Mass Spectrometry Analysis on the Breakage of Allergens in High-Molecular-Mass Polymer of Roasted Peanuts.

Peanut allergy is a prevalent and concerning food allergy. Roasting can introduce structural changes to peanut allergens, affecting their allergenicity, but the structure on the primary structure is unclear. Here, the breakage sites were identified by mass spectrometry and software tools, and structural changes were simulated by molecular dynamics and displayed by PyMOL software. Results revealed that the appearance frequencies of L, Q, F, and E were high at the N-terminal of the breakage site, while S and E were dominant at the C-terminal. In the conformational structure, breakage sites were found close to disulfide bonds and the Cupin domains of Ara h 1 and Ara h 3. The breakage of allergens destroyed linear epitopes and might change the conformation of epitopes, which could influence peanuts' potential allergenicity.

Sensitive ELISA and lateral flow immunoassay for the detection of walnut traces in processed food and working surfaces.

Walnut represents one of the most allergenic nuts that can be found as a hidden allergen. In this study, sandwich ELISA and lateral flow immunoassay (LFIA), based on the determination of Jug r 1, were developed to detect walnut. Cross-reactivity was only found with Pecan nut among a panel of 88 food ingredients tested. ELISA and LFIA could detect 0.25 and 0.5 µg/g of walnut protein in complex food matrices spiked with walnut extract, respectively. Furthermore, walnut was detected in blended (chocolate) and incurred foods (ice cream and bread) added with ground walnut at levels of 0.5 and 1.5 µg protein/g by ELISA and LFIA, respectively. LFIA could also detect 0.1 µg of walnut protein in working surfaces. ELISA displayed acceptable precision and high recovery (71-97 %) and both tests were robust. This study shows that developed ELISA and LFIA are reliable tools to be applied in allergen control programs.

Mechanistic insights into silica nanoparticle-allergen interactions on antigen presenting cell function in the context of allergic reactions.

The incorporation of nanomaterials into consumer products has substantially increased in recent years, raising concerns about their safety. The inherent physicochemical properties of nanoparticles allow them to cross epithelial barriers and gain access to immunocompetent cells. Nanoparticles in cosmetic products can potentially interact with environmental allergens, forming a protein corona, and together penetrate through damaged skin. Allergen-nanoparticle interactions may influence the immune response, eventually resulting in an adverse or beneficial outcome in terms of allergic reactivity. This study determines the impact of silica nanoparticle-allergen interactions on allergic sensitization by studying the major molecular mechanisms affecting allergic responses. The major birch pollen allergen Bet v 1 was chosen as a model allergen and the birch pollen extract as a comparator. Key events in immunotoxicity including allergen uptake, processing, presentation, expression of costimulatory molecules and cytokine release were studied in human monocyte-derived dendritic cells. Using an in vivo sensitization model, murine Bet v 1-specific IgG and IgE levels were monitored. Upon the interaction of allergens with silica nanoparticles, we observed an enhanced uptake of the allergen by macropinocytosis, improved proteolytic processing, and presentation concomitant with a propensity to increase allergen-specific IgG2a and decrease IgE antibody levels. Together, these events suggest that upon nanoparticle interactions the immune response is biased towards a type 1 inflammatory profile, characterized by the upregulation of T helper 1 (Th1) cells. In conclusion, the interaction of the birch pollen allergen with silica nanoparticles will not worsen allergic sensitization, a state of type 2-inflammation, but rather seems to decrease it by skewing towards a Th1-dominated immune response.

Aptamer based point of care diagnostic for the detection of food allergens.

Aptamers, due to their small size, strong target affinity, and ease of chemical modification, are ideally suited for molecular detection technologies. Here, we describe successful use of aptamer technology in a consumer device for the detection of peanut antigen in food. The novel aptamer-based protein detection method is robust across a wide variety of food matrices and sensitive to peanut protein at concentrations as low as 12.5 ppm (37.5 µg peanut protein in the sample). Integration of the assay into a sensitive, stable, and consumer friendly portable device will empower users to easily and quickly assess the presence of peanut allergens in foods before eating. With many food reactions occurring outside the home, the type of technology described here has significant potential to improve lives for children and families.

Allergen Profile of London Plane Tree Pollen: Clinical and Molecular Pattern in Central Spain.

