Journal of Chromatography B: Biomedical Sciences and Applications
Simplified method for purification of colostrum to obtain secretory component of immunoglobulin A, using secretory component as a reference protein in tracheal aspirate fluid
Introduction
Airway fluid analysis remains an important tool for assessment of many aspects of pulmonary function. Interpretation of bioactive substances measured in alveolar lavage fluid is limited because of the absence of a reliable reference marker with which to standardize the concentrations of relevant substances in the retrieved epithelial lining fluid (ELF) [1]. This limitation has prompted attempts to identify reliable and reproducible reference substances. Candidate substances have included methylene blue [2], inulin [3], urea [4], total protein [5]and albumin 4, 6. Each of these reference standards can introduce error when values are calculated from epithelial lung fluid [1]. Difficulties encountered with putative reference standards include: diffusion of substances into or from instilled lavage fluid during the process of collection of ELF, uptake of the reference material by the cells lining the airways of the lungs, release of the reference material into lavage fluid because of cell death or because of interruption of the integrity of the alveolar capillary barrier. The latter is a particular problem in diffuse pulmonary diseases in which alterations in permeability are likely to play an important role. This problem is encountered in virtually all of the important neonatal pulmonary disorders. An additional limitation for studies in neonates is the inability selectively to obtain distal airway or alveolar space ELF without variable dilution. Thus, an ideal reference standard would be a substance not found in plasma, but present in or secreted into ELF in a constant concentration relatively unaffected by disease states.
A recent study in preterm neonates [7]supports the use of the glycosolated polypeptide, soluble secretory component (SSC) of immunoglobulin A (IgA), as a useful reference substance. This substance is secreted by the bronchial and bronchiolar epithelium of the lung as early as after 16 weeks of gestation [8]. It is also secreted in the lymphoid tissues of mammary glands and is found in human colostrum. SC of IgA is a single polypeptide chain of approximately 500 amino acid residues with a large glycosolated region. It is found on the basolateral surfaces of secretory epithelial cells where it mediates the transport of polymeric immunoglobulin (pIg) into external secretions. SC is released into secretions either in the free form or bound to a pIg. Secretion of the free peptide appears to occur at a constant rate. In addition, its concentration in serum of newborns is very low and unchanging over the first weeks of life [7]. Thus, SC of IgA may be a useful reference protein for standardizing airway fluid measurements.
The first purpose of this report is to develop a simplified method for the purification of SSC as reference standard. We used commercially available polyclonal rabbit antisera in an enzyme immunoassay (EIA) for the detection of SSC, after SC was purified from a readily available source, human colostrum. The second purpose of the report is to compare values of SSC measured in samples of tracheal aspirate fluids obtained from term infants with respiratory failure with total protein and albumin measurements made in the same ELF layer samples. We thereby provide a simple means of establishing a reference standard for SSC of IgA, which proved useful for its application to the measurement in full term infants.
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Human volunteers
Clinical studies were performed after obtaining approval from the combined pediatrics institutional review board of the University of Missouri–Kansas City and The Children's Mercy Hospital. Specific written consent was not obtained from the parent(s) of each patient because no experimental manipulation was performed. Secretions obtained in the course of routine respiratory toilet were analyzed.
Reagents
The following chemicals were obtained from the sources indicated:
Polyclonal rabbit anti SC antibody
Results
The purification of SSC from human colostrum according to the basic methods of Klingmuller and Hilschman [9]was modified to take advantage of high-pressure liquid chromatography techniques. The initial size-exclusion separation on Sephacryl S-300 (Fig. 1) indicates that the majority of the SSC is in the intermediate molecular mass range of the column. There is a minor amount of SSC in the initial volume indicating a slight propensity of the SSC either to polymerize or to aggregate with other
Discussion
A simplified method to provide a reliable preparation of SSC of IgA, which can serve as a reference protein for measurements of a variety of substances of biological interest in tracheal and pulmonary epithelial secretions is described. The clinical relevance of this substance has recently been set forth by Watts and Bruce [7]and by Goil et al. [14]. In these studies, 7, 14conducted in both preterm and full term infants, both shortly after birth and over a variety of postnatal ages, it was
Acknowledgements
Supported in part by grants from The Katherine B. Richardson Fund of Children's Mercy Hospital (C.B. and S.G.), The Children's Mercy Hospital Physician Scientist Award (W.E.T.) and The Wyeth Pediatrics Neonatology Fellows Research Award (S.G.).
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Preparation of the porcine secretory component and a monoclonal antibody against this protein
2015, Protein Expression and PurificationCitation Excerpt :SC improves the ability of the mouse respiratory tract to defend itself against bacterial affection [15], interacts with the surface proteins of Salmonella typhimurium to inhibit its invasion of and adherence to HeLa cells [16], and reduces giardia infection [17]. Soluble SC was isolated from human colostrum as a reference protein in tracheal aspirate fluid, using serial centrifugation, size-exclusion fractionation and ion-exchange chromatography [18]. The human SC gene was also successfully amplified for the construction of a prokaryotic expression plasmid, and monoclonal antibodies (MAbs) against SC were produced [19].
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