Clin. exp. Immunol. (1979) 36, 183-188.

Human immunoglobulin D in colostrum, saliva and amniotic fluid H. F. SEWELL, J. B. MATTHEWS, VICTORIA FLACK & R. JEFFERIS Department of Immunology and Department of Oral Pathology, The Medical School, Vincent Drive, Birmingham

(Accepted for publication 21 September 1978)

SUMMARY

An antiserum raised to a partially purified preparation of secretory IgA isolated from human colostrum was shown to contain antibodies directed against human IgD. The inferred presence of IgD in the human colostrum was confirmed and also its association with antibody activity, as demonstrated by the presence of anti-E. coli antibodies. IgD was also shown to be present in whole saliva, parotid saliva and amniotic fluid, but could not be detected in jejunal juice.

INTRODUCTION The presence of immunoglobulins and specific antibodies in non-vascular body fluids has been well documented. Hanson (1961) reported the presence of immunoglobulins in human colostrum and noted a quantitative predominance of IgA. Chodirker & Tomasi (1963) clearly established the quantitative abundance of IgA in human saliva, colostrum and other body fluids. Other studies have documented the presence of IgM (Brandtzaeg, 1970), IgG and traces of IgE (Salmon, 1970) in various body fluids. Using the Mancini technique developed to detect IgD at concentrations >30 ,ug/ml, Rowe, Crabbe & Turner (1968) reported this immunoglobulin class to be undetectable in a number of body fluids and secretions. Specific antibody activity has also been demonstrated to be associated with the presence of immunoglobulin in such fluids. Thus antibodies to various enterobacteria have been detected in colostrum obtained from several mammalian species. Although colostral antibodies have been shown to be predominantly of the secretory IgA (sIgA) class, Ahlstedt et al. (1975), using a sensitive enzyme-linked immunoabsorbent assay, demonstrated the presence of IgG and IgM antibodies to E. coli in human colostrum. The secretory form of IgA is a complex protein polymer, formed from two IgA monomer molecules joined together through a J chain (the secreted product of a plasma cell), which specifically binds a product of epithelial cells called secretory component (Tomasi et al., 1965; Halpern & Koshland, 1970). During some preliminary studies of immune responses in the gut, we observed that an antiserum raised to a partially purified colostral sIgA preparation gave a precipitin line in gel diffusion with a purified human IgD myeloma protein. This was shown to be due to the presence of antibodies to IgD present in the antiserum and implied that IgD was present in the original colostrum sample and was isolated with the sIgA. We now report the results ofour studies to investigate the presence of IgD in human colostrum, saliva and amniotic fluid.

MATERIALS AND MATERIALS Colostrum. We are indebted to Dr A. Flynn of the Birmingham Maternity Hospital for the supply of both fresh and stored colostral samples, obtained from volunteers. Enzyme activities were inhibited by the addition of 8-amino caproic acid (Koch-

Correspondence: Dr R. Jefferis, Department of Immunology, The Medical School, Vincent Drive, Birmingham B15 2TJ. 0099-9104/79/0040-0183$02.00 (@ 1979 Blackwell Scientific Publications

