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Human Antibodies 22 (2013) 67–71 DOI 10.3233/HAB-140272 IOS Press
Production of Anti-CD14 monoclonal antibody using synthetic peptide of human CD14 as immunizing antigen Leili Aghebati Maleki1 , Dariush Shanehbandi1, Jafar Majidi, Mehdi Yusefi, Jalal Abdolalizadeh, Mona Orangi and Behzad Baradaran∗ Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
Abstract. BACKGROUND: CD14 is a myeloid differentiation antigen expressed primarily on peripheral blood monocytes, dendritic cells and macrophages. It is a key regulator of inflammatory responses to gram-negative bacteria, oxidative burst and septic shock. OBJECTIVE: The aim of this study was to produce and characterize monoclonal antibody against CD14 for use in detection and diagnosis of monocytes. METHODS: To produce MAb against CD14 protein, mice were immunized with two KLH-conjugated CD14 peptides. The spleen cells of the immunized mice were then fused with SP2/0 by hybridoma technique. Fused cells were grown in selective medium and cloned by limiting dilution method. The desired clones were selected and supernatants of hybridoma cells were screened by ELISA for antibody. Monoclonal antibody was purified by chromatography and confirmed by SDS-PAGE. Finally, immunoblotting and flowcytometry were recruited to explore the specificity of the MAb. RESULTS: Our results showed successful production and characterization of anti CD14 monoclonal antibody. The MAb was IgG2a with Kappa light chain and immunobloting and flowcytometry results demonstrated specific reactivity of this MAb with CD14. CONCLUSIONS: The results show that, the produced anti- CD14 MAb is highly specific and functional in biomedical applications such as flow cytometry and western blotting and could be utilized for identification of monocytes. Keywords: Monoclonal antibody, CD14, synthetic peptide, flow cytometry, western blotting
1. Introduction The CD14 glycoprotein is a high affinity, GPI-linked receptor for lipopolysaccharide (LPS) and LPS binding protein (LPB) which is expressed at high levels on peripheral blood monocytes, macrophages and dendritic cells and some Langerhans cells [1,2]. CD14, the monocyte differentiation antigen, is anchored to the cell membrane by a phosphatidylinos1 These
authors contributed equally to this paper study. author: Behzad Baradaran, Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. Tel.: +98 411 3364665; Fax: +98 411 3364665; E-mail: behzad_ [email protected]. ∗ Corresponding
itol linkage. Although its function is unknown, the CD14 gene maps to a region encoding several myeloid growth factors and receptors [3]. Interestingly, CD14 role is known in phagocytic clearance of apoptotic cells. CD14 isoforms with molecular weights of 52–55 kDa, expressed on the surfaces of monocytes and neutrophils are attached to the cell surface by a glycosylphosphatidylinositol (GPI) anchor, membrane protein (mCD14) and the serum soluble 48–56 kDa (sCD14, an acute phase protein). Soluble CD14 found in human serum has been attributed to the shedding of mCD14 from monocytes, macrophages and PMN. Membrane CD14 as a receptor for lipopolysaccharide (LPS) on the membrane of the mononuclear phagocyte (MPS) binds to LPS-binding
c 2013 – IOS Press and the authors. All rights reserved ISSN 1093-2607/13/$27.50
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L.A. Maleki et al. / Production of monoclonal antibody against CD14
protein (LBP) in plasma and transfers to the cell surface receptor CD14 [4]. It has been commonly used in normal tissue or blood and in leukemia as a marker for myeloid cells. LPS stimulates the human monocytes activation via several intracellular signaling pathways that involves the proinflammatory factors [5]. On the cell surface, CD14 associates with Toll-like receptor 4 (TLR4). Binding of the LPS and LPSbinding protein complex to CD14 induces signal transduction through TLR4, which then triggers the synthesis and release of proinflammatory chemokine (IP10) and cytokines (TNF-α, IL-6 and IL-1) [6]. In this study, we have employed a peptide-based antibody generation protocol for producing antibody against human CD14 glycoprotein for diagnostic and research applications. 2. Materials and methods 2.1. Preparation of antigen and coupling to carrier protein The amino acid sequence of human CD14 was carefully analyzed. Then, based on local hydrophilicity predicted by Hopp and Woods method [7], two 14mer synthetic peptides from extracellular part of human CD14 protein were designed and considered as immunogen. The immunogen sequences were KRVDADADPRQYAD and LAELQQWLKPGLKV. A cysteine residue was added to the C terminus of the peptide to facilitate the conjugation to carrier protein. The designed peptide was conjugated to Keyhole Lympet Hemocyanin (KLH) and Bovine Serum Albumin (BSA) (Thermo, USA), separately. subsequently, the efficiency of conjugation was checked by SDS-PAGE method [8]. 2.2. Immunization of mice Initially, 8-week-old female Balb/c mice were immunized by intraperitoneal injection with the KLHconjugated peptide dissolved in normal saline solution and emulsified with an equal volume of Freund’s complete adjuvant (CFA) (Sigma-Aldrich Co. Louis, USA). All animal experiments followed the guidelines of the Laboratory Animal Ethical Commission of Tabriz University of Medical Sciences. Subsequent immunizations (booster) at 2 weeks intervals were given with Freund’s incomplete adjuvant. Each mouse was immunized 4 times. Blood samples were collected by tail bleeding to determine antiserum titer using ELISA. The mice with high antiserum titer were given a final booster immunization 4 days before the cell fusion.
