**3. Results**

30 Current Insights in Pollen Allergens

1988)

PoAb

Mouse anti-olive Ole e 1 mAb (Lauzurica et al.

Rabbit anti-olive Ole e 2

Rabbit anti-Cu/Zn SOD PoAb (Agrisera Prod.

Rabbit anti-olive Ole e 9

**2.4. Absolute and relative quantitation of allergens** 

specific spots, etc., two different methods for quantitation were used:

PoAb 1:10,000

(Morales et al. 2008)

No. AS06 170)

Quantity One v4.6.2 software (Bio-Rad).

density, which was assigned 100%.

which was re-assigned 100%.

**Ole e 1** 

**Ole e 2** 

**Ole e 5** 

**Ole e 9 (N- domain)** 

PoAb: polyclonal antibody.

**Target Primary antibody Dilution Secondary antibody Dilution** 

Goat anti-mouse IgG Ab, Alexa fluor 488-conjugated

1:10,000

1:10,000

1:10,000

1:10,000

(Molecular Probes)

Donkey anti-rabbit IgG (Fab fragment) Ab, Cy3 conjugated (Jackson ImmunoResearch)

Goat anti-rabbit IgG Ab, Alexa fluor 633-conjugated

Goat anti-rabbit IgG Ab, Alexa fluor 633-conjugated

(Molecular Probes)

(Molecular Probes)

1:20,000

1:20,000

1:250

**Table 1.** Antibodies and dilutions used for immunoblotting experiments. mAb: monoclonal antibody;

Imaging was carried out with a Pharos FX Plus Molecular Imager (Bio-Rad) using the

The intensity of each fluorescent band was calculated using the quantitation tools of the Quantity One v4.6.2 software. In order to increase sensitivity of measurements and to avoid disturbing factors like the intensity of the background, the presence of individual non-

 For each allergen studied, reactive bands were identified, their optical density individually measured and then their absolute values added for each cultivar. Relative percentages of each allergen were then calculated for each cultivar, taking the cultivar

 Simultaneous measurement of the optical density corresponding to all reactive bands from a given allergen in each cultivar was also performed. As before, relative percentages were also calculated, referred to the cultivar with the highest optical

Finally, average of the percentages calculated by both methods was worked out, and the resulting percentages were newly made relative to the cultivar with the highest percentage,

with the highest optical density as the reference, which was assigned 100%.

## **3.1. SDS-PAGE protein profiles**

Figure 1 shows the protein profiles of the extracts analysed after SDS-PAGE and Coomassie staining. The patterns observed for the major protein species were somewhat similar for all the cultivars tested. However, clear quantitative differences were distinguished, from which the most conspicuous were those in the protein range of 17-20 kDa. Proteins within this range were relatively abundant in the extracts corresponding to the cvs. ΄Picual΄, ΄Manzanilla΄, ΄Cornicabra΄, ΄Hojiblanca΄, ΄Loaime΄, ΄Blanqueta΄ and ΄Lucio΄.

When the commercial pollen extract was assayed by SDS-PAGE, a protein profile similar to the profile corresponding to the individual cultivars was observed, although several bands were absent or poorly resolved. Proteins in the range 17-20 kDa represented a low proportion of the total protein for this extract.

**Figure 1.** Coomassie stained SDS-PAGE gel of the univarietal pollen extracts and the commercial extract (*Olea europaea*) after using denaturing, reducing conditions. Gels contained 30 µg total protein per lane.

#### **3.2. Immunoblot detection and quantitation of Ole e 1**

Immunoblots probed with the monoclonal antibody to Ole e 1 resulted in the presence of two major immunoreactive bands of 18 and 20 kDa, corresponding to the monomeric nonglycosylated and mono-glycosylated forms (Figure 2). Other immunoreactive bands with low quantitative relevance were observed (36, 40 y 44 kDa) in several lanes.

Bands corresponding to the Mw of 18 and 20 kDa were quantitated according to the methods described above. Absolute measurements of the intensity of each individual band and both bands simultanously are displayed in Table 2, as well as the relative percentages calculated as described above.

