Journal of Immunological Methods 413 (2014) 45–56

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Research paper

Comparative characterization of mAb producing hapten-specific hybridoma cells by flow cytometric analysis and ELISA Maren Kuhne a, Martin Dippong a,b, Sabine Flemig a, Katrin Hoffmann a, Kristin Petsch a, Jörg A. Schenk c,d, Hans-Jörg Kunte a, Rudolf J. Schneider a,⁎ a b c d

BAM Federal Institute for Materials Research and Testing, Richard-Willstätter-Str. 11, D-12489 Berlin, Germany University of Potsdam, Institute for Biochemistry and Biology, Karl-Liebknecht-Str. 24-25, 14476 Potsdam, Germany UP Transfer GmbH, Am Neuen Palais 10, D-14469 Potsdam, Germany Hybrotec GmbH, Am Mühlenberg 11, D-14476 Potsdam-Golm, Germany

a r t i c l e

i n f o

Article history: Received 20 June 2014 Received in revised form 13 July 2014 Accepted 14 July 2014 Available online 21 July 2014 Keywords: Immunization Hapten Monoclonal antibodies Hybridoma Flow cytometry ELISA

a b s t r a c t A novel method that optimizes the screening for antibody-secreting hapten-specific hybridoma cells by using flow cytometry is described. Cell clones specific for five different haptens were analyzed. We selectively double stained and analyzed fixed hybridoma cells with fluorophore-labeled haptens to demonstrate the target-selectivity, and with a fluorophore-labeled anti-mouse IgG antibody to characterize the level of surface expression of membrane-bound IgGs. ELISA measurements with the supernatants of the individual hybridoma clones revealed that antibodies from those cells, which showed the highest fluorescence intensities in the flow cytometric analysis, also displayed the highest affinities for the target antigens. The fluorescence intensity of antibody-producing cells corresponded well with the produced antibodies' affinities toward their respective antigens. Immunohistochemical staining verified the successful double labeling of the cells. Our method makes it possible to perform a high-throughput screening for hybridoma cells, which have both an adequate IgG production rate and a high target affinity. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Their high affinity, selectivity and specificity make monoclonal antibodies (mAbs) a very important tool in research, diagnostics and therapy. Antibody production is routine. When the target is a protein or a microorganism, immunization can be done directly and an immune response is elicited that finally results in mAb-producing B cells in the spleen and antibodies in the serum. Low-molecular compounds with molecular weights below 1.000 Da do not cause an immune response and are called haptens. In order to initiate an

⁎ Corresponding author. Tel.: +49 30 8104 1151; fax: +49 30 8104 1157. E-mail address: [email protected] (R.J. Schneider).

http://dx.doi.org/10.1016/j.jim.2014.07.004 0022-1759/© 2014 Elsevier B.V. All rights reserved.

immune response, haptens have to be linked to a carrier protein such as bovine serum albumin (BSA) (Dutton and Bulman, 1964; Walters et al., 1972; Fasciglione et al., 1996; Ramin and Weller, 2012). After triggering an immune reaction, the immune system produces B cells, which can be isolated from the spleen and fused with “immortal” myeloma cells in order to obtain so-called hybridoma cells (Köhler and Milstein, 1975). The hybridoma technique nowadays is applied almost in the same way as back then. The efficiency of polyethylene glycol (PEG)-stimulated fusion and electrofusion is very low. It ranges from 0.0001% to 0.01% successfully fused cells per initial cell number. Great care has to be given to dilution factors and cultivation conditions such as the use of feeder cells (De Blas et al., 1981). The indispensable identification and isolation of the desired antibody-producing

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hybridoma clone is still far from being trivial (Pasqualini and Arap, 2004; Chiarella and Fazio, 2008). The approaches are mostly empirical and guided by non-standardized protocols and individual experience. The immunoassay-based screening for single hybridoma cell clones that produce an antibody with the desired selectivity is mainly done by enzyme-linked immunosorbent assays (ELISAs). To isolate the cell clone of interest, multiple rounds of limiting dilution have to be carried out, which result in a high number of cells being lost during the process. This makes the entire procedure extremely time- and labor-intensive (Cervino et al., 2008; Zhang and Wang, 2009). Selective labeling of hybridoma cells with fluorophore-tagged antigens has been described, which allows for screening and isolation of such cells with the help of their cell-surface associated antibodies (Parks et al., 1979). Furthermore, it is also possible to use fluorescein-tagged antibodies directed against immunoglobulin G (IgG) antibodies to determine IgG on the surface of hybridoma cells and to screen for hybridoma cells, which secrete IgG variants (Liesegang et al., 1978; Kromenaker and Srienc, 1994). It was also reported that there is a relationship between the antibody production rate and the amount of IgG on the cell surface (Sen et al., 1990; Cherlet et al., 1995). However, no attempts have been made so far to correlate the amount of antibody production of the cell with the target-selectivity of the produced antibodies. The aim of our work was to optimize the screening by flow cytometry for hybridoma cells producing specific antibodies of interest. The presented method is taking advantage of the fact that desired hybridoma clones carry antibodies on their cell surface. The membrane-based antibodies allow for double labeling of these cells by anti-mouse IgG antibodies, which are coupled to a fluorophore and should help to estimate the antibody expression of the cell. The second label is a fluorescein– hapten conjugate that characterizes the target-selectivity of the produced antibodies. In order to validate our newly developed flow cytometry method, ELISAs had to be performed using purified soluble antibodies from the supernatant of individual hybridoma clones. The presented studies were carried out with cell clones derived from immunizations against (i) the two mycotoxins aflatoxin B1 (Afla) and zearalenone (ZON), (ii) the steroid digoxigenin (DIG), and (iii) the natural estrogenic hormones, estrone (E1) and 17β-estradiol (E2). 2. Materials and methods 2.1. Materials All solvents and chemicals were obtained from Merck KGaA (Darmstadt, Germany), Sigma-Aldrich (Taufkirchen, Germany), and Steraloids (Newport, RI, USA) and were of best available quality. 5-(Aminoacetamido)fluorescein (FITC) was obtained from Invitrogen (Carlsbad, California, USA). Horseradish peroxidase (HRP) was EIA-grade and obtained from Roche (Mannheim, Germany). Bovine serum albumin (BSA) was purchased from Protea Biosciences (Morgantown, WV, USA). The immunogens ZON coupled with keyhole limpet hemocyanin (KLH) and Afla-KLH and the conjugates ZON–FITC and Afla–FITC were obtained from aokin AG (Berlin, Germany). All buffers and solutions were prepared with ultrapure water from a Synthesis A10 Milli-Q® water purification system.

