3Cg was made from defatted German cockroaches. New Zealand White rabbits were immunized with rBla g 1 or rBla g 2. Optimal dilutions of anti-Bla g 1 and anti-Bla g 2 sera were established by ELISA. E3Cg was selectively depleted of Bla g 1 or Bla g 2 by immunoabsorption with anti-Bla g 1 or anti-Bla g 2 attached to Protein G agarose beads. Competition ELISA using pooled human sera, or mixed anti-Bla g 1 and anti-Bla g 2 serum, was performed on the depleted extracts, and on depleted extracts reconstituted with rBla g 1 or rBla g 2.

Results Unlike pooled human-allergic IgE sera, anti-Bla g 1 and anti-Bla g 2 IgG – in dilutions as low as 10−6, could be used in the competition ELISA to measure the loss of allergen in depleted E3Cg. As little as 0.001 μg/mL of added rBla g 1 and 0.1 μg/mL of added rBla g 2, could be detected.

Conclusion The competition ELISA can be highly sensitive to compositional differences in complex allergen mixtures, even when the specific detecting antibody is present in relatively small amounts.