An oral screening procedure to determine the sensitizing capacity of infant feeding formulae

Allergy to cows' milk is an important and frequent cause of cows' milk protein-intolerance in infants. Many infants within days of birth are fed cows' milk or cows' milk-based formula feeds. It is thought desirable to have a test available that would indicate the allergenic potency per os of milk formulae and of milk substitutes intended for infant feeding. Such a test, based on the finding that guinea-pigs drinking cows' milk become anaphylactically sensitized to milk proteins, is described. After drinking pasteurized milk for only 4 days, 88% of guinea-pigs were susceptible to fatal anaphylaxis on intravenous challenge on the 22nd day; drinking milk for 8 days resulted in 100% of guinea-pigs becoming sensitive to fatal anaphylaxis. Following milk drinking for 7 days, a subsequent interval of 7 days was required before anaphylactic sensitivity could be shown in 100% of animals. With this information the effect of heat on the per os allergenic potency of cow's milk could be examined; likewise the allergenic potency of supposedly hypo-allergenic infant feed formulations. Pregestimil induced no anaphylactic sensitivity even after 37 days of drinking, and no anaphylactic antibodies, detectable by PCA, were formed to the milk proteins β-lactoglobulin and casein. In contrast, AL110 likewise drunk by guinea-pigs for 37 days resulted in 90% fatal anaphylactic reactions in animals challenged with the same product. Anaphylactic antibodies were found to casein but not to β lactoglobulin. Comminuted Chicken and Prosobee (a soya milk), two alternative ‘hypo-allergenic’ cows' milk substitutes, rendered approximately 50% guinea-pigs anaphylactically sensitive to the product fed, but neither product stimulated cross-reactive anaphylactic antibodies to either casein or β-lactoglobulin as tested by PCA. There is some indirect evidence that results of the guinea-pig test are relevant to responses in infants with cows' milk protein intolerance.     

IgE Cross-reactivity with Caseins from Different Species in Humans Allergic to Cow's Milk

Fifty-eight sera from humans allergic to cow's milk proteins were analysed for the specificity of their IgE response to the whole casein fraction of milk from different ruminant and nonruminant species (e.g. cow, sheep, goat, rabbit and rat). IgE-specific responses were determined by an enzyme allergosorbent test using the purified casein fractions as immobilized antigen and an anti-human IgE monoclonal antibody labelled with acetylcholinesterase. Co-and/or cross-sensitizations to caseins of the different ruminant species occurred extensively, though IgE responses to ovine and caprine casein appeared to be lower than that obtained with bovine casein. Cross-reactivity is suggested by the significant reactivity of rat and rabbit casein toward human IgE. In terms of specificity and intensity, the IgE response to caseins demonstrates a great variability. Structural homologies in caseins of such different species, that can share common epitopes for the IgE of some patients, suggest that prevention of cow's milk allergy cannot be achieved by using milk from other species as substitutes.     

Natural history of cows' milk allergy in children: immunological outcome over 2 years

In this investigation 98 children (median age 24 months) with cows' milk allergy (CMA) were studied over a median period of 2 years to see whether acquisition of clinical tolerance to cows' milk was associated with the changes in levels of IgG and IgE anti-cows' milk antibodies, and skin test reactivity to a cows' milk extract. Two groups of CMA patients were examined. The first were IgE sensitized and responded rapidly to small volumes of cows' milk with urticaria, and/or exacerbations of eczema, and/or wheeze, and/or vomiting (n= 69). The second, a late reacting group (n= 29) demonstrated coughing, diarrhoea, eczematoid rashes, and/or a combination of these which developed more than 20 hr after commencing normal volumes of cows' milk. Significant immunological changes were confined to the 69 IgE sensitized immediate-reacting-group of patients. Of these, there were 15 children who achieved clinical tolerance to cows' milk and they showed a significant fall in the levels of skin test reactivity to cows' milk over the study period (P <0.01). In addition, these 15 children had lower serum IgE antibodies to cows' milk proteins both at the outset and the final follow-up compared with the 54 patients whose CMA persisted. No consistent change in the IgG antibody responses to cows' milk proteins was seen in either group of patients over the study period. The findings suggest patients with immediate type hypersensitivity to cows' milk proteins whose disease persists for more than 2 years have a more severe dysregulation of IgE synthesis to cows' milk proteins from the outset. The role of humoral immune mechanisms in the pathogenesis of late reacting CMA remains unclear.     

