Isolation of Monoclonal Antibodies Monospecific for Bovine P-Lactoglobulin KONRAD M. KUZMANOFF and CRAIG W. BEATTIE' Spedalized Center for Cancer Research and Education University of Illinois School of Medicine at Chicago Chicago 60612 ABSTRACT
Monoclonal antibodies were generated against purified bovine plactoglobulin A. Six antibodies reactive only with 0-lactoglobulin were selected. At least one of the antibodies appeared to be species monospecific for bovine j3lactoglobulin. The remaining five antibodies recognized proteins in caprine and porcine whey fractions. One monoclonal antibody (59-1) exhibited a distinct 2:l preference for p-lactoglobulin B over A at near neutral pH (7.5). These antibodies should prove extremely useful adjuncts in structural studies of bovine p-lactoglobulin. (Key words: bovine, P-lactoglobulin, monoclonal antibodies) Abbreviation key: P-LG = p-lactoglobulin, BSA = bovine serum albumin, Kd = dissociation constant, MAb = monoclonal antibody, PI = isoelectric point, PBS = phosphate-buffered saline, RIA = radioimmunoassay.
human placental protein 14, which shares AA sequence similarity with retinol-binding protein, also exhibits N-terminal AA sequence similarity with p-LG from several species, including the bovine (13). Commercially available polyclonal antibodies suitable for quantitative determination of pLG do not allow for topological or conformational analysis of the protein. Recently, Kaminogawa et al. (14) reported production of monoclonal antibodies (MAb) prepared against heat-denatured P-LG derivatives. Unfortunately, the modified topology induced during the formation of derivatives of 0-LG generally is unsuitable for use in conformational analysis of the native protein. Monoclonal antibodies directed against nonderivatized, nonheatdenatured PLG have not been reported. This report describes characteristics of murine MAb specific for nonderivatized native bovine 0-LG prepared by immunization after prior immunosuppression. The epitopes fecognized by these antibodies exhibit substantial pHdependent conformational changes that alter the binding of individual antibodies.
The 0-lactoglobulin (P-LG) present in the whey protein fractions of milks from several species, including cow, pig, and sheep, has been characterized only recently (2, 25). The bovine p-LG message has been sequenced (2) and has 93% sequence similarity with ovine PLG (10). Although the specific function of this low molecular weight protein (-18 kDa) remains unknown, the presence of receptors for a P L G retinol complex (25) in the microvilli of neonate calf intestine suggests that p-LG may be involved in the transport of vitamin A. The
Received February 14, 1991. Accepted June 21, 1991. 'To whom correspondence should be addressed. 1991 I Dairy Sci 74:3731-3740
MATERIALS AND METHODS Antibody Production
Casein and whey proteins were repurified by gel filtration over a Sephadex G-50 (Pharmacia, Piscataway, NO column (1 x 35 cm) eluted with phosphate-buffered saline (PBS). Female BALBK mice were initially immunized with bovine a-, p-, and Kcasein and a-lactalbumin (Sigma Chemical Co., St. Louis, MO). Two days later, the mice were injected intraperitoneally with cyclophosphamide (40 m a g , Neosar, Adria Labs, Columbus, OH) in PBS. nYee weeks after the initial immunization, mice were immunized with bovine j3-LG A (20) using RIB1 adjuvant (RIB1 Jmmunochemical Research, Hamilton, MT). A secon-
WZMANOIT AND BEATIlE
dary immunization with p-LG A was given after an additional 3 wk. Three days prior to fusion, the mouse spleen was removed, dissociated, and sensitized in vitro with 15 ng of bovine p-LG A (5). Mouse spleen cells were fused with myeloma cells (X63-Ag8.653) using polyethylene glycol (6, 15), and hybridomas were selected with hypoxanthine, aminopterin, thymidine medium (16, 24). Monoclonal cell cultures were prepared by limiting dilution subcloning (18) and screened for antibody avidity and crossreactivity to ensure homogeneity with the parent culture. Antibody-producing cultures were assayed using goat anti-mouse [1251JF(ab> fragments (Jackson Immunomearch Laboratories, Avondale, PA) according to the indirect solid phase radioimmunoassay (RIA) method of Howard et al. (12). Antibody binding to test antigen was determined as the value of the observed counts corrected for nonspecific binding of the tracer antibody [anti-mouse [125rJF(ab’)2] in the absence of test antibody (counts per minute 10% fetal bovine serum). Antibody isotype was determined using micro-Ouchterlony diffusion plates (Miles Scientific, Naperville, IL) and confirmed using an isotype- and subtype-specific ELISA (Bio-Rad Laboratories, Richmond, CA). Ascites fluid from pristaneprimed BALBIC mice (15) was used for larger scale production of antibodies. Antibodies were purified using immobilized protein G (Genex, Gaithersburg, MD) according to the manufacturer’s promdure.
