Isolation and Characterization of Monoclonal Antibodies Monospecific for Bovine a-Casein and f3-Casein KONRAD M. KUZYANOFF; JOHN ANDRESEN, and CRAIG W. BEAlTlE Speaalized Center for Cancer Research and Education University of Illinois School of Medicine at Chicago Chicago, IL 60612

of clustered serine phosphate residues (2). The exact structure of the CN micelle and the preTwo monoclonal antibodies monospecise way in which calcium phosphate contribcific for bovine %lcasein, which recogutes to micelle struchm has not been elucidatnize three genetic variants of ql-casein, ed. Although serine phosphate clusters are have been isolated and their binding clearly essential to micelle formation (2), surproperties characterized. Antibodies face features of the calcium-sensitive phos57-115 and 57-310 recognize different phoproteins may also play a significant role. antigenic determinants on the o&l protein Antigenic determinants on bovine o&l-CN have with affinity constants of 1.63 x 10" and been identified with polyclonal antitxxiies to 2.13 x 10" W 1 , respectively. peptides isolated by tryptic digest of the %IFive monoclonal antibodies, 58-409, CN protein, but little information is available 58-416, 58488, 58-504, and 58-557, on epitope location within each Eragment or on monospecific for bovine p-casein with the crossreactivity with Q-CN or other caseins affinity constants >lo9 M-l, which recog(1, 15). High affinity and epitope-specific monize a similar epitope(s) also were isolatnoclonal antibodies (MAb) to the %I- and %2ed. All seven antibodies are of isotype CN are currently not available. A similar probIgGl and recognize both the denatured lem exists for bovine p-CN. Monoclonal antiand undenatured forms of their antigen, bodies to human p-CN either crossreact with making them suitable for qualitative and other human milk proteins (4) or p-CN from quantitative radioimmunoassay. species other than the bovine (6). Antibodies (Key words: monoclonal antibodies, aSlthat may specifically bind to and identify gecasein, p-casein) netic variants of the calcium-sensitive CN that Abbreviation key: CN = casein, FBS = fetal affect important parameters such as renneting bovine serum, MAb = monoclonal antibodies, (16) are also lacking. We have been developing NEAA = nonessential amino acids, NTCP = panels of MAb specific for bovine %I- and pnitracellulose, PBS = phosphate-buffered sa- CN for topological studies and quantitation of line, RAMIgG = ['"Ilrabbit anti-mouse IgG, these proteins. The present report describes a series of specific, high affiity MAb to o&land RIA = radioimmunoassay. and p-CN suitable for such studies. ABSTRACT


The calcium-binding phosphoproteins %I-, %2-, and pcaseins (CN)occur in milk in a suspension of micelles stabilized primarily by Antigen PurHicatlon K-casein (17, 18). The structural characteristic Samples of bovine %I-, %2- and P-CN used of %I- and p-CN central to their function in to establish MAb specificity were kindly protransporting calcium phosphate is the presence vided by H. Farrell (USDA, Philadelphia, PA). Additional samples of bovine a- and p-CN were obtained from Sigma Chemical (St. Louis, MO). Murine CN were a gift from A. MackinReceived April 6, 1990. Accepted July 5, 1990. lay (University of New South Wales, Kensing~

1991 J Dairy Sci 74803-810




ton, Australia). Dephosphorylated a-and PCN were a gift from C. Brooks (OhioState University, Columbus, OH). All additional proteins were purchased from Sigma Chemical. The purity of bovine a- and p-CN samples used for immunization (Sigma Chemical, St. Louis, MO) and crossreactivity assays was determined by SDS-PAGE (11) on an 18% polyacrylamide gel with silver staining (13).


the [1251JRAMlgGtracer antibody in the absence of test antibody. Monoclonal cell cultures were prepared by limiting dilution subcloning (3, 12). These cultures were screened for antibody avidity and cmssreactivity to ensure homogeneity with the parent cultures. i-typing

