Scope for growth and dietary needs of Mediteranean Pinnids maintained in captivity.

BACKGROUND: The measurement of the energy available for growth (scope of growth, SFG) can be used in bivalves to make a long-term prediction in a short-term experiment of the condition of the individual. In order to tackle the best conditions for captive maintenance of Mediterranean Pinnids, a SFG study was conducted using Pinna rudis as a model species. Three diets were examined to test the viability of live microalgae and commercial products: i) a control diet using 100% of live microalgae based on the species Isochrysis galbana (t-ISO), ii) a 100% of commercial microalgae diet based on the product Shellfish Diet 1800®, and iii) a 50/50% mix diet of I. galbana (t-ISO) and Shellfish Diet 1800®. RESULTS: SFG results showed significant differences among diets in the physiological functions measured and suggested lower acceptability and digestibility of the commercial product. Negative SFG values were obtained for the commercial diet which indicates that it should be rejected for both Pinnid maintenance. The mixed diet showed improved physiological performance compared to the commercial diet, resulting in a higher SFG that had no significant differences with the control diet. However, in the long-term, the lower digestibility of the mixed diet compared to the control diet could lead to a deterioration of individuals' conditions and should be considered cautiously. CONCLUSIONS: This work represents the first case study of SFG in Pinna spp. and provides fundamental data on dietary needs for the critically endangered species, P. nobilis.

Nature more than nurture affects the growth rate of mussels.

We tested the hypothesis that environmental trophic conditions prominent during the growing period (nurture conditions) can modify the differing physiological profiles between fast (F)- and slow (S)-growing juveniles of the mussel Mytilus galloprovincialis. Approximately 200 individuals were fed a high organic content diet dosed below the pseudofaeces threshold (BP), whereas another 200 were fed a low organic content diet dosed above the pseudofaeces threshold (AP), forcing them to maintain a continuous production of pseudofaeces. After 3 months, F and S individuals in each rearing condition were selected and used in feeding experiments. We measured the physiological parameters of the energy balance of selected F and S mussels fed on 4 different diets and tested the effects of the rearing condition (BP vs AP) and growth condition (F vs S) upon the physiological variables. Irrespective of the rearing condition, F-mussels attained higher values of scope for growth with the four experimental diets due to their capacity to display higher clearance rates and preingestive selection efficiencies. F-individuals also had higher gill-surface areas than S individuals. We discussed the role of the gills in determining inter-individual growth rate differences in the mussel.

Mytilus galloprovincialis fast growing phenotypes under different restrictive feeding conditions: Fast feeders and energy savers.

The present study aims to test if the environmental conditions prevailing during the growing period can determine the physiological profiles of specimens differentiated as fast (F) or slow (S) growers in the mussel Mytilus galloprovincialis. We reared mussel spats in the laboratory under two different conditions. In Treatment I (continuous feeding during discontinuous immersion), two mussel groups were submitted to a daily air exposure of 8 h and fed continuously during immersion-time, with either high-quality food dosed below the pseudofaeces threshold (BP group) or low organic content food dosed above the pseudofaeces threshold (AP group). In Treatment II (discontinuous feeding during continuous immersion), mussels were continuously immersed but fed only 1 day per week (RC group). Mussels were reared for 7 and 11 months (time required for size-differentiation) in Treatments I and II, respectively, and the smallest and largest individuals from each group were selected as S and F specimens. A series of feeding experiments (with different food quality, food ration and under continuous food supply) were performed to analyse the physiological performance of selected F and S mussels. In Treatment I, no significant differences were found in the metabolic rates between F and S mussels, and the faster growth rate of F-mussels resulted from their capacity to display higher clearance-ingestion rates and pre-ingestive selections. The physiological basis of growth rate differences between F and S mussels were found to be the same in mussels reared with diets below or above a pseudofaeces threshold (FBP, FAP, SBP and SAP). In contrast, the mussels from Treatment II had no significant differences in the feeding rates between FRC and SRC mussels. However, F individuals were found to have a 33% lower standard metabolic rate, indicating that fast growth under severe feeding restriction stemmed from a higher capacity of F-mussels to save energy during long periods of starvation. Despite the differences in the physiological basis explaining fast growth between the two treatments, F-mussels were found to possess significantly higher gill-surface area in both cases. It is thus concluded that endogenous factors affecting the gill-surface area play a major role in determining inter-individual growth rate differences in the mussel, Mytilus galloprovincialis.

