Acta Paediatr 81: 113-18. 1992
Immunoglobulin G subclasses in human colostrum, milk and saliva Kyuchan Kim, Margaret A Keller and Douglas C Heiner Department ?J-Pediatrics. UCLA School of Medicine, Harbor-UCLA Medical Center, Torrance. California, USA
Kim K, Keller MA, Heiner DC. Immunoglobulin G subclasses in human colostrum, milk and saliva. Acta Paediatr 1992;81:113-18. Stockholm. ISSN 0803-5253 Previous studies have suggested local production of IgG4 in human colostrum and mature milk. We extended these observations to examine all IgG subclasses in mammary secretions and in saliva, a mucosal secretion. In human colostrum and milk, the geometric mean percentages of IgG contributed by IgG2 were 44% and 43%, respectively, and by IgG4,6% in both. These percentages are significantly increased compared to the contributions in matched plasma, 29% for IgG2 and 2% for IgG4. The contribution of IgGl (47%)and IgG3 ( < 4 % ) were decreased compared to plasma which contained 64% IgGl and 6% IgG3. Similarly, in salivary secretions the percentages of IgG contributed by IgG2 and IgG4 were increased compared to serum while the percentage of IgGl was decreased. IgG3 was not measurable in any saliva specimen by the technique used. These data demonstrate that IgG subclass distribution in two separate mucosal secretions is uniquely different from that in matching plasma or serum. 0 Colostrum. IgC subclasses, milk, mucosal immunity, saliva K Kim, Harbor-UCLA Medical Center, Department of Pediatrics, Division ImmunologylAllergy. 1000 West Carson, 5-4, Torrance, CA 90509, USA
Human breast milk contains many immunologically active components that may protect suckling infants from infection. Secretory IgA is the major mucosal immunoglobulin, but there is some contribution from IgM and IgG antibodies (14)Local . mammary production of IgG4 was suggested in our previous study where it comprised 15% of total IgG in colostrum compared to 4% in matched plasma (1). In a second study of both colostrum and milk, IgG4 comprised an average of 100/0 of IgG vs 2% for plasma (2). The enhancement of IgG4 in mammay secretions was true when total IgG and albumin contents were used as the reference. Moreover, specific IgG4 to one or more of four mucosal antigens tested was clearly enhanced compared to serum in six of 27 subjects, supporting the concept of local production in the mammary gland. Because of these findings and other evidence that IgG subclasses may play an important role in mucosal immunity (5-8), the levels of all IgG subclasses were determined in mammary secretions and in another mucosal secretion, saliva. We investigated whether the proportional contribution to total IgG of each subclass in mucosal secretions was consistently different from that in the systemic circulation.
Materials and methods Specimens Colostrum was obtained with an electric suction pump (Egnell) one to five days postpartum (mean 1.8 days) from 14 lactating women. Specimens were stored at -70°C until assayed after cells and fat were removed. Milk samples were processed similarly but were collected 22-36 days postpartum (mean 29.8 days). Plasma
specimens were obtained simultaneously with colostrum and milk. All subjects signed an informed consent approved by the institutional Human Subjects Committee. Unstimulated whole saliva and matched serum were collected from seven healthy laboratory workers and were stored at - 70°C following centrifugation.
Methods IgG I , IgG2 and IgG3 were measured by commercial radial immunodiffusion (RID) kits (ICN Biomedicals, Costa Mesa, CA). The specificity of each antibody was verified by reaction with only the appropriate purified myeloma proteins from a panel comprising each immunoglobulin class and subclass. Standard curves were constructed with the WHO standard serum or a secondary standard established in our laboratory, as well as with reference standards provided by the manufacturer. Plasma or serum (5 p l ) diluted in 0.1 M phosphate buffer with 20'X fetal calf serum was placed in wells. Previous studies in our laboratory demonstrated no difference in immunoglobulin isotype values when serum vs plasma from the same subject was assayed. Colostrum and milk were applied undiluted three successive times at 10-min intervals. Thus, the volume of mucosal secretions placed in wells was three times as much as plasma or serum samples. It was demonstrated previously in our laboratory that this method increased the sensitivity of RID without affecting accuracy. To verify this technique, plasma samples were diluted three-fold further than the usual dilutions and applied three times in the same manner as mucosal secretions on a parallel plate. The concentrations of each subclass measured by these two methods differed by less than 5%. The diameter of each
1 14 K Kim et a/.