The isotype of the antibodies was determined using radioiodinated isotype, subtypeFemale BALB/C mice were immunized with specific, rabbit anti-mouse standards, (Bio-Rad bovine a-or p-CN using RIB1 adjuvant (RIB1 Laboratories, Richmond, CA) in an indirect Immunochemical Research, Hamilton, MT). solid-phase RIA as described. The fusion of mouse spleen cells with myeloma cells (X63-Ag8.653), using polyethylene Determination of liter, Avidity, glycol, was done essentially according to the Speciflclty, and Affinity procedure of De St. Groth and Scheidegger (5). Dilution curves were constructed in order to Viable spleen cell and myeloma hybridomas were selected with hypoxanthine-aminopterin- determine the optimal, nonsaturating concentrathymidine medium (5, 14). Antibody-producing tion of antigen and the linear portion of the cultures were assayed by solid-phase indirect binding w e of antibody for antigen. These radioimmunoassay. Affinity purified curves were generated by maintaining a fixed [lZsI]rabbit anti-mouse IgG (RAMIgG) was concentration of either antibody (titer) or antiused as the radiolabeled tracer (Jackson Im- gen (avidity) while the other was varied. Inmunoresearch Labs, Avondale, PA). The direct RIA, as described, was used to measure RAMIgG was iodinated using the chloramine T the binding of antibody to antigen. Antibody procedure described by Hunter and Greenwood specificity for a-and p-CN was determined in (10). The radioimmunoassay (RIA) procedure crossreactivity studies using indirect RIA as was based upon the indirect solid-phase method described. Western blot analysis was according of Howard et al. (9). Briefly, 20 pl of the to the method of Towbin et al. (19). Casein immunogen (a-or P-CN) at 25 pg/d was samples were loaded at 10 p g a n e with all gels applied to the wells of a polyvinyl chloride containing the same set of 10 samples. The microtiter plate m a t e c h Laboratories, Alex- samples were transferred to .45 pn nitrocelluandria, VA) and incubated overnight at 4'C. lose (NTCP) (Schleicher and Schuell, Keene, Subsequently, the wells of the plates were NH) after SDS-PAGE on a 12.5% gel. Antigen specificity was assessed in two washed three times with .l% BSA in phosphate-buffered saline (PBS). The wells were stages. Antibody-producing hybridomas, which then blocked with 10% fetal bovine serum recognized their immunogen, were initially (FBS) for 1 h at 25'C and washed with .l% screened against a panel of seven antigens that p-, K-) and whey BSA in PBS. Hybridoma culture medium con- included the bovine CN (a-, taining test antibody was applied at 20 WweU proteins (a-lactalbumh, p-lactoglobulin A, pand incubated for 1 h at 25'C. The wells were lactoglobulin B; Sigma Chemical, St. Louis, washed, as described, then incubated with MO) and a mixed antigen sample. The mixed 80,000 cpdwell [1251JRAMIgG in 20 pl at sample contained BSA, actin, and hemoglobin 25'C for 1 h. The wells were then washed as (Sigma Chemical, St. Louis, MO). Low crossdescribed, dried, separated with a hot wire cut- reactivity was defined as the ratio of binding ter, and counted using a Model 1285 Gamma for immunogen:nonimmunogen that exceeded Counter (TM Analytic, Elk Grove Village, E). 10. In initial screens, this ratio in some inThe binding of antibody to test antigen was stances was dependent upon the concentration calculated as the value of the observed counts of antibody used for the assay. Antibodies that per minute corrected for nonspecific binding of passed the initial assay for crossreactivity were Antibody Production

Joumal of Dairy Science Vol. 74, No. 3, 1991


subjected to a second, larger panel of potential antigens. The second crossreactivity panel contained 23 antigens, which included CN and whey proteins from bovine, murine, and human sources. The murine CN sample was composed of mixed CN and whey proteins. Several additional proteins present in serum (actin, albumin, fibrinogen, fibronectin, and hemoglobin) (Sigma Chemical, St. Louis, MO) and cell culture media (transferrin) were included as part of the panel in order to eliminate antibodies with general nonspecificity. All initial assays were performed using culture media Supernatant. Culture media was 4 mM glutamine, 2 mM sodium pyruvate, 5 mM oxalacetic acid, .2 W/ml bovine insulin, 50 IU/ ml penicillin, 50 pg/ml streptomycin, 2 mg/ml glucose, 1% nonessential amino acids (NEAA), 10% NCTC-135, 15% FBS in RPMI-1640 (Gibco, Grand Island, NY), pH 7.4. The FBS, NCTC-135, NEAA, and WMI-1640were purchased from Gibco (Grand Island, NY). All other reagents were purchased from Sigma Chemical (St. Louis, MO). Ascites fluid from pristane-primed BALB/C mice (14) was used for larger scale production of antibodies. Antibodies were purified using immobilized protein A (Pierce Chemical, Rockford, IL.) according to the procedure provided by the manufacturer. Antibody affinity was determined according to the method of Friguet et al. (7).