2.3. Hybridization and cloning Mouse myeloma SP2/0 cell line was used as fusion partner. Thus, one week before the fusion cells were cultured in RPMI (GIBCO) with 10% FBS until reaching to more than 70% confluence in logarithmic phase. The spleen cells of the immune mouse were removed under sterile condition. Spleen cells were fused with SP2/0 cells at a 5:1 ratio by PEG1450 (Sigma-Aldrich Co. Louis, USA) as fusogen. The fused cells suspended in 50ml of pre-warmed HAT medium (Gibco Co, Scotland, UK) and distributed into five culture plates (96 wells, Nunc) containing feeder layer, in the amount of 100 μl/well (1.3 × 104 cells to each well). Supernatants of the wells were exchanged with HAT 4X medium in the forth, seventh, tenth and fourteenth days after the fusion. After fourteen days, well reproduced clones were transported into the new plates containing feeder layer [9]. After screening, the clones with high absorbance were selected for cloning by limiting dilution (L.D) method. The cells were diluted so that contained only one cell in each 10 μl. About twelve days after L.D, the supernatants of monoclones were screened for production of antibody. Suitable monoclones possessing high absorbance were selected for characterization of antibodies. 2.4. Mass production For Mass production of monoclonal antibodies in ascitic fluid, three Female BALB/c mice (4–6 weeks old) were obtained from Pasteur institute of Iran. Initially, 0.5 ml Pristane (2, 6, 10, 14 tetra methyl pentadecane, Sigma) was administered intraperitoneally into each mouse. Twelve days after priming with Pristane, high cell densities of a desired mono clone (106 cells/0.5 ml PBS) were injected intraperitoneally into each mouse. Their ascitic fluids were harvested by 19-gauge needle. The titer of ascitic fluid was assessed by ELISA method. 2.5. Purification of the immunoglobulins and isotyping Immunoglobulins from the ascetic fluids were recovered by ammonium sulfate precipitation. The precipitated fraction was dialyzed against 10 mM PBS (pH = 7.4) and purified using Sepharose beads conjugated with Protein A column affinity chromatography. The purified immunoglobulins were stored at −70◦ C. Isotyping of purified immunoglobulins was performed with a commercial isotyping kit (Thermo, USA) according to the manufacturer’s guideline.
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2.6. SDS-PAGE
2.9. Flow cytometry analysis
Purity of MAb was assessed by SDS-PAGE in both reduced and non-reduced conditions. 100 μg of purified MAb was mixed with 20 μl of sample buffer, then boiled for 5 minutes and cooled on ice. SDS-PAGE was carried out using 12.5% polyacrylamide gel in 100 mA for 1 hr. The gel was stained with Coomassie Brilliant Blue R-250 (Sigma).
Reactivity of anti CD14 monoclonal antibodies was assessed using indirect flow cytometry. Briefly, Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density-gradient centrifugation and washed twice with washing buffer containing PBS (0.15 M), BSA (0.5%), NaN3 (0.1%). After washing, 1 × 106 PBMCs were resuspended in 100 μl staining buffer and stained with 1 μg FITC-conjugated anti CD14 (prepared in our lab) and appropriate amount of commercial anti-CD14 monoclonal antibody (Dako, F0844) as a positive control. Then cells incubated for 40 min at 4◦ C in the darkness followed by twice extra washing step. Finally, cells scanned with a FACSCalibur flow cytometer (Becton- Dickinson, Mountain View, CA) and data were analysed using CellQuest 3.1 software (Becton-Dickinson) [12].
