Peach allergen Pru p 1 content is generally low in fruit but with large variation in different varieties

Abstract Background Pru p 1 is a major allergen in peach and nectarine, and the different content in varieties may affect the degree of allergic reactions. This study aimed to quantify Pru p 1 levels in representative peach varieties and select hypoallergenic Pru p 1 varieties. Methods To obtain monoclonal and polyclonal antibodies, mice and rabbits, respectively, were immunized with recombinant Pru p 1.01 and Pru p 1.02. The Pru p 1 levels in fruits from 83 representative peach varieties was quantified by sandwich enzyme‐linked immunosorbent assay (sELISA). nPru p 1 was obtained through specific monoclonal antibody affinity purification and confirmed by Western blot and mass spectrometry. The variable Pru p 1 content of selected varieties was evaluated by Western blot and the expression level of encoding Pru p 1 genes by quantitative polymerase chain reaction. Results A sELISA method with monoclonal and polyclonal antibodies was built for quantifying Pru p 1 levels in peach. Pru p 1 was mainly concentrated in the peel (0.20–73.44 μg/g, fresh weight), being very low in the pulp (0.05–9.62 μg/g) and not detected in wild peach. For the 78 peach and nectarine varieties, Pru p 1 content varied widely from 0.12 to 6.45 μg/g in whole fruit. We verified that natural Pru p 1 is composed of 1.01 and 1.02 isoallergens, and the Pru p 1 expression level and Pru p 1 band intensity in the immunoblots were in agreement with protein quantity determined by ELISA for some tested varieties. In some cases, the reduced levels of Pru p 1 did not coincide with low Pru p 3 in the same variety in whole fruit, while some ancient wild peach and nectarines contained low levels of both allergens, and late‐ripening yellow flesh varieties were usually highly allergenic. Conclusion Pru p 1 content is generally low in peach compared to Pru p 3. Several hypoallergenic Pru p 1 and Pru p 3 varieties, “Zi Xue Tao,” “Wu Yue Xian,” and “May Fire,” were identified, which could be useful in trials for peach allergy patients.

varied widely from 0.12 to 6.45 μg/g in whole fruit. We verified that natural Pru p 1 is composed of 1.01 and 1.02 isoallergens, and the Pru p 1 expression level and Pru p 1 band intensity in the immunoblots were in agreement with protein quantity determined by ELISA for some tested varieties. In some cases, the reduced levels of Pru p 1 did not coincide with low Pru p 3 in the same variety in whole fruit, while some ancient wild peach and nectarines contained low levels of both allergens, and late-ripening yellow flesh varieties were usually highly allergenic.
Conclusion: Pru p 1 content is generally low in peach compared to Pru p 3. Several hypoallergenic Pru p 1 and Pru p 3 varieties, "Zi Xue Tao," "Wu Yue Xian," and "May Fire," were identified, which could be useful in trials for peach allergy patients. health. It is a native fruit crop of China but is grown worldwide with high levels of consumption. 1 However, it is one of the fruits most frequently reported as allergenic, with sensitization prevalence in Europe increasing from 5.4% to 7.9% in 2014, and it poses a potential major risk to some individuals. 2,3 To date, allergens identified in peach fruit and pollen include Pru (gibberellin-regulated protein), 2 Pru p 9 (PR-1, pollen allergen), 4 and ENEA (the first four N-terminal residues, similar to latex Hev b 5), a recently identified allergen. 5 Of these, the two main allergens Pru p 1 and Pru p 3 together account for more than 95% of peach allergies in Europe and China. 3,6,7 They induce two different allergy patterns: in central Europe and north China the symptoms are mild and local, such as oral allergy syndrome (OAS) related to Pru p 1, while in southern Europe and China the symptoms are mostly OAS and/or systemic due to Pru p 3. 3,8 Hypersensitivity caused by Pru p 1 is mostly induced by cross-reactions with Fagales pollen group 1 allergenic proteins, such as Bet v 1. [9][10][11] Three naturally occurring isoforms of Pru p 1, Pru p 1.0101, Pru p 1.0201, and Pru p 1.0301, have been identified in the "Earlygold" peach cultivar and their immunoglobulin E (IgE)-binding efficiency has been studied. Pru p 1.0301 is mainly expressed in peach pollen, and Pru p 1.0101 (DQ251187) and Pru p 1.0201 (KM350692) isoallergens are present in the fruit, with Pru p 1.0201 having the highest binding capacity to IgE compared with Pru p 1.0101 and Pru p 1.0301. 12,13 This means that Pru p 1.0101 and Pru p 1.0201 can essentially represent the total Pru p 1 content in peach fruit.
Allergen levels in various fruits have been extensively studied to help patients avoid high-risk varieties, especially in Rosaceae fruits such as apple and peach. The PR-10 protein levels are highly dependent on the varieties and also influenced by storage conditions and duration. 14-16 LTP and PR-10 proteins have been most studied due to their high sensitization rate and cross-reactivity. LTP is stable, mainly concentrated in the peel and the levels vary greatly in different varieties, related to ripening, aroma, and sugar content. [17][18][19] The content of PR-10 proteins has been found to be lower than that of LTP in peach, and is usually affected by genotype, storage conditions and storage time. 14,20 A recent study selected Pru p 3 hypoallergenic varieties after measuring over 100 varieties using sensitive monoclonal antibody ELISA. 19 Quantification of the Pru p 1 levels is necessary to analyze whether there is a relationship between fruit quality (aroma, sugar content, ripening date) and Pru p 1 content, to evaluate the potential risk level of Pru p 1 and to screen out hypoallergenic varieties for both Pru p 1 and Pru p 3.
The aim of this study was to establish a sandwich enzyme-linked immunosorbent assay (sELISA) method, to quantify Pru p 1 content in 83 peach varieties and define varieties with low levels of Pru p 1 allergen to benefit peach allergenic patients.

