Scaitd. J. Itiimunoi., Vol. 6, 1977.

IgE in Human Urine and Milk M. W. TURNER, D. B, L. McCLELLAND, A. R. MEDLEN & C. R. STOKES Department of Immunology, Institute of Child Health, London, and Department of Therapeutics, The Royal Infirmary, Edinburgh, Great Britain

Turner, M. W., McClelland, D. B, L., Medlen, A. R. & Stokes, C. R. IgE in Human Urine and Mi!k. Scand. J. Imtnuttot. 6, 343-348, 1977. IgE was found in urine from healthy adult volunteers at very low levels {approximately 0.003-0.010 IU/ml), corresponding to a 24-h excretion rate of 3-16 IU. IgE was not detected in 36 out of 47 samples of milk and colostrum. In samples from six women the protein was present at low concentration 1-2 days postpartum but was not detected in later samples (usually day 5 or 6). Mammary secretions from four allergic donors were studied, and IgE was detected at Iow concentrations in samples from the two most severely affected individuals. The levels of IgE observed in both urine and milk suggest that there is no significant synthesis of the protein in either the urinary tract or in mammary tissue. M. W. Turner, Ph.D., Department of Immtinology, 30 Guilford St., London WClN JEH, England

In Our earlier studies of IgE levels in human urine (1, l4) we used the solid-phase radioimmunosorbent assay, which we have later shown to be an unsatisfactor}' procedure for this biological fluid (12). Technical improvements of the radioimmunoassay for IgE prompted the present investigation of normal human urine. In addition, we have studied the content and molecular size of IgE in colostrum and milk from both allergic and non-allergic women.

MATERIALS AND METHODS Urine samples. Urine was collected from nine healthy male volunteers for a 24-h period into containers with 200 mg sodium azide as preservative. The urine was passed through Whatman grade l l l V filter paper and the volume measured. An aliquot (100 ml) of each pool was dialysed, using 23/32-in. Visking tubing, against running cold tap water overnight and then against three changes of distilled water for 24 h at -f-4°C. The aliquot was

Institute of Child

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Iyophilized and then reconstituted in the smallest practical volume (500-900 ^1) of IgE assay buffer (containing 50% horse serum) and stored at -7O''C until ret^uired for assay. Serum samples were also obtained from eight of the subjects approximately synchronously with the urine samples. These samples were also stored at -7O''C until required. Milk samples. Women admitted to the Simpson Memorial Maternity Pavilion, Edinburgh, were interviewed by one of us (D. B. L. McClelland), and those willing to participate in the investigation were questioned about their allergic histor)'. Those with a historjof asthma, eczema, or hayfever were classified as allergic. No skin tests were peformed on the patients. Paired serum and colostrum samples were obtained from the women, and in most cases two samples several days apart were collected. Milk and colostrun] samples were defatted by centrifuging for 1 h at 13,000 g at 4''C. All samples were fro2en at -70°C, and IgE analyses were performed on the total group i n one batch.

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M. W. Turner, D. B. L. McClelland, A. R. Medlen & C. R. Stokes

IgE determhiat/ons. IgE was assayed b)' a double antibody technique ( U ) , using rabbit anti-human IgE (Hoechst Pharmaceuticals, England) in the first stage and donkey antirabbit antiserum (Wellcome Reagents Ltd.) in the second. Results were expressed in international units per inilHIitre, using a commercial calibrated standard (Pharmacia). The concentration of the first antibody was adjusted to permit either a high or a low sensitivit}- assay. Thus serum samples were assayed using standards in the range 1-200 IU/ml, whereas samples of urine and milk were assayed using standards within the range 0.0625-16.0 IU/ ml. The threshold levels of detectability in the two assays were 2 and 0.125 IU/ml, respectively. Some samples of colostrum were prediluted 1:3; and for these the threshold level was 0.375 IU/ml.

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Gel filtration of milk sample. A sample of milk obtained 11 months postpartum from a 28-year-old woman with eaema and asthma and a high serum level of IgE (4,400 Il.I/ml) was subjected to gel filtration to delineate the molecular size of any IgE present. 1.5 ml of milk was centrifuged at 3,000 rpm for 15 min and the fat-free supernatant removed. After Millipore filtration (0.22 ^m) 0.75 ml of sample was recovered and applied to a column of Sephadex G-200 (2.8 X 41.4 cm; bead size, 40-120 ^m). Elutioii was performed at room temperature, using a buffer of O.lM Tris-HQ0.2M NaCl-2mM EDTA Nag, pH 7.6, containing 0.02% sodium azide. A constant elution rate of 10 ml,fh was used, and the eluant was monitored continuously at 280 nm. The column had previously been calibrated with serum proteins, and milk fractions corresponding to the moleailar weight range 69,000-1,000,000 were selected for IgE analysis. Alternate fractions were concentrated by ultrafiltration (8/32-in. Visking tubing) back to the initial sample volume (0.75 ml).

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RESULTS concentrates from 9 healthy subjects were studied, but duplicate concentrates were prepared from 4 of these individuals, giving

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IgB in Human Urine and Milk a total of 13 samples. As shown in Table I, IgE was detected in all the samples except the

two concentrates from Subject E.S. The agreement between duplicate concentrates was good

Table II. IgE levels in colostrum and milk Patient

Allergic history

2

Serum IgE, IU/ml

Colostrum/milk. days postpartum

Colostra!/mi Ik IgE, IU/ml

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2

0.75

IGE in human urine and milk.

Scaitd. J. Itiimunoi., Vol. 6, 1977. IgE in Human Urine and Milk M. W. TURNER, D. B, L. McCLELLAND, A. R. MEDLEN & C. R. STOKES Department of Immunol...
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