Biomedical subjects

L Boluda

Publications and source records attributed to L Boluda.

10 recordsLinked to original sources

PubMed2002

Allergenicity and immunochemical characterization of six varieties of Olea europaea.

BACKGROUND: The inhalation of Olea europaea pollen is one of the most important causes of allergic respiratory diseases in the Mediterranean basin. The objective of this study was to investigate the antigenic and allergenic composition of six different O. europaea varieties collected in southern Spain. METHODS: The varieties included in the study were: Acebuche (wild olive), Carrasqueño, Nevado, Hojiblanco, Manzanillo and Picual. Extracts of these six varieties were prepared. Twenty-nine olive individuals with an immunoglobulin(Ig)E-mediated allergy to olive pollen were skin tested with these extracts. The antigenic profile of these extracts was evaluated by SDS-PAGE; the allergenic profile was investigated by immunoblotting using the serum of these 29 individuals. The Ole e 1 content was established by ELISA inhibition using purified Ole e 1 and rabbit polyclonal antibodies and by scanning densitometry. RESULTS: The extracts that induced the smallest wheal size were Acebuche and Hojiblanco, being significantly different from the rest of the extracts. The antigenic and allergenic profiles of the extracts also varied. The Ole e 1 content ranged from 0.050 in Hojiblanco to 0.232 in Manzanillo, measured by ELISA inhibition and from 0.153 in Hojiblanco to 0.677 in Nevado, measured by scanning densitometry. CONCLUSIONS: The different varieties of O. europaea pollen studied demonstrated great differences in the in vivo and in vitro potency of the extracts. There were significant differences in the Ole e 1 content, while the protein content remained very similar in these extracts. This study confirms previous observations of a great variability in the antigenic and allergenic composition of O. europaea pollen extracts and establishes significant differences in Ole e 1 content.

Allergens↗

PubMed2002

Rapid release of Ole e 1 from olive pollen using different solvents.

BACKGROUND: Solubility is an important characteristic of allergenic molecules. The aim of this study was to investigate the solubility of Ole e 1, a major allergen of Olea europaea, using different solvents. MATERIAL AND METHODS: Olea europaea pollen was placed in a glass column and extracted using three different solvents: deionized water, phosphate buffer 0.01 M (PBS) and normal saline (NaCl 0.9%). Several fractions were collected after extraction with each solvent and pooled based on individual protein content. Each fraction corresponded to a different elution profile, as determined by linear regression analysis. After 130 min of extraction, the pollen that remained in the column was further extracted overnight. A control olive pollen extract was also prepared with each solvent. The antigenic and allergenic profiles of all the eluted and pooled fractions were analysed by SDS-PAGE and inmmunoblots. Protein and Ole e 1 content and the amount of protein needed to produce 50% inhibition were also calculated. Ten patients were skin prick tested with the fractions obtained with deionized water. RESULTS: Four elution profiles were obtained using deionized water as the extracting solution and three with the two other solvents. The three solvents produced different kinetics of allergen release. Ole e 1 was rapidly released when water was used, obtaining a total of 256 micro g of Ole e 1/ml after only 7 min of extraction (fraction EC1). Using PBS, or NaCl 0.9%, the release of Ole e 1 started after 4 and 9 min of extraction, respectively. The highest amount ofOle e 1 was eluted after 44 and 26 min, with a total concentration of 162 and 203 micro g of Ole e 1/ml, respectively. The presence of Ole e 1 in each phase was verified by SDS-PAGE and immunoblot analyses. CONCLUSIONS: The extracting solution seems to determine the antigenic profile of olive pollen extracts. Ole e 1 is rapidly released from the pollen grain after extraction in deionized water. The solubility seems to be affected by the use of other solvents. These techniques could be used to manipulate the Ole e 1 content in O. europaea extracts.

Allergens↗

PubMed2002

Trisetum paniceum (wild oats) pollen counts and aeroallergens in the ambient air of Madrid, Spain.

BACKGROUND: Madrid has a short but intensive grass pollen season, in which 79% of the total grass pollen load is released from the middle of May to the middle of June. The main objectives of this study were to quantify Trisetum paniceum (wild oats) aeroallergen in the atmosphere in Madrid from February to December 1996 and to correlate the aeroallergen concentrations with grass pollen counts. METHODS: Two different samplers were used to assess allergen exposure; a Burkard spore trap was used to collect pollen grains and a high-volume air sampler to collect airborne particles. A total of 182 air filters were collected and extracted in 1 ml of PBS and analysed by ELISA inhibition, using pooled sera from highly allergic individuals. RESULTS: T. paniceum aeroallergens were detected not only during the grass pollen season, but also before and after. Wild oat allergens had two main peaks of 1 and 1.9 microg/m(3), occurring in late May and July, respectively. The time series analysis established the existence of lags between the two main variables pollen counts and aeroallergen activity. Analysis of the data by the Spearman rank test and linear regression showed a weak correlation between grass allergenic activity and grass pollen counts (Spearman's rho = 0.29). Data obtained from time series analysis demonstrated that grass allergenic activity correlated strongly with current and 5-week-old grass pollen grain counts (r(2) = 0.73). CONCLUSIONS: Wild oats allergenic activity was detected during the entire year and not only during the pollen season. This fact is an important aspect to be considered in the clinical follow-up and treatment of grass pollen-sensitised patients in Madrid.

