A method for measuring specific IgE in sera by direct ELISA without interference by IgG competition or IgG autoantibodies to IgE.

BACKGROUND: In measuring specific IgE levels in sera by direct ELISA, competition with coexisting IgG often impedes an exact IgE determination; additionally, IgG autoantibodies to IgE (IgG-IgE) in sera affect the assay. In this paper, we attempt to determine accurate specific IgE levels by selective removal of IgG with a protein G-immobilized gel (PG) and by acid treatment of the PG to compensate for the unintended removal of IgE, probably due to the PG binding IgG-IgE. METHODS: IgG in sera was removed using PG at pH 7.0. Then, the PG was treated with citrate buffer at pH 3.0 for 5 min to liberate IgE from IgG-IgE complexes, after IgG-binding sites on the PG were saturated with bovine IgG, since PG came to bind IgE at acidic pHs. IgE levels were then measured by ELISA. RESULTS: The PG treatment of sera removed the effect of inhibitory competition by coexisting IgG, especially at higher concentrations of sera, to improve specific IgE detection by direct ELISA. However, PG treatment alone sometimes reduced IgE levels (39% of sera tested), even though PG does not bind IgE at pH 7.0, which indicated the presence of IgG-IgE complexes. The reduction in IgE returned almost to their original levels in the sera by acid treatment of the PG. By combining the PG treatment with acid treatment, specific IgE measurement in sera was improved significantly (p < 0.01, Wilcoxon signed rank test). CONCLUSION: Measurement of specific IgE in sera by direct ELISA was improved by using the PG and acid treatment technique.

Crude protein content and amino acid composition in Taiwanese human milk.

Breast milk provides the essential nutrients for infants in readily available form. The content of nitrogen in human milk is of great importance because it relates to the growth of infants in the early stage, and the composition of nitrogenated compounds varies according to the lactational stage. Three-hundred-and-three human milk specimens were obtained from 240 healthy mothers living in two different districts in Taiwan, and 264 specimens were used for the analysis. The crude protein content, total and free amino acid compositions as well as urea content were evaluated using pooled milk samples according to different lactational stages and geographical location. The crude protein content decreased sharply from colostrum (2.51 g/100 mL) to mature milk (1.25 g/100 mL). Total amino acids account for 80-85% of the crude protein throughout the whole lactation period. Crude protein also contained 30 to 35 mg/ 100 mL urea and 41 to 48 mg/ 100 mL free amino acids as non-protein nitrogen components. The ratio of essential to non-essential amino acids remained constant throughout the lactation period in spite of a decline in amino acid content. The amino acid composition per 1 g of nitrogen varied during the lactation period. The differences of these lactational changing patterns of individual amino acids were probably reflected by variation of the protein composition during lactation. The sum of free amino acid content ranged from 43 to 50 mg/100 mL in Taipei and 40 to 45 mg/100 ml, in Kaohsiung. Although the variations of free amino acids during the lactation period differed among amino acids, glutamic acid predominated in mature milk while phosphoethanolamine was predominant in colostrum.

In vitro and in vivo effects of exogenous nucleotides on the proliferation and maturation of intestinal epithelial cells.

To determine the nutritional role of nucleotides, the in vitro and in vivo effects of exogenous nucleotides on the development of intestine were investigated. First, the in vitro effects of nucleotides on the proliferation and maturation of enterocytes were studied by using a human colon tumor cell line (Caco-2) and a rat normal small intestinal crypt cell line (IEC-6). Second, the in vivo effects of nucleotides were also studied in early weaned rats fed nucleotide-unsupplemented or high-nucleotide-supplemented diet. Nucleotide composition resembled that of human milk (CMP:UMP:AMP:IMP:GMP = 10:1:1:1:1, in weight). Nucleotide supplement did not enhance Caco-2 cells proliferation; however, it significantly enhanced maltase and sucrase activities. In contrast, nucleotides supplement enhanced ICE-6 cells proliferation and maltase activity. CMP, predominantly contained in the mixture, enhanced most effectively the proliferation and maturation of cells. In the in vivo experiment, nucleotides significantly enhanced sucrase activity in the intestinal mucosa of early weaned rats. The results presented here suggest that a nucleotide supplement may enhance enterocyte proliferation and/or maturation in vivo and in vitro. Therefore exogenous nucleotides may play an important role in the development of the intestine.

