A comparison of the binding of secretory component to immunoglobulin A (IgA) in human colostral S-IgA1 and S-IgA2 (original) (raw)
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A Sensitive Method for Specific Quantitation of Secretory IgA
Scandinavian Journal of Immunology, 2008
A method to quantitate specifically secretory IgA (SIgA) has been developed using the enzyme-linked immunosorbent assay. The IgA in the test sample was adsorbed to anti-alpha antibodies attached to plastic tubes via a cost of IgA myeloma protein. The reacted SIgA was determined using anti-secretory component antiserum conjugated with alkaline phosphatase. The technique permitted quantitation of secretory IgA in biological fluids like milk, urine, and saliva with a reproducibility of +/-7%, down to 0.03 mg/l. In contrast to earlier techniques, the presence of up to 157% of serum IgA without secretory component (SC) and free SC did not disturb the measurements of SIgA. Furthermore, variations in pH and osmolarity, within biological ranges in secretions, did not influence the estimations.
Bovine Secretory Component * ISOLATION
Bovine free secretory component was purified from whey by salt precipitation, gel filtration, DEAEcellulose and phosphocellulose chromatography, and immunoadsorption. It was obtained in immunologically pure form and in 56% yield. The Stokes radius of pure free secretory component was found to be 4.3 nm by gel filtration, and an .& of 4.1 S was determined by the ultracentrifuge. The molecular weight was 79,000 by sodium dodecyl sulfate gel electrophoresis and by sedimentation equilibrium in the ultracentrifuge, using a d of 0.73 determined by ultracentrifugation in D,O and H,O. A minimal axial ratio of approximately 5 was calculated. Amino acid analysis of bovine free secretory component showed remarkable similarity to that of human, dog, and rabbit but carbohydrate analysis showed significant differences. In contrast to the human, bovine free secretory component has 2 methionine residues/mol. The NH&rminal sequence was found to be Lys-Ser-Pro-Ile-Phe-Gly-Pro-Glu-Glu-Val-Asp-Ser-Val. This sequence is identical with that of the human and dog. However, the poor immunological crossreactivity between the dog, human, and bovine proteins suggests that significant structural differences will be found in other regions of the molecule. Secretory immunoglobulin A is the predominant class of immunoglobulin in mucous membrane secretions (1). Unlike serum immunoglobulin A (IgA), it contains an additional specific polypeptide chain called the secretory component, which has been demonstrated in several species (for review see Ref. 2). The secretory component is synthesized in epithelial cells and becomes disulfide-linked to (Y chains of dimeric IgA as the IgA is transported across the epithelial mucous membrane (2). In addition to secretory component bound to IgA, this polypepide chain is present in secretions unbound to other proteins, and this constitutes the free secretory component. The exact biological significance of the secretory component is not yet clear, but evidence is available suggesting that bound secretory component protects the secretory IgA molecules against proteolytic enzymes present in secretions (3-5). It has also been suggested that secretory component plays a role in the transport of IgA across the epithelial lining (6, 7) and, more recently, that it acts as a surface receptor for dimeric IgA and IgM in epithelial cells (8). Chemical and physicochemical studies of both free secretory component and the released bound secretory component in
Anti-alpha-galactosyl immunoglobulin A (IgA), IgG, and IgM in human secretions
Clinical and diagnostic laboratory immunology, 1995
Anti-alpha-galactosyl (anti-Gal) is a natural human serum antibody that binds to the carbohydrate Gal alpha 1,3Gal beta 1,4GlcNAc-R (alpha-galactosyl epitope) and is synthesized by 1% of circulating B lymphocytes in response to immune stimulation by enteric bacteria. We were able to purify secretory anti-Gal from human colostrum and bile by affinity chromatography on silica-linked Gal alpha 1,3Gal beta 1,4GlcNAc. We found similar secretory anti-Gal antibodies in human milk, saliva, and vaginal washings. Secretory anti-Gal from milk and saliva was exclusively immunoglobulin A (IgA); that from colostrum and bile also contained IgG and IgM isotypes. Serum was also found to contain anti-Gal IgM and IgA in addition to the previously reported IgG. Anti-Gal IgA purified from colostrum and bile had both IgA1 and IgA2. Secretory anti-Gal from saliva, milk, colostrum, and bile agglutinated rabbit erythrocytes (RRBC) and bound to bovine thyroglobulin, both of which have abundant alpha-galactos...
Purification and characterization of secretory IgA from baboon colostrum
Journal of Immunological Methods, 1997
. Ž . In this report, we describe a method for purifying secretory immunoglobulin A sIgA from baboon Papio anubis colostrum. The colostrum was first clarified by centrifugation and then analyzed with various anti-human Ig-specific immunologic reagents. Cross-reactive IgA in the baboon colostrum was identified by ELISA. Western blot analysis also Ž . Ž . demonstrated cross-reactive epitopes associated with human IgA1, IgA2, secretory component SC , and joining J chain. To purify the sIgA, colostrum was separated into 4 distinct fractions by gel filtration chromatography. Analysis of the individual fractions by ELISA indicated that the IgA elutes over one peak. The IgA fraction was compared with purified Ž . Ž . human sIgA on SDS-PAGE, and exhibited heavy H chains, light L chains, SC, and J chain. The baboon colostrum was Ž . also analyzed by ELISA for specific IgG H and L chain epitopes utilizing monoclonal antibodies MAbs . No significant quantity of IgG was detected in the baboon colostrum or in the individual 4 fractions, while L chain reactivity was observed in the sIgA fraction. The sIgA fraction was pooled, concentrated, and was found to contain approximately 7 mgrml sIgA. To determine if the baboon sIgA was dimeric like human sIgA, the purified sIgA was sized by molecular sieve Ž . chromatography. The molecular size of the sIgA preparation 350 kDa was determined empirically by comparison to known molecular species used to calibrate the column. In addition, native SDS-PAGE indicated that baboon sIgA, like human sIgA, migrates between IgG and IgM, suggesting it has a dimeric form. The purified baboon sIgA preparation should prove Ž dine; HRP, horseradish peroxidase; ABTS, 2,2 -azino-bis 3-ethylbenzthaizoline-6-sulfonic acid; kDa, kilodaltons ) Ž . Ž . Corresponding author. Tel.: q1 405 271-5630; Fax: q1 405 271-6339. 0022-1759r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved.