2.7. Western blot analysis of anti-CD14 MAb in identification of peripheral blood monocytes The capability of the produced antibodies in identifiation and enrichment of human monocytes was evaluated by western blotting. Initially, proteins ofmonocytes were extracted using RIPA buffer (50 mM Tris– HCl (pH 7.4), 1% Triton X-100, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, 0.1% SDS and a protease inhibitor cocktail) and electrophorized and transferred to a PVDF membrane. Nonspecific reactivity was blocked by incubating the membrane in 10 mM Tris–HCl (pH 7.5), 150 mM NaCl, 2% Tween 20 and 4% skim milk. After adding the primary antibody, the membrane was incubated overnight at 4◦ C. Subsequent to three times of washing, the membrane was exposed to the HRP (Horse Radish Peroxidase) conjugated secondary antibody for one hour at room temperature. Protein detection was achieved with the enhanced chemiluminescence (ECL) system (ECL, Amersham Phamacia Biotech Inc, USA) [10]. 2.8. Conjugation with fluorescein isothiocyanate (FITC) For conjugation of monoclonal antibody, 200 μl of MAb (5 mg/ml) was added in 800 μl of reaction buffer (500 Mm Carbonate, pH = 9.2) and dialyzed against PBS buffer for 24 hours. The antibody concentration was measured after buffer equilibration in 280 nm. 10 mg of FITC was dissolved in 1 mL of anhydrous DMSO immediately before use. FITC (SIGMA, Germany) was added to give a ratio of 80 μg per mg of antibody and mixed immediately. The tubes were wrapped in a foil and shaken at room temperature for 1 hour. The un-reacted FITC was removed and the antibody exchanged into storage buffer (10 mM Tris, 150 mM Nacl, 0.1% Nah3, pH = 8.2) by dialysis overnight [11].
3. Results Two synthetic peptides, corresponding to human CD14 N-terminus sequence, were conjugated to carrier protein. The peptide–KLH was used for mice immunization and peptide-BSA was used for conjugation assessment and specificity test of the antibody. The change in mobility shift of conjugated BSA compared to non-conjugated one, revealed the suitable efficiency of the coupling [13]. After four times immunization of four Balb/c mice against KLH-conjugated peptides, sera from these mice were evaluated by indirect binding assays for antibody reactivity with BSApeptide conjugate. The serum of the immune mouse at 1/8000 dilution, displayed high absorbance in reaction with BSA-peptide. Table1 represents the results of indirect ELISA. Based on the outcomes, the mouse with higher titer of specific antibody (Mouse 2) was chosen for hybridoma production. The spleen cells of the immunized mouse were fused with SP2/0 myeloma cells as fusion partner. The fused cells were then suspended in HAT medium and distributed into five 96 well culture plates containing feeder layer. Several anti-CD14 monoclonal antibody producing hybridomas were obtained. Among them, “2E8” had a high reactivity with immunogenic peptide in ELISA (Table1). Due to stronger reactivity of “2E8” in ELISA, all subsequent tests were carried out on this clone. Further characterization of the produced antibody showed that it was an IgG2b isotype with a kappa light chain. The purity of the antibody was assessed by SDS-PAGE. In reduc-
70
L.A. Maleki et al. / Production of monoclonal antibody against CD14 Table 1 Comparison of the mean absorbance of Ascitic fluid and purified antibody at 450 nm NC (SP2/0) 0.08
NC∗ (Non-immune mouse serum) 0.15
PC** (Immune mouse serum) 1.32
Ascitic fluid/BSAPeptide (1/8000 dilution) 1.12
2E8 clone 1.32
Purified Antibody/BSAPeptide (1/8000 dilution) 1.20
Purified Antibody/Raji cell (1/8000 dilution) 1.06
*: Negative control with 1/8000 dilution. **: Positive control with 1/8000 dilution.
4. Discussion
Fig. 1. SDS-PAGE of purified Anti CD14 MAb in reduced form. Two bands of 25 and 50 kDa, corresponding to the heavy and light chains were detected.
Fig. 2. Western blot analysis of immunorereactivity between anti-CD14 MAb and monocytes lysate.
ing experiment, two bands of 50 and 25 kDa representing the heavy and light chains were detected (Fig. 1). In Non-reducing SDS-PAGE, only one 150 kDa band was appeared which demonstrates the successful production and purification of the target antibody. In addition, the purified antibody showed an acceptable activity with the immunizing peptide in ELISA (Table 1). In addition, western blotting to evaluate the immunorereactivity between the produced anti-CD14 monoclonal antibody and monocytes resulted in a single specific band (Fig. 2). Then, reactivity of anti-CD14 MAb was analyzed using indirect flow cytometry analysis. As shown in Fig. 3, reactivity of this MAb with PBMCs isolated from healthy volunteers was 8 percent.