Profile of nucleotides and nucleosides of human milk.

The content of nucleotides and nucleosides of human milk was analyzed using a newly developed method for high performance liquid chromatography. By this method it is possible to analyze nucleotides and nucleosides simultaneously. Human milk was pooled according to season, lactation period, and geographical area. Three kinds of nucleosides--cytidine, uridine, and adenosine--and 6 kinds of nucleotides--5'-CMP, 5'-UMP, 5'-AMP, 5'-GMP, 5'-IMP, and 5'-CDP--were detected. Cytidine, 5'-CMP, and 5'-CDP predominated throughout lactation. Also there seemed to be geographical differences in nucleoside composition. The overall amounts of nucleotides and nucleosides were higher in winter than in summer. No nucleosides were detected in bovine milk, nor in bovine milk-based infant formula, and bovine milk contained much less nucleotides than human milk. These results suggest that nucleosides and nucleotides found in human milk may play some important roles in the development of infants.

Effects of dietary nucleotides on lipid metabolism and learning ability of rats.

To investigate the effects of dietary nucleotides on lipid metabolism and learning ability, male Sprague-Dawley rats were fed with a nucleotides-supplemented diet or a nucleotides-free diet for 5 weeks. The content of nucleotides in the diet was 1.0% and their composition resembled that in human milk. The content of phosphatidylcholine (PC) and the ratio of PC to phosphatidylethanolamine (PE) in the cerebral cortex of rats fed the nucleotides-supplemented diet were significantly higher than that of rats fed the nucleotides-free diet. However, there was no difference in the content of PC and the ratio of PC to PE in the liver between the two groups. The levels of docosahexaenoic acid (C22:6n-3) and arachidonic acid (C20:4n-6) in the cerebral PC fraction were higher in rats fed the nucleotides-supplemented diet. The learning ability of rats fed the nucleotides-supplemented diet, which was evaluated by the water-filled multiple T-maze test and passive avoidance test, was superior to the of rats fed the nucleotides-free diet. The results presented here suggest that dietary nucleotides may influence lipid metabolism of the cerebral cortex and contribute to the rise in learning ability of rats.

The concentration of epidermal growth factor in Japanese mother's milk.

The concentration of epidermal growth factor (EGF) in human milk was measured by radioreceptor assay (RRA). Human milk samples collected from healthy Japanese mothers who delivered at full term were divided into 30 groups according to differences in the duration of lactation, seasons of the year and place of residence. Human milk collected from 3 to 5 days after delivery in winter and summer contained 15.03 micrograms/100 ml and 15.46 micrograms/100 ml of EGF, respectively. The concentrations decreased rapidly to 8.04 micrograms/100 ml and 8.12 micrograms/100 ml in milk from 31 to 60 days, and to 5.10 micrograms/100 ml and 5.44 micrograms/100 ml in milk from 241 to 481 days. Seasonal and regional differences in EGF concentration were observed to some extent, but no clear tendency could be defined. These results suggest that EGF is an essential and fundamental factor for the growth of infants in the very early stages after delivery, because it was high only at the beginning of lactation and then it decreased.

Inhibition of cholera toxin by human milk fractions and sialyllactose.

