Survey of immunoglobulin G content and antibody specificity in cows’ milk from British Columbia

The immunoglobulin G (IgG) concentrations of 254 raw milk samples collected from the province of British Columbia (Canada) in 1990 and 1991 ranged from 0.030 to 0.71 mg ml^‐1, with mean and median values of 0.28 and 0.27 mg ml^‐1 respectively in 1990, and 0.25 and 0.24 mg ml^‐1 respectively in 1991. Although significant differences were noted in IgG contents of milks from five geographical areas and 14 dairies for each of the 2 years, there were also significant differences between milks from year to year. All of the milks showed measurable antigen‐binding activity in enzyme‐linked immunosorbent assay (ELISA) against the lipopolysaccharide (LPS) fractions of five bacteria (Shigella flexneri 1A, Escherichia coli O111:B4, Escherichia coli O128:B12, Salmonella typhimurium and Salmonella enteritidis). An immune milk obtained from vaccinated cows showed significantly higher anti‐S. enteritidis LPS activity than any of the raw milk samples. However, levels of specific anti‐LPS activity against the other four bacteria were roughly of the same order of magnitude for raw milk samples and the immune milk.     

Allergy to bovine β-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.     

Quantification of bovine IgG in milk using enzyme‐linked immunosorbent assay

A double sandwich enzyme‐linked immunosorbent assay (ELISA) procedure for the quantification of IgG in bovine milk was developed for detecting the concentration of IgG in various homogenized HTST, UHT, evaporated and raw milk samples as well as skim milk powder. ELISA results were consistently lower than radial immunodiffusion (RID) values for the same sample, suggesting interference by an unknown constituent in the milk matrix. This difficulty was overcome by using a reference milk, quantified previously for IgG by RID. A significant linear relationship (y=2.03x‐0.034, r=0.891, n= 150, p<0.001) was obtained for IgG content in unknown raw milk samples determined by ELISA using serial dilutions of the reference milk (y) or commercial IgG (x) to construct standard curves. The slope of the regression line could be used as a correction factor. Alternatively, IgG in unknown samples could be directly quantified from absorbance values of reference milk serial dilutions assayed on the same ELISA plate. Using this procedure, homogenized, HTST pasteurized milk contained from 65 to 79% of the IgG found in raw milk. Skim milk powder also retained a major portion of IgG, while evaporated and UHT pasteurized milk were virtually devoid of IgG.     

Sensitization to human milk

Background Allergy to milk is one of the earliest manifestations of IgE‐mediated allergies and affects about 2.5% of newborn children. Several reports indicate that milk‐allergic patients may be sensitized also to human milk proteins. Objective To analyse the specificity and possible biological relevance of IgE reactivity to human milk antigens in milk‐allergic patients. Methods The specificity of IgE reactivity to cow's milk and human milk antigens was analysed with sera from milk‐allergic children and adults by IgE immunoblotting. IgE cross‐reactivity between milk antigens was studied by immunoblot inhibition experiments. That IgE reactivity to human milk antigens is not due to alloreactivity or due to the transmission of foreign antigens into mother's milk was demonstrated through the analysis of milk samples from genetically unrelated mothers before and after intake of dietary milk products. The biological relevance of IgE reactivity to human milk was confirmed by skin testing. Results IgE antibodies to human milk were found in more than 80% of the tested milk‐allergic patients. Cross‐reactive IgE‐reactive human antigens such as α‐lactalbumin and non‐cross‐reactive human milk antigens were identified. Immediate‐type skin reactions could be elicited with human milk samples in patients with IgE reactivity to human milk. Conclusion IgE reactivity to human milk in milk‐allergic patients can be due to cross‐ sensitization and genuine sensitization to human milk and may cause allergic symptoms. IgE‐mediated sensitization to human milk is common in milk‐allergic patients and may require diagnostic testing and monitoring.     