8 9 10 11


4 45 4


29 424 4






Pigure 1. Purity of bovine casein and whey milk proteins used for immanization (u-and Jhseins) and crossreactivity assays. Lanes 1, 5, 11: Molecular weight markers with relative molecular weight as k i l d t o n s indicated in the margins; lane 2: ctcaseiu(s);lane 3: fhasein; lane 4: Kcasein; lane 6 a-lactalbumiq lane 7: a-lactalbumin (human); lane 8: i3-lactoglobulin A; lane 9: klactoglobulin B; and lane 10: calmodulin.

ent in the samples of bovine a-,p-, and IC-CN, little contamination of individual caseins was apparent (Figure 1). The minor bands present in each of the caseins were ascribed to posttranslational modification (K-CN), degradation products, or minor contaminants. Antigen Specificity

Characterization of Epitope Similarity

Two U A b specific for bovine a-CN (MAb 57-115 and 57-310) and five MAb specific for In order to clarify the similarity of the bind- bovine p-CN (MAb 58-409, 58416, 58-488, ing regions recognized by the monoclonal anti- 58-504, and 58-557) were selected. All seven bodies, competitive solid-phase RIA (7, 8) were MAb are of isotype IgGl and are specific for used to determine the similarity of epitopes their immunogen with virtually no crossreactirecognized by the antibodies. A fixed concen- vity to selected milk or other proteins (Table tration (80,000cpm in 20 PI) of isotopically 1). labeled lZI test antibody was incubated with The two a-CN MAb selected (57-115 and antigen in the presence of increasing concmtra- 57-310) do not bind with the murine CN samtions of unlabeled competing antibody. Both ple. The five bovine p-CN MAb (58-409, the test and competing antibody were applied 58-416, 58-488, 58-504, 58-557) also appear simultaneously and incubated with the bound monospecies-specific for bovine P-CN, since antigen overnight at 4'C. Washes and protein they do not crossreact with either murine CN or blocking steps were identical to those used in human PCN. Interestingly, all MAb show a the standard solid-phase assay. high level of binding with the dephosphorylated form of their antigen, suggesting they bind to RESULTS AND DISCUSSION nonphosphorylated regions of the protein. Solid phase RIA of MAb 57-115 and Antigen Purity 57-310 binding demonstrated significant speciAlthough silver-stained gels showed minor ficity for &l-CN. The preference of MAb bands of lower relative molecular weight pres- 57-115 for %I- over Q-CN is 8:l (%I- $470 Journal of Dairy Science Vol. 74, No. 3, 1991



1 2 3 4 5

6 7 8 910


ob,-+ 0










d Figure 2. Binding of a-and &casein (CN)monoclonal antibodies (MAb) to a- and f3-CN genetic variants. Purified genetic variants of bovine %I-. %T, and PCN were probed by Western blot analysis. a) h b e d with MAb 57-115; b) probed with hfAb 57-319 c) probed with MAb 58-488; d) probed with MAb 58-557. Lane 1: %2-CN 11R lane 2: g2-CN 9P; lane 3: q1-C; Jane 4: g l - B lane 5: %I-& lane 6 &CN B-5P lane 7: f3-CN A1-5P; lane 8: & CN C-5P; lane 9 &CN Ab-5P; and lane 1 0 PCN A2-5P.

. I > . *



15 I on(













entralion ()hq/rnl)

Figure 3. Dekmhation of mcasein concentration-dependent bleding of antibody. The a-monoclonal antibodies (MAb), 57-115 (0) and 57-310 (e), at 1 ~ g / m l ,were assayed for antigen concentmion-dependent binding over the range of 1 to 25 pgbl. Corrected binding was calculated as the difference between total counts p a minnte and counts per minute in the absencc. of test antibody. All assays were performed in triplicate. The SD is shown as double mor bars.