Ingestion and absorption of particles derived from different macrophyta in the cockle Cerastoderma edule: effects of food ration.

We analyzed the capacity of the common cockle Cerastoderma edule to utilize detrital food particles obtained from three different macrophytes: the vascular plant Juncus maritimus and two green macroalgae (Ulva lactuca and Enteromorpha sp.). We measured feeding and digestive parameters at three concentrations of detritus (0.5, 1.0 and 3.0 mm(3) l(-1)), so that functional relationships between ingestive and digestive processes could be assessed. Increasing concentrations of detritus (food) resulted in a reduction in filtering activity (clearance rate l h(-1)), but an increase in ingestion rate. Consequently, gut content also increased with increasing food concentration, irrespective of food type. In contrast, the trend followed by absorption efficiency with increasing ingestion rate was determined by food type, being significantly reduced (from 0.63 to 0.11) with Juncus but remaining almost constant with the green macroalgae (0.58 ± 0.07 with Ulva) or only minimally reduced (from 0.66 to 0.48 with Enteromorpha). This differential response had clear consequences for energy uptake: absorption rate increased with increasing particulate organic matter with Enteromorpha but decreased with Juncus. We discuss the possible role of digestive parameters such as digestibility, gut content and gut-residence time in the differential utilization of detrital matter from different vegetal origins by cockles.

The Russian Thistle (Salsola kali) pollen major allergen, Sal k 1, can be quantified in allergenic extracts and airborne pollen.

BACKGROUND: Russian thistle (Salsola kali) pollen is an important cause of pollinosis in areas where rainfall is not abundant. Our aim was to develop an ELISA for quantification of the major allergen of S. kali extracts, Sal k 1, and to assess the correlation of this allergen content with the allergenic activity of extracts. METHODS: Sal k 1 was purified by ion exchange and gel permeation chromatography and identified by mass spectrometry. Monoclonal antibody 4C11 was used for capture at 5 microg/ml and biotin-labeled specific antiserum at 0.25 microg/ml served for detection. The allergenic activity of the pollen extracts was measured by enzyme allergosorbent test inhibition. RESULTS: Sal k 1 reacted to 85% of sera from 40 S. kali-allergic patients and was able to inhibit 92% of the IgE-binding capacity of patients' serum pool to the whole extract. The ELISA had a lineal range between 1.25 and 20 ng/ml of purified Sal k 1. The intra- and interassay coefficients of variation were lower than 5 and 10%, respectively. The assay was very sensitive since it had a detection limit of 0.08 ng/ml. No reactivity was found outside the Amaranthaceae family where only Kochia and Salicornia sp. gave significant reactivity. A good correlation (Spearman's rho = 0.92) was obtained between Sal k 1 content of different S. kali extracts and their IgE-binding activity. CONCLUSIONS: The results proved the usefulness of the two-site sandwich ELISA for aeroallergen control and for the standardization of S. kali pollen extracts intended for clinical use.

Engineering of major house dust mite allergens Der p 1 and Der p 2 for allergen-specific immunotherapy.

BACKGROUND: Specifically designed recombinant allergens with reduced IgE reactivity are promising candidates for a more defined, effective, and safer specific immunotherapy (SIT). OBJECTIVE: We sought to obtain hypoallergenic hybrid molecules which could potentially be applied to house dust mite (HDM) allergy treatment. METHODS: Two hybrid molecules (QM1 and QM2) derived from the two major Dermatophagoides pteronyssinus allergens, Der p 1 and Der p 2, were engineered by PCR, produced in Escherichia coli, and purified. The overall IgE-binding capacity of the hybrids was compared with their single components by Western blot, specific IgE, skin prick test (SPT), and IgE-inhibition assays. T cell proliferation assay were performed to confirm their retention of T cell reactivity. Immune responses to the hybrid molecules were studied in BALB/c mice. RESULTS: The IgE reactivity of both hybrid proteins was strongly reduced as evaluated by in vitro methods. Furthermore, in vivo SPTs performed on 106 HDM-allergic patients showed that the hybrid proteins had a significantly lower potency to induce cutaneous reactions than the individual components. Hybrid molecules induced higher T cell proliferation responses than those produced by an equimolecular mixture of Der p 1 and Der p 2. Immunization of mice with the hybrid proteins induced Der p 1- and Der p 2-specific IgG, which inhibited the binding of allergic patients' IgE to these natural allergens. CONCLUSION: QM1 and QM2 hybrids exhibited less IgE-binding activity but preserved immunogenicity and fulfilled the basic requirements for hypoallergenic molecules suitable for a future SIT of HDM allergy.