Scatchard (26). Evidence of site-site interaction was evaluated according to the method of Hill (11). The RIA-determined specificity of anti$LG antibodies for their immunogen was assessed in two stages. The MAb that recognized bovine &U; were initially screened against a panel of seven antigens, which included the bovine casein (a-, P, and K-casein) and whey proteins (a-lactalbumin, p-LG A, and p-LG B) (Sigma Chemical Co.) and a mixed antigen sample, containing bovine actin, serum albumin (BSA), and hemoglobin. Antibodies binding with only bovine p-LG or showing only low crossreactivity were used in a second assay for immunogen specificity. The second crossreactivity panel contained 15 antigens, which included several proteins present in bovine serum and cell culture media, as well as casein and whey proteins from bovine and murine sources. Additional species speciticity was determined for casein and whey fractions (22) from goat, pig, and sheep milk. Whole milk samples @orcine, caprine, and ovine) were kindly provided by R. T. Stone (USDAMARC, Clay Center, NE). Competitive RIA (7, 9, 15) were performed using one radiolabeled MAb at fixed concentration (100,000 f 2000 cpm per well, in 20 pl: 3.5 f .2 ng, .18 f .02 pg/ml, 12.8 pCi/pg) as the labeled, competing antibody. Concentration of unlabeled test antibody was varied from .04to 45.44 pg/ ml. Test and competing antibodies were applied simultanmusly and incubated with previously bound antigen overnight at 4‘C. Antigen concentration was 25 pg/d (.5 pg per well). Washes and protein-blocking steps were idenDeterrnlnatlon of Antlgen Tlter, tical to those used in the standard solid phase Antlbody Avldlty, and SpecMclty RIA (15). Curves for titer and avidity were generated Antigen-antibody complexes were imfrom dilutions of antigen (.02 to 25 pg/ml) munoprecipitated with immobilized protein G with a fixed concentration of purified MAb (1 (Genex) essentially as described (8). Briefly, a pg/ml) and from dilutions of antibody (.01 to radiolabeled antigen sample containing 5 pg/ 10 pg/ml) with a fixed concentration of anti- ml each of bovine a-,P, and Kcasein, agen (15 pg/ml), respectively. These a w e s lactalbumin, and PLG A was incubated for 30 were used to determine the optimal, non- min with test antibody bound to protein G saturating concentration of antigen and the lin- agarose. After five washes with .l% BSA in ear portion of the binding c w e of antibody PBS, bound radiolabeled antigen was counted for antigen. Antibcdy specificity for PLG was using a model 1285 gamma counter (’I’M Anaassessed by RIA (12) and immunoprecipita- lytic, Elk Grove Village, E).Bound antigen tion. Antibody affinity for bovine PLG was was visualized by autoradiography after SDSdetermined according to the method of Frankel PAGE (17) on a 12.5% polyacrylamide gel. and Gerhard (7) and plotted according to The pH dependence of antibody-antigen Journal of Dairy Science Vol. 74, No. 11, 1991
MONOCLONALS TO BOVINE &LAC"OGL.OBULIN
TABLE 1. Moooclonal antibody (MAb) specificity for fHactoglobulial
binding was examined at pH 5.5, 6.5,and 7.5. In order to reduce aberrant binding resulting from pH shift, all reagents used for the indirect solid phase RIA (12), prior to the addition of radiolabeled second antibody, were maintained at the chosen pH. Second antibody, at pH 7.5, was applied after three washes at the experimental pH, followed by three washes at pH 7.5. The final washes to remove unbound seo ond antibody were also at pH 7.5.
