Milk sIgA Research Articles

Hiv-specific secretory IgA in breast milk of HIV-positive mothers is not associated with protection against HIV transmission among breast-fed infants

To test whether secretory immunoglobulin A (sIgA) to human immunodeficiency virus (HIV) antigens in breast milk of HIV-positive women is associated with protection against HIV transmission among breast-fed infants. Nested, case-control design in which HIV-specific sIgA was measured in breast milk collected from 90 HIV-positive women enrolled in a study in Lusaka, Zambia. Milk samples were selected to include 26 HIV-positive mothers with infected infants (transmitters) and 64 mothers with uninfected infants (nontransmitters). HIV-specific sIgA was detected more often in breast milk of transmitting mothers (76.9%) than in breast milk of nontransmitting mothers (46.9%, P = .009). There were no significant associations between HIV-specific sIgA in breast milk and other maternal factors, including HIV RNA quantities in breast milk, CD4 count, and plasma RNA quantities. HIV-specific sIgA in breast milk does not appear to be a protective factor against HIV transmission among breast-fed infants.

Human milk antibodies with polysaccharide kinase activity

It was shown for the first time that a small fraction of milk secretory IgA (sIgA) is tightly bound to oligosaccharides (oligoSACs) and polysaccharides (polySACs). The ability of sIgA to phosphorylate oligo- and polysaccharides was shown to be an intrinsic property of this antibody. In contrast to known kinases, sIgAs with polysaccharide kinase activity can transfer phosphoryl group to oligo- and polysaccharides not only from [γ- 32P]ATP but can also use [ 32P] orthophosphate as a substrate of phosphorylation reaction. An extremely unusual property of polysaccharide kinase Abs is their high affinity for orthophosphate ( K m = 15–77 μM), and orthophosphate is a better substrate than ATP. Two first examples of natural abzymes (Abzs) with synthetic activity were milk sIgA with protein and lipid kinase activities. Polysaccharide kinase sIgA of human milk is the third example of natural antibodies (Abs) with synthetic activity.

Neuroendocrine and Immune Relationships in Postpartum Fatigue

Purpose To explore relationships between fatigue, depression, stress, and infection in the postpartum. Study design and methods This was a cross-sectional, correlational design with a sample of 119 new mothers at 4 to 6 weeks postpartum. Mothers completed fatigue, mood, and stress instruments as well as maternal and infant infection checklists. Morning blood and hindmilk (in breastfeeders) samples were collected. ELISA was used to measure prolactin, cortisol, melatonin, and secretory IgA (sIgA). Correlations between psychosocial, health, and biologic variables were explored. Results Fatigue was correlated with symptoms of infection in both mothers and babies, with perceived stress and postpartum stress as well as with depression. Serum prolactin was inversely associated with depression. Milk sIgA was related to milk prolactin and inversely related to stress. Milk melatonin and prolactin were inversely related, and fatigue scores were correlated with melatonin and inversely with prolactin. Clinical implications The data suggest that fatigue, stress, depression, and infection are related in postpartum mothers and that these relationships extend to levels of serum hormones and milk factors. It is possible that a cycle is established that multiplies the effects of fatigue when mothers experience stress and that this cycle of stress and fatigue may lead to immune deficits and infectious illnesses.

Lipid kinase activity of antibodies from milk of clinically healthy human mothers

We have shown recently that polyclonal human milk sIgA contains a subfraction of antibodies (Abs) tightly bound to unusual minor milk lipids containing sialic acid. Here, we show that a small subfraction of milk IgG is tightly bound to the similar or the same minor lipids. The ability of small fractions of sIgA and IgG from human milk to phosphorylate selectively two minor lipids in the presence of [γ- 32P]nucleoside triphosphates was shown here for the first time to be an intrinsic property of these antibodies. In contrast to known kinases, antibodies with lipid kinase activity can transfer phosphoryl group to lipids not only from ATP but also from other different nucleotides (dATP, GTP, dGTP, UTP, TTP) with comparable efficiencies (30–100%). To our knowledge, there are no examples of enzymes using orthophosphate as a substrate of phosphorylation reactions. An extremely unusual property of lipid kinase Abs is their high affinity for orthophosphate ( K m = 1.6–5.6 μM) and capability to phosphorylate minor lipids using [ 32P] orthophosphate as donor of phosphate group. The relative specific activity and affinity of abzymes for orthophosphate and ATP depend significantly on donor milk. However, the levels of Ab-dependent phosphorylation of lipids for all Abs in the case of ATP (100%) and orthophosphate (60–80%) as substrates are comparable. The first example of natural abzymes with synthetic activity was milk sIgA with protein kinase activity. Most probably, lipid kinase sIgA and IgG of human milk are the second example of Abs with synthetic activity.