| Plant materials
Based on peach genetic diversity previously identified in China, a core collection of 19 nectarine and 64 peach varieties (60 cultivated and 4 wild), a total of 83, were selected (Table S1) (Table S1) based on the Descriptors and Data Standard for Peach. 23 Peach fruit were pitted and separated into peel, pulp and whole (including peel and pulp). The samples were stored at −40°C.

| Preparation of protein extracts and protein determination
Protein extraction was carried out as described previously. 18

| Mouse monoclonal antibodies
To produce anti-Pru p 1.0101 and anti-Pru p 1.0201 mAbs, four Bal b/c mice were immunized subcutaneously intraperitoneally with 10-40 μg (interval of 10 μg) rPru p 1.0101 and rPru p 1.0201, obtained from previous research. 13 The mice were boosted five times twice at 2-week intervals with 50 μg antigens in Incomplete Freund's adjuvant. Antibody producing hybridoma cells secreting anti-rPru p 1 monoclonal antibodies were selected by ELISA and Western blot with two isoallergen antigens and peach peel extracts. Antibodies were purified using HiTrap Protein-A affinity chromatography.
Peach peel extract and eluate samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Liquid chromatography-mass spectrometry (LC-MS/MS) after in-gel trypsin digestion was used for identitymatching of the purified protein to deduced allergens from peach extract.

| sELISA quantification of Pru p 1 allergen
Monoclonal and polyclonal antibodies specific for the two Pru p 1 isoallergens were used to find the suitable pair for sELISA. A0-A7-G11 was selected as the capturing antibody due to its high affinity to both isoallergens, with bound proteins detected by biotinylated

| Statistical analyses
Quantitative data were expressed as mean ± SE by Prism 6.0 (GraphPad Software). Significant differences among groups were assessed using the Kruska-Wallis nonparametric test. All statistical tests with p < 0.05 were considered as significant. Realtime PCR data were presented according to the comparative method (2 −ΔΔCt ), where ΔC t is the difference in threshold cycles for the target (C t sample) and reference (C t ACTIN). Excel and Prism 6.0 were used for qPCR statistical analyses and figure plotting.

| Selection of mAbs and pAbs
Screening by ELISA against rPru p 1.01 and rPru p 1.02 resulted in five mAbs and two pAbs. The mAbs A0-A7-G11, B6-A1-B11, A2-D8, and pAb P1 were obtained against Pru p 1.0101, and two mAbs, C7-C4, 5-D10, and the pAb P2 against Pru p 1.0201. All mAbs detected Pru p 1 at 17 kDa in Western blot (Figure 1). Preliminary testing indicated that mAb A0-A7-G11 had high binding capacity and specificity, making it a suitable capturing antibody. As binding of the polyclonal antibodies P1 and P2 was similar for both isoallergens, they were mixed at a ratio 1:1 as the detecting antibodies. Pair matching and antibody specificity tests were also carried out (Tables 1 and 2).

| Dose-response standard curve
The dose-response standard curve, obtained with rPru p 1.0101 and rPru p 1.0201 (1:1 mixed), ranged from 0 to 500 ng/ml with a linear section between 4 and 32 ng/ml (Figure 2). The intra-assay F I G U R E 1 Western blot of selected mAbs specific to rPru p 1.

| Natural Pru p 1 purification and protein identity
The natural Pru p 1 purified through affinity chromatography was confirmed by immunoblot and LC-MS ( Figure 3). SDS-PAGE and Western blot gave a band at 17 kDa ( Figure 3A,B). Peptide spectrum matching gave the sequence coverage of 75.0% for Pru p 1.0101 and 83.1% for Pru p 1.0201 ( Figure 3C).