serum proteins or bovine milk proteins other than fl-LG were isolated (Table 1). AU six MAb are of isotype IgG1. Based on RIA, antibodies 59-5,59-6.and 59-7bound 3% or less, with bovine a-, p-, and casein. The remaining three MAb (59-1, 59-2, and 59-3) did not bind with the bovine caseins. None of the MAb crossreacted with bovine alactalbumin. Immunoprecipitation of a radiolabeled mixed casein and whey sample revealed that the MAb bound only with B-LG (Figure RESULTS AND DISCUSSION 1). Under standard RIA conditions (PBS, pH Antlgen Speclflclty 7.4), using culture Supernatant, all six MAb Purity of casein and whey protein samples recognized both the A and B variant of PLG. was determined by SDS-PAGE (17) on an However, one MAb (59-1)exhibited nearly a 18% polyacrylamide gel and visualized by sil- 2 1 preference for &LG A at this pH. The ver staining (23) prior to use of the samples for remaining five MAb bound B-LG A and B in a immunization (f3-LG) and in crossreactivity as- ratio of -1:l. Five of the MAb (59-2, 59-3, 59-5, says. Although lower molecular weight bands were present in a-,b, and K-casein, possibly 59-6,and 59-7)crossreacted with whey fracresulting from degradation, no cross contami- tions from goat and pig but did not recognize nation between casein and whey proteins was sheep whey protein. However, MAb 59-1 exhibited only minor (4 to 6%) crossreactivity apparent (15). Six MAb that bound bovine B-LG >50 with any of these species. Binding to the portimes background with little or no binding to cine whey fraction ranged from 4% for MAb Jolunal of Dairy Science Vol. 74, No. 11, 1991
1 2 3 4 5 6
Figure 1. ImmMoprccipitation of pLactoglobalin Monoclonal antibodies (MAb) directed against PLG, bound to immobilized protein G, wae hubatcd with samplcs containing radiolabcl4 bo* OG, p, K-cILsein, CG lactalbumin, and Autoradiography after SDS-PAGE on a 12.5% gel showed binding only with fLLG. Lane 1: MAb 59-1; lane 2 MAb 59-2; lane 3: MAb 59-3; lane 4 MAb 59-5; lane 5: MAb 5 9 6 , lane 6 MAb 59-7;lane 7: MAb 59-7 (second sample); lam 8: bovine PLG standard. Lanes 6 and 7, MAb 59-7, parifid from different ascites samples.
59-1 to 26% for MAb 59-7, whereas binding B-LG A at the recognition sites for these antiwith the caprine whey fraction ranged from 4% bodies. for MAb 59-7 to 41%for MAb 59-2 (Table 2). There was no crossreactivity with the casein Binding Affinity fractions of ovine, caprine, or porcine milks. Each of the six MAb bound &LG over a The observation of antibody binding with caprine and porcine whey fractions, both of concentration range of .024 to 25 pg/ml which have been reported to contain p-LG (3, (Figure 2a). Antigen concentrations above 1 4). suggests that the MAb recognize a similar pg/d (20 ng) resulted in a near-saturating topology and, thus, sequence. The noticeable response (plateau) for MAb 59-1 and 59-3, absence of binding with the ovine whey k-whereas concentrations greater than 3 ~dml tion, which also contains J3-LG (lo), suggests (MAb 59-5) and 10 clg/ml (MAb 59-2, strong dissimilarity between ovine and bovine 59-6, and 59-7) were required to saturate the
TABLE 2. Antibody species specificity. Values arc presented as a perocntage of lactoglobulin (PLG). Cross reactant w h q and Carein samples were at 50
the bhling observed for bovine p bovine was at 25 pg/ml.
Antibody Bpecics cnmsnactivityl Specits
pis sheep Goat
59-I 5 6 4 3 1
59-2 3 41 14 2 0 0 100
pig CaJCin 2 COW BJ-G A 100 'Values arc corrected for faal b o v i i s a w n blank. Journal of Dairy Science Vol. 74, No. 11. 1991
59-3 2 28 2 . 5 1 1
59-5 3 20 6 4 0 1 100
0 5 15 0 0 1 100
59-7 0 4 26
2 0 0 100
MONOCLONALS TO BOVINE ~LAcroGLOBULIN sa
Antigen Concentration t.pg/ml)
Antiger Concentration (pg/rnl)
Figure 2. Binding of moncclonaI antibodies (MAb) to f%lactoglobulin (BLG). Antibody titer of purified 59-1, 59-2, 59-3, 59-5, 59-6, and 59-7 (at 1 [email protected]
) was determined in triplicate against increasing Concentrations of bovine BLG. Measurable binding was observed as low as .5 ng of applied antigen (25 nghnl). (a) B e as counts per minute (cpm) corrected for baclground; (b) binding for each MAb as a percentage of the maximum binding (100%) observed for that mtibodx MAb 59-1 (0);MAb 59-2 (e);MAb 59-3 (A); MAb 59-5 (A);Computer (0); MAb 59-7 0.
