Andrea Brazdova1, Oumsaad Naas2, Nicolas Visez2, Jean-Pierre Sutra1, Hélène Sénéchal1 et Pascal Poncet1,3
1. Hôpital d’Enfants Armand Trousseau, Laboratoire de Biochimie, Equipe «Allergie & Environnement», 26 avenue du Dr Arnold Netter,
75012 - Paris.
2. Université de Lille, Laboratoire de Physico-chimie des Processus de Combustion et de l’Atmosphère, Lille.
3. Institut Pasteur, département Infection & Epidémiologie, Paris.

Introduction

Allergy is a hypersensitivity disorder of the immune system. According to epidemiological studies, at present, 20-30% of the population in many countries around the world suffers from allergies, and this percentage is growing trend.

Pollen are significant sources of clinically relevant out door aeroallergens, recognized as both a major trigger for, and cause of, allergic respiratory diseases.

Allergens are proteins with a broad range of molecular weights (5-50 kDa) exhibiting different features of solubility and stability, able to cause IgE-mediated hypersensitivity after contact with the immune system. The development of new types of allergy treatments needs diverse and well-characterized allergenic source materials. This study describes an effective method for allergen characterization.

Overview

  • Key word: Allergen, IgE immunoreactivity, pollen homogenization, hypersensitivity community, DNA extraction
  • Aim of the study: identification of fast method for protein extraction from pollen grains
  • Application: Western blot analysis
  • Sample name: Birch, Nettle, Wall Pellitory pollens
  • Sample type: Pollen
  • Material: FastPrep-24™ instrument, CoolPrep adapter, 2 ml Lysing Matrix C & E tubes
  • Buffer: PBS

Protocols & Parameters

A- Incubation method
  1. Add 50 mg of pollen and 500 µl of PBS in a tube
  2. Place the tube in a shaker for 18 hours, in cold room
  3. Centrifuge the suspension 20 mins at 18 000 x g, 4°C
  4. Keep the supernatant at -20°C prior to analyses

B - Grinding method
  1. Add 50 mg of pollen and 500µl of PBS in 2 ml Lysing Matrix C or E tube.
  2. Load Lysing Matrix tubes in a CoolPrep Adapter, containing dry ice.
  3. Process with the FastPrep-24 5G: 40 sec at a speed setting of 6.0 m/s.
  4. Centrifuge the Lysing Matrix tubes 20 mins at 18 000 x g, 4°C to pellet debris.
  5. Keep the supernatant at -20°C prior to analyses

Results

Optical microscope observation of pine pollen(X 200) before and after grinding with the FastPrep-24™ 5G System. Left: pollen grain before grinding. Right: homogenized pollen grain with FastPrep-24™ 5G, 40s at speed 6 m/s with Lysing Matrix C.

Comparison of 8 pine pollen protein extracts obtained by standard
or FastPrep® method. Experience is repeated 4 times using 4
different pollen batches. Protein concentration is determined using
Bradford assay.
Comparison of protein extraction with standard (OVN) or FastPrep method, using Lysing Matrix C or E. Coomassie blue gel staining.

Conserved IgE immunoreactivity in pollen extracts obtained with FastPrep-24™ 5G instrument. Left, IgE immunoreactivity of 6 birch pollen allergic patients tested against birch pollen protein (9-14 strips). Middle and right, IgE immunoreactivity of 12 patients, allergic to herbaceous pollen, tested against nettle and wall pellitory pollen protein (17-22 & 25-30). Relative masses expressed in kDa. 15, 16, 23, 24, 31 & 32 strips correspond to negative controls. Protein profiles comparison of nettle and wall pellitory pollen obtained by FastPrep-24™ 5G (MP Bio) homogenization or by overnight incubation (OVN). The 4 extracts are used without dilution or concentration. SDS-PAGE 8-18 %. Coomassie blue gel staining.
Conclusion

  • Protein extraction from pollen samples with the FastPrep-24™ 5G showed to be highly effective compared to the standard method based on overnight incubation.
  • The effectiveness of the FastPrep® method is quantitative, higher protein yield, and qualitative, wide variety composition of protein extracts.
  • The FastPrep® system is a powerful tool to get rapidly and with a very high reproducibility protein extracts ready for electrophoresis (SDS-PAGE) analysis.
  • IgE immunoreactivity is conserved in protein extracted with the FastPrep-24™ 5G instrument.