Clustering of Olive Pollens Into Model Cultivars on the Basis of Their Allergenic Content 33

**Figure 3.** Immunoblot probed with the anti-Ole e 2 polyclonal antiserum. Five major bands were observed (orange arrows), corresponding to apparent molecular weights of 14, 13.7, 14.2, 14.9 and

*Cornicabra* 

**13.0 kDa** 357152 277747 248153 405554 165942 394519 537187 356024 910640 476889 350778 **13.7 kDa** 398025 305569 261364 312528 198300\* 250846 484703 463371 1004748 454397 486318 **14.2 kDa** 264210 198300\* 198300\* 223593 147305 198300\* 198300\* 198300\* 987772 410790 198300\* **14.9 kDa** 198300\* 198300\* 198300\* 198300\* 290636 198300\* 198300\* 198300\* 198300\* 198300\* 198300\* **15.7 kDa** 198300\* 198300\* 198300\* 217632 198300\* 198300\* 198300\* 198300\* 198300\* 198300\* 198300\*

**above** 1415987 1178216 1104417 1357607 1000483 1240265 1616790 1414295 3299760 1738676 1431996

**%** 42.91 35.70 33.47 41.14 30.31 37.59 49 42.86 100 52.70 43.39

**bands** 2281004 2385713 2464789 2550651 2499993 2472138 2437099 2312637 2913894 2092950 1973168

**%** 78.3 81.87 84.59 87.53 85.80 84.84 83.64 79.37 100 71.83 67.71

**Table 3.** Quantitation of the five major bands cross-reactive to the anti Ole e 2 antibody. Absolute data in volume units (INT\*mm2). \*: band not present. The indicated value corresponds to the average of 5

60.605 58.78 59.03 64.335 58.055 61.215 66.32 61.115 100 62.265 55.55

*Verdial* 

*Lechín* 

*Hojiblanca* 

*Loaime* 

*Blanqueta* 

*Lucio* 

15.7 kDa.

**Σ bands** 

**Relative** 

**Relative** 

**Average relative %** 

**All** 

*O. europaea* 

*Picual* 

measurements made in the background.

*Manzanilla* 

*Arbequina* 

**Figure 2.** Immunoblot probed with the anti-Ole e 1 monoclonal antibody. Two major bands were observed (yellow arrows), corresponding to apparent molecular weights of 18 and 20 kDa.


**Table 2.** Quantitation of the two major bands cross-reactive to the anti Ole e 1 antibody. Absolute data in volume units (INT\*mm2).

#### **3.3. Immunoblot detection and quantitation of Ole e 2**

Immunoblots probed with the polyclonal antiserum to Ole e 2 resulted in the presence of up to five major immunoreactive bands of c.a. 14, 13.7, 14.2, 14.9 and 15.7 kDa (Figure 3).

Bands corresponding to the five Mws were quantitated according to the methods described above. Absolute measurements of the intensity of each individual band and all five bands simultanously are displayed in Table 3, as well as the relative percentages calculated as described above.

**Σ 18 and** 

**Average** 

in volume units (INT\*mm2).

described above.

**Figure 2.** Immunoblot probed with the anti-Ole e 1 monoclonal antibody. Two major bands were observed (yellow arrows), corresponding to apparent molecular weights of 18 and 20 kDa.

*O. europaea Picual Manzanilla Arbequina Cornicabra Verdial Lechín Hojiblanca Loaime* 

**18 kDa** 3672 14385 16746 11894 20033 12174 17668 30706 41345 40388 17436 **20 kDa** 2348 10666 11233 7983 11716 3204 11804 22780 21143 32768 13087

**20 kDa** 6021 25051 27979 19877 31749 15378 29472 52784 62489 73156 30523 **Relative %** 8.23 34.24 38.25 27.17 43.40 21.02 40.28 72.15 85.41 100 41.72 **18 and 20 kDa** 6659 26741 29626 22044 34260 20950 32493 54095 60906 73900 30345 **Relative %** 9.01 36.18 40.09 29.83 46.36 28.35 43.97 73.21 82.42 100 41.06

**relative %** 8.62 35.21 39.17 28.5 41.88 24.68 42.125 72.68 83.915 100 41.39

**Table 2.** Quantitation of the two major bands cross-reactive to the anti Ole e 1 antibody. Absolute data

Immunoblots probed with the polyclonal antiserum to Ole e 2 resulted in the presence of up

Bands corresponding to the five Mws were quantitated according to the methods described above. Absolute measurements of the intensity of each individual band and all five bands simultanously are displayed in Table 3, as well as the relative percentages calculated as

to five major immunoreactive bands of c.a. 14, 13.7, 14.2, 14.9 and 15.7 kDa (Figure 3).

