Heat treatment modifies the allergenicity of beef and bovine serum albumin

The effect of heat on the allergenicity of beef and bovine serum albumin was investigated among 10 toddlers skin prick test (SPT)-positive to raw and cooked beef. The meat-allergy diagnosis was confirmed during double-blind, placebo-controlled food challenge (DBPCFC) with 180 g of beef cooked for 5 min at 100°C. SPT with homogenized and freeze-dried beef, and heated and unheated bovine serum albumin were performed. Both heated and unheated bovine serum albumin, homogenized beef, and freeze-dried beef were used in trial DBPCFC. All children were SPT-positive to unheated bovine serum albumin. Seven were positive to heated bovine serum albumin, one to freeze-dried beef, and none to homogenized beef. DBPCFCs were negative for homogenized beef and freeze-dried beef, positive for unheated bovine serum albumin in five patients, and positive for heated albumin in four children. We conclude that heating reduces sensitization to beef and bovine serum albumin but does not abolish reactivity to albumin under home conditions. However, industrially heat-treated and sterilized homogenized beef and freeze-dried beef may be suitable substitutes in beef-allergic children's diets.     

Characterization of bovine serum albumin epitopes and their role in allergic reactions

Objective:  This review provides updated information on conformational and sequential epitopes identified in bovine serum albumin (BSA) and summarizes available data about the role of structural modifications on BSA antigenicity/allergenicity.
Data sources:  Data on beef allergy and BSA antigenicity are reported, with reference both to the basic literature and to clinical results obtained by our group.
Results and discussion:  BSA is an important allergen involved in milk and beef allergy. The presence of conformational epitopes has been suggested by indirect evidence, while at least one sequential epitope has been experimentally identified. The role of structural modifications on BSA antigenicity is discussed as well as the increased tolerance observed in allergic subjects consuming beef as strained (homogenized) and freeze-dried derivatives.
Conclusion:  Study of the molecular characteristics of a known major allergen allows the identification of technological processes that may be capable of improving the tolerance of allergic subjects to a specific food. Even though any hoped for reduced allergenicity must be verified under medical supervision, the use of new products could obviate the need to avoid important foods such as meat in childhood.     

Immune recognition of serum albumin—XIII. Autoreactivity with rabbit serum albumin of rabbit antibodies against bovine or human serum albumins and autoimmune recognition of rabbit serum albumin

Recently, this laboratory reported an autoreactivity of myoglobin (Mb)3 antisera with the Mb of the species in which the antisera are raised. Also, animals injected with autologous Mb mounted an autoimmune antibody and T-lymphocyte proliferative response against this protein. This posed the possibility that autoimmune recognition might be a general phenomenon not confined only to sequestered proteins such as Mb. Using RSA, we have demonstrated unambiguously that RSA cross-reacted with rabbit antisera to bovine (BSA) or to human (HSA) albumin. In exchange experiments, ¹²⁵I-labelled RSA was bound by the IgG in the immune complexes isolated from rabbit antisera to BSA or HSA. Also, ¹²⁵I-antibodies were bound by RSA-adsorbents. Both binding activities were inhibited specifically by RSA. RSA was isolated from three rabbits and each rabbit was immunized with its own RSA. In each rabbit autoantibodies were found by exchange between immune complexes and the rabbit's own [¹²⁵I]-RSA. Also, ¹²⁵I-antibodies from each rabbit were bound by adsorbents of the rabbit's own RSA. Inhibition studies, data on preimmune sera and on antisera against other proteins confirmed the specificity of the binding. The findings confirm the universality of autoimmune recognition and lend support to our previous suggestion that antigenic sites are ‘structurally-inherent’ in the protein.     

Occupational asthma caused by inhalation of bovine serum albumin powder

Bovine serum albumin (BSA), which is present in bovine plasma, is one of the major allergens affecting patients with food allergies induced by milk and meat. It is also commonly used in research laboratories. Although some reports have documented food allergies associated with BSA, BSA-induced occupational asthma has not been reported. We report a case of occupational asthma and rhinitis in a laboratory worker caused by the inhalation of BSA powder, in which an IgE-mediated response was suggested as the pathogenic mechanism.     

Sodium cromoglycate in the treatment of eosinophilic gastroenteritis

Two patients suffering from eosinophilic gastroenteritis (EG) were treated with sodium cromoglycate (SCG). Before treatment they showed enteric and cutaneous symptoms, such as abdominal pain, nausea, vomiting, diarrhoea and recurrent urticaria and angioedema. The histological findings were a notable amount of eosinophilic infiltration in the lamina propria and gastric glands, a villous shortening and thickening and weak eosinophilic inflammation in the duodenum. The patients were treated with 300 mg SCG, 4 times daily, for 4/5 months. During treatment, the clinical symptoms disappeared and at the end of treatment a reduced inflammation with an almost complete decrease of eosinophilic infiltration was observed. The results provide evidence of SCG efficacy in the treatment of EG and suggest its employment as an alternative to the steroids commonly used in EG.     