BACKGROUND AND OBJECTIVES: Platanus acerifolia (London plane tree) is a deciduous tree of the Platanaceae family. Sensitization to this plant varies with geography. Madrid, located in central Spain, has one of the highest London plane tree pollen concentration levels on the Iberian Peninsula. We evaluated both the clinical characteristics and the molecular sensitization pattern of patients with allergy to London plane tree pollen in the region of Madrid. METHODS: Thirty-eight patients allergic to London plane tree pollen were selected according to their clinical symptoms and positive results in skin prick testing and/or specific IgE determination. Serum was collected, and allergen components were evaluated using immunodetection techniques as well as ImmunoCAP. The IgE-binding proteins detected were identified and characterized using mass spectrometry. RESULTS: Analysis of serum samples from allergic patients revealed 9 IgE-binding bands in London plane tree pollen extract. Among these, the 45-kDa protein, which corresponded to Pla a 2, was detected in 76.3% of patients. However, the 18-kDa (Pla a 1) and 9-kDa (Pla a 3) bands were detected in 44.7% and 23.7% of sera, respectively. These results were confirmed using purified proteins. Characterization of the allergen revealed the 27-kDa protein to be glutathione-S-transferase. CONCLUSIONS: The molecular profile of patients sensitized to London plane tree pollen differs from that reported in studies from other locations. In the population we studied, the prevalence of Pla a 2 was higher than that of Pla a 1 and Pla a 3. In addition, the minor allergen previously referred to as Pla a 4 was characterized as glutathione-S-transferase.

Allergen Profile of London Plane Tree Pollen: Clinical and Molecular Pattern in Central Spain

Background: Platanus acerifolia (London plane tree) is a deciduous tree of the Platanaceae family. Sensitization to this plant varies withgeography. Madrid, located in central Spain, has one of the highest London plane tree pollen concentration levels on the Iberian Peninsula.Objectives: We evaluated both the clinical characteristics and the molecular sensitization pattern of patients with allergy to London planetree pollen in the region of Madrid.Patients and Methods: Thirty-eight patients allergic to London plane tree pollen were selected according to their clinical symptoms andpositive results in skin prick testing and/or specific IgE determination. Serum was collected, and allergen components were evaluatedusing immunodetection techniques as well as ImmunoCAP. The IgE-binding proteins detected were identified and characterized usingmass spectrometry.Results: Analysis of serum samples from allergic patients revealed 9 IgE-binding bands in London plane tree pollen extract. Among these,the 45-kDa protein, which corresponded to Pla a 2, was detected in 76.3% of patients. However, the 18-kDa (Pla a 1) and 9-kDa (Pla a 3)bands were detected in 44.7% and 23.7% of sera, respectively. These results were confirmed using purified proteins. Characterization ofthe allergen revealed the 27-kDa protein to be glutathione-S-transferase.Conclusions: The molecular profile of patients sensitized to London plane tree pollen differs from that reported in studies from otherlocations. In the population we studied, the prevalence of Pla a 2 was higher than that of Pla a 1 and Pla a 3. In addition, the minorallergen previously referred to as Pla a 4 was characterized as glutathione-S-transferase. (AU)

Antecedentes: Platanus acerifolia es un árbol de hoja caduca de la familia Platanaceae. La sensibilización frente a esta planta varía enfunción de la zona geográfica. Madrid, ubicada en el centro de España, tiene uno de los mayores niveles de concentración de polen deeste árbol en la Península Ibérica.Objetivo: Evaluar las características clínicas y los patrones moleculares de sensibilización en pacientes con alergia al plátano de sombraen la región de Madrid.Pacientes y Métodos: Treinta y ocho pacientes alérgicos al polen del plátano de sombra fueron seleccionados de acuerdo con los síntomasclínicos, pruebas cutáneas positivas y/o IgE específica. El suero se recogió y se evaluaron los componentes alérgicos mediante técnicas deinmunodetección, así como ImmunoCAP. Las proteínas que unían IgE fueron identificadas y caracterizadas por espectrometría de masas.Resultados: El análisis de los sueros de los pacientes alérgicos reveló 9 bandas que captaban IgE en los extractos de polen de plátano desombra. Entre estas, la proteína de 45 kDa, correspondiente a Pla a 2, se detectó en el 76,3% de los pacientes. Sin embargo, las bandasde 18 kDa (Pla a 1) y 9 kDa (Pla a 3) fueron reconocidas en el 44,7% y 27,3%, respectivamente. Estos resultados se confirmaron usandoproteínas purificadas. La caracterización de los alérgenos identificó la proteína de 27 kDa como una glutatión S-transferasa.Conclusiones: El perfil molecular de los pacientes sensibilizados al polen del plátano de sombra varía respecto al descrito en estudios deotras localizaciones. Nuestra población muestra una mayor prevalencia de Pla a 2 comparado con Pla a 1 y Pla a 3. Además, el alérgenominoritario previamente denominado Pla a 4 fue caracterizado como una glutatión-S-transferasa. (AU)