Bovine β-lactoglobulin levels in hydrolysed protein formulas for infant feeding

A bovine milk protein, β-lactoglobulin (βLG), was measured by an enzyme-linked immunosorbent assay (ELISA) in seven different infant formulas based on protein hydrolysates from cows' milk whey or casein, and from bovine collagen and soy. βLG levels in the formulas were 1/100 to 1/4800 000 lower than in cows' milk (CM). There was a great difference in the βLG level between the partly and the extensively hydrolysed formulas; the amount of βLG was 40 000-fold higher in the partial hydrolysates vs the extensively hydrolysed formulas. Residual βLG may have been responsible for the allergic reactions described in some children with cows' milk allergy (CMA) receiving these formulas.     

Bovine milk, including pasteurised milk, contains antibodies directed against allergens of clinical importance to man.

Pasteurised and raw bovine milk and bovine colostrum samples were assayed by enzyme-linked immunoassay for the presence of antibodies directed against a selection of allergens of importance in human atopic disease. Samples were tested for the presence of antibodies directed against or cross-reacting with ryegrass pollen, house dust mites, Aspergillus mould and wheat proteins. Antibodies of each specificity were detected in every sample tested, including all samples of commercial pasteurised milk. The results are discussed with reference to a hypothesis that dietary xenogeneic antibodies may play a role in the emergence of some human atopic disease, and the recent demonstration that oral immunisation with xenogeneic antibodies may lead to the production of anti-immunoglobulin antibodies including anti-idiotypic antibodies.     

Allergen-Specific IgE Antibodies against Antigenic Components in Cow Milk and Milk Substitutes

Crossed radioimmunoelectrophoresis (CRIE) was used to study the presence of serum IgE against antigenic components of cow milk in 21 selected milk-allergic patients. The amount of each IgE specificity was estimated by a scoring system. The milk-allergic children had mainly IgE against alpha-lactalbumin, beta-lactoglobulin, albumin and immunoglobulin, the four major proteins of bovine whey, as well as IgE against three casein components. A serum pool from 1000 normal adults had IgE against the same whey protein, but in smaller amounts, and no IgE against the casein components. Eight cow milk-based formulae, commonly used for infant feeding, and goat milk were studied by the same method. It was found that six of the milk substitutes did not differ significantly from cow milk in antibody binding, but the two hydrolysed casein products, Nutramigen and Pregestimil, consisted of such small molecules that the rabbit antisera could not precipitate the hydrolysed proteins in the gels on the CRIE plates. It was therefore not possible to study their IgE binding, if any, by this method.     

Development of a One Step Strip Test for the Detection of (Dihydro)streptomycin Residues in Raw Milk

The one step strip test described is a competitive immunoassay in which the detector reagent consists of colloidal gold particles coated with affinity purified monoclonal anti(dihydro)streptomycin antibodies. The capture reagent in the assay is a streptomycin- bovine serum albumin conjugate which is immobilised on the lateral flow membrane of the test device. In the test procedure, three drops of raw milk are brought into the sample well of the test device and allowed to migrate over the membrane. The more analyte present in the sample, the more effectively it will compete with the streptomycin immobilised on the membrane for binding to the limited amount of antibodies of the detector reagent. A sufficient amount of (dihydro)streptomycin in the sample will thus prevent the binding of the detector reagent to the streptomycin immobilised on the membrane, resulting in disappearance of the test line in the read out zone. Using raw milk samples, spiked with either streptomycin or dihydrostreptomycin, complete disappearance of the test line was obtained with 160 ng ml^-1 and 190 ng ml^-1, respectively. This demonstrates that the test is applicable for screening raw milk samples for the presence of (dihydro)streptomycin residues at MRL level (EU: 0.2 mg (dihydro)streptomycin kg^-1 milk). The major advantages of the one step strip test are that results can be obtained within 10 min and that all reagents are included in the test device.     

