Anaphylaxis from cow's milk casein

The case of a 35-year-old woman who suffered anaphylaxis after the ingestion of milk or milk-containing products is presented. Symptoms also appeared after the patient used a cosmetic which contained casein. Skin prick tests, specific IgE measurement, and immunoblotting were carried out with cow's milk extract, and its purified proteins: casein, β-lactoglobulin, and α-lactalbumin. Specific IgE to cow's milk casein was detected with the three tests. Immunoblotting revealed one band of 24 kDa approximately corresponding to the β-fraction of casein. Negative results to β-lactoglobulin and α-lactalbumin were obtained with the three methods. Symptoms were controlled by rigorous dietary and behavior measures.     

Immunogenicity and antigenicity of a partially hydrolyzed cow's milk infant formula

We evaluated the immunogenicity and antigenicity of a formula based on partially hydrolyzed cow's milk whey protein in infants at risk of atopy and in controls. Total IgE and specific IgE, IgG, and IgG4 subclass antibodies against egg albumin and cow's milk α-lactalbumin, casein, and β-lactoglobulin were measured by radioimmunoassay of cord blood and of peripheral blood at 5 days and 6 months of life in five groups of infants: 16 breast-fed infants at risk of atopy (group 1), 21 partially hydrolyzed whey formula-fed infants at risk of atopy (group 2), 14 formula-fed infants at risk of atopy (group 3), 10 breast-fed control infants (group 4), and 13 formula-fed control infants (group 5). Total IgE concentration was significantly lower in group 2 at 6 months than in groups 3 and 5 infants and similar to that observed in groups 1 and 4 infants. The concentration of specific antiegg and anti-cow's milk protein IgG and of specific anti-cow's milk α-lactalbumin and β-lactoglobulin IgG4 subclass antibodies was significantly reduced in group 2 as compared to group 3 infants and similar to that found in breastfed infants. In conclusion, the partially hydrolyzed formula was less immunogenic and antigenic than a traditional formula and was as immunogenic and antigenic as breast milk.     

Cow's milk allergy in adults is rare but severe: both casein and whey proteins are involved

Background Studies on cow's milk allergy (CMA) in adults are scarce. Little is known about the clinical symptoms, eliciting doses (ED), and allergens involved.
Objective The aim of this study was to analyse the clinical symptoms, ED and allergen recognition in adult CMA patients, compared with cow's milk (CM)-sensitized, but tolerant controls.
Methods Adult CMA patients were evaluated by standardized questionnaires ( n =30), skin prick tests (SPTs) and specific IgE for CM allergens ( n =18), and a double-blind placebo-controlled food challenge (DBPCFC, n =10). A control group ( n =25) of CM-sensitized, but tolerant adults was included.
Results The majority of CMA patients (20/30, 67%) reported severe symptoms. In all patients participating in DBPCFC, CMA was confirmed. ED for subjective symptoms (0.3–300 mg CM protein) were significantly lower than that for objective symptoms (300–9000 mg CM protein). The severity of CMA by history and ED was not correlated with SPT or IgE. Patients had higher SPT reactivity than controls for CM, α-lactalbumin and β-lactoglobulin ( P =0.002, P =0.014 and P =0.004) but not for casein. Specific IgE to CM tended to be higher ( P =0.068) and IgE to casein was higher in patients than that in controls ( P =0.016). No difference was observed for IgE to α-lactalbumin and β-lactoglobulin.
Conclusion Adult CMA is severe in nature. ED are low, starting from 0.3 mg CM protein. Patients with CMA recognize the same major allergens (casein and whey proteins) as controls, but display a stronger SPT and IgE reactivity.     

