Immunology Letters, 33 (1992) 217-222
0165 - 2478 / 92 / $ 5.00 © 1992 Elsevier Science Publishers B.V. All rights reserved IMLET 01824
Construction of a quadroma to a-endorphin/horseradish peroxidase using an actinomycin D-resistant mouse myeloma cell line Yu.S. M a s s i n o a, E.A. Kizim a, N . N . D e r g u n o v a b, V . M . V o s t r i k o v b a n d A . D . D m i t r i e v a aInstitute of Higher Nervous Activity and Neurophysiology, Russian Academy of Sciences, Moscow, Russia; and bThe Mental Health Center of the Russian Academy of Medical Sciences, Moscow, Russia
(Received 7 April 1992; accepted 7 April 1992)
1. Summary
2.
A hybrid hybridoma (quadroma), secreting antibodies with double specificity to ~-endorphin (~EP) and horseradish peroxidase (HRP), has been produced. The bispecific antibodies constituted about 28-29% of all immunologically active IgG, produced by quadroma. The quadroma was isolated by fusion of two mouse hybridomas (antiHRP and anti-0t-EP) with distinct phenotypes: double mutant AMDR/HAT s, and wild type (AMDS/HATR). A novel strategy for the construction of a double-mutant was applied, based on the use of an actinomycin D-resistant (AMD R) mouse myeloma for initiation of one of the parental hybridomas.
Bifunctional antibodies, which contain two different antigen combining sites, have been shown to be promising tools for immunohistochemistry, immunoassay and cell targeting [1,2]. Earlier, we have produced hybridomas secreting monoclonal antibodies to the 17-amino acid-residue peptide ~-EP [3,4]. Endorphins (ct, fl and y) are a group of related peptides, showing opioid activity and occurring in certain subpopulations of nervous, neuroendocrine and immune cells as a result of the processing of the high-molecular weight precursor proopiomelanocortin (POMC) [5]. The resulting antibodies bound ~-, r- and ?-endorphins and POMC in RIA, immunoblotting and immunohistology [3,4]. In the present work one of these hybridomas (Fs/40E9) has been fused with a newly established anti-HRP hybridoma to produce antibodies with double specificity to ct-EP and HRP. In the course of this work we have elaborated upon the original approach, facilitating the construction of quadromas - hybrid hybridomas, secreting bispecific antibodies. This approach, based on the use of the AMD R mouse myeloma cell line, is described.
Key words: ct-Endorphin; Peroxidase; Bifunctional antibody;
Actinomycin D resistance; Quadroma Correspondence to: Dr. A.D. Dmitriev, Russian Academy of Sciences, Institute of Higher Nervous Activity and Neurophysiology, 5-a Butlerov Str., Moscow, 117865, Russia. Abbreviations: AMD ~, actinomycin D-resistant; 8-AzaR, 8azaguanine resistant; BSA, bovine serum albumin; DAE, double antigen ELISA; ELISA, enzyme-linked immunosorbent assay; ct-EP, ct-endorphin; HAT s, hypoxanthine aminopterin thymidine-sensitive; HRP, horseradish peroxidase; IFA, incomplete Freund's adjuvant; LDso, lethal dose 50; PAP, peroxidase-antiperoxidase; PBS, phosphate-buffered saline; POMC, proopiomelanocortin; RIA, radioimmunoassay; SAE, single antigen ELISA.
Introduction
217
3.
3.1.
Materials and Methods
Isolation of a mouse myeloma cell line resistant to A M D
The X63-AMD R cell line, resistant to 30 ng/ml of AMD in culture medium, was isolated from X63-Ag8.653 mouse myeloma [6] by stepwise selection. Resistant cells were generated by adding AMD to the growth medium (DMEM + 1020% fetal calf serum (FCS) + 2 mM glutamine + 100 U/ml gentamycin) for 4 months at a concentration which was gradually raised from 0.2 ng/ml (LDs0 for X63-Ag8.653) to 30 ng/ml. The X63-AMD a line was HAT-sensitive (HATS), like the ancestral cells.
3.2.
Generation of parent hybridomas
The mouse hybridoma, secreting antibodies to ~-EP (alone F8/40Eg) had been produced earlier [3,4]. To obtain the anti-HRP hybridoma, spleen lymphocytes from immune BALB/c mouse were fused in the usual way [3] with X63-AMD R cells. Mice were immunized by numerous injections of HRP (total dose 200 #g per mouse). The selection was performed in HAT medium (1 x 10 - 4 N hypoxanthine, 4 x 10 -7 M aminopterin, 1.6 x 10 -5 M thymidine). Anti-HRP antibodies were tested as described below.
3.3.
Back selection to the HA f phenotype
HAT sensitivity (HAT s) was introduced into the cells by cloning hybridomas in the presence of 20 #g/ml 8-azaguanine (8-Aza) at cell density 10-100 cells per well of a 96-well plate.
3.4.
Quadroma construction
Quadromas were generated from the fusion of hybridoma F8/40E9 to ~-EP (IgG2a) and of hybridoma 36F9 to HRP (IgG1). Clone 36F9 had two selective markers: HAT s and AMD R. 1 x 106 cells from each parental line were fused in a usual way [3] and seeded in selective medium into one 96-well plate (2 × 104 cells per well) with a macrophage feeder layer. The selective medium contained AMD (3 ng/ml) and HAT. 218
3.5.
