Abstract
Immunoglobulins are the primary protective products in human milk and are responsible for transferring maternal pathogen memory to the infant, providing protection by binding to recognized pathogens and inhibiting virulence. To better understand potentially protective/anti-infective compounds in human milk, the establishment of human milk–tailored analytical approaches is crucial, as most contemporary analytical methods have been optimized for plasma or serum. One of the most prominent immunoglobulins in human milk is secretory immunoglobulin A (sIgA), which may be relevant for the protection of breastfed infants from harmful pathogens. Advanced sIgA detection methods can help monitor the immune status and development of the mother-infant dyad. We therefore developed an enzyme-linked immunosorbent assay (ELISA) sIgA method for the quantitative analysis of IgA plus secretory component (SC), validated with sIgA standards and substantiated by mass spectrometry (MS)–based proteomics. A very strong correlation was observed between the MS-detected IgA1 and the human milk–specific sIgA ELISA (r = 0.82). Overall, the MS data indicate that the developed human milk sIgA ELISA does not differentiate between sIgA1 and sIgA2 and is, therefore, a reflection of total sIgA. Furthermore, our MS data and the human milk–derived sIgA ELISA data are better correlated than data derived from a standard serum IgA ELISA kit (relative to MS IgA1 r = 0.82 and r = 0.42, respectively). We therefore propose our human milk–specific sIgA ELISA as an ideal quantitative indicator of total sIgA with advantages over current serum IgA ELISA kits.
Highlights
IntroductionBiomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Padualaan 8 Utrecht 3584 CH The Netherlands
Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Padualaan 8 Utrecht 3584 CH The NetherlandsNetherlands Proteomics Center, Padualaan 8 Utrecht 3584 CH The NetherlandsDanone Nutricia Research, Uppsalalaan 12 Utrecht 3584 CT The NetherlandsBeijing Institute of Nutritional Resources, Beijing 100069 ChinaChemical Biology & Drug Discovery, Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Universiteitsweg 99 Utrecht 3584 CG The NetherlandsHuman milk is a highly complex biological matrix of fats, carbohydrates and proteins, cells, bacteria, and metabolites
This is important as the production of endogenous IgGs in the infant develops after birth [28]
Summary
Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Padualaan 8 Utrecht 3584 CH The Netherlands. Chemical Biology & Drug Discovery, Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Universiteitsweg 99 Utrecht 3584 CG The Netherlands. The complexity of the human milk matrix is often overlooked and methods that have not been validated for human milk are used to assess its components. This has been shown to be problematic, in micronutrient analysis [1], and when assessing human milk glycoproteins [2]. There are varying Ig isotypes in human milk, with secretory IgA (sIgA) being by far the most abundant [3]
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