The Secretory Immunoglobulin A Response in the Gut
Biochemical Society Transactions, 1977
Humoral immunity manifest in the mammalian gut is often due to the presence there of sIgA (secretory immunoglobulin A) antibodies. A considerable proportion of antibodies of this immunoglobulin isotype are synthesized locally by plasma cells, which are abundant in intestinal lamina propria. B-lymphocyte precursors for IgAsecreting plasma cells ('IgA plasma cells') appear to derive from the Peyer's patches (aggregated lymphatic follicles) of the small intestine. The Peyer's patches of rabbits contain a subpopulation of B-lymphocytes that can repopulate the spleen and lamina propria of irradiated allogeneic recipients with IgA plasma cells (Craig & Cebra, 1971). Pokeweed mitogen stimulates this subpopulation in vitro to generate IgA plasma cells (Jones et al., 1974). The immediate precursors for IgA plasma cells have been isolated in the subpopulation of Peyer's-patch cells bearing membrane Fab, and lacking membrane IgM by fluorescence-activated cell-sorting (Jones et al., 1974; Jones & Cebra, 1974). In addition to supplying precursors for IgA plasma cells, an 'antigen-sampling' role has been ascribed to Peyer's patches (Bockman & Cooper, 1973). Whole protein molecules may pass intact across their specialized dome epithelial cells and arrive in the midst of B-lymphocyte follicles. Thus IgA precursors may have their first encounter with antigen in the Peyer's patches, be stimulated to divide, and migrate in lymph/blood to intestinal lamina propria and generate plasma cells. We have devised an adoptive transfer system to compare the antigen-sensitivity of Peyer's-patch cells with cells from peripheral lymph nodes and spleen (Cebra et al., 1977a,b). Syngeneic or congeneic (CB20+Balb/c) cells were transferred into sublethally irradiated (600rad) recipients which were then challenged 1 day later with 5 x lo8 sheep erythrocytes. Antibody-forming cells in the recipients' spleens were enumerated at various times thereafter by the Jerne plaque assay and by facilitating antisera to distinguish cells making IgM, IgGl, IgG2 and IgA isotopes. Fluorochrome-labelled alloantisera, which distinguished IgA from CB20 (Ig-2*) and Balb/c (Ig-2") congeneic mice, were used to determine the origin of IgA plasma cells in recipient mice. The Peyer's-patch cells were as effective in adoptively transferring IgM and IgG responses as cells from peripheral lymph nodes or spleen. However, only Peyer's-patch cells transferred a significant IgA response, which began at about day 10 after transfer and reached a maximum of IgA Vol. 5
Infection and Immunity, 2003
The cleavage of human serum monomeric immunoglobulin A1 (IgA1) and human secretory IgA1 (S-IgA1) by IgA1 proteinase of Neisseria meningitidis and cleavage by the proteinase from Proteus mirabilis have been compared. For serum IgA1, both proteinases cleaved only the ␣ chain. N. meningitidis proteinase cleaved only in the hinge. P. mirabilis proteinase sequentially removed the tailpiece, the CH3 domain, and the CH2 domain. The cleavage of S-IgA1 by N. meningitidis proteinase occurred only in the hinge and was as rapid as that of serum IgA1. P. mirabilis proteinase predominantly cleaved the secretory component (SC) of S-IgA1. The SC of S-IgA1, whether cleaved or not, appeared to protect the ␣1 chain. Purified Fc fragment derived from the cleavage of serum IgA1 by N. meningitidis proteinase stimulated a respiratory burst in neutrophils through Fc␣ receptors, whereas the (Fc␣1) 2 -SC fragment from digested S-IgA1 did not. The loss of the tailpiece from serum IgA1 treated with P. mirabilis proteinase had little effect, but the loss of the CH3 domain was concurrent with a rapid loss in the ability to bind to Fc␣ receptors. S-IgA1 treated with P. mirabilis proteinase under the same conditions retained the ability to bind to Fc␣ receptors. The results are consistent with the Fc␣ receptor binding site being at the CH2-CH3 interface. These data shed further light on the structure of S-IgA1 and indicate that the binding site for the Fc␣ receptor in S-IgA is protected by SC, thus prolonging its ability to activate phagocytic cells at the mucosal surface.
Protein A Reactivity of Various Mammalian Immunoglobulins
Scandinavian Journal of Immunology, 1978
Serum samples and immunoglobulin fraciions of eight mammalian species were applied to a Sepharose-protein A column. As with the human immunoglobulin subclasses IgGl, lgG2 and IgG4, all examined animal IgG classes and subclasses were bound to a greater or lesser extent lo protein A. However, the binding of IgGl of ruminants was very poor. Polyclonal IgM and IgA of the pig, the dog and the cat may be separated in protein A reactive and protein A non-reaciive fractions. In addition, monoclonal canine IgM and IgA partially reacted with protein A. In combination with methods such as ammonium sulphate precipitation, ion exchange chromatography and gel-filtration, affinity chromatography with protein A is recommended for the rapid purification of certain Ig (sub)classes of a number of mammalian species.