Induction of ascites
5 x 10 6 hybrid cells were inoculated intraperitoneally into BALB/c mice primed as described [3,4] with pristane or incomplete Freund's adjuvant (IFA) or with both agents.
3.6.
Detection of anti-HRP antibodies
Anti-HRP activity was detected in peroxidaseantiperoxidase (PAP) test. The 96-well ELISA plates were coated with affinity isolated rabbit antibodies against mouse IgG (10 #g/ml in 0.01 M Na-phosphate buffer, pH 7.5 at 0°C, overnight). Then the wells were successively incubated with 1% BSA, 100/zl of hybridoma supernatants, diluted 1:2 by PBS with 0.05% Tween 20, and 100-/~1 aliquots of PBS, containing 0.5 #g of HRP (for 90 min, at 37°C). After each step the wells were several times washed with PBS+0.05% Tween-20. O-Phenylenediamine (Sigma) was used for the color reaction. Absorbance was measured at 472 nm in a Microplate reader (Titertek Multiskan ME).
3.7.
Screening of anti-~-endorphin antibody by single-antigen ELISA
Single-antigen ELISA assays (SAE) were carried out in 96-well ELISA plates, coated with ~EP, conjugated to bovine serum albumin. Conjugate was prepared as previously described using bis-diazotinated benzidine [7]. The coating was achieved by incubating the wells overnight at 0°C with 100-/~1 aliquots of 0.01 M sodium phosphate buffer, containing 0.5 #g of ~-EP-BSA conjugate. Next, the wells were incubated with supernatants followed by the HRP-conjugated rabbit antimouse IgG antibodies. The incubation conditions and washing were as described for the PAP test. O-Phenylenediamine was used for the color reaction.
3.8.
Bispecific antibody detection by double antigen ELISA
Bifunctional activity was tested using double antigen ELISA (DAE). In the first step, the wells of the ELISA plate were coated (as described in
section 3.7) with the first antigen, ~-EP, conjugated to BSA [7]. Next, the wells were incubated with hybridoma supernatants, followed by the second antigen, HRP, as described for the PAP test. The reaction was developed with O-phenylenediamine. Supernatants from anti-HRP and anti-0t-EP hybridomas were used as the negative controls.
3.9. Radioimmunological detection of anti-~tendorphin antibodies Anti-0t-EP antibodies were detected as described [3], using synthetic ~-EP (Serva), radioiodinated by the chloramine method [8].
3.10. Immunoblotting The extract of bovine pituitary, prepared as described [9], was separated by electrophoresis in 15% SDS-polyacrylamide gel and electrophoretically transferred to a 0.45-#m nitrocellulose membrane [10]. The blot was cut into strips, each containing one sample lane. Non-specific binding was blocked with 3% BSA. The strips were then individually incubated for 1 h at 20°C with a mixture of bispecific antibodies and HRP, 10 #g each. Peroxidase staining was performed with diaminobenzidine. Buffer systems were as in [9].
3.11.
Purification of quadroma produced antibodies from ascitic fluid by affinity chromatography
H R P and ct-EP were conjugated to CNBr-activated Sepharose 4B (Pharmacia) according to Pharmacia recommendations. 10 mg of H R P was bound to 1 g of CNBr-activated Sepharose 4B. 100 #g of ~-EP was bound to 100 mg of CNBractivated Sepharose 4B. Ascitic fluid (0.5 ml) was diluted 1:10 in PBS with 0.05% Tween-20 and applied to a Protein A-Sepharose column (Pharmacia). IgG was eluted with 0.1 M acetic acid. IgG containing fractions were pooled, neutralized with concentrated ammonia and applied to a HRP-Sepharose column. Antibodies not bound to H R P were washed off by PBS with 0.05 Tween 20. Elution from the affinity column was done using 0.05 M acetic acid-HCl (pH 2.5). The frac-
tions of HRP-binding antibodies were collected, immediately neutralized with concentrated ammonia, pooled and passed in the same way over ctEP-Sepharose column (to separate monospecific anti-HRP IgG and antibodies with double specificity to ~t-EP and HRP). The fraction containing antibodies not bound to HRP was also passed over 0t-EP-Sepharose (to separate anti-~-EP and inactive immunoglobulins). Each fraction was tested for the activity against ~-EP (in RIA and SAE), H R P (in PAP test) and bifunctional activity (in DAE).
3.12. Immunohistochemistry Rats was perfused under nembutal anesthesia through the aorta with 4% phosphate-buffered paraformaldehyde. Slices of perfused, fixed pituitary (40 #m thick) were incubated overnight at 0°C in PBS, containing 15/~g/ml of affinity isolated bifunctional antibodies, 15 #g/ml HRP, 2% neutral goat serum and 0.2% Triton X-100. Diaminobenzidine was used for peroxidase reaction. 4.