Expression of a recombinant protein immunochemically equivalent to the major Anisakis simplex allergen Ani s 1.

BACKGROUND: Anisakis simplex is a nematode which can parasitize humans, producing anisakiasis and can induce immunoglobulin-(Ig)-E-mediated allergic symptoms. Parasite recombinant proteins, such as the major allergen Ani s 1, may be useful tools to avoid misdiagnosis of A simplex allergy due to cross-reactivity when whole parasite extracts are used. OBJECTIVE: To obtain Ani s 1 allergen as a recombinant protein with IgE-binding properties similar to its natural counterpart. METHODS: Ani s 1-encoding cDNA was amplified by polymerase chain reaction and cloned. The allergen was expressed in Escherichia coli as a nonfusion protein. Natural and recombinant Ani s 1 were investigated by means of Western blotting, enzyme allergosorbent test, enzyme-linked immunosorbent assay (ELISA), and ELISA inhibition using sera from 53 patients with A simplex allergy. RESULTS: Residues of the amino acid sequence of the encoded protein were 99.4% identical to the reported one. Purified rAni s 1 was obtained with a yield of 2 mg/L of culture while the yield of the natural counterpart was only 50 micro/g of larvae. rAni s 1 reactivity was not significantly different from that of the natural allergen; the correlation was excellent (p = 0.92, P < .001). ELISA-inhibition experiments showed that the dose-response inhibition curve obtained with rAni s 1 overlapped with that of nAni s 1. In an enzyme allergosorbent analysis, 86.8% of the A simplex-allergic patient sera reacted to rAni s 1. CONCLUSION: Recombinant Ani s 1 is immunochemically equivalent to its natural counterpart and therefore might be useful for the in vitro diagnosis of anisakiasis and A simplex-mediated allergy.

An antibody-based ELISA for quantification of Ani s 1, a major allergen from Anisakis simplex.

Anisakis simplex is a nematode parasite that can infect humans who have eaten raw or undercooked seafood. Larvae invading the gastrointestinal mucosa excrete/secrete proteins that are implicated in the pathogenesis of anisakiasis and can induce IgE-mediated symptoms. Since Ani s 1 is a potent secreted allergen with important clinical relevance, its measurement could assess the quality of allergenic products used in diagnosis/immunotherapy of Anisakis allergy and track the presence of A. simplex parasites in fish foodstuffs. An antibody-based ELISA for quantification of Ani s 1 has been developed based on monoclonal antibody 4F2 as capture antibody and biotin-labelled polyclonal antibodies against Ani s 1 as detection reagent. The dose-response standard curves, obtained with natural and recombinant antigens, ranged from 4 to 2000 ng/ml and were identical and parallel to that of the A. simplex extract. The linear portion of the dose-response curve with nAni s 1 was between 15 and 250 ng/ml with inter-assay and intra-assays coefficients of variation less than 20% and 10%, respectively. The assay was specific since there was no cross-reaction with other extracts (except Ascaris extracts) and was highly sensitive (detection limit of 1.8 ng/ml), being able to detect Ani s 1 in fish extracts from codfish and monkfish.

Effects of body-size and season on digestive organ size and the energy balance of cockles fed with a constant diet of phytoplankton.

