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ANTIGEN USED FOR IMMUNIZATION
Progesterone 3(o-carboxymethyl) oxime conjugated to bovineserum albumin (BSA.P) (steroids:BSA, 38:1) was obtainedfrom the Sigma (St. Louis, MO). All other steroids, as well as,the antigens b-estradiol 6-(o-carboxymethyl) oxime conjugatedto bovine serum albumin (BSA.E), testosterone conjugated tobovine serum albumin (BSA.T), aldosterone conjugated tobovine serum albumin (BSA.A), corticosterone conjugated tobovine serum albumin (BSA.C), and dexamethazone were alsoobtained from Sigma.
METHOD OF IMMUNIZATION
Progesterone was dissolved in Phosphate-buffered saline (PBS:10 mM K2HPO4, 10 mM KH2PO4, 0.15 mol/L NaCl, pH 7.2)and mixed an equal volume of Freund’s complete adjuvant and0.2 mL (50 mg of progesterone) was given intraperitoneal in-jection to five 8-week-old BALB/c mice. Booster injectionswere performed with three weekly intervals by using of Fre-und’s incomplete adjuvant (Sigma). Four days before fusion,an intravenous injection of 25 mg of progesterone in 0.1 mLPBS without adjuvant was given.
The indirect enzyme-linked immunosorbent assay(ELISA)(1,2) was used for the screening of hybridoma super-natant and plasma of the mice. Briefly, 96-well polystyreneplates (NUNC immunoplates; Denmark) were coated with 200ng BSA.P and, parallel with BSA in 100 mL PBS. Coating ofplates was carried out at 4°C overnight. Then 1% milk powderin PBS was added to the wells and the plates were incubatedfor 1 h at 37°C. The diluted mouse plasma (in PBS) or hy-bridoma supernatant was added. Each hybridoma supernatantwere tested with each steroids (BSA.P, BSA.E, BSA.T, BSA.C,BSA.A, and dexamethzone) and the plates were incubated at37°C for 1 h. Alkaline phosphatase conjugate of polyvalentgoat-anti-mouse Ig (Sigma) at 1:1000 dilution buffer (PBS) wasadded to each well and incubated for 1 h at 37°C. Para-nitro-phenyl phosphate mg/mL in substrate buffer (1 mM ZnCl2,MnCl2, 0.1 M glycine pH 10.4) was added subsequently. After45 min, the reaction was measured by the absorbance at 405nm on a Bio-Tech EIA reader. PBS-Tween-20 was used aswashing buffer between each incubation.
The spleen cells of the BALB/c mouse immunized with pro-
gesterone, as described above, and mouse myeloma cells F0(ATTC CRL 1646) were used in fusion. The myeloma cellswere cultivated in Dulbecco’s minimum essential medium(DMEM) medium (Gibco, Grand Island, NY) supplementedwith 10% fetal bovine serum (TBS) (Biochrom, Berlin, Ger-many) under humidified atmosphere of 5% CO2.
SELECTION AND CLONING PROCEDURE
1.107 myeloma cell line F0 (ATCC CRL 1646) and 1.108 spleencells (immunized with progesterone) were fused between 1/10ratio using polyethylene glycol 4000 as fusing agents.(3–5) Af-ter fusion, the cells were distributed into the wells of 96-cul-ture plates (Nunc) at a cell density 25,000 cells per well andcultivated in the standard medium (DMEM) supplemented with20% fetal calf serum (FCS, Biochrom), 100 mM hypoxantine,0.4 mM aminopterine and 16 mM thymidine (HAT). After14–21 days, hybrid cells showing antibody activity againstprogesterone were cultured further using a layer of feeder cells(macrophages) from a nonimmunized BALB/c mouse to assistin the early stages of growth. Positive hybrid clones producingantibody with the highest specificity were subcloned by limit-ing dilution.(6) At each stage of growth, aliquots of hybrid cul-tures (3–5.106 cells) were frozen in liquid nitrogen in 80%DMEM, 20% FCS, and 10% dimethylsulphoxide.