remaining antibodies. Half maximal binding for each antibody ranged from -.05 pg/ml for MAb 59-1, 59-2, and 59-3 to -.9 pg/ml for MAb 59-6 and 59-7. Monoclonal antibody 59-5 exhibited half maximal binding of -.17 pg/ml with a distinct supraoptimal saturation above 3.1 pg/ml. Binding at the lowest antigen assayed (.48 ng, .024 pg/ml) exceeded background values by at least 100% (>loo% for 59-2, 59-6, and 59-7; >2W% for 59-3 and 59-5; and >300% for 59-1). Normalization of the titration curves revealed the presence of three distinct groups of antigen concentrationdependent anti'body binding (Figure 2b). Antibodies 59-1, 59-2, and 59-3 exhibited a rapid rise in binding over antigen concentrations of
.02 to .2 pg/d. A second group, MAb 59-6 and 59-7, showed a more gradual increase in antigen concentrationdependent binding, which ranged from .02 to 6.25 pg/mL The final group, which contained MAb 59-5, exhibited an increase in binding simiIar to that of MAb 59-2. However, maximal binding exceeded that of MAb 59-2 by 76% and was supraoptimal at concentrations above 3.1 pg/ ml. Examination of avidity revealed two distinct groups (Figure 3, a and b). Antibody 59-5 appeared to have the lowest maximal binding. All of the antibodies began to saturate near 3.0 pg/d of antibody. However, because none of the MAb showed complete saturation over the concentrations tested (.01 to 10 pg/ ml), half maximal binding could not be determined. The MAb affiity constants (&) for p-LG (26) ranged from 5.5 x 109 M-' to 1.7 x 1O'O M-l. As observed for avidity, two groups were present. Antibody 59-5, with the lowest Kd value (5.5 x 109 W ) ,appeared distinct from the other five antibodies (Figure 3c). The affinity of the remaining MAb clustered near 1 x 1Olo W 1(59-1, 1.3 x lolo; 59-2, 1.5 x lolo; 59-3, 1.3 x lolo; 59-6, 1.3 x 1O'O; and 59-7, 1.7 x 1O'O W1).When site-site interaction was analyzed (ll), five of the antibodies
(59-1, 1.06, 59-2, 1.07; 59-3, 1.07; 59-6, 1.06; and 59-7, 1.06) gave coefficients of -1.1. In contrast, MAb 59-5 gave a higher coefficient of 1.49. These observations suggest that, except for MAb 59-5, only one distinct binding site is present for each MAb. Antibody 59-5 appears to recognize a second site, as ill coefficient >1. The presindicated by a H ence of such a site is supported by the observed decrease in apparent binding observed for MAb 59-5 above antigen concentrations of 3.1 pg/ml. However, because Scatchard analysis strongly suggests the presence of only one epitope site, it is unlikely that MAb 59-5 recognizes two nonadjacent, spatially distinct regions, but rather a region susceptible to conformational alteration. Epltoplc Slmllarltles
Competition curves (Figure 4) illustrate the epitope specificity of each antibody compared with MAb 59-5. Each curve was generated at a Journal of Dauy Science Vol. 74, No. 11, 1991
KUZMANOFF AND BEATIlE
., * . . . - . l o
ARatio of Unlabeled
txBrmned ' for their ability to inhibit the binding of ['zIIMAb 59-5. Only MAb 59-5 showed nearly complete inhibition (self-inhibition). MAb 59-1 (0);MAb 59-2 (0); MAb 59-3 (A); MAb 59-5 (A);MAb 59-6 0;MAb 59-7 0. Were
Figure 4. Competitive inhibition: epitope grouping. The jMactoglobulin-specific monoclonal antibodies (MAb)
Antibody Concentration (pg/rnl)
Antibody Concentmtion. w/rnl
Bound ( ~ 1 02,' Figure 3. Avidity of monoclonal antibodies (h4Ab) specific for j3-lactoglobulin @E)Antibody . (.l to 10 pg/ ml) avidity was determined at fmed antigen concentration (25 Wd). (a) The concentration dependence upon antibody with binding given as background corrected counts per minute (cpm); (b) the avidity m ' e s on a semi-log scale and more clearly dernonstraW that MAb 59-5 is distinct in its concentration-dependent binding; (c) the Scatchard plots for the six &LG-specifz MAb. MAb 59-1 (0); MAb 59-2 (0); MAb 59-3 (A); MAb 59-5 (A);MAb 59-6 (0); MAb 59-7 0. Jouroal of D m Science Vol. 74. No. 11. 1991