Differential binding of IgG and IgA antibodies to antigenic determinants of bovine serum albumin

The aim of this study was to investigate the recognition pattern of bovine serum albumin (BSA), a major dietary protein by serum IgG and IgA antibodies. Anti-BSA IgG and IgA antibodies were measured by ELISA technique in 3 different cohorts: 578 unselected persons, 84 new-onset insulin-dependent diabetes mellitus (IDDM) patients and 103 atopic persons. In order to characterize the recognition pattern of the different BSA domains, recombinant BSA and recombinant fragments covering the 3 BSA domains were produced. BSA digestion was monitored in simulated gastric fluid experiments by means of domain specific monoclonal antibodies. IgG and IgA antibody titres to native BSA were highest in IDDM patients. The three major BSA domains were equally well recognized by IgG antibodies of the three cohorts. Interestingly all three study groups showed a dissociation of their IgG and IgA antibody response to the first BSA domain. The ratio of IgG to IgA antibodies recognizing this domain was 93%/42% in controls, 92%/37% in IDDM patients and 80%/47% in atopic persons. In simulated gastric fluid experiments, the first BSA domain was the first to become undetectable to specific monoclonal antibodies during digestion. In conclusion humoral IgG and IgA antibodies recognize the major BSA domains with different frequencies. The N-terminal domain of BSA, the first to be degraded during simulated gastric digestion is less well recognized by IgA antibodies. This suggests that early digestion is negatively correlated to the IgA antibody response and that the IgA response associated to the gut associated lymphoid tissue (GALT) and the systemic IgG antibody responses are independent.     

Synthesis of the antigen bovine serum albumin‐ergosterol and its immunocharacterization

Fungal contamination of agricultural commodities leads to their spoilage and renders them unfit for human consumption. Ergosterol, a predominant sterol in most fungi and a major constituent of the cell membrane, has been established as a reliable biochemical marker for fungal growth. Various chemical and physico‐chemical methods to quantify ergosterol as an index of fungal contamination are in practice. Yet, immunoassays are the methods of choice in food analysis due to their increased specificity, sensitivity and rapidity. This paper reports the synthesis of an antigen, bovine serum albumin‐ergosterol conjugate, and its immunocharacterization. Ergosterol was converted to ergosterol hemisuccinate (EHS) by succinylation. Subsequently, EHS was conjugated to bovine serum albumin by the mixed anhydride reaction. The molar ratio was found to be 1:28. Antisera raised against the synthesized antigen in rabbits was characterized by the Ouchterlony double‐diffusion technique and an antibody capture assay. Ouchterlony analysis showed a titre of 1:2. Further, characterization by an antibody capture assay, using 50 ng well (10 ng equivalent of ergosterol) of the antigen, gave a titre of 1:4000 dilution of antiserum, with an absorbance of 1.0 at 405 nm. The synthesized antigen may find an application in the development of an immunoanalytical method for ergosterol quantification as a measure of food quality in relation to fungal contamination.     

Immune recognition of serum albumin--XII. Evidence for time-dependent immunochemical cross-reactivity of subdomains 3, 6 and 9 of bovine serum albumin by quantitative immunoadsorbent titration studies

Antisera were raised in rabbits against bovine serum albumin (BSA) or against fragment 377–571 (domain 3) of BSA. Serial bleedings were obtained at different times after immunization. Fragments 115–184, 307–385 and 504–581 were isolated in pure form from BSA. These corresponded essentially to subdomains 3, 6 and 9 respectively of BSA. The capacity of each fragment to bind ¹²⁵I-labelled antibodies of rabbit anti-BSA or anti-domain 3 antisera was determined by a quantitative immunoadsorbent titration technique. With anti-BSA antisera from each rabbit, the maximum (plateau) amount of antibodies bound by each fragment was found to increase with time antisera are obtained after the first immunization. With antisera against domain 3, considerable amounts of antibodies were bound by subdomains 3, 6 and 9 and the fraction of cross-reacting antibodies also increased with time after the initial immunization. Antibodies against each subdomain fragment were isolated on fragment adsorbents and were investigated for their cross-reaction with the other two fragments. Specific antibodies against each subdomain were found to cross-react with the other two subdomains. The degree of cross-reaction increased remarkably with the duration of immunization and reached as high as 100% for the cross-reaction between subdomains 3 and 6. The results are consistent with an unusual functional equivalence of antigenic sites within the same protein molecule. This functional equivalence of the antigenic sites of albumin, which we had previously proposed, improves with time after immunization.     