**3.3. Immunoblot detection and quantitation of Ole e 2** 

*Blanqueta* 

*Lucio* 

**Figure 3.** Immunoblot probed with the anti-Ole e 2 polyclonal antiserum. Five major bands were observed (orange arrows), corresponding to apparent molecular weights of 14, 13.7, 14.2, 14.9 and 15.7 kDa.


**Table 3.** Quantitation of the five major bands cross-reactive to the anti Ole e 2 antibody. Absolute data in volume units (INT\*mm2). \*: band not present. The indicated value corresponds to the average of 5 measurements made in the background.

#### **3.4. Immunoblot detection and quantitation of Ole e 5 and Ole e 9**

Immunoblots probed with the commercial antibody to Cu,Zn SOD (Ole e 5) and the polyclonal antiserum to Ole e 9 resulted in the presence of up to five major immunoreactive bands of c.a. 16, 16.5, 22, 26 and 50 kDa for Ole e 5, and two immunoreactive bands of c.a. 36 and 46.5 kDa for Ole e 9 (Figure 4).

Clustering of Olive Pollens Into Model Cultivars on the Basis of Their Allergenic Content 35

*Lechín* 

*Hojiblanca* 

*Loaime* 

*Blanqueta* 

*Lucio* 

*O. europaea* 

**Σ bands** 

**Average** 

**Average relative to 100 %** 

**Σ 36 and** 

**36 and 46.5** 

**Average** 

in volume units (INT\*mm2).

*Picual* 

different cultivars (΄Hojiblanca΄ and ΄Lechín΄).

*O. europaea Picual Manzanilla Arbequina* 

*Manzanilla* 

*Arbequina* 

*Cornicabra* 

**16.0 kDa** 12522 10472 13810 16174 8515 16875 20320 19682 1054 793 714 **16.5 kDa** 33503 21491 23191 27710 33630 28904 36422 39682 27218 23811 4304 **22.0 kDa** 9451 7946 9623 14385 12453 9703 13812 17211 6380 5201 5041 **26.0 kDa** 6221 3389 3688 7134 7617 4447 7775 11424 12698 9191 7098 **50.0 kDa** 5611 7474 9643 4818 3941 5794 8238 19507 12979 5625 4200

**above** 67308 50772 59955 70221 66156 65723 86567 107506 47350 44621 21357 **Relative %** 62.61 47.23 55.77 65.32 61.54 61.13 80.52 100 44.04 41.51 19.87 **All bands** 122909 95529 107694 136847 128879 118503 157103 155113 136354 107779 79135 **Relative %** 78.23 60.81 68.55 87.11 82.03 75.43 100 98.73 86.79 68.60 50.37

**relative %** 70.42 54.02 62.16 76.215 71.785 68.28 90.26 99.365 65.415 55.055 35.12

**Table 4.** Quantitation of the five major bands cross-reactive to the anti Cu,Zn SOD (Ole e 5) antibody. Absolute data in volume units (INT\*mm2). In this case, the average relative percentage was again referred to 100%, as the maximun relative percentages previously calculated corresponded to two

*Cornicabra* 

**36 kDa** 20241 18902 25868 18912 15583 17605 24746 44273 29766 28377 20736 **46.5 kDa** 39567 23419 17751 16764 13584 13916 20517 53338 22784 13160 12730

**46.5 kDa** 59808 42322 43619 35677 29167 31521 45264 97612 52550 41538 33467 **Relative %** 61.27 43.36 44.68 36.55 29.88 32.29 46.37 100 53.83 42.55 34.28