The production of monoclonal antibodies was first discovered by Cesar Milstein and Georges Köhler in 1975. Since then, a few CD14-specific monoclonal antibodies have been developed. For instance,a high affinity Anti-CD14 monoclonal antibody was produced and developed by Moonsom et al. They used CD14 expressing COS Cells as immunizing antigen and generated CD14 MAbs which could be utilized to enumerate monocytes of peripheral blood as well as using referent CD14 MAb [13]. In the present study, an immunization strategy based on synthetic peptide was employed for production of MAb against human CD14. In this regard, two 14-mer peptides, adapted from CD14 structure, were designed and synthesized. The use of synthetic peptides as immunogens is an attractive alternative where the complete protein is not available in adequate quantities for immunization. In addition, this method could be employed for production of antibodies to recognize specific regions of a polypeptide [9]. Synthetic peptides provide the opportunity to overcome the lack of protein antigen. But it should be noted that , factors such as the secondary and tertiary configuration, length, hydrophilicity and localization of the peptides in the native molecule, must be considered in generating efficient MAbs [14]. A major problem with utilizing peptides as antigen is their small sizes, which appropriate carriers could be a helpful solution [15]. KLH has been demonstrated to be an effective carrier protein for immunization with short peptides in the high-yield production of antibodies for research, biotechnology and therapeutic applications [16]. However, because of very high molecular weight of KLH, it is not possible to run the KLH conjugate on SDS-PAGE. Consequently, BSA was utilized to evaluate the conjugation efficacy. At the next step, mice were immunized with synthetic peptide – KLH conjugates to raise antibodies. The serum of the mouse 2, indicated high absorbance in reaction with BSA-peptide at 1/8000 dilution. Among several antiCD14 MAb producing hybridomas resulted from this mouse, “2E8” clone demonstarted high reactivity with immunogenic peptide in ELISA and considered for
L.A. Maleki et al. / Production of monoclonal antibody against CD14
A
References [1]
[2]
[3]
B
[4]
[5]
[6]
[7] [8] Fig. 3. Representative data for indirect flow cytometry of anti CD14 MAb. A: Isotype control and B: Anti CD14 MAb reaction with PBMCs isolated from healthy volunteers. (Colours are visible in the online version of the article; http://dx.doi.org/10.3233/ HAB-140272)
further characterization. The produced antibody was recognized as IgG2b isotype with kappa light chain. Immunoblot analysis showed a strong reactivity with CD14. The western blotting result was also in conformity with ELISA from the viewpoint of specificity. According to flow cytometry results, our generated MAb had an acceptable reactivity compared to commercial anti CD14 MAb. These findings suggest that, the produced antibody is highly specific and functional for use in applications such as western blotting and flow cytometry. Furthermore, the two synthetic peptides included in this study were highly efficient immunogens for development of monoclonal antibodies.
[9]
[10]
[11]
[12]
[13]
[14]
[15]
Acknowledgements We would like to thank for Immunology Research Center (IRC) for kind assistance. This work was supported by a grant from Immunology Research Center (IRC).
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[16]
A.H. Werners et al., Genotyping of Toll-like receptor 4, myeloid differentiation factor 2 and CD-14 in the horse: an investigation into the influence of genetic polymorphisms on the LPS induced TNF-alpha response in equine whole blood, Veterinary Immunology and Immunopathology 111 (2006), 165– 173. R. Landmann, B. Muller and W. Zimmerli, CD14, new aspects of ligand and signal diversity, Microbes and Infection/Institut Pasteur 2 (2000), 295–304. K.L. Lohmann, M.L. Vandenplas, M.H. Barton, C.E. Bryant and J.N. Moore, The equine TLR4/MD-2 complex mediates recognition of lipopolysaccharide from Rhodobacter sphaeroides as an agonist, Journal of Endotoxin Research 13 (2007), 235–242. S. Mauel, F. Steinbach and H. Ludwig, Monocyte-derived dendritic cells from horses differ from dendritic cells of humans and mice, Immunology 117 (2006), 463–473. P. Antal-Szalmas, J.A. Strijp, A.J. Weersink, J. Verhoef and K.P. Van Kessel, Quantitation of surface CD14 on human monocytes and neutrophils, Journal of Leukocyte Biology 61 (1997), 721–728. H.W.L. Ziegler-Heitbrock and R.J. Ulevitch, CD14: Cell surface receptor and differentiation marker, Immunology Today 14 (1993), 121–125. P. Hornbeck, Assays for antibody production, Current Protocols in