Secretory antibodies against Giardia intestinalis in lactating Nicaraguan women

Secretory IgA (sIgA) antibodies are important in the host defence against the intestinal protozoan parasite Giardia intestinalis. However, few antigens have been identified. In this study 100 milk and saliva samples from lactating women, living in an endemic region (León, Nicaragua), were screened for the presence of antibodies against G. intestinalis. Most milk and saliva samples contained anti-Giardia antibodies (59% and 52%, respectively), with a mean sIgA content 50 times higher in milk than in saliva. The positive samples reacted with trophozoite membrane, flagella and cytoplasmic antigens. Western blot analysis showed that milk and saliva anti-Giardia sIgA recognized up to 16 different Giardia proteins in the molecular weight region 20-165 kDa. Two-dimensional Western blotting showed that the major immunoreactive proteins were the same as the immunoreactive proteins identified by serum from acute giardiasis patients in a non-endemic country. The major difference was a stronger reactivity against the variant surface proteins (VSPs) in the milk samples. Milk sIgAs also recognized recombinant Giardia proteins such as alpha-1 giardin, ornithine carbamoyl transferase, VSP-4EX, arginine deaminase and alpha-enolase. These antigens will be important targets in the development of new immunodiagnostic tools and vaccines.

Neuroendocrine and Immune Relationships in Postpartum Fatigue

To explore relationships between fatigue, depression, stress, and infection in the postpartum. This was a cross-sectional, correlational design with a sample of 119 new mothers at 4 to 6 weeks postpartum. Mothers completed fatigue, mood, and stress instruments as well as maternal and infant infection checklists. Morning blood and hindmilk (in breastfeeders) samples were collected. ELISA was used to measure prolactin, cortisol, melatonin, and secretory IgA (sIgA). Correlations between psychosocial, health, and biologic variables were explored. Fatigue was correlated with symptoms of infection in both mothers and babies, with perceived stress and postpartum stress as well as with depression. Serum prolactin was inversely associated with depression. Milk sIgA was related to milk prolactin and inversely related to stress. Milk melatonin and prolactin were inversely related, and fatigue scores were correlated with melatonin and inversely with prolactin. The data suggest that fatigue, stress, depression, and infection are related in postpartum mothers and that these relationships extend to levels of serum hormones and milk factors. It is possible that a cycle is established that multiplies the effects of fatigue when mothers experience stress and that this cycle of stress and fatigue may lead to immune deficits and infectious illnesses.

Micronutrient deficiency and supplementation in Indonesian infants. Effects on immune function.

Health of infants in developing countries is constantly challenged by pathogens. Unfortunately, the immune system of newborn infants is immature, developing during the first year of life to attain adequate immunocompetence at about one year of age. During the first few months of life, maternal antibodies (especially IgG) acquired in utero circulate and protect the newborn. In addition, via breast milk, the infant acquires considerable amounts of immunoglobulins (mostly sIgA) and possibly other humoral and cellular components of the mother’s immune defence. The protective effect of sIgA in breast milk is restricted to the gut. The precise role of the other immunomodulatory components of breast milk, remains unclear.

Induction of functional secretory IgA responses in breast milk, by pneumococcal capsular polysaccharides.

Capsule-specific secretory IgA (s-IgA) in breast milk may enhance protection against pneumococcal disease in infants. After immunization of 3 lactating mothers with 23-valent polysaccharide vaccine, specific s-IgA, but not IgG, increased by >2-fold in milk of at least 1 subject for 6 of 7 serotypes. The s-IgA was predominantly IgA1, in secretory form, and highly specific with avidity distinct from serum IgA and IgG. Milk whey from 2 immunized women supported dose- and complement-dependent killing of Streptococcus pneumoniae serotypes 19F and 14 by human neutrophils, as did purified s-IgA to serotype 19F. These data reveal that capsule-specific human s-IgA in breast milk can initiate killing of S. pneumoniae, providing proof of concept that vaccine-induced human mucosal s-IgA can support functional bactericidal activity. Determining the biologic role for s-IgA in killing and inhibiting adherence of S. pneumoniae in vivo will contribute to the development of mucosal vaccines against S. pneumoniae.