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Light Laboratories Ltd.) and phenylmethylsulphonyl fluoride (Sigma Chemical Co. Ltd.), (Jefferis & Matthews, 1977). Samples were clarified by centrifugation at 10,000 g for 30 min and the immunoglobulin-rich layer was separated and stored at - 20'C (Tomasi et al., 1965). Parotid salivas. These were collected from normal donors by the use of a Curby Cup arrangement. This was placed over the orifice of Stensens duct and maintained in position by slight negative pressure. Donors were given acid-lemon drops to stimulate salivary flow. The saliva was collected into bijoux and treated as described above for colostrum. Whole saliva. Unstimulated whole saliva was collected from normal volunteers and treated as described above. Saliva from an IgD myeloma patient (Ro) was similarly treated. Amniotic fiuids. These were kindly supplied by Dr B.D. Williams (The Royal Postgraduate Medical School, Hammersmith). Jejunal juices. These were kindly donated by Dr R.A. Thompson (see below). Antigens and antisera. Purified IgD, Fab5, Fc5 and corresponding antisera were prepared as described previously (Jefferis & Matthews, 1977). Sheep antisera specific for IgA, IgM and IgG were obtained from the Immunodiagnostic Research Laboratory of the Department of Immunology (formerly Department of Experimental Pathology). These antisera were processed to obtain y-globulin fractions and their specificity was established as described previously (Sewell et al., 1978). An antiserum to secretory IgA and free secretory component isolated from a pool of whole human saliva and jejunal juice was kindly donated by Dr R.A. Thompson of the Regional Immunology Laboratory, East Birmingham Hospital. We also possessed another antiserum to 'secretory IgA' obtained by immunization of a sheep with an IgA-enriched fraction obtained from a Sephadex G200 separation of human colostrum. Whilst testing the specificity of this antiserum, it was noted that it agglutinated sheep red blood cells sensitized with purified IgD preparations. The agglutination could be specifically inhibited by Fc3 preparations, but not by myeloma IgA or free secretory component. The anti-secretory IgA obtained from Dr R.A. Thompson did not agglutinate IgD-sensitized sheep red blood cells. Determination ofanti-E. coli antibody titres. These were determined essentially as described previously (Sewell et al., 1978). Sheep red blood cells (SRBC) were sensitized with E. coli antigen (Lipopolysaccharide extract E. coli 0127, Difco Ltd.) by simple adsorption or by chemically coupling using glutaraldehyde. All samples assayed were pre-absorbed with SRBC. With some colostral samples a non-specific 'stickiness' with glutaraldehyde-sensitized cells was observed. The standard procedure used to overcome similar problems encountered with other mucilaginous samples, e.g. saliva, was adopted. Thus, a dilution of the sample (1/3; v/v) with 8% sodium bicarbonate solution was heated at 50°C for 15-20 min. All such samples were assayed for antibody activity using SRBC sensitized by simple absorption. Red cell-linked antigen-antiglobulin reactions (RCLAAR). These were performed as described previously (Sewell et al., 1978) to determine classes of anti-E. coli antibodies. Assays giving positive results using anti-FcO antisera were checked for specificity by inhibition of agglutination using purified Fc5 preparations. Assayfor IgD in non-vascular bodyfluids. SRBC were sensitized with a Yi preparation of a sheep anti Fc5 antiserum by the method of Ling, Bishop & Jefferis (1977) or Goding (1976). Essentially identical results were obtained with each technique, but the procedure of Goding (1976) was generally found to be most convenient. A 1% or 0-5% suspension of sensitized SRBC were used in a microtitre system. The fluids to be investigated were titred out in 0-1% ovalbumin-PBS diluent, using Cooke microtitre 'u' trays and standard 25 j1 droppers and diluter. For quantification of IgD levels, standards containing 1 ug, 5 ,ug, 10 ug, 20 ug, 50 ,ug, 100 ,ug and 1000 jg/ml of a purified IgD paraprotein were titrated against the appropriate anti-IgD sensitized SRBC. The titres of test samples were compared with those of the appropriate standards.

RESULTS Immunoglobulin D in body fluids Fig. 1 illustrates the presence of antibody activity to IgD in an antiserum raised to partially purified preparation of colostral 'sIgA'. The antiserum was also shown to agglutinate Fc3-coated SRBCs; the reaction was completely inhibited by Fc6. The immunogen 'sIgA' at 20 mg/ml gave a weak line on immunodiffusion analysis with a specific anti-Fc5 antiserum which showed identity with a purified Fc6 preparation (Fig. 1). The presence of IgD in colostrum was further investigated by fractionation of several samples on Ultrogel AcA34. Pooled fractions in the region of the elution profile corresponding to the elution position of IgD in myeloma sera were concentrated and shown to contain IgD on gel diffusion analysis. IgD estimations on concentrated pooled fractions showed that in addition, a significant proportion of the IgD was eluting in a position indicating a higher molecular weight-possibly resulting from aggregation. Table 1 summarizes the results for the detection of IgD in non-vascular body fluids using anti-IgDsensitized SRBC. The demonstration of IgD in whole saliva from normal volunteers was further confirmed by analysis of whole saliva samples from three IgD myeloma patients (Ro, We and No). Fig. 2 shows the immunodiffusion analysis of the saliva from patients Ro and No.