Immunology, John Wiley & Sons, (2001). G. Kohler and C. Milstein, Continuous cultures of fused cells secreting antibody of predefined specificity, Nature 256 (1975), 495–497. L.A. Maleki, J. Majidi, B. Baradaran, J. Abdolalizadeh and A.M. Akbari, Production and characterization of murine monoclonal antibody against synthetic peptide of CD34, Human Antibodies 22 (2013), 1–8. L. Aghebati Maleki et al., Large scale generation and characterization of anti-human CD34 monoclonal antibody in ascetic fluid of Balb/c mice, Advanced Pharmaceutical Bulletin 3 (2013), 211–216. B. Baradaran et al., Development and characterization of monoclonal antibodies against human epidermal growth factor receptor in Balb/c mice, Human Antibodies 18 (2009), 11– 16. D. Schrama, R.A. Reisfeld and J.C. Becker, Antibody targeted drugs as cancer therapeutics, Nat Rev Drug Discov 5 (2006), 147–159. S. Moonsom and W. Kasinrerk, Production of anti-CD14 monoclonal antibodies using CD14 expressing COS cells as immunizing antigen, Asian Pacific Journal of Allergy and Immunology/Launched by the Allergy and Immunology Society of Thailand 18 (2000), 53–61. R. Hadavi et al., Production of monoclonal antibody against human Nestin, Avicenna Journal of Medical Biotechnology 2 (2010), 69–77. J.B. Bussel et al., Update on therapeutic monoclonal antibodies, Current Problems in Pediatric and Adolescent Healthcare 37 (2007), 118–135. H. Modjtahedi, Monoclonal antibodies as therapeutic agents: Advances and challenges, Iranian Journal of Immunology 2 (2005), 3–20.
Human Antibodies 22 (2013) 67–71 DOI 10.3233/HAB-140272 IOS Press
Production of Anti-CD14 monoclonal antibody using synthetic peptide of human CD14 as immunizing antigen Leili Aghebati Maleki1 , Dariush Shanehbandi1, Jafar Majidi, Mehdi Yusefi, Jalal Abdolalizadeh, Mona Orangi and Behzad Baradaran∗ Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
Abstract. BACKGROUND: CD14 is a myeloid differentiation antigen expressed primarily on peripheral blood monocytes, dendritic cells and macrophages. It is a key regulator of inflammatory responses to gram-negative bacteria, oxidative burst and septic shock. OBJECTIVE: The aim of this study was to produce and characterize monoclonal antibody against CD14 for use in detection and diagnosis of monocytes. METHODS: To produce MAb against CD14 protein, mice were immunized with two KLH-conjugated CD14 peptides. The spleen cells of the immunized mice were then fused with SP2/0 by hybridoma technique. Fused cells were grown in selective medium and cloned by limiting dilution method. The desired clones were selected and supernatants of hybridoma cells were screened by ELISA for antibody. Monoclonal antibody was purified by chromatography and confirmed by SDS-PAGE. Finally, immunoblotting and flowcytometry were recruited to explore the specificity of the MAb. RESULTS: Our results showed successful production and characterization of anti CD14 monoclonal antibody. The MAb was IgG2a with Kappa light chain and immunobloting and flowcytometry results demonstrated specific reactivity of this MAb with CD14. CONCLUSIONS: The results show that, the produced anti- CD14 MAb is highly specific and functional in biomedical applications such as flow cytometry and western blotting and could be utilized for identification of monocytes. Keywords: Monoclonal antibody, CD14, synthetic peptide, flow cytometry, western blotting
1. Introduction The CD14 glycoprotein is a high affinity, GPI-linked receptor for lipopolysaccharide (LPS) and LPS binding protein (LPB) which is expressed at high levels on peripheral blood monocytes, macrophages and dendritic cells and some Langerhans cells [1,2]. CD14, the monocyte differentiation antigen, is anchored to the cell membrane by a phosphatidylinos1 These
authors contributed equally to this paper study. author: Behzad Baradaran, Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. Tel.: +98 411 3364665; Fax: +98 411 3364665; E-mail: behzad_ [email protected]. ∗ Corresponding
itol linkage. Although its function is unknown, the CD14 gene maps to a region encoding several myeloid growth factors and receptors [3]. Interestingly, CD14 role is known in phagocytic clearance of apoptotic cells. CD14 isoforms with molecular weights of 52–55 kDa, expressed on the surfaces of monocytes and neutrophils are attached to the cell surface by a glycosylphosphatidylinositol (GPI) anchor, membrane protein (mCD14) and the serum soluble 48–56 kDa (sCD14, an acute phase protein). Soluble CD14 found in human serum has been attributed to the shedding of mCD14 from monocytes, macrophages and PMN. Membrane CD14 as a receptor for lipopolysaccharide (LPS) on the membrane of the mononuclear phagocyte (MPS) binds to LPS-binding
