Effect of various stabilizing agents on Imperata cylindrica grass pollen allergen extract


Naveen Arora, Allergy and Immunology Section, Centre for Biochemical Technology, Near Jubilee Hall, Mall Road, Delhi-110007, India. E-mail: naveen@cbt.res.in


Background Allergen extracts are unstable, heat labile or susceptible to proteases. Stability of allergen extracts is important for proper diagnosis and therapy of allergic disorders.

Objective The present study was undertaken to determine the preservation and stabilization conditions of Imperata cylindrica (Ic) grass pollen extract.

Methods The Ic extract was kept with 0.1 mε-aminocaproic acid (EACA), 0.75 m sucrose, 5% glycerol, 0.03% human serum albumin (HSA) or 0.4% phenol for different time periods. The extracts were stored for 3, 6 and 12 months each at 4 °C, 4 °C with daily exposure to room temperature (RT) for 1 h, and RT. The quality of extracts was analysed by SDS-PAGE, Western blot, ELISA, ELISA inhibition and skin test.

Results Extracts kept with EACA and sucrose retained most of the protein bands followed by glycerol as determined by SDS-PAGE and Western blot during all storage periods and conditions in comparison with standard extracts. The extracts kept with HSA, phenol and without preservative (WP) showed protein degradation below 33 kDa after 3 months storage at all conditions. However, a 67-kDa allergen was stable in these extracts. EACA extract required 75 to 120 ng of protein for 50% inhibition in IgE binding under different conditions, whereas standard extract required 70 ng for the same. ELISA also demonstrated high allergenic reactivity of EACA extract. ID test on allergy patients with EACA extract demonstrated same allergenic potency as that of standard extract.

Conclusion EACA is the best preservative/stabilizing agent of Ic pollen extract, followed by sucrose and glycerol. Ic extract kept with phenol, HSA and without preservative showed degradation within 3 months. EACA preserved extract is equally potent as that of standard extract up to 1 year's storage.