Human Antibodies 23 (2014/2015) 7–12 DOI 10.3233/HAB-140277 IOS Press

Development and characterization of monoclonal antibodies against human IgA in Balb/c mice Fatemeh Ezzatifara,b,c , Jafar Majidia,b,c,∗ , Behzad Baradaranb,c, Leili Aghebati Malekib,c , Jalal Abdolalizadeha,b and Mehdi Yousefib,c a

Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran c Department of Immunology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran b

Abstract. OBJECTIVE: Immunoglobulin A (IgA) is the second immunoglobulin in human serum. Monoclonal antibodies have many potential uses in diagnosis, treatment and purification. The conjugated monoclonal antibodies against human IgA are utilized in diagnostic kits. METHODS: For production of monoclonal antibodies against human Immunoglobulin A, spleen cells of the immune mouse were fused with SP2/0 as myeloma cells and Poly Ethylene Glycol (PEG). Supernatant of hybridoma cells was screened to determine the antibody by ELISA. The appropriate clones were selected for limiting dilution (L.D). Ultimately, appropriate monoclone was injected intraperitoneally into the mouse that has been primed with Pristane. RESULTS: In current study, 175 clones were obtained of which 5 clones had absorbance values of about 3 were selected for limiting dilution. Between these clones, 3-D5monoclone with IgG1 subclass was selected as appropriate one and it was reproduced in FCS free RPMI 1640. For large scale production in invivo, the appropriate clone was implanted in the peritoneum of the Balb/c mouse and its titer was determined, which showed 1/100,000 dilution for ascitic fluid, having no cross reactivity with IgM & IgG. CONCLUSIONS: Monoclonal antibody was purified by chromatography, confirmed by SDS-PAGE and then conjugated with enzyme and used for diagnostic kits. Keywords: Production, monoclonal antibody, ELISA, limiting dilution, IgA

1. Introduction Monoclonal antibodies (mAbs) are extensively used in fundamental medical researches, diagnosis and treatment of the diseases like infectious diseases and cancers In diagnostic kits of the infectious diseases like Toxoplasmosis, Helicobacter pylori, Cytomegalovirus and etc, monoclonal antibodies against human IgA conjugated with enzymes, fluorochromes or radioactive labels plays an important role in the diagnostic kits [1–3]. Immunoglobulin (Ig) A is being the most ∗ Corresponding author: Jafar Majidi, Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. Tel.: +98 411 3364665; Fax: +98 411 3364665; E-mail: [email protected].

important antibody class at mucosal sites, IgA is the second common antibody in the circulation. In the human body, more immunoglobulin (Ig) A is produced per day (66 mg kg −1 day −1) than all other antibody isotypes [4]. Detection of serum IgA may be another practical alternative for the diagnosis of infections. The diagnosis of acute infection should be based upon the demonstration of a rise in antibody titres in sequential serum samples [2,3,5]. Additionally, pathogen specific IgM and IgA antibodies tend to persist long. IgG avidity has demonstrated its efficacy in diagnosis of acute Toxoplasma infections in several studies [6]. As a sign of initial infection, even seroconversion able to in some cases is stimulated by a sero-reversion following latent infection. Serological diagnosis of acute infection

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F. Ezzatifar et al. / Development and characterization of monoclonal antibodies against human IgA in Balb/c mice

has usually been made by detecting specific IgM antibodies or by the demonstration of a considerable increase in specific IgG antibody levels, or both [7]. On the other hand, because of the prevalence of high IgG of infectious agents antibody titers in normal subjects and because, in some people, specific IgM antibodies can stay for several months or even years subsequent the acute infection, the interpretation of serological test results is challenging when acute toxoplasmosis is suspected [8]. Several reports have emphasized the significance of the detection of infectious agents specific IgA antibodies for the diagnosis of acute human infection [9,10]. The aim of the current study was to produce and characterize a monoclonal antibody against human IgA as a tool for diagnostic and research applications.

the lymphocytes from the mouse spleen cells at a ratio of 1:5 (1 SP2/0 and 5 spleen cells). The cell mixture was pelleted at 1000 rpm and fused by adding 50% (w/v) pre-warmed (37◦ C) PEG 1450 by stirring over 1 min. Selective HATmedium was added to the pellet (2 × 106 cells/ml) and cells were seeded into a 96-well plate. Cell growth and colony formations were investigated daily. Colonies were appeared after 4–7 days. The presence of antibody against the immunized human IgA was determined by ELISA technique. Selected antibody producing cultures were cloned by limiting dilution process according to the conventional methods. Ultimately, appropriate monoclones possessing high absorbance were selected for large scale production and characterization of antibodies [12]. 2.3. Procedure of determining antibodies by ELISA

2. Materials and methods 2.1. Immunization protocol and screening of immunized mouse Four eight-week-old female Balb/c mice were obtained from Pasteur institute of Iran and immunized intraperitoneally (I.P) and subcutaneously (S.C) with immunoglobulin A at 4 times with three weeks intervals. 50 μg/50 μl of the immunoglobulin A was emulsified with identical volumes of Freund’s complete adjuvant (CFA) (Sigma-Germany). Following immunizations (booster) were used for the 3 times consecutively with Incomplete Freund’s adjuvant (IFA) (Sigma-Germany). 7 days after second booster, Blood samples were collected with eye bleeding and the serum titer was investigated using indirect ELISA. 4 days before a cell fusion, the final injection was carried out with 50 μg of immunoglobulin A without any adjuvant into the vein and peritoneum of immune mouse [11].