2.2. Derivate synthesis and conjugation 2.2.1. Immunogen The immunogen estrone (E1) was synthesized in our laboratory by coupling of E1 to the carrier protein BSA. The coupling of E1-6-CMO (1,3,5(10)-estratrien-3-ol-6,17-dione6-O-carboxymethyloxime) to BSA was performed via the carbodiimide method adapted from Schneider and Hammock (1992). First, the hapten is activated by NHS/DCC followed by the coupling to BSA. Ten micromole (3.6 mg) E1-6-CMO was dissolved in 430 μl anhydrous N,N-dimethylformamide, and 12 μmol N-hydroxysuccinimide (24 μl from a 0.5 M stock solution) was added, followed by 12 μmol dicyclo hexylcarbodiimide (24 μl from a 0.5 M stock solution) under nitrogen atmosphere. After stirring for 18 h at room temperature, the solution was centrifuged for 10 min at 14,000 rpm and 20 °C. The clear supernatant was collected. The molar ratio of BSA and the activated ester was 1 : 50. BSA (13.4 mg) was dissolved in 4.8 ml carbonate buffer (0.13 M NaHCO3) and 480 μl of the supernatant containing the activated ester was added dropwise to the BSA solution and stirred for 4 h at room temperature. The solution was pipetted onto a G-25 Sephadex column (GE Healthcare, Munich, Germany) conditioned with 1/10 PBS buffer (10 mM sodium dihydrogen phosphate, 70 mM disodium hydrogen phosphate, 145 mM sodium chloride, pH 7.6). The same buffer was used for the elution of the conjugate. Fractions were collected in a microtiter plate (UV star, Greiner Bio-One, Frickenhausen, Germany) and their absorbance was measured photometrically at 280 nm. The degree of labeling (DOL) was determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/MS) (Autoflex III, Bruker Daltonics, Bremen, Germany). Sample preparation, measurement and data processing were performed according to Bahlmann et al. (2009). The mean coupling ratio of the E1– BSA conjugate was 20 (±6.6) molecules of E1 per BSA molecule. The concentration of the E1–BSA was determined photometrically at 280 nm and 620 nm using a calibration curve of BSA. The concentration was 7.3 mg/ml. 2.2.2. Enzyme tracer synthesis For ELISA enzyme tracers are required. Several conjugates with HRP were synthesized. The synthesis of E1–HRP was performed via coupling of E1-6-CMO to HRP. The method was adapted from Munro and Stabenfeldt (1984) for the conjugate synthesis of progesterone. First, the hapten is activated followed by the coupling to HRP. Five micromole (1.8 mg) E1-6-CMO, in the presence of 1 μl N-methylmorpholine, was dissolved in 100 μl anhydrous N,N-dimethylformamide (DMF) and activated by 1 μl iso butyl chloroformate at a temperature of − 21 °C under nitrogen atmosphere. The molar ratio of HRP to the activated ester was 1 : 50. After stirring for 30 min at room temperature, the solution was added dropwise to 4 mg HRP dissolved in 50 μl H2O and 30 μl DMF at a temperature of − 21 °C. The mixture was stirred for 1 h at − 21 °C and additional 2 h at 0 °C. Afterwards, the solution was pipetted onto a G-25 Sephadex column conditioned with 1/10 PBS buffer (10 mM sodium dihydrogen phosphate, 70 mM disodium hydrogen phosphate, 145 mM sodium chloride, pH 7.6). The same buffer was used for the elution of the

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conjugate. The synthesis of E2–HRP from E2-6-CMO (1,3,5(10)-estratrien-3,17β-diol-6-one-6-carboxylmethyloxime) and HRP was performed analogously to the synthesis of E1– HRP. The synthesis of DIG–HRP was also performed via the carbodiimide method, starting with a commercially available activated NHS ester of DIG (Sigma-Aldrich), dissolved in dry tetrahydrofuran. The molar ratio of HRP to the activated ester was 1 : 50. HRP (1.1 mg) was dissolved in 600 μl carbonate buffer (0.13 M NaHCO3). Afterwards, 60 μl of the clear supernatant of the activated ester was added dropwise to the HRP solution and stirred for 4 h at room temperature. Following this, the solution was pipetted onto a G-25 Sephadex column conditioned with 1/10 PBS buffer (10 mM sodium dihydrogen phosphate, 70 mM disodium hydrogen phosphate, 145 mM sodium chloride, pH 7.6). The same buffer was used for the elution of the conjugate. The degree of labeling was determined analogously to E1– BSA. The mean DOL of the E1–HRP conjugate was 0.7 molecules of E1 per HRP molecule, the ratio of the E2–HRP conjugate was 2.2 molecules of E2 per HRP molecule and the ratio of DIG–HRP was 1.9 molecules of DIG per HRP molecule. The concentrations of the HRP conjugates were determined photometrically at 405 nm and 620 nm using a calibration curve derived from HRP dilutions. The concentrations were as follows: E1–HRP 2.7 mg/ml, E2–HRP 2.8 mg/ml, and DIG– HRP 1.1 mg/ml. 2.2.3. Fluorescein conjugates The coupling of the E1-6-CMO to FITC was performed via a slightly modified carbodiimide method compared to the one described in Section 2.2.1. First, 2.5 μmol (0.9 mg) E1-6-CMO was dissolved in 2.5 mmol (108 μl) dry tetrahydrofuran at a temperature of 30 °C. Three micromole (0.4 mg) N-hydroxysuccinimide (6 μl from a 0.5 M stock solution) was added. Approximately 1.5 mg di-(N-succini midyl)carbonate was added. Afterwards, 3 μmol (0.6 mg) dicyclohexylcarbodiimide (6 μl from a 0.5 M solution) was added. After stirring and centrifugation the clear supernatant was collected. The molar ratio of fluorescein to the activated ester was 1 : 1.5. Sixty microliters of the collected supernatant was added dropwise to 1.7 μmol (0.7 mg) fluorescein, dissolved in 600 μl carbonate buffer (0.27 M NaHCO3) and stirred for 3 h at room temperature. The conjugate was directly used for flow cytometric analysis. The synthesis of E2–FITC from E2-6-CMO and FITC was performed analogously to the synthesis of E1–FITC. The synthesis of DIG–FITC was also performed via the carbodiimide method starting with the activated ester of DIG. All conjugates were purified via preparative high-performance liquid chromatography (HPLC) (Series 1200, Agilent Technologies, Waldbronn, Germany). An Agilent 1200 quaternary pump, a Phen RP-18 analytical column (250 × 3 mm + 10 × 3 mm pre-column; Sepserv, Berlin, Germany), an Agilent 1200 column thermostat at 40 °C and an Agilent 1200 diode array detector were used. The flow rate was 0.4 ml/min and the pressure was set to 300 bar. The solvents were (A) ultrapure water containing 10 mM ammonium acetate and 0.1% acetic acid, and (B) methanol containing 10 mM ammonium acetate and 0.1% acetic acid. At the beginning 80% solvent (A) was used. After 3 min, the percentage of solvent (B) was linearly

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increased to 95% within 20 min. After 28 min, the percentage of solvent (B) was decreased to 20% within 1 min. Then the composition was kept constant until the end of the run (40 min). The fraction containing the main peak was evaporated to dryness under a stream of nitrogen and the residue was redissolved in phosphate buffered saline (PBS). 2.3. Cultivation of hybridoma cells Cell culture media, plastics and various additives were supplied by Biochrom (Berlin, Germany). Cells were grown under comparable culture conditions in hybridoma media composed of RPMI media, 10% fetal bovine serum, 1% gluta mine, 0.1% β-mercaptoethanol and 1% CellShield™ and cultivated at 37 °C in a humidified 5% CO2 atmosphere. Cells were routinely passed every two days. 2.4. Generation of monoclonal antibodies The hybridoma technology was applied for the generation of murine monoclonal hapten-specific antibodies. Female BALB/c mice were immunized with 100 μg corresponding immunogen in Freund's complete adjuvant supplied by Difco (Lawrence, KS, USA). The animals were boosted regularly at 4 to 6 week intervals for at least 12 weeks with 50 μg immunogen in PBS. Four days later, the spleen cells of mice were fused with X63-Ag8.653 myeloma cells by an electrofusion technique (Schenk et al., 2004). Briefly, the spleen/myeloma cell ratio was approximately 2:1 in 10% PEG 8000 and the voltage ranged from 3000 to 3500 V/cm. Following fusion, the cells were plated into ten 96-well plates (Nunc, Wiesbaden, Germany) on peritoneal feeder cells and cultured in HAT selection medium as described in Micheel et al. (1994). Selected hybrids were cultivated, subcloned by limiting dilution and stored in liquid nitrogen according to common methods. 2.5. Hybridoma clones Mouse hybridoma clones producing monoclonal antibodies of IgG1 subclass (Afla-GE9, ZON-EA6-D5, DIG-DA5, DIG-DA5-E9, DIG-HC9-F9, E2-DE9, E2-BF1and E1-BH5) and of IgG2 subclass (E1-HC7 and E1-BH7) were used for the experiments. Subclasses were determined as described in Schenk et al. (2004). Clones, negative for the analyzed antigen, were used as control cells for flow cytometry, immunohistochemical staining and ELISA measurements. We used sulfamethoxazole (SMX)-specific clone SMX-HF3 or Afla-GE9 as a control. 2.6. Flow cytometry To study the surface expression of antibody production, 1 × 106 hybridoma cells were fixed for 10 min with 2% buffered formalin (J.T. Baker, Deventer, The Netherlands) and washed with PBS. Cells were simultaneously stained with anti-mouse-IgG-AlexaFluor®-647 (BD Biosciences, Heidelberg, Germany) and fluorescein-labeled antigen for 30 min in the dark. Antibody expression was measured by multicolor flow cytometry using a CyFlow® space cytometer (Partec, Münster,

