BIOCHEMICAL
MEDICINE
AND
METABOLIC
BIOLOGY
47, 189-194 (1992)
BRIEF COMMUNICATION Enzyme-Linked
Immunoassay of Haptocorrin: Analysis of Milk and Granulocytes
An enzyme-linked, sandwich-type immunoassay method was described for quantitive assay of haptocorrin. The ranges of haptocorrin concentrations measurable by the method were comparable with those by radioimmunoassay. Using the method, the mean + SD of haptocorrin was 4.83 5 1.23 pg/ml in the colostrum (4th and 5th days after parturition), 3.17 “_ 1.77 &ml in the mature milk, and 21 ng/104 granulocytes (634 ng/mg protein). Haptocorrin from milk showed a homogeneous band at 66 kDa on SDS polyacrylamide electrophoresis followed by immunoblotting, while that from the granulocytes exhibited several closely aligned bands at 80 kDa and one or two bands at around 20 kDa. o IWZ Academic PIW, I~C.
Haptocorrin (R binder), a member of the family of cobalamin-binding glycoproteins, is present in a variety of external secretions, including breast milk, saliva, and tears, and in granulocytes (1,2). The physiological role of haptocorrin remains to be studied. The tissue distribution of haptocorrin is quite similar to that of lactoferrin, an iron-binding protein with antimicrobial activity (3,4). The prevailing method for measurement of haptocorrin is radioimmunoassy (5). The present study describes an enzyme-linked immunoassay method (ELISA) for haptocorrin, its application to the measurement of this protein in colostrum, mature milk, and granulocytes. The comparison between the levels of haptocorrin and lactoferrin in colostrum and mature milk was also made.
MATERIALS
AND METHODS
Preparation of Antiserum against Haptocorrin in Human Milk
Colostrum was collected from 28 donors at the 4th or 5th day after parturition, and mature milk from another 28 donors at 4 weeks. Haptocorrin was extracted from these samples with affinity chromatography (6,7). The purity of the extract was proven by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Polyclonal antiserum was prepared by immunizing a rabbit with the purified extract, emulsified in Freund’s complete adjuvant (8). The antiserum was rendered specific for haptocorrin by absorbing it with normal human serum coupled to 189 0885-4505192 $3.00 Copyright 0 PI92 by Academic Press, Inc. All rights of reproduction in any form reserved.
190
BRIEF
COMMUNICATION
Sepharose 4B. The IgG fraction of the antiserum was prepared with diethylaminoethyl cellulose chromatography as described elsewhere (9). Enzyme-Linked
Immunoassay
Polystyrene balls (6.5 mm diameter, Ichiko Co., Japan) were coated at 4°C overnight with 10 pg/ml anti-R binder IgG in 0.05 M carbonate buffer, pH 9.5. The IgG-coated balls were immersed in 1% bovine serum albumin (BSA) in 0.04 M phosphate buffer (PBS), pH 7.4, to block the residual binding sites, and washed with PBS containing 0.5 M NaCl. The balls were then placed one each in a test tube either with 0.2 ml, 1:25 diluted milk or with purified haptocorrin in PBS with 0.1% BSA, capped, placed on a to-and-fro shaker, and incubated at 30°C for 16 h. The balls thus treated were washed twice with PBS containing 0.1% BSA. The washed balls were then incubated at 25°C for 2 h each with 0.2 ml horseradish peroxidase-labeled anti-haptocorrin IgG antibody solution, and again washed twice with PBS. The anti-haptocorrin IgG antibody was labeled with horseradish peroxidase (HRP Grade I, Boehringer-Mannheim, GmbH) as described by Nakane and Pierce (10). The HRP activity on balls was visualized by incubation at 25°C for 30 min each with a 300~~1 colorant consisting of 0.7 mg/ml 2,2-azino-di-3-ethylbenzothiazolin-6-sulfonate (Boehringer-Mannheim GmbH) and 0.008% H202 in 0.1 M citrate buffer (pH 4.0). The reaction was stopped with 3 ml of 0.1 M citrate buffer, pH 2.8, which contained 0.05% NaN3. The absorbance at 415 nm was measured with a spectrophotometer (Model 200-10, Hitachi, Japan). Each determination was carried out in duplicate. Concentrations of Lactoferrin The purification of lactoferrin from human milk was described elsewhere, as was the preparation of its antiserum (11). The levels of lactoferrin were measured by the single radial immunodiffusion method (12). Preparation of Granulocyte Lysate Granulocytes were separated from heparinized blood samples pooled from six healthy donors, using gravity sedimentation with methylcellulose (13), followed by Ficoll-Hypaque centrifugation. The granulocytes were treated with 5 mM diisopropyl fluorophosphate methylsulfonylsulfate in PBS, to inhibit the action of serine protease in the granulocytes. Granulocytes in the suspension were counted, lysed using a sonicator at 0°C for 10 s, and centrifuged at 8000 g at 4°C for 10 min. The granulocyte protein in the supematant was measured by the method of Lowry et al. (14), using bovine serum albumin as a standard. SDS-PAGE and Immunoblotting SDS-PAGE and immunoblotting
were performed
as described elsewhere (15).