Pasteurization of milk proteins promotes allergic sensitization by enhancing uptake through Peyer's patches

Background:  The underlying mechanisms responsible for allergic sensitization to food proteins remain elusive. To investigate the intrinsic properties (as well as the effect of pasteurization) of the milk proteins α-lactalbumin, β-lactoglobulin and casein that promote the induction of milk allergy.
Methods:  Alteration of structure and immune-reactivity of native and pasteurized proteins was assessed by gel filtration and ELISA. Uptake of these proteins was compared in vitro and in vivo . The biological effect was assessed by orally sensitizing C3H/HeJ mice with milk proteins followed by a graded oral challenge. Required dose to induce anaphylaxis, symptoms and mean body temperature was recorded. Antigen-specific antibodies and cytokine production by splenocytes were analyzed.
Results:  Soluble β-lactoglobulin and α-lactalbumin but not insoluble casein were readily transcytosed through enterocytes in vitro and in vivo . Pasteurization caused aggregation of β-lactoglobulin and α-lactalbumin inhibiting uptake by intestinal epithelial cells in vitro and in vivo. Furthermore, aggregation redirected uptake to Peyer's patches, which promoted significantly higher Th2-associated antibody and cytokine production in mice than their native counterparts. Despite this only the soluble forms of β-lactoglobulin and α-lactalbumin elicited anaphylaxis (following priming) when allergens were administered orally. Aggregated β-lactoglobulin and α-lactalbumin as well as casein required systemic administration to induce anaphylaxis.
Conclusions:  These results indicate that triggering of an anaphylactic response requires two phases (1) sensitization by aggregates through Peyer's patches and (2) efficient transfer of soluble protein across the epithelial barrier. As the majority of common food allergens tend to form aggregates, this may be of clinical importance.     

Identification of a masked allergen, α-lactalbumin, in baby-food cereal flour guaranteed free of cow's milk protein

We report on a baby food, cereal flour P (CFP), which, although guaranteed free of cow's milk protein, caused failure to thrive and diarrhea, vomiting, and coughing fits in a 22-month-old child. The purpose of this study was to identify the allergen involved. The investigation used prick tests, RAST, and the RAST inhibition method. Specific IgE was elevated to 100 kU/l for cow's milk and to 15.3 kU/1 for a-lactalbumin (2.5 kU/1 for casein, 0.7 kU/1 for β-lactoglobulin). Antibovine IgG IgEs were associated. RAST inhibition experiments demonstrated the presence of α-lactalbumin in "food-quality" lactose used in this flour, at a dose of 1–5 μg/g of CFP. The daily intake of a-lactalbumin was found to be less than 70 μg. This exquisite clinical sensitization was attributed to the intestinal hyperpermeability (IH) which favors the access of milk allergen to the blood, leading to an ever-growing state of hypersensitivity. It could have been due to egg- and mustard-associated allergies as well as to giardiasis and intestinal candidosis. This work underlines the risk of masked food allergens and the need of thoroughly informative labeling.     

ADCC-Mediating Capacity in Children with Cow's Milk Protein Intolerance in Relation to IgG Subclass Profile of Serum Antibodies to β-Lactoglobulin

In a previous study sera from children with cow's milk protein intolerance (CMPI) exhibiting gastrointestinal symptoms were found to efficiently induce antibody-dependent cell-mediated cyto-toxicity (ADCC) to β-lactoglobulin-coated cells. In contrast, sera from children with coeliac disease showed a low ADCC-mediating capacity, despite high levels of IgG anti-β-lactoglobulin antibodies. The study described here was undertaken to evaluate whether differences in IgG subclass profile of anti-β-lactoglobulin antibodies could explain the observed variations in the ADCC-mediating capacity. Forty-eight sera from the following groups of children were investigated: CMPI with predominantly gastrointestinal symptoms, CMPI with skin symptoms of immediate-onset, children with untreated coeliac disease and healthy references. Absorption experiments indicated that primarily IgGl antibodies were responsible for the ADCC-mediating capacity of the sera. Accordingly, the ADCC reactivity of individual sera correlated with their IgGl antibody levels. Sera from CMPI children with gastrointes tinal symptoms, most of which had a high ADCC reactivity, also demonstrated a distinctive subclass pattern of their anti-β-lactoglobulin antibodies with higher relative proportions of IgGl (ratios: IgGl/IgG, IgGl/IgG3 and IgGl/IgG4) than those from the other diagnostic groups. Using logistic regression analysis, the diagnostic potential of ADCC as well as of different IgG subclass variables for the recognition of gastrointestinal symptoms caused by CMPI was evaluated. The ADCC reactivity of sera was found to be the best predictor in this model.     

