Immunodominant semen proteins II: contribution of seminal proteins to female immune infertility.

Seminal fluid is a protective medium for sperm, but it also represents potential immunogenic structures for the female immune system. Anti-seminal antibodies may threaten early fertilization. The aim of our work is to detect and identify seminal proteins that are related to female isoimmunization. In this report, we quantified serum anti-seminal IgG antibodies. Seminal proteins were analysed by two-dimensional gel electrophoresis followed by immunoblotting. To identify IgG-binding proteins of interest, a proteomic approach was selected. The dominant seminal antigens were detected within the relative molecular mass ranging from 25 to 85 kDa and the isoelectric point from 5 to 7. The detected proteins were further identified as prostate-specific antigen, prostatic acid phosphatase, zinc-α-2-glycoprotein and zinc finger protein 778. Since these proteins were recognized by IgGs produced by infertile women and not by fertile women, we presume that major seminal antigens may play an important role in the pathogenesis of female immune infertility. Our study suggests the pattern of seminal proteins for further therapeutic attempts in the diagnosis of female immune infertility.

Characterization of peptidic and carbohydrate cross-reactive determinants in pollen polysensitization.

BACKGROUND: Cross-reactivity may be due to protein sequence or domain homologies and/or the existence of cross-reactive carbohydrate determinants (CCDs). The clinical relevance of peptidic cross-reactivities is well known, whereas that of CCDs is still a question of debate. The aim of this study is to characterize the IgE specificity of various patients suffering from pollen polysensitization to identify both peptidic and carbohydrate cross-reactive determinants. MATERIAL AND METHODS: Rapeseed, grass and Arabidopsis proteins were separated by isoelectric focusing, followed by SDS-PAGE, and transferred to a nitrocellulose sheet. The sheets were incubated either with an individual serum from a birch+grass-sensitive patient, followed by anti-human IgE, or with labelled Concanavalin A (ConA). Binding inhibition was tested by incubation of the sera with a mixture of sugar residues. RESULTS: The results showed two different patterns of cross-reacting sera: a pattern that implies few proteins, not always glycosylated and known as allergens, and a pattern that implies numerous proteins with molecular masses over 30 kDa. This second pattern was very close to the ConA -binding pattern. The IgE binding was abolished by pre-incubation with sugar residues only in the case of the second pattern. DISCUSSION: This study shows that multiple pollen sensitizations could result from multiple sensitizations to specific proteins or from a cross-sensitization to a wide range of glycoproteins. Two-D blots allow to characterize a cross-sensitization due to carbohydrate determinants, and thus to improve the diagnosis of allergy and its medical treatment.

Allergy to bovine beta-lactoglobulin: specificity of human IgE to tryptic peptides.

BACKGROUND: Bovine beta-lactoglobulin (Blg) is a major cow's milk allergen. It is the main whey protein, without any counterpart in human milk. Blg chemical hydrolysates appeared to retain most of the immunoreactivity of the native protein. Allergenicity of Blg has already been shown to be associated with the four peptides derived from cyanogen bromide cleavage of Blg. OBJECTIVES: To map the major allergenic epitopes (e.g. regions of the molecule able to bind IgE) on Blg using specific IgE from sera of 46 milk-allergic patients as a probe. METHODS: Direct and competitive inhibition enzyme immunoassays involving immobilized native protein or purified peptides derived from Blg tryptic cleavage. RESULTS: Several peptides capable of specifically binding human IgEs were identified and were classified according to the intensity and frequency of the responses. The major epitopes appeared to be fragments (41-60), (102-124) and (149-162) recognized by 92, 97 and 89% of sera, respectively, whilst a second group which contained the fragments (1-8) and (25-40) was recognized by 58 and 72% of the population. A third group, comprising peptides (9-14), (84-91) and (92-100), was still detected by more than 40% of sera. CONCLUSION: Three peptides were identified as major epitopes, recognized by a large majority of human IgE antibodies. Numerous other epitopes are scattered all along the Blg sequence.

Allergy to bovine beta-lactoglobulin: specificity of human IgE using cyanogen bromide-derived peptides.

BACKGROUND: Bovine beta-Lactoglobulin (Blg) is a major allergen involved in allergy to cow's milk proteins. Hydrolyzing Blg did not totally suppress its allergenicity; moreover its immunoreactivity may be increased. The aim of this work was to evaluate the specificity of serum IgE to different fragments of Blg in a group of 19 individuals allergic to cow's milk. METHODS: This study was performed using both direct and competitive inhibition ELISA involving immobilized native protein or peptides derived from Blg cyanogen bromide cleavage. RESULTS: Analyses of responses to each peptide revealed a large number of epitopes recognized by specific IgE of human allergic sera. However, there were differences in the specific determinants recognized, depending on the serum. Generally, peptides (25-107) and (108-145) retained substantial proportions of the immunoreactivity of the whole protein. Two other peptides, i.e. (8-24) and (146-162), were less recognized but were not inert. CONCLUSION: The main conclusion is that many epitopes were identified all along the Blg sequence by specific anti-Blg IgE from allergic humans.

Monoclonal anti-human IgE: histamine release capacity and effect on in vitro sensitization of human basophils.

IgE-anti-IgE complexes were formed by preincubation of a human myeloma IgE with a monoclonal anti-human IgE antibody at different concentrations. Binding of IgE onto the anti-IgE inhibited the histamine release capacity of anti-IgE from basophils. The IgE cell-binding capacity was altered by the IgE/anti-IgE ratio in the preincubation buffer. Heating of the complexes gave a partial recovery of the anti-IgE degranulation capacity, suggesting that the monoclonal anti-IgE is specific for an epitope present on the D epsilon 2 domain.

Biological effect of transferrin on mast cell mediator release during the passive cutaneous anaphylaxis reaction: a possible inhibition mechanism involving iron.

A purification procedure for passive cutaneous anaphylaxis inhibitory factor from BALB/c mouse serum has been previously described. In the present work, this inhibitory activity was found to be related to transferrin. No activity was obtained using iron-unsaturated transferrin, whereas iron-saturated transferrin appeared to be potent. The in vivo inhibition of IgE-dependent mediator release from rat mast cells was also obtained using free iron. This effect was observed when iron was injected prior to or simultaneously with mouse IgE in rat skin. Iron and iron-saturated transferrin could play a role in the mechanism of desensitization by modulating the responsiveness of mast cells.

Rapid visual detection of Escherichia coli and Vibrio cholerae Heat-labile enterotoxins by nitrocellulose enzyme-linked immunosorbent assay.

A modification of the enzyme-linked immunosorbent assay for a sensitive and rapid visual detection of heat-labile enterotoxins from Escherichia coli and Vibrio cholerae is described. Small amounts of bacterial supernatant fluids are bound to nitrocellulose filters which are used as sorbents in the nitrocellulose enzyme-linked immunosorbent assay. The test is based on the immunological similarity between V. cholerae and E. coli heat-labile enterotoxins. Six isolates of V. cholerae and 48 isolates of E. coli were examined for heat-labile enterotoxins by the nitrocellulose enzyme-linked immunosorbent assay and the Vero cell bioassay. With some strains, the nitrocellulose enzyme-linked immunosorbent assay was found to be more sensitive for detection of E. coli heat-labile enterotoxin than the Vero cell test. A similar result was obtained by endpoint titration of heat-labile enterotoxin-positive E. coli H10407 culture fluid in both assays. The sensitivity of the nitrocellulose enzyme-linked immunosorbent assay for the detection of purified cholera toxin was at a total level of 1 ng, which is a good result when compared with other serological assays.