Effect of cooked and raw egg consumption on ovalbumin content of human milk: a randomized, double-blind, cross-over trial

BACKGROUND: Maternal avoidance of egg intake has been recommended to treat egg allergy in breastfed infants. OBJECTIVE: To determine if the concentration of ovalbumin (OVA) in human milk is directly related to the quantity and form of egg consumed by breastfeeding mothers. METHODS: Randomized, blinded, cross-over, intervention trial. Breastfeeding women (n = 41) attended four clinic days between 11 and 14 weeks of lactation and on each day were randomly allocated to receive a test breakfast, identical except for the egg content (no egg, one raw egg, half a cooked egg or one cooked egg). Breast milk samples were collected at two hourly intervals for 8 h and their OVA concentration measured by ELISA. RESULTS: There was a direct, dose-response between the amount of cooked egg ingested and the peak OVA concentration (no egg 0.05 ng/mL [95% confidence interval (CI), 0.01-0.11], half a cooked egg 2.24 ng/mL [95% CI, 0.57-3.91], one cooked egg 3.16 ng/mL [95% CI, 1.41-4.91], n = 41, P<0.05) as well as the total OVA excretion (no egg 0.18 ng/mL/h [95% CI, 0.04-0.39], half a cooked egg 4.93 ng/mL/h [95% CI, 1.40-8.46], one cooked egg 9.14 ng/mL/h [95% CI, 4.25-14.03], n = 41, P<0.05). The peak concentration and total OVA excretion in response to one raw egg did not differ from ingesting half a cooked egg. There was no detectable OVA in the breast milk of 24% (10/41) women up to 8 h after any egg challenge. CONCLUSION: OVA was detected in the breast milk of lactating women up to 8 h after a controlled intake of egg. A dose-response correlation was indicated. As excretion of OVA in human milk appears to be a normal phenomenon, further studies need to determine the threshold of OVA excretion that leads to symptoms in egg-allergic breastfed infants.     

“Latent” healthy human serum autoantibodies cross-reacting with DNA and bacterial lipopolysaccharides

Department of Radiation Biochemistry, Research Institute of Medical Radiology, Academy of Medical Sciences of the USSR, Obninsk. (Presented by Academician of the Academy of Medical Sciences of the USSR V. A. Nasonova.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 111, No. 5, pp. 516–518, May, 1991.     

抗人c-erbB2单克隆抗体的制备及其特异性鉴定

目的:制备小鼠抗人c-erbB2mAb,并进行特异性鉴定。方法:应用计算机软件分析人源c-erbB2抗原表位,人工合成羧基端含优势表位的13肽,与钥孔戚血蓝蛋白(KLH)偶联后,免疫BALB/c小鼠。取免疫小鼠脾细胞与Sp2/0骨髓瘤细胞常规融合,依次经HAT选择培养、间接ELISA法、克隆化和免疫组化染色法筛选出稳定分泌抗天然人源c-erbB2mAb的杂交瘤细胞株。用交叉反应试验和阻断试验检测mAb的特异性。结果:获得1株可稳定分泌抗天然人源c-erbB2抗体的杂交瘤细胞株。该mAb与已知的c erbB2抗原阳性的乳腺癌标本起反应;与其他不表达c-erbB2分子的细胞不起反应。用合成的13肽阻断后,失去与c-erbB2抗原的反应性。结论:用合成的13肽作为免疫原成功地制备出1株抗c-erbB2的mAb。     

Detection and comparison of neuraminidase activities in human and bovine group B streptococci

Human and bovine group B streptococcus (GBS) isolates were serotyped and amounts of released N‐acetylneuraminic acid from N‐acetylneuraminyl‐lactose by extracellular neuraminidase were colorimetrically assessed. According to serotyping by co‐agglutination method, 30 of bovine GBS and 43 of human GBS could be serotyped (ST) by monospecific antisera coated with protein A. The remaining GBS strains were designated as nontypeable (NT). The released N‐acetylneuraminic acid was determined in 90.9% of bovine GBS and 47.1% of human GBS isolates. The differences between the total bovine and human GBS isolates were statistically significant (p < 0.001). In comparison with detected N‐acetylneuraminic acid level in bovine and human groups, significant decrease was observed in the bovine NT group according to increased human NT (p < 0.01) and bovine ST groups (p < 0.01). However, N‐acetylneuraminic acid level in bovine ST and bovine total groups significantly (p < 0.001) increased with respect to the human ST group and human total group. Neuraminidase activity was detected more frequently in bovine GBS isolates. Considerable differentiations were observed between typeable and nontypeable isolates.     