Results and Discussion
The most simple and effective way to produce bifunctional antibodies is to fuse two hybridomas, secreting monoclonal antibodies to different antigens [1,2]. Theoretically, the quadroma can produce ten species of hybrid and parental immunoglobulins, resulting from random combinations of the heavy and light chains produced in parental cells [1]. In order to produce quadromas, the selective markers should be introduced into the parental cell lines, permitting the use of metabolic selective systems for isolation of hybrids. One possibility is to fuse two single-mutant hybridomas with different dominant markers [11]. In the other method, which is most frequently employed, a double mutant cell line carrying two selective markers (dominant and recessive), is fused with a wild type hybridoma [2,12,13]. The recessive marker is usually HAT s , the dominant marker resistance to a drug, e.g., oubain [2], neomycin [12] or AMD [13]. The latter approach with certain modifications, described below, was applied by us for the construction of antibodies with double specificity to 219
~-EP and HRP by fusion of anti-~-EP and antiHRP hybridomas. The advantage of this method is that one marker, the recessive marker HAT s , can be easily conferred to the parental cell line with the help of 8-aza back selection, due to the high incidence of 8-aza R variants in the established hybridomas [12]. However, the introduction of the dominant marker requires special procedures: stepwise selection of the established hybridomas for drug resistance [2,13] or retrovirus transfection of resistance genes into parental cell lines [11,12]. In the present work we have overcome this difficulty by creating a mouse myeloma cell line with an AMDR/HAT s phenotype. It was isolated from the HAT s mouse myeloma X63Ag8.653 by stepwise selection, used to develop AMD resistance in other cell systems [14,15]. AMD R is due to the increase in drug efflux system activity, resulting in the so-called 'multidrug resistance' (reviewed in [16]). In somatic cell hybrids it is expressed as codominant trait [14]. These cells were used for the initiation of the newly established anti-HRP parental hybridoma. About 300 hybrids arose as the result of the fusion of 1 x 107 X63-AMD R cells, resistant to 30 ng/ml of AMD in culture media, and 4 x 10 7 anti-HRP immune mouse splenocytes. The fusion efficiency was the same as was usually obtained by us with the ancestral X63-Ag8.653 cells. 28 hybrid clones actively secreted antibodies to HRP, as detected in the PAP test. All derived the resistance to the drug from myeloma cells, though it was not added to HAT medium during the selection period. AMD in doses as high as 10--15 ng/ml did not affect their growth, while the wild type cells were completely killed at 0.8-1 ng/ml. The hybridomas kept the resistance to high doses of AMD (10 ng/ml) after prolonged (at least 2.5-3 months) cultivation in the absence of the drug. Therefore, the use of AMD R myeloma cells in this fusion resulted in the automatic introduction of the dominant selective marker into the large number of hybridomas. This modification greatly facilitated the further construction of a double mutant. Thus, to obtain double mutant AMDR/ HAT s phenotype it was sufficient to subject the anti-HRP clones to one-step back selection for HAT s by 8-Aza. The frequency of 8-Aza R variants wasabout 1 x l0 -2. 220
One of the derived AMDR/HAT s anti-HRP clones (36F9) was fused with wild type (AMDS/ HAT R) anti-~-EP-hybridoma F8/40E9 [3,4] to construct quadromas. Both hybridomas produced immunoglobulins of the IgG class (IgG1 and IgG2a, respectively). The quadromas were isolated by growing the cells after fusion in selective medium containing AMD and HAT, which was lethal for both parents. As a result of the fusion, more than 300 putative quadroma clones were generated in a 96-well plate, 3-4 clones per well (fusion efficiency about 1.5 × 10-4). In all wells both parental antibody activities were found. To facilitate the detection of truly bifunctional clones, the cultures from five random wells were pooled and the mixed population was cloned by the method of limiting dilution. The resulting single quadroma clones were tested for anti-~-EP parental activity (in RIA and SAE), for anti-HRP activity (in PAP test) and for bifunctional activity (in DAE). Among 36 clones tested only two (52Clo and 51G12) were positive in all tests for the parental activities (absorbance in ELISA tests was > 2). These two clones also demonstrated bifunctional activity in the DAE test (absorbance 0.4-0.6 in the negative controls (see Materials and Methods) 0.02-0.05). Further subcloning resuited in 90% bispecific clones. One of bispecific subclones, 56C2, isolated from quadroma 52C10, was used for ascites production in vivo. With the help of a combination of ~-EP-Sepharose and HRP-Sepharose affinity chromatography, the antibodies produced by clone 56C2 in ascites (Protein A-Sepharose purified IgG) were separated into 4 fractions according to their immunological activities: bispecific, anti-HRP, anti-~-EP or inactive (Table 1). The immunological activity of the fractions was confirmed in radioimmunological and ELISA tests, as summarized in Table 1. The proportion of bispecific antibodies constituted as much as 2829% of the immunologically active quadroma IgG in different ascites (Table 1). In a totally random association of two H chains and two L chains the bifunctional antibodies would be expected to constitute only one-sixth of the possible immunologically active combinations. However, a number of investigators have demonstrated a preferential association of 'autologous' H and L
TABLE 1 The results of affinity fractionations of IgG produced by quadroma 56C 2 in ascites tumors Antibody content is indicated relative to 1 ml of ascitic fluid. Concentration of antibodies was measured by absorption at 280 nm, considering that A28o= 1.3 corresponds to concentration 1 mg/ml. Fraction
I aseites pool a
II ascites pool
mg
mg
Whole IgG (Protein A-Sepharose purified)
5.74
Bispecific antibodies Anti-HRP antibodies Anti-g-EP antibodies Inactive antibodiesd
1.0 1.49 0.96 1.48
Total active IgG (bispecific + anti-HRP + anti-~-EP)
3.45
%c
Immunological activityb %¢
3.7
28.9 43.2 27.8
100
0.45 0.74 0.43 1.52 1.62
27.7 45.7 26.5
anti a-EP
anti HRP
bispecific
+
+
+
+ + -
+ + -
+ -
100
aThe I ascites pool (2 ml) was collected from two pristane-primed mice, the II pool (7 ml) from two pristane + I F A primed animals. bAnti-g-EP activity was detected in R I A and SAE, anti-HRP in PAP, bispecific in the D A E test. ¢% in relation to total active IgG (100%). dThis fraction includes the immunologically inactive quadroma antibodies and nonspecific mouse IgG.