Seasonal variation in size-dependence of seawater clearance rate, absorption efficiency, oxygen consumption, gill area, length of the crystalline style and dry weight of digestive gland was analyzed in cockles Cerastoderma edule from the Mundaka Estuary, Spain. Experimental determinations were performed monthly (from July 1998 to November 1999) in cockles being fed with Tetraselmis suecica (organic content: 87.84 +/- 1.95%) at a concentration of 3 mm(3)/l for 3 days. Analysis of covariance reveals no seasonal differences in both size-dependence of seawater clearance rate and oxygen consumption, which were found to scale to dry body weight with mass-exponents of 0.56 and 0.62, respectively. No significant correlation was found between absorption efficiency and body weight. Mass-exponents for gill area, dry weight of the digestive gland and length of the crystalline style remained constant among seasons showing values of 0.62, 0.34 and 0.82, respectively. Seasonal trends for every physiological determination were calculated for a standard size (200 mg) cockle: standardized clearance rates and oxygen consumptions followed a similar trend with minimum values in winter ( approximately 0.5 l/h and approximately 100 microl O2/h, respectively) and maximum values during spring-summer ( approximately 1.7 l/h and approximately 250 microl O2/h, respectively), whereas absorption efficiency and food throughput time showed both the opposite pattern with highest values corresponding to winter months ( approximately 50-60% and approximately 5-6 h, respectively), and lowest ( approximately 30% and approximately 3-4 h, respectively) to summer-autumn. Scope for growth exhibited minimum values in winter followed by a rapid increase along the winter-spring transition, maximum values being attained in spring (May) and summer (July). Exponential decline of seasonal values of absorption efficiency associated to rising ingestion rates of organic matter presented an asymptotic minimum at 0.35. Absorption efficiency was positively related to food throughput time, whereas the latter fell to a minimum of 3.548 h with increasing food intake. So, maintenance of throughput time-and consequently absorption efficiency-along with enhanced filtering activity provided cockles with higher absorption rates improving scopes for growth registers during spring and summer. These dynamics might be explained as the consequence of the seasonal digestive adjustments in cockles, which, in fact, were found to increase the size of the digestive organs during that period.

Cloning, expression and characterization of mugwort pollen allergen Art v 2, a pathogenesis-related protein from family group 1.

Mugwort (Artemisia vulgaris) belongs to the Compositae family, and is one of the main causes of allergy in late summer and autumn. The aim of the study was to characterize the allergen Art v 2 from mugwort pollen. Skin prick tests, performed in 19 patients allergic to mugwort and 10 control patients, showed an Art v 2 sensitization prevalence of 58%, whereas none false-positives were detected among control patients. Art v 2 was purified by standard chromatography and binding to Concanavalin A column and had an apparent molecular mass of 33 and 20 kDa, calculated by gel permeation and SDS-PAGE under denaturing conditions, respectively, showing that the allergen is composed of two identical subunits. Art v 2-encoding cDNA was amplified by PCR using degenerate primers based on reported partial amino acid sequences. Cloned cDNA encoding Art v 2 contains 140 bp that codify for a polypeptide of 15.8 kDa, with a predicted pI value of 5.2, and one potential N-glycosylation site. Protein homology search demonstrated that Art v 2 share 55-42% identical residues with pathogenesis-related protein PR-1 of tomato, potato, rape, wheat and rice. Homology was also found to Ves v 5 (41% identical residues). Bacterial-expressed recombinant Art v 2 was recognized only by 21% of mugwort-allergic patients. In conclusion, Art v 2 from mugwort is the first weed pollen allergen that belongs to the pathogenesis-related protein PR-1 and its recombinant form could help molecular diagnosis of mugwort associated allergy.

Diagnosis of Parietaria judaica pollen allergy using natural and recombinant Par j 1 and Par j 2 allergens.

BACKGROUND: Parietaria judaica pollen is one of the main causes of allergic diseases in the Mediterranean area and contains two major allergens, called Par j 1 and Par j 2. OBJECTIVE: To evaluate the diagnostic potential of natural and recombinant forms of Par j 1 and Par j 2 in comparison with standardized P. judaica pollen extract. METHODS: Thirty patients allergic to P. judaica pollen and 15 control patients were investigated. Skin prick tests and determination of specific IgE levels were performed with commercial P. judaica extract, natural Par j 1 and Par j 2, and recombinant forms of both allergens expressed in P. pastoris. RESULTS: The whole group of patients with allergy to P. judaica had a positive skin test reaction to purified nPar j 1-Par j 2 and rPar j 2 at 5 microg/mL, and no false-positive reactions were detected. Natural and recombinant Par j 1 and Par j 2 showed no significantly different responses in skin tests compared with P. judaica extract. A high correlation was found between the serum-specific IgE levels to P. judaica extract vs. natural (R=0.996; P<0.001) and recombinant allergens (R=0.887 and 0.982 for rPar j 1 and rPar j 2, respectively; P<0.001). rPar j 2 displayed a 100% sensitivity and specificity among P. judaica-allergic patients. CONCLUSIONS: In vivo and in vitro diagnosis of P. judaica pollen allergy could be simplified using rPar j 2. This protein showed comparable IgE response and skin prick reactivity with those produced by P. judaica pollen extract.

