Quantification of a 24 kd major allergen of culex quinquefasciatus whole body extract by immunological techniques

Introduction:

In an earlier study, we have reported the identification, isolation and purification of a 24 kd major allergenic protein of crude mosquito (Culex quinquefasciatus; Cq) extract¹. We further quantified this 24 kd purified protein in crude Cq extract which may serve as a reference reagent for the quality control of commercially available Cq extract.

Methods:

ELISA and ELISA inhibition assays were developed for quantification of 24 kd purified protein in crude Cq extract. 

Results:

Dose-related inhibition was obtained in Cq-FrIb ELISA with the purified protein (24 kd) as well as crude Cq WBE. Dose of purified protein and crude Cq WBE required for 50% inhibition (calculated from inhibition curves) was 144 ng and 1145 ng, respectively; i.e. 125.8 μg/mg of crude lyophilized Cq WBE. Since, 1 ml of 1:20 w/v Cq WBE solution contains 3.7 mg of crude Cq WBE, it is recommended that the amount of purified protein in this solution should be 465.5 μg/ml of 1:20 w/v mosquito extract. Further, for performing ID tests (1:500 w/v), the Cq extract solution should contain 18.6 μg/ml of the purified protein. Accordingly, the concentration of purified protein in the Cq WBE for various dilutions of immunotherapy vaccine may be also calculated for quality control purposes.

Conclusion:

The 24 kd purified major allergenic proteins of Cq may be used as a reference reagent for the standardization of Cq extract used for effective diagnosis and efficacious immunotherapy of millions of patients of respiratory allergy in our country. It is recommended that this purified protein may be used as a reference reagent for quality control of commercially available mosquito WBE.

Reference:

1.      Kausar MA, Vijayan VK, Bansal SK, Vermani M and Agarwal MK. Isolation and purification of a 24 kd major allergen of Culex quinquefasciatus whole body extract. Annals of Allergy, Asthma and Immunology 2009;102:23.