Detection and identification of a soy protein component that cross-reacts with caseins from cow's milk

Soy-based formulas are the most employed cow's milk substitutes in the treatment of cow's milk allergy in our country. Since adverse reactions have been reported in allergic patients as a consequence of exposure to soy proteins, we have investigated the possible cross-reactivity between components from soybean and cow's milk. A cow's milk specific polyclonal antiserum and casein specific monoclonal antibodies were used in immunoblotting and competitive ELISA studies to identify a 30-kD component from soybean that cross-reacts with cow's milk caseins. Its IgE binding capacity was tested by EAST, employing sera from cow's milk allergic patients, not previously exposed to soy proteins. The 30 kD protein was isolated and partially sequenced. It is constituted by two polypeptides (A5 and B3) linked by a disulphide bond. The protein's capacity to bind to the different antibodies relies on the B3 poly-peptide. These results indicate that soy-based formula, which contains the A5-B3 glycinin molecule, could be involved in allergic reactions observed in cow's milk allergic patients exposed to soy-containing foods.     

Effect of heat treatment and enzymatic digestion on the B cell epitopes of cow's milk proteins

Background Processing milk leads to changes in clinical allergenicity. However, the mechanism by which heat treatment affects the allergenicity of milk proteins is not fully understood.
Objective We investigated the effect of heat treatment and enzymatic digestion on the allergenicity of B cell epitopes of milk proteins using a histamine release assay.
Methods Human basophils were passively sensitized using sera from 10 patients with allergies to cow's milk. All the patients experienced symptoms immediately after ingesting milk. The human basophils were obtained from umbilical cord blood mononuclear cells after culturing the mononuclear cells for 3–4 weeks in the presence of IL-3. After sensitization with 10% patient sera for 48 h, the cells were stimulated with untreated, heat-treated, or heat-treated and pepsin-and-trypsin-digested β-lactoglobulin or α-casein for 1 h. The histamine concentrations in the supernatants were then measured by radioimmunoassay.
Results Heat treatment alone did not alter the molecular weight of β-lactoglobulin or α-casein. Heat treatment of β-lactoglobulin significantly increased its susceptibility to enzymatic digestion in a time- and temperature-dependent manner and reduced its ability to induce histamine release from sensitized basophils. Similar findings were not observed for α-casein. The combination of heat treatment and enzymatic digestion reduced the abilities of both β-lactoglobulin and α-casein to induce histamine release from passively sensitized basophils.
Conclusions Heat treatment reduced the allergenicity of β-lactoglobulin by inducing conformational changes and by increasing its susceptibility to enzymatic digestion, both of which disrupted B cell epitopes, whereas heat treatment alone did not alter the allergenicity of α-casein.     

Allergenic epitopes of bovine αS1-casein recognized by human IgE and IgG

B-cell epitopes of bovine α_S1-casein, one of the major allergens of cow's milk, were identified by a screening method based on synthetic peptides. According to the known amino acid sequence of α_S1-casein, a set of 188 overlapping sequential decapeptides shifted by one amino acid was manually synthesized on polyethylene pins by the 9-fluorenyl-methoxycarbonyl (Fmoc) method. Peptides were screened by an enzyme-linked immunosorbent assay (ELISA) specific for human IgE and IgG. Bound antibodies were detected by successive incubation with up to three polyclonal antibodies, the last one conjugated to horseradish peroxidase. Tested sera were from 15 patients with acute clinical reactions to cow's milk and IgE-specific reactions to bovine α-casein in the ELISA and immunoblot. Sera from 10 healthy subjects without remarkable reactions to cow's milk proteins were used as controls. All sera from allergic subjects showed reactions with three regions of α_S1-casein, corresponding to amino acids 19–30, 93–98, and 141–150. Furthermore, individual sera showed reactions with other parts of the protein. No essential differences in the epitope specificity of IgE and IgG were found. Inhibition of IgE binding to α_S1-casein with soluble synthetic peptides confirmed the results and revealed peptide CN-2 as the most inhibiting one.     

