Isolation of Monoclonal Antibodies Monospecific for Bovine lC-Casein KONRAD M. KUZMANOFF, JOHN W. ANDRESEN, and CRAIG W. BEATTIE SpecIalized Center for Cancer Research and Education University of illinois SChool of Medicine at Chicago 840 South Wood Street
Chicago, IL 60612 ABSTRACT ~ovine lC-casein represents a major portion of the total protein present in milk and is required for formation of the caseinate micelles responsible for the transportation of both calcium and phosphorous. Two monoclonal antibodies directed against bovine lC-casein have been isolated. Both monoclonal antibodies are highly specific for bovine lccasein. Western analysis of denatured lccasein suggests epitope specificity is, in part, conformationally dependent Additional epitope mapping with chymosin and neuraminidase also suggest antibody binding is in the region of the amino acid sequence Pro-Thr-Thr at positions 92 to 94 and 134 to 136. (Key words: monoclonal antibodies, lccasein, milk proteins)
INTRODUCTION
Bovine lC-casein represents nearly 15% of the total protein content in mature milk (18). This relatively small casein milk protein (M = 19.5 kDa) interacts with both cx- and Ikasehts, as well as the whey protein ~-lactoglobulin (9) to form caseinate micelles that function as carriers of both calcium and phosphorous. lcCasein-dependent micelles are responsible for ~e. ~uced sensit~vity to calcium-induced preCIpItatIon. lC-Casero has been identified in human (24) and rat milk (12) and is the most evolutionarily conserved of the caseins (21). The primary stnIcture of para-lC-casein the insoluble fragment released by the acti~ of chymosin upon lC-casein, has been reported (2, 4, 8, 13, 16) and the complete amino acid sequence deduced from the reported cDNA se-
quence (14). Preliminary information on the secondary stnIcture of lC-casein is based upon predictive models (11) and may not reflect the true secondary structure. Similarly, little information is available on the quaternary structural interactions of the lC-casein-dependent micelle. One approach to the characterization of the secondary structure and quaternary interaction of a macromolecule is to use epitope-specific monoclonal antibodies (MAb) to map the surface of the molecule. Monoclonal antibodies for human (5) and rat (12) lC-casein have been reported. In both cases, the MAb were not monospecific for lC-casein. Recently, a MAb against bovine lC-casein was reported (1). However, no data was shown for purity of immunizing antigen or erossreactivity with specific bovin~ caseins or other bovine proteins, such as fibrmogen, either by competitive RIA or Western blot analysis. This report describes the isolation and characterization of two MAb specific for bovine lCcasein as a first step in the development of a lCcasein specific panel of MAb that may be used to assist in mapping the surface of this protein. MATERIALS AND METHODS Antigen Purification
Bovine cx-, ~-, and lC-casein and bovine exlactalbumin and ~-laetoglobulins A and B were obtained from Sigma Chemical Co. (St. Louis, MO). Proteins used for crossreactivity assays were also obtained from Sigma Chemical. Murine casein was a gift from A. G. MacKinlay. The purity of casein and whey protein samples used for immunization (lC-casein) and crossreactivity assays was determined by SDS-PAGE (15) on an 18% polyacrylamide gel with silver staining (17). Antibody Production
Received December 18, 1989.
Accepted April 6, 1990. 1990 J Dairy Sci 73:2741-2748
Female BALB/C mice were immunized with repurified bovine lC-Casein (St. Louis, MO) us2741
2742
KUZMANOFP ET AL.
