Monoclonal Antibodies to Progesterone: Characterization and Selection for Enzyme Immunoassay in Bovine Milk

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<ul><li><p>HYBRIDOMAVolume 18, Number 3, 1999Mary Ann Liebert, Inc.</p><p>Monoclonal Antibodies to Progesterone: Characterization andSelection for Enzyme Immunoassay in Bovine Milk</p><p>A. WALDMANN</p><p>ABSTRACT</p><p>Thirty-one stabile murine monoclonal antibody (MAb) producing cell lines to progesterone were generatedby using a short and a long immunization protocol. Long-term immunization with high doses of 11a-hy-droxyprogesterone-hemisuccinate-bovine serum albumin (11a-OH-P-HS-BSA) antigen led to very good anti-body response in Balb/c mice. The donor mouse produced antiserum with a high titre of 1/250,000. ElevenMAbs were selected for further characterization since they showed high sensitivities ( , 35 pg/well to inhibit50% of the tracer) in bridge homologous enzyme immunoassay (EIA). The results were compared to the donormouse polyclonal antiserum. The MAbs and the donor mouse antiserum were generally found to be highlyspecific, when tested with 30 different steroids. Employing MAb 9C11, with affinity constant, Ka , to 11a-OH-P-HS of 1.1 3 1010 M 2 1, a bridge heterologous microtitre plate EIA for milk progesterone was developed, us-ing the second-antibody coating technique and horseradish peroxidase (HRP) as an enzyme label. The assayis simple and convenient to use, as it permits direct addition of undiluted milk samples, at the same time main-taining high sensitivity, high precision, and a wide range of optical density (OD) values. The major advantageof the assay developed, compared to previously published direct addition milk progesterone immunoassays,is that progesterone concentrations, measured by the EIA, were not influenced by changing milk fat concen-trations, even when milk samples containing up to 10% of milk fat were used for analysis.</p><p>289</p><p>INTRODUCTION</p><p>PRO GESTER ON E IN BLO O D AN D M ILK in cyclic cows reflect theactivity of the corpus luteum and thus provide a precise in-dicator of ovarian function. (1) Besides the importance of pro-gesterone analysis for scientific investigations, determination ofthe hormone is considered a useful parameter for veterinary andanimal production requirements. (2,3 ) Due to its noninvasive na-ture, simplicity of sample collection, and stability of proges-terone in milk, the determination of milk progesterone has be-come a sample of choice in lactating animals.</p><p>Several enzyme immunoassays (EIAs) for milk progesteronehave been described using sheep, rabbit, and goat polyclonalantisera. (411) To improve affinity and obtain substantial quan-tity of antibodies, many groups have prepared mouse mono-clonal antibodies (MAbs) to progesterone. (1221) Some of theseMAbs have been utilized as reagents in different progesteroneimmunoassays developed for human plasma,(19,2224) horse</p><p>plasma,(25) human saliva (18) and human urine. (26) Most an-tiprogesterone antibodies derived by hybridom a technologyhave affinities far below the corresponding conventional anti-sera. This might explain the relatively small number of publi-cations reporting the use of antiprogesterone MAb in quantita-tive progesterone immunoassays , compared to the use ofpolyclonal antibodies. However, the use of MAbs should leadto improvem ents in test standardization and avoid the depen-dency on animals producing high-quality antisera.</p><p>So far, only three MAb based nonisotopic quantitative milkprogesterone immunoassay s have been reported on.(2729) Suf-ficient sensitivity of the milk progesterone assay, developed byStanley et al.,(28) was gained by the use of an enzyme amplifi-cation system of alkaline phosphatase. The amplification pro-cedure, in turn, has made the assay more complicated and ex-pensive than the conventional EIA. Groves et al.