affinity purified bispecific antibody 56C2-Bi (from quadroma 56C2). The pituitary is the main site of endorphin synthesis and secretion in mammals [5]. Fig. 1 demonstrates the single-step immunoperoxidase staining of tissue section of rat pituitary with 56C2-Bi antibody. It is known that in anterior lobe of pituitary about 10% of cells produce endorphins, in the intermediate lobe 90% of cells,
chains in competitive studies in vitro [17], as well as in hybrid hybridomas in vivo [1]. The ability of bispecific antibodies to simultaneously bind two antigens, ~-EP and HRP, would be of great use for immunohistology and immunoblotting, permitting immunoperoxidase staining in a single step. In this work it was demonstrated in rat and bovine pituitary using the
/
....iii
a
b
Fig. 1. Immunolocalizati0n of ~-endorphin in rat pituitary by single-step PAP method using bispecific antibodies. The slices were incubated with the mixture of bispecific antibodies and HRP. Diaminobenzidine was used for peroxidase reaction. (a) Anterior lobe of the pituitary; (b) intermediate (Int.) and posterior (P) lobes of the pituitary. Magnification: x 2500.
221
bridoma. AMDR mouse myeloma cell line can be widely used in quadroma technology. Acknowledgements The authors will always be grateful to the late Prof. A.M. Ugolev for continuous support and fruitful discussions of this work. References Ill Milstein, C. and Cuello, A.C. (1984) Immunol. Today 5, 299.
121Staerz, U.D. and Bevan, M.J. (1986) Proc. Natl. Acad. Sci. Fig. 2. Immunobiotting detection of POMC in bovine pituitary. The blot corresponding to the electrophoregram of pituitary extract was cut into parallel strips and incubated with the mixture of bispecitic antibodies and HRP (10 pg/ml each). The positions of POMC, g-lipotropin and endorphins are indicated.
and in the posterior lobe none [5]. Correspondingly, rare immunoreactive cells can be observed in the anterior lobe, vigorous immunostaining in the intermediate lobe, and no immunoreactivity in posterior lobe (Fig. 1). In immunoblotting of the extract of bovine pituitary with the 56Cz-Bi antibody (Fig. 2) there immunoreactive bands are observed with molecular weights corresponding to the endorphin precursor POMC (MI = 3 1000) and the products of its processing, j?-lipotropin (M, = 10 000) and endorphins (MT = 1700-3400, all types of endorphins in one band) [5]. Similar pictures were obtained with the monospecific anti-ol-EP antibodies [4]. Thus, the use of the AMDa mouse myeloma cell line allowed us to shorten and simplify the process of quadroma construction. In this work, AMD: myeloma cells were fused with the immune mouse splenocytes. Another way to introduce AMDR to the parental hybridoma with the help of this cell line may be the fusion of AMDR myeloma cells with the wild type established hy-
222
USA 83, 1453. 131Massino, Yu.S., Tsibezov, V.V., Dmitriev, A.D. and Kolyaskina, G.I. (1987) Biotechnology 3, 730. [41 Massino, Yu.S., Tsibezov, V.V., Dmitriev, A.D., Vostricov, V.M., Soldatova, LA. and Kolyaskina, G.I. (1988) Bull. Exp. Biol. Med. 11, 578. t51 Eipper, B. and Mains, R. (19t32) Endocrinol. Rev. 1, 1. 161Kearney, J.F., Radbruch, A., Liesegang, B. and Rajewski, K. (1979) J. Immunol. 123, 1548. 171 Bassiri, R.M. and Utiger, R.D. (1978) Endocrinology 90 722. PI Orth, D.N. (1975) Methods Enzymol. 37, 22. 191Pshenichkin, SF., Zozulya, A.A., Kizim, E.A. and Dmitriev, A.D. (1988) Biokhimiya 53, 309. UOI Towbin, H., Staehelin, T. and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76, 4350. [Ill Moran, T.M., Usuba, O., Shapiro, E., Rubinstein, L.J., Ito, M. and Bona, C.A. (1990) J. Immunol. Methods 129, 199. 1121De Lau, W.B.M., Van Loon, A.E., Heije, K., Valerio, D. and Bast, B. J.E.G. (1989) J. Immunol. Methods 117, 1. [I31 Chervonsky, A.V., Faerman, A.I., Evdonina, L.V., Jasova, A.K., Kazarov, A.R. and Gussev, A.I. (1988) Mol. Immunol. 25, 913. I141 Sobel, J.S., Albreeht, A.M., Riehm, H. and Biedler, J.L. (1971) Cancer Res. 31, 297. LlSl Massino, Yu.S., Kakpakova, ES., Kopnin, B.P. and Pogosianz, E. (1981) Genetika 17, 1253. WI Gottesman, M.M. and Pastan, I. (1988) J. Biol. Chem. 263, 12163. [I71 Hamel, P.A., Klein, M.H. and Dorrington, K.J. (1986) Mol. Immunol. 23, 503.