**kDa** 72676 51950 56389 47169 39589 45075 59672 102195 74531 52213 39717 **Relative %** 71.11 50.83 55.17 46.15 38.73 44.10 58.39 100 72.93 51.09 38.86

**relative %** 66.19 47.095 49.925 41.35 34.305 38.195 52.38 100 63.38 46.82 36.57

**Table 5.** Quantitation of the two major bands cross-reactive to the anti Ole e 9 antibody. Absolute data

70.87 54.36 62.55 76.70 72.24 68.72 90.83 100 65.83 55.41 **35.34** 

*Verdial* 

*Lechín* 

*Hojiblanca* 

*Loaime* 

*Blanqueta* 

*Lucio* 

*Verdial* 

**Figure 4.** Immunoblot probed with the anti- Cu,Zn-SOD (Ole e 5) commercial antibody and the polyclonal antiserum to Ole e 9. Five major bands were observed (blue arrows), corresponding to apparent molecular weights of 16, 16.5, 22, 26 and 50 kDa for Ole e 5, and two immunoreactive bands of c.a. 36 and 46.5 kDa for Ole e 9 (red arrows).

Bands corresponding to the five Mws of Ole e 5 and two of Ole e 9 were quantitated according to the methods described above. Absolute measurements of the intensity of each individual band and all five bands simultanously are displayed in Tables 4 and 5, as well as the relative percentages calculated as described above.

## **4. Clustering of cultivars according to their relative allergenic content**

Table 6 summarizes the final relative averages of reactivity calculated for each cultivar and allergen. Relative values present a wide range in the case of allergens Ole e 1 and Ole e 9, whereas Ole e 2 and Ole e 5 allergens maintain values relatively constant, higher than 50% for all cultivars, with a single exception (Ole e 5 in the cultivar ΄Lucio΄).

Therefore, the following thresholds have been defined in order to divide cultivars into cultivars with high/average/low allergenic content for the allergens Ole e 1 and Ole e 9. In the case of Ole e 1, we have considered that percentages of 30% and 35% may represent reasonable limits, taking into account the extremely high content of some cultivars in this allergen, which may represents up to 23% of the total protein content for these cultivars (Castro et al. 2003). For Ole e 9, the percentages of 40% and 60% were selected as the thresholds.

Clustering of Olive Pollens Into Model Cultivars on the Basis of Their Allergenic Content 35


and 46.5 kDa for Ole e 9 (Figure 4).

c.a. 36 and 46.5 kDa for Ole e 9 (red arrows).

the relative percentages calculated as described above.

**3.4. Immunoblot detection and quantitation of Ole e 5 and Ole e 9** 

Immunoblots probed with the commercial antibody to Cu,Zn SOD (Ole e 5) and the polyclonal antiserum to Ole e 9 resulted in the presence of up to five major immunoreactive bands of c.a. 16, 16.5, 22, 26 and 50 kDa for Ole e 5, and two immunoreactive bands of c.a. 36

**Figure 4.** Immunoblot probed with the anti- Cu,Zn-SOD (Ole e 5) commercial antibody and the polyclonal antiserum to Ole e 9. Five major bands were observed (blue arrows), corresponding to apparent molecular weights of 16, 16.5, 22, 26 and 50 kDa for Ole e 5, and two immunoreactive bands of

Bands corresponding to the five Mws of Ole e 5 and two of Ole e 9 were quantitated according to the methods described above. Absolute measurements of the intensity of each individual band and all five bands simultanously are displayed in Tables 4 and 5, as well as

**4. Clustering of cultivars according to their relative allergenic content** 

for all cultivars, with a single exception (Ole e 5 in the cultivar ΄Lucio΄).

Ole e 9, the percentages of 40% and 60% were selected as the thresholds.