(27) producedan ovine MAb to progesterone for a bridge heterologous mi-crotitre-plate EIA. The described assay showed high sensitiv-</p><p>University of Tartu, Institute of General and Molecular Pathology, Veski 34, 51014 Tartu, Estonia.</p></li><li><p>ity, but utilized long incubation steps and elevated temperatureto obtain higher endpoint signal. This again has made the as-say unpractical. In a previous study Waldmann (29) reported abridge homologous MAb-based milk progesterone EIA usinghorseradish peroxidase as an enzyme label.</p><p>The present study aims to characterize 11 high-affinity an-tiprogesterone MAbs for use in EIA, in terms of sensitivity andspecificity, and compare the results with the polyclonal anti-serum obtained from the same mouse. We also report on a de-velopment of a simple direct-addition bridge heterologous mi-crotitre-plate EIA of progesterone in bovine milk using thesecond antibody coating technique. This technique, first re-ported by Meyer and Gven, (30) improves precision and over-comes time dependent drift, observed in microplate im-munoassays when hormone specific antibody is directly coatedto the wells.(31)</p><p>MATERIALS AND METHODS</p><p>Reagents</p><p>Unless mentioned otherwise, all chemicals, steroids, steroidderivates for immunization and synthesis, reagents, and mediaused in cell fusion and cell culture were purchased from SigmaChemical Co. (St. Louis, MO). Tween-20 was obtained fromFerak (Berlin, Germany), Ovalbumin (OVA) from Fluka(Switzerland). The labelled and unlabelled affinity purified sec-ond antibodies were prepared at the Institute of General andMolecular Pathology, University of Tartu.</p><p>Buffers</p><p>Phosphate-buffer ed saline (PBS) 0.15 M pH 7.27.4 con-tained per liter 8 g of NaCl, 0.1 g KCl, 1.15 g Na2HPO4, 0.2 gKH2PO4. PBS with casein, Tween, and Thimerosal (PBSCTT),and PBS with Tween (PBST) were prepared in the same man-ner, but PBSCTT contained, per liter, 0.8 g of casein, 0.5 mLof Tween-20, and 0.2 g of Thimerosal. PBST contained, perliter, 0.5 mL of Tween-20.</p><p>Preparation of hormone conjugates</p><p>11 a -OH-P-HS was conjugated with OVA as described byBacigalupo et al.(32) 11 a -OH-P-HS-HRP and progesterone 3-(O-carboxymethyl) oxime HRP (P-3-O-CMO-HRP) were syn-thesized by the mixed anhydride method according to Munroand Stabenfeldt. (31) The conjugates were chromatographe d ona Sephadex G-25 colum n, then tested for their titer and storedat 2 40C.</p><p>Enzyme substrate solutions</p><p>The substrate solution containing o-phenylened iamine(OPD/H2O2) was prepared by dissolving 1.5 mg of OPD in10 mL 0.1 M citrate/phosphate buffer, pH 5.0 and by adding10 m L of 10% H2O2 . The substrate solution containing3.3 9 .5.5 9 -tetramethylbenzidine (TMB/H2O2) was prepared byadding of 100 m L TMB stock solution (10 mg TMB dissolvedin 1 mL of dimethysul foxide) and 8 m L of 10% H2O2 to 10</p><p>mL of 0.1 M acetate/ citrate buffer pH 4.5 containing 1 mMethylenedia minetetraa cetic acid disodium salt (Reanal, Bu-dapest, Hungary).</p><p>Immunization</p><p>Short-term immunization . Male BALB/c mice received anintraperitoneal (i.p.) injection of 50 m g of 11 a -OH-P-HS-BSA(molar ratio for steroid: BSA, 22:1). The antigen was dissolvedin 50 m L of PBS and emulsified in an equal volum e of com-plete Freunds adjuvant. After an interval of 1 week, 50 m g ofantigen was given as above, except for the use of incompleteFreunds adjuvant. Five days later, final boosts with 400 m g ofantigen in 0.1 mL PBS were given i.p. at 4, 3, and 2 days be-fore fusion was performed.