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Germany). Excitation at wavelengths of 488 nm and 638 nm, detecting fluorescence at 520 nm and 682 nm and a processing rate of ~700 cells per second were used. Data were analyzed using the Partec FlowMax 2.70 software. As sheath fluid sterile-filtered phosphate-buffered saline (Partec, Münster, Germany) with 0.01% potassium azide was used. For analysis, all samples were adjusted to a volume of 1 ml. Electronic gating was set to specify the analyzed cell populations. 50,000 cells from each sample were analyzed. 2.7. Immunohistochemical analysis of hybridoma cells Glass slides were cleaned by immersion in 70% ethanol for 10 min and autoclaved for 20 min at 121 °C. Clean slides were submerged into diluted poly-L-lysine for 10 min, washed in PBS and water and dried. For the biological experiments, cells were cultured on poly-L-lysine coated coverslips for at least 48 h in a cell density of 70% to 80%. Cells were fixed with 4% paraformaldehyde for 10 min and washed with PBS. Nonspecific antibody binding was blocked with donkey serum (Chemicon, Schwalbach, Germany). In order to visualize the antigen-binding on cells, the slides were subsequently incubated with FITC-labeled antigens and AlexaFluor®647-labeled antibodies specific for mouse-IgG (Life Technologies, Darmstadt, Germany) for 30 min. After counterstaining the nuclei using 4′,6-diamidino-2-phenylindole (DAPI) (Roche, Mannheim, Germany), the cells were embedded in Fluoromount G (Southern Biotech, Birmingham, USA). Images were obtained and analyzed with a confocal laser scanning microscope Olympus FluoView™ FV1000 using an objective UPLSAPO 40 ×, numerical aperture NA = 0.90 (Olympus, Hamburg, Germany). The excitation wavelengths were 351/364 nm (argon-UV laser), the 488 nm line of an argon-ion laser, and 633 nm (red Helium-Neon). Emission was detected from 400 nm to 475 nm (DAPI channel), from 500 nm to 600 nm (FITC channel), and in the channel AlexaFluor®647 (barrier filter BA 650IF). 2.8. ELISA 2.8.1. Competitive direct ELISA The immunoassays were performed in the direct competitive format adapted from Zettner and Duly (1974), using the enzyme HRP as label and the chromogenic substrate TMB (3,3′,5,5′tetramethylbenzidine). All incubation steps were performed at room temperature on a Titramax 101 plate shaker (Heidolph, Schwabach, Germany) at 750 rpm (Grandke et al., 2013). Between individual incubation steps, the plates were washed with an automatic 96-channel plate washer (BioTek Instruments, ELx405 Select™, Bad Friedrichshall, Germany). Three-cycle washing steps were carried out with a PBS-based washing buffer (0.75 mM potassium dihydrogen phosphate, 6.25 mM dipotassium hydrogen phosphate, 0.025 mM sorbic acid potassium salt, 0.05% (v/v) Tween™ 20, pH 7.6). Each well of transparent high-binding microtiter plates was coated with 200 μl of 1 mg/l anti-mouse IgG (R1256P) (Acris Antibody GmbH, Herford, Germany) in PBS (10 mM sodium dihydrogen phosphate, 70 mM disodium hydrogen phosphate, 145 mM sodium chloride, pH 7.6) and incubated for 18 h on a plate shaker. The plates were sealed with Parafilm®

to prevent evaporation. After a three-cycle washing step, 200 μl anti-E1 antibody (M631, Calbioreagents, San Mateo, California, USA), diluted 1:10,000 in TRIS buffer (13.7 μg/l; 10 mM tris(hydroxymethyl)aminomethane (TRIS), 150 mM sodium chloride, pH 8.5) or supernatant, diluted up to 1:10,000 in TRIS, was added and incubated for 1 h. Following another washing step, 100 μl of the calibrators in the range of 0 to 1000 μg/l was added to each well and incubated for 10 min. Calibrators were obtained by sequential dilution of the stock solution with ultrapure water. Following this, 100 μl tracer (E1–HRP conjugate diluted 1:100,000 in TRIS buffer) was added in each well and incubated for 30 min. After another washing step, 200 μl substrate solution was added. A protocol according to Frey et al. (2000) was used for the preparation of the HRP substrate TMB. For one plate, 22 ml citrate buffer (220 mM potassium dihydrogen citrate, 0.5 mM sorbic acid potassium salt, pH 4.0) with 8.5 μl H2O2 (30%) and 550 μl TMB solution (40 mM TMB, 8 mM tetrabutylammonium borohydride, in N,N′-dimethylacetamide) were mixed and 200 μl was added to each well. Following a 30 min incubation step, the reaction was stopped by adding 100 μl H2SO4 (1 M). Absorbance was measured photometrically at 450 nm and referenced to 620 nm. The immunoassays for E2 and DIG were performed as for E1, only differing in the HRP conjugate (E2–HRP, DIG–HRP). I¼

A−D
þD c B

1þ

ð1Þ

C

Sigmoidal calibration curves were obtained by fitting a four-parameter logistic function (Eq. (1)) (Dudley et al., 1985) to the mean absorbance values obtained from the calibration standards. The test midpoint (≈ IC50) of the calibration curve is given by parameter C and represents a measure for assay sensitivity (Grandke et al., 2012). 2.8.2. Sandwich-ELISA We developed a sandwich ELISA for the measurement of mouse IgG in the supernatant of hybridoma cell cultures. In order to compare the amounts of soluble antibodies, each culture was treated under the same conditions. Thirty microliter hybridoma media were inoculated with 1 ∗ 106 cells and cultivated for 3 days. Two hundred microliter supernatant of each clone was used for ELISA measurements. The microtiter plates were coated with 200 μl of 2 mg/l anti-mouse IgG (R1256P) (Acris Antibody GmbH, Herford, Germany) in PBS and incubated for 18 h on a plate shaker. The plates were sealed with Parafilm® to prevent evaporation. Following a three-cycle washing step, the remaining binding sites on the MTP were blocked with 200 μl casein solution (0.1%) in PBS buffer. After incubating for 1 h, the plates were washed again. Standards (200 μl anti-E1 mAb, M631, Calbioreagents, San Mateo, California, USA) and cell culture supernatants diluted in a range of 1 : 100 – 1 : 10,000 in TRIS buffer were incubated for 45 min. After a three-cycle washing step, 200 μl HRP-conjugated anti-mouse-IgG (R1256HRP) (Acris Antibody GmbH, Herford, Germany) diluted 1 : 10,000 in TRIS buffer was added in each well and incubated for 1 h. After another washing step, 200 μl substrate solution was added. The protocol for the preparation