Purified allergens vs. complete extract in the diagnosis of plane tree pollen allergy.

BACKGROUND: Plane tree pollen allergy is a clinical disorder affecting human population in cities of Europe, North America, South Africa, and Australia. OBJECTIVE: To compare IgE-reactivity of the natural and recombinant forms of two major plane allergens, Pla a 1 and Pla a 2, with the reactivity of Platanus acerifolia pollen extract. METHODS: Forty-seven patients with P. acerifolia allergy, 15 of them monosensitized, and 24 control subjects were included in the study. Natural Pla a 1 and Pla a 2 were purified by standard chromatographic methods and recombinant proteins were expressed in Escherichia coli. Skin prick test and determination of specific IgE were performed with commercial P. acerifolia extract and natural and recombinant purified allergens. RESULTS: Pla a 1 and Pla a 2 were responsible for 79% of the IgE-binding capacity against P. acerifolia pollen extract. A high correlation has been found between the IgE response to nPla a 1 (R = 0.80; P < 0.001) or nPla a 2 (R = 0.79; P < 0.001) vs. P. acerifolia extract as well as between natural and recombinant Pla a 1 (R = 0.89; P < 0.001). Skin testing showed no significant differences between extract and nPla a 2, whereas a higher reactivity was found with nPla a 1. In contrast, rPla a 1 revealed markedly reduced sensitivity in comparison with extract by skin prick test and specific IgE. The sensitivity of the mix Pla a 1+Pla a 2 was 100% and 87.5% for monosensitized and polysensitized patients, respectively, with no false-positive reactions detected. Conclusion Pla a 1 and Pla 2 are sufficient for a reliable diagnosis of P. acerifolia in most patients and induce comparable skin test reactivity as a whole extract.

A sensitive monoclonal antibody-based enzyme-linked immunosorbent assay to quantify Parietaria judaica major allergens, Par j 1 and Par j 2.

BACKGROUND: Parietaria pollen is one of the most important causes of pollinosis in Mediterranean countries. Parietaria judaica pollen extract presents two major allergens, Par j 1 and Par j 2, that belong to the lipid transfer protein family. OBJECTIVE: To develop an ELISA for quantification of both major allergens of P. judaica pollen extracts, and to assert correlation of these allergens content with the allergenic activity of extracts. METHODS: Natural Par j 1-Par j 2 allergens were purified by gel filtration, ion exchange, and affinity chromatography and identified by mass spectrometry. Rabbit antisera were obtained using this protein preparation as antigen and used for immunoaffinity purification of nPar j 1-Par j 2. BALB/c mice were immunized with the immunopurified nPar j 1-Par j 2 and after fusion and screening by direct ELISA, 5D4 monoclonal antibody was selected as capture antibody to develop a quantitative two-site ELISA. Bound proteins were detected by a biotinylated Par j 1-Par j 2-specific polyclonal antibody. RESULTS: The optimized ELISA was developed from 25 to 8000 pg/mL of purified Par j 1-Par j 2, and a linear portion of 200-1000 pg/mL. The intraassay and interassay coefficients of variation were lower than 7% and 14% respectively. The assay was very sensitive and specific as it had a detection limit of 25 pg/mL and did not detect reactivity with the same family plants, as Urtica. Par j 1-Par j 2 allergens content was measured in 14 P. judaica and two P. officinalis pollen extracts showing a significant correlation with their allergenic activity measured by enzyme allergosorbent test inhibition. CONCLUSIONS: The results proved the usefulness of the two-sandwich ELISA for the standardization of Parietaria pollen extracts intended for clinical use, because of its good correlation with allergenic potency.

Development of a sandwich-type ELISA for measuring Pla a 1, the major allergen of Platanus acerifolia pollen.