58488 (Figure 2c) and 58-557 ( F i p 2d). The intensity of binding by p-CN variant A3-5P for both MAb 58488 and 58-557 appeared lower than for the other genetic variants and may represent a slightly decreased binding of the anti-fi-CN antibody for this variant. Antigen Titer

Bovine a-CN MAb 57-115 and 57-310 exhibit saturable binding at 10 pg/d of bovine aCN (Figure 3). Maximum binding occurs at 8 pg/ml, with half-maximal binding occurring at cpm +/- 37 (mean and SD); %2- :805 cpm +/- immunogen concentrations of 1 pdml (MAb 72, n = 3). The MAb 57-310 bound %I- and 57-310) and 1.5 pdml (MAb 57-115). Detectact&3I in a ratio of 29:l (%I- :8349 cprn +/ble binding exceeding 500 cpm (background493 (mean and SD); q 2 - :286 cprn +/- 2, n = corrected) was observed at a concentration of 3). 50 ndml (1 nglwell) a-CNfor both antibodies. Similar binding was observed for MAb The monospecies-specific bovine p-CN 57-115 and 57-310 for % l € N when examined MAb appear to form two p u p s (Figure 4). by Western blot. Only the SI- genetic variants Group I, which is represented by MAb (%l-A, Q-B, %1-C; courtesy of H. Farrell) bound MAb 57-115 (Figure 2a) and 57-310 58-409 and 58-557, requires a threefold higher (Figure 2b). Posttranslationally modified sam- concentration of antigen for saturation of bindples of Q- (Q-CN A-9P and a&N A-11P ing and yields a lower maximum level of bindcourtesy of H. F m l l ) were not recognized. ing at saturating concentrations of antigen. AnTwo of the p-CN MAb (58-488 and tibodies 58-509 and 58-557 exhibited saturable 58-557) were used for Western analysis of five binding at 7 pdml of immunogen. In contrast, p-CN genetic variants. All five P-CN variants MAb 58416, 58488, and 58-504, which com(p-CN A1-5P, &CN A2-5P, &CN A3-5P, PCN prise Group II, reached saturation at a lower B-5P, p-CN C-5P) were recognized by MAb antigen concentration. Binding was saturable at Journal of Dairy Science Vol. 74, No. 3, 1991




TABLE 1. Monoclonal antibody (MAb) CrosSeaCtivily as determioed by radioimmunoassay (RIA). Binding was corrected for background resulting from nonspecific binding of the ['25I]rabbit anti-mouse 0 IgG in the absence of test antibody [phosphate-buffered saline (PBS)vdues]. Test antigen was used at 25 pghd with antibody at 1 Values represent the mean of triplicate assays. Binding is shown as corrected counts per minute.


&Casein MAb

a-Casein MAb Antigen








(cpmlwell) Actin

Albumin Fibrinogen Fibronectin y-Globulin Hemoglobin Lactoferrin Lactoferrin 0' Lactoperoxidase Transferrin WLactalbumiIl

a-LactalbumiIl 0' &Lactoglobulin A &Lactoglobulin B OGCasein a - k i n (-P)~ K-CaSeh K-CaSeh 0' Pcasein &casein ( C B ~ Bcasein (H) &casein (-P)* Murine casein PBS

0 0 0 0 13 12 3

34 0 0 0 0 0 0 10,956 10,862 0 0 9 34 0 286

0 0 0 0 0 0 0 0 0 0 0 0 0 0 9567 11,616 0 0 0 0 0 47





0 0 0 0 17 0 0

7 11 0 0 0 16 0 0 0 0 0


0 0 1 26 0 0 68 3% 0 0 7107 6521 0 7103 58 134

0 0 0 0 3 0 0 0 0 0 0 0 0 0 15 181 0 0 8870 8073 0 8993 69 126


20 0 37 70 0 0 8264 7749 21 8404 55 94

0 0 0 3 0 0 0 0

0 0 0 3 0 0 0 0 0 0 0 0 49 0 0 83 0 0 7108 6375 0 7199 38 132

0 0 0 0 49 0 0 83 0 0 7108 6375 0 7199 38 132

'H = Human. *-P = Dephosphorylated. = cist from C. B K O ~ .

-2 pg/d bovine 0-CN for these MAb. All Group II antibodies exhibit half-maximal binding near 1.5 pg/ml with detectable significant binding of p-CN at 75 ng/ml.




Antibody Avidity

The a-CN MAb have similar dilution curves. Saturable binding occurred at dilutions between 1/20 to 1/100 of cell supernatant (Figure 5). Supraoptimal concentrations exceeding 1/20 dilution of antibody resulted in a slight decline in observed binding. Half maximal binding 0ccum.d at 1/3300 dilution for MAb 57-115 and at 1/5W for MAL, 57-310. Detectable binding (in excess of 500 cpm or 5% of maximal binding) was observed at dilutions exceeding 10,000 for both a-CN monoclonals.