Voltammetric Immunosensor to Track a Major Peanut Allergen (Ara h 1) in Food Products Employing Quantum Dot Labels.

Tracking unreported allergens in commercial foods can avoid acute allergic reactions. A 2-step electrochemical immunosensor was developed for the analysis of the peanut allergen Ara h 1 in a 1-h assay (<15 min hands-on time). Bare screen-printed carbon electrodes (SPCE) were used as transducers and monoclonal capture and detection antibodies were applied in a sandwich-type immunoassay. The short assay time was achieved by previously combining the target analyte and the detection antibody. Core/shell CdSe@ZnS Quantum Dots were used as electroactive label for the detection of the immunological interaction by differential pulse anodic stripping voltammetry. A linear range between 25 and 1000 ng·mL-1 (LOD = 3.5 ng·mL-1), an adequate precision of the method (Vx0 ≈ 6%), and a sensitivity of 23.0 nA·mL·ng-1·cm-2 were achieved. The immunosensor was able to detect Ara h 1 in a spiked allergen-free product down to 0.05% (m/m) of peanut. Commercial organic farming cookies and cereal and protein bars were tested to track and quantify Ara h 1. The results were validated by comparison with an ELISA kit.

Selenium biofortification of different varieties of apples (Malus domestica) - Influence on protein content and the allergenic proteins Mal d 1 and Mal d 3.

As allergy towards apples is widespread, the evaluation of various cultivation and postharvest influences on the allergenic potential is of great importance. Therefore, the analysis of the Mal d 1 content was the focus of this study, originally dealing with investigating the influence of a selenium biofortification on apple quality. The Mal d 1 content of apples was in most cases reduced when the fruits were biofortified with selenium. Apple variety and climatic conditions were identified as further influencing factors for the Mal d 1 content of the fruits. The separate analysis of the peel and the fruit flesh showed that the content of Mal d 1 in the fruit flesh was significantly lower in the biofortified samples than in the controls. In conclusion, the results indicate that the selenium biofortification of apples and biochemical mechanism behind can reduce the allergenic potential regarding the content of Mal d 1.

Visible on-site detection of Ara h 1 by the switchable-linker-mediated precipitation of gold nanoparticles.

Biosensors have been widely applied in tests for allergens, but on-site detection remains a challenge. Herein, we proposed a detection procedure for peanut Ara h 1 as a representative allergen, which was extracted from a cookie, thereby minimising the need for any complex pretreatment that was difficult to perform, and enabling the visual detection of the target without the use of analytical equipment. The extraction procedure was performed in less than 30 min using a syringe and filter (0.45 µm). The detection method for Ara h 1 was based on the aggregation of switchable linkers (SL) and gold nanoparticles (AuNP), and the presence of 0.19 mg peanut protein per 30 g of cookie could be confirmed within 30 min based on the AuNP/SL concentration ratio by the precipitation. This proposed procedure could be successfully applied to the detection of a wide range of food allergens.

The influence of source maps on SILAM performance in modeling ragweed pollen concentrations in the area of a major European source.