Cow's milk IgE and IgG antibody responses to cow's milk formulas

For comparison of the antigenicity and allergenicity of three cow's milk formulas, serum IgE antibodies to cow's milk, β‐lactoglobulin and casein, and IgG antibodies to β‐lactoglobulin were analyzed in 94 infants with a family history of allergy. They were participating in a randomized trial comparing the allergy prophylactic effect of feeding an extensively hydrolyzed (N), a partially hydrolyzed (PH), and a regular cow's milk formula (RM). Only infants who had been formula‐fed for 3 months or more were included. IgE antibodies to cow's milk proteins were more common in the RM group (22/34) than in the N (2/31) and PH groups (3/29). There was a strong correlation between sensitization to cow's milk and β‐lactoglobulin ( r =0.85, P <0.001). The IgG responses to β‐lactoglobulin were low in the N group, intermediate in the PH group, and high in the RM group. High responses, as well as detection of IgE antibodies, were associated with development of atopic disease. The low antigenicity and allergenicity of the extensively hydrolyzed formula support its use in allergy prophylaxis. The partial hydrolysate seemed to be less suitable for this purpose.     

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.

     

A hydrolysed rice-based formula is tolerated by children with cow''s milk allergy: a multi-centre study

BACKGROUND: Children allergic to cow's milk are fed a soy- or a hydrolysed cow's milk-based substitute. Neither can rule out a sensitization risk. Previous studies have shown that hydrolysed rice is tolerated by animals and children with multiple food hypersensitivities. OBJECTIVE: A prospective clinical assessment of tolerance to a rice-based hydrolysed formula was carried out in children allergic to cow's milk. Patients and methods One hundred children (42 girls and 58 boys, mean age 3.17+/-2.93 years, median 2.20, range 0.18-14.6 years) with a history of immediate reactions to cow's milk and confirmed at double-blind, placebo-controlled food challenge (DBPCFC) when not contraindicated were assessed for clinical tolerance to cow's milk proteins. Their allergy work-up included skin prick tests with whole milk, alpha-lactalbumin (ALA), beta-lactoglobulin (BLG) and total caseins, and specific IgE determinations using CAP technology were performed against whole milk, ALA, BLG and casein. Sensitization to rice and rice-based hydrolysed formula was similarly investigated. Patients' sera were evaluated at immunoblotting for specific IgE to cow's milk proteins, rice and rice-based hydrolysed formula. DBPCFC was carried out with increasing doses of a rice-based hydrolysed formula. RESULTS: All patients were sensitized to cow's milk and/or at least one cow's milk protein fraction. Eighty-seven out of 99 were positive to cow's milk and/or a cow's milk protein fraction at skin prick test. Positive (>0.35 kUA/L) specific IgE determinations were found for cow's milk and/or milk fractions (92/95), rice (21/91) and hydrolysed rice infant formula (4/91). At immunoblotting, sera from 96 children were positive to alpha-casein (n=54), beta-casein (n=38), ALA (n=57), BLG (n=37) and bovine serum albumin (n=61). Similarly, although patients' sera often contained specific IgE against rice proteins at CAP (21/91) and immunoblotting (70/96), only six very weakly positive responses were observed against rice-based hydrolysed formula. All DBPCFC with rice-based hydrolysed formula were negative. CONCLUSIONS: Rice-based hydrolysed formula is a possible alternative not only for children with multiple allergies, but also for children with cow's milk allergy.     

Molecular basis of IgE cross-reactivity between human beta-casein and bovine beta-casein, a major allergen of milk

Twenty patients allergic to cow's milk proteins and with high levels of specific IgE directed against bovine whole casein were selected to evaluate reactivity of their IgE antibodies with human beta-casein. Highly purified human and bovine beta-caseins were prepared by selective precipitations and FPLC separation. Their identity and purity were assessed by HPLC, analysis of amino acid composition, sequencing of the five N-terminal amino acid residues and immunochemical tests. Direct and indirect ELISAs were performed using human and bovine beta-casein coated into microtiter plates and monoclonal anti-human IgE antibody AChE labelled for revelation. Seven sera contained specific IgE directed against human beta-casein. Inhibition studies using native human and bovine beta-caseins as well as bovine beta-casein-derived peptides demonstrated that, depending on the sera, one or several common epitopes located in different parts of the molecule were shared by the two homologous proteins.     