0165 - 2478 / 92 / $ 5.00 © 1992 Elsevier Science Publishers B.V. All rights reserved IMLET 01824
Construction of a quadroma to a-endorphin/horseradish peroxidase using an actinomycin D-resistant mouse myeloma cell line Yu.S. M a s s i n o a, E.A. Kizim a, N . N . D e r g u n o v a b, V . M . V o s t r i k o v b a n d A . D . D m i t r i e v a aInstitute of Higher Nervous Activity and Neurophysiology, Russian Academy of Sciences, Moscow, Russia; and bThe Mental Health Center of the Russian Academy of Medical Sciences, Moscow, Russia
(Received 7 April 1992; accepted 7 April 1992)
1. Summary
2.
A hybrid hybridoma (quadroma), secreting antibodies with double specificity to ~-endorphin (~EP) and horseradish peroxidase (HRP), has been produced. The bispecific antibodies constituted about 28-29% of all immunologically active IgG, produced by quadroma. The quadroma was isolated by fusion of two mouse hybridomas (antiHRP and anti-0t-EP) with distinct phenotypes: double mutant AMDR/HAT s, and wild type (AMDS/HATR). A novel strategy for the construction of a double-mutant was applied, based on the use of an actinomycin D-resistant (AMD R) mouse myeloma for initiation of one of the parental hybridomas.
Bifunctional antibodies, which contain two different antigen combining sites, have been shown to be promising tools for immunohistochemistry, immunoassay and cell targeting [1,2]. Earlier, we have produced hybridomas secreting monoclonal antibodies to the 17-amino acid-residue peptide ~-EP [3,4]. Endorphins (ct, fl and y) are a group of related peptides, showing opioid activity and occurring in certain subpopulations of nervous, neuroendocrine and immune cells as a result of the processing of the high-molecular weight precursor proopiomelanocortin (POMC) [5]. The resulting antibodies bound ~-, r- and ?-endorphins and POMC in RIA, immunoblotting and immunohistology [3,4]. In the present work one of these hybridomas (Fs/40E9) has been fused with a newly established anti-HRP hybridoma to produce antibodies with double specificity to ct-EP and HRP. In the course of this work we have elaborated upon the original approach, facilitating the construction of quadromas - hybrid hybridomas, secreting bispecific antibodies. This approach, based on the use of the AMD R mouse myeloma cell line, is described.
Key words: ct-Endorphin; Peroxidase; Bifunctional antibody;
Actinomycin D resistance; Quadroma Correspondence to: Dr. A.D. Dmitriev, Russian Academy of Sciences, Institute of Higher Nervous Activity and Neurophysiology, 5-a Butlerov Str., Moscow, 117865, Russia. Abbreviations: AMD ~, actinomycin D-resistant; 8-AzaR, 8azaguanine resistant; BSA, bovine serum albumin; DAE, double antigen ELISA; ELISA, enzyme-linked immunosorbent assay; ct-EP, ct-endorphin; HAT s, hypoxanthine aminopterin thymidine-sensitive; HRP, horseradish peroxidase; IFA, incomplete Freund's adjuvant; LDso, lethal dose 50; PAP, peroxidase-antiperoxidase; PBS, phosphate-buffered saline; POMC, proopiomelanocortin; RIA, radioimmunoassay; SAE, single antigen ELISA.
Introduction
217
3.
3.1.
Materials and Methods
Isolation of a mouse myeloma cell line resistant to A M D
The X63-AMD R cell line, resistant to 30 ng/ml of AMD in culture medium, was isolated from X63-Ag8.653 mouse myeloma [6] by stepwise selection. Resistant cells were generated by adding AMD to the growth medium (DMEM + 1020% fetal calf serum (FCS) + 2 mM glutamine + 100 U/ml gentamycin) for 4 months at a concentration which was gradually raised from 0.2 ng/ml (LDs0 for X63-Ag8.653) to 30 ng/ml. The X63-AMD a line was HAT-sensitive (HATS), like the ancestral cells.
3.2.
Generation of parent hybridomas
The mouse hybridoma, secreting antibodies to ~-EP (alone F8/40Eg) had been produced earlier [3,4]. To obtain the anti-HRP hybridoma, spleen lymphocytes from immune BALB/c mouse were fused in the usual way [3] with X63-AMD R cells. Mice were immunized by numerous injections of HRP (total dose 200 #g per mouse). The selection was performed in HAT medium (1 x 10 - 4 N hypoxanthine, 4 x 10 -7 M aminopterin, 1.6 x 10 -5 M thymidine). Anti-HRP antibodies were tested as described below.
3.3.
Back selection to the HA f phenotype
HAT sensitivity (HAT s) was introduced into the cells by cloning hybridomas in the presence of 20 #g/ml 8-azaguanine (8-Aza) at cell density 10-100 cells per well of a 96-well plate.
3.4.
Quadroma construction
Quadromas were generated from the fusion of hybridoma F8/40E9 to ~-EP (IgG2a) and of hybridoma 36F9 to HRP (IgG1). Clone 36F9 had two selective markers: HAT s and AMD R. 1 x 106 cells from each parental line were fused in a usual way [3] and seeded in selective medium into one 96-well plate (2 × 104 cells per well) with a macrophage feeder layer. The selective medium contained AMD (3 ng/ml) and HAT. 218
3.5.