c 2013 – IOS Press and the authors. All rights reserved ISSN 1093-2607/13/$27.50
68
L.A. Maleki et al. / Production of monoclonal antibody against CD14
protein (LBP) in plasma and transfers to the cell surface receptor CD14 [4]. It has been commonly used in normal tissue or blood and in leukemia as a marker for myeloid cells. LPS stimulates the human monocytes activation via several intracellular signaling pathways that involves the proinflammatory factors [5]. On the cell surface, CD14 associates with Toll-like receptor 4 (TLR4). Binding of the LPS and LPSbinding protein complex to CD14 induces signal transduction through TLR4, which then triggers the synthesis and release of proinflammatory chemokine (IP10) and cytokines (TNF-α, IL-6 and IL-1) [6]. In this study, we have employed a peptide-based antibody generation protocol for producing antibody against human CD14 glycoprotein for diagnostic and research applications. 2. Materials and methods 2.1. Preparation of antigen and coupling to carrier protein The amino acid sequence of human CD14 was carefully analyzed. Then, based on local hydrophilicity predicted by Hopp and Woods method [7], two 14mer synthetic peptides from extracellular part of human CD14 protein were designed and considered as immunogen. The immunogen sequences were KRVDADADPRQYAD and LAELQQWLKPGLKV. A cysteine residue was added to the C terminus of the peptide to facilitate the conjugation to carrier protein. The designed peptide was conjugated to Keyhole Lympet Hemocyanin (KLH) and Bovine Serum Albumin (BSA) (Thermo, USA), separately. subsequently, the efficiency of conjugation was checked by SDS-PAGE method [8]. 2.2. Immunization of mice Initially, 8-week-old female Balb/c mice were immunized by intraperitoneal injection with the KLHconjugated peptide dissolved in normal saline solution and emulsified with an equal volume of Freund’s complete adjuvant (CFA) (Sigma-Aldrich Co. Louis, USA). All animal experiments followed the guidelines of the Laboratory Animal Ethical Commission of Tabriz University of Medical Sciences. Subsequent immunizations (booster) at 2 weeks intervals were given with Freund’s incomplete adjuvant. Each mouse was immunized 4 times. Blood samples were collected by tail bleeding to determine antiserum titer using ELISA. The mice with high antiserum titer were given a final booster immunization 4 days before the cell fusion.
2.3. Hybridization and cloning Mouse myeloma SP2/0 cell line was used as fusion partner. Thus, one week before the fusion cells were cultured in RPMI (GIBCO) with 10% FBS until reaching to more than 70% confluence in logarithmic phase. The spleen cells of the immune mouse were removed under sterile condition. Spleen cells were fused with SP2/0 cells at a 5:1 ratio by PEG1450 (Sigma-Aldrich Co. Louis, USA) as fusogen. The fused cells suspended in 50ml of pre-warmed HAT medium (Gibco Co, Scotland, UK) and distributed into five culture plates (96 wells, Nunc) containing feeder layer, in the amount of 100 μl/well (1.3 × 104 cells to each well). Supernatants of the wells were exchanged with HAT 4X medium in the forth, seventh, tenth and fourteenth days after the fusion. After fourteen days, well reproduced clones were transported into the new plates containing feeder layer [9]. After screening, the clones with high absorbance were selected for cloning by limiting dilution (L.D) method. The cells were diluted so that contained only one cell in each 10 μl. About twelve days after L.D, the supernatants of monoclones were screened for production of antibody. Suitable monoclones possessing high absorbance were selected for characterization of antibodies. 2.4. Mass production For Mass production of monoclonal antibodies in ascitic fluid, three Female BALB/c mice (4–6 weeks old) were obtained from Pasteur institute of Iran. Initially, 0.5 ml Pristane (2, 6, 10, 14 tetra methyl pentadecane, Sigma) was administered intraperitoneally into each mouse. Twelve days after priming with Pristane, high cell densities of a desired mono clone (106 cells/0.5 ml PBS) were injected intraperitoneally into each mouse. Their ascitic fluids were harvested by 19-gauge needle. The titer of ascitic fluid was assessed by ELISA method. 2.5. Purification of the immunoglobulins and isotyping Immunoglobulins from the ascetic fluids were recovered by ammonium sulfate precipitation. The precipitated fraction was dialyzed against 10 mM PBS (pH = 7.4) and purified using Sepharose beads conjugated with Protein A column affinity chromatography. The purified immunoglobulins were stored at −70◦ C. Isotyping of purified immunoglobulins was performed with a commercial isotyping kit (Thermo, USA) according to the manufacturer’s guideline.
