Technology Transfer
Production of recombinant allergens from ragweed pollen within the INSPIRED project
7 types of E.coli cells and 7 types of insect cells (Spodoptera frugiperda Sf9) infected with genetically modified baculoviruses for the production of recombinant ragweed pollen allergens
Details
cell Escherichia coli Genetically modified BL21 Gold capable of producing recombinant allergens:
- Both 3
- Both 5
- Both 8
- Both the 9th
- Both 10
- Both 11
- Both 12
baculovirus Californic autograph containing the gene sequence for ragweed allergens capable of infecting insect cells Sf9 to produce recombinant allergens:
- Both 1
- Both 3
- Both 4
- Both 5
- Both 6
- Both 11
- Both 12
Publications/ Patent
cell Escherichia coli Genetically modified BL21 Gold capable of producing recombinant allergens:
- Both 3
- Both 5
- Both 8
- Both the 9th
- Both 10
- Both 11
- Both 12
baculovirus Californic autograph containing the gene sequence for ragweed allergens capable of infecting insect cells Sf9 to produce recombinant allergens:
- Both 1
- Both 3
- Both 4
- Both 5
- Both 6
- Both 11
- Both 12
Recombinant allergens from ragweed pollen
Details
Allergens from ragweed pollen:
- produced in sheets:
- Both 3
- Both 5
- Both 8
- Both the 9th
- Both 10
- Both 11
- Both 12
- produced in Sf9:
- Both 1
- Both 3
- Both 4
- Both 5
- Both 6
- Both 11
- Both 12
Purity in SDS-PAGE > 95%
Publications/ Patent
- Tamas et al. 2023 – Ragweed Major Allergen Amb a 11 Recombinant Production and Clinical Implications
– Impact Factor: 6.064 – https://www.mdpi.com/2218-273X/13/1/182
- Grijincu et al. 2023 – Physicochemical and immunological characterization of Amb a 12, a novel ragweed (Ambrosia artemisiifolia) pollen allergen – Impact factor 4.174 –
https://www.sciencedirect.com/science/article/pii/S0161589023000548
- Zbîrcea et al. 2023 – Relationship between IgE levels specific for ragweed pollen extract, Amb a 1 and cross-reactive allergen molecules – Impact factor 6.208
ELISA Kit for Ragweed Pollen Allergy Diagnosis by Detection of IgE Specific to Ragweed Pollen Allergen Panel
Details
ELISA plates coated with recombinant ragweed pollen allergens. Serum from allergic patients is added to the allergen and detected with anti-human IgE antibodies labeled with horseradish peroxidase.
Publications/ Patent
Panaitescu C., Chen KW., Păunescu V., Buzan RM. "Kit and method of use for ragweed allergy diagnosis", No.A /00615 /2020, filing date 09/30/2020, State Office for Inventions and Trademarks (OSIM), Directorate of Invention Patents and Technological Information.
ELISA kit for the detection and quantification of ragweed allergens from the environment or from allergenic extracts for diagnosis or immunotherapy
Details
ELISA plates coated with serial dilutions of recombinant ragweed pollen allergens to which dilutions of the extract to be tested are added. Allergen-specific sera from rabbits immunized with recombinant ragweed pollen allergens are added to the allergen and sample. Binding of these allergen-specific antibodies is detected with anti-rabbit IgG antibodies labeled with horseradish peroxidase.
Publications/ Patent
Panaitescu C., Chen KW., Buzan RM., Grijincu M., Zbircea LE., Tamas TP., Haidar L., Tanasie G., Hutu I., Anghel S. "Test kit with allergen-specific antibodies for the detection and quantification of allergens from Ambrosia pollen from the environment and from allergenic diagnostic/therapeutic extracts", no. A/ 00759/ 2020, filing date 19/11/2020, State Office for Inventions and Trademarks (OSIM), Directorate of Invention Patents and Technological Information.
Within the INSPIRED project (Innovative strategies for the prevention, diagnosis and therapy of respiratory diseases induced by ragweed pollen) the IgE sensitization profile of allergic patients to ragweed pollen was identified (Ambrosia artemisiifolia).
Sensitization profile identified among ragweed pollen allergic patients according to Buzan et al. 2022
Based on this profile, allergens from ragweed pollen were identified and characterized (Ambrosia artemisiifolia). To perform the characterization, the protein sequence of these allergens was identified from the databases (allergen.org) and a construct was made for their expression in prokaryotic cells, Escherichia coli, and eukaryotes, Spodoptera frugiperda (Sf9).
3D model of ragweed pollen allergens obtained in https://swissmodel.expasy.org/
The expression system was chosen according to the structural complexity of the protein.
The construct encoding the protein of interest was inserted into the genome of prokaryotic cells by heat shock administration. A transcription factor was then added to induce protein expression. To produce the proteins in insect cells, the gene construct was introduced into the cells E. coli for amplification and obtaining a bacmid with the sequence of interest. This was then used for insect cell transfection, baculoviral stock amplification and protein production. The proteins were then isolated and purified by affinity chromatography. Purity was checked on SDS-PAGE. More detailed protein production can be found in Zbircea et al. 2023, Tamaș et al. 2023, Grijincu et al. 2023.
Steps taken to produce recombinant allergens
Recombinant ragweed pollen allergens (SDS-PAGE)
These proteins have been included in a patent for the development of a ragweed pollen allergy diagnostic kit. They were also used to obtain allergen-specific antibodies that were used in a second patent to detect ragweed pollen allergens in
Patents resulting from the INSPIRED project
Panaitescu C., Chen KW., Păunescu V., Buzan RM.
"Kit and method of use for the diagnosis of ragweed allergy", no.A /00615 /2020, date of deposit 30/09/2020, State Office for Inventions and Trademarks (OSIM), Directorate of Invention Patents and Technological Information .
Panaitescu C., Chen KW., Buzan RM., Grijincu M., Zbircea LE., Tamas TP., Haidar L., Tanasie G., Hutu I.
"Test kit with allergen-specific antibodies for the detection and quantification of allergens from ragweed pollen in the environment and from diagnostic/therapeutic allergenic extracts", no. A/ 00759/ 2020, filing date 19/11/2020, State Office for Inventions and Trademarks (OSIM), Directorate of Invention Patents and Technological Information.
Publications resulting from the INSPIRED project
Chen et al. 2018 – Ragweed Pollen Allergy: Burden, Characteristics, and Management of an Imported Allergen Source in Europe – Impact factor 2.932 – https://karger.com/iaa/article/176/3-4/163/168362
Panaitescu et al. 2022 – Precision medicine in the allergy clinic: the application of component resolved diagnosis – Impact Factor: 4.47 – https://www.tandfonline.com/doi/full/10.1080/1744666X.2022.2034501
Bumbacea et al. 2022 - Mechanisms of allergen immunotherapy supporting its disease-modifying effect – Impact Factor: 4.19 – https://www.futuremedicine.com/doi/abs/10.2217/imt-2021-0325
Marusciac et al. 2021 – Symptom patterns and comparison of diagnostic methods in ragweed pollen allergy – Impact factor 2.751 – https://www.spandidos-publications.com/10.3892/etm.2021.9957
Buzan et al. 2022 – Complex IgE sensitization patterns in ragweed allergic patients: Implications for diagnosis and specific immunotherapy – Impact Factor: 5.657– https://onlinelibrary.wiley.com/doi/full/10.1002/clt2.12179
Zbircea et al. 2023 – Relationship between IgE Levels Specific for Ragweed Pollen Extract, Amb a 1 and Cross-Reactive Allergen Molecules – Impact factor 6.208 – https://www.mdpi.com/1422-0067/24/4/4040
Tamas et al. 2023 – Ragweed Major Allergen Amb a 11 Recombinant Production and Clinical Implications – Impact Factor: 6.064 – https://www.mdpi.com/2218-273X/13/1/182
Grijincu et al. 2023 – Physicochemical and immunological characterization of Amb a 12, a novel ragweed (Ambrosia artemisiifolia) pollen allergen – Impact factor 4.174 – https://www.sciencedirect.com/science/article/pii/S0161589023000548
The results of this research were and will be included in the following doctoral theses under the coordination of Prof. Univ. Dr. Panaitescu Carmen:
| Name surname | Thesis title |
| Tamas Tudor Paul | The major allergen Amb a 11 from ragweed pollen (Ambrosia artemisiifolia): expression in prokaryotic and eukaryotic systems, immunoproteomic aspects and clinical correlations |
| Buzan Maria Roxana | Immunoproteomic characterization of recombinant allergens from pollen Ambrosia artemisiifolia |
| Zbîrcea Lauriana-Eunice | Evaluation of the cross-reactivity reaction and testing the clinical relevance of some allergens from Ambrosia artemisiifolia |
| Careful Manuela | Characterization of a new allergenic protein from the lyase family from ragweed pollen (Ambrosia artemisiifolia) and evaluating its clinical relevance |
| Bison Razvan | Evaluation of markers of allergic inflammation by noninvasive methods |