JgD in ccrtain boly jluilds

FIG. 1. Gel diffusion analysis demonstrating (a) anti-IgD activity in the sheep anti-sIgA antiserum (Z207 concentrated x 3), and (b) IgD in the antigen preparation (human colostral sIgA at 20 mg/ml) used for raising Z207. The reactions are compared with a sheep anti-Fc5 antiserum (Z400), bovine serum albumin, Fc6, and normal human serum (CHA) possessing a high level of IgD.

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H.F. Sewell et al. TABLE 1. Detection of IgD in non-vascular body fluids

Body fluid

Number of samples

Positive agglutination of anti-IgD-SRBC

Colostrum

12

12

Concentration range of IgD (fig/ml) 8/12 (1-20)

4112 (20-50) Whole saliva (normals) Parotid saliva (normal stimulated) Jejunal juice Amniotic fluid

7 5 5 20

3 4 0 7

n.d. n.d. 0 4/7 (005-1)* 3/7 (1-5)

n.d. = Not determined. * Tests performed on samples concentrated x 20 and results expressed for unconcentrated samples.

Anti-E. coli antibodies in colostruini The colostral samples were initially screened for anti-E. coli antibodies. All the samples tested together with some matching serum samples possessed antibodies. Table 2 shows the results obtained from the agglutination of the E. coli-SRBC, and the classes of antibodies detected by RCLAAR assays.

_ A:. '' 'WZEX '._. :. ..

A.

Is_.

..

*:w

-

. A.: ::

A:.

A_

1IG. 2. Gel diffusion analysis of whole saliva samples from tw o IgD myeloma patients Ro and No against neat (nt) and dilutions of sheep anti-Fed antiserum (Z400).

DISCUSSION We would like to emphasize once again the starting point for this study, namely the finding that an antiserum raised to a partially purified sIgA preparation isolated from human colostrum had a fairly high titre of antibody directed against human IgD. The antiserum was otherwise specific with respect to the other human immunoglobulin classes and serum proteins. This experience may sound a cautionary note that will be of interest to others. This finding automatically points to the presence of IgD in human colostrum and in this studvy We have positively demonstrated this fact. When colostrum xras fractionated by gel filtration on Ultrogel AcA34, concentrated fractions w ere obtained in M hich IgD w as

IgD in -certain body fluids

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TABLE 2. Antibodies to E. coli detected in colostrum and serum and their corresponding isotypes

Samples HR (Col) HR (Ser) DA (Col) DA (Ser)* RA (Col) RS (Ser) HK (Col) HK (Ser) ER (Col) ER (Ser) SK (Col) SK (Ser) MY (Col) PE (Col)

Anti-E. coli titre detected by passive haemagglutination 1:32 1:16 1:16 1:8 1:32 1:32 1:128 1:8 1:64 1:32 1:8 1:8 1:64 1:128

Classes of antibodies detected by RCLAAR

IgA IgM IgG IgD + + + + + + + -

+ + + A + +

+ + + + + + + + + + + + + +

+ + + + + + + + + + + +

4-

+ + + + + + +

+I+

* Serum DA, which was negative for IgD by RCLAAR, was absorbed with polyvalent anti-human IgG, IgM and IgA as described (Sewell et al., 1978). On re-assay IgD antibodies could be demonstrated in the serum. (+) Positive agglutination with the relevant antiglobulin. (-) No. Col = Colostrum. Ser = Serum.