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of the HRP substrate TMB is described in Section 2.8.1. Absorbance was measured photometrically at 450 nm and referenced to 620 nm. 3. Results 3.1. Antigen-specific labeling of hybridoma cells In this work we studied hybridoma cells and their produced antibodies against (i) the mycotoxins zearalenone and aflatoxin B1, (ii) the steroid digoxigenin and (iii) the estrogens estradiol and estrone (Fig. 1). All five antigens are haptens. In order to estimate the amount of membrane-bound antibodies on hybridoma cells and to characterize their specificity toward the haptens ZON or Afla, we carried out flow cytometric measurements and immunohistochemical analyses using confocal laser scanning microscopy (CLSM). Two different hybridoma clones and a control clone were incubated with the FITC-labeled haptens ZON and Afla,

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respectively. A criterion in optimization of the staining procedure is to maximize the differentiation between the positive stain from the negative control. For this, FITC-labeled target was titrated out by non-specific staining. The staining was monitored by multicolor flow cytometry and CLSM. Flow cytometry was able to discriminate between hybridoma cells carrying target-selective antibodies against ZON and Afla, respectively and non-specific hybridoma cells (Fig. 2). The hybridoma clone ZON-EA6-D5 showed two distinct populations after incubation with FITC-labeled ZON (Fig. 2A). Only 60% of the cells expressed ZON-specific antibodies on their surface. This was confirmed by CLSM, where only a part of the cell population is stained with the ZON–FITC-conjugate (Fig. 2B upper panel). These findings indicate that the ZON-EA6-D5 hybridoma cells are not monoclonal. In contrast, the hybridoma cell clone Afla-GE9 carries only antibodies specific for Aflatoxin (Fig. 2). Only one maximum was detected by flow cytometry and the

Fig. 1. Chemical structures and molecular weights of studied haptens. (A) Zearalenone (ZON), (B) aflatoxin B1 (Afla), (C) digoxigenin (DIG), (D) estradiol (E2) and (E) estrone (E1).

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fluorescence intensity was higher compared to the fluorescence intensity obtained from the control cells. Apparently, Afla-GE9 cells are monoclonal. This finding was confirmed by CLSM (Fig. 2B). The data presented here demonstrate the power of our newly developed flow cytometry method to detect monoclonal hybridoma cells that produce hapten-specific antibodies.

3.2. Analysis of the specificity of the antigen labeling The aim of the experiment was to determine the specificity of the FITC-labeled hapten conjugates in tagging the hybridoma cells of interest. We tested the hapten conjugates with the help of two hybridoma cell clones with different antibody specificities. The Afla-GE9 hybridoma cells expressing Afla-specific antibodies and the E2-BF1

Fig. 2. Identification of antibody-producing hybridoma cells by flow cytometric analysis and immunohistochemical staining. (A) Formalin fixed hybridoma cells were incubated with FITC-labeled antigen. Staining of the surface-bound antigen was monitored by flow cytometry. Percentages of all cells within the respective range are indicated. Bottom panels are from a control clone (SMX-HF3) with the same staining. Histograms show the results of 3 comparable experiments. (B) Hybridoma cells grown on coverslips were stained with FITC-conjugates specific for ZON and Afla, respectively. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole. Stained cells were identified by confocal laser scanning microscopy (CLSM).

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hybridoma cells producing antibodies specific for estradiol were incubated with the FITC-labeled E2 conjugate and analyzed by flow cytometry (Fig. 3). The incubation of the Afla-GE9 hybridoma cell clone with the E2–FITC conjugate shows almost no positive fluorescence signals by flow cytometric analysis (Fig. 3A) and only a negligible amount (b1%) of cells was classified as positive in the FITC range. After incubation of the E2-BF1 hybridoma cells with E2–FITC conjugate and applying the same range as in the experiment seen in Section 3.1, almost all cells were assigned as positive (Fig. 3C). Finally, we mixed the two different hybridoma cell lines. Here, two distinct populations of cells were observed, which displayed different fluorescence intensities (Fig. 3B). In addition, the signal ratio corresponds rather well to the pipetted ratio (50%) of E2-specific hybridoma cells using the same range. This clearly demonstrates the power of hapten–FITC conjugates to discriminate between cells expressing two different antibodies (E2-BF1 and Afla-GE9). 3.3. Correspondence of flow cytometry and ELISA In these experiments we analyzed DIG-specific hybridoma cell clones. The aim of the analysis was to test (i) whether the amount of antigen-specific cell-bound IgG corresponds to the amount of soluble IgG and (ii) whether the specificity of the cell-bound antibodies is similar to the specificity of the soluble antibodies. In order to determine the antibody specificity for the DIG hapten we applied a DIG–FITC conjugate. The additional incubation of DIG-specific hybridoma cell clones with antimouse-IgG-AlexaFluor®647 allows estimating the relative amount of IgG on the surface of hybridoma cells. Fluorescence intensities of DIG–FITC are plotted against the fluorescence intensities of anti-IgG-AlexaFluor®647 to discriminate the double positive cells (Fig. 4). Almost 90% of both cell clones, DIG-DA5 and DIG-DA5-E9, express DIG-specific antibodies on the cell surface (Fig. 4A).