Allergenic and antigenic properties of bovine serum albumin

The antigenic sites on bovine serum albumin were studied utilizing peptic and tryptic fragments of the molecule. Rabbits were immunized with small doses of bovine serum albumin and their serum antibody response measured by a bincling assay and the IgE response by antigen-induced histamine release from their basophils. The basophils from two bovine serum albumin allergic individuals were also used for histamine release studies. Serum antibodies bound large fragments of bovine serum albumin; these fragments also induced histamine release from basophils. Although about half of the antibody bincling activity was recovered in the two halves of the albumin molecule only about 10% of the histamine releasing activity was present in the same fragments. The loss of activity of the bovine serum albumin molecule on proteolytic cleavage into two halves could be due to the breakup of the molecule in the middle of the third domain with loss of antigenic sites and/or due to minor conformational changes in the fragments as compared to the intact molecule. Large fragments of bovine serum albumin induced basophil histamine release, thus demonstrating the presence of at least two antigenic determinants on each of these peptides. This data therefore suggests the presence of at least four antigenic IgE-bincling sites on the bovine serum albumin molecule. By basophil desensitization experiments unique IgE-reactive antigenic sites were demonstrated on each half of the molecule; however, some of the sites on the COOH-terminal half cross-reacted with antibodies directed towards the NH₂-terminal part of the molecule. The IgE-response of rabbits to bovine serum albumin was specific; there was no cross-reactivity with rat or mouse albumin. The present finclings indicate a substantial loss in the IgE-reactive determinants of bovine serum albumin by cleavage into large fragments.     

IgE against ethylene oxide-altered human serum albumin in patients with anaphylactic reactions to dialysis

We have measured total antibody and IgE directed against ethylene oxide-altered human serum albumin (ETO-HSA) in the sera of 24 patients who have experienced anaphylaxis during hemodialysis and of 41 patients who have not had such episodes during hemodialysis. ETO is used to sterilize dialyzers and other medical equipment. The geometric mean level of IgE to ETO-HSA in patients with reactions (0.9 ng ETO-HSA bound to IgE per milliliter of serum) is significantly higher than in nonreacting patients (0.1 ng/ml, p less than 0.0001). Sixteen of 24 patients with reactions had detectable levels of IgE to ETO-HSA, whereas only three of 41 nonreacting patients had detectable levels (p less than 0.0001 chi-square). The geometric mean level of total antibody to ETO-HSA is also significantly higher in patients with reactions (270 ng ETO-HSA bound per milliliter) than in nonreacting patients (31 ng/ml, p less than 0.0001). Fourteen of 24 patients with reactions but only four of 39 nonreacting patients had total antibody binding of ETO-HSA (p less than 0.0001 chi-square). These data extend our previous observations on a small group of 13 patients receiving hemodialysis (seven patients with reactions, and six nonreacting patients) and clearly demonstrate an association between the presence of IgE or total antibody to ETO-HSA and immediate anaphylactic reactions in this group of 65 patients receiving hemodialysis.     

Influence of total serum IgE levels on the in vitro detection of β-lactams-specific IgE antibodies

Background Allergic reactions to β-lactams are a frequent cause of adverse drug reactions; the diagnosis is based on history, clinical examination, skin testing (prick and intradermal) and demonstration of serum-specific IgE antibodies (Abs).
Objective We compared the diagnostic performance of the Phadia CAP system for the detection of IgE to β-lactams carried out using the new test with cut-off limits of 0.10 kUA/L and the old test with cut-off limits of 0.35 kUA/L for positive results; subsequently, we analysed the effect of total serum IgE values and of atopic phenotype on the diagnostic performance of the tests.
Methods The study comprised a total of 34 patients with a history of immediate adverse reactions to β-lactams, which were confirmed by positive skin testing, and 115 control subjects with tolerance to β-lactams over the last year. The Phadia CAP System was used for the determination of serum total and specific IgE Abs towards penicilloyl G (c1), penicilloyl V (c2), ampicilloyl (c5) and amoxicilloyl (c6). The overall diagnostic performance was assessed as a diagnostic odds ratio (DOR).
Results The new test showed a higher sensitivity (85% vs. 44%) than the old test and a lower specificity (54% vs. 80%) but the overall diagnostic performance was poor (DOR 6.78 vs. 3.16, P =0.333) in both tests. The total IgE value influences the DOR of both tests; DOR was better for values under 200 kU/L [DOR=66; 95% confidence interval (CI): 11.3–384.1] or 500 kU/L (DOR=45.7; 95% CI: 5.3–394.4) for the new and old tests, respectively.
Conclusions The reduction in the positive cut off value has not significantly improved the overall diagnostic performance of the β-lactams-specific IgE assay. Because of the influence of serum total IgE on the detection of β-lactam-specific IgE Abs, the combination of both tests is mandatory in the in vitro diagnostic approach of β-lactam allergy.     