Human serum antibodies reactive with dietary proteins. Antigenic specificity

The antigenic specificity of human serum IgG antibodies reactive with common dietary proteins has been evaluated by competitive binding using a solid-phase immunoassay (ELISA). Antibodies reactive with bovine milk antigens were shown to be reactive predominantly with casein, rather than alpha-lactalbumin, beta-lactoglobulin, gamma-globulin or albumin. Furthermore, sera containing antibodies reactive with bovine casein, wheat gliadin and chicken ovalbumin showed competitive binding only by each respective dietary protein antigen. IgG4 antibodies specifically reactive with ovalbumin, gliadin or casein were also not cross-reactive in competitive binding studies. Furthermore, both IgG2 and IgG4 anti-milk antibodies showed significant inhibition only with bovine casein, and not with alpha-lactalbumin or beta-lactoglobulin. These data are relevant to concepts regarding the immunobiological role of antibodies of the IgG4 isotype.     

In vitro allergenicity of cows' milk substitutes

There are numerous alternatives to cows' milk formula for allergic children. We have investigated the allergenicity of several of these using RAST and RAST inhibition on serum from 16 patients with a known history of cows' milk protein intolerance (CMPI) and 16 atopic controls. A RAST grade of ≥3 for cows' milk was present in all those with CMPI, whilst all the controls gave RAST of ≤1 - Modified cows' milk formula, goats' infant formula, sheep and goats' milk produced similar results to cows' milk. Only two patients had RAST ≥3 for soya milk and the soy/beef hydrolysate gave positive results in only three patients. One had positive RAST to Nutramigen and two to Pregestimil. Of the whey hydrolysates investigated, Pepti-junior gave seven positive RASTs whilst we were unable to hind Alfare to the sepharose in sufficient quantities to interpret the results which were negative in all cases. RAST inhibition data on pooled sera from the same patients agreed with the RAST results. The inhibition curves showed high inhibition with goats', sheep, modified cows' milk formula and the casein formula. AL110 (50%). Soy and soy/beef hydrolysate showed a much lower inhibition pattern. Casein hydrolysates showed low inhibition while the whey hydrolysate produced higher inhibition. We have shown that despite claims of low allergenicity. some of these alternative formulae are antigenically recognized in vitro by some cows' milk intolerant patients.     

Identification of Activin A and Follistatin in Human Milk

Activin A is a dimeric protein member of the transforming growth factor- &#103 (TGF- &#103 ) family: it is synthesized by a variety of organs and follistatin is an activin-binding protein. A sensitive and specific assays for bioactive dimeric activin A and follistatin have recently allowed to measure these proteins in blood and other biological fluids, giving a new insights into their possible physiological role. Since human breast is able to produce activin A, the aim of the present study was to evaluate whether it and follistatin are measurable in breast milk of women during lactation. Concentrations of activin A and follistatin were measured in milk samples collected at 3, 5 and 30 days after delivery by using specific and sensitive two-site ELISAs. For the first time the presence of immunoreactive activin A and follistatin in human milk has been shown; no significant different concentration between the third and the fifth day after delivery was found. Furthermore, no difference of activin A and follistatin concentration between the whole and the skim milk or between spontaneous delivery and cesarean section was found. Milk activin A and follistatin concentrations after 1 month of lactation were significantly decreased (P <0.01). Activin A and follistatin are present in human milk in high concentrations in the first week of lactation, while decrease after a month suggesting a possible role as growth factors in human milk.     

Identification of casein as the major allergenic and antigenic protein of cow's milk

The objective of this study was to analyze both the allergenicity and immunogenicity of cow's milk proteins. To this end, 80 milk-atopic patients were selected on the basis of the presence of cow's milk-specific IgE antibodies in serum and compatible clinical history. Fifteen patients allergic to other allergens and 10 nonatopic subjects were studied as controls. The specificity of serum IgG and IgE antibodies was determined by immunoblotting, employing both cow's milk and milk components, i.e., α- and β-casein, β-lactoglobulin, and α-lactalbumin separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The experiments showed that casein-specific IgE antibodies were present in all (80/80) sera examined; 10/80 showed reactivity to α-lactoglobulin, and 5/80 showed reactivity to α-lactalbumin. None of the 25 negative control sera analyzed showed the presence of specific IgE antibodies against milk proteins. These results were similar to those corresponding to the detection, by the radioallergosorbent test, of IgE antibodies against the milk components coupled to paper disks. All sera from milk-atopic patients also showed IgE reactivity against a high-molecular-mass fraction that hardly enters the gel. This fraction, after separation by gel filtration and treatment with β-mercaptoethanol and urea, was shown by SDS-PAGE analysis to be formed by casein monomers. All sera analyzed by immunoblotting reacted against the components corresponding to casein monomers. Inhibition of immunoblotting by adsorption with different milk components confirmed that those high-molecular-mass aggregates are formed by casein components. The results presented here strongly suggest that casein is the major allergenic component of cow's milk.     

