Journal of Immunological Methods, 156 (1992) 69-77 © 1992 Elsevier Science Publishers B.V. All rights reserved 0022-1759/92/$05.00
Bispecific IgA/IgM antibodies and their use in enzyme immunoassay O. Behrsing, G. Kaiser, L. Karawajew and B. Micheel Max Delbriick Centre for Molecular Medicine, Robert-RiSssle-Str. 10, 0-1115 Berlin-Buch, Germany (Received 2 April 1992, revised received 28 May 1992, accepted 15 June 1992)
Two hybrid hybridomas secreting polymeric bispecific antibodies to human chorionic gonadotropin and calf intestinal alkaline phosphatase were produced by fusion of IgA- and IgM-secreting mouse hybridomas. Both hybrid antibodies were purified from ascitic fluid by size exclusion chromatography. An IgM-like fraction was shown to exhibit bispecific activity. Bispecificity was completely lost following mild reduction and alkylation. Both bispecific antibodies were used to develop a sensitive enzyme immunoassay for hCG. Key words." Bispecific antibody; IgA/IgM hybrid; Enzyme immunoassay; Human chorionic gonadotropin
Introduction Bispecific monoclonal antibodies have elicited much interest in biomedical research during the last few years because, in contrast to naturally occurring antibodies, they are able to link two different antigens (Nolan and O'Kennedy, 1990; Songsivilai and Lachmann, 1990). Bispecific anti-
Correspondence to: B. Micheel, Max Delbriick Centre for Molecular Medicine, Robert-R6ssle-Str. 10, O-1115 BerlinBuch, Germany. Abbreviations: AP, alkaline phosphatase; DMSO, dimethyl sulfoxide; EIA, enzyme immunoassay; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; HAT, hypoxanthine-aminopterin-thymidine; hCG, human chorionic gonadotropin; Ig, immunoglobulin; IU, international unit; kDa, kilodalton; OD, optical density; PBS, phosphate-buffered saline; PEG, polyethylene glycol; RPMI, Roswell Park Memorial Institute cell culture medium; SD, standard deviation; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SEC, size exclusion chromatography; TRIS, Tris(hydroxymethyl)aminomethane; TRITC, tetramethylrhodamine isothiocyanate.
bodies can be produced by biochemical (Nisonoff and Rivers, 1961) and cell fusion methods (Milstein and Cuello, 1983). The general method in use today is production by hybrid hybridomas obtained by fusing two different hybridoma lines. Various antibody chain combinations have been described and an association of different heavy chains within one Ig class to give a functional bispecific antibody appears to be possible (Lebegue et al., 1990). Fusion of hybridomas producing different Ig classes have also resulted in Igproducing hybrid cell lines but hybrid molecules comprising 3' and/x chains (Takahashi and Fuller, 1988), 3' and E chains (Van Dijk et al., 1989) or 3' and a chains (Urnovitz et al., 1988) have not been detected. Hybrid IgA/IgM molecules were first found in myeloma patients (Tung et al., 1981; Zhang et al., 1983). Polymeric bispecific antibodies containing a and ~ chains were then produced by cell fusion (Shinmoto et al., 1988; Urnovitz et al., 1988; Ju et al., 1989) using the hybrid hybridoma technique. The established hybrid clones produced IgA/IgM
70 hybrid antibodies resulting from mouse hybridoma x mouse myeloma fusions (Urnovitz et al., 1988; Ju et al., 1989) and a fusion of a human lymphoblastoid cell line with human B lymphocytes (Shinmoto et al., 1988). In all these cases the bispecific molecules were found to be IgMlike. Here we describe hybrid hybridomas obtained from fusions of mouse hybridomas producing IgM antibodies against calf intestinal alkaline phosphatase (AP) and IgA antibodies against the hormone human chorionic gonadotropin (hCG).
Materials and methods
(Kearney et al., 1979) using a modified standard procedure (Karsten and Rudolph, 1985; see also Table I). Cell lines were maintained in RPMI 1640 medium containing 15% FCS, 2 x 10 -3 M L-glutamine and 5 x 10 -5 M 2-mercaptoethanol.
Antibodies B9-AB9, a mouse IgG2b antibody recognizing the a chain of hCG, human follicle stimulating hormone, thyrotropic hormone and luteinizing hormone was produced in our laboratory (B6ttger et al., in preparation). Goat anti-mouse Ig antibodies were produced and immunopurified in our laboratory. HRP-labeled goat anti-mouse IgM (~ chain-specific) and goat anti-mouse IgA (a chainspecific) antibodies were purchased from Sigma.
Materials Reagents The following reagents were used: calf serum, FCS (Staatliches Institut fOr Immunpr~iparate und N~ihrmedien, Berlin, Germany), Coomassie brilliant blue R-250, dithiothreitol, FITC, iodoacetamide, p-nitrophenylphosphate, Tris, TRITC (Serva, Heidelberg, Germany), PEG 1500, PEG 6000 (Ferak, Berlin, Germany), hCG standard, HPLC standard, RPMI 1640 (Boehringer Mannheim, Mannheim, Germany), pristane (2,6, 10,14-tetramethylpentadecane, Sigma Chemical Co., St. Louis, MO, USA), Kieselgur (Merck, Darmstadt, Germany), low molecular weight electrophoresis calibration kit, Sephadex G-25 (Pharmacia-LKB, Uppsala, Sweden) and alkaline phosphatase from calf intestine (Sigma and Forschungsinstitut fOr Medizinische Diagnostik (FMD), Dresden, Germany). Cells and cell lines Feeder cells were obtained from the peritoneal cavity of BALB/c mice. Hybridoma B9-BA8 secreting mouse IgA antibody specific for human chorionic gonadotropin (hCG) and hybridomas E1E7 and ElF1 secreting mouse IgM antibodies to different epitopes on calf intestinal alkaline phosphatase (EC 22.214.171.124, AP) were produced in our laboratory (B6ttger et al., in preparation; Behrsing et al., in preparation) by fusion of spleen ceils from hCG- or AP-immunized BALB/c mice and HAT sensitive X63-Ag 8.653 myeloma cells
Production and characterization of bispecific antibodies Fusion protocol and production of bispecific antibodies Cells were fused as described earlier (Karawajew et al., 1987) with minor modifications. In short, cells from one hybridoma (E1E7 or ELF1) were washed twice in serum-free RPMI 1640 medium and stained with FITC (10 /xg/ml in RPMI 1640, pH 6.8). Cells from hybridoma B9BA8 were stained with TRITC (13 /zg/ml in RPMI 1640, pH 7.2). Then 3 X 106 labeled B9BA8 cells were mixed with 3 X 106 FITC-labeled cells and fused using PEG 1500 according to the standard technique (Galfr~ et al., 1977). The fusion mixtures were cultivated for 3 h in RPMI 1640 containing 15% FCS. After this procedure heterofluorescent cells were sorted with a fluorescence activated cell sorter FACS III (Becton Dickinson, Sunnyvale, CA, USA). Sorted cells from the two fusions were then plated as single cells in four 96-well tissue culture plates (Nunc, Denmark) containing murine peritoneal feeder cells. Supernatants from cultures were tested for bispecific antibody activity 2-3 weeks after fusion as described under 'solid phase antibody binding assays' below. Cells from positive wells were recloned immediately by the limiting dilution technique. Positive hybrid hybridomas were cultured as normal hybridoma lines. For the production of ascitic fluid BALB/c mice were inoculated in-
71 traperitoneally with 0.2 ml pristane and one or two weeks later with 2 - 1 0 x 106 hybrid hybridoma cells.
Purification of bispecific antibodies The precipitation of antibodies from ascitic fluid was carried out as described by Neoh et al. (1986) using a final concentration of 6% PEG 6000. Size exclusion chromatography was performed using a Pharmacia-LKB FPLC system and a Superose 6 HR 10/30 column (PharmaciaLKB, Uppsala, Sweden). The running buffer was 50 mM Tris-HCl, pH 8.0, containing 0.5 M NaCI. Prior to chromatography ascitic fluid was delipidated with Kieselgur (15 m g / m l ascitic fluid) and defibrinated by the addition of 25 mM CaCI 2. After an incubation at room temperature for 30 min, Kieselgur and fibrin were removed by centrifugation (10 min, 5000 x g) and the supernatant was precipitated twice with ammonium sulfate at 50% and 40% saturation sequentially. The last precipitate was dissolved in a minimum volume of running buffer and a sample volume of 0.5 ml was used for chromatography. The flow rate was 0.25 ml/min and fractions of 0.5 ml were collected. To calibrate the column, an HPLC standard was used and the following profile was obtained: fl-galactosidase (E. coli, M~ 465 kDa) eluted at 13.0 ml, IgG (sheep, M r 150 kDa) at 14.5 ml, Fab fragment from IgG (sheep, M r 50 kDa) at 16.4 ml, myoglobin (horse, M r 17 kDa) at 17.1 ml and Gly-Tyr dipeptide (Mr 238 Da) at 21.3 ml. All size exclusion chromatograms (Figs. 1, 2 and 4) were reproduced as computer graphics.