Induction of ascites
5 x 10 6 hybrid cells were inoculated intraperitoneally into BALB/c mice primed as described [3,4] with pristane or incomplete Freund's adjuvant (IFA) or with both agents.
3.6.
Detection of anti-HRP antibodies
Anti-HRP activity was detected in peroxidaseantiperoxidase (PAP) test. The 96-well ELISA plates were coated with affinity isolated rabbit antibodies against mouse IgG (10 #g/ml in 0.01 M Na-phosphate buffer, pH 7.5 at 0°C, overnight). Then the wells were successively incubated with 1% BSA, 100/zl of hybridoma supernatants, diluted 1:2 by PBS with 0.05% Tween 20, and 100-/~1 aliquots of PBS, containing 0.5 #g of HRP (for 90 min, at 37°C). After each step the wells were several times washed with PBS+0.05% Tween-20. O-Phenylenediamine (Sigma) was used for the color reaction. Absorbance was measured at 472 nm in a Microplate reader (Titertek Multiskan ME).
3.7.
Screening of anti-~-endorphin antibody by single-antigen ELISA
Single-antigen ELISA assays (SAE) were carried out in 96-well ELISA plates, coated with ~EP, conjugated to bovine serum albumin. Conjugate was prepared as previously described using bis-diazotinated benzidine [7]. The coating was achieved by incubating the wells overnight at 0°C with 100-/~1 aliquots of 0.01 M sodium phosphate buffer, containing 0.5 #g of ~-EP-BSA conjugate. Next, the wells were incubated with supernatants followed by the HRP-conjugated rabbit antimouse IgG antibodies. The incubation conditions and washing were as described for the PAP test. O-Phenylenediamine was used for the color reaction.
3.8.
Bispecific antibody detection by double antigen ELISA
Bifunctional activity was tested using double antigen ELISA (DAE). In the first step, the wells of the ELISA plate were coated (as described in
section 3.7) with the first antigen, ~-EP, conjugated to BSA [7]. Next, the wells were incubated with hybridoma supernatants, followed by the second antigen, HRP, as described for the PAP test. The reaction was developed with O-phenylenediamine. Supernatants from anti-HRP and anti-0t-EP hybridomas were used as the negative controls.
3.9. Radioimmunological detection of anti-~tendorphin antibodies Anti-0t-EP antibodies were detected as described [3], using synthetic ~-EP (Serva), radioiodinated by the chloramine method [8].
3.10. Immunoblotting The extract of bovine pituitary, prepared as described [9], was separated by electrophoresis in 15% SDS-polyacrylamide gel and electrophoretically transferred to a 0.45-#m nitrocellulose membrane [10]. The blot was cut into strips, each containing one sample lane. Non-specific binding was blocked with 3% BSA. The strips were then individually incubated for 1 h at 20°C with a mixture of bispecific antibodies and HRP, 10 #g each. Peroxidase staining was performed with diaminobenzidine. Buffer systems were as in [9].
3.11.
Purification of quadroma produced antibodies from ascitic fluid by affinity chromatography
H R P and ct-EP were conjugated to CNBr-activated Sepharose 4B (Pharmacia) according to Pharmacia recommendations. 10 mg of H R P was bound to 1 g of CNBr-activated Sepharose 4B. 100 #g of ~-EP was bound to 100 mg of CNBractivated Sepharose 4B. Ascitic fluid (0.5 ml) was diluted 1:10 in PBS with 0.05% Tween-20 and applied to a Protein A-Sepharose column (Pharmacia). IgG was eluted with 0.1 M acetic acid. IgG containing fractions were pooled, neutralized with concentrated ammonia and applied to a HRP-Sepharose column. Antibodies not bound to H R P were washed off by PBS with 0.05 Tween 20. Elution from the affinity column was done using 0.05 M acetic acid-HCl (pH 2.5). The frac-
tions of HRP-binding antibodies were collected, immediately neutralized with concentrated ammonia, pooled and passed in the same way over ctEP-Sepharose column (to separate monospecific anti-HRP IgG and antibodies with double specificity to ~t-EP and HRP). The fraction containing antibodies not bound to HRP was also passed over 0t-EP-Sepharose (to separate anti-~-EP and inactive immunoglobulins). Each fraction was tested for the activity against ~-EP (in RIA and SAE), H R P (in PAP test) and bifunctional activity (in DAE).
3.12. Immunohistochemistry Rats was perfused under nembutal anesthesia through the aorta with 4% phosphate-buffered paraformaldehyde. Slices of perfused, fixed pituitary (40 #m thick) were incubated overnight at 0°C in PBS, containing 15/~g/ml of affinity isolated bifunctional antibodies, 15 #g/ml HRP, 2% neutral goat serum and 0.2% Triton X-100. Diaminobenzidine was used for peroxidase reaction. 4.
