HYBRIDOMA Volume 10, Number 1, 1991 Mary Ann Liebert, Inc., Publishers
Monoclonal Antibodies to Gelonin: Production and Characterization JOACHIM ZIMMERMANN and WOLFGANG E. TROMMER Fachbereich Chemie der Universität Kaiserslautern, FRG
ABSTRACT BALB/c mice were immunized with gelonin, a 30kD glycoprotein (type 1 RIP) from the seeds of Gelonium multiflorum. By polyethylene glycol-induced fusion of isolated spleen cells with the myeloma cell line NS-1, three different hybridomas were obtained. Two of them were found to secrete antibodies of the IgG^ subclass, whereas the third cell line produced antibodies of the IgM type. The IgG^ secreting cell lines were adapted to serum-free medium conditions, and the antibodies were isolated from the culture supernatant. The isolated antibodies recognize independent epitopes on the gelonin molecule. The toxicity of gelonin in reticulocyte lysates was not affected when the protein was incubated with the antibodies. The lgG-|S exhibit average affinity constants of about 109 M~1 and 1010 M respectively, as determined by a solid-phase EIA using the avidin-biotin system. .
INTRODUCTION In view of their
of autoimmune diseause the of immunotoxins years (1,2). Among the various toxins which could be used in the preparation of such conjugates, ribosome-inactivating proteins (RIPs) are of growing importance (3,4.5). These proteins can be isolated from many plants (6) and exist either as single chain (type 1 RIPs) or as double-chain proteins (type 2 RIPs). The latter are extremely toxic towards intact cells, because the B-chain of these toxins binds to cell surfaces and allows the A-chain to enter the cytoplasm where it damages the 60S-subunit of the ribosomes irreversibly (7.8,9). The best known example of a type 2 RIP is ricin (10,11). Type 1 RIPs lack the B-chain and are much less toxic to intact cells. On the other hand, type 1 RIPs inhibit in-vitro translation (reticulocyte lysate) nearly as strongly as type 2 RIPs, making them attractive for the application as ¡mmunoconjugates (16). Gelonin, a 30 kD glycoprotein from the seeds of Gelonium multiflorum is an example of a type 1 RIP, and several immunotoxins were produced with gelonin as In
ses, much interest has been focused in recent
toxic component (12-15). In order to facilitate the purification and identification of such immunotoxins, we have prepared monoclonal anti-gelonin antibodies which may be utilized for the production of an affinity column.
MATERIALS AND METHODS
Reagents Peroxidase-conjugated rabbit immunoglobulins to mouse immunoglobulins were from DAKOPAZ (Glostrup, Denmark). Anti-mouse immunoglobulin isotype-specific monoclonal antibodies and anti-mouse immunoglobulin alkaline phosphatase -conjugate were obtained from PharMingen (San Diego, USA). N-hydroxysuccinimidobiotin, avi-
din-peroxidase-conjugate, 2,2'-azino-di(3-ethylbenzthiazoline sulfonic acid) (ABTS) and Coomassie Brilliant Blue R250 are commercially available from Sigma (St. Louis, USA). Immuno-microtiter plates were from Dynatech. RPMI 1640 medium, DMEM/ F12 Ham's Nutrient Mix (1:1), fetal bovine serum, sodium pyruvate (100 mM), penicillin (10000 u/ml)/streptomycin (10000 ug/ml) were from Gibco (Eggenstein, FRG). Nutridoma NS serum substitutent is a product of Boehringer (Mannheim, FRG). Protein G Sepharose 4 Fast Flow was purchased from Pharmacia (Uppsala, Sweden). Reticulocyte lysates (untreated), SERVALYTE Preocotes and marker proteins for IEF were obtained from Serva (Heidelberg, FRG). Cellulose acetate filters 20 urn were from Sartorius (Göttingen, FRG). Fractogel CM 650-(S) and polyethylene glycol (m. w. 1500) are products of Merck (Darmstadt, FRG). L-(U-14C)-valin (50 uCi/ml) was from Amersham International (Little Chalfort, UK).