Solid phase antibody binding assays Microtiter plates with 96 wells (MLW Polyplast, Halberstadt, Germany) or polyvinylchloride pill blisters were used as a solid phase. The wells were washed between the incubations with tap water. Incubations were performed in general at room temperature for 1 h, and the sample volumes were 50 /xl. Except for coating and substrate incubations all dilutions were made in PBS containing 20% calf serum. AP from Sigma was used for antibody T6F, AP from FMD was used for T6E.
For the detection of alkaline phosphatase activity 10 mM p-nitrophenylphosphate in 0.1 M diethanolamine buffer, pH 10.0 was used as substrate. The reaction was stopped by adding 200 /zl 1 N NaOH per well and evaluated visually or by measuring absorbance at h - - 4 0 5 nm with a Dynatech MR 700 Microplate Reader (Dynatech Labs., USA). For the detection of horseradish peroxidase activity 1 m g/ m l o-phenylenediamine and 0.01% H 2 0 2 in 0.1 M citrate buffer, pH 5.0, were used as substrate. The reaction was stopped by the addition of 200/zl 2 N H2SO 4 supplemented with 50 mM Na2SO 3 and evaluated visually or by measuring absorbance at ~ = 492 nm. Detection of anti-AP antibodies. Plates were coated overnight with affinity-purified polyclonal goat anti-mouse Ig antibody ( 1 0 / x g / m l in PBS). Then the wells were incubated with diluted antibody supplemented with 4/~ g/ m l AP. Finally the wells were incubated with AP substrate solution for 10 min and the reaction evaluated. Detection of anti-hCG antibodies. Purified hCG was diluted in 50 mM ammonium acetate buffer, pH 7.0, to about 4 I U / m l and added to polyvinylchloride pill blisters or microtiter plates (50/zl/well). The wells were dried overnight and could thus be stored for several weeks. Then antibody-containing solutions were added. Antibodies bound to the solid phase were detected using peroxidase-labeled goat anti-mouse IgA antibody at the dilution recommended by the manufacturer. Finally the substrate solution for HRP was added and the plate incubated for 30 min.
Detection of bispecific anti-hCG-anti-AP antibodies. Plates were coated with purified hCG (4 I U / m l ) as described for the above assay. Then the plates were washed, cell culture supernatant was added and supplemented immediately with 4 /~g/ml AP. The wells were incubated for 2 h at room temperature, washed again and then the substrate for AP detection was added. Screening for hybrid hybridoma clones was carried out using this enzyme immunoassay.
Determination of antibody isotypes after binding of bispecific antibody to antigen. Two different isotype assays were carried out in order to detect heterologous heavy chain binding to solid-phase adsorbed AP or hCG. Microtiter plates were
coated with AP (4 ~ g / m l in 50 mM ammonium acetate buffer, pH 7.0, 2 h, room temperature) or hCG (see anti-hCG assay). Diluted fractions from bispecific antibodies T6E or T6F were then added. In the next step the AP-coated wells were incubated with HRP-labeled goat anti-mouse IgA antibodies and the hCG-coated wells were incubated with HRP-labeled goat-anti-mouse IgM antibodies. The wells were developed with peroxidase substrate and the reaction was stopped after 30 min.
bated with dilutions of hCG standard (5-400 IU/1) for 1 h at 20°C. The wells were washed again and purified preparations of bispecific antibodies T6E or T6F, diluted to 10 /zg/ml and supplemented with 8 /xg/ml AP, were added. After an incubation at 20°C for 1 h the plates were washed and substrate was added. The reaction was stopped after 30 min and absorbance was measured at 405 nm. The detection limits were determined according to Richardson et al. (1983).
Mild reduction and alkylation Mild reduction and alkylation were performed according to Milstein et al. (1975) with minor modifications. Chromatographically purified T6E, T6F, E1E7, ElF1 (IgM-like fractions) and B9BA8 (monomeric IgA fraction) antibodies were treated sequentially with dithiothreitol (0.6 mM, 1 h, 20°C) and iodoacetamide (3.0 mM, 1 h, 4°C). All manipulations were performed under N 2. The product was then fractionated by FPLC size exclusion chromatography as described above.