Characterization of a folate receptor in parotid gland and a folate binding protein in saliva from humans. Epitope relatedness to human milk folate binding protein

The present study was performed to establish the antigenic identity and origin of the folate binding protein in human saliva. We identified a folate receptor in human parotid and submandibular gland which immunoreacted with antibodies against human milk folate binding protein, as evidenced by ELISA and immunostaining of ductal epithelium and secretory glandular material. The receptor concentration was 0.4-1.4 nmol 3H-folate bound/g protein. Ligand binding was of a high-affinity (K=10(10) M(-1)) type, exhibited positive cooperativity, a slow radioligand dissociation at pH 7.4, and inhibition by folate analogues. The concentration of immunoreactive folate binding protein in saliva as determined by ELISA with antibodies against human milk folate binding protein was several fold higher than that determined by radioligand binding (nil - 1 nM). This indicates that a major fraction of the immunoreactive material does not bind 3H-folate, and could represent a precursor form of the protein. In conclusion, the folate binding protein in human saliva seems to be a secretory product of the salivary glands. The protein is also epitope-related to folate binding proteins in other human mucosal secretions.     

Development of ic-ELISA and lateral-flow immunochromatographic assay strip for the detection of vancomycin in raw milk and animal feed

A highly sensitive monoclonal antibody (mAb) 3H4 against vancomycin (VAN) was prepared. Indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and lateral-flow immunochromatographic assay (ICA) were developed based on the mAb. The 50% inhibition concentration (IC₅₀) value and limit of detection (LOD) value of ic-ELISA method for vancomycin were 0.59 and 0.06?ng/mL, and for norvancomycin were 1.51 and 0.13?ng/mL under optimized conditions as pH 7.4, 0.4% (m/v) NaCl, and 5% (v/v) acetonitrile. In lateral-flow ICA, the visual limit of detection (vLOD) value and cut-off values for vancomycin were 1 and 2.5?ng/mL, and for norvancomycin were 5 and 10?ng/mL under optimized conditions as pH 8.6 with 1?mg/mL coating antigen and 1?µg/mL gold nanoparticle-labeled mAb. In raw milk and animal feed samples, recovery rates from ic-ELISA ranged from 89.2% to 121.6%. The vLOD and cut-off value were 5–10?ng/g and 100–200?μg/kg, respectively. Therefore, both methods were sensitive, rapid, and effective for the on-site detection and rapid mass screening of samples.     

Secretion of respiratory syncytial virus inhibitors and antibody in human milk throughout lactation

Neutralising inhibitors to respiratory syncytial (RS) virus have been demonstrated in the whey of most samples of human milk tested. Although high titres were secreted in colostra of some mothers (1/10–1/2,560; median 1/40) inhibitor levels in milk collected after the first week of lactation were uniformly low (median 1/10). High neutralising titres correlated with high colostral levels of specific antiviral IgA but, unlike neutralising activity, IgA antiviral antibody persisted in the milk of only four of 18 mothers. Similarly, antiviral IgG and IgM antibodies were not generally detected after the first postpartum week. Differences in antibody secretion among mothers did not correlate with differences in total protein or total immunoglobulin secretion, and appeared to reflect maternal immune status. In one mother a marked rise in specific antiviral IgA and IgG secretions during the second and third months of lactation suggested a response to virus infection. The relevance of maternal immunity and colostral and milk antiviral antibody to protection of breast-fed babies from RS-virus bronchiolitis is discussed.     

Allergic reactions to raw, pasteurized, and homogenized/pasteurized cow milk: a comparison

Five children aged 12-40 months with IgE-mediated adverse reactions to cow milk (immediate onset clinical pattern of cow milk allergy) were orally challenged double-blind in random order with three different milk preparations processed from the same batch of milk 1) raw untreated cow milk, 2) pasteurized cow milk, 3) homogenized and pasteurized cow milk, and 4) Nutramigen (a commercial hypoallergenic infant formula based on hydrolysed casein) as placebo. Skin prick tests with the same preparations were also performed. On oral challenge the three different processed milk types provoked significant and similar allergic reactions in each child, and no adverse reactions followed the challenge with placebo (Nutramigen). Skin prick test with the same milk products were positive in all children and comparable to the results with an extract of purified raw cow milk protein (Soluprick), whereas Nutramigen did not elicit any skin reactions. A tendency towards a lower threshold of reaction and larger skin reactions induced by the processed milk preparations might indicate an increased ability of pasteurized and homogenized/pasteurized milk to evoke allergic reactions in patients allergic to milk.     