demonstrable by precipitation with a specific antiserum in gel diffusion. The elution volume of a major proportion of this colostral IgD was very similar to that observed for IgD paraproteins in myeloma sera. The IgD levels in unfractionated colostrum were assessed using an agglutination of anti-IgD-coated sheep red blood cells technique which had been 'standardized' using purified human IgD myeloma proteins. IgD could be detected in all twelve samples examined. In eight out of twelve it was present at a level of 1-20 ug/ml and in the remaining four at the higher level of 20-50 pg/ml. These levels lie just at or below the limit of detectability in the Mancini assay used by Rowe et al. (1968) and this presumably explains why these authors failed to detect the immunoglobulin. Quantification of IgD in a number of body fluids, including colostrum, has been performed by Dunnette et al. (1977) using a sensitive radioimmunoassay system. In this study all eleven colostral samples were positive for IgD with an upper level of concentration of 13 pug/ml. Matched serum and colostrum samples were screened by the sensitive RCLAAR assay for antibodies to E. coli. Interestingly, IgD anti-E. coli antibodies could be readily demonstrated to be present in all of eight colostral samples examined, whilst it could not be demonstrated initially in five out ofsix matched serum samples. The latter result has previously been shown (Sewell et al., 1978) to be due to the presence of overwhelming amounts of anti-E. coli antibody of classes other than IgD. The inference from the studies on colostrum must be that IgD forms a relatively higher proportion of the total antibody present than is the case in serum. Recent studies have demonstrated that the adult human intestine is not impermeable to macromolecules and that the human newborn intestine shows an appreciable permeability for a period of 18-48 hr after birth (Warshaw, Walker & Isselbacher, 1974; Walker, Isselbacher & Bloch, 1975; Sewell, 1978). Ogra, Weintraub & Ogra (1977) have clearly demonstrated the presence of colostrum-derived IgA in the serum of breast-fed babies. Therefore, one may speculate that maternal colostral IgD may also gain

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access to the circulation of the new-born. The possible significance of such passive transfer of immunoglobulin in the face of the quantitatively greater concentrations of maternal IgG transmitted by the placental route is unclear. Using simple gel diffusion we were able to demonstrate the presence of IgD in the saliva of three patients with myelomatosis having an IgD paraproteinaemia. Also, using the sensitive agglutination of the SRBC anti-IgD-SRBC system, we were able to detect IgD in three out of seven normal whole saliva samples and also in four out of five parotid saliva samples. Rowe et al. (1968), using the less sensitive Mancini technique, could not detect the presence of IgD in whole saliva. However, Dunnette et al. (1977) reported that IgD in parotid saliva could not be detected by radioimmunoassay at concentrations > 0 003 pg/ml. Whilst we did not quantify the IgD in whole or parotid saliva, the sensitivity of the techniques employed are comparable. Unfortunately Dunnette et al. (1977) did not test whole saliva. Care was taken in our study to obtain parotid saliva free of whole saliva, but contamination must remain as a possible explanation of our findings. A somewhat surprising finding was the detection of IgD in seven out of twenty of the amniotic fluids assayed. Proteins present in amniotic fluid are most likely to be of foetal rather than maternal origin and since Rowe et al. (1968) and Johansson & Berg (1967) failed to detect IgD in cord blood (N.B., one out of 116 was positive in the study of Johansson & Berg) it was concluded that the foetus does not synthesize this immunoglobulin class. However, Dunnette et al. (1977) were able to detect and quantify IgD present in cord blood, using their radioimmunoassay system capable of detecting IgD at levels of > 1 0 pg. It is not surprising to discover that as more sensitive techniques are applied, so IgD (doubtless among many other serum proteins) can be positively detected and quantified in a number of body fluids. The agglutination of antibody-coated cells technique that we have employed will routinely detect levels of IgD down to < 1 0 ng/ml. In spite of this sensitivity we were unable to detect IgD in the five jejunal juices tested. The possible significance of the low levels of IgD present in body fluids is unresolved. However, it does appear that the level detected in colostrum and its association with anti-E. coli activity is of interest and merits further definitive study of its origin and possible significance. REFERENCES AHLSTEDT, S., CARLSSON, B., HANSON, L.A. & GOLDBLUMN, R.G. (1975) Antibody production by human colostral cells. I. Immunoglobulin class, specificity and quantity. Scanid. 7. Immunzol. 4, 535. BRANDTZAEG, P. (1970) Human secretory immunoglobulins. II. Salivary secretions from individuals wvith selectively excessive or defective synthesis of serum immunoglobulins. COiM. exp. Immunol. 8, 69. CHODIRKER, W.B. & ToNMASI, T.B. (1963) Gamma globulins: Quantitative relationships in human serum and nonN ascular fluids. Scienice, 142, 1080. DUNNETTE, S.L., GLEICH, G.J., MILLER, D.R. & KYLE, R.A. (1977) Measurement of IgD by a double antibody radioimmunoassay: demonstration of an apparent trimodal distribution of IgD levels in normal human sera. 7. Immunol. 119, 1727. GODING, J.W. (1976) The chromic chloride method of coupling antigens to erythrocytes. Definition of some important parameters. ]. Immuzol. Methods, 10, 61. HAI.PERN, NI.S. & KOSHLAND, M.E. (1970) Novel subunit in secretory IgA. Nature (Loizd.), 228, 1276. HANSON, L.A. (1961) Comparative immunological studies of the immune globulins of human milk and of blood serum. IJut. Arch. Allergy ApPi. Immuniol. 18, 241. JEFFERIS, R. & MATTHEWS, J.B. (1977) Studies of IgD myeloma proteins. Proteolytic digestion patterns. Imnmuwiochemistry, 14, 171. JOHANSSON, S.G.O. & BERG, T. (1967) Immunoglobulin levels in healthv children. .1cta. Paedtatr. Scantd. 56, 572.