The Level of Antibodies to the Proteins of Cow's Milk in the Serum of Normal Human Infants

The coated tanned red cell technique has been used to measure the level of serum antibody in normal infants to the proteins of cow's milk.

In 286 sera of infants between the ages of 7 and 97 weeks titres up to 1000 were found with the mode around 64. This distribution was quite different from that found with adult and infant cord sera. Antibodies specific for casein, α-lactalbumin and bovine plasma albumin were shown to be present but none could be shown to β-lactoglobulin.

The object of the study was to get evidence that some normal infants were sufficiently sensitized to cow's milk proteins to support the hypothesis that sudden `cot death' in infants is due to an anaphylactic type of reaction consequent on inhalation of cow's milk.

     

Casein is an essential cofactor in autoantibody reactivity directed against the C-terminal SmD1 peptide AA 83-119 in systemic lupus erythematosus

The C-terminal peptide SmD1(83-119) has been identified as an important autoantigen in systemic lupus erythematosus (SLE). ELISA studies have shown that roughly 70% of all sera from patients with SLE react with this peptide. Previous findings revealed that the addition of blocking agents and sample dilution buffers influences the discrimination between positive and negative anti-SmD1(83-119) sera in SLE. The aim of the present study was to identify possible cofactors in the anti-SmD1(83-119) reactivity. We therefore tested SLE sera (n=6) for anti-SmD1(83-119) reactivity by ELISA and analysed the effects of different blocking agents (1% skim milk, 1% gelatin, and 1% BSA). In our investigation, lipids were extracted from skim milk using dichlomethane, and the putative fraction was tested to assess the assay's ability to discriminate between positive and negative sera. The effects of enzymatic digestion by casein were analyzed, and different concentrations of casein were used to determine the role of this protein in the detection of anti-SmD1(83-119) antibodies by ELISA. Furthermore, rabbits were immunized with SmD1(83-119) adsorbed to casein and control proteins. One percent skim milk was the most effective blocking agent and sample dilution buffer for the discrimination between positive and negative sera. As demonstrated by SDS electrophoresis, the discriminative capacity was influenced by enzymatic digestion of skim milk proteins, but not by lipid extraction. Differences in anti-SmD1(83-119) reactivity upon variation of the casein concentration suggest that the protein plays a significant role in the detection of anti-SmD1(83-119) antibodies. However, our immunisation studies did not show any effect of casein on anti-SmD1(83-119) reactivity, suggesting that it has no immunogenic effect on the anti-SmD1(83-119) response. In conclusion, casein seems to be an important cofactor in autoantibody reactivity directed against the C-terminal SmD1(83-119) peptide and probably functions by changing the conformation of the peptide's critical epitope.     

Reduction of the Antigenicity of Whey Proteins by Lactic Acid Fermentation

Lactic acid bacteria (399 meso- and thermophilic strains or mixed cultures) were screened for their ability to reduce the antigenicity of bovine alpha -lactalbumin ( alpha -la) and beta -lactoglobulin ( beta -lg). Residual antigenicity of these proteins in whey from the fermented milks was determined by indirect ELISA and competitive ELISA for alpha -la and beta -lg, using rabbit polyclonal antibodies. The antigenicity was related to allergenicity, as determined by skintesting with selected samples of whey from the fermented milks. The antigenicity of whey proteins was found to be reduced by over 99% as compared to raw milk after the lactic acid fermentation of sterilized cow's milk. The allergenicity of alpha -la and beta -lg was not, however, eliminated, being only slightly attenuated.     

Antigenic stimulation with goat and cow milk by oral and parenteral route in guinea pigs

The aim of this study was to examine the sensitisation capacity of goat milk (GM) and cow milk (CM) by the oral or the parenteral route, and thus determine whether the serologic responses developed are different, identify the proteins targeted by the antibodies thus formed, and determine whether these antibodies depend on the immunisation route employed. Enzyme-linked immunosorbent assay was used to determine the specific antibodies of the isotypes IgG1 and IgG(Fc) developed against GM and CM. The sera from the animals immunised parenterally developed a stronger serologic response than did those immunised by the oral route. When CM was used as the immunising agent, the specific isotopic response was stronger than that obtained with GM. There were no differences in the antigen profile revealed by the sera of the animals immunised by the oral or parenteral routes. A marked degree of cross-reactivity between the two types of milk was found.     

Human serum antibodies reactive with dietary proteins. IgG subclass distribution

The isotype distribution of human IgG antibodies reactive with common dietary proteins has been evaluated in sera from adult patients with the irritable bowel syndrome and with bronchial asthma using a solid-phase immunoassay (ELISA). In both these medical disorders, serum antibodies reactive with ovalbumin or gliadin were restricted predominantly to the IgG4 isotype; however, IgG antibodies reactive with bovine milk antigens, notably casein, were often restricted to both the IgG2 and IgG4 isotypes. A similar serum IgG antibody isotype distribution for these dietary protein antigens was also demonstrated in IgG antibody-positive healthy adults. These data amplify the view that production of antibodies of the IgG4 isotype may reflect a normal immune response to dietary protein antigens presented at mucosal surfaces.     

Effect of Conjugation of Cow Milk Whey Protein with Polyethylene Glycol on Changes in their Immunoreactive and Allergic Properties

Polyethylene glycol (PEG) was conjugated with milk proteins to determine the changes of the immunoreactive properties of α-lactalbumin (α-la) and β-lactoglobulin (β-lg). The competitive ELISA method was applied to determine the antigenicity of modified protein, using rabbit polyclonal antibodies. The immunoreactivity decreased to 0.90 μg ml-1 α-la (which corresponded with 0.05%) and 0.14 μg ml^-1 β-lg (which corresponded with 0.08% of the immunoreactivity observed in raw milk). The determination of allergenicity of modified whey proteins was carried out on a group of 10 allergic patients and it was insignificantly weaker than that of a control sample of milk allergens.     

Effect of heating and enzymatic hydrolysis on casein cow milk sensitivity in Moroccan population

The objectives of the present work were first to evaluate the sensitivity to cow raw milk of the population of Fez, and then to study the effect of heating and pepsin hydrolysis on the allergenicity of casein. A cross-sectional study was carried out in Fez Hospitals, in which 1000 patients were recruited to establish a sera bank used to evaluate specific IgE to cow milk and to casein. Then, we evaluated the reaction of human IgE to heated and pepsin-hydrolysed casein. The results showed that 11.5% of the population studied self-reported reactions to foods. From them, 3.6% reported allergy to milk. Evaluation of specific IgE to cow raw milk showed that 11.9% of patients presented higher specific IgE levels. The treatments of casein indicated that both heating and pepsin hydrolysis totally decreased its binding on the human IgE.     

Usefulness of Measurement of Antibodies in Serum in Diagnosis of Sensitivity to Cow Milk and Soy Proteins in Early Childhood

A noteworthy feature of this study is that comparisons were made between specific, sensitive, quantitative measurements of serum antibodies to food proteins and objective appraisal of clinical manifestations buy double-blind food challenges. Over 50 children, 4–30 months of age, with suspicious histories of adverse reactions to cow milk or soy products were investigated. Levels of serum antibodies to cow-milk proteins were clearly higher in children with adverse reactions to milk, confirmed by blind challenge; there was no overlap with the lower levels of serum antibodies in children without confirmed reactions. Findings with serial determinations of serum antibodies to cow milk in selected cases are also presented.
The levels of serum antibodies evoked by cow milk and soy formulae fed from birth until 4 months of age were similar. The levels of serum antibodies to soy protein in children with and without adverse reactions to soy products were not definitive.
The bearing of these data on the comparative "antigenicity" of cow milk and soy products and the distinction between asymptomatic and clinically significant, symptomatic, sensitivity are discussed.     

Monoclonal antibodies to human casein

Two monoclonal antibodies, LICR-LON-32.2 (32.2) and LICR-LON-14.1 (14.1), are described which react with human casein. 32.2 reacts with human beta-casein and 14.1 with human kappa-casein. 32.2 also reacts with rat band 2 casein and bovine beta-casein, but 14.1 appears to be specific for human kappa-casein. These monoclonal antibodies do not cross-react with other milk proteins.