BACKGROUND: Platanus acerifolia is an important cause of pollinosis in Western European cities. Pla a 1, a nonglycosylated 18-kDa protein with a prevalence of 80%, is a major allergen in P. acerifolia pollen extracts. Our aim was to develop a Pla a 1-specific ELISA to quantify this protein in allergenic extracts and preparations for clinical use. METHODS: Pla a 1 was purified by cation exchange at pH 7.0, gel filtration, and anion exchange chromatography at pH 10.0. Monoclonal (mAb) and polyclonal antibodies were obtained by immunizing mice and rabbits with nPla a 1. One (5C1) of the 13 mAb obtained was used as capture antibody at 5 mug/ml and biotin-labeled specific polyclonal antiserum at 0.63 microg/ml served for detection. RESULTS: The prevalence of Pla a 1-specific IgE to purified Pla a 1 among 47 P. acerifolia-allergic patients was 79%. The Pla a 1-ELISA developed has a linear range of 3-25 ng/ml, high sensitivity with a detection limit of 0.5 ng/ml and is highly specific as none of the 24 pollen, mite, mold, and plant food extracts tested gave positive results. The assay could quantify Pla a 1-like proteins in other planetree pollen extracts. A good correlation was obtained between Pla a 1 content of 11 P. acerifolia pollen extracts (average content 0.69% of the total protein) and their IgE-binding activity. CONCLUSIONS: The described two-site sandwich ELISA to measure Pla a 1 is useful for standardization of planetree pollen extracts intended for clinical use.

Pho d 2, a major allergen from date palm pollen, is a profilin: cloning, sequencing, and immunoglobulin E cross-reactivity with other profilins.

BACKGROUND: Up to now, some date palm pollen (DPP) allergens have been described but very few data are available about their molecular nature. The aim of this study was to identify and characterize Pho d 2, a major allergen from this pollen. METHODS: Sera from 25 patients allergic to DPP were analysed by immunoblotting. Purification of DPP profilin was performed by poly-l-proline affinity chromatography. Profilin-encoding cDNA from DPP was cloned by using a RT-PCR strategy and recombinant allergen was expressed as a non-fusion protein in Escherichia coli. Natural and recombinant Pho d 2 were investigated by means of enzyme allergosorbent test to compare the immunologic properties of both allergens and to analyse cross-reactivity with other profilins. RESULTS: A 14.4 kDa protein was identified as a major allergen in DPP extract. Purification, cloning, heterologous expression, and inhibition experiments identified it as profilin (Pho d 2). Pho d 2 comprises 131 amino acids and has high sequence identity with other allergenic food and pollen profilins. The prevalence of specific IgE antibody reactivity to natural Pho d 2 by ELISA was 56% and 64% by skin prick test (SPT). Pho d 2 is an important allergen as it is responsible for more than 70% of the IgE reactivity to the pollen extract. IgE directed against Pho d 2 showed a strong cross-reactivity with other profilins such as those from olive tree and grass pollens. CONCLUSION: Pho d 2, a 14.4 kDa protein identified as profilin, is a major and relevant allergen in DPP, as confirmed by SPT and thereby may elicit clinical symptoms in sensitized patients.

Quantification assay for the major allergen of Cupressus sempervirens pollen, Cup s 1, by sandwich ELISA.

BACKGROUND: The Cupressaceae are an important cause of pollinosis, particularly in Mediterranean countries. Cypress pollen allergenic extracts are difficult to produce since they have a low protein and a high carbohydrate content and consequently accurate standardization of these extracts is essential for diagnosis and immunotherapy. METHOD: Natural Cup s 1 was purified by a combination of hydrophobic interaction, gel filtration and ion exchange chromatographies and its enzymatic activity was analyzed. The allergen was used as reference material in the ELISA standard curve. The assay was based on a specific monoclonal antibody (3D2) immobilized on ELISA plates and used to capture Cup s 1. Bound proteins were detected by a combination of biotinylated specific antiserum and peroxidase-conjugated streptavidin. RESULTS: Purified Cup s 1 is a functional pectate lyase enzyme with a specific activity of 750 U/mg protein. The developed ELISA measured Cup s 1 concentrations ranging from 31.25 to 250 ng/ml in the lineal portion of the standard curve. The intra-assay and inter-assay variation coefficients in the working range were less than 8.1 % and 16 %, respectively. The assay was highly sensitive, with a detection limit of 3.8 ng/ml. The dose-response curves obtained with C. sempervirens pollen extracts and extracts belonging to other species from the Cupressaceae family showed a good parallelism compared with those obtained using the purified allergen, indicating that the same protein was measured. CONCLUSIONS: The assay described is sensitive, specific and reproducible for the quantification of Cup s 1 in C. sempervirens pollen extracts for clinical use. This ELISA could also be useful for other Cupressaceae-related pollen extracts.