Electrophoresis SDS-polyacrylamide gel electrophoresis of immunoglobulin samples was carried out according to Laemmli (1970) under reducing conditions using a linear gradient of 7.5-15% acrylamide. Phosphorylase B (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20.1 kDa) and a-lactalbumin (14.4 kDa) were used as molecular weight standards. Protein staining was performed using Coomassie brilliant blue R-250. Antibody samples for electrophoresis were dialysed against PBS and then concentrated by exposing the dialysis tubing to dry Sephadex G-25 fine for 2 h.
Use of bispecific IgA / IgM antibodies for the determination of hCG Enzyme immunoassay for the detection and determination of hCG Microtiter plates (96 wells, MLW Polyplast, Halberstadt, Germany) were coated overnight at 4°C with antibody B9-AB9 (5 /xg/ml in PBS). After washing with tap water the wells were incu-
Cell fusion, screening for hybrid hybridomas, and antibody production 1.5% of all cells from E1E7 x B9-BA8 and 0.5% of all cells from ElF1 × B9-BA8 fusion mixtures were found to exhibit double fluorescence and were sorted. About 1500 sorted cells from each fusion were then plated. Growth was observed in all wells. The screening was complicated by the high non-specific adsorption of IgM (especially antibody E1E7) to plastic surfaces. Thus it was necessary to perform parallel tests with calf serum-blocked wells. Two clones of each fusion were found to produce bispecific antibod-
TABLE I CHARACTERISTICS OF HYBRIDOMAS AND HYBRID HYBRIDOMAS Designation Hybridomas B9-BA8
EIE7 ElF1 B9-AB9
Human chorionic gonadotropin (hCG) Alkaline phosphatase (AP) AP a chain of hCG and related hormones (capture antibody in EIA)
Hybrid hybridomas hCG/AP T6E (B9-BA8 × E1E7) hCG/AP T6F (B9-BA8 x ELF1)
IgG2b IgA/IgM IgA/IgM
73 ies. The hybrid hybridomas were designated T 6 E (from E1E7 × B9-BA8) and T6F (from E l F 1 × B9-BA8; see Table I). The hybrid hybridoma cells had to be recloned several times before ascitic fluid could be produced. Ascitic fluids from parental and hybrid hybridomas were produced in B A L B / c mice. Ascitic fluid from the hybrid hybridomas had a titer of approximately 1/1000 in the bispecific assay (titer refers here to the lowest dilution still showing positive results). This was about 100 times higher than the titer of culture supernatant of the same clone. Only one out of two or three inoculated mice produced ascites and a retransplantation of ascites cells was always unsuccessful.
Purification and analysis of bispecific antibodies T6E and T6F from ascitic fluids
Precipitates from ascitic fluid containing bispecific antibodies T6E or T6F were fractionated by size exclusion chromatography (SEC). The fractions obtained were then tested for anti-hCG, anti-AP and bispecific activity. The optical densities produced by the 1/100 dilutions of each fraction in these tests were taken as a measure of activity of the fraction. Chromatograms of T6E and T6F (Fig. 1) showed three major peaks at 7.4 ml (peak I, excluded material), 10.5 ml (peak II, IgM-like) and 14.5 ml (peak III, IgG-like) eluate. The test results for T 6 E and T6F fractions (Fig. 1) were very similar. Peak I showed no detectable antibody activity. Bispecific activity could be demonstrated only in the IgM-like fractions (peak II) of both T 6 E and T6F. Two different isotype assays were carried out with SEC fractions in order to check whether a chain-containing antibodies could bind to solid-phase adsorbed AP and whether p, chain-containing antibodies could bind to adsorbed hCG. These tests indicated that molecules containing both a and IZ chains were present only in the IgM-like fractions (data not shown). The IgG-like fractions (peak III) showed high anti-hCG activity. For T6E some anti-AP activity could also be detected in peak III. For comparison F P L C profiles of the parental antibodies Ei'E7, E l F 1 and B9-BA8 are shown in Fig. 2. For E1E7 and E l F 1 anti-AP activity was found mainly in peak II (pentameric IgM) but
o OD4~ 1'2
B )D~ o
~ . ,
8 10 12 14 16 18 ~uale (ml)
8 10 12 14 16 18
Fig. 1. Antibody activity in different fractions of ascitic fluid from the hybrid hybridomas T6E and T6F. A and E: ascitic fluids from clones T6E and T6F were subjected to size exclusion chromatography. Absorbance at 280 nm shows relative protein concentration. B and F, C and G, D and H: relative bispecific, anti-AP and anti-hCG activities of SEC fractions (diluted 1/100) were measured by solid phase EIA. The incubation sequences were: B, F: hCG --*diluted fraction+ AP ~ substrate; C, G: goat-anti-mouse Ig antibody ~ diluted fraction+AP ~ substrate; D, H: hCG ~ diluted fraction goat-anti-mouse IgA antibody-HRP ~ substrate.