The effects of human milk fractions on clolera toxin B subunit binding to monosialoganglioside 1 (GM1) were investigated. Human milk, human defatted milk, whey, and a low-molecular-weight fraction of human milk inhibited the binding, but casein did not inhibit it. The inhibitory activity of whey from bovine-milk-based infant formula was less than that of whey from human milk. Differences in composition between human and bovine whey seemed to influence the extent of the inhibitory activity. Sialylated oligosaccharides were considered to be the possible components that inhibited cholera toxin. The effects of sialyllactose, a predominant sialylated component of human milk, on cholera toxin-induced diarrhea were investigated by the rabbit intestinal loop method. Sialyllactose inhibited the cholera toxin inducing fluid accumulation, although neither sialic acid nor lactose had an effect on it. The results suggest that sialyllactose is responsible for the inhibitory activity of milk on cholera toxin.

Inhibitory effects of milk gangliosides on the adhesion of Escherichia coli to human intestinal carcinoma cells.

The effects of milk gangliosides and their derivatives on the adhesion of enterotoxigenic and enteropathogenic Escherichia coli to Caco-2 cells, a human intestinal carcinoma cell line, were investigated. Human milk gangliosides inhibited the adhesion of enterotoxigenic E. coli to Caco-2 cells in the same proportion, regardless of the lactational stage, but bovine milk gangliosides were less effective. The most effective inhibitor was monosialoganglioside 1 (GM1); the adhesion rate of enterotoxigenic E. coli in the presence of GM1 was less than 20% of the positive control. The adhesion of E. coli was also depressed to 31.4% by monosialoganglioside 3 (GM3). However, the inhibitory effect of disialoganglioside 3 (GD3) was less than that of GM3. GD3 lactone, ceramide lactoside, and N-acetylneuraminic acid did not inhibit E. coli adhesion to Caco-2 cells. GM3 also inhibited the adhesion of enteropathogenic E. coli to Caco-2 cells. Thus, these results suggest that GM3 possibly behaves as a physiological component in the intestinal tract of infants to protect them against enteric infections.

Cryptic B cell determinant in a short peptide: T cells do not induce antibody response of B cells when their determinants entirely overlap each other.

A synthetic peptide fragment encompassing residues 21-40 (F21-40) of bovine beta-lactoglobulin was used as a model antigen to investigate the relationship between T and B cell determinants. The establishment of fine specificity for these determinants in F21-40 in BALB/c mice revealed that this short peptide contained two T cell determinants, Td1 (residues 21-32) and Td2 (28-40), and that Td2 entirely overlapped the dominant B cell determinant (Bd) of residues 28-40. A truncated peptide, F25-40, was capable of strongly restimulating F21-40-primed T cells in vitro, and its binding affinity to anti-F21-40 antibodies was the same as that of F21-40, indicating that F25-40 maintained the same antigenic structure as that of region 25-40 in F21-40. However, immunization with F25-40 could not elicit specific antibodies despite priming specific T cells. From these results, Bd in F25-40 can be regarded as a cryptic B cell determinant. The absence of antibody response to F25-40 was probably caused by the complete overlapping of Td2 and Bd. This overlapping may have caused the presentation of Td2 only by professional antigen-presenting cells, but never by Bd-specific B cells possessing the potential to be differentiated to antibody-producing cells by T cell help. These findings demonstrate that the co-existence of T and B cell determinants within a single molecule does not always assure specific antibody production, which depends upon the spatial relationship between these determinants.

Inhibition by lactoferrin and kappa-casein glycomacropeptide of binding of Cholera toxin to its receptor.

Inhibition from binding of Cholera toxin (CT) to Chinese hamster ovary (CHO)-K1 cells and ganglioside GM1 by lactoferrin (Lf) and kappa-casein glycomacropeptide (GMP) from cow's milk was examined. Both Lf and GMP effectively reduced the CT-derived morphological changes in CHO-K1 cells. The competitive binding assay demonstrated that both Lf and GMP inhibited the binding of CT to GM1, although their affinity for CT was lower than that of GM1. The inhibitory effect of Lf and GMP seemed to be attributed to their terminal sialic acid, although the sugar chain sequence only partially fitted to the CT-receptor.