Table 6 summarizes the final relative averages of reactivity calculated for each cultivar and allergen. Relative values present a wide range in the case of allergens Ole e 1 and Ole e 9, whereas Ole e 2 and Ole e 5 allergens maintain values relatively constant, higher than 50%

Therefore, the following thresholds have been defined in order to divide cultivars into cultivars with high/average/low allergenic content for the allergens Ole e 1 and Ole e 9. In the case of Ole e 1, we have considered that percentages of 30% and 35% may represent reasonable limits, taking into account the extremely high content of some cultivars in this allergen, which may represents up to 23% of the total protein content for these cultivars (Castro et al. 2003). For **Table 4.** Quantitation of the five major bands cross-reactive to the anti Cu,Zn SOD (Ole e 5) antibody. Absolute data in volume units (INT\*mm2). In this case, the average relative percentage was again referred to 100%, as the maximun relative percentages previously calculated corresponded to two different cultivars (΄Hojiblanca΄ and ΄Lechín΄).


**Table 5.** Quantitation of the two major bands cross-reactive to the anti Ole e 9 antibody. Absolute data in volume units (INT\*mm2).


Clustering of Olive Pollens Into Model Cultivars on the Basis of Their Allergenic Content 37

antibodies with higher specificity) will undoubtedly improve the present type of studies,

This work was supported by the Spanish Ministry of Science and Innovation (MICINN) (ERDF-cofinanced projects AGL2008-00517, BFU2011-22779 and PIE-200840I186) and the Junta de Andalucía (ERDF-cofinanced projects P2010-CVI5767 and P2010-AGR6274). The authors acknowledge the availability of plant material and the collaboration of the staff of the IFAPA center "Venta del Llano" (Mengíbar, Spain) depending from the Andalusian

which has to be considered still preliminary.

Sonia Morales, Antonio Jesús Castro, Carmen Salmerón, María Isabel Rodríguez-García and Juan de Dios Alché *Estación Experimental del Zaidín (CSIC), Granada, Spain* 

*R&D Inmunal S.A.U. Tecnoalcalá, Alcalá de Henares, Madrid, Spain* 

*Plantarum*, Vol. 104, No. 4, pp. 772-776, ISSN 1399-3054

*Proteomic Research Service, Hospital Universitario San Cecilio, Granada, Spain* 

Alché, J.D., Castro, A.J., Jiménez-López, J.C., Morales, S., Zafra, A., Hamman-Khalifa, A.M. & Rodríguez-García, M.I. (2007). Differential characteristics of olive pollen from different cultivars: biological and clinical implications. *Journal of Investigational* 

Alché, J.D., Cismondi, I.D., Castro, A.J., Hamman Khalifa, A., & Rodríguez García, M.I. (2003). Temporal and spatial gene expression of Ole e 3 allergen in olive (*Olea europaea* L.) pollen. *Acta Biologica Cracoviensia, Series Botanica*, Vol. 45, No. 1, pp. 89-95, ISSN 0001-

Alché, J.D., Corpas, F., Rodríguez-García, M.I., & del Río, L.A. (1998). Identification and immunolocalization of superoxide dismutase isoenzymes of olive pollen. *Physiologia* 

Alché, J.D., M'rani-Alaoui, M., Castro, A.J., & Rodríguez-García, M.I. (2004). Ole e 1, the major allergen from olive (*Olea europaea* L.) pollen, increases its expression and is

*Allergology & Clinical Immunology*, Vol. 17, Suppl 1., pp. 63-68, ISSN 1018-9068 Alché, J.D., Castro, A.J., Olmedilla, A., Fernández, M.C., Rodríguez, R., Villalba, M. & Rodríguez-García, M.I. (1999). The major olive pollen allergen (Ole e I) shows both gametophytic and sporophytic expression during anther development, and its synthesis and storage takes place in the RER. *Journal of Cell Science*, Vol. 112, No. 15, pp. 2501-

**Acknowledgements** 

Regional Government.

Francisco Manuel Marco

2509, ISSN 0021-9533

**Author details** 

Sonia Morales

**5. References** 

5296

**Table 6.** Abstract of the relative percentages of reactivity corresponding to the cultivars analysed for each allergen. High reactivity is marked by using bold text, and low reactivity is marked by italics, after considering the thresholds indicated.

The following categories were established after following the above mentioned criteria:


The *Olea europaea* commercial extract doesn't match any of the tested cultivars, corresponding to an extract with a relative high proportion of Ole e 9 and low proportion of Ole e 1.