"Latex-fruit syndrome": frequency of cross-reacting IgE antibodies

An association between allergies to latex proteins and to various foods has been reported and confirmed by RAST and immunoblotting inhibition. However, no significant data had been collected on the frequency of specific IgE antibodies to fruits in these patients and the frequency of a history of fruit intolerance. Serum samples of 136 patients with well-documented, clinically relevant, immediate-type hypersensitivity against latex proteins were analyzed for IgE antibodies against a panel of different fruits. Patient history of food intolerance was documented by a standardized questionnaire. Fruit-specific IgE antibodies were detected in 69.1% of serum samples. Cross-reacting IgE antibodies recognizing latex and fruit allergens (papaya, avocado, banana, chestnut, passion fruit, fig, melon, mango, kiwi, pineapple, peach, and tomato) were demonstrated by RAST-inhibition tests. Of our patients, 42.6% reported allergic symptoms after ingestion of these fruits and a total of 112 intolerance reactions were recorded. However, fruit-specific IgE antibodies were detected only in serum samples from 32.1% of the patients who perceived symptoms due to these fruits. Thus, serologic tests seem to be of low significance for prediction of food allergy in latex-allergic patients.     

Decoration of heparin and bovine serum albumin on polysulfone membrane assisted via polydopamine strategy for hemodialysis

Renal failure brings about abnormality of waste and toxins and deposition in the body. In clinic, the waste and toxins in vitro are eliminated by hemodialysis device with polysulfone (PSF) porous membranes. In the work, decoration of heparin (Hep) and bovine serum albumin (BSA) on PSF membranes would be beneficial to improve the hemocompatibility and reduce the anaphylatoxin formation during hemodialysis. The PSF porous membranes are surface-modified by simply dipping them into dopamine aqueous solution for 8 h. Then, Hep and BSA are immobilized covalently onto the resultant membrane. Attenuated total reflectance Fourier transform infrared spectra (ATR-FTIR) confirms that Hep and BSA are successfully introduced onto the surface of PSF membranes. Scanning electronic microscopy (SEM) and atomic force microscopy (AFM) display the changes of surface morphologies after modification. The result of water contact angle measurement shows that the hydrophilicity of PSF membranes is remarkably improved after coating polydopamine (pDA) and binding Hep and BSA. The experiments of hemocompatibility indicate that Hep and BSA grafted onto membranes suppress the adhesion of platelet and enhance the anticoagulation ability of PSF membranes. Furthermore, the protein adsorption tests reveal that Hep and BSA immobilized onto membranes depress the protein absorption and develop antifouling-protein ability of pristine membrane. This study proves a convenient and simple approach to graft two functional organic polymers which, respectively, play a vital role and then improve the hemocompatibility and biocompatibility of PSF membranes for their biomedical and blood-contacting applications.     

Effects of bovine serum albumin on antibody determination by the enzyme-linked immunosorbent assay

The effect of bovine serum albumin (BSA) post-coating and addition of BSA to serum diluent was studied in an IgG and IgA Candida antibody enzyme-linked immunosorbent assay (ELISA). BSA post-coating decreased the background readings and increased the specific Candida antibody activity in most sera. However, in a few sera post-coating alone increased background readings and this effect was probably due to the occurrence of BSA antibodies. It could be abolished by the addition of BSA to serum diluent. It is therefore suggested that BSA post-coating should only be used in combination with BSA addition to serum dilution buffer in ELISA.     

Low temperature incorporation of bovine serum albumin into a bead formed macroporous hydrophilic polymer matrix with potential for sustained release

A process of freeze-thaw polymerization involving the low temperature photopolymerization of a mixed solution of monomers and bovine serum albumin around frozen ice crystals has been used to generate a bead formed macroporous hydrophilic matrix with potential for sustained release. Beads over the size range 100-3000 μm were fabricated with surface and internal pores of between 0.7-2.6 μm whose diameter could be controlled by manipulation of the monomers to solvent ratio. Increasing both the proportion of monomers in the monomer solution and the percentage of BSA incorporated reduced the EWC of beads. The BSA release profile was characterized by an initial burst followed by a lower but sustained release lasting up to 1 month. The total cumulative release of BSA and the proportion of the incorporated BSA load subsequently released were both reduced in physiological saline compared with distilled water but enhanced by freeze drying, mild agitation and incubation at 37°C.     

Haematological characteristics of Catla catla against the treatment of bovine serum albumin

The haematological characteristics of Catla catla were investigated against the treatment of bovine serum albumin. A significant increase in haemoglobin, mean cell haemoglobin and mean cell haemoglobin concentration were found in both sexes, indicating a marked immune response by the host system. Factors that effect the haematological parameters of fish are discussed.