</p><p>Long-term immunization . Male BALB/c mice received ani.p. injection of 100 m g of 11 a -OH-P-HS-BSA (molar ratio forsteroid, 22:1) dissolved in 50 m L of PBS and emulsified in anequal volume of complete Freunds adjuvant. After an intervalof 2 months and 1 month, booster injections were given asabove. One month after the third injection, final boosts with200 m g of antigen in 0.1 mL of PBS were given i.p. on foursuccessive days. Fusion was performed one day after the lastinjection with the spleen cells of the donor mouse showing thehighest serum antibody titer.</p><p>Generation of MAbs</p><p>In each experiment, cells from one spleen were fused withSP2/0 myeloma cells from one 280-mL tissue culture flask(8090% confluence). The splenocytes and the myeloma cellswere washed twice with Hanks solution. The final pellet wasmixed by tapping the tube and 1 mL 50% (v/v) polyethyleneglycol (mol. wt. 4000 E. Merck AG, Darmstadt, Germany) inRPMI-1640 was added over 1 min with gentle shaking. Themixture was centrifuged at 130 3 g for 4 min. The supernatantwas poured off and the mixture was diluted by slow (1 min)addition of 10 mL of RPMI-1640 followed by slow addition (1min) of 10 mL of RPMI-1640 supplem ented with 10% FCS.The mixture was centrifuged for 10 min at 200 3 g, the super-natant was discarded and the fused cell pellet was resuspendedin hypoxantine-ami nopterin-thymidi ne (HAT) medium and dis-tributed (150 m L per well) in the 96-well tissue culture plates.After 20% confluence was reached, aliquots of hybridom a su-pernatants (50 m L) were tested by EIA to detect antiproges-terone antibodies. Selected clones were subcloned by the lim-iting dilution. Ascitic fluids were produced in pristane-prim edBALB/c mice.</p><p>Primary screening of positive clones</p><p>Two-fold diluted cell culture supernatants in PBSCTT (100m L) were incubated for 1 h at room temperature in the wells ofan 11 a -OH-P-HS-OVA immobilized plate (Titertek, Flow Lab-oratories, Irvine, Scotland). After washing with PBSCTT, in-cubation was continued for 1 h with 100 m L of 1/1000 goatanti-mouse immunoglobu lins (GAM Ig) coupled to HRP. Fol-lowing a second washing 100 m L of OPD/H2O2 substrate wasadded. The color reaction was terminated after 30 min by theaddition of 50 m L of 2 M H2SO4 and ODs were read at 492nm with a Twin Reader Plus (Labsystem, S. Turku, Finland).</p><p>WALDMANN290</p></li><li><p>Determination of specificity</p><p>Microtiter plates (Titertek) were first coated by dispensing100 m L of 3.2 m g/mL affinity purified GAM Ig in PBS. Theplates were incubated overnight at 4C. After threefold wash-ing with PBSCTT, plates were incubated with 100 m L of suit-able diluted (1/5001/2500) positive cell culture supernatantsor the donor mouse antiserum (1/250,000) in PBSCTT for 2 hat room temperature (the supernatants and the donor mouse an-tiserum dilutions were selected by 50% inhibition of tracer bind-ing). After three washes with PBSCTT, 10 m L of progesteronestandards and the potentially cross-reacting steroids in the rangeof 0 to 2187 nmol/L in PBS were added to the wells, followedby 100 m L of 11 a -OHP-HS-HRP conjugate (1/30,000). Theplates were incubated for 2 h at room temperature under con-stant shaking, then washed three times with PBSCTT. The colorwas developed with OPD/H2O2 substrate. The cross-reactivi-ties were calculated according to Abraham.(33)</p><p>Typing and subtyping of immunoglobu lins</p><p>Immunoglobul in classes and subclasses were determined bydouble antibody microtiter-plate EIA. The antiserum raisedagainst mouse m -chain coupled to HRP was prepared at this in-stitute. Rabbit antibodies raised against mouse immunoglobu -lins IgG1, IgG2a, IgG2b, and IgG3 were obtained from MilesLaboratories (Elkhart, IN).