This initial proposal should be implemented by further analyzing additional olive pollen allergens (some of them highly relevant from a clinical point of view like Ole e 7), and by analyzing the allergen profiles of other agronomically relevant cultivars. However, the classification obtained here is in good agreement with the genetic relationships among cultivars already described on the basis of Ole e 1 and Ole e 2 polymorphism (Hamman-Khalifa et al. 2008; Jiménez-López et al., 2012). Moreover, this classification also supports clinical findings describing sharp differences in patient's reactivity to commercially available extracts depending on their place of residence in Spain, where these model cultivars are differentially predominant (Casanovas et al. 1997). Providing that sensitization to specific allergens can be determined in individual patients, the application of the concept of allergenic profile to allergen extracts could be considered a major adventage. This concept would therefore open the posibility of choosing the allergen extract matching the sensitivity of each patient. Moreover, the continuous development of new molecular tools (e.g. new antibodies with higher specificity) will undoubtedly improve the present type of studies, which has to be considered still preliminary.

## **Acknowledgements**

36 Current Insights in Pollen Allergens

*O. europaea Picual Manzanilla* 

considering the thresholds indicated.

in this same group.

Ole e 1.

*Arbequina* 

cultivar ΄Loaime΄ could be included in this same group.

΄Blanqueta΄ could be included in this same group.

*Cornicabra* 

*Verdial* 

**Ole e 1** 8.62 35.21 39.17 28.5 41.88 24.68 42.125 72.68 83.915 100 41.39 30%-

**Ole e 2** 60.605 58.78 59.03 64.335 58.055 61.215 66.32 61.115 100 62.265 55.55 - **Ole e 5** 70.87 54.36 62.55 76.70 72.24 68.72 90.83 100 65.83 55.41 35.34 - **Ole e 9** 66.19 47.095 49.925 41.35 34.305 38.195 52.38 100 63.38 46.82 36.57 40%-

**Table 6.** Abstract of the relative percentages of reactivity corresponding to the cultivars analysed for each allergen. High reactivity is marked by using bold text, and low reactivity is marked by italics, after

The following categories were established after following the above mentioned criteria:

΄Hojiblanca΄-type extract, characterized by high contents of Ole e 1 and Ole e 9. The

 ΄Picual΄- type extract, characterized by high content of Ole e 1 and low to average contents of Ole e 9. The cultivars ΄Manzanilla΄, ΄Lucio΄, ΄Cornicabra΄, ΄Lechín΄ and

 ΄Arbequina΄ -type extract, characterized by low content in both Ole e 1 and Ole e 9, with average to high contents of Ole e 5 and Ole e 2. The cultivar ΄Verdial΄ could be included

The *Olea europaea* commercial extract doesn't match any of the tested cultivars, corresponding to an extract with a relative high proportion of Ole e 9 and low proportion of

This initial proposal should be implemented by further analyzing additional olive pollen allergens (some of them highly relevant from a clinical point of view like Ole e 7), and by analyzing the allergen profiles of other agronomically relevant cultivars. However, the classification obtained here is in good agreement with the genetic relationships among cultivars already described on the basis of Ole e 1 and Ole e 2 polymorphism (Hamman-Khalifa et al. 2008; Jiménez-López et al., 2012). Moreover, this classification also supports clinical findings describing sharp differences in patient's reactivity to commercially available extracts depending on their place of residence in Spain, where these model cultivars are differentially predominant (Casanovas et al. 1997). Providing that sensitization to specific allergens can be determined in individual patients, the application of the concept of allergenic profile to allergen extracts could be considered a major adventage. This concept would therefore open the posibility of choosing the allergen extract matching the sensitivity of each patient. Moreover, the continuous development of new molecular tools (e.g. new

*Lechín* 

*Hojiblanca* 

*Loaime* 

*Blanqueta* 

*Lucio* 

*Thresholds* 

*low/average/high* 

35%

60%

This work was supported by the Spanish Ministry of Science and Innovation (MICINN) (ERDF-cofinanced projects AGL2008-00517, BFU2011-22779 and PIE-200840I186) and the Junta de Andalucía (ERDF-cofinanced projects P2010-CVI5767 and P2010-AGR6274). The authors acknowledge the availability of plant material and the collaboration of the staff of the IFAPA center "Venta del Llano" (Mengíbar, Spain) depending from the Andalusian Regional Government.