Allergy to goat and sheep cheese with good tolerance to cow cheese

BACKGROUND: We report on a patient who experienced allergic reactions after eating goat cheese and after touching goat and sheep cheese, but not after consuming cow's milk dairy products. OBJECTIVE: To assess the allergenicity and IgE-binding capacity of the caseins from the three different species. METHODS: Skin prick tests were carried out using whole milk and caseins from three different species (goat, sheep and cow), and whey fractions of cow's milk. Total serum IgE and specific IgE to cow's milk proteins were measured by CAP system and specific IgE against caseins and whole milk were determined by ELISA technique. To evaluate allergenic cross-reactivity, inhibition of the IgE ELISA activity to goat's milk and goat casein was tested for the three caseins. SDS-PAGE and immunoblotting was used to determine IgE binding bands in caseins. RESULTS: Skin tests were positive to sheep and goat's milk, sheep and goat casein, as well as to sheep and goat cheese. Total serum IgE was 66 kU/L and IgE determinations by CAP were negative. IgE ELISA against the caseins from goat and sheep was strongly positive, whereas it was negative to cow casein. ELISA inhibition assays revealed a high degree of cross-reactivity between goat casein and sheep casein. Immunoblotting showed three IgE-binding bands in goat casein at 31, 27 and 22 kDa, which may correspond to alpha-, beta- and gamma-caseins. A band at about 31 kDa was observed in sheep casein and another band at 34 kDa was recognized in cow casein. CONCLUSION: This patient developed allergy to goat and sheep cheese with good tolerance to cow's milk. We identified goat casein as the main allergen causing sensitization in this patient as demonstrated by in vivo and in vitro tests. A high degree of cross-reactivity between goat and sheep casein was observed.     

Detection of reaginic and short-term sensitizing anaphylactic or anaphylactoid antibodies to milk in sera of allergic and normal persons

Sera of 50% of persons extremely sensitive and of 30% of persons moderately sensitive to milk had heat-labile reaginic antibodies conferring passive cutaneous anaphylactic sensitivity on monkeys for 24 hr. More persons in each group had IgE antibodies that bound radio-isotope labelled β-lactoglobulin and passively sensitized monkey basophils for anaphylactic degranulation. These tests, however, are not certain evidence of clinical allergy, because of the sera of nineteen topic children, three mediated monkey PCA tests and five had γE anti-$bt-lactoglobulin antibodies detected by antigen binding or basophil degranulation, though the serum donors were not clinically allergic to milk. Similarly few normal, milk-tolerant adults also had γE anti-β-lactoglobulin antibodies. Sera of nine children with milk-induced gastroenteritis occurring 12–36 hr after exposure had no anti-milk reagins, and only one had γE binding β-lactoglobulin. This allergy may be mediated by heat-stable IgG antibodies, because three sera conferred milk sensitivity on monkey skin for 4 hr and two others for 2 hr. The three sera had high titres of IgG agglutinins, one to β-lactoglobulin, one to α-lactalbumin and one to casein. All three fixed complement in the presence of milk, and one had precipitins. They did not passively sensitize human mast cells or monkey basophils for anaphylactic degranulation, but they prepared human lung for anaphylactic release of hist-amine, though in small amounts compared to that released from lung prepared with reagins. The sera of two of three cases of cot death had IgG antibodies with similar properties.     

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.     

Evaluation of the residual antigenicity and allergenicity of cow's milk substitutes by in vitro tests