L.A. Maleki et al. / Production of monoclonal antibody against CD14
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2.6. SDS-PAGE
2.9. Flow cytometry analysis
Purity of MAb was assessed by SDS-PAGE in both reduced and non-reduced conditions. 100 μg of purified MAb was mixed with 20 μl of sample buffer, then boiled for 5 minutes and cooled on ice. SDS-PAGE was carried out using 12.5% polyacrylamide gel in 100 mA for 1 hr. The gel was stained with Coomassie Brilliant Blue R-250 (Sigma).
Reactivity of anti CD14 monoclonal antibodies was assessed using indirect flow cytometry. Briefly, Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density-gradient centrifugation and washed twice with washing buffer containing PBS (0.15 M), BSA (0.5%), NaN3 (0.1%). After washing, 1 × 106 PBMCs were resuspended in 100 μl staining buffer and stained with 1 μg FITC-conjugated anti CD14 (prepared in our lab) and appropriate amount of commercial anti-CD14 monoclonal antibody (Dako, F0844) as a positive control. Then cells incubated for 40 min at 4◦ C in the darkness followed by twice extra washing step. Finally, cells scanned with a FACSCalibur flow cytometer (Becton- Dickinson, Mountain View, CA) and data were analysed using CellQuest 3.1 software (Becton-Dickinson) [12].
2.7. Western blot analysis of anti-CD14 MAb in identification of peripheral blood monocytes The capability of the produced antibodies in identifiation and enrichment of human monocytes was evaluated by western blotting. Initially, proteins ofmonocytes were extracted using RIPA buffer (50 mM Tris– HCl (pH 7.4), 1% Triton X-100, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, 0.1% SDS and a protease inhibitor cocktail) and electrophorized and transferred to a PVDF membrane. Nonspecific reactivity was blocked by incubating the membrane in 10 mM Tris–HCl (pH 7.5), 150 mM NaCl, 2% Tween 20 and 4% skim milk. After adding the primary antibody, the membrane was incubated overnight at 4◦ C. Subsequent to three times of washing, the membrane was exposed to the HRP (Horse Radish Peroxidase) conjugated secondary antibody for one hour at room temperature. Protein detection was achieved with the enhanced chemiluminescence (ECL) system (ECL, Amersham Phamacia Biotech Inc, USA) [10]. 2.8. Conjugation with fluorescein isothiocyanate (FITC) For conjugation of monoclonal antibody, 200 μl of MAb (5 mg/ml) was added in 800 μl of reaction buffer (500 Mm Carbonate, pH = 9.2) and dialyzed against PBS buffer for 24 hours. The antibody concentration was measured after buffer equilibration in 280 nm. 10 mg of FITC was dissolved in 1 mL of anhydrous DMSO immediately before use. FITC (SIGMA, Germany) was added to give a ratio of 80 μg per mg of antibody and mixed immediately. The tubes were wrapped in a foil and shaken at room temperature for 1 hour. The un-reacted FITC was removed and the antibody exchanged into storage buffer (10 mM Tris, 150 mM Nacl, 0.1% Nah3, pH = 8.2) by dialysis overnight [11].
3. Results Two synthetic peptides, corresponding to human CD14 N-terminus sequence, were conjugated to carrier protein. The peptide–KLH was used for mice immunization and peptide-BSA was used for conjugation assessment and specificity test of the antibody. The change in mobility shift of conjugated BSA compared to non-conjugated one, revealed the suitable efficiency of the coupling [13]. After four times immunization of four Balb/c mice against KLH-conjugated peptides, sera from these mice were evaluated by indirect binding assays for antibody reactivity with BSApeptide conjugate. The serum of the immune mouse at 1/8000 dilution, displayed high absorbance in reaction with BSA-peptide. Table1 represents the results of indirect ELISA. Based on the outcomes, the mouse with higher titer of specific antibody (Mouse 2) was chosen for hybridoma production. The spleen cells of the immunized mouse were fused with SP2/0 myeloma cells as fusion partner. The fused cells were then suspended in HAT medium and distributed into five 96 well culture plates containing feeder layer. Several anti-CD14 monoclonal antibody producing hybridomas were obtained. Among them, “2E8” had a high reactivity with immunogenic peptide in ELISA (Table1). Due to stronger reactivity of “2E8” in ELISA, all subsequent tests were carried out on this clone. Further characterization of the produced antibody showed that it was an IgG2b isotype with a kappa light chain. The purity of the antibody was assessed by SDS-PAGE. In reduc-
70
L.A. Maleki et al. / Production of monoclonal antibody against CD14 Table 1 Comparison of the mean absorbance of Ascitic fluid and purified antibody at 450 nm NC (SP2/0) 0.08
NC∗ (Non-immune mouse serum) 0.15
PC** (Immune mouse serum) 1.32
Ascitic fluid/BSAPeptide (1/8000 dilution) 1.12
2E8 clone 1.32
Purified Antibody/BSAPeptide (1/8000 dilution) 1.20
Purified Antibody/Raji cell (1/8000 dilution) 1.06
*: Negative control with 1/8000 dilution. **: Positive control with 1/8000 dilution.