191
BRIEF COMMUNICATION
w
P
2ME C--J MG
2ME (+) MG -92.5KD -66.2KD -45.OKD -31 .OKD -21.5KD -14.4KD
B
A
FIG. 1. (A) SDS-PAGE of haptocorrin purified from human milk (P) and whole human milk (W), stained by Coomassie brilliant blue. (B) Immunoblotting patterns of haptocorrin from milk (M) and the granulocyte-lysate (G) on SDS-PAGE, developed by anti-milk haptocorrin. 2ME( +) and (-) respectively indicate the presence and absence of 2-mercaptoethanol in the electrophoresed samples. Numbers at the right margin represent molecular weight markers.
RESULTS Purification
of the Milk
Haptocorrin
Haptocorrin purified from human milk showed a single band on SDS-PAGE at a position corresponding to a molecular weight of 66 kDa (Fig. 1A). However, by immunoblotting with anti-haptocorrin, the purified haptocorrin exhibited an extra band at around 80 kDa together with the main band of 66 kDa (Fig. 1B). ELBA of Haptocorrin The standard curve for the milk haptocorrin was obtained by the ELISA method, using the purified haptocorrin as a standard. The range measurable with the method was 5 to 200 rig/ml. Haptocorrin and Lactoferrin in Milk The individual distribution of haptocorrin and lactoferrin concentration in the colostrum and the mature milk is given in Fig. 2. The mean + SD of the haptocorrin in mature milk, 3.17 2 1.77 pg/ml, was 66% of that in colostrum (4.83 + 1.22 lug/ml). The corresponding value for lactoferrin was 28%. Haptocorrin in the Granulocytes The milk and granulocyte haptocorrin revealed different banding patterns on immunoblotting with the anti-milk haptocorrin antiserum (Fig. 1B). The milk haptocorrin showed a main band of 66 kDa, both without and with 2-mercap-
192
BRIEF COMMUNICATION
.. 22 \\ 1; .g.; 2Q”0 “8
I -p
MEDICINE
AND
METABOLIC
BIOLOGY
47, 189-194 (1992)
BRIEF COMMUNICATION Enzyme-Linked
Immunoassay of Haptocorrin: Analysis of Milk and Granulocytes
An enzyme-linked, sandwich-type immunoassay method was described for quantitive assay of haptocorrin. The ranges of haptocorrin concentrations measurable by the method were comparable with those by radioimmunoassay. Using the method, the mean + SD of haptocorrin was 4.83 5 1.23 pg/ml in the colostrum (4th and 5th days after parturition), 3.17 “_ 1.77 &ml in the mature milk, and 21 ng/104 granulocytes (634 ng/mg protein). Haptocorrin from milk showed a homogeneous band at 66 kDa on SDS polyacrylamide electrophoresis followed by immunoblotting, while that from the granulocytes exhibited several closely aligned bands at 80 kDa and one or two bands at around 20 kDa. o IWZ Academic PIW, I~C.
Haptocorrin (R binder), a member of the family of cobalamin-binding glycoproteins, is present in a variety of external secretions, including breast milk, saliva, and tears, and in granulocytes (1,2). The physiological role of haptocorrin remains to be studied. The tissue distribution of haptocorrin is quite similar to that of lactoferrin, an iron-binding protein with antimicrobial activity (3,4). The prevailing method for measurement of haptocorrin is radioimmunoassy (5). The present study describes an enzyme-linked immunoassay method (ELISA) for haptocorrin, its application to the measurement of this protein in colostrum, mature milk, and granulocytes. The comparison between the levels of haptocorrin and lactoferrin in colostrum and mature milk was also made.
MATERIALS
AND METHODS
Preparation of Antiserum against Haptocorrin in Human Milk
Colostrum was collected from 28 donors at the 4th or 5th day after parturition, and mature milk from another 28 donors at 4 weeks. Haptocorrin was extracted from these samples with affinity chromatography (6,7). The purity of the extract was proven by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Polyclonal antiserum was prepared by immunizing a rabbit with the purified extract, emulsified in Freund’s complete adjuvant (8). The antiserum was rendered specific for haptocorrin by absorbing it with normal human serum coupled to 189 0885-4505192 $3.00 Copyright 0 PI92 by Academic Press, Inc. All rights of reproduction in any form reserved.