Air Pollutants↗

PubMed1999

Determination of Ole e 1 by enzyme immunoassay and scanning densitometry. Validation by skin-prick testing.

Ole e 1 is an important allergen in Olea europaea pollen extracts. This study describes the development of two new methods that can be used to estimate the Ole e 1 content in olive tree pollen extracts. They are based on (1) an enzyme immunoassay that uses rabbit polyclonal, monospecific antibodies and purified Ole e 1, and (2) scanning densitometry of SDS-PAGE gels. Twelve extracts were evaluated by in vivo and in vitro methods. The in vivo biological potency was estimated by prick skin testing 17 allergic individuals; the in vitro allergenic potency by direct IgE and IgE inhibition assays. The enzyme immunoassay showed an operative range of 0.03-100 microg/ml and demonstrated to be specific for Ole e 1. The Ole e 1 content ranged from 1% to 5% of the total protein in the 12 extracts. The amount of Ole e 1, assessed by gel scanning densitometry significantly correlated with the Ole e 1 content obtained by the immunoassay (r = 0.92; p < 0.001). The Ole e 1 content showed a significant correlation with the total allergenic potency of the extracts, evaluated by direct IgE, specific IgE inhibition and skin-prick testing. These two methods can be used to determine the Ole e 1 content in olive pollen extracts. The content of Ole e 1 can vary from 1% to 5% of the total protein in the extracts.

Allergens↗

PubMed1998

Purification, characterization, and partial sequencing of two new allergens of Olea europaea.

BACKGROUND: The inhalation of olive (Olea europaea) pollen is an important cause of allergic respiratory diseases in southern Europe and California. OBJECTIVE: The aims of this study were to characterize the allergenic composition of O. europaea pollen collected in California and to purify two important allergens. METHODS: One hundred grams of O. europaea pollen was extracted dialyzed in 10 kd cut-off membranes and lyophilized. Allergen characterization was done by sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotting. Two allergens were isolated by gel filtration, ion exchange, and hydrophobic interaction chromatography and sequenced. RESULTS: Ole e 4 has an apparent molecular weight, under reducing conditions, of 32 kd and pIs between 4.65 and 5.1. The N-terminal was blocked and the analysis of the amino acid sequence of two internal regions revealed no homology with other known proteins. Ole e 5 has a molecular weight of 16 kd and pIs between 5.1 and 6.5. The amino acid sequence of the N-terminal showed a high degree of homology with superoxide dismutase of several plant species. Ole e 4 and Ole e 5 had an IgE binding frequency by immunoblot of 80% and 35%, respectively. CONCLUSIONS: Olive pollen extracts have a heterogeneous composition, with several important allergens. One of these allergens showed a high degree of homology with a superoxide dismutase.

Allergens↗

PubMed1996

Binding affinities of allergens from pollen, mites, and house dust for specific IgG subclass antibodies.

The distribution and affinity of IgG subclasses against various aeroallergens were assessed by inhibition of specific antibody binding. Two parameters from the dose-response curves were taken as indicative of antibody affinity: the point of 50% inhibition and the value of the slopes on double-log plots. It was found that IgG4 antibody specific for aeroallergens (i.e., from pollens of several species of Gramineae, Olea europaea, and Parietaria judaica and from house dust) usually exhibits high affinity, except for Dermatophagoides pteronyssinus. High binding affinity was also displayed by IgG1 subclass antibodies against the allergens of O. europaea and P. judaica. Distinct IgG subclass affinity profiles were observed for the allergens of grass pollen (i.e., Holcus lanatus) and dust mites (i.e., D. pteronyssinus). These results demonstrate that IgG subclass distribution, as well as antibody affinity, depends on the nature of the sensitizing allergen.

Adult↗

PubMed1995

Do IgE-IgG complexes occur in the circulation?