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All DIG–FITC-binding cells were IgG-producing cells, as shown by double staining with Alexa®647-labeled antimouse-IgG. Flow cytometric analysis of the hybridoma cell clone DIG-HC9-F9 showed that almost all cells are IgGspecific, but only 5% are specific for the antigen DIG. The incubation of the control cell clone with the DIG–FITC conjugate and anti-mouse-IgG-AlexaFluor®647 showed no antigen-specific staining. To confirm the ability of hybridoma cells to express hapten-specific antibodies on their surface with the ability to secrete antibodies, ELISA experiments were performed. The antibody concentration in the cell culture supernatant was determined for the clones DIG-DA5-E9 (43 mg/l), DIG-DA5 (65 mg/l), and DIG-HC9-F9 (not assessable) and the control cell clone (27 mg/l). The subsequent ELISA experiments were performed with an adjusted antibody concentration of 20 μg/l and provided the following results: (i) The amount of antigen-specific soluble IgG corresponds to the amount of antigen-specific cell-bound IgG. Soluble antigen-specific IgG (ELISA response curves) were only obtained from flow cytometric positive clones DIG-DA5 and DIG-DA5-E9. The hybridoma cell clone DIG-HC9-F9 did not secrete a detectable amount of DIG-specific antibodies into the supernatant (Fig. 4B) and shows only a low amount of DIG-specific antibodies on the cell surface (Fig. 4A). The control cell clone neither secretes DIG-specific antibodies as seen in ELISA response curves (Fig. 4B) nor carries antigen-specific antibodies as shown in flow cytometric analysis (Fig. 4A). (ii) The test-midpoint or C-value of the antibody response curve which provides a measure of antibody affinity was 93 μg/l for the clone DIG-DA5-E9 and 75 μg/l for DIG-DA5. The C-value of soluble antibodies was inversely proportional to the mean fluorescence intensity (Mean-X) of membrane-bound antibodies obtained by flow cytometric analysis (DIG-DA5-E9: 2.49 and DIG-DA5: 3.38).

Fig. 3. Mixed cultures of anti-E2 producing and anti-Afla producing hybridoma cells incubated with an E2–FITC-conjugate. Formalin-fixed hybridoma cell clones E2-BF1 and Afla-GE9 were mixed and incubated with the E2–FITC conjugate. Staining of the surface bound antigen was monitored by flow cytometry. (A) Afla-GE9, (B) E2-BF1 : Afla-GE9 = 1 : 1, and (C) E2-BF1.

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Fig. 4. Correspondence between the amount of antigen-specific surface IgG measured by flow cytometry (A) and antigen-specificity of secreted IgGs determined by ELISA (B). (A) Flow cytometric analysis of three different formalin-fixed hybridoma clones specific for DIG and a control cell clone (Afla-GE9). Cells were double stained with the DIG–FITC conjugate and anti-mouse-IgG-AlexaFluor®647. Percentages of all cells within the respective gate are indicated. (B) Antibody response curves specific for DIG. Supernatant of the same clones was analyzed by a direct competitive ELISA. The comparison between three different clones and the control is shown. Parameters of the 4-parameter fitting are shown in the table.

In summary, the different antibody affinities of three stable DIG-specific hybridoma cell clones determined by ELISA correspond to the amount of antibodies monitored via flow cytometry.

In order to increase the repertoire of tested haptens, hybridoma cell clones producing antibodies specific for the estrogens E2 (Fig. 5) and E1 (Fig. 6) were included in our analysis.

Fig. 5. Comparative analysis of mAb producing hybridoma clones by ELISA (A), immunohistology (B) and flow cytometry (C). (A) Calibration curves were obtained using supernatant from hybridoma cell cultures specific for E2. The response of six different clones is shown. In the table the parameters of the 4-parameter fitting are given. (B) Hybridoma cell clones E2-DE9, E2-BF1 and a control cell clone (Afla-GE9) grown on coverslips were stained with the FITC-labeled E2 conjugate and anti-mouse-IgG-Alexa®647. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole. Positive cells were identified by CLSM. (C) Different hybridoma clones specific for E2 and the control clone were fixed with formalin and incubated with the same dyes. Staining by the surface-bound antigen was monitored by flow cytometry. Percentages of all cells within the respective gates are indicated.