Breast milk transmission of HIV-1. Laboratory and clinical studies.

Breast milk transmission of HIV-1 can occur at any time during the entire duration of breastfeeding. The risk of late postnatal transmission (after 2.5 months of age) is 3.2 per 100 child/years of breastfeeding, but early postnatal transmission may be more frequent than previously thought. Exclusive breastfeeding has been suggested to be less risky than mixed feeding. Breast milk contains immunoactive cells, antiinfectious substances, immune globulins, cytokines, and complement factors. HIV-1 has been found in breast milk from HIV-infected mothers as both cell-associated and cell-free particles. Mastitis has been suggested to facilitate transmission of HIV-1. The portal of entry of HIV-1 in the infant mucosae may involve tonsilar lymphoepithelium, M cells, and enterocytes from intestinal surfaces. Anti-HIV-1 SIgA and IgM in breast milk and intestinal fluid may confer some protection. Transmission of HIV-1 by breastfeeding has to be taken into account in designing interventions to reduce/prevent mother-to-child transmission in developing countries.

Characterization of the mucosal immune response in breast milk after peroral immunization of chimpanzees ( Pan troglodytes) with Streptococcus mutans

The characteristics of the mucosal immune response to Streptococcus mutans cells, antigen A, antigen B, glucosyltransferases and glucan-binding proteins were examined in four pregnant chimpanzees that had been immunized perorally with Strep. mutans. Six pregnant chimpanzees served as non-immunized controls. None of the chimpanzees harbored S. mutans. Samples of milk were collected from all animals throughout the experiment. Peroral immunization resulted in an overall 17-fold median increase in SIgA in milk. Although SIgA1 comprised almost two-thirds of milk SIgA, Strep. mutans whole-cell antibody activity was contained predominantly in the SIgA2 subclass. The difference between the specific activities of anti- Strep. mutans SIgA1 and SIgA2 antibodies compared over time reached the borderline of statistical significance ( p=0.08). The avidity of anti- Strep. mutans antibodies was low in three of four chimpanzees and there was no evidence of affinity maturation. SIgA antibodies from the milk of all four immunized chimpanzees recognized antigen A. In three animals these antibodies were restricted to the SIgA1 subclass and, in one animal, anti-A antibodies were confined to SIgA2. Antibodies from all of the immunized chimpanzees recognized degradation products of antigen B in both the SIgA1 and the SIgA2 subclasses. Only two of four immunized chimpanzees responded to glucosyltransferases and these antibodies were restricted to the SIgA1 subclass. None of the chimpanzees responded to the 74-kDa glucan-binding protein. However, three animals produced SIgA1 antibodies against the 59-kDa glucan-binding protein and two of these also produced SIgA2 antibodies against this protein.

Secretory immunoglobulin A is a component of the human milk fat globule membrane.

Secretory immunoglobulin A (sIgA), the predominant antibody fraction of human milk, represents a major protective factor against neonatal infection. Until now, sIgA had been identified only in the humoral fraction of human milk. The aim of this study was to assess whether in human milk sIgA is also associated with the milk fat globule (MFG). Using anti-sIgA-agglutinated human MFG and immune fluorescence microscopy, we could demonstrate that sIgA is, in fact, associated with human MFG. In human MFG membranes separated by Western blotting, sIgA bands were specifically stained, suggesting that sIgA is strongly associated with the human MFG membrane. This may be of physiologic relevance, inasmuch as earlier we could show that a quantity of undigested and functional human MFG are in fact found in the stools of the newborn. This would allow an additional extension of the protective mechanisms of sIgA throughout the whole intestine.