Quantification assay for the major allergen of Cupressus sempervirens pollen, Cup s 1, by sandwich ELISA

Background: The Cupressaceae are an important cause of pollinosis, particularly in Mediterranean countries. Cypress pollen allergenic extracts are difficult to produce since they have a low protein and a high carbohydrate content and consequently accurate standardization of these extracts is essential for diagnosis and immunotherapy. Method: Natural Cup s 1 was purified by a combination of hydrophobic interaction, gel filtration and ion exchange chromatographies and its enzymatic activity was analyzed. The allergen was used as reference material in the ELISA standard curve. The assay was based on a specific monoclonal antibody (3D2) immobilized on ELISA plates and used to capture Cup s 1. Bound proteins were detected by a combination of biotinylated specific antiserum and peroxidase-conjugated streptavidin. Results: Purified Cup s 1 is a functional pectate lyase enzyme with a specific activity of 750 U/mg protein. The developed ELISA measured Cup s 1 concentrations ranging from 31.25 to 250 ng/ml in the lineal portion of the standard curve. The intra-assay and inter-assay variation coefficients in the working range were less than 8.1 % and 16 %, respectively. The assay was highly sensitive, with a detection limit of 3.8 ng/ml. The dose-response curves obtained with C. sempervirens pollen extracts and extracts belonging to other species from the Cupressaceae family showed a good parallelism compared with those obtained using the purified allergen, indicating that the same protein was measured. Conclusions: The assay described is sensitive, specific and reproducible for the quantification of Cup s 1 in C. sempervirens pollen extracts for clinical use. This ELISA could also be useful for other Cupressaceae-related pollen extracts (AU)

Antecedentes: Las cupresáceas son una importante causa de polinosis, particularmente en los países Mediterráneos. Los extractos alergénicos de polen de ciprés son difíciles de producir ya que tienen bajo contenido de proteínas y alto de carbohidratos, por lo que es esencial una precisa estandarización de estos extractos para su uso en diagnóstico e inmunoterapia. Método: El alergeno natural Cup s 1 fue purificado mediante interacción hidrofóbica, tamizado molecular, e intercambio iónico, y se analizó su actividad enzimática. Este alergeno fue utilizado como referencia en la realización de la curva de calibrado del ensayo. El ensayo se basó en un anticuerpo monoclonal (3D2) inmovilizado en la plaza y usado para capturar a Cup s 1. Posteriormente un antisuero es- pecífico contra Cup s 1 marcado con biotina unido a estreptavidina conjugada con la enzima peroxidasa sirvieron como detectores del alergeno unido. Resultados: Se determinó que Cup s 1 es una enzima funcional con actividad pectato liasa y una actividad específica de 750 U/mg. La parte lineal de la curva estándar del ELISA fue considerada la zona óptima de ensayo y se situaba entre 250 y 31,25 ng/ml, siendo en este intervalo los coeficientes de variación intraensayo e interensayo menores del 8,1 por ciento y del 16 por ciento respectivamente. Además de reproducible, el ensayo resultó sensible con un límite de detección de 3,8 ng/ml. Las curvas obtenidas con los extractos de C. sempervirens y otros extractos de polen de géneros de la familia Cupressaceae fueron paralelas a la del alergeno purificado indicando que se estaba midiendo la misma proteína. Conclusiones: Se describe un ensayo que resulta específico y de gran sensibilidad para la cuantificación de Cup s 1 en extractos de polen de C. sempervirens de uso clínico, y que puede ser aplicable a otros géneros de la familia Cupressaceae (AU)

PCR-based cloning and immunological characterization of Parietaria judaica pollen profilin.