IgG anti-IgE autoantibodies in human allergic sera have been investigated using an enzyme immunoassay on microplates coated with anti-human IgE MoAb. After incubation with serum, the plates were developed with peroxidase-labelled anti-human IgE MoAb for the determination of total IgE levels, or with anti-IgG MoAb for evaluating IgG anti-IgE autoantibodies. Using this methodology, no correlation was found between total IgE and IgG anti-IgE levels in groups of sera of allergic individuals. Although the results obtained with enzyme-labelled anti-IgG are often interpreted as indicative of IgE-IgG complexes captured from the serum, molecular sieving on gel columns as well as direct ultrafiltration experiments through 300-kD membranes demonstrate that such complexes do not occur preformed in the circulation, but arise de novo on the anti-IgE-coated solid phase during in vitro incubation with human serum. It is suggested that IgG anti-IgE autoantibodies react with IgE only after the latter has undergone a conformational change, either by colloidal manipulation or after reaction with allergen.

Adult↗

PubMed1994

Evaluation of the potency of allergenic extracts by inhibition of IgG-antibody binding.

A method is described for the in vitro potency evaluation of allergenic extracts by using their capacity of binding to specific human IgG antibodies. The results obtained with this inhibition-type enzyme immunoassay are compared with the analyses by the customary method of IgE antibody binding. A large series of allergenic protein fractions from the pollen of the Gramineae, Olea europea, Parietaria judaica, and from the dust mite Dermatophagoides pteronyssinus as well as from cat dander and peanuts were examined for inhibition of specific antibodies of both IgG and IgE isotypes. Potency evaluation by inhibition assays for IgE- and IgG-binding showed a significant correlation for the points of 50% inhibition (r = 0.92, p = 0.0001), but not for the slopes of the inhibition curves, i.e. the respective antibody avidities. Evidence is provided that the strict relationship between IgE- and IgG-inhibition by allergens could not be explained by possible cross-contaminations of the anti IgE- or anti IgG-reagents employed in the immunoassays. It is concluded that the inhibition of IgG-antibody binding presents a fast, reliable and low-cost alternative for the potency control of allergens used in clinical practice.

Allergens↗

PubMedDate not supplied

The role of IgG in type-I allergy: an unsolved problem.

The role of antiallergen IgG antibodies in allergy remains unclear. In this review we present evidence for and against the hypothesis that IgG (IgG4) could act as a sensitizing antibody. After considering the available data, we conclude that the possible sensitizing ability of IgG4 may depend on the nature and origin of the antibodies and cells used in the various experiments and/or is related to the differential induction of two subtypes of IgG4 (anaphylactic vs. blocking). It still remains to be settled whether the observed increase in specific IgG during immunotherapy is causally related to the relief of symptoms, or whether it merely represents an epiphenomenon due to high antigen exposure. Furthermore, the presence of IgG anti-IgE antibodies in the serum of allergic patients and their possible pathophysiological relevance is discussed in the light of recent evidence suggesting that the raised levels of these antibodies may likewise represent an epiphenomenon of the immune response. There exists a tight relationship between antiallergen IgE and IgG antibodies. This correlation has led to the suggestion that IgG determinations might be useful for diagnostic purposes. There also exists a good correlation between these antiallergen isotypes in inhibition assays. It has therefore been proposed to employ inhibition of IgG binding for the standardization of allergenic extracts. More recent studies explore the relevance of the binding affinities or avidities of allergen-specific IgG antibodies. Antibody affinity may have important repercussions with respect to the biological effects. It has been pointed out that the affinity of specific IgG subclasses in allergic patients depends, among other things, on the nature of the sensitizing allergen. Further studies on IgG binding to the so-called "major" and "minor" allergens may help clarify the role of IgG in allergic disorders.

Animals↗

PubMedDate not supplied

Determinations of Par j 1 by a competitive enzyme immunoassay using human specific IgE and IgG. Validation by skin prick testing.

Par j 1 is the major allergen of Parietaria judaica. The objectives of this study were the following: 1) to purify Par j 1; 2) to develop an enzyme immunoassay based on the bivalent properties of specific IgE and IgG to determine the Par j 1 content in several batches of P. judaica extracts; and, 3) to study the contribution of Par j 1 to the total allergenicity and antigenicity of P. judaica extracts. P. judaica pollen was extracted and subjected to hydrophobic interaction and gel filtration chromatography for the purification of Par j 1. Inhibition enzyme immunoassays, SDS-PAGE and immunoblotting were used to characterize the allergen content. The in vivo biological potency of the extracts was estimated by skin prick testing 26 P. judaica clinically sensitive patients. The new enzyme immunoassay showed a high degree of specificity and sensitivity, detecting from 2 to 100 ng Par j 1/ml. The range of Par j 1 content in nine batches ranged from 23% to 78% of the total protein in the extracts. The Par j 1 content showed a significant correlation with the allergenic potency of these extracts evaluated by specific IgE inhibition and skin prick testing; the correlation with the specific IgG inhibition capacity was not significant. Purified Par j 1 shows great specific IgE and IgG binding capacity; its content can be determined using this newly developed enzyme immunoassay. Par j 1 levels exhibit a significant correlation with the biological potency of the extracts. This method allows the detection of Par j 1 isoforms.

Allergens↗