Mold Allergy Science Fair Projects
Allergen Molds in Indoor Environments

"Penicillium brevicompactum and Penicillium viridicatum culture grown on synthetic medium."

Mentors: H.M.Vijay, Ph.D, FAAAAJ and V. Kumar, M.Sc.
Environmental Health Directorate, Health Canada, Ottawa, Ontario, Canada
Student: Prashanthi
Grade 10
Ontario, Canada
[ 1 ]: brackets with numbers in them give you the reference numbers at the bottom of this page.

Allergy Science Fair Projects Research
Penicillium is one of the most prevalent indoor fungi and has been associated with an increased incidence of childhood asthma and pulmonary bleeding [1-4]. It has been considered as one if the most important causes of extrinsic bronchial asthma [5].

Penicillium brevicompactum (PB) has been identified as the most prevalent indoor species of the genus [1-4]. It has been recognized that little is known about the allergens of these common airborne species [5]. Also, Penicillium viridicatum (PV) has been reported to be the most potent of allergenic species [6].

Allergy Science Fair Projects Hypothesis
The two species, PB and PV, were cultured on completely synthetic revised tobacco medium under laboratory conditions to be obtain more controlled material for characterization of allergenic and biochemical properties of the extracts.

Allergy Science Fair Projects Methods and Materials Used
The isolates of PB (231273) and PV (231275) (Department of Agriculture, Canada) were transferred to potato-dextrose agar plates of 9cm diameter.

When Penicillium colonies were 5-6cm in diameter, 1cm2 blocks were cut from the colonies and transferred to flasks containing 250ml of synthetic revised tobacco medium (RTM). Sixty-three flasks of each of the two cultures were incubated at 22ºC without shaking for 26 days with 8 hours day light exposure everyday.

The mold pellicles were harvested washed with acetone and air dried. The broth extracts from both cultures, PB and PV were prepared from a suspension of a 26 day mold culture. The broth filtrate (15L) was centrifuged at 16, 000rpm for 30 minutes.

The supernatant was then concentrated by ultra-filtration through Amicon using YM-5 membrane (5 kDA cutoff), washed with water, centrifuged at 16 000rpm at 4ºC for 30 minutes.

The supernatant was filtered through Millipore 0.45 µm filter and lyophilized.

Mycelia and spores extracts of PB and PV were prepared by stirring with double distilled water at 1:10 w/v, for 48 hours at 4ºC for 30 minutes. The suspension was centrifuged at 3,000rpm at 4ºC for 30 minutes.

The resulting supernatant was concentrated and washed with water using an Amicon YM-5 membrane. The retentate was filtered through 0.45µm filter and lyophilized.

Protein contents of the broth extracts and mycelia and spore extracts were determined by commercial dye-binding method of Bradford [7] using bovine serum albumin.

The protein components of the extracts were separated by sodium dodecyl/sulphate polycrylamide gel electrophoresis (SDS-PAGE) and electro blotted on to PVDF membrane.

Proteins were visualized on the membrane by staining with Comassie Brilliant Blue R-250.

Click on image to enlarge.
Comassie Stain
The IgE antibody preparation was made by mixing equal volumes of sera from 8 patients who were highly allergic to Penicillum allergens as judged by skin tests reaction of 4+ and a direct RAST [8] filter of 3+ using discs coupled with PB and PV extracts > 5kDa.

Proteins binding to specific human IgE antibodies against Penicillium allergens (PB and PV) were detected by incubating the PVDF membranes with atopic sera (an eight patient pool), followed by incubation with horse radish peroxide conjugated to goat anti-human IgE.

The membranes were washed and stained with the substrate (OPTI-4CN).

Allergy Science Fair Projects: Data/Results
The yields of dry deflated pellicles from both, PB and PV grown on RTM were found similar i.e.: for PB (1.28 g/ flask) and PV (1.10 g/flask). Similarly, broth extracts of PB and PV gave yields of 1.76mg and 1.10mg per flask, respectively.

However, significant difference was observed between the yields of soluble materials of the extracts of mycelia and spores of PB and PV; PB gave 75.8mg and PV 22.21mg. The findings suggest that PV gave 30% less yield compare to that obtained with PB.

No difference in the protein content was found between the broth extracts of the two species (8.5 to 9.5%). However, significant difference was observed between the protein contents of the soluble extracts of mycelia and spores of PB and PV; PV gave 8.5% whereas PB gave only 3.7%.

Click on image to enlarge.
Weatern Blotting
The number of discrete atopic IgE reacting bands in immunoblots of Penicillium extracts ranged from 8-9 bands (PV-8; 10, 11, 25, 30, 35, 45, 50 and 93 kDa) (PB-9; 8, 17, 20, 22, 25, 29, 37, 40, 45 kDa). The mycelia and spore extracts of both species gave very weak allergenic bands. This could possibly be due to leaching of allergenic components in broth during culture process. Certain allergens should be considered for potential cross-reactivity between species.

Allergy Science Fair Projects: Conclusions
The findings suggest that Penicillium brevicompactum and Penicillium viridicatum could be the candidates for cloning studies since these species offer a large numbers of strongly immunoreactive bands in Western blots.

It would also be interesting to study the broth extracts for isolation, characterization and purification of allergenic components. Moreover, it would be important to optimize the culture conditions of the Penicillium species grown on RTM.

Future Study / Goals for Allergy Science Fair Projects
To determine the cross-reactivity within the species immunodiffusion test will be carried out. This test will allow the determination of specie cross-reactivity with each other and with sera from a patient pool. Photos will be taken using immunodiffusion camera.

Immnoduffusion Petri plates set with Noble Agar will be used to incubate samples. Enzyme screening and isoelectric focusing tests are also possibilities that are being considered for future study.

References that you can use for furture Allergy Science Fair Projects

1. Salvaggio J. and Aukrust l.: 1981, Mold-induced asthma, J. Allergy Clin, Immunol. 68, 327-346.

2. Al-Doory Y. and Domson J.F.: 1984. Mould Allergy. Philadelphia: Lea & Febiger.

3. Gravesen S.: 1985, Indoor airborne mold spores. Allergy 40, (Suppl. 1), 247.

4. Licorish K., Novey H.S., Kozak P., Fairshter R.D. and Wilson A.F.:1985, Role of Alternaria and Penicillium spores in the pathogenesis of asthma. J. Allergy Clin. Immunol. 76, 819-825.

5. Shen H., Lin W., Tsai J., Liaw S., and Han S.: 1996, Allergenic components in three difference species of Penicillum: crossreactivity among major allergens. Clinical and Experimental Allergy. 26, 444-451.

6. Vijay. H. M., Abebe M., Kumar V., DeVouge M., Schrader T., Thaker A., Comtois P., and Garcia E.B.: 2005, Allergenic and mutagenic characterization of 14 Penicillium species. Aerobiologia. 21, 95-103.

7. Bradford M.: 1976, A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principal of protein-dye binding. Anal. Biochem. 72, 248-254.

8. Vijay. H. M., Huang. H, Yong N.M, and Bernstein I.L.: 1984, Studies on Alternaria allergens. I.V. Comparative Biochemicla and Immunological Studies of Commercial Alternaria tenuis Batches. Int. Arch. Allergy Immun. 74, 256-261.

Key Words for Allergy Science Fair Projects
Mold allergens, Penicillium brevicompatum, Penicillium viridicatum, Protein determination, SDS-PAGE Immunoblot, IgE antibodies

To see the abstract,go to science fair projects on allergen molds.