The Pannonian Plain is one of the centers of ragweed distribution in Europe. The province of Vojvodina (Serbia) is located on the southern part of the Pannonian Plain, representing a highly infested region. In this study, we have used the SILAM atmospheric dispersion model to simulate ragweed pollen concentrations during the season 2016 in the Vojvodina region. SILAM was tested with three different source maps of ragweed distribution in Vojvodina only: (1) map used in operational SILAM, which was calibrated with the SILAM model and observations, (2) map derived using "top-down" approach with land cover data inventory, and (3) map obtained with "top-down" approach using crop classification from the satellite data. Additionally, the sensitivity studies were done using two modified maps to study the effect of the source strength and long-range transport. Results of simulations were validated with the bi-hourly, daily, and seasonal pollen concentrations measured at five stations in Vojvodina. Overall Pearson correlation coefficients were 0.51 (Map 1), 0.50 (Map 2), and 0.42 (Map 3), while debiased scores were 232.95 pollen m-3 (Map 1), 245.59 pollen m-3 (Map 2), and 258.24 pollen m-3 (Map 3). Even though Vojvodina is in the area of a major European source, regional transport of ragweed pollen from a few hundred kilometers of the surrounding area was important in explaining the presence of pollen in the afternoon hours, although it could not completely explain total pollen quantity. The results confirmed that it is vital to calibrate source maps using atmospheric dispersion model with the observed pollen data.

Evaluation of two commercial peach extracts for skin prick testing in the diagnosis of hypersensitivity to lipid transfer protein. A multicenter study.

Summary: The clinical usefulness of two commercial peach extracts for SPT (by Lofarma SpA and ALK-Abellò, respectively) was compared in a multicenter study carried out in Italy. Peach allergic patients were tested with the two extracts in parallel and underwent the detection of IgE specific for all three peach allergens currently available (Pru p1, Pru p3, and Pru p4, respectively). The two extracts were almost identical in terms of sensitivity and specificity, being able to detect virtually all patients sensitized to stable peach allergens (lipid transfer protein (LTP) and, presumably, peamaclein) but scoring negative in patients exclusively sensitive to labile allergens (either PR-10 and/or profilin). Thus, the two extracts represent an excellent tool to carry out a preliminary component-resolved diagnosis of peach allergy at the first patient visit.

Bacillus velezensis DP-2 isolated from Douchi and its application in soybean meal fermentation.

BACKGROUND: Soybean meal (SBM) is the most common protein source used in the poultry and livestock industries. It has high-quality protein, an excellent amino acid (AA) profile, and positive isoflavone properties. However, the antigen proteins in SBM are unsuitable for young animals. The objective of this study was to identify a Bacillus strain that can degrade soybean antigen proteins, and to evaluate the feasibility of its application in SBM fermentation. RESULTS: Bacillus velezensis DP-2 was isolated from Douchi, a fermented Chinese food. It degraded 96.14% and 66.51% of glycinin and ß-conglycinin, and increased the trichloroacetic acid-soluble protein (TCAN) content by 5.46 times in the SBM medium. DP-2 could secrete alkaline protease and neutral protease, with productivities of 5.85 and 5.99 U mL-1 . It had broad-spectrum, antibacterial activities against Rhizopus nigricans HR, Fusarium oxysporum ACCC37404, Penicillium digitatum SQ2, Aspergillus flavus C1, Aspergillus niger ACCC30005, Trichoderma viride YZ1, Candida tropicalis CICC1630, and Salmonella sp. ZY. For SBM fermentation, the optimal inoculum rate, temperature, and fermentation time of DP-2 were 2.21 × 107 CFU g-1 , 37 °C, and 48 h, respectively. The fermented soybean meal (FSBM) was cream-colored and glutinous. Its crude protein (CP), soluble protein, and TCA-N content were improved by 13.45%, 12.53%, and 6.37 times, respectively. The glycinin and ß-conglycinin content were reduced by 78.00% and 43.07%, respectively, compared with raw SBM. CONCLUSIONS: Bacillus velezensis DP-2 has potential as a starter culture for SBM fermentation. © 2020 Society of Chemical Industry.

Development of Pretreatment Protocols for Determination of Soybean ß-Conglycinin in Processed Soybean Foods Using Commercial ELISA Kits.

ß-Conglycinin is the major storage protein in soybeans. Pre-clinical animal models and human clinical studies have demonstrated the triglyceride-lowering effect of this protein, suggesting that it could be put into practical use as a functional food material. To date, however, there are no accurate and simple assays for quantification of ß-conglycinin. In this study, samples were pretreated by mixing them with rice flour powder prior to extraction of proteins. Then, we used commercially available ELISA kits for detection of allergens that could be present in any contaminating soybean residue. This enabled accurate and highly reproducible quantitation of ß-conglycinin content in several processed soybean foods.

Tiered approach for the identification of Mal d 1 reduced, well tolerated apple genotypes.

A rising proportion of the world population suffers from food-related allergies, including incompatibilities to apples. Although several allergenic proteins have been found in apples, the most important proteins that cause allergic reactions to apples in Central-Northern Europe, and North America are the Mal d 1 proteins, which are homologues of the birch pollen allergen Bet v 1. As the demand for hypoallergenic fruits is constantly increasing, we selected apple genotypes with a low total content of Mal d 1 by enzyme-linked immunosorbent assay analysis from segregating populations and tested the tolerability of these fruits through a human provocation study. This tiered approach, which exploited the natural diversity of apples, led to the identification of fruits, which were tolerated by allergic patients. In addition, we found a significant correlation (coefficient >0.76) between the total Mal d 1 content and flavan-3-ol amount and show that the isoform composition of the Mal d 1 proteins, which was determined by LC-MS/MS has a decisive effect on the tolerability of apple genotypes. The approach presented can be applied to other types of fruit and to other allergenic proteins. Therefore, the strategy can be used to reduce the allergen content of other plant foods, thereby improving food safety for allergy subjects.

Evaluation of the phenolic profile and immunoreactivity of Mal d 3 allergen in ancient apple cultivars from Italy.

BACKGROUND: Since the second half of the 20th century, the cultivation of ancient and local apple cultivars has almost disappeared from orchards in Italy. Some of these ancient apple cultivars often possess high nutraceutical values and display lower allergenicity than the modern ones, supporting the so-called 'green revolution' theory. RESULTS: In this study, the phenolic composition and the antioxidant activity of five ancient apple cultivars ('Belfiore', 'Pomella Genovese', 'Gravenstein', 'Bella del Bosco', and 'Piatlin') were compared with a 'Golden Delicious' commercial cultivar. Additionally, apples were tested for their potential allergenicity by detecting the presence of Mal d 3, a non-specific lipid transfer protein that represents the main apples' allergen. All apples came from northern Italy (Trentino Region) and were organically produced. Results showed that, for all cultivars, the skins contained more polyphenols than the pulps. 'Bella del Bosco' had the highest amount of polyphenols and antioxidant activity, whereas 'Piatlin' had the lowest phenolic content. All ancient cultivars presented a higher amount of pulp phenolic compounds than 'Golden Delicious'. Immunoblotting techniques showed that 'Bella del Bosco' and 'Piatlin' had very low quantities of Mal d 3 allergen; hence, they can be considered hypoallergenic cultivars. CONCLUSIONS: The preservation of ancient apple cultivars would be of great importance, not only to maintain the biodiversity but also for their nutritional properties. The hypoallergenic activity of some of these cultivars could be of interest also for the preparation of different apple-based products. © 2020 Society of Chemical Industry.

Influence of different extraction conditions on the detection of glycinin and ß-conglycinin in model processed foods by ELISA.

The presence of undeclared soy proteins in food can cause severe reactions in soy allergic individuals. The extraction of target proteins from processed foods is a crucial step in allergen detection by immunoassays, as only successfully extracted target proteins can be detected by the specific antibodies. The effectiveness was studied of different conditions (type of buffer, temperature and time of incubation) on the extraction of total protein, and concentration of glycinin and ß-conglycinin from different food matrices. The yields were determined using a soy protein isolate and three processed foods (sausage, bread and pâté) incurred with soy proteins. The yields were affected by the processing of analysed products and the composition and pH of the extraction buffers. Neutral and alkaline buffers (pH from 7.4 to 10.6) exhibited good protein extraction capacity and detectability of the specific target proteins. Denaturing additives and highly alkaline buffer (pH 12) extracted more crude protein but they were incompatible with the ELISA assay. Overall, the best results were obtained using phosphate (pH 7.4) and Tris/HCl (pH 8.5) buffers in the presence of 0.5 M NaCl. Crude protein yield of food extracts did not correlate with that of glycinin and ß-conglycinin, whereas a good relationship was found between the yields of the two proteins.

Proteome Analysis of Hordein-Null Barley Lines Reveals Storage Protein Synthesis and Compensation Mechanisms.