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Judging from the ELISA calibration curves (Fig. 5A), it appears that the hybridoma cell clones E2-DE9 and E2-BF1 produced antibodies with high affinities. However, flow cytometric analysis revealed that the E2-DE9 cells are not monoclonal (Fig. 5C). Two cell populations were distinguishable in the dot blot. This result was confirmed by the immunohistochemical analysis of double labeled hybridoma cells (Fig. 5B). Hapten-specific antibodies were found on all E2-BF1 cells, as shown by staining with the E2–FITC-conjugate and anti-mouse-IgG-Alexa®647. Immunofluorescence microscopy revealed limited binding to IgG on the surface of E2-DE9 cells, although almost all cells are positive for E2–FITC. With the analyte E2 we could show that the antibody amount of five different E2-specific hybridoma cell clones could be determined by ELISA or flow cytometry. Both methods show comparable results. Four of five E2 hybridoma clones secreted antibodies (E2-DE9: 17 mg/l; E2-BF1: 14 mg/ l; E2-DA10: 3 mg/l; E2-PA0: 2 mg/l and Afla-GE9: 27 mg/l) whereas clone E2-AA3 did not secrete any antibodies. This can also be seen in flow cytometric analysis. Except for clone E2-AA3 all hybridoma clones carried IgG on the cell surface as shown by anti-mouse-IgG-Alexa®647 staining. Concerning the antibody specificity only hybridoma cell clones E2-DE9 and E2-BF1 secreted E2-specific antibodies as seen by the C-values of the antibody response curves (E2-DE9: 64 μg/l and E2-BF1: 51 μg/l) (Fig. 4A). Both clones carry antigen-specific antibodies on their cell surface as indicated by the double positive cell staining (E2-DE9: 44% and E2-BF1: 88%) and the Mean-X value (E2-DE9: 3.1 and E2-BF1: 6.1) obtained by flow cytometric analysis. In contrast, the other four clones were negative in both experiments (Fig. 4C). In summary, the antibody affinities of E2-specific hybridoma cell clones, determined by ELISA (C-value), correspond inversely proportional to the antibody affinity monitored via flow cytometry (Mean-X). For the hapten E1, we used the method of double staining to screen for freshly fused hybridoma cells. A panel of 768 E1 hybridoma cell clones was studied. For the comparison of antibody characteristics by flow cytometry and ELISA, we analyzed 12 pre-selected E1 hybridoma clones and a control clone. Here, 5 clones are shown as an example. The relative amount of antigen-specific surface IgG was determined by flow cytometric analysis and three of twelve E1 hybridoma cell clones carried antigen-specific antibodies on their cell surface (Fig. 6A). These clones showed positive signals after E1–FITC and anti-IgG-AlexaFluor®647 labeling and the mean fluorescence intensities of these double positive clones were 2.6 for E1-BH5, 2.5 for E1-BH7, and 4.3 for the clone E1-HC7 in sequential analyses. The clone E1-AA3 produced high amounts of surface IgG, but the antibodies were not specific for E1, whereas the clone E1-CH1 was negative for E1 and anti-IgG-AlexaFluor®647. In order to validate the antigen-specificity of the mAbs, cell culture supernatants of growing hybridoma cells were tested by direct competitive ELISA (Fig. 6B). We determined the concentrations of the cell culture supernatants (E1-BH7: 17 mg/l; E1-BH5: 16 mg/l, E1-HC7: 47 mg/l; E1-AA3: 106 mg/l, E1-CH1: 0.1 mg/l and Afla-DE9 (control): 27 mg/l). Since the antibody concentration influences the C-value of the antibody response curves, we applied all E1-antibodies from the 5 different clones at the

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Fig. 6. Comparison of antibody expression of different hybridoma clones, specific for E1, from the same fusion experiment. (A) Flow cytometric analysis of five different E1 hybridoma clones and a control clone (Afla-GE9). Hybridoma cells were stained with E1–FITC and anti-IgG-AlexaFluor®647. Percentages of all cells within the respective gates are indicated. (B) Calibration curves of direct competitive ELISAs using the same six different antibodies from the immunization for E1 as in A. Parameters of the 4-parameter fitting are shown in the table.

same concentration (20 μg/l). The three positive clones (E1-BH5, E1-BH7 and E1-HC/) that were identified by flow cytometry before showed the typical sigmoidal ELISA

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calibration curves. The C-value of the antibody response curve was 1.7 μg/l for clone E1-BH5, 0.4 μg/l for E1-HC7 and 5.43 μg/l for E1-BH7. The C-value for E1-CH1 and the control clone was not assessable. For all three positive E1 clones the amount of soluble antigen-specific IgG corresponds to the amount of antigen-specific cell-bound-IgG. All three positive E1 clones showed that fluorescence intensity in flow cytometric analysis was inversely proportional to the C-value of the ELISA calibration curves. For example, the lowest C-value determined by ELISA was 0.4 μg/l for clone E1-HC7 which corresponds to the highest mean fluorescence intensity value of 4.3, observed by flow cytometry for the same clone. 4. Discussion Antibodies for haptens are required in many fields, especially in food analysis and environmental analysis. Aflatoxin B1 and zearalenone are mycotoxins and their determination is crucial for food safety. Therefore, analytical methods including immunoanalytical methods are continuously being improved (Chun et al., 2009). Major concerns are hormonally active compounds, such as estrogens, which can already be found in surface water. The occurrence of estrogens in water asks for simple, reliable, fast and cost-effective methods for their detection and analysis (Zhang et al., 2014). The generation of anti-hapten antibodies requires the coupling of haptens or their derivatives to carrier proteins (Dutton and Bulman, 1964; Walters et al., 1972; Fasciglione et al., 1996; Ramin and Weller, 2012). An often occurring problem in hapten-specific antibody generation is the occurrence of antibodies that are not specific to the hapten but to epitopes found on the carrier protein (Clementi et al., 1991). This reduces the total amount of hybridoma cells that produce hapten-specific antibodies and more cells have to be screened in order to obtain the desired hybridoma clone that produces a high-affinity, highly selective antibody against the hapten (Hock et al., 1995). In this article we describe a method to monitor the affinity and monoclonality of hybridoma cells specific for different haptens. The developed staining procedure for hybridoma cells allows the direct assessment of the pattern of antigen-specific surface IgG. For labeling, we used fluorescein-labeled haptens to measure the antibody specificity and secondary antibodies labeled with AlexaFluor®647 directed against mouse immunoglobulin. The “secondary” antibodies help in detecting antibody producers. In flow cytometric analyses we showed that the identification of double-positive cells (haptenbinding + IgG-producing) worked for all tested antigens aflatoxin and zearalenone (Fig. 2), digoxigenin (Fig. 4), estradiol (Fig. 5) and estrone (Fig. 6). To simplify the measuring procedure we fixed the hybridoma cells with formaldehyde. It has been shown before that the fixation has no influence on the fluorescence of surface IgG (Sen et al., 1990; Cherlet et al., 1995). Previous flow cytometric studies of hybridoma cells aimed to detect antibodies on the cell surface using only fluorescein-labeled anti-mouse IgG (Marder et al., 1990; Sen et al., 1990; McKinney et al., 1991; Kromenaker and Srienc, 1994; Cherlet et al., 1995; Borth et al., 2000). The lack of information on the specificity of the hybridoma cells,

however, is the major disadvantage of this method and therefore it has not been used on a routine basis, yet. The antigen-specific labeling of hybridoma cells has been rarely described, with the exception of Parks et al. (1979), who used antigen-specific microspheres. In another context genetically modified hybridoma cells were sorted antigen-specifically via a streptavidin/biotin system (Price et al., 2009). However, the antigen-specific detection of hapten-specific antibodies on the surface of B cells was reported before (McHeyzer-Williams et al., 1991; Lalor et al., 1992; McHeyzer-Williams et al., 2000). Due to the lack of published reports on flow cytometric detection of hapten-specific hybridoma cells, our first experiments reported here were designed to determine the specificity of the surface staining procedure with the fluorescein-labeled haptens ZON–FITC and Afla–FITC (Fig. 2A). To achieve this goal, a 1 : 1 mixture of equal concentrations of Afla- and E1hybridoma cells were analyzed and both cell populations could be distinguished and their respective proportion of the total population was correctly quantified (Fig. 3B). Immunofluorescence microscopy of membrane immunoglobulin expression showed the same level of surface antigen-specific antibodies (Fig. 2B) compared to flow cytometric analysis (Fig. 2A). Also, the double staining by fluoresceinlabeled E2 and anti-mouse-IgG-AlexaFluor®647 reveals a good correspondence between microscopy and flow cytometry analysis. We demonstrated that several hybridoma cell clones specific for different haptens carried antibodies on their surface. From these findings we conclude that our optimized immunofluorescence staining procedure is specific. Price et al., 2009 showed different amounts of cell surface IgG on transfected cells by immunofluorescence microscopy. However, microscopic images of antigen-specific antibodies on the surface of hybridoma cells have not been shown in the literature so far. Care should be taken to generalize these results, because not all hybridoma cell clones express surface IgG (Matsuuchi et al., 1992; Seegmiller et al., 2007). To guarantee precise measurements, a high antibody affinity is crucial for immunoanalytical measurements, especially when analyte concentrations are at trace levels. Several publications have reported some correlation between surface antibody content and antibody secretion rate (Marder et al., 1990; Sen et al., 1990; McKinney et al., 1991; Kromenaker and Srienc, 1994). Sen et al. (1990) found a linear correlation between mean surface fluorescence intensity and the specific antibody production rate, while Marder et al. (1990) used FACS to isolate clones derived from high fluorescent intensity sorting gates and found enhanced immunoglobulin secretion from these clones. Up to now, there are no reports about a correlation between signal intensities in flow cytometry and affinity of antibodies displayed in ELISA measurements. Using ELISA technique, it is possible to determine the antibody affinity (Friguet et al., 1985; Underwood, 1993; Loomans et al., 1995). Here we show the possibility to correlate the fluorescence intensity of antibody-producing cells with the antigen affinities of the produced antibodies. Monoclonality of a hybridoma culture is of great importance. One of the most commonly used methods to get a true single

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clone is the subcloning of a hybridoma culture, which is based on repeated cycles of limiting dilution. The Poisson distribution is used to calculate the cell dilution that will result in a high degree of probability in monoclonality (Coller and Coller, 1983; Staszewski, 1984). However, this statistical approach cannot ensure that a culture is really monoclonal (Coller and Coller, 1986; Underwood and Bean, 1988). For this reason it is still possible that non-producers overgrow producing clones because non-producers usually have a higher growth rate than producing cells (Richieri et al., 1991) and the desired clones will be lost (Frame and Hu, 1990; Ozturk and Palsson, 1990). This can also be seen in Figs. 4A and 2A. Clones ZON-EA6-D5 and E2-DE9 show two distinct populations with different fluorescence intensities. This demonstrates that both clones are not monoclonal. Using the flow cytometer, it is possible to check for monoclonality of a cell culture. There is often only a small minority of cells differing in the antibody expression level. In order to identify these cells thousands of clones need to be analyzed, which is impossible by traditional methods (Browne and Al-Rubeai, 2007). A high-throughput technique such as flow cytometry and cell sorting would be suitable in the selection of these rare cells (Borth et al., 2000). Our approach employs the technique of antigen-specific labeling of cells, which allows fluorescence activated cell sorting and thereby eliminate the abovementioned problems.

5. Conclusions The conventional hybridoma screening and subcloning process for appropriate hybridoma cells has two major drawbacks. Firstly, it is time-consuming because several rounds of limiting dilution are required to reach monoclonality and even then, monoclonality is not guaranteed. Secondly, the number of clones that can be screened is limited resulting in a low efficiency of the screening (Holmes and Al-Rubeai, 1999). In this study, we overcome these limitations of the screening process by using flow cytometry and ELISA to screen for high-producing hybridoma cells. In our study we could show that it is possible to specifically double label cells according to their affinity and target selectivity. ELISA measurements showed that the clones with the highest antigen-specific fluorescence intensity in the flow cytometer have the highest antigen-specific binding affinities against the target. Our double staining procedure permits a detailed analysis of hybridoma clones during the screening process. This system should be suitable for other analytes helping to optimize the conventional screening process.

Acknowledgments The authors want to thank BAM Federal Institute for Materials Research and Testing for a PhD grant to M. Dippong. We thank A. Lehmann and A. Grasnick, both from BAM for hapten–fluorophore conjugate purification and characterization. We thank aokin AG for granting some hybridoma cell clones. We are grateful to A. Andersson and C. Lenz from UP Transfer GmbH for hybridoma generation.

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