Purification of Gelonin
Gelonin was isolated according to Stirpe et al. (12) with the modifications as described in (13). Peeled and homogenized seeds were extracted with 5 mM sodium phosphate buffer (pH 7.4) containing 140 mM NaCl (18h, 4°C). The extract was centrifuged and dialyzed against 5mM sodium phosphate containing 5mM Na-EDTA (pH 6.5). The supernatant was applied on Fractogel CM-650 (S), and gelonin was eluted with 0.17M NaCl in starting buffer. Yields ranged from 2-3 mg gelonin per g of shelled seeds as based on UV-absorption (12).
Production of MAbs Four female BALB/c mice were injected intraperitoneally (i.p.) with 20 (xg gelonin in 50 (il PBS emulsified with 100 ul Freund's complete adjuvant. After 14 and 28 days, respectively, 10 ug gelonin in 50 (il PBS emulsified with 100 (il Freund's incomplete adjuvant were given i.p. Final boosts with 10 (ig gelonin in 100 |il PBS were given i.p. on days 58, 59 and 60 (17). On day 61 the spleen was removed and 108 washed spleen cells were fused with P3-NS1-Ag 4-1 (abbreviated NS-1) myeloma cells (ratio 4:1) according to the method of Galfrè and Milstein (18,19,20). The selection of hybrids was carried out by growth in HAT medium (21). The culture supematants of wells with
growing hybrids were assayed by an ELISA on gelonincoated plates according hybrids were cloned by the limiting dilution technique.
Serum-Free Medium Conditions
Antibody producing with sodium
grown in F12/DMEM (1:1) medium
pyruvate (1 mM), mercaptoethanol (50 uM), penicillin/streptomycin
(1000 u/ml, 1000 ug/ml) and FCS (10%) together with Nutridoma NS stitute (1X). The hybridomas were adapted to serum-free conditions decrease in the serum content over two weeks.
Determination of Antibody Subclasses The immunoglobulin subclasses of the produced antibodies were determined by ELISA procedures as described in (24) using anti-mouse isotype-specific antibodies for coating of the microtiter plates and anti-mouse antibody alkaline-phosphatase conjugates for detection of bound mouse antibodies.
Purification of MAbs
500 ml of serum-free culture supernatant was centrifuged, filtered through a 20 |im cellulose acetate filter and applied on Protein-G Sepharose (flow: 0.5 ml/min; gel volume: 0.5 ml)(34). After washing the column with 20 mM sodium phosphate pH 7.2 the MAbs were eluted with 0.1 M glycine/HCI (pH 2.7), immediately neutralized with 1 M Tris/HCI pH 9.0, and dialyzed against PBS. Yields ranged from 5-7 mg IgG per 500 ml supernatant.
Determination of Isoelectric Points
10 til of the purified antibodies (1-2 mg/ml) in PBS were applied to SERVALYTE Precotes (12,5 x 12,5 cm, 150 i_m, pH 3-10 ). The isoelectric focusing was performed as described in (36) and was completed when the current was constant at 1 mA for at least 15 min at 1500 V. The focused antibodies were stained with Coomassie Blue R250. Serva protein test mixture 9 was used as standard.
equal amounts (20 ng per well) of the proteins performed as described in (24). The MAbs (2 mg/ml in PBS) were diluted with PBS-Tween to about 20 ug/ml. To detect the binding of the antibodies, an anti-mouse immunoglobulin peroxidase conjugate was used. Microtiter
tested, and ELISA experiments
Biotinvlation of MAbs The purified MAbs were biotinylated with biotin-N-hydroxysuccinimide ester (25). 2 ml of MAb in PBS (2 mg/ml) were dialyzed overnight (4°C) against 0.1 M NaHC03 buffer containing 0.15 M NaCl, pH 8.3. The solution was mixed with 200 ul N-hydroxysuccinimidobiotin (1 mg/ml in DMSO) and reacted for 2-3 h at room temperature followed by dialysis against PBS (18 h, 4°C).
Epitopes Recognized by
plate was coated with 2 (ig gelonin per well as described in biotinylated antibody (100 ul) was added to serial 2-fold
(24). A constant amount of
dilutions of unlabeled MAb in PBS (100 ul), and the mixtures were added to the wells of a microtiter plate (100 ul/well). The ELISA was performed as described in (24). The bound biotinylated MAbs were detected by means of an avidin-peroxidase conjugate (Sigma). The amount of biotin-labeled antibody was chosen to give an absorption of about 1-1.5 in the absense of unlabeled antibody.