Hordeins are the major barley seed storage proteins and are elicitors of celiac disease. Attempts to reduce the hordein level in barley have been made; however, the resultant pleiotropic effects are less understood. Here, data-independent acquisition mass spectrometry was used to measure proteome-wide abundance differences between wild-type and single hordein-null barley lines. Using comparative quantitative proteomics, we detected proteome-wide changes (∼59%) as a result of the specific reduction in hordein proteins. The comparative analysis and functional annotation revealed an increase in non-gluten storage proteins, such as globulins and lipid transfer proteins, and proteins rich in essential amino acids in the null lines. This study yields an informative molecular portrait of the hordein-null lines and the underlying mechanisms of storage protein biosynthesis. This study indicates the extent to which protein content can be manipulated without biological consequence, and we envision its wide-scale application for studying modified crops.

Effectiveness of different proteases in reducing allergen content and IgE-binding of raw peanuts.

The effectiveness of some non-specific proteases in reducing raw peanut allergenicity was investigated. Peanut kernels were treated by Alcalase, papain, Neutrase and bromelain, respectively. The residues of major peanut allergens Ara h 1, Ara h 2 and Ara h 6 were determined by sandwich ELISA and SDS-PAGE, and the allergenicities of treated peanuts were compared to that of untreated peanuts by western blot. All tested proteases were effective in reducing Ara h 1, but their effectiveness in hydrolyzing Ara h 2 and Ara h 6 varied greatly. The maximal reductions of extractable Ara h 1, Ara h 2 and Ara h 6 were 100%, 100% and 99.8%, respectively, achieved by Alcalase hydrolysis. Alcalase was more effective in overall allergenicity reduction; bromelain and Neutrase were the least effective in reducing Ara h 2 and Ara h 6, respectively. The hydrolysis of original allergens also produced some smaller peptides with strong IgE-binding.

Identification of Kiwellin-like Proteins in Fruits by Using In Silico Tools.

Identification of allergen proteins by using wet-lab technology is a time-consuming and also costly process. In recent years, thanks to the developments in the field of bioinformatics, it is now possible to estimate the allergen proteins by using in silico tools. In the present study, it is aimed to find kiwellin-like proteins from different fruits samples by using bioinformatics tools. According to the results of the study, six proteins from Corchorus olitorius, Cucumis sativus, Capsicum chinense, Carica papaya, Morus notabilis and Jatropha curcas were defined as the allergens. In conclusion, in silico tools developed under the field of bioinformatics can provide a big contribution to the estimation of unknown allergen proteins in different fruits. Based on the in silico results, physicians can suggest people who have allergenicity to kiwellin not to consume the fruits that contain kiwellin-like proteins.

Extraction Conditions Affect the Immunoreactivity of Peanut Allergens.

Peanut allergic consumers rely on food package labels to avoid foods containing peanut. The inadvertent presence of peanut in foods due to cross-contact can be fatal if ingestion of such food leads to an allergic reaction. Analytical methods are available to detect undeclared peanut in foods. However, depending on the type of food matrix and food processing parameters, method performance can be adversely affected due to reduction in the extraction efficiency of peanut proteins. Temperature and probe sonication were used as a preincubation treatment for peanut flour slurries to assess their effect on the total peanut protein solubility from raw, light-roasted, and dark-roasted peanut flours. The effect of these treatments on the immunoreactivity of peanut allergens (Ara h 1, Ara h 2, Ara h 3, and Ara h 6) was determined by an indirect enzyme-linked immunosorbent assay using antibodies raised against these individual peanut proteins. Preincubation at 50 °C did not significantly improve the peanut protein solubility, whereas an increase in protein solubility was observed when light- and dark-roasted peanut flour slurries were preincubated at 90 °C or sonicated. The immunoreactivity of peanut allergens varied depending on the degree of peanut flour roasting and type of preincubation treatment. Overall, the immunoreactivity of peanut allergens from most peanut flour slurries was unaffected when preincubated at 50 °C for up to 60 min or sonicated with a probe for up to 5 min, whereas preincubation at 90 °C resulted in a time-dependent reduction in immunoreactivity of peanut allergens. Sonication treatment may improve peanut protein extraction without markedly affecting their immunoreactivity. PRACTICAL APPLICATION: Extraction of peanut proteins is vital for developed analytical methods to estimate peanut allergens in foods. The manuscript describes the effect of two different temperatures (50 and 90 °C) and probe-type sonication on peanut protein solubility. The findings suggest sonication can improve peanut protein solubility without markedly affecting their immunoreactivity.