ACTA PEDIATR 8 I ( 1992)
Table I. The geometric mean levels (g/l) of IgG subclasses in colostrum (C), milk (M) and matching plasma (PLC and PLM). ~
Total IgG
IgG 1
IgG2
IgG3
IgG4
Colostrum PLC CjPLC (%)
0.0372 6.209 0.6
0.0349 2.585 1.1c
< 0.0034 0.577 < 0.4
0.0049 0.194 1.4d
0.0804 9.986 0.8
Milk PLM M/PLM (%)
0.025 1 7.546 0.3
0.0196 3.204 0.6'
~0.0016 0.786 c 0.2
0.0042 0.201 0.9
0.0469 12.280
ND < 0.05
ns ns
p" Pb
< 0.05 ns
< 0.05 < 0.05
0.4
c0.05 ns
a Paired 1-tests of mean levels in colostrum compared to milk (ND=not determined due to many unmeasurable IgG3 levels; ns=not significant); paired 1-testsof mean levels in PLC compared to PLM; mucosal/systemicratios for IgG2 in both colostrum (p < 0.0005) and milk (p
Immunoglobulin G subclasses in human colostrum, milk and saliva Kyuchan Kim, Margaret A Keller and Douglas C Heiner Department ?J-Pediatrics. UCLA School of Medicine, Harbor-UCLA Medical Center, Torrance. California, USA
Kim K, Keller MA, Heiner DC. Immunoglobulin G subclasses in human colostrum, milk and saliva. Acta Paediatr 1992;81:113-18. Stockholm. ISSN 0803-5253 Previous studies have suggested local production of IgG4 in human colostrum and mature milk. We extended these observations to examine all IgG subclasses in mammary secretions and in saliva, a mucosal secretion. In human colostrum and milk, the geometric mean percentages of IgG contributed by IgG2 were 44% and 43%, respectively, and by IgG4,6% in both. These percentages are significantly increased compared to the contributions in matched plasma, 29% for IgG2 and 2% for IgG4. The contribution of IgGl (47%)and IgG3 ( < 4 % ) were decreased compared to plasma which contained 64% IgGl and 6% IgG3. Similarly, in salivary secretions the percentages of IgG contributed by IgG2 and IgG4 were increased compared to serum while the percentage of IgGl was decreased. IgG3 was not measurable in any saliva specimen by the technique used. These data demonstrate that IgG subclass distribution in two separate mucosal secretions is uniquely different from that in matching plasma or serum. 0 Colostrum. IgC subclasses, milk, mucosal immunity, saliva K Kim, Harbor-UCLA Medical Center, Department of Pediatrics, Division ImmunologylAllergy. 1000 West Carson, 5-4, Torrance, CA 90509, USA
Human breast milk contains many immunologically active components that may protect suckling infants from infection. Secretory IgA is the major mucosal immunoglobulin, but there is some contribution from IgM and IgG antibodies (14)Local . mammary production of IgG4 was suggested in our previous study where it comprised 15% of total IgG in colostrum compared to 4% in matched plasma (1). In a second study of both colostrum and milk, IgG4 comprised an average of 100/0 of IgG vs 2% for plasma (2). The enhancement of IgG4 in mammay secretions was true when total IgG and albumin contents were used as the reference. Moreover, specific IgG4 to one or more of four mucosal antigens tested was clearly enhanced compared to serum in six of 27 subjects, supporting the concept of local production in the mammary gland. Because of these findings and other evidence that IgG subclasses may play an important role in mucosal immunity (5-8), the levels of all IgG subclasses were determined in mammary secretions and in another mucosal secretion, saliva. We investigated whether the proportional contribution to total IgG of each subclass in mucosal secretions was consistently different from that in the systemic circulation.
Materials and methods Specimens Colostrum was obtained with an electric suction pump (Egnell) one to five days postpartum (mean 1.8 days) from 14 lactating women. Specimens were stored at -70°C until assayed after cells and fat were removed. Milk samples were processed similarly but were collected 22-36 days postpartum (mean 29.8 days). Plasma
specimens were obtained simultaneously with colostrum and milk. All subjects signed an informed consent approved by the institutional Human Subjects Committee. Unstimulated whole saliva and matched serum were collected from seven healthy laboratory workers and were stored at - 70°C following centrifugation.
Methods IgG I , IgG2 and IgG3 were measured by commercial radial immunodiffusion (RID) kits (ICN Biomedicals, Costa Mesa, CA). The specificity of each antibody was verified by reaction with only the appropriate purified myeloma proteins from a panel comprising each immunoglobulin class and subclass. Standard curves were constructed with the WHO standard serum or a secondary standard established in our laboratory, as well as with reference standards provided by the manufacturer. Plasma or serum (5 p l ) diluted in 0.1 M phosphate buffer with 20'X fetal calf serum was placed in wells. Previous studies in our laboratory demonstrated no difference in immunoglobulin isotype values when serum vs plasma from the same subject was assayed. Colostrum and milk were applied undiluted three successive times at 10-min intervals. Thus, the volume of mucosal secretions placed in wells was three times as much as plasma or serum samples. It was demonstrated previously in our laboratory that this method increased the sensitivity of RID without affecting accuracy. To verify this technique, plasma samples were diluted three-fold further than the usual dilutions and applied three times in the same manner as mucosal secretions on a parallel plate. The concentrations of each subclass measured by these two methods differed by less than 5%. The diameter of each
1 14 K Kim et a/.
ACTA PEDIATR 8 I ( 1992)
Table I. The geometric mean levels (g/l) of IgG subclasses in colostrum (C), milk (M) and matching plasma (PLC and PLM). ~
Total IgG
IgG 1
IgG2
IgG3
IgG4
Colostrum PLC CjPLC (%)
0.0372 6.209 0.6
0.0349 2.585 1.1c
< 0.0034 0.577 < 0.4
0.0049 0.194 1.4d
0.0804 9.986 0.8
Milk PLM M/PLM (%)
0.025 1 7.546 0.3
0.0196 3.204 0.6'
~0.0016 0.786 c 0.2
0.0042 0.201 0.9
0.0469 12.280
ND < 0.05
ns ns
p" Pb
< 0.05 ns
< 0.05 < 0.05
0.4
c0.05 ns
a Paired 1-tests of mean levels in colostrum compared to milk (ND=not determined due to many unmeasurable IgG3 levels; ns=not significant); paired 1-testsof mean levels in PLC compared to PLM; mucosal/systemicratios for IgG2 in both colostrum (p < 0.0005) and milk (p