A r t i q e r i C o n c e n t r a t i o n (ug/rnl)

Figure 4. Determination of &casein concentration-dependent bioding of antibody. The &casein monoclonal 58416 (A), 58-488 (A), antibodies W b ) 5 8 4 9 (e), 58-504 0.and 58-557 (O),at 1 WmI, were assayed for antigen conccntration-dependentbmding over the range of 1 to 25 cle/m. Values represent the mean (and SD) of triplicate assays. Journal of Dairy Science Vol. 74, No. 3, 1991






Antibody C o n c e n t r a i i o r . Log(D luiion Factor)

Figure 5. Avidity of wcasein monoclonal antibodies (MAb). Concentration-dependentantibody binding was determined using an indirect, solid phase radioimmunoassay. Antigen was at 15 Culture supernatant from log phase MAb 57-115 (0) and 57-310 (0) cultures was diluted over thc range 1 to 1 x I$. values me thc mean of triplicate assays. ’zhe “dilution factor” is the reciprocal of the dilution (l/dilution).


The dilution curves for the bovine p-CN monoclonals appear to represent a family of similar antibodies with classical sigmoid concentration dependent binding curves (Figure 6). As observed with antigen titration, the PCN MAb segregate loosely into two groups consisting Of MAb 58409 and 58-557 (Group I) and of MAb 58416, 58488, and 58-504 (Group 11). Group I MAb 58-409 and 58-557 exhibit maximal binding at supernatant dilutions of 1/3 and 1/9, (Figure 6) with half-maximal binding at 1/25 and 1/90 and detectable binding at 1/ 250 and 1/800, respectively. These antibodies represent the lower range of avidity for the five anti-kCN antibodies. The Group II UAb (58416, 58488, 58-504) exhibit higher maximal and half-maximal binding with detection of binding at higher dilutions. The maximal binding occurs between 1/30 (MAb 58488 and 58-504)and 1/80 (MAb 58416) dilution of supernatant. The half-maximal binding for these three MAb was less than 1/200 (58409: 1/27; 58-557:1/162)while detectable binding was 1/ loo0 dilution of culture supernatant. Epltope Slmllarlty and Antlbody Afflnlty




Figure 6. Avidity of j3-caseiu monoclonal antibodies (MAb). Cowemation-dependentantitdy binding was determined using an indirect, solid phase radioimmunoassay. Culture supernatant from actively growing, log phase MAb 58-409 (0). MAb 58416 (A), MAb 58-488 (U), MAb 58-504 (0).and MAb 58-557 0 cultures were diluted over tbe range 1 to 1 x lo6. Antigen was at 15 pg/rnl. AU assays were performed in triplicate. Ihe “dilution factor” is the reciprocal of the dilution (l/dilution).

antibody, even at concentrations of competing, unlabeled antibody, which exceeded the labeled test antibody by 200:l (Figure 7). In self-competition assays, MAb 57-115 inhibited binding of [lZI]57-115 by 90% at a ratio of 75:l of unlabeled to labeled antibody. Half maximal inhibition of binding was observed at a ratio of 6:l. In contrast, the self-


Ratio of Unlabe1ed:Labeled Monoclonal

Figure 7. Epitope determination by antibody competition using a-casein monoclonal antibody (MAb). The ratio of radiolabeled a-casein specific MAb competed with unlaperformed in

MAb 57-310 (0) Competition studies clearly indicated that with labeled 57-115 (o),Ii MAb 57-115 AND 57-310 antibody did not beled 57-115 (A), and block the binding of the other labeled, s1-CN beled 57-310 (A). Journal of Dairy Science Vol. 74, No. 3, 1991


Antlbody Concentration, Log(Dilution Factor)




conditions, destroyed their immunologic reactivity. The affinity of the bovine p-CN MAb was 2.39 x lo9 W1,1.33 x 1Olo W 1 , and 5.9 x lo9 W1 for 58-409, 58-416, and 58-557, respectively.