LING, N.R., BISHOP, S. & JEFFERIS, R. (1977) Use of antibody-coated red cells for the sensitive detection of antigen and in rosette tests for cells bearing surface immunoglobulins. 3. Immunol. Methods, 15, 279. OGRA, S.S., WEINTRAUB, D. & OGRA, P.1L. (1977) Immunologic aspects of human colostrum and milk. Y. Immunol. 119, 245. ROWE, D.S., CRABBE, P.A. & TURNER, M.N. (1968) Immunoglobulin D in serum, body fluids and lymphoid tissues. C/in. exp. Immunol. 3, 477. SALMON, S.E. (1970) IgE globulin in secretions. Clin. Res. 18, 135 (abstract). SEWELL, H.F. (1978) Aspects of oral immunization and the immune response. Ph.D. thesis. Department of Immunology (formerly Experimental Pathology), University of Birmingham, England. SEWELL, H.F., CHAMBERS, L., MAXWELL, V., MATTHEWS,

J.B. & JEFFERIS, R. (1978) The natural antibody response to E. coli includes antibodies of the IgD class. C/in. exp. Immunol. 31, 104. TOMASI, T.B., TAN, E.M., SOLOMON, A. & PRENDERGAST,

R.A. (1965) Characteristics of an immune system common to certain external secretions. 3. exp. Med. 121, 101. WALKER, W.A., ISSELBACHER, K.J. & BLOCH, K.U. (1975) Intestinal uptake of macromolecules III. .7. Immuniol. 115, 854. WARSHAW, A.L., WALKER, W.A. & ISSELBACHER, K.J. (1974) Protein uptake by the intestine. Evidence for absorption of macromolecules. Gastroeitterol/oj', 66, 987.

Human immunoglobulin D in colostrum, saliva and amniotic fluid.

Clin. exp. Immunol. (1979) 36, 183-188. Human immunoglobulin D in colostrum, saliva and amniotic fluid H. F. SEWELL, J. B. MATTHEWS, VICTORIA FLACK &...
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