also in peak IV (monomeric IgM). For B9-BA8 anti-hCG activity was detected in peak I (dimeric IgA) and peak II (monomeric IgA). Electrophoresis under reducing conditions (Fig. 3) showed the presence of a and Iz chains in IgM-iike fractions of T 6 E and T6F. Two different light chains could also be distinguished in both T6E and T6F. Whereas T6F contained more or less proportional quantities of the heavy and
8 10 12 14 16 18
Fig. 3. Electrophoretic analysis of bispecific and parental antibodies in SDS-PAGE under reducing conditions, Lane 1: molecular weight markers. Molecular mass is shown in kDa. Lane 2: IgM-like fraction of ascitic fluid from clone E1E7. Lane 3: IgM-like fraction of ascitic fluid from clone T6E. Lane 4: monomeric fraction of ascitic fluid from clone B9-BA8. Lane 5: IgM-like fraction of ascitic fluid from clone T6F. Lane 6: IgM-like fraction of ascitic fluid from clone ELF1./z, position of /z chains; a, position of ~ chain; LI, position of light chain from antibody ELF1; L 2, position of light chain from antibody EIE7; L3, position of light chain from antibody B9-BAS.
8 10 12 14 16 18 Fig. 2. Fractionation of ascitic fluids from clones B9-BA8 (A), E1E7 (B) and ElF1 (C) obtained by size exclusion chromatography. Protein concentration was estimated by measuring optical density at 280 nm.
T h e s e p e a k fractions were tested for anti-hCG, anti-AP and bispecific activity. T h e tests showed that bispecific activity of the T 6 E and T 6 F antibodies was completely lost after this treatment. Isotype assays showed that no /x chains were attached to solid phase b o u n d h C G and no a chains were attached to solid-phase
light chains the p r o p o r t i o n of a chains in T 6 E was r a t h e r low. T h e antibodies T6E, T6F, E l F 1 and E 1 E 7 w e r e also purified by precipitation with 6% P E G 6000. T h e preparations o b t a i n e d had a lower purity c o m p a r e d to the IgM-like fractions f r o m FPLC.
Mild reduction and alkylation IgM-like fractions of T 6 E and T 6 F antibodies were mildly r e d u c e d and carboxymethylated. T h e p r o d u c t s were fractionated by size exclusion chrom a t o g r a p h y (Fig. 4). O n e major IgG-like p e a k was f o u n d after the t r e a t m e n t in b o t h cases.
8 10 12 14 16 18
Fig. 4. Analysis of mildly reduced and carboxymethylated IgM-like fractions of T6E (A) and T6F (B) ascites by size exclusion chromatography.
° o I ~~o4,0o ~ ~ oI ~ o , o o
eonoontraUon of I ' ~ G (IU/I)
' ' ' eoneenlration of h C G (IU/I)
Fig. 5. Standard curves for the demonstration hCG in an hCG-containing standard solution. The data were obtained from an enzyme immunoassay which used purified bispecific antibodies T6E (A) and T6F (B) as tracers. Each point represents the mean _+SD of four replicates. The dashed line represents background absorbance with no hCG present.
bound AP. Anti-AP and anti-hCG antibody titers decreased approximately 10 fold during mild reduction and alkylation. For comparison, purified fractions of antibodies E1E7, E l F 1 and B9-BA8 were reduced and alkylated under the same conditions. After size exclusion chromatography on Superose 6 all products showed an IgG-like major peak. The anti-AP activity of reduced and carboxymethylated E1E7 and E l F 1 decreased as had the activity of T6E and T6F. The anti-hCG activity of B9-BA8 remained unaffected.