2 i 0







Ratio of unlabeled to labeled Antibody




In summary, seven monospecific MAb to %I- and P-CN have been isolated and their affinity and binding specificity characterized. Two MAb (57-115 and 57-310), specific for bovine q l - C N by both solid-phase RIA and Western blot analysis, recognize two different epitopes on asl-CN. Five MAb, 58-409, 58416,58-488,58-504, and 58-557, are monospecific for bovine p-CN and recognize a similar, if not identical, epitopic region or regions, which are apparently exposed in both undenatured antigen (RIA) and on Western blots after exposure of antigen to SDS.

Ratio of unlabeled to labeled Antibody


Supported by a grant from the National Dairy Promotion and Research Board to C. W. Beattie. REFERENCES












Ratio of unlabeled to labeled Antibody

Figure 8. Epitope determination by monoclonal antibody (h4Ab) competition using &casein specific MAb. l k e ratio of radiolabeled basein specific MAb competed with unlabeled MAb was varied from 0 to 100. a) MAb 58-409 was radiolabeled.b) MAb 58-416 was radiolabeled. c) MAb 58-557 was radiolabeled. Symbols: MAb 58-409 (0), 58-416 (0).58-488 (A), 58-504 (A), 58-557 (0).

inhibition of MAb 57-310 required a ratio of 150:l in order to block [lZ5r]57-310binding at the 50% level. The affiity of the bovine CN antibodies was 1.63 x 10" M - I (57-115) and 2.13 x 10" M-' (57-310). Nearly complete inhibition of labeled binding was achieved for the p-CN MAb 58-409, 58-416, and 58-557 at a ratio of 1OO:l (Figure 8). Self competition assays for MAb 58488 and 58-504 could not be evaluated as isotopic labeling of these antibodies, even under mild

lAmetani, A., S. Kaminogawa. S. Shimizu, and K. Yamauchi. 1987. Rapid Screening of antigenically reactive fragments of ql-casein using HPLC and ELISA. J. Biochem. 102:421. 2Bonsing, J., and A. G. MacKfiday. 1987. Recent studies on nucleotide sequences encoding the caseins. J. Dairy Res. 54447. 3Brown, J. M. 1983. Detection of a human sarcomaassociated antigen with monoclonal antibodies. Cancer Res. 43:113. 4 B ~ h e l lJ., , J. Bartek, and J. Taylor-PapadimitriOU. 1985. Production and characterbtion of monoclonal antitdies to human castin. A monoclonal antiidy that can react with casein and a-lactalbumin. Hybridoma 4341. 5 De St. Groth, F., and D. Scheidegger. 1980. Production of monoclonal antibodies: strategy and tactics. J. Immunol. Methods 351. 6 Earl, H.M., and R.A.J. McIlhenny. 1985. Monoclonal antibodies to human casein. Mol. h u n o l . 22:981. 7Pnguet, B., L. Djavadi-Ohauiance, J. Pages, A. Bussard, and M. Goldberg. 1983. A convenient enzymelinked immunosorbent assay for testing whether monoclonal antibodies recognize the same antigenic site. Application to hybridomas specific for the BZ-subunit of Escherichia coli tryptophan synthase. J. Immunol. Methods 60:351. SFrankel, M. E.,and W. Gerhard. 1979. The rapid

Journal of Dairy Science Vol. 74. No. 3, 1991



determination of binding constants for antiviral antibodies by a radioimmunoassay. An analysis of the interaction between hybridoma proteins and influenza Viros. Mol. Immunol. 16:lOl. 9 Howard, P.D., J. A. Ledbetter,S. Q. Mehdi, and L. A. Haenberg. 1980. A rapid method for thc detection of antibodies to cell surface antigens: a solid phase radioimmunoassay using cell membranes. J. Immunol. Methods 3875. 10 Hunter, W. M., and P.C. Greenwood. 1962. Reparation of 131iodine-labelledhuman growth hormone of high specific activity. Nature (Lond.) 194:495. 11 Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of the bacteaiophage T4. Nature (Land.) 222680. 12 Letkovits, I., and H. Waldman 1979. Ldting dilution analysis. Page 60 in Limiting dilution analysis of cells in the immune system. Lefkovitz, I., and H. Waldman, ed. Cambridge Univ. Press, Richmond, VA. 13 Merril, C. I&., D. Goldmann, and M.L. Van Keuren. 1984. Gel protein stains. Silva stain. Methods En-

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Isolation and characterization of monoclonal antibodies monospecific for bovine alpha-casein and beta-casein.

Two monoclonal antibodies monospecific for bovine alpha s1-casein, which recognize three genetic variants of alpha s1-casein, have been isolated and t...
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