4. Discussion
Fig. 1. SDS-PAGE of purified Anti CD14 MAb in reduced form. Two bands of 25 and 50 kDa, corresponding to the heavy and light chains were detected.
Fig. 2. Western blot analysis of immunorereactivity between anti-CD14 MAb and monocytes lysate.
ing experiment, two bands of 50 and 25 kDa representing the heavy and light chains were detected (Fig. 1). In Non-reducing SDS-PAGE, only one 150 kDa band was appeared which demonstrates the successful production and purification of the target antibody. In addition, the purified antibody showed an acceptable activity with the immunizing peptide in ELISA (Table 1). In addition, western blotting to evaluate the immunorereactivity between the produced anti-CD14 monoclonal antibody and monocytes resulted in a single specific band (Fig. 2). Then, reactivity of anti-CD14 MAb was analyzed using indirect flow cytometry analysis. As shown in Fig. 3, reactivity of this MAb with PBMCs isolated from healthy volunteers was 8 percent.
The production of monoclonal antibodies was first discovered by Cesar Milstein and Georges Köhler in 1975. Since then, a few CD14-specific monoclonal antibodies have been developed. For instance,a high affinity Anti-CD14 monoclonal antibody was produced and developed by Moonsom et al. They used CD14 expressing COS Cells as immunizing antigen and generated CD14 MAbs which could be utilized to enumerate monocytes of peripheral blood as well as using referent CD14 MAb [13]. In the present study, an immunization strategy based on synthetic peptide was employed for production of MAb against human CD14. In this regard, two 14-mer peptides, adapted from CD14 structure, were designed and synthesized. The use of synthetic peptides as immunogens is an attractive alternative where the complete protein is not available in adequate quantities for immunization. In addition, this method could be employed for production of antibodies to recognize specific regions of a polypeptide [9]. Synthetic peptides provide the opportunity to overcome the lack of protein antigen. But it should be noted that , factors such as the secondary and tertiary configuration, length, hydrophilicity and localization of the peptides in the native molecule, must be considered in generating efficient MAbs [14]. A major problem with utilizing peptides as antigen is their small sizes, which appropriate carriers could be a helpful solution [15]. KLH has been demonstrated to be an effective carrier protein for immunization with short peptides in the high-yield production of antibodies for research, biotechnology and therapeutic applications [16]. However, because of very high molecular weight of KLH, it is not possible to run the KLH conjugate on SDS-PAGE. Consequently, BSA was utilized to evaluate the conjugation efficacy. At the next step, mice were immunized with synthetic peptide – KLH conjugates to raise antibodies. The serum of the mouse 2, indicated high absorbance in reaction with BSA-peptide at 1/8000 dilution. Among several antiCD14 MAb producing hybridomas resulted from this mouse, “2E8” clone demonstarted high reactivity with immunogenic peptide in ELISA and considered for
L.A. Maleki et al. / Production of monoclonal antibody against CD14
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[7] [8] Fig. 3. Representative data for indirect flow cytometry of anti CD14 MAb. A: Isotype control and B: Anti CD14 MAb reaction with PBMCs isolated from healthy volunteers. (Colours are visible in the online version of the article; http://dx.doi.org/10.3233/ HAB-140272)
further characterization. The produced antibody was recognized as IgG2b isotype with kappa light chain. Immunoblot analysis showed a strong reactivity with CD14. The western blotting result was also in conformity with ELISA from the viewpoint of specificity. According to flow cytometry results, our generated MAb had an acceptable reactivity compared to commercial anti CD14 MAb. These findings suggest that, the produced antibody is highly specific and functional for use in applications such as western blotting and flow cytometry. Furthermore, the two synthetic peptides included in this study were highly efficient immunogens for development of monoclonal antibodies.
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Acknowledgements We would like to thank for Immunology Research Center (IRC) for kind assistance. This work was supported by a grant from Immunology Research Center (IRC).
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