Relaxation Training and Breast Milk Secretory IgA

To evaluate the hypothesis that breast-feeding women who participate in relaxation training will have increased secretory IgA (sIgA) levels in their breast milk compared with women not receiving training. Nonrandomized control trial of a convenience sample. Women were recruited from the postpartum floor of a university teaching hospital. The intervention took place in the women's homes. Women in the first 48 hours after delivery who were planning to breast-feed their healthy newborn infants for at least 8 weeks were approached for enrollment. Women were excluded if they had previous experience with relaxation training. At 4 to 6 weeks postpartum, we enrolled 38 women still breast-feeding their infants. Women were allocated into 3 groups. Women in group 1 were taught relaxation and had breast milk samples collected before and after the teaching. Women in group 2 had conversation with similar breast milk sample collection, and women in group 3 had 1 breast milk sample collected. Women in group 1 were encouraged to practice the relaxation once or twice a day for 2 weeks, and a second visit was made to all mothers with repeated breast milk collections. Women who were still breast-feeding at 6 to 8 weeks after study end had a final breast milk sample collected. Breast milk was analyzed for secretory IgA levels. Stress was assayed using the Symptom Checklist-90-R and open-ended questions. There was no difference in sIgA levels among the 3 groups at any time. Women who reported stress present between visit 1 and visit 2 increased their sIgA levels at the final sample collection (+0.16 g/L) compared with women who reported no stress (-0.09 g/L; P= .03). The ratings of success in relaxation in women in group 1 were related to the following sIgA levels in sample 4: poor relaxation, 0.67 g/L; fair relaxation, 0.41 g/L; good relaxation, 0.35 g/L; and very good, 0.30 g/L (P= .006). Self-reported stress appears to increase breast milk sIgA levels. Success at relaxation was inversely related to sIgA levels in the group learning relaxation.

Human immunodeficiency virus type 1 IgA antibody in breast milk and serum.

Breast-feeding plays a potentially significant role in mother to child transmission of human immunodeficiency virus type 1 (HIV-1). The additional transmission risk attributable to breast-feeding and the factors that enhance or inhibit transmission are presently unknown. One mechanism by which breast milk might inhibit HIV-1 transmission is the presence of specific antibodies directed against HIV-1 in breast milk of seropositive mothers. In this study serum and breast milk samples from women in Nairobi, Kenya, were tested to determine the prevalence of HIV-1 IgA antibodies. A Western blot test developed in our laboratory was used to detect anti-HIV-1 immunoglobulin A in serum and anti-HIV-1 secretory IgA (sIgA) in breast milk. Ninety-four percent of 63 HIV-1 seropositive women had anti-HIV-1 IgA in serum and 59% had anti-HIV-1 sIgA in their breast milk. No significant associations with maternal characteristics or serum anti-HIV-1 IgA or IgG banding patterns and the presence of anti-HIV-1 sIgA in breast milk were found. No protective effect of anti-HIV-1 sIgA was seen regarding mother to child transmission; however, further studies are necessary to determine the effect of these antibodies in maternal sera or in breast milk on the efficacy of HIV-1 transmission.

Characterizations and psychoneuroimmunologic implications of secretory immunoglobulin A and cortisol in preterm and term breast milk

This article combines data from two separate investigations. One study examined relationships between psychosocial factors and preterm milk immune variables. The other examined relationships between psychosocial and breastfeeding satisfaction factors and perceived milk sufficiency in term mothers. Milk samples were collected on the fifth postpartum day and frozen. Both studies collected data on anxiety, but other psychosocial variables differed. Mood states and social support were studied in preterm mothers, while breastfeeding satisfaction, milk maturation, and infant suckling characteristics were studied in term mothers. Milk samples were assayed for secretory immunoglobulin A (sIgA) and cortisol. sIgA and cortisol levels were higher in the milk from preterm mothers and were inversely related to each other in both preterm and term milk. In preterm mothers, anger and vigor were positively correlated with higher milk sIgA. The findings suggest that cortisol is present in milk and may potentially influence the secretion of milk sIgA. The relationships that were found when comparing psychosocial, dyadic, and stress factors with milk sIgA and cortisol are provocative and suggest new paradigms for studying lactation.

Salivary anti‐RSV IgA antibodies and respiratory infections during the first year of life in atopic and non‐atopic infants

Salivary SIgA antibodies against RS virus were studied in 105 children during the first year of life. The infants were divided into groups according to their risk of atopy. At birth 13 neonates showed measurable amounts of SIgA to RS virus. In another 26 children specific antibodies were detected but in concentrations too low for quantitative analysis. During the first year of life this increased to 29 antibody-positive samples with measurable amounts of antibody and 39 with concentrations too low for quantitative determination. At this time 8 children of the high risk group had developed symptoms of allergy. None of these children had measurable amounts of SIgA anti-RSV in their saliva. In comparison, 10 of the remaining 26 high risk infants without symptoms of allergy did have such antibodies. Atopic infants had significantly more respiratory infections during the first year of life than nonatopic infants. The avidity of SIgA anti-RSV in neonatal samples was significantly higher than avidity determined in breast milk SIgA but comparable to the avidity of serum IgG. During the first year of life a continuing decrease of salivary SIgA avidity was observed.

