'Present address: John Innes Institute, Colney Lane, Norwich NR4. 7UH, U.K.. 2Abbreviations: McAb, monoclonal antibody; KLH, keyhole limpet hemocyanin ...
Plant Physiol. (1988) 88, 959-960 0032-0889/88/88/0959/02/$0 1.00/0
Communication
Monoclonal Antibodies to 13-Deoxy-Gibberellins Received for publication February 21, 1988
J. PAUL KNOX', MICHAEL H. BEALE, GEOFFREY W. BUTCHER, AND JAKE MACMILLAN*
Monoclonal Antibody Centre, AFRC Institute ofAnimal Physiology and Genetics Research, Cambridge Research Station Babraham, Cambridge CB2 4AT, U.K. (J.P.K., G.W.B.), and School of Chemistry, University of Bristol, Bristol BS8 I TS, U.K. (M.H.B., J.M.) munogen and the isolation of two different McAbs that recognize 1 3-deoxy-GAs.
ABSTRACT The production and characterization of two high affinity rat monoclonal antibodies to 13-deoxy-gibberellins is described. Hybrid myelomas were derived from rats immunized with an immunogenic keyhole limpet hemocyanin-gibberellin conjugate, linked at carbon-3 to gibberellin A4 via a hemisuccinate bridge. The selected monoclonal antibodies were characterized by a competitive radioimmunoassay.
We have recently described (5) the production of a set of McAbs2 which recognize different structural features of the GAs. Our approach was based on the premise that linkage of GAs to immunogenic protein carriers at different positions on the GA molecule would expose different GA-epitopes, thereby providing a range of McAb specific to these epitopes. Use of the protein conjugate of GA, linked at C-3 via a hemisuccinate (Scheme I, structure 1) led to the production of a McAb (MAC 136) which
O9-1
CH2
H0 CO CH2
CH2 I
CORH
(1) R- NH-KLH, R1 OH (2) R=OH, R1 H (3) R=OCOOCH2CHMe2, R1 H (4) R-NH-KLH ,RX-H SCHEME I
recognized 1 3-hydroxy-GAs that contain a 7-CO2H group and a 16,17-double bond. Thus, this antibody binds efficiently to GA53, GA,9, GA20, GA,, GA29, and GA8 and will be useful in the immunoaffinity purification from plant extracts of these members of the early-13-hydroxylation biosynthetic pathway. This paper describes the preparation of McAb using the analogous GA4-3-hemisuccinyl conjugate (Scheme I, structure 4) as im-
MATERIALS AND METHODS Chemicals. [1,2 -3H2]GA4 (1.41 x IO'5 Bq mol-') was obtained from Amersham International (Cardiff, U. K.). The preparation of GA4-3-hemisuccinate (2), by standard procedures, has been outlined (1, 6h) and will be described in detail elsewhere. Preparation of GA4-3-Hemisuccinyl-KLH Conjugate. To [1,23H2]GA4-3-hemisuccinate (2) (35 mg, 6400 Bq) in dry dioxane (2 mL) was added tributylamine (23 uL) and isobutylchloroformate (13 ,sL). After 15 min, formation of the less polar mixed anhydride (3) was confirmed by analytical TLC. The reaction mixture was then added dropwise over 5 min to a stirred solution of KLH (40 mg) in 25 mm borate buffer, pH 9.0 (30 mL), and dioxane (15 mL) at 4°C. After 24 h at 4°C the solution was dialyzed, initially against 25 mm borate buffer, pH 8.0, and then against deionised water, until no more [3H] was released. Lyophilization yielded 50 mg of GA4-3-KLH conjugate (4) (20 Bq mg-', equivalent to 251 nmol GA4, mg-' conjugate). Cell Fusions, Hybridoma Selection, and Antibody Screening. Three procedures were carried out as previously described (5). Radioimmunoprecipitation Assay. Rat antisera were monitored, and hybridoma cell culture supernatants were screened, by a radioimmunoprecipitation assay using [1,2-3H]GA4 as already described (5). Derivation of Hybrid Myelomas. Hybrid myelomas secreting McAbs MAC 213 and 214 were derived from a fusion of spleen cells of a female F344 rat immunized with GA4-3-KLH. The initial injection was performed intramuscularly with 200 jg of conjugate dissolved in PBS and emulsified with complete Freund's adjuvant. A boost of 200 ,Ag of immunogen in incomplete Freund's adjuvant was performed on d 42. A pre-fusion boost of 100 ,ug ofconjugate in 1 ml PBS was given intravenously on d 84, 3 d before the fusion. MAC 213 was derived from a fusion of spleen cells with the rat myeloma line Y3 Agl.2.3 (Y3) (3). MAC 214 arose from a fusion of spleen cells with mouse myeloma NSO/unc (4). Characterization of McAb. McAb classes were determined by Ouchterlony immunodiffusion. The affinities of the McAb were derived from the standard curve of binding inhibition by GA4 as described by Muller (8). The cross-reactivities of other compounds were determined by competitive radioimmunoassay and expressed as a percentage on a molar basis relative to GA4 as described previously (5).
'Present address: John Innes Institute, Colney Lane, Norwich NR4
RESULTS AND DISCUSSION Two fusions were performed with spleen cells from a rat immunized with GA4-3-KLH (4). One half of the cells were fused
7UH, U.K.
2Abbreviations: McAb, monoclonal antibody; KLH, keyhole limpet hemocyanin (Sigma); GA, = gibberellin A". 959
960
-KNOX ET AL.
Table I. Characteristics ofMcAb Developedfrom Immunization with GA4-3-KLH McAb
Characteristic Myeloma Immunoglobulin class Affinity for GA4 (L mol-')
MAC 213 Y3 IgG2a 6.7 x 108
MAC 214 NSO IgG2a 3.5 x 10'0
Table II. Specificities of McAb For structures, see Crozier (2) and MacMillan (6, 7). Specificity MAC 213 MAC 214 Amount GA4 equivalent to 100% 2 pmol 0.23 pmol cross-reaction 0. 1% 0.3% C,g-GAs GA, GA3 0.1 0.1 100 100 GA4 0.3 0.5 GA5