fixed concentration of antigen (25 pdml). Only unlabeled MAb 59-5 competed effectively with [1251JMAb59-5 (self-competition) for binding with &LG. Antibodies 59-1, 59-6, and 59-7 inhibited binding of MAb 59-5 by 18, 19, and 13%, respectively, whereas MAb 59-2 and 59-3 did not compete at any concentration tested. The inability of MAb 59-2 and 59-3 to inhibit the bmding of MAb 59-5 suggests that the epitope recognized by MAb 59-5 shares no commonalty with the MAb 59-2 or 59-3. Similarly, the small reduction in binding observed for MAb 59-1, 59-6, and 59-7 suggests that these antibodies recognize epitopes substantially different from the epitope of MAb 59-5. The conformational similarity and, thus, AA sequence similarity of the 59-5 epitope with the epitopes for MAb 59-1, 59-6, and 59-7 would appear to be less than 20%,based on the level of competition. The negligible level of competition argues against overlapping epitopes, which would exhibit higher inhibition of MAb59-5 as a result of steric hindrance. Antlbody Binding Dependence on pH
p-LG A as Antigen. Antibody 59-1 bound maximally at pH 5.5 at a saturating antigen concentration of 12 pg/d (Figure 5a). Binding decreased in the order MAb 59-1 > 59-7,
U a, +J U
F i 5. Dependence of antigen binding on pH. B W g of the lact tog lo^ (&LG)-specific monoclonal antibodies (h4Ab) was examined at pH 5.5,6.5, and 7 5 for both the A and B variant. Each antibcxly was assayed for titer o v a the range .39 to 25 pghd at a fixed antibody concentration (2 pghl). AU assays were in triplicate with standard deviation indicated by the vertical e m bars. Plales are in pairs (e.& a, b) for direct comparison of the differences in bindiug between the bovine A and B variants at the three assay p H values used. (a, b): MAb 59-1; (c, d): MAb 59-2; (e, f): MAb 59-3; (g, h): MAb 59-5; (i, j): MAb 596; (4 1): h¶Ab59-7. Plates a, c, e, g, 6 are k and &LG A. Plates b, d, f, h, j, and 1 are PLG B pH 5.5 (0);pH 6.5 (A); pH 7.5 0.
59-2, and 59-3 and 59-6 > 59.5. Antibodies 59-2,59-3, 59-5, and 59-6 bound to p-LG A at -14% of the maximum observed for MAb 59-1. At pH 6.5, a similar order of binding was observed. However, the binding of MAb 59-2 (Figure 5c) and 59-3 (Figure 5e) decreased 31 and 33%. whereas the binding of MAb 59-5 (Figure 5g) and 59-6 (Figure 5i) increased 193 and 138%, respectively. Maximal binding of MAb 59-7 (Figure 5k) increased 22%. At pH 7.5, MAb 59-1 and 59-5 (cf. Figure 5 , a and g) showed the highest level of binding. The binding of MAb 59-6 increased 39% (Figure Si) over the level observed at pH 6.5 (230% above the value at pH 5.5). Antibodies 59-2 (Figure 5c) and 59-3 (Figure 5e) showed a further decrease in the level of binding (58 and 53% less than observed at pH 6.5). whereas MAb 59-7 (Figure 5k) exhibited a slight decrease of 11%.At all three assay pH, MAb 59-1 showed the highest level of binding to p-LG A with higher binding at acidic pH. Both MAb 59-2 and 59-3 had much higher binding at acidic pH, whereas MAb 59-5 and
59-6 showed their highest binding at pH 7.5. Antibody 59-7 bound highest at pH 6.5 with an 8 and 11% decrease at pH 5.5 and 7.5, respectively. p-U; B us Antigen. At pH 5.5, binding of all MAC, to p-LG B, except 59-1, was higher than that observed for p-LG A. Antibody 59-1 had the highest level of binding (Figure 5b); MAb 59-3 showed a threefold increase in binding compared with p-LG A. whereas binding of MAb 59-1 decreased 18%. The binding of MAb 59-1 and 59-3 decreased dramatically at pH 6.5 (Figure 5 , b and f), representing only 43 and 63%, respectively, of the binding at pH 5.5. The remaining four MAb showed increased binding at pH 6.5. Antibodies 59-5,59-6, and 59-7 showed the highest binding at pH 7.5 (Figure 5 , h, j, and I), whereas the binding of MAb 59-2 and 59-3 decreased (Figure 5, d and f). Antibody 59-1 (Figure 5b), which bound maximally at pH 5.5 and decreased at pH 6.5, showed high binding at pH 7.5, representing 76% of the value observed at pH 6.5 and 206% of that at pH 6.5. For P-LG B, MAb 59-1 and 59-3 showed highest binding under acidic conJournal of Dairy Science Vol. 74, No. 11, 1991
ditions (Figure 5, b and f), whereas MAb 59-5 and 59-6 had highest binding at pH 7.5 (Figure 5, h and j). Antibody 59-2 and 59-7 exhibited highest bmding at pH 6.5 (Figure 5, d and 1). Comparison of binding to the p-LG A and B variants showed a clear change in magnitude, pH optimum, or both of binding for each antibody. Antibody 59-1 had higher binding to p-LG A at all pH (Figure 5a). Binding decrearred with an increase in pH. In contrast, binding with &LG B was lowest at pH 6.5 (Figure 5b). Antibody 59-2 showed a clear binding optimum to p-LG A at pH 5.5 (Figure 5 ~ ) Optimal ; binding to p-LG B occurred at PH 6.5 and represented a fivefold increase in magnitude (Figure 5d). Antibody 59-3 also showed optimal binding at acidic