The indirect ELISA was used for the monitoring of hybridoma supernatant and screening mouse immune responses for determination of antibody against human IgA. The wells of ELISA plate (Nunc,Roskilde, Denmark) were coated with 0.5 μg/100 μl of immunoglobulin A per well and incubated at 37◦ C for 45 min. Then, the plate was washed 2 times with PBS (pH 7.3) containing Tween 20 (PBST 0.05%); non-specific binding sites were blocked and incubated with 2% BSA for 90 min at 37◦ C. After two times washing, 100 μl of hybridoma supernatants or mice sera were added to the wells and incubated 45 min at 37◦ C. Rabbit Anti-mouse IgG conjugated with HRP (100 μl, 1/4000 dilution) (Sigma, Germany) was added to each well and incubated for 45 min at 37◦ C. 100 μl of Tetramethylbenzidine (TMB) substrate solution (Sigma, Germany) was added into each well to develop the color reaction and the plate incubated in dark place at room temperature. After 20 min, the reaction was stopped by adding 100 μl stopping solution (0.16 M H2 SO4 ) and absorbance (OD) was measured at 450 nm by an ELISA reader (STAT FAX 303+, USA) [13].

2.2. Cell fusion with polyethylene glycol (PEG), selection of hybridoma and cloning

2.4. Classification of antibody

Mouse myeloma SP2/0 cell line was used as fusion partner. 7 days before the fusion, SP2/0 (NCBI 129, National Cell Bank of Iran, Pasteur Institute of Iran, Tehran) was cultured in RPMI 1640 media (Sigma, Germany) and 10% FBS until reaching to > 70% confluency. The feeder-layer cells were prepared from peritoneal cavity of unimmunized Balb/c mouse two days before fusion. SP2/0 cells were combined with

The class and subclass of the mAbs were identified by using ELISA mouse mAb isotyping Kit (Thermo Scientific, USA). First, the supernatant was diluted (1/10) by Tris Buffer Saline (TBS) and 50 μl of diluted antibody was added to each well of the 8-well strip. At the subsequently step, 50 μl of the anti-mouse IgG1, IgG2a, IgG2b, IgG3, IgA and IgM, kappa, lambda light chains HRP conjugated (secondary antibody) was

F. Ezzatifar et al. / Development and characterization of monoclonal antibodies against human IgA in Balb/c mice

added to each well and incubated for one hour at room temperature. Subsequently, isotypes of mAbs in culture supernatants were determined according to the ELISA technique mentioned above. 2.5. Mass production Four female Balb/c mice (4–6 weeks old) were obtained from Pasteur institute of Iran. 0.5 ml Pristane (2, 6, 10, 14 tetra methyl pentadecane, Sigma, UK) was injected intraperitoneally into each mouse. 10 days after priming with Pristane, high cell densities of desired mono clone (3–4 × 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 [14]. 2.6. Purification of mAbs by ion exchange chromatography The ascetic fluids were diluted two times with PBS and fractionated with 40% saturated ammonium sulfate. Ion exchange chromatography was carried out using DEAE-cellulose (DE-52, Whatman, England) equilibrated with 0.01 M phosphate buffer PH 8.1. IgG were frequently eluted as breakthrough fractions with the equilibration buffer. IgG was eluted with 0.02M– 0.05 M phosphate buffer at a flow rate of 4 ml/min and collected in 8-ml fractions [15]. 2.7. Cross-reactivity studies In order to examine the ascitic fluid for cross reactivity with IgM, IgG as nonspecific and IgA as specific antigens, the micro titer plates were coated with each class of the purified immunoglobulin (0.5 μg/ml in coating buffer, 100 μl/well) by incubating for 1 hour at 37◦ C. The plates were blocked with 2% BSA as described above. The rest of the assay was preceded as described for the ELISA procedure. 100 μl of mAb (1/100,000 dilution) was added to each well as primary antibody. The ELISA assay was continued as described above. 2.8. Confirmation of the mAb purity by SDS-PAGE SDS-PAGE analysis was carried out for the purity assessment of the eluted protein. The collected protein fractions were subjected to electrophoresis in 12.5% SDS-PAGE on non-reducing condition and reducing form. Protein bands were imagined by Coomassie blue staining technique.


