Isolation of Monoclonal Antibodies Monospecific for Bovine a-Lactalbumin KONRAD M. KUZMANOFF, JOHN W. ANDRESEN, and CRAIG W. BEATTIE SpecfaJized Center for Cancer Researc? .and Edu~n University of Illinois School of MediCIne at Chicago

Chicago 60612

against bovine a-lactalbumin (1, 2, 18), although some crossreactivity with bovine 13casein was noted (1). Heterogeneity of polyclonal antibodies generally reduces epitope specificity, which limits the ability to characte~­ ize antigenic determinants. Monoclonal antibodies (MAb) against rat (16) and human alactalbumin (5) have been generated also; however, the latter antibody exhibited significant crossreactivity with human J3-casein. As part of our studies on hormonal and developmental regulation of casein and whey proteins during lactogenesis, we have developed panels of MAb to investigate structural aspects of these proteins and to quantitate their .synthesis d~g lactogenesis. This report descnbes charactenstics of MAb specific for bovine a-lactalbumin.

ABSTRACT

Monoclonal antibodies were generated against purified bovine a-lactalbumin. Three antibodies reactive only to a-lactalbumin were selected. Two of the antibodies appeared to be species monospecific and bound with only bovine a-lactalbumin. The third monoclonal recognized both bovine and human a-lactalbumin with a 2:1 preference for bovine a-lactalbumin. Two of the monoclonals (48-60, 48-68) bound to different epitopes with high affinity (Ka = 8 x 1011, 5.3 X 10 12 M-l). The third monoclonal (48-22), which was of isotype 19A, had an epitope similar to that of 48-60 but with an apparent affinity of -4 x 1012 M-l. These antibodies should prove extremely useful adjuncts in structural studies of bovine alactalbumin. (Key words: bovine, a-lactalbumin, monoclonal antibodies)

MATERIALS AND METHODS AnUbody ProductIon

INTRODUCTION

Considerable research has been directed toward characterization of the bovine milk whey protein a-lactalbumin. This small mol~ar weight metalloprotein (Mr -14.1 kDa) functions as a regulatory component of the Golgi localized galactosyl transferase system responsible for lactose synthesis (8) and shares greater than 40% amino acid sequence identity with c-type lysozyme (3, 12, 21). a-Lactalbumin contains one high affmity calcium binding site (7, 13, 15) which is dissimilar to the calcium-binding site~ of other known low molecular weight calcium binding proteins (24). Polyclonal antibodies have been produced with relatively high sensitivity and specificity

Received November 20, 1989. Accepted May 11, 1990. 1990 J Dairy Sci 73:3077-3083

Female BALB/C mice were immunized with repurified bovine a-lactalbumin (Sigma Chemical Co., St. Louis, MO). Mouse spleen cells were fused with myeloma cells (X63-Ag8.653) using polyethylene glycol (4, 6) and selected with HAT (hypoxanthine-aminopterin-thymidine) medium (4, 20). Monoclonal cell cultures were prepared by limiting dilution subcloning (4). Cultures were screened for antibody avidity and crossreactivity to ensure homogeneity with the parent culture. Antibody-producing cultures were assayed using goat anti-mouse [l25I]F(ab'h fragments (Jackson Immunoresearch Labs., Avondale, PA) according to the indirect solid phase radioimmunoassay method of Howard et al. (14). Antibody binding to test antigen was determined as the value of the observed counts corrected for nonspecific binding of the tracer antibody (anti-~ouse [l25I]F(ab'h) in the absence of test antIbody (counts per minute, 10% fetal bov~e seru~). Antibody isotype was detemuned usmg micro-Ouchterlony diffusion plates (Miles Sci-

3077

3078

KUZMANOPF ET AL.

entific, Naperville, IL) and confinned using an isotype- and subtype-specific enzyme-linked immunoassay (BioRad Laboratories, Richmond, CA). Determination of Antigen Titer, Antibody Avidity, and SpeCificity

Curves for avidity and titer were generated from dilutions of antigen with fixed concentration of antibody and from dilutions of antibody with fixed concentration of 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 antibodies for a-lactalbumin was determined using both indirect radioimmunoassay (14) and Western blot analysis (25). Antibody affinity for bovine a-lactalbumin was determined according to the method of Friguet et al. (10) and plotted according to Scatchard (22). The RIA determined specificity of anti-alactalbumin antibodies for their immunogen was assessed in two stages. Antibodies that recognized bovine a-lactalbumin were initially screened against a panel of seven antigens, which included the bovine caseins (a-, 13-. and lC-casein) and whey proteins (a-lactalbumin, ~­ lactoglobulin A, J3-lactoglobulin B) (Sigma Chemical Co., St. Louis, MO) and a mixed antigen sample containing bovine serum albumin (BSA), bovine actin, and hemoglobin. Antibodies binding with only bovine a-lactalbumin or showing low cross reactivity were used in a second assay for immunogen specificity. Low crossreactivity (high specificity) was defined as a ratio of binding for immunogen to nonimmunogen that exceeded 10. The second crossreactivity panel contained 22 antigens, which included several proteins present in serom and cell culture media as well as casein and whey proteins from bovine, murine, and human sources. Competitive RIA (4, 9, 10), were performed using one monoclonal ([1251]48-60) at fixed concentration (80,000 ± 2000 cpm/well, in 20 1Jl: 9.8 ± .2 ng, .49 ± .01 ~g/ml, 3.7 ~Ci/~g), as the labeled, competing antibody. Concentration of unlabeled test antibody was varied from 5 x 10-5 to 1.25 x 1()3 ~g/ml. Test and competing antibodies were applied simultaneously and incubated with previously bound antigen overnight at 4°C. Antigen concentration was 15 Ilg/ml (20 J.ll/well). Iournal of Dairy Science Vol. 73,

No. II, 1990

Washes and protein blocking steps were identical to those used in the standard solid phase assay. Western blot analysis of antigen-antibody specificity was performed essentially as described by Towbin et al. (25). A BioRad Trans Blot (Richmond, CA) was used for electrophoretic transfer of SDS-PAGE (17) separated samples to nitrocellulose paper (NTCP). Transfer conditions were 100 V, 4"C, overnight with a buffer of 25 roM Tris, 192 mM glycine, pH 8.3, 20% (vol/vol) methanol, and .1% (voV vol) lithium dodecyl sulfate. Monoclonal antibody at titers determined by RIA were incubated with NTCP after blocking with 10% (wt/vol) BSA. Radiolabeled goat anti-mouse [125I]F(abn fragments were used as secondary antibody and antibody-antigen binding was visualized by autoradiography. RESULTS AND DISCUSSION Antigen SpeCificity

Purity of casein and whey protein samples used for immunization (a-lactalbumin) and crossreactivity assays was determined by SDSPAGE (17) on an 18% polyacrylamide gel and visualized by silver staining (19). Although lower Mr bands were present in a, ~, and lCcasein, no crosscontamination between casein and whey proteins was apparent. Minor bands present in each of the caseins were ascribed either to degradation products or genetic variants (Figure 1). Fourteen hybridomas with high affinity for bovine a-lactalbumin were isolated. Ten monoclonals were specific for a-lactalbumin with no significant binding to the bovine caseins (fable 1). Monoclonal antibody 48-16 bound to both bovine ~-casein and ~­ lactoglobulin, and MAb 48-17 and 48-25 bound to both a-casein and ~-lactoglobulin in addition to a-lactalbumin. Monoclonal 48-27 bound to a- and ~asein. Five of the MAb identified as having high affmity for bovine a-lactalbumin during the initial screen (fable 1) showed low or no binding to bovine caseins and ~-lactoglobulin and were rescreened in a second panel. Three MAb (48-60, 48-68, and 48-22) were selected that exhibited an antigen specificity that exceeded 50:1 for a-lactalbumin over other antigens tested (fable 2). Two of the MAb (48-60 and

3079

MONOCLONALS TO BOVINE a-LACfALBUMIN

8 9 1011 -

.

45 " 31 ,29 , 24

;l

;'

......, •..... ~·421 ..

lh· Figure 1. Purity of bovine casein and whey milk proteins used for immunization (a-lactaIbumin) and crossreactivity assays. Lanes I, 5, and 11: molecular weight III81'kers with molecular weights as indicated in tbc 1IllUgins; lane 2: a-casein; lane 3: jkasein; lane 4: K-easein; lane 6: alactalbumin; lane 7: a-laeta1bumin (human); lane 8: ~ lactoglobulin A:, lane 9: ~lactoglobulin B; lane 10: calmodulin.

48-68) are apparently monospecific for bovine (X-lactalbumin with no significant affinity for bovine or murine caseins or human (X-lactalbumin. Both 48-60 and 48-68 are of ootype IgGl. The third monoclonal (48-22) differed from 48-60 and 48-68 in its species specificity. Antibody 48-22, isotype IgA, recognized both b0vine and human (X-lactalbumin with a 2:1 preference for bovine a-lactalbumin. Additional studies, which used concentrations of competing antigens that exceeded 25 Jlg/ml (500 ng

applied/well), showed that these three MAb recognized only (X-lactalbumin. Despite the similarity in amino acid regions (3) and cDNA (11, 23, 26) sequence between lysozyme and alactalbumin, no binding to chicken lysozyme was observed, even at saturating concentrations of antigen (25 Jlg/ml). Thus, the epitopes of (Xlactalbumin recognized by these antibodies are not present in lysozyme, either because the regions recognized are not part of the lysozyme amino acid sequence, or because binding is dependent upon the topology of the native protein. These observations suggested that, except for the binding of MAb 48-22 with human alactalbumin, these monoclonals appear monospecies-specific for bovine a-lactalbumin. Western blot analysis with MAb 48-60 and 48-68 revealed no change in the binding patterns observed by RIA (Figure 2). Binding Affinity

Each of the three MAb selected by the second roood of screening (fable 2) bound (Xlactalbumin at concentrations ranging from .1 to 25 J1g/ml (2 to 500 ng, Figure 3). Concentrations of antigen above 5 Jlg/ml (100 ng) resulted in a saturating response (plateau). Half maximal binding for each antibody (Figure 3) was approximately 1 Jlg/ml (20 ng). Binding at the lowest antigen concentration assayed (2 ng, .1 Jlg/ml) exceeded backgrOWld values by at

TABLE 1. Specificity of monoclonal antibodies for a-Iactalbumin. 1 Bovine caseins Antibody 48-3 11 14 16 17 22 25 27 53 54 60 68 73 50-24

a. 11 3

0 0 1349 9 589 995 0 14 0 0 4 Jl

Bovine

p

K

11 0 15 4202 35 40 10 4399 14 125 4 136 9 14

3 0 6 0 0 27 0 11 0 5 0 27 0 0

3693 2844 2197 6282 4175 2096 4085 393 2843 4264 5415 7669 683 43]9

~Lactoglobulin

Human

a-lactalbumin 758 2701 2409 7464 4977 1248 3962 0 2918 61 7 0 0 7557

A

284 2489 2615

6990 3888 9 4162 27 3246 12 4 4 0 497

B 258 1763 2523 7062 3620 41 4154 53 2817 35 15 20 13 375

IVaJues are counts per minute, corrected for fetal bovine serum blank. Assayed in triplicate using 25 J1g/ml antigen. Journal of Dairy Science Vol. 73,

No. II, 1990

3080 TABLE

KUZMANOPF ET AL.

2. Antibody specificity: crossreactivity panel. I Antibody

48-22 (1) 2

Antigen Actin Albumin Fibrinogen Fibronectin "(-Globulins Hemoglobin Transferrin Lactoferrin cx-Lactalbumin ~Lactoglobulin ~Lactoglobulin

0

A B

Lactoperoxidase lC-Casein I-Casein

a..

~Casein

ES 2 Murine caseins ~Casein (H)2 lC-Casein (H) Lactoferrin (H) cx-Lactalbumin (H) Lysozyme (C)

48-60

22

(0)

0 2

(0)

11

(1)

0

0 0 16 1 4 0 4108 4 15 9 1 0 3 0

(I)

(2) 15 1 (1) (1) 6 1207 (100) (1) 9 (3) 41 17 (1) (3) 6 (2) 15 (1) 3 4 (1) (4) 40 (0) 0 (1) 6 (1) 7 590 (49) 12 (1)

44

7 0

(0) (0) (0) (0) (0) (0) (0) (0) (100) (0) (0) (0) (0) (0) (0) (0) (0) (0) (0) (0) (0) (0)

48-68 (0) 12 (0) 1 (0) 0 (0) 11 (0) 9 (0) 2 (0) 0 (0) 0 6212 (100) (0) 4 (0) 20 (0) 14 (0) 3 (0) 17 (0) 0 (0) 0 (0) 37 (0) 8 (0) 0 (0) 6 (0) 6 (0) 0

5 13 1 lYalues are corrected for fetal bovine serum blank. Assayed in triplicate using 25 !J.g/ml antigen. Yalues in parentheses

are percentage binding relative to binding of bovine a-lactalbumin.

2H = Human; C = chicken; ES = estrogen sulfotransferase.

least 100% (100% for 48-60: 121 ± 18 cpm; 500% for 48-22 and 48-68: 543 ± 12 cpm, 522 ± 8 cpm; mean ± SD; P