Results and Discussion
The most simple and effective way to produce bifunctional antibodies is to fuse two hybridomas, secreting monoclonal antibodies to different antigens [1,2]. Theoretically, the quadroma can produce ten species of hybrid and parental immunoglobulins, resulting from random combinations of the heavy and light chains produced in parental cells [1]. In order to produce quadromas, the selective markers should be introduced into the parental cell lines, permitting the use of metabolic selective systems for isolation of hybrids. One possibility is to fuse two single-mutant hybridomas with different dominant markers [11]. In the other method, which is most frequently employed, a double mutant cell line carrying two selective markers (dominant and recessive), is fused with a wild type hybridoma [2,12,13]. The recessive marker is usually HAT s , the dominant marker resistance to a drug, e.g., oubain [2], neomycin [12] or AMD [13]. The latter approach with certain modifications, described below, was applied by us for the construction of antibodies with double specificity to 219
~-EP and HRP by fusion of anti-~-EP and antiHRP hybridomas. The advantage of this method is that one marker, the recessive marker HAT s , can be easily conferred to the parental cell line with the help of 8-aza back selection, due to the high incidence of 8-aza R variants in the established hybridomas [12]. However, the introduction of the dominant marker requires special procedures: stepwise selection of the established hybridomas for drug resistance [2,13] or retrovirus transfection of resistance genes into parental cell lines [11,12]. In the present work we have overcome this difficulty by creating a mouse myeloma cell line with an AMDR/HAT s phenotype. It was isolated from the HAT s mouse myeloma X63Ag8.653 by stepwise selection, used to develop AMD resistance in other cell systems [14,15]. AMD R is due to the increase in drug efflux system activity, resulting in the so-called 'multidrug resistance' (reviewed in [16]). In somatic cell hybrids it is expressed as codominant trait [14]. These cells were used for the initiation of the newly established anti-HRP parental hybridoma. About 300 hybrids arose as the result of the fusion of 1 x 107 X63-AMD R cells, resistant to 30 ng/ml of AMD in culture media, and 4 x 10 7 anti-HRP immune mouse splenocytes. The fusion efficiency was the same as was usually obtained by us with the ancestral X63-Ag8.653 cells. 28 hybrid clones actively secreted antibodies to HRP, as detected in the PAP test. All derived the resistance to the drug from myeloma cells, though it was not added to HAT medium during the selection period. AMD in doses as high as 10--15 ng/ml did not affect their growth, while the wild type cells were completely killed at 0.8-1 ng/ml. The hybridomas kept the resistance to high doses of AMD (10 ng/ml) after prolonged (at least 2.5-3 months) cultivation in the absence of the drug. Therefore, the use of AMD R myeloma cells in this fusion resulted in the automatic introduction of the dominant selective marker into the large number of hybridomas. This modification greatly facilitated the further construction of a double mutant. Thus, to obtain double mutant AMDR/ HAT s phenotype it was sufficient to subject the anti-HRP clones to one-step back selection for HAT s by 8-Aza. The frequency of 8-Aza R variants wasabout 1 x l0 -2. 220
One of the derived AMDR/HAT s anti-HRP clones (36F9) was fused with wild type (AMDS/ HAT R) anti-~-EP-hybridoma F8/40E9 [3,4] to construct quadromas. Both hybridomas produced immunoglobulins of the IgG class (IgG1 and IgG2a, respectively). The quadromas were isolated by growing the cells after fusion in selective medium containing AMD and HAT, which was lethal for both parents. As a result of the fusion, more than 300 putative quadroma clones were generated in a 96-well plate, 3-4 clones per well (fusion efficiency about 1.5 × 10-4). In all wells both parental antibody activities were found. To facilitate the detection of truly bifunctional clones, the cultures from five random wells were pooled and the mixed population was cloned by the method of limiting dilution. The resulting single quadroma clones were tested for anti-~-EP parental activity (in RIA and SAE), for anti-HRP activity (in PAP test) and for bifunctional activity (in DAE). Among 36 clones tested only two (52Clo and 51G12) were positive in all tests for the parental activities (absorbance in ELISA tests was > 2). These two clones also demonstrated bifunctional activity in the DAE test (absorbance 0.4-0.6 in the negative controls (see Materials and Methods) 0.02-0.05). Further subcloning resuited in 90% bispecific clones. One of bispecific subclones, 56C2, isolated from quadroma 52C10, was used for ascites production in vivo. With the help of a combination of ~-EP-Sepharose and HRP-Sepharose affinity chromatography, the antibodies produced by clone 56C2 in ascites (Protein A-Sepharose purified IgG) were separated into 4 fractions according to their immunological activities: bispecific, anti-HRP, anti-~-EP or inactive (Table 1). The immunological activity of the fractions was confirmed in radioimmunological and ELISA tests, as summarized in Table 1. The proportion of bispecific antibodies constituted as much as 2829% of the immunologically active quadroma IgG in different ascites (Table 1). In a totally random association of two H chains and two L chains the bifunctional antibodies would be expected to constitute only one-sixth of the possible immunologically active combinations. However, a number of investigators have demonstrated a preferential association of 'autologous' H and L
TABLE 1 The results of affinity fractionations of IgG produced by quadroma 56C 2 in ascites tumors Antibody content is indicated relative to 1 ml of ascitic fluid. Concentration of antibodies was measured by absorption at 280 nm, considering that A28o= 1.3 corresponds to concentration 1 mg/ml. Fraction
I aseites pool a
II ascites pool
mg
mg
Whole IgG (Protein A-Sepharose purified)
5.74
Bispecific antibodies Anti-HRP antibodies Anti-g-EP antibodies Inactive antibodiesd
1.0 1.49 0.96 1.48
Total active IgG (bispecific + anti-HRP + anti-~-EP)
3.45
%c
Immunological activityb %¢
3.7
28.9 43.2 27.8
100
0.45 0.74 0.43 1.52 1.62
27.7 45.7 26.5
anti a-EP
anti HRP
bispecific
+
+
+
+ + -
+ + -
+ -
100
aThe I ascites pool (2 ml) was collected from two pristane-primed mice, the II pool (7 ml) from two pristane + I F A primed animals. bAnti-g-EP activity was detected in R I A and SAE, anti-HRP in PAP, bispecific in the D A E test. ¢% in relation to total active IgG (100%). dThis fraction includes the immunologically inactive quadroma antibodies and nonspecific mouse IgG.