Determination of hCG by using bispecific antibodies T6E and T6F in enzyme immunoassay SEC purified bispecific antibodies were used as tracers in an E I A for hCG. Typical standard curves for h C G using T 6 E and T6F as tracers are shown in Fig. 5. The graphs showed good linearity over a range from 15-200 IU/1. H C G concentrations between 200 and 400 I U / 1 could also be measured without sample dilution. The detection limit was about 7 I U / 1 with T6F and 15 I U / I with T6E as tracer. Results were similar when PEG-precipitated antibodies were used as tracers. Sensitivities were much lower when unpurifled ascitic fluid was used (data not shown).
Two hybrid hybridomas were established which produced bispecific monoclonal antibodies reac-
tive with human chorionic gonadotropin (hCG) and calf intestinal alkaline phosphatase (AP). For the selection of hybrid hybridomas the parental hybridoma cells were stained before fusion with two different fluorescent dyes and fused hybrids were isolated by fluorescence activated cell sorting (Karawajew et al., 1987). This method" proved to be as efficient for these cell lines as for I g G / I g G - p r o d u c i n g hybrid hybridomas. To establish hybrid hybridoma T6E two fusion experiments were necessary whereas the establishment of T6F was already successful during the first run. To avoid the overgrowth of hybrids by non-fused parental cells quick and sensitive assays were necessary. Solid-phase binding assays turned out to be the most effective. The difficulties which originated from non-specific binding of the antiAP antibodies were easily overcome by performing parallel tests on serum-blocked and hCGcoated wells. Ascitic fluids from hybrid hybridomas T 6 E and T6F transplanted as ascitic tumors in syngeneic B A L B / c mice were fractionated by size exclusion chromatography to obtain isolated bispecific molecules. As was demonstrated by solid-phase antibody binding assays, the hybrid hybrid0mas secreted an IgM-like I g A / I g M hybrid molecule, IgA monomers and traces of IgM monomers. Dimeric IgA may also have been present in the shoulder of peak III (elution volume 13.0 ml, Figs. 1A and 1E). The presence of parental
76 monospecific IgM in the IgM-like fraction could not be definitely proven. Bispecific activity was only detected in the IgM-like fraction and these findings are in accordance with the experiments of Urnovitz et al. (1988) and Shinmoto et al. (1988). This fraction was therefore studied in more detail. The IgM-like fraction of T6E contained approximately equal amounts of the parental light chains but much fewer a chains than /z chains (Fig. 3). Obviously in this fraction a high proportion of B9-BA8-1ight chains was associated with E1E7-/z chains. The bispecific antibodies produced by hybrid hybridomas T6E and T6F were used together with alkaline phosphatase as tracers in enzyme immunoassay for the demonstration of hCG. Purified IgM-like fractions of T6E and T6F resulted in a much better sensitivity and detection limit in enzyme immunoassay than unfractionated ascitic fluid or culture supernatant. This indicates that interfering monomeric and, perhaps, dimeric IgA were eliminated during chromatography or P E G precipitation. The assay characteristics of the antibodies were comparable to those observed with bispecific I g G 1 / I g G 1 antibodies constructed in our laboratory (Karawajew et al., 1988; Behrsing et al., in preparation) and to those obtained using enzyme-labelled antibodies (B6ttger et al., in preparation). The assays developed using bispeciflc T6E or T6F antibodies showed sufficient sensitivity for use as pregnancy or tumor marker tests (Norman et al., 1990). Thus I g A / I g M - h y brids should find many possible immunoassay applications. Mild reduction and alkylation of bispecific antibodies produced almost e~clusively IgG-like m o l e c u l e s w h i c h were presumed to be the monomers of the polymeric bispecific antibodies. These monomers showed no bispecific activity. Monomers containing a chains did not bind to AP and monomers containing /z chains did not bind to hCG. Smaller Ig fragments (e.g., single chains or H-L pairs) were not found. It was therefore concluded that bispecific I g A / I g M antibodies consist of IgG-like monomers organized in a polymeric IgM-like structure. The presence and possible role of the J chain (Koshland, 1985) in this structure is not yet clear and will be the subject of further experiments.
Our work demonstrates the simple straightforward production of bispecific I g A / I g M antibodies and suggests that these antibodies are probably organized as IgM-like polymers of monospecific IgG-like antibodies. Thus the structure of bispecific I g A / I g M antibodies differs profoundly from that of bispecific IgG antibodies which are comprised of heterologous heavy chain pairs. It can be assumed that these differences in structure as well as in Ig class features will enlarge the spectrum of potential applications for bispecific antibodies, particularly for immunotargeting.
Acknowledgements This work was supported in part by a grant from the Bundesministerium fiir Forschung und Technologie.
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