BACKGROUND: This study aimed the to investigate presence of residual allergenic cow's milk proteins (CMP) in some milk substitutes employed in the treatment of cow's milk allergy (CMA). These allergens may interfere with the treatment, and elicit allergic reactions in sensitized individuals. METHODS: The protein composition of the different extracts was evaluated by Lowry's method and tricine SDS-PAGE. Different immunoenzymatic methods were used (ELISA, EAST and immunoblotting) to quantify total serum IgE and specific serum IgE, as well as to detect the presence of antigenic and allergenic components. RESULTS: The results showed a higher protein content in mammalian milks (cow, sheep, mare, goat, and human) than in hydrolyzed substitutes (partially or extensively hydrolyzed casein or whey proteins). Residual native, processed, or contaminant polypeptides have been identified in the moderate hydrolysates, whereas extensive hydrolysates did not show the presence of residual components by immunoblotting. However, specific antibodies with capacity to bind to peptides have been detected by EAST and ELISA, suggesting that extensive hydrolysates contain residual peptides that preserve immunoreactive epitopes. We were unable to demonstrate either residual antigenicity or allergenicity in an amino-acid-based formula. CONCLUSIONS: Immunoenzymatic methods were used to detect the presence of cross-reactive components in mammalian milks. Residual allergenic components from cow's milk could be identified in both the moderate and extensive hydrolysates analyzed. This information may be relevant to the treatment of CMA.     

Clinical manifestations of cows' milk allergy in childhood. I. Associations with in-vitro cellular immune responses

In this study 51 children who presented with symptoms of cows' milk allergy (CMA) were categorized clinically by their response to cows' milk challenge. Forty-two patients had unequivocal evidence of CMA and nine were milk tolerant. Of the patients with CMA two groups were identified. The first, made up of 32 patients, had immediate-type hypersensitivity reactions to milk associated with both positive skin-prick test (SPT) and RAST. The second group of 10 late reacting patients developed symptoms of CMA over several hours or days; significant increases in irritability, frequency of bowel actions, and rhinitis following milk ingestion were noted in this group. Leucocyte inhibition factor (LIF) produced in response to α-lactalbumin, β-lactoglobulin and α-casein was assessed in the immediate and late reacting CMA patients as well as in the milk-tolerant group. There was no difference in LIF production between the milk-tolerant group and those with immediate reactions. However, these two groups produced less LIF than the late reacting patients for α-lactalbumin ( P = 002), α-casein ( P = 0.03) and β-lactoglobulin ( P = 0.05). A clinical diary score card was found to be a useful instrument to assess the response of non-immediate reactions to milk ingestion.     

Allergenicity of α-caseins from cow, sheep, and goat

The allergic potential of α-caseins from bovine, ovine, and goat's milk sharing more than 85% identical amino acids was compared. Caseins were purified by anion-exchange chromatography and used for a specific IgE and IgG ELISA with diluted human sera. Sera were from 17 children with immediate-type allergy to cow's milk, from 59 children with atopy but without food allergy, and from 27 healthy children without atopic disease. The sera of cow's milk-allergic children showed a significantly higher IgE and IgG binding to α-caseins from all three species than the sera of the other groups. All groups showed an increased antibody binding to bovine a-casein compared to the sheep and goat proteins, but the differences were significant only in the groups of atopic children and of healthy controls. Furthermore, inhibition of the IgE binding to bovine α-casein with α-casein from cow, goat, and sheep revealed that the a-caseins from these species are highly cross-reactive, on the basis of the small differences in their primary structure. In conclusion, the milk of goat and sheep harbor an allergic potential and is not suitable for the nutrition of milk-allergic patients.     

A sensitive enzyme-linked immunosorbent assay for determination of bovine β-lactoglobulin in infant feeding formulas and in human milk

We developed a sensitive sandwich-type ELISA for measuring low levels of cow's milk (CM) β-lactoglobulin. Purified anti-β-lactoglobulin was used as coating antibody and also as second antibody conjugated with alkaline phosphatase. Polyethylene glycol 6000 was added to the incubation buffers to improve sensitivity. The detection limit of the assay was 0.002 μg/l, which is much better than sensitivities reported for other β-lactoglobulin assays. The sensitivity was not impaired by the presence of other CM proteins. The recovery from breast milk was 93% and from the diluting buffer 127%. The coefficient of variation within day was 5–15% and between days 10%. One hour after oral intake of milk, P-lactoglobulin could be detected in the breast milk of three mothers at concentrations of about 1–2 μg/l. Widely different concentrations of β-lactoglobulin were measured in two protein hydrolysates based on CM whey and casein proteins; the observed concentrations were 200 and 0.0056 μg P-lactoglobulinμ/g dry weight, respectively.