pH for both 8-LG A (Figure 5e) and P-LG B (Fgure 50. However, the magnitude of binding at pH 5.5 was three times greater for p-LG B than for A. Antibodies 59-5 (Figure 5, g and h) and 59-6 (Figure 5, i and j) exhibited clear optima at pH 7.5 for both p-LG A and B with the lowest binding at pH 5.5. Antibody 59-7 was the least sensitive to variations in assay pH. Although optimal binding to P-U;A was at pH 6.5 (Figum 5k). the binding at pH 5.5 and 7.5 varied by only 8 and 11%. respectively. Similarly, maximum binding for FLG B was at pH 5.5 F i g u r e 51) with variation of 4 and 8% at pH 6.5 and 7.5. Interspecies-specific binding was also used to localize potential epitopes for PLG-specific MAb. None of the antibodies recognized ovine whey protein, and all of the antibodies, excluding MAb 59-1 and 59-5, recognized,to V ~ U Y ing degrees (59-7, 59-3 > 59-2, 59-6 >> 59-1, 59-3, porcine whey protein. Therefore, the regions of similarity of the deduced AA sequence between bovine and porcine (63%) (1) and between bovine and ovine (93%) (2) would not be expected to contain the epitopes of these MAb in bovine P-LG A. The AA differences reduce MAb binding to porcine pLG and abolish it in ovine B-LG. Those regions in bovine &LG A that differ in AA sequence from porcine and ovine P-LG should contain the epitopic regions for these MAb. For maximal antigenicity. these regions 1) should be hydrophilic in nature and 2) would be expected to contain or be near a proline residue (19). Based on the deduced M sequence of bovine (2), ovine (10). and porcine Journal of Dairy Science VoL 74, No. 11, 1991
(1) FLG, there are five hydrophilic regions in bovine &LG that differ in the pig and sheep. Four of these regions contain or are near at least one proline residue (Figure 6). Antibodies 59-1 and 59-5 do not bind with either porcine or ovine whey protein (-5% of bovine value) and would be expected to bind to a region altered in sheep and either absent from or severely altered in pig. The bovine AA sequence LSFN (AA 165 to 168) of region V (AA 165 to 178) is missing in porcine and contains two AA changes (AA 166, 174) in ovine j3-LG. Region V is separated by a proline residue (AA 169) into two subregions, Va and Vb. Although the calculated overall isoelectric point (PI) of region V is acidic at pH 5.3, the PI of the subregions are 7.0 (AA 166 to 169) for region Va and 5.3 (AA 170 to 178) for region Vb. Antibody 59-5 shows highest binding at pH 7.5 and lowest binding at pH 5.5, whereas the reverse is true for MAb 59-1. Antibody 59-5 would be expected to bind to region Va (AA 166 to 169), and MAb 59-1 binds to region Vb (AA 170 to 178). Antibodies 59-2 and 59-3, which exhibit very similar pH-dependent binding to bovine p-LG A, bind maximally at pH 5.5. Epitopes for these MAb should be similar and either adjacent or partially overlapping with a PI near or below pH 5.5. The binding of MAb 59-3 to swine whey protein was nearly twice the binding of MAb 59-2. This suggests that the region (epitope) "cognized by 59-3 in swine has fewer AA changes than the region recognized by MAb 59-2. Region 111 (AA 121 to 134) contains one proline residue (AA 129), which separates the observed swine AA changes into two groups @la: AA 121, 128; IIlb: AA 131). Both groups also contain AA alterations present in sheep (AA 121, 134). Both subregions have acidic PI values of 3.8 with symmetrical hydropathy. One would predict that MAb 59-2 and 59-3 bind to region ID. Region IIIa (AA 121, 128) contains two AA alterations in swine, one of which is coincident with the AA change found in sheep. Region IIIa should be the epitope for MAb 59-2, which shows lower recognition of swine. Region IIIb (AA 128 to 134). with only one AA change in swine (AA 131), is topologically separated from region IIIa by the proline residue at AA 129 and should be the epitope of MAb 59-3. Additional evidence for region III
MONOCL0NAL.S TO BOVINE pLA(JT0GLoBULIN
Amino Acid Position Numoer
Figure 6. Hydropathy plot of bovine lactoglobulin (BLG) A. Five hydrophilic regions with amino alterations in porcine and ovine pU; that contain or are bounded by proline residues and possess isoelectric point @I) values suitable for the observed binding of the PLG-specific monoclonal antibodies are shown. Region I AA 165 to 178; region II: AA 79 to 87; region m:AA 121 to 134; region IV AA 141 to 151; region V: AA 165 to 178.