In-v itro Translation
mg/ml) in PBS was incubated for 2 h at room temperature with amounts of purified MAbs (0.6 mg/ml). After incubation the mixtures were diluted to a gelonin concentration of 1 ug/ml. The inhibition of in-vitro translation caused by these gelonin-antibody complexes was tested in reticulocyte lysates ac-
cording to the method of Pelham and Jackson by pure gelonin under identical conditions. Calculation of
The calculation of the affinity constants by an ELISA method the RIA procedure described by Clark and Wagener (27,28).
a.) Serial 2-fold dilutions of gelonin from 256 (ig/ml to 0.5 ug/ml in coating buffer (24) were pipetted into the wells of a microtiter plate. After 18 h at 4°C the liquid was removed by flicking the plate, and the latter was incubated with 1X bovine serum albumin in PBS at room temperature for at least 2 h to block non-specific binding. A constant amount of biotinylated antibody was added, and after 90 minutes the bound biotinylated antibodies were determined by an avidin-peroxidase conjugate (Sigma, 1000x diluted). Between each step the plate had to be carefully washed with PBS-tween buffer. The amount of biotin-labeled antibody was chosen to give a maximal absorbtion of 2. The gelonin concentration for half maximal absorption was used for the coating of the next microtiter plate (20 ug/ml). b.) A constant amount of biotinylated antibody (tracer, 100 ul) was added to serial 2-fold dilutions of unlabeled MAb (inhibitor) in PBS (100 ul), and 100 ul of the mixtures were added to the wells of a microtiter plate which had been coated according to a. The remaining rows contained the same amount of biotin-labeled MAb without the inhibitor. The ELISA was performed as described above. After the incubation the supematants of the wells without inhibitor were transferred to another microtiter plate which had been coated with the same amount of gelonin (20 ug/ml). The concentrations of unlabeled MAbs that led to half maximal inhibition were evaluated. c.) Determination of the b-factor was carried out as follows: The supematants from wells without inhibitor were transferred to another microtiter plate. Serial 2-fold dilutions of biotinylated MAbs were pipetted into the remaining wells, beginning with the concentration used under b. The ELISA was performed as described above. By comparison of the absorption of the supernatant with the absorption of the standard dilution, the fraction of the bound biotin labeled MAb could be determined.
The concentration of unlabeled MAbs leading to half maximum inhibition and the b-factor were used to calculate the affinity constant according to the following
where l( is the inhibitor concentration (unlabeled MAb) at 50 % inhibition; T. is the total concentration of biotinylated MAb; b, the fraction of bound biotinylated MAb in absense of inhibitor.
BALB/c mice were immunized with purified gelonin, and blood probes were still positive in an ELISA on 2x104-fold dilution when the immunization schedule was complete. Spleen cells were fused with NS-1 myeloma cells with polyethylene glycol (PEG 1500) as fusing agent, whereby best results could be obtained when the ratio of spleen cells to myeloma cells was about 4:1. The selection of growing hybrids was carried out in HAT (hypoxanthine, aminopterin thymidine) medium. Spots of growing hybrids were visible after eight days and the supematants were assayed every other day for the production of anti-gelonin antibodies. Three positive hybrids were cloned twice according to the limiting dilution In order to increase the probability for single clones, the cells were diluted as to allow for growth in about 403! of the wells only (statistically one cell per well). The culture medium used for the fusion, selection and cloning was RPMI 1640 (20 X fetal bovine serum). After cloning, the RPMI 1640 medium was replaced by a 1:1 mixture of DMEMHam's F12 (10% fetal bovine serum) because of the somewhat higher content of amino acids and lipid precursors essential for the growth of hybridomas in the absense of serum (22,23). The medium was additionally supplemented with NutridomaNS serum substitute (1%), and after successive reduction of the serum content during two weeks the hybridomas grew in its absence. Two of the hybridomas were found to secrete antibodies of the lgG-| type (ZAG1, ZAG2) whereas the third hybridoma secreted IgM antibodies (ZAG3) as determined by an ELISA using anti-mouse isotype-specific antibodies for coating of the microti-
IgGi's were purified directly from the culture supernatant by protein G affinity chromatography. The apparent molecular weights of the light and heavy chains as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis Both
found to be 28 kD and 59 kD for ZAG1 and 28 kD and 55 kD for ZAG2,
spectively (Figure Isoelectric
of the antibodies
three bands in the range from 6.4
to 6.6 for ZAG1 and three bands from 6.7 to 6.9 for ZAG2 (data not shown) establishing their structural difference. Attempts to seperate the three bands by means
of ion exchange chromatography were unsuccessful. To assess their specificity with respect to gelonin, the cross-reactivities with several other proteins were tested. Table 1 summarizes the results of the corresponding ELISA experiments. As can be seen in Table 1, ZAG1 and ZAG2 are highly specific with respect to gelonin. Interestingly cross reactivity occurs only with the glycoprotein thyroglobulin, whereas there is no reaction with all the other tested proteins, indicating that the
Myoglobin FIGURE 1. SDS-PAGE of ZAG1.
MAbs. Lane A, marker mix; lane B, ZAG2; lane C,
TABLE 1 Cross reactivities with different proteins as determined by an ELISA. Bound antibodies were detected with an anti-mouse immunoglobulin peroxidase conjugate. Values are B, C, inactivaexpressed as optical density units at 414 nm. A, ted peroxidase ; D, ovalbumin*; E, deglycosylated gelonin; F, bovine serum albumin; G, myoglobin; H, chymotrypsinogen; I, catalase; J, insulin. (»: Glycoproteins).
MAb ZAG1 ZAG2 ZAG3
0.72 1.1 1.0
0.28 0.36 1.28
0.02 0.0 0.38
0.0 0.0 0.81
E 0.03 0.01 0.01
F 0.0 0.0 0.5
G 0.01 0.0 0.83
0.02 0.03 0.83
0.0 0.01 1.45
J 0.01 0.01 0.65
MAbs are directed against carbohydrate moieties of gelonin. This conclusion is supported by the fact that there is no reaction with gelonin that had been deglycosylated. ZAG3 reacted with all of the tested proteins to a varying degree. Consequently the following experiments were performed with purified ZAG1 and ZAG2 only. The results of the epitope analysis obtained by competitive ELISA are shown in Figure 2 Clearly, both MAbs can compete with themselves but do not affect each other leading us to the conclusion that the two MAbs recognize independent epitopes. In order to decide whether the epitopes as recognized by ZAG1 and ZAG2 are involved in the catalytic mechanism or are at least located near the active site of gelonin the ribosome inactivation by gelonin was tested in reticulocyte lysates after incubation with each of the MAbs (Figure 3). Obviously, the catalytic activity of gelonin is not affected by the presence of the antibodies, indicating epitopes distinct from the active site. reZAG1 and ZAG2 exhibit average affinity constants of 109M"1 and spectively, as determined according to equation (1) by a modification of the procedure described by Clark and Wagener (28). .
Comparison of epitopes recognized by ZAG1 and ZAG2: The wells of a microtiter plate were coated with gelonin and incubated with biotinylated ZAG1 (•, Fig. 2a) or ZAG2 (», Fig. 2b), respectively, together with decreasing dilutions of unlabeled MAb. The bound biotin-labeled antibodies were determined by
4A shows the
at half maximal binding of the MAbs plates in further experiments.
for both MAbs. The concentration of gelonin then used for coating of the microtiter
Figure 4B shows the inhibition curves that were obtained in a competitive inhibition ELISA involving the addition of increasing amounts of unmodified MAbs which competed with a given amount of biotinylated MAbs. The concentration of unlabeled IgG leading to 50X inhibition was used to calculate the average affinity constants according to equation [ID. To decide, whether the MAbs were bound unspecifically to the plate the following negative control was performed. Microtiter plates that had been coated with bovine serum albumin (no gelonin) were incubated with the biotinylated MAbs. After incubation with an avidin-peroxidase conjugate and the substrate the absorption at 492 nm did not exceed 0.05 units, indicating that unspecific binding was negligible. Table 2 summarizes the properties of the produced antibodies.