### **Author details**

Sonia Morales, Antonio Jesús Castro, Carmen Salmerón, María Isabel Rodríguez-García and Juan de Dios Alché *Estación Experimental del Zaidín (CSIC), Granada, Spain* 

Francisco Manuel Marco *R&D Inmunal S.A.U. Tecnoalcalá, Alcalá de Henares, Madrid, Spain* 

Sonia Morales *Proteomic Research Service, Hospital Universitario San Cecilio, Granada, Spain* 

### **5. References**


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Geller-Bernstein, C., Arad, G., Keynan, N., Lahoz, C., Cardaba, B., & Waisel, Y. (1996). Hypersensitivity to pollen of Olea europaea in Israel. *Allergy*, Vol. 51, No. 5, pp. 356-359,

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Hamman-Khalifa, A.M., Alché, J.D., & Rodríguez-García, M.I. (2003). Discriminación molecular en el polen de variedades españolas y marroquíes de olivo (Olea europaea

Hamman Khalifa, A.M., Castro, A.J., Rodríguez García, M.I., & Alché, J.D (2008). Olive cultivar origin is a major cause of polymorphism for Ole e 1 pollen allergen. *BMC Plant* 

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Jiménez-López, J.C., Morales, S., Castro, A.J., Volkmann, D., Rodríguez-García, M.I. & Alché, J.D. (2012). Characterization of profilin polymorphism in pollen with a focus on

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Morales, S., Jiménez-López, J.C., Castro, A.J., Rodríguez-García, M.I., & Alché, J.D. (2008). Olive pollen profilin (Ole e 2 allergen) co-localizes with highly active areas of the actin cytoskeleton and is released to the culture medium during in vitro pollen germination.

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**Chapter 3** 

© 2012 Zienkiewicz et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 Zienkiewicz et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Detection and Quantitation of Olive Pollen** 

Krzysztof Zienkiewicz, Estefanía García-Quirós, Juan de Dios Alché,

María Isabel Rodríguez-García and Antonio Jesús Castro

and to evaluate their allergenic activity in sensitized patients.

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/46171

**1. Introduction** 

**Allergen Isoforms Using 2-D Western Blotting** 

The use of biological extracts for allergy diagnosis and immunotherapy has some disadvantages, including the high variability in their allergenic composition and the presence of allergens to which the patient is not allergic. As the result, wrong diagnosis, new sensitizations and/or systemic reactions often occur, limiting their use for specific immunotherapy. One of the strategies to overcome these problems is the standardization of biological extracts in order to control their allergenic composition. For this purpose, it is highly recommended to identify the allergenic molecules in the extract, to quantify them

The allergenic pollen used for the preparation of natural extracts or recombinant allergens may contain different allergenic isoforms and/or variable amounts of each allergen, depending of its genetic origin among other factors (Castro et al. 2003, Hamman-Khalifa et al. 2008, Castro et al. 2010 & Jiménez-López et al. 2012). Consequently, allergic patients from different geographical areas may exhibit differential sensitization to a given allergen (Movérare et al. 2002). This fact can hinder the diagnosis of an allergic patient in response to a particular extract. Therefore, the allergenic variability in standardized protein extracts should resemble as much as possible to that observed in the natural sources in order to

Olive pollen produces seasonal respiratory allergy in the Mediterranean area, as well as in other temperate regions where it is intensively cultivated. Eleven olive pollen allergens, called Ole e 1 to Ole e 11, have been identified and characterized so far (Esteve et al. 2012). Many of these proteins exhibit a significant polymorphism as a consequence of the existence of point substitutions in the amino acid sequence, posttranslational modifications (e.g. glycosylation), and/or multimeric forms (Castro et al. 2010). In addition, some allergens

assure the efficiency and safety in the diagnosis and immunotherapy procedures.