| Quantification of Pru p 1 in 83 peach varieties
Even though Pru p 1 was generally low in the 83 cultivars, the variation ranged from 0.12 to 6.45 μg/g in whole fruit. There was an undetectable level of Pru p 1 content in four individual plants of the wild peach variety used for rootstock, and it was higher in nectarine (2.58 μg/g) than in pubescent peaches (2.16 μg/g) ( Figure 4A). Pru p 1 was mainly concentrated in the peel (0.20-73.44 μg/g), less in the pulp (0.05-9.62 μg/g). There was no significant difference in Pru p 1 content between peel from nectarine and peach cultivars (shown in Figure 4B). Variety group, ripening date, and aroma intensity all had no significant effect on Pru p 1 (p > 0.05). Representative hypoallergenic Pru p 1 varieties were "Zi Xue Tao," "Wu Yue Xian," "May Fire," and the high Pru p 1 allergen varieties were "Zhong You 7," "Zao Zhen Bao," "Chun Lei."

| Immunoblot of 10 representative varieties
Ten varieties representing wild, low/medium/high-Pru p 1 content groups based on the sELISA results were immunoblotted. Figure 5A shows that Pru p 1 was not detected in wild varieties. In peach and nectarine varieties, the 17 kDa bands were stronger with increasing Pru p 1 content, which indicated that immunoblot results were generally consistent with sELISA. Specific details, including concentration of peach crude extract, quantitative concentration of Pru p 1 and quantities of samples added are given in Table S2.  Note: Coated with 100 μl of 3 μg/ml A0-A7-G11, antigen 100 μl at 1 μg/ml (peach peel extract 100 μl), detector pAb 100 μl at 3 μg/ml (P1 and P2 1:1 mixed).
-5 of 10 4 | DISCUSSION sELISA is commonly used in fruit allergen quantification, for orange, apple, and peach. 15,[17][18][19]25 This method is advantageous in that it reacts directly with allergens in food and is highly sensitive and accurate. Owing to the similar epitopes of Pru p 1.01 and Pru p 1.02, our method was developed using a monoclonal (A0-A7-G11) and two polyclonal antibodies (P1 and P2), able to effectively identify these two major isoforms to determine total Pru p 1 level in peach fruit.
Results from ELISA showed that the binding capacity of mAb A0-A7-G11 to the target antigens was high, but very low to other antigens ( Table 2). The recovery rate, CVs, intra-and inter-assay precision were acceptable (Table 3)   The expression of allergen encoding genes is affected by many factors. We have previously found that Pru p 3 content is related to ripening date, sugar content, and aroma, while this correlation was not identified in Pru p 1. 19 Our quantification data showed that most nectarines contained high levels of Pru p 1, some up to 5.97 μg/g. The level of Pru p 1 in peel has been found to be more than 50 times that in the pulp. 24 A previous study has shown that Pru p 1.01 and Pru p 1.02 gene expression are predominant and constitutively expressed with a maximum peak of expression in the S2 phase, and vary greatly in different cultivars in mature fruits. 32 Here, we also found variation in its expression, with Pru p 1.02 predominant, especially in the ancient varieties "Mao Tao" and "Xue Bu Dai." In apple, a similar phenomenon has been found, with differing expression of three major Mal d 1 isoforms, Mal d 1.02 being the most highly expressed isoform. 26 Gene expression by qPCR is only indicative of possible higher protein presence: our initial aim to quantify Pru p 1 .01 and Pru p 1.02 and correlated with the gene expression was not successful. Till now, no protein quantification to the isoallergen level by Sandwich ELISA in fruit has been available, perhaps requiring mass spectrometry aided with sensitive isoform-specific peptide markers.

Measured (ng/ml) SD (ng/ml) CV (%) Recovery rate Measured (ng/ml) SD (ng/ml) CV (%)
Our aim was to screen for hypoallergenic peach varieties, to allow breeders and growers to produce fruit with lower allergenic potential, which might be tolerated by patients with peach allergy.
Results from this study may have implications for medical diagnostics, immunotherapy, clinical research, and breeding schemes for new hypoallergenic cultivars. We also identified peach varieties with high levels of Pru p 1 and Pru p 3 allergens, such as "Jin Shuo," "Jin Feng," and "Zao Zhen Bao" (shown in Table S1), which could be used as source material for diagnosis and for purification of natural Pru p 1 and Pru p 3 allergens.

| CONCLUSION
A large variation of Pru p 1 content, ranging from 0.12 to 6.45 μg/g in FW, was observed among 83 peach/nectarine varieties, and mainly concentrated in the peel. The content of Pru p 1 in the whole fruits of nectarines was slightly higher than that of peaches, and the content of the wild peach "Mao Tao" was the lowest.
Although the reduced levels of Pru p 1 do not always coincide with low Pru p 3, the ancient and early ripening red flesh varieties usually contained low Pru p 1 and Pru p 3 like "Zi Xue Tao," "Wu Yue Xian," and "May Fire." This knowledge will help breeders select hypoallergenic cultivars for agricultural production, as well as medical practitioners for clinical trials.