Bovine milk IgG,but not serum IgG,inhibits pokeweed mitogen-induced antibody secretion by human peripheral blood mononuclear cells

Bovine milk IgG markedly inhibits the pokeweed mitogen (PWM)-induced secretion of immunoglobulins from human peripheral blood mononuclear cells. Heat-aggregated bovine milk IgG is even more inhibitory, demonstrating significant inhibition when levels as low as 5–9 µg/ml are continuously present in thein vitro 14-day culture system. However, bovineserum IgG, regardless of its state of aggregation, and control proteins have little effect on PWM-induced secretion of human IgG, IgA, and IgM. In a similar fashion, goat milk IgG, especially when aggregated, inhibits human antibody secretion whereas goat serum IgG does not. Inhibition appears to be mediated by Fc receptors since F(ab)₂ fragments of milk-derived bovine IgG do not inhibit PWM-induced antibody secretion. The continuous presence of bovine milk IgG is not essential since preincubation of milk IgG with PWM and human mononuclear cells for 24 hr also results in inhibition of human immunoglobulin secretion. In examining potential mechanisms of inhibition, it was found that bovine milk IgG, bovine serum IgG, and another chitincontaining protein, bovine thyroglobulin, each caused a small and equal inhibition of the binding of¹²⁵I-labeled PWM to human mononuclear cells, yet only the milk IgG inhibited antibody production. These studies raise the question of whether bovine milk IgG might modulate the human immune systemin vivo.     

Rapid and sensitive immunoassays for the detection of lomefloxacin and related drug residues in bovine milk samples

The misuse and illegal use of fluoroquinolones (FQs) in animal-based food products have drawn considerable attention in several countries. As a result, there has been an increased demand for efficient detection methods of FQs in food products. In this study, we developed an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and immunochromatographic strip device based on a monoclonal antibody against lomefloxacin (LFLX), a second generation FQ. Ic-ELISA had an IC₅₀ value of 0.19?ng/mL with a limit of detection of 0.04?ng/mL in 0.01?M phosphate-buffered saline (PBS). The intra- and inter-assay recovery rates of LFLX in bovine milk samples were 98.02–107.40% and 100.65–107.82%. The immunochromatographic assay of LFLX in PBS and spiked bovine milk samples had visual cutoff values at 1 and 5.0?ng/mL, respectively, with significant cross-reactivity with norfloxacin and enoxacin. The developed ic-ELISA and strip method may assist in the detection of FQs in foods.     

A comparative study of attachment of human, bovine and mouse blastocysts to uterine epithelial monolayer

The in-vitro attachment of human, bovine and murine blastocyststo monolayer cultures of uterine epithelium were studied bytransmission electron microscopy. The human trophoblastic cellsintrude between uterine epithelial cells forming a multilayerduring attachment in vitro, thus resembling the intrusive typeof penetration observed in vivo. The bovine trophoblastic outgrowthresembled an epithelio-chorial attachment as the trophoblastformed an attachment plate on top of the endometrial cells withoutpenetration. In the murine attachment study, the trophoblastcells immediately displaced the uterine cells and formed contactwith the culture vessel.     

Allergy to bovine β-lactoglobulin: specificity of immunoglobulin E generated in the Brown Norway rat to tryptic and synthetic peptides