Out of 17 clones, 2, MAM-2A9 and MAM4H10 producingantibody with the highest specificity were subjected to threesubsequent subcloning steps by limiting dilution.(4)
SPECIFIC ANTIGEN IDENTIFIED
The affinity of these monoclonal antibodies (MAbs) for pro-gesterone were determined by equilibrium dialysis. The affin-ity constants (Kd,M) measured for the purified MAbs are shownin Table 1.
HEAVY AND LIGHT CHAIN OFIMMUNOGLOBULIN
Two progesterone-specific MAbs were obtained. MAbs wereidentified as the IgG class, which were IgG1 (MAM-2A9) and
HYBRIDOMAVolume 20, Number 1, 2001Mary Ann Liebert, Inc.
MAbs Against Progesterone Hormone
69
Monoclonal Antibodies
MAbs AGAINST PROGESTERONE HORMONE70
IgG2a (MAM4H10) subtype, using hybridoma subisotyping kit(Behring Diagnostics, La Jolla, CA) system.
SPECIFICITY
Two MAbs had higher affinity for the progesterone used in theimmunization did not show any cross-reaction with the othersteroid hormones (Table 2).
AVAILABLITY
Tissue culture supernatant Yes 3 NoAscitic fluid Yes No 3Hybridoma cells Yes No 3
ACKNOWLEDGMENT
This work was supported in the framework of the NATO Sci-ence for Stability Programme (Project NATO-Tu-Biotechnol-
ogy II). The authors are grateful to A.I. Manav for technical as-sistance.
ADDRESS CORRESPONDENCE TO:
F. YücelInstitute for Genetic Engineering and BiotechnologyTUBITAK Marmara Research CenterGebze, KocaeliTurkeyTelephone: 90-262-6412300Fax: 90-262-6412309E-mail: [email protected]. ÇirakogÏ luDepartment of Medical BiologyMedical Faculty Marmara UniversityHaydarpasa-IstanbulTurkeyTelephone: 90-216-4144737Fax: 90-216-3480848E-mail: [email protected]
REFERENCES
1. Sauer MJ, Foulkes JA, Worsfold A, and Morris BA: Use ofprogesterone 11-glucuronide-alkaline phosphatase conjugatein a sensitive microtitre-plate enzyme immunoassay of prog-esterone in milk and its application to pregnancy testing indairy cattle. J Reprod Fertil 1986;76:375–391.
2. Engvall E: Enzyme immunassay ELISA and EMIT. Meth-ods Enzymol 1980;70:419–439.
3. Galfre G, and Milstein C: Preparation of monoclonal anti-bodies: strategies and procedures. In: Methods in Enzymol-ogy. Langone JJ, Vunakis H (Eds.). Academic Press, NewYork, 1981, pp. 3–46.
4. Fazekas S, Groth ST, and Scheidegger D: Production ofmonoclonal antibodies strategy and tactics. J Immunol Meth-ods 1980;35:1–21.
5. Liddell JE and Cryer A: A Practical Guide to MonoclonalAntibodies. John Wiley & Sons. Ltd., England, 1991, p. 67.
6. Lietzke R, and Unsicker K: A statistical approach to deter-mine monoclonality after limiting cell population of a hy-bridoma clone. J Immunol Methods 1985;76:223–228.
TABLE 1. APPARENT DISSOCIATION CONSTANTS (Kd) OF MABS ANTIBODIES OBTAINED USING
IMMUNIZATION WITH PROGESTERONE
Monoclonal antibody KdM
MAM-2A9 0.1 3 1028
MAM-4H10 1.6 3 1027
TABLE 2. COMPARISON OF THE REACTIVITY (OD405) OF THE MABS WITH DIFFERENT STEROIDS
Hybrids
Steroids used in ELISA MAM-4H10 MAM-2A9
Progesterone 1.31 1.41Estradiol 0.11 0.10Testosterone 0.13 0.12Aldosterone 0.11 0.11Corticosterone 0.10 0.10Dexamethazone 0.20 0.23BSA 0.21 0.18No antigen (blank) 0.10 0.10