ing RIBI adjuvant (RIBI Imrnunochemical Research, Hamilton, M1). Fusion of mouse spleen cells with myeloma cells (X63-Ag8.653) using polyethylene glycol was essentially according to the procedure of De St. Groth and Scheidegger (3) as modified by Brown (1). Viable spleen cell myeloma hybridomas were selected with HAT medium (1, 19). Antibody-producing cultures were ass~ed using affinity-purified, goat, antimouse [1 5I]F(abn fragments (Jackson Imrnunoresearch Labs., Avondale, PA) as a tracer. The F(ab'h fragments were iodinated using Iodo-Beads (Pierce Chemical, Rockford, ll..) according to the procedure recommended by the manufacturer. Radioimmunoassay (RIA) procedure was a modification of the indirect solid phase method of Howard et al. (10). Briefly, 20 ~ of the immunogen (lC-casein) at 25 Ilg/ml were applied to the wells of a polyvinyl chloride (pVC) microtiter plate (Dynatech Laboratories, Alexandria, VA) and incubated overnight at 4"C. Subsequently, the wells of the plate were washed three times with .1% bovine serum albumin (BSA) in 10 mM phosphate, 350 roM NaCI, pH 7.4 (PBS). The wells were then blocked with 10% newborn calf serum for 1 h at 25°C and then washed with .1% BSA in PBS. Hybridoma culture medium containing test antibody was applied at 20 ul/well and incubated with 80,000 cpm!well [12~:tiF(ab'h in 20 ~ at 25"C. After a I-h incubation, the wells were washed as described, dried, separated with a hot wire cutter, and counted using a model 1285 gamma counter (TM Analytic, Elk: Grove Village, ll..). Binding of antibody to test antigen was determined as the value of the observed counts (as counts per minute) corrected for nonspecific binding of the tracer antibody [antimouse [l25I]F(ab')i) in the absence of test antibody. Monoclonal cell cultures were prepared by limiting dilution subcloning (19) using dilutions of 5, 1, and .5 cells/well. These cultures were screened for antibody avidity and crossreactivity to insure homogeneity with the parent culture. Isotyplng
Isotype of the antibodies was determined using micro-Ouchterlony diffusion plates (Miles Scientific, Naperville, ll..) and confirmed Journal of Dairy Science Vol 73,
No. 10, 1990
using an isotype-subtype specific enzymelinked immunosorbent assay (ELISA), (BioRad Laboratories, Richmond, CA). Determination of Antigen Titer, Antibody AVidity, and Specificity
Initial assays were performed using hybridoma culture media. Ascites fluid from pristane primed BALB/C mice (19) was used for larger scale production of antibodies. Antibodies were purified using immobilized protein A (Pierce Chemical, Rockford, ll..) according to the procedure recommended by the manufacturer. Protein concentrations were determined according to Peterson (20). Curves for avidity and titer were generated from dilutions of antigen with fixed antibody and antibody with fixed antigen, respectively. These curves were used to determine the optimal, nonsaturating concentration of antigen and the linear portion of the binding curve of antibody for antigen. Specificity of the antibodies for K-casein was determined using both indirect RIA, (1, 10) and Western blot analysis (23). The RIA-determined specificity of anti-Kcasein antibodies for their immunogen was assessed in two stages. Antibodies that recognized bovine K-casein were initially screened against a panel of 7 antigens which included the bovine casein and whey proteins (a.-, ~-, and K-casein; a-lactalbumin, ~-lactoglobulin A, and ~lactoglobulin B) and a mixed antigen sample containing bovine actin, hemoglobin, and BSA. Antibodies binding with only bovine K-casein or showing low crossreactivity were used in a second assay for immunogen specificity. Low crossreactivity (high specificity) was defined as the ratio of binding for immunogen: nonimmunogen, which exceeded 10. In some instances, this ratio was dependent upon the concentration of antibody used for the assay. Antibodies that passed the initial assay for crossreactivity were subjected to a second, larger panel of potential antigens. The second crossreactivity panel contained 20 antigens, which included casein and whey proteins from bovine, murine, and human sources. Several bovine proteins present in serum and cell culture media were included as part of the panel in order to eIiminate MAb with general, nonspecific reactivity.
2743
MONOCLONAL ANTIBODIES FOR BOVINE lC-CASEIN
Western Blot Analysis
Antibodies with high specificity for K-casein by RIA were analyzed for binding specificity by Western blot analysis according to the method of Towbin et al. (23). The SOS-PAGE (15) gel system was modified for multiple loads of sample. Repurified bovine K-casein (10 ~g) was loaded and electrophoresed 4 cm into a 12.5%, .75-mm preparative polyacrylamide gel (75 min, .6 mNcm at constant current). Electrophoresis was stopped, and 10 J.lg of bovine 13-casein were loaded on the gel and electrophoresed 4 cm. A third sample of bovine
Chicago, IL 60612 ABSTRACT ~ovine lC-casein represents a major portion of the total protein present in milk and is required for formation of the caseinate micelles responsible for the transportation of both calcium and phosphorous. Two monoclonal antibodies directed against bovine lC-casein have been isolated. Both monoclonal antibodies are highly specific for bovine lccasein. Western analysis of denatured lccasein suggests epitope specificity is, in part, conformationally dependent Additional epitope mapping with chymosin and neuraminidase also suggest antibody binding is in the region of the amino acid sequence Pro-Thr-Thr at positions 92 to 94 and 134 to 136. (Key words: monoclonal antibodies, lccasein, milk proteins)
INTRODUCTION
Bovine lC-casein represents nearly 15% of the total protein content in mature milk (18). This relatively small casein milk protein (M = 19.5 kDa) interacts with both cx- and Ikasehts, as well as the whey protein ~-lactoglobulin (9) to form caseinate micelles that function as carriers of both calcium and phosphorous. lcCasein-dependent micelles are responsible for ~e. ~uced sensit~vity to calcium-induced preCIpItatIon. lC-Casero has been identified in human (24) and rat milk (12) and is the most evolutionarily conserved of the caseins (21). The primary stnIcture of para-lC-casein the insoluble fragment released by the acti~ of chymosin upon lC-casein, has been reported (2, 4, 8, 13, 16) and the complete amino acid sequence deduced from the reported cDNA se-
quence (14). Preliminary information on the secondary stnIcture of lC-casein is based upon predictive models (11) and may not reflect the true secondary structure. Similarly, little information is available on the quaternary structural interactions of the lC-casein-dependent micelle. One approach to the characterization of the secondary structure and quaternary interaction of a macromolecule is to use epitope-specific monoclonal antibodies (MAb) to map the surface of the molecule. Monoclonal antibodies for human (5) and rat (12) lC-casein have been reported. In both cases, the MAb were not monospecific for lC-casein. Recently, a MAb against bovine lC-casein was reported (1). However, no data was shown for purity of immunizing antigen or erossreactivity with specific bovin~ caseins or other bovine proteins, such as fibrmogen, either by competitive RIA or Western blot analysis. This report describes the isolation and characterization of two MAb specific for bovine lCcasein as a first step in the development of a lCcasein specific panel of MAb that may be used to assist in mapping the surface of this protein. MATERIALS AND METHODS Antigen Purification
Bovine cx-, ~-, and lC-casein and bovine exlactalbumin and ~-laetoglobulins A and B were obtained from Sigma Chemical Co. (St. Louis, MO). Proteins used for crossreactivity assays were also obtained from Sigma Chemical. Murine casein was a gift from A. G. MacKinlay. The purity of casein and whey protein samples used for immunization (lC-casein) and crossreactivity assays was determined by SDS-PAGE (15) on an 18% polyacrylamide gel with silver staining (17). Antibody Production
Received December 18, 1989.
Accepted April 6, 1990. 1990 J Dairy Sci 73:2741-2748
Female BALB/C mice were immunized with repurified bovine lC-Casein (St. Louis, MO) us2741
2742
KUZMANOFP ET AL.
ing RIBI adjuvant (RIBI Imrnunochemical Research, Hamilton, M1). Fusion of mouse spleen cells with myeloma cells (X63-Ag8.653) using polyethylene glycol was essentially according to the procedure of De St. Groth and Scheidegger (3) as modified by Brown (1). Viable spleen cell myeloma hybridomas were selected with HAT medium (1, 19). Antibody-producing cultures were ass~ed using affinity-purified, goat, antimouse [1 5I]F(abn fragments (Jackson Imrnunoresearch Labs., Avondale, PA) as a tracer. The F(ab'h fragments were iodinated using Iodo-Beads (Pierce Chemical, Rockford, ll..) according to the procedure recommended by the manufacturer. Radioimmunoassay (RIA) procedure was a modification of the indirect solid phase method of Howard et al. (10). Briefly, 20 ~ of the immunogen (lC-casein) at 25 Ilg/ml were applied to the wells of a polyvinyl chloride (pVC) microtiter plate (Dynatech Laboratories, Alexandria, VA) and incubated overnight at 4"C. Subsequently, the wells of the plate were washed three times with .1% bovine serum albumin (BSA) in 10 mM phosphate, 350 roM NaCI, pH 7.4 (PBS). The wells were then blocked with 10% newborn calf serum for 1 h at 25°C and then washed with .1% BSA in PBS. Hybridoma culture medium containing test antibody was applied at 20 ul/well and incubated with 80,000 cpm!well [12~:tiF(ab'h in 20 ~ at 25"C. After a I-h incubation, the wells were washed as described, dried, separated with a hot wire cutter, and counted using a model 1285 gamma counter (TM Analytic, Elk: Grove Village, ll..). Binding of antibody to test antigen was determined as the value of the observed counts (as counts per minute) corrected for nonspecific binding of the tracer antibody [antimouse [l25I]F(ab')i) in the absence of test antibody. Monoclonal cell cultures were prepared by limiting dilution subcloning (19) using dilutions of 5, 1, and .5 cells/well. These cultures were screened for antibody avidity and crossreactivity to insure homogeneity with the parent culture. Isotyplng
Isotype of the antibodies was determined using micro-Ouchterlony diffusion plates (Miles Scientific, Naperville, ll..) and confirmed Journal of Dairy Science Vol 73,
No. 10, 1990
using an isotype-subtype specific enzymelinked immunosorbent assay (ELISA), (BioRad Laboratories, Richmond, CA). Determination of Antigen Titer, Antibody AVidity, and Specificity
Initial assays were performed using hybridoma culture media. Ascites fluid from pristane primed BALB/C mice (19) was used for larger scale production of antibodies. Antibodies were purified using immobilized protein A (Pierce Chemical, Rockford, ll..) according to the procedure recommended by the manufacturer. Protein concentrations were determined according to Peterson (20). Curves for avidity and titer were generated from dilutions of antigen with fixed antibody and antibody with fixed antigen, respectively. These curves were used to determine the optimal, nonsaturating concentration of antigen and the linear portion of the binding curve of antibody for antigen. Specificity of the antibodies for K-casein was determined using both indirect RIA, (1, 10) and Western blot analysis (23). The RIA-determined specificity of anti-Kcasein antibodies for their immunogen was assessed in two stages. Antibodies that recognized bovine K-casein were initially screened against a panel of 7 antigens which included the bovine casein and whey proteins (a.-, ~-, and K-casein; a-lactalbumin, ~-lactoglobulin A, and ~lactoglobulin B) and a mixed antigen sample containing bovine actin, hemoglobin, and BSA. Antibodies binding with only bovine K-casein or showing low crossreactivity were used in a second assay for immunogen specificity. Low crossreactivity (high specificity) was defined as the ratio of binding for immunogen: nonimmunogen, which exceeded 10. In some instances, this ratio was dependent upon the concentration of antibody used for the assay. Antibodies that passed the initial assay for crossreactivity were subjected to a second, larger panel of potential antigens. The second crossreactivity panel contained 20 antigens, which included casein and whey proteins from bovine, murine, and human sources. Several bovine proteins present in serum and cell culture media were included as part of the panel in order to eIiminate MAb with general, nonspecific reactivity.
2743
MONOCLONAL ANTIBODIES FOR BOVINE lC-CASEIN
Western Blot Analysis
Antibodies with high specificity for K-casein by RIA were analyzed for binding specificity by Western blot analysis according to the method of Towbin et al. (23). The SOS-PAGE (15) gel system was modified for multiple loads of sample. Repurified bovine K-casein (10 ~g) was loaded and electrophoresed 4 cm into a 12.5%, .75-mm preparative polyacrylamide gel (75 min, .6 mNcm at constant current). Electrophoresis was stopped, and 10 J.lg of bovine 13-casein were loaded on the gel and electrophoresed 4 cm. A third sample of bovine