Profilin has been described as an allergen present in pollen of trees, grasses and weeds. Since Parietaria judaica profilin has a molecular mass similar to other Parietaria allergens (Par j 1 and Par j 2) in the 14-10 kDa range, it is difficult to assess the prevalence of profilin by immunoblotting or to obtain sufficient amounts of purified native profilin for investigation and diagnosis. The aim of this study was to identify P. judaica profilin by PCR-based cDNA cloning and to elucidate its allergenic characteristics. Two cDNA clones encoding P. judaica pollen profilin were isolated by polymerase chain reaction (PCR) amplification using degenerate primers. Sequencing of both clones (Par j 3.0101 and Par j 3.0102) demonstrated a high amino acid sequence homology. Immunodetection of P. judaica pollen after isoelectrofocusing and incubation with rabbit antiserum against profilin indicated the existence of at least 2 isoforms. Expression in Escherichia coli BL21 (DE3) was carried out using a vector based in the T7 expression system, and the recombinant allergen was isolated by affinity chromatography on poly-(L-proline)-Sepharose. Cross-reactivity has been found between recombinant P. judaica pollen profilin and profilins from other botanical unrelated plants.

Quantification of the major allergen from cypress (Cupressus arizonica) pollen, Cup a 1, by monoclonal antibody-based ELISA.

BACKGROUND: Cypress pollen allergy is an important cause of rhinoconjunctivitis and asthma in Mediterranean countries. Cypress allergenic extracts are difficult to produce since they have low protein and high carbohydrate content, thus accurate standardization of them is essential to guarantee their quality. The aim of this study is to develop a sandwich ELISA for the quantification of Cup a 1, the major allergen of cypress (Cupressus arizonica) pollen extract. METHODS: Monoclonal antibodies directed to purified Cup a 1 were produced. Two of them (9C7 as capture antibody and 3D2 as the tracer) were selected to develop a quantitative sandwich ELISA. This ELISA was subsequently evaluated and compared with other techniques. RESULTS: The described ELISA is very sensitive with a detection limit of 8.7 ng/ml and a practical working range of 62.5-1,000 ng/ml. The assay is also highly reproducible with intra-assay and interassay coefficients of variation of less than 10%. The purified Cup a 1, used as standard, presents pectate lyase enzymatic activity. The assay also detected Cup a 1-like proteins in pollen from other Cupressaceae. A good correlation was obtained between Cup a 1 content of 12 C. arizonica pollen extracts and their IgE-binding activity. CONCLUSIONS: The described Cup a 1 ELISA is sensitive, specific and reproducible and can be used for the quantification of Cup a 1 in C. arizonica and other related pollen extracts. It also provides a reliable indication of the allergenic activity of the whole cypress pollen extract.

Biological characterization of glutaraldehyde-modified Parietaria judaica pollen extracts.

BACKGROUND: Allergoids are widely used in specific immunotherapy (SIT) for the treatment of IgE-mediated allergic diseases, but all techniques for standardization of conventional allergic extracts may not be appropriate for standardization of a glutaraldehyde (GA)-modified extract because of the unique characteristics of these extracts. OBJECTIVE: To assess an accurate methodology for standardization of chemically modified extracts. METHODS: GA-modified extracts from Parietaria judaica pollen were purified by diafiltration. Biochemical properties were investigated by determination of amino groups, chromatography, and SDS-PAGE. The IgE-binding activity was determined by skin prick test, enzyme allergosorbent test inhibition, basophil activation, and histamine release tests. Peripheral blood mononuclear cells (PBMCs) from P. judaica pollen-allergic subjects were stimulated with either native or allergoid extracts, and proliferation was measured. RESULTS: Biochemical data indicated a high degree of allergen polymerization resulting in extract components higher than 100 kDa. IgE-binding activity, both in vivo and in vitro, was reduced by more than 99.8%. Both allergen and allergoid induced PBMC proliferation and synthesis of blocking IgG antibodies at similar rates. Moreover, no evidence of introduction of new determinants by chemical modification was found. CONCLUSIONS: The preparation of GA-modified extracts by diafiltration is faster and more reliable than previous chromatographic methods. These modified extracts have drastically reduced their allergenicity while maintaining their immunogenicity, and therefore they can be used in safer and shortened schedules of SIT.