tions from pig and goat. One antibody, 59-1. is specific for bovine P-LG. At least three different epitope regions on p-LG A are recognized by these six antibodies. Based on titer, avidity, affiity constants, and pH alteration of binding, all three epitopes should be present on both FLG A and B. The epitope recognized by MAb 59-5 is clearly different from the sites recognized by the other five antibodies. The epitopes recognized by MAb 59-2 and 59-3 appear similar and may either overlap or be adjacent. In contrast, the epitOpe(S) recognized by MAb 59-6 and 59-7 distinct from the epitopes for MAb 59-2 and 59-3. All epitopes appear to exhibit pHdependent alteration of conformation; MAb 59-1,59-2, and 59-3 show the largest variation with assay pH. ACKNOWLEDGMENTS
as the binding site for MAb 59-2 and 59-3 relies upon the considerable increase in binding to P-LG B at acidic pH exhibited by these MAb. Bovine p-LG B has been reported to have a substitution at AA 120 (21). This alteration is within the region predicted for the epitopes of MAb 59-2 and 59-3 and may account for the alteration in binding to bovine pLG B. Antibody 59-6 exhibits highest binding to bovine p-LG A at near neutral pH with moderate (15%) recognition of porcine whey protein. Region II (AA 79 to 87) is the only hydrophilic region other than the epitope of UAb 59-5, at region Va, with a PI of 7.0. Region II contains two AA changes in swine (AA 77, 81) and one change (AA 80) in sheep. This region is bounded by proline residues at AA 66 and 95, which could limit conformational perturbation from pH-induced protonation, suggesting that MAb 59-6 binds to region II. Region IV, with an acidic PI of 4.17, is bounded by proline residues at 142 and 160 and may be the region recognized by MAb 59-7. CONCLUSIONS
Six MAb specific for p-LG have been isolated and characterized. Five of the MAb also recognize, albeit to a lesser degree, whey frac-
This research was supported by a grant from the National Dairy Research and Promotion Board to C. W. Beattie. Murine caseins were kindly supplied by A. G. MacKinlay. Whole milk samples from goat, sheep, and cow were kindly provided by R. T. Stone, USDA-ARS-Meat Animal Research Center, Clay Center, NE. REFERENCES 1 Alexander, L. J., and C. W. Beattie. 1991. Sequence of porcine ~lactoglobuliicDNA. Anim. Genet. (in
press). 2Alexander, L. J.. G. Hayes, M. J. Pearse, C. W. Beattie, A. F.Stewart, I. M. Willis, and A. G. Mackinlay. 1989. Complete sequence of the bovine & lactoglobulin cDNA. Nucleic Acids Res. 17:6739. 3 Bell, K., H. A. McKenzie, W. H. Murphy, and D. C. Shaw. 1970. Beta-lactoglobulin - a unique protein variant. Biochim. Bi=214 427. 4&U, K., H. A. McKenzie, and D. C. Shaw. 1968. Amino acid composition and peptide maps of beta lactoglobulin variants. Biochim. Biophys. Acta 154 284. 5 De Boer, M., G. Ten Voorde, F. A. Ossendorp, G. Van Duijn, P. F. Brunin& and J. M. Taga. 1988. Stimulation in vitro of sensitized splenocytes for the generation of antigen-specific hybridomas. Page 59 in Progress in biotechnology 5: in vitro immunization in hybridoma technology. CAK. Borrebaeck, ed. Elsevier Sci. Publ., New York, NY. 6 De St. Groth, F., and D.Scheidegger. 1980. production of monoclonal antibodies: strategy and tactics. J. Immuml. Methods 353. 7Franke1, M. E., and W. Gerhard. 1979. Ihe rapid determination of binconstants for antiviral antiJournal of Dairy Science Vol. 74, No. 11, 1991