affinity purified bispecific antibody 56C2-Bi (from quadroma 56C2). The pituitary is the main site of endorphin synthesis and secretion in mammals [5]. Fig. 1 demonstrates the single-step immunoperoxidase staining of tissue section of rat pituitary with 56C2-Bi antibody. It is known that in anterior lobe of pituitary about 10% of cells produce endorphins, in the intermediate lobe 90% of cells,
chains in competitive studies in vitro [17], as well as in hybrid hybridomas in vivo [1]. The ability of bispecific antibodies to simultaneously bind two antigens, ~-EP and HRP, would be of great use for immunohistology and immunoblotting, permitting immunoperoxidase staining in a single step. In this work it was demonstrated in rat and bovine pituitary using the
/
....iii
a
b
Fig. 1. Immunolocalizati0n of ~-endorphin in rat pituitary by single-step PAP method using bispecific antibodies. The slices were incubated with the mixture of bispecific antibodies and HRP. Diaminobenzidine was used for peroxidase reaction. (a) Anterior lobe of the pituitary; (b) intermediate (Int.) and posterior (P) lobes of the pituitary. Magnification: x 2500.
221
bridoma. AMDR mouse myeloma cell line can be widely used in quadroma technology. Acknowledgements The authors will always be grateful to the late Prof. A.M. Ugolev for continuous support and fruitful discussions of this work. References Ill Milstein, C. and Cuello, A.C. (1984) Immunol. Today 5, 299.
121Staerz, U.D. and Bevan, M.J. (1986) Proc. Natl. Acad. Sci. Fig. 2. Immunobiotting detection of POMC in bovine pituitary. The blot corresponding to the electrophoregram of pituitary extract was cut into parallel strips and incubated with the mixture of bispecitic antibodies and HRP (10 pg/ml each). The positions of POMC, g-lipotropin and endorphins are indicated.
and in the posterior lobe none [5]. Correspondingly, rare immunoreactive cells can be observed in the anterior lobe, vigorous immunostaining in the intermediate lobe, and no immunoreactivity in posterior lobe (Fig. 1). In immunoblotting of the extract of bovine pituitary with the 56Cz-Bi antibody (Fig. 2) there immunoreactive bands are observed with molecular weights corresponding to the endorphin precursor POMC (MI = 3 1000) and the products of its processing, j?-lipotropin (M, = 10 000) and endorphins (MT = 1700-3400, all types of endorphins in one band) [5]. Similar pictures were obtained with the monospecific anti-ol-EP antibodies [4]. Thus, the use of the AMDa mouse myeloma cell line allowed us to shorten and simplify the process of quadroma construction. In this work, AMD: myeloma cells were fused with the immune mouse splenocytes. Another way to introduce AMDR to the parental hybridoma with the help of this cell line may be the fusion of AMDR myeloma cells with the wild type established hy-
222
USA 83, 1453. 131Massino, Yu.S., Tsibezov, V.V., Dmitriev, A.D. and Kolyaskina, G.I. (1987) Biotechnology 3, 730. [41 Massino, Yu.S., Tsibezov, V.V., Dmitriev, A.D., Vostricov, V.M., Soldatova, LA. and Kolyaskina, G.I. (1988) Bull. Exp. Biol. Med. 11, 578. t51 Eipper, B. and Mains, R. (19t32) Endocrinol. Rev. 1, 1. 161Kearney, J.F., Radbruch, A., Liesegang, B. and Rajewski, K. (1979) J. Immunol. 123, 1548. 171 Bassiri, R.M. and Utiger, R.D. (1978) Endocrinology 90 722. PI Orth, D.N. (1975) Methods Enzymol. 37, 22. 191Pshenichkin, SF., Zozulya, A.A., Kizim, E.A. and Dmitriev, A.D. (1988) Biokhimiya 53, 309. UOI Towbin, H., Staehelin, T. and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76, 4350. [Ill Moran, T.M., Usuba, O., Shapiro, E., Rubinstein, L.J., Ito, M. and Bona, C.A. (1990) J. Immunol. Methods 129, 199. 1121De Lau, W.B.M., Van Loon, A.E., Heije, K., Valerio, D. and Bast, B. J.E.G. (1989) J. Immunol. Methods 117, 1. [I31 Chervonsky, A.V., Faerman, A.I., Evdonina, L.V., Jasova, A.K., Kazarov, A.R. and Gussev, A.I. (1988) Mol. Immunol. 25, 913. I141 Sobel, J.S., Albreeht, A.M., Riehm, H. and Biedler, J.L. (1971) Cancer Res. 31, 297. LlSl Massino, Yu.S., Kakpakova, ES., Kopnin, B.P. and Pogosianz, E. (1981) Genetika 17, 1253. WI Gottesman, M.M. and Pastan, I. (1988) J. Biol. Chem. 263, 12163. [I71 Hamel, P.A., Klein, M.H. and Dorrington, K.J. (1986) Mol. Immunol. 23, 503.