FIGURE 3. Toxicity of gelonin in reticulocyte lysates: After incubation of gelonin with equimolar amounts of ZAG1 (•) or ZAG2 (w), respectively, inhibition of in vitro translation was measured. Pure gelonin ( ) and BSA ( , no inhibition) were used as controls.
gelonin (|ig/ml) FIGURE 4A. Calculation of affinity constants: The wells of a microtiter plate were coated with decreasing dilutions of gelonin and incubated with constant amounts of biotinylated antibodies (ZAG1», ZAG2»). The bound antibodies were detected with an
avidin-peroxidase conjugate. lOOr
IgGLng] FIGURE 4B. Calculation of affinity constants: The wells of a microtiter plate were coated with gelonin and incubated with the same quantities of biotinylated ZAG1 (•) or ZAG2(~), respectively, as used in 4A, together with decreasing dilutions of unlabeled antibody. The quantitiy of gelonin chosen was sufficient to give 50-70% of the maximal absorption (calculated from 4A).
DISCUSSION a single chain plant toxin (type 1 ribosome-inactivating protein) is in the construction of ¡mmunoconjugates and of antigen-coupled utilized widely immunosuppressants. Hence, specific antibodies against gelonin may be useful in the identification and preparation of such conjugates, as, e.g., for the development of an affinity matrix. Moreover, the intracellular trafficking of the conjugates could be studied by means of labeled antibodies. Three hybridoma cell lines secreting distinct antibodies against gelonin were obtained. The cells were adapted to a serum-free medium in order to simplify the purification of the antibodies. The properties of these antibodies are summarized in
ZAG1 ZAG2 ZAG3
59 kD 55 kD
lightchain 28 kD 28 kD
focusing 6.4-6.6 6.7-6.9
reactivity none none
^Aff ZAG1 ZAG2 ZAG3
toxicity of gelonin
109 M~1 1010 M"1
Table 2. ZAG1 and ZAG2, immunoglobulins of the IgG-. subtype, appear to be rather specific for gelonin and to recognize sugar moieties as shown in Table 1. In contrast, ZAG3, an IgM antibody, exhibits high cross-reactivity and hence, was not characterized further. An apparent problem is the heterogeneity of ZAG1 and ZAG2 as revealed by isoelectric focusing. Both antibodies are resolved in three bands each, however within a narrow pH range. This heterogeneity is most likely due to the fact that the NS-1 cell line used in the fusion experiment still produces light chains, one, or even two of which might be substituted in the antibodies for the light chain originating from the spleen lymphocytes. Although isolated heavy chains are known to bind sometimes as tightly to their epitope as the complete antibodies (35), the affinity of such hybrid antibodies is likely to be impaired. Hence, affinity constants as given in Table 2 are average values for the corresponding mixtures. In this paper we also describe a modified technique for the estimation of affinity constants of antibodies based on an ELISA instead of a solution phase radioimmunoassay. A general problem associated with solid phase techniques is that one of the components of the interaction is attached to a plate and so is immobile. The measured avidity might therefore not be the same as in solution phase where both antibody and antigen are mobile. There have been few direct comparisons between antibody binding to liquid phase and immobilized (solid phase) antigens (37,38), indicating that there is no marked difference between the liquid phase and immobilized avidity constants. However, in most cases the antibody was found to bind more readily to the antigen in liquid phase. Another problem that might occur is that both the antigen-binding sites of the antibody could react with antigens on the plate, leading to an artificially high avidity. In practice, as discussed in (37) the solid phase surface is very far from being saturated with the antigen. Consequently, the probability for such cross links is very low. Since we had shown that unspecific binding of the antibodies to the plate was negligible, our affinity constants rather
A critical parameter in these calculations is the b factor, i.e.. the concentration of free biotinylated antibody (the tracer) in the absence of its unlabeled form, the inhibitor in equation 1. Instead of simply taking the maximum absorption as shown in Figure 4A as a measure for complete binding of the tracer, the finite residual free fraction was determined in a second ELISA and was corrected. In summary, ZAG1 and ZAG2 should prove rather useful as probes for native gelonin in consideration of their high affinity as well as specificity.
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Reprint requests: Prof. Dr. Wolfgang E. Trommer FB Chemie der Universität Kaiserslautern Postfach 3049 6750 Kaiserslautern FRG