Secretory Anti-Giardia lamblia Antibodies in Human Milk: Protective Effect Against Diarrhea

To determine whether anti-Giardia lamblia secretory IgA (sIgA) antibodies in human milk protect infants from acquisition of or symptoms associated with Giardia infection. One hundred ninety-seven Mexican mother/infant pairs were followed weekly from birth for diarrheal disease and feeding status. Infant stool specimens were collected weekly and were cultured for bacterial pathogens and tested for Giardia and rotavirus by enzyme-linked immunosorbent assay. Maternal milk samples were collected weekly for 1 month postpartum and monthly thereafter. To determine the protective effect of anti-Giardia sIgA in milk against infection and against diarrhea due to Giardia, milk samples from mothers of infected infants and appropriately matched controls were assayed for anti-Giardia sIgA by enzyme-linked immunosorbent assay. Asymptomatic, infected infants ingested significantly (P = .046) higher amounts of milk anti-Giardia sIgA compared with symptomatic, infected infants. However, milk anti-Giardia sIgA concentrations did not differ between Giardia-infected and noninfected infants. The amount of anti-Giardia sIgA in human milk was associated with prevention of symptoms of diarrhea due to Giardia, but not with acquisition of the organism.

Lysozyme, lactoferrin, and secretory immunoglobulin A content in breast milk: influence of duration of lactation, nutrition status, prolactin status, and parity of mother

Milk lactoferrin (LF), lysozyme (LZ), and secretory IgA (sIgA) were measured cross-sectionally in 127 Zaïrean mothers, lactating greater than or equal to 18 mo. The 54 urban mothers were of marginal nutrition status [body mass index (BMI) 22.6 +/- 2.6 kg/m2 and albumin 33.1 +/- 4.5 g/L]. The neighboring rural mothers were of significantly (P less than 0.001) poorer nutrition status (BMI 20.5 +/- 2.2 kg/m2 and albumin 27.7 +/- 5.4 g/L). In both urban and rural mothers, as lactation progressed LF decreased by 33% and 55% whereas sIgA remained unchanged and LZ steadily increased. There was more LZ and sIgA in rural milk, contrasting with the poorer maternal nutrition. As calculated from individual milk yields, the urban infants were fed daily with twice as much LF and sIgA but with similar amounts of LZ as were the rural infants. In the early stage of lactation, the milk of both groups of Zaïrean mothers contains more sIgA than that of a group of west European (Belgian) mothers (n = 20), but the LF and LZ contents were rather similar.

INHIBITION OF ADHESION OF ENTEROTOXIGENIC E.OOLT (ETHC) TO HUMAN SMALL INTESTINAL EOTEROCYTES BY HUMAN MILK: EVIDENCE FOR A SPECIFIC ANTIBODY MEDIATED MECHANISM

Adhesion of ETEC to the small intestinal mucosa is an essential event in colonisation and the development of diarrhoeal disease. Special classes of protein fimbriae termed Colonisation Factor Antigens (CFA) promote mucosal adhesion of ETEC. We have previously demonstrated secretory IgA (sIgA) antibody to ETEC CFA/I in human milk. In this study we tested the anti-adhesive effect of such antibody using an in vitro isolated human enterocyte adhesion assay. Bacterial adhesion was quantitatively assessed by counting the nunber of bacteria adhering to the brush border of 100 enterocytes and an adhesion index defined as the number of adherent bacteria/brush border. ETEC strain M109C2 producing CFA/I had an adhesion index of 2.2 (range 2.1-2.4) whereas the same strain lacking CFA/I had an adhesion index of 0.02 (range 0-0.03). At a dilution of 1:10, 6 human milk specimens from Sri Lankan women with demonstrable CFA/I-specific sIgA antibody produced a marked reduction in adhesion index from 2.2 to 0.16 (range 0.05-0.25) whereas 6 milk specimens with no demonstrable CFA/I-specific sIgA showed no reduction in adhesion index (mean 2.2, range 2.1-2.6). These results strongly suggest an anti-adhesive protective role for the CFA/I-specific sIgA in human milk. The presence of anti-CFA/I antibody only in milk from Sri Lankan women and not in any of 75 Caucasian, U.K. women tested presumably reflects prior exposure only of the Sri Lankan mothers to CFA/I-positive ETEC.