BACKGROUND: Animal models which reflect the induction and development of food-allergic reactions are important in the identification of allergenic potential of food proteins and peptides. A number of rat strains, including PVG, Hooded Lister and Brown Norway have been shown to produce immunoglobulin (Ig) E antibodies to food proteins as well as to inhaled allergens. Previous work in our laboratory using the Brown Norway (BN) rat has shown that specific IgE is produced following administration of ovalbumin and milk products via both enteral and parenteral route; this has allowed us to rank ovalbumin, lactoferrin and bovine serum albumin in terms of their inherent allergenic potential and has enabled us to demonstrate that milk protein allergens recognized by the systemically-sensitized animal are consistent with those recognized by sera from cow's milk-allergic patients (the most common allergens recognized were beta-lactoglobulin and the alpha, beta and kappa-caseins). OBJECTIVE: To demonstrate that the BN rat model can be used to identify the major IgE-binding peptides from beta-lactoglobulin, a known human food allergen, and that those IgE-binding peptides are similar to those recently identified using sera from cow's milk-allergic patients. METHODS: BN rats were exposed to beta-lactoglobulin or to semiskimmed milk via the intraperitoneal route in the presence of the adjuvant carrageenan. Specific IgE raised against beta-lactoglobulin was determined by a direct enzyme immunoassay using acetyl-cholinesterase substrate; specific IgG responses were also monitored. Overlapping synthetic peptides and tryptic peptides were used within the ELISA to identify the major and minor IgE-binding immunoreactive sequences. RESULTS: In terms of comparative immunogenicity, there appeared to be sequences that were predominantly IgE- or IgG-reactive. IgE-dominant regions were amino acid sequences 21-40, 41-60, 107-117 and 148-168 whereas sequences 1-24, 67-77, 82-92, 85-95 and 117-127 appeared more selective for IgG antibody recognition. An increased capacity to induce specific IgE was observed when the allergen was present in the context of whole food. CONCLUSIONS: These studies provide evidence that the immune system of the BN rat and humans - at least in the case of milk allergens - is recognizing similar protein allergens and indeed, at the molecular level, similar peptide epitopes.     

Phenotypic characterization and functional activity of human milk macrophages

A large population (about 80%) of the cells obtained from colostrum and early human milk were considered to be macrophages by the following criteria: nonspecific esterase stain, adherence, phagocytosis and IgG-Fc receptor expression. The majority of freshly isolated human milk macrophages (HMM phi) stain for the monocyte antigen OKM1. Another monocyte antigen, 61D3, was expressed only by 30% of HMM phi. Class II antigens were expressed by HMM phi. About 85% of the cells were DR-positive whereas 50% were DS-positive as assessed with a panel of monoclonal antibodies directed against class II antigens. Monocyte and class II antigens were gradually lost during in vitro culture. HMM phi can support proliferative response to antigens and mitogens when cocultured with autologous peripheral T cells. The proliferative response was significantly reduced when monoclonal antibodies to DR or DS were added to the assay. These results indicate that HMM phi have the phenotype and functional characteristics of antigen presenting cells.     

Binding of fibronectin, fibrinogen and type II collagen to streptococci isolated from bovine mastitis

Binding of 125I-labelled fibronectin, fibrinogen and type II collagen to group B (S. agalactiae), group C (S. dysgalactiae and S zooepidemicus), group E (S. uberis) and nontypable streptococci isolated from bovine mastitis was studied. S. agalactiae and S. uberis were found to bind low levels of all three proteins, while S. zooepidemicus bound high levels. Binding of the proteins to S. dysgalactiae varied, i.e. fibronectin was high, fibrinogen moderate and collagen low. Nontypable strains showed moderate or low binding of all proteins. Both hydrophobic and hydrophilic strains were found to bind fibronectin. For S. dysgalactiae the specific fibronectin binding ranged from 70% to 10% and for S. zooepidemicus it was more than 80% and this binding was sensitive to papain treatment. The binding of 29K-fibronectin fragment to one S. dysgalactiae strain showed an affinity of KD = 2.6 x 10(-8) M and the number of binding sites per colony forming unit (CFU) was calculated at 11,000.     

Enzyme immunoassay of specific human IgE to purified cows’ milk allergens

An immunometric enzyme immunoassay for specific immunoglobulin E (IgE) against five purified cows’ milk allergens, β‐lactoglobuHn, α‐lactalbumin, bovine serum albumin, lactoferrin and whole casein fraction, has been developed. Allergens were immobilized on microtitration plates. After incubations with sera from allergic patients, specific IgE bound to the plastic were detected using a monoclonal anti‐human IgE antibody labelled with acetylcholinesterase. Quantitative determinations were made by comparison with a dose‐response curve obtained under the same conditions with standard total IgE. A quantification limit of 0.08 IU ml^‐1 can thus be obtained with a coefficient of variation of lower than 5%. This allows specific IgE determinations of clinical significance in sera from allergic patients at a dilution of at least 1/10 (i.e. in a few μl of serum) with good precision and reproducibility. The determinations of specific IgE in sera from 11 patients allergic to cows’ milk showed an excellent correlation of this assay with clinical data.