</p><p>Determination of relative affinity constant</p><p>First, MAbs were purified from ascitic fluid by ammoniumsulfate precipitation and ion exchange chromatography on BlueDEAE-Toyopearl 650S, using the Pharmacia standard FPLCsystem. (34) The IgG concentration was estimated at 280 nm us-ing an extinction coefficient of 14. The affinity constants weredetermined by incubation of serial dilutions of each MAb (from2 m g/mL to 2 ng/mL) in the wells of 11 a -OH-P-HS-OVA im-</p><p>MONOCLONAL ANTIBODIES FOR MILK PROGESTERONE EIA 291</p><p>TABLE 1. CHARACTER ISTIC S O F MABS RA ISED AG AIN ST 11 a -H YDROX YPRO GES TER ON E-11-HE M ISUCCINAT E-BSA</p><p>Cross-reactivitie s (%)</p><p>AntibodySteroids 9C11 8F12 2C9 12A2 3G6 10H3</p><p>Progesterone 100 100 100 100 100 100Progesterone 3-O-carboxymethylo xime 33.9 31.1 19.3 24.3 11 17.311 a -OH-Progesterone 40.7 22.2 79.7 33.5 39.8 50.511 a -OH-Progesterone-11-hem isuccinate 47.5 154 129.7 97.1 86.8 94.511 b -OH-Progesterone 7.6 7.1 9.9 9 8.4 5.217 a -OH-Progesterone 8.1 5.3 5.6 7.7 6.7 8.119-OH-Progesterone 24.7 14.5 8.1 7.8 4.1 3Pregnenolone 12.5 43.6 21.2 5.3 11 48.73 b , 17 a -dihydroxy-5-preg nane-20-dione , 0.1 , 0.1 , 0.1 , 0.1 , 0.1 , 0.15 a -pregnane-3,20-di one 45.1 34.6 26.5 35.5 35.9 66.95 a -pregnane-3 b ,20 b -diol , 0.1 , 0.1 , 0.1 , 0.1 , 0.1 , 0.15 b -pregnane-3 b ,20 b -diol , 0.1 , 0.1 , 0.1 , 0.1 , 0.1 , 0.15 a -pregnane-3 b ,20 a -diol , 0.1 , 0.1 , 0.1 , 0.1 , 0.1 , 0.15 b -pregnane-3 a ,20 a -diol , 0.1 , 0.1 , 0.1 , 0.1 , 0.1 , 0.1Oestradiol-17 b , 0.1 , 0.1 , 0.1 , 0.1 , 0.1 , 0.1Oestriol , 0.1 , 0.1 , 0.1 , 0.1 , 0.1 , 0.1Oestrone , 0.1 , 0.1 , 0.1 , 0.1 , 0.1 , 0.1Cortisol , 0.1 , 0.1 , 0.1 , 0.1 , 0.1 , 0.11-dehydrocortiso l , 0.1 , 0.1 , 0.1 , 0.1 , 0.1 , 0.1Corticosterone , 0.1 , 0.1 , 0.1 , 0.1 , 0.1 , 0.1Deoxycorticoste rone , 0.1 , 0.1 , 0.6 , 0.1 , 0.2 , 0.1Aldosterone , 0.1 , 0.1 , 0.1 , 0.1 , 0.1 , 0.1Aethiocholanol one 6 79.8 35.3 41.4 7.6 11.75 a -dihydrotestostero ne , 0.1 , 0.1 , 0.1 , 0.1 , 0.1 , 0.15 b -dihydrotestostero ne , 0.1 , 0.1 , 0.1 , 0.1 , 0.1 , 0.8Androsterone 0.4 3 0.8 1.3 0.4 0.45 a -androstane-3, 17-dione , 0.1 1.3 0.2 0.7 , 0.1 , 0.15 b -androstane-3, 17-dione , 0.1 8.4 0.9 7.4 0.9 , 0.1Dehydroepiand rosterone 0.1 1.1 , 0.1 0.3 , 0.1 , 0.1Testosterone , 0.1 , 0.1 , 0.1 , 0.1 , 0.1 , 0.14-androstene-3, 17-dione , 0.1 1.3 0.2 0.7 , 0.1 , 0.1Sensitivity pg/wella 6.7 8.3 9.5 9.9 9.9 12Affinity constant expressed as 1010 M 2 1 1.07 NT 0.5 0.65 0.94 NTImmunoglobu lin class IgG2b IgM IgG2b IgG2b IgG2b IgG2a</p><p>aSensitivity, defined as the concentration of progesterone that causes a 50% reduction of the initial binding in the standardcurve in bridge homologous EIA.</p><p>NT 5 not tested.</p></li><li><p>mobilized plates. After 1 h incubation, the saturation curveswere evaluated by peroxidase labelled anti-m ouse IgG anti-bodies, as described above. The relative affinity constants werecalculated as the reciprocal of the MAb concentration giving50% saturation binding.</p><p>Evaluation of the performance of MAb 9C11 for EIAin bovine milk</p><p>Nunc-Immuno Plates MaxiSorp F-96 (Roskilde, Denmark)were coated by dispensing 200 m L of 3 m g/mL affinity puri-fied GAM Ig in PBS. The plates were incubated overnight at4C. After being washed two times with PBST, plates weretreated for 10 min with PBS containing sucrose (50 g/L), BSA(5 g/L), and Thimerosal (0.2 g/L). The plates were air dried atroom temperature, sealed and stored at 4C until use. Before</p><p>the assay, the plates were brought to room temperature. Twentymicroliters of milk samples or standards were added to thewells, followed by 100 m L of suitable diluted (1/12 500) P-3-O-CMO-HRP conjugate and 100 m L of MAb 9C11 (supernatantdiluted 1/2000) in PBSCT. The plates were coated with platesealing tape and incubated for 40 min at room temperature un-der constant shaking. The wells were washed four times withPBST. Two hundred microliters of TMB/H2O2 substrate wereadded and the color reaction stopped after 30 min by adding 50m L of 4 M H2SO4. The ODs were read at 450 nm with a Twin-Reader Plus (Labsystem, S. Turku, Finland).</p><p>Ovucheck method</p><p>Milk progesterone EIA kit based on polyclonal antiserumand alkaline phospha...</p></li></ul>