Mucosal immunity.

Mucosal defense is provided by a number of host factors countering the specific virulence factors of the many microorganisms infecting the mucous membranes. Secretory IgA antibodies presumably play an important role. Increase of the sIgA antibodies may most advantageously be attained by parenteral immunization, following mucosal priming. This was demonstrated in a rat model, where it was also noted that antigen injection into PP induced high milk IgA antibody levels. In man, parenteral vaccination against polio increased the sIgA antibody levels in the milk of mothers previously exposed naturally to the poliovirus. The response was relatively short-lived. In the previously unexposed, there was little or no response. By contrast peroral immunization with live poliovirus vaccine did not increase, or even decrease, the milk sIgA poliovirus antibody levels. Although salivary sIgA antibodies against antigens of colonizing E. coli appear during the first days of life, they are slow to increase. This deficiency is richly compensated for by all the sIgA antibodies that are provided the baby through the milk. No transfer of dimeric IgA into the milk could be shown in lactating rats, in contrast to what has been reported in mice. There is no evidence for a contribution to milk sIgA from serum in man. Close to parturition, human milk often contains some 7S IgA and various sizes of free SC, in addition to the dominating 11S sIgA. A few days later there is almost exclusively monomeric SC and 11S sIgA. IgG antibodies also play a role at the mucosal level. IgG2 antibodies against the bacterial polysaccharide capsule are as slow to appear as sIgA in ontogeny, possibly explaining the prevalence of infections with encapsulated bacteria and the poor response to polysaccharide vaccines in early childhood. Other defense factors preventing infections by way of mucous membranes may be important. Thus, oligosaccharides present in human milk seem to specifically prevent pneumococcal attachment to retropharyngeal cells. This anti-attachment capacity, in addition to that provided by milk and salivary IgA antibodies, may explain why breast-fed babies have less otitis media than formula-fed ones.

Sequential purification of lactoferrin, lysozyme and secretory immunoglobulin A from human milk

Human milk protects the immunologically immature infant from gastro-intestinal invasion of pathogenic microbes by providing significant quantities of 3 proteins: sIgA, LF, and Lys [l-4]. sIgA and LF constitute 5% each of the total milk protein, while Lys represents < 0.5% [l]. LF, a 75 000-80 000 Mr single polypeptide chain [5], binds 2 atoms of ferric iron with a K1 260 x larger than that of serum transferrin [6]. By sequestering iron so strongly, LF acts to deprive the gut flora of the free iron required for proliferation and is strongly bacteriostatic in vitro for many common gut-associated pathogens [7,8]. Lys (EC 3.2.1.17; N-acetylmuramide glycanohydrolase) is an enzyme which hydrolyzes the glycosidic bonds between N-acetylglucosamine and N-acetylmuramic acid, components of the bacterial cell wall peptidoglycan. Breast milk [Lys] are 3-10 mg% compared with serum levels of 0.6-l mg%, and the human milk enzyme [l] has been reported to have a specific activity 3.5 X that of egg white lysozyme [4]. The mechanism of its role in the defense of the infant gut from pathogens, yet to be fully explained, most probably is intricately interrelated with antibody and possibly complement components for full effectiveness [9]. Our long-range purpose is to systematically study such concertive or synergistic relationships involving host defense factors in human milk. Both LF and Lys represent very effective, however non-specific, antimicrobial activities. However, milk sIgA confers upon the infant specific antibacterial, antiviral, antitoxic antibodies [lo] reflective of the spectrum of the mother’s antigenic experience. sIgA in milk is 150 mg% [I]. The intake of specific antimicrobial antibodies is quite high throughout the nursing period thus providing passive immunity during the time in which the infant immune system is maturing. By mechanisms which are not yet clear, sIgA potentiates the antibacterial activity of LF 171. We describe here a protocol for sequential isolation of all 3 of these proteins from the same sample of pooled human milk. The very high isoelectric pH-values of Lys and LF enable them to be separated from the remainder of milk (whey) proteins and from each other in a single chromatographic separation on heparin-Sepharose, a technique described in [ 1 l] for the isolation of pure LF. From the remaining proteins, pure sIgA has been isolated. It is of great interest to begin to examine the factors in human milk which, both singly and in concert, protect against a battery of potential pathogens.