bodies by a radioimmunoassay. An analysis of the interaction between hybridoma proteins and influenza virus. Mol. Immunol. 16:lOl. IFredrikson, G., S. Nilsson, H. Olsson, L. Bjorck, B. Akerstrom, and P. Belfrage. 1987. Use of protein G for preparation and characterization of rabbit antibodies against rat adipose tissue hormone-sensitive lipase. J. Immunol. Methods 9765. 9Friguet. B., L. Djavadi-Ohaniance, J. Pages, A. Bussard. and M. Goldberg. 1983. A convenient enzymeliuked immunosorbent assay for testing whether noclonal antibodies recognize the same antigenic site. Application to hybridomas specifii for the &-subunit of Escherichia coli hyptophm synthase. J. Immunol. Methods 60:351. 10Gaye. P., D. Hue-Delahaie, J.-C. Mercier, S. Soulier, J.-L. Violette, and J.-P. m t.1986. Ovine b lactoglobulin messenger RNA nucleotide seqnence and mRNA levels during differentiation of the mammary gland. Biochimie 68:1097. 11 Hill,A. V. 1909. The mode of action of nicotine and curari, determined by the form of the contraction cwve and the method of temperature coefficients. I. Physiol. 39:361. 12 Howard, P. D., J. A. Ledbetter. S. Q. Mehdi, and L. A. Herzcnberg. 1980. A rapid method for the detection of antibodies to cell surface. antigens: a solid phase radioimmunoassay using cell membranes. J. Immunol. Methods 38:75. IfHUhtala, M.-J.. M. Seppata, A. NWVP. Palomaki, M.Jullcunen. and H. Bohn. 1987. Amino acid sequence homology between human placental protein 14 and fLlactoglobalins from various species. Endocrinology 1202620. 14Kaminogawa, S.. M. Hattori, 0. Ando, J. Kurisaki, and K. Yamauchi. 1987. Reparation of monoclonal antibody against bovine lactoglobulin and its miqne binding aflinity. Agric. Biol. Chem. 51:797. 15 Kuunan~ff.K. M., J. W. Andresen, and C. W. Beattie. 1990. Isolation of monoclonal antibodies mone specific for bovine K-c&. J. Dairy Sci. 73:2741. and C. W. Bat16Ku~t~110ff. K. M., J. W. tie. 1990. Isolation of monoclonal antibodies mono-
Journal of Dairy Science Vol. 74, No. 11, 1991
specific for bovine a-lactalbumin. J. Dairy Sci. 73: 3077. 17 Laemmh’,U. K. 1970. Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature (tond.) 227:680. 18 Lefltovits, I., and Waldman H. 1979. Limiting dilution analysis. Page 60 in Limiting dilution analysis of cells in immune systems. I. Lefkovitz and H. W a l d m a ~ed. Cambridge Univ. Press, Richmond, Surrey, Engl. 19Lemer. R A., N. Green, H. Alexander. F. Lin. I. G. Sutclif€e, and T. Shinnick. 1981. Chemically synthe sized peptides predicted from the nucleotide sequence of the hepatitis B virus genome elicit antibodies reactive with the native envelope of Dane particles. Proc. Natl. Acad. Sci. USA 783403. 20Mathew. W. D., and P. H. Patterson. 1983. The production of a monoclonal antibody that blocks the action of a neurite outgrowth-promoting factor. Cold Spring Harbor Symp. Quant. Biol. 48:625. 21 McKenzie, H. A. 1971. ~Lactoglobulins.Page 25 in Milk proteins: chemistry and molecular biology. Vol. II. H. A. McKenzie. ed. Academic Press, New York.
NY. 22 McKenzie, H.A. 1971. Whole casein: isolation, prop d e s , and zone electrophoresis. Page 87 in Milk proteins: chemistry and molecular biology. Vol. II.H. A. McKenzie, ed. Academic Press, New York, NY. 23 Merril, C. R. Goldmann, D., and M. L. Van Keuren. 1984. Gel protein stains. Silver stain. Methods Enm o l . 104441. 24 Oi, V. T., and L. A. H m b e r g . 1980. Immun0glOb~lin producing hybrid cell lines. Page 351 in Selected metbods in cellular immmology. 8. B. Mishell and S. M.Shiigi, ed. W. H. Freeman and Co., San Prancisco, CA. 25 Papiz, M. Z., L. Sawyer, E. E. Eliopoalos, A.C.T. North, J.B.C. Findlay, R Sivaprasdaro, T. A. Jones, M. E. Newcomer, and P. J. Kraulis. 1986. The s t r u e ture of fLlactoglobulin and its similarity to plasma retinol-binding protein. Nature (Lond.) 3 x 3 8 3 . 26Scatchiud. G. 1949. The attraction of proteins for small molecules and ions. Ann. New York Acad Sci. 51:660.