190
BRIEF
COMMUNICATION
Sepharose 4B. The IgG fraction of the antiserum was prepared with diethylaminoethyl cellulose chromatography as described elsewhere (9). Enzyme-Linked
Immunoassay
Polystyrene balls (6.5 mm diameter, Ichiko Co., Japan) were coated at 4°C overnight with 10 pg/ml anti-R binder IgG in 0.05 M carbonate buffer, pH 9.5. The IgG-coated balls were immersed in 1% bovine serum albumin (BSA) in 0.04 M phosphate buffer (PBS), pH 7.4, to block the residual binding sites, and washed with PBS containing 0.5 M NaCl. The balls were then placed one each in a test tube either with 0.2 ml, 1:25 diluted milk or with purified haptocorrin in PBS with 0.1% BSA, capped, placed on a to-and-fro shaker, and incubated at 30°C for 16 h. The balls thus treated were washed twice with PBS containing 0.1% BSA. The washed balls were then incubated at 25°C for 2 h each with 0.2 ml horseradish peroxidase-labeled anti-haptocorrin IgG antibody solution, and again washed twice with PBS. The anti-haptocorrin IgG antibody was labeled with horseradish peroxidase (HRP Grade I, Boehringer-Mannheim, GmbH) as described by Nakane and Pierce (10). The HRP activity on balls was visualized by incubation at 25°C for 30 min each with a 300~~1 colorant consisting of 0.7 mg/ml 2,2-azino-di-3-ethylbenzothiazolin-6-sulfonate (Boehringer-Mannheim GmbH) and 0.008% H202 in 0.1 M citrate buffer (pH 4.0). The reaction was stopped with 3 ml of 0.1 M citrate buffer, pH 2.8, which contained 0.05% NaN3. The absorbance at 415 nm was measured with a spectrophotometer (Model 200-10, Hitachi, Japan). Each determination was carried out in duplicate. Concentrations of Lactoferrin The purification of lactoferrin from human milk was described elsewhere, as was the preparation of its antiserum (11). The levels of lactoferrin were measured by the single radial immunodiffusion method (12). Preparation of Granulocyte Lysate Granulocytes were separated from heparinized blood samples pooled from six healthy donors, using gravity sedimentation with methylcellulose (13), followed by Ficoll-Hypaque centrifugation. The granulocytes were treated with 5 mM diisopropyl fluorophosphate methylsulfonylsulfate in PBS, to inhibit the action of serine protease in the granulocytes. Granulocytes in the suspension were counted, lysed using a sonicator at 0°C for 10 s, and centrifuged at 8000 g at 4°C for 10 min. The granulocyte protein in the supematant was measured by the method of Lowry et al. (14), using bovine serum albumin as a standard. SDS-PAGE and Immunoblotting SDS-PAGE and immunoblotting
were performed
as described elsewhere (15).
191
BRIEF COMMUNICATION
w
P
2ME C--J MG
2ME (+) MG -92.5KD -66.2KD -45.OKD -31 .OKD -21.5KD -14.4KD
B
A
FIG. 1. (A) SDS-PAGE of haptocorrin purified from human milk (P) and whole human milk (W), stained by Coomassie brilliant blue. (B) Immunoblotting patterns of haptocorrin from milk (M) and the granulocyte-lysate (G) on SDS-PAGE, developed by anti-milk haptocorrin. 2ME( +) and (-) respectively indicate the presence and absence of 2-mercaptoethanol in the electrophoresed samples. Numbers at the right margin represent molecular weight markers.
RESULTS Purification
of the Milk
Haptocorrin
Haptocorrin purified from human milk showed a single band on SDS-PAGE at a position corresponding to a molecular weight of 66 kDa (Fig. 1A). However, by immunoblotting with anti-haptocorrin, the purified haptocorrin exhibited an extra band at around 80 kDa together with the main band of 66 kDa (Fig. 1B). ELBA of Haptocorrin The standard curve for the milk haptocorrin was obtained by the ELISA method, using the purified haptocorrin as a standard. The range measurable with the method was 5 to 200 rig/ml. Haptocorrin and Lactoferrin in Milk The individual distribution of haptocorrin and lactoferrin concentration in the colostrum and the mature milk is given in Fig. 2. The mean + SD of the haptocorrin in mature milk, 3.17 2 1.77 pg/ml, was 66% of that in colostrum (4.83 + 1.22 lug/ml). The corresponding value for lactoferrin was 28%. Haptocorrin in the Granulocytes The milk and granulocyte haptocorrin revealed different banding patterns on immunoblotting with the anti-milk haptocorrin antiserum (Fig. 1B). The milk haptocorrin showed a main band of 66 kDa, both without and with 2-mercap-
192
BRIEF COMMUNICATION
.. 22 \\ 1; .g.; 2Q”0 “8
I -p