2.9. Immunoblotting for analysis of specificity of mAb Western blotting technique was used for confirming the result of SDS-PAGE and to observe pattern of specificity and cross-reactivity of anti-IgA monoclonal antibody. IgA, IgG and IgM was subjected to SDS-PAGE and then immunoblotting assay. The nitrocellulose membrane and several thicknesses of Whatman chromatography papers were soaked for 20 min in the transfer buffer (25 mM Tris, 192 mM glycine, 20% V/V methanol, pH 8.3). The wet nitrocellulose membrane was overlaid on the wet Whatman sheets by taking precaution to avoid bubbles. Subsequently, the gel of SDS-PAGE was placed on the wet nitrocellulose membrane and then several wet Whatman papers were placed on it. Transfer of the proteins from gel to nitrocellulose membrane was done in 100V for 3 hours. Subsequently, non-specific sites were blocked with 2% BSA solution. After three times of washing, the membrane was cut into strips and incubated for 2 hours at 37◦ C with the supernatants of appropriate clones. Once more, after five times of washing, the strips were incubated for 2 hours at 37◦ C with Rabbit Anti- Mouse IgA conjugate (1/2000 dilution). The strips were washed and detected by ECL (Amersham Phamacia Biotech Inc, USA) hyperfilm after exposure for 5 min. 2.10. Conjugation with horse radish peroxidase (HRP) Four mg of HRP was dissolved in 1 ml of distillated water. Subsequently 0.2 ml freshly prepared sodium Periodate solution (0.1M) was added to the enzymatic solution and incubated on shaker for 20 minutes at room temperature. The solution was dialyzed against acetate buffer (pH 4.4), overnight at 4◦ C. 8 mg of the purified monoclonal antibody was dissolved in 1 ml sodium carbonate (10 mM, pH 9.5). The pH of the dialyzed enzyme was reached to 9 and immediately the solution containing monoclonal antibody was added to it and shaked for 2 hours at room temperature. Then 0.1 ml of the freshly prepared sodium brohydrate was added and incubated for 30 minutes at room temperature. The final solution was precipitated with ammonium sulfate and then was dialyzed against PBS buffer [15].

3. Result Among the four immunized Balb/c mice against human IgA, the serum of the immune mouse at


F. Ezzatifar et al. / Development and characterization of monoclonal antibodies against human IgA in Balb/c mice Table 1 The presence of antibody against Human IgA in serum of the mice. The serum of the immune mouse and the non-immune mouse negative control were diluted 1/64000 Mouse Optical density

Negative control 0.09

Mouse 1 1.2

Mouse 2 0.64

Mouse 3 0.11

Mouse 4 0.83

Table 2 Determination of the isotype of mAb by ELISA. Class and subclasses of monoclonal antibodies of 3-D5 monoclone Lambda 0.124

kappa 1.724

IgM 0.119

IgA 0.114

IgG2b 0.101

Table 3 Examining the cross-reactivity of the mouse mAbs with indirect ELISA. Mouse mAbswith 1/100,000 dilution IgM 0.33

IgG 0.21

IgA 1.81

IgG3 0.137

IgG2a 0.139

IgG1 1.053 M


Class Clone 3-D5 2



Class Optical density 50KD



25KD A


4 150KD



Fig. 1. 3-D5 monoclone with higher OD as desired monoclone. Monoclone in the growing form (A), Monoclone in the highly proliferated form (B). (Colours are visible in the online version of the article; http://dx.doi.org/10.3233/HAB-140277)

1/64000 dilution, showed highest absorbance in reaction with human IgA using ELISA method (Table 1). Consequently, the immune mouse was selected for the fusion. The final result of the successful fusion of the immune mouse spleen cells with myeloma SP2/0 cells were about 174 wells containing positive clones with high absorbance in reaction with IgA. Among them, 5 wells had absorbance more than 3 and among them, 1 well had absorbance approximately 3 which were selected for cloning by Limiting Dilution method. The final result of cloning with L.D was approximately 50 wells containing monoclones. Among them, 3 monoclones had absorbance approximately 1.5–2, out of which, one suitable monoclone (Fig. 1) with high mAb concentration was selected for mass production and ascitic fluid antibodies. Supernatants of 1 appropriate clones and ascitic fluid were considered for cross reactivity with IgM & IgG by ELISA. The results showed that the mAb secreted by the 3D5 monoclone and its ascitic fluid display no cross reactivity with IgM &


Fig. 2. SDS-PAGE analysis non-reducing SDS-PAGE (A) and reducing SDS-PAGE (B) of produced monoclonal antibody. In reduced form (2 and 3), two bands were seen in 50 and 25 kDa but in non-reducing SDS-PAGE condition, only one band was seen in about 150 kDa. The SDS-PAGE gel lane 1 contains a band at 150 kDa is in non-reducing condition, in lane 2, 3 contain two band at 25 and 50 kDa in reducing condition and lane 4 produced antibody before purification. (Colours are visible in the online version of the article; http://dx.doi.org/10.3233/HAB-140277)

IgG. The mentioned clone was expanded in the culture medium in large scale and its supernatant was precipitated with 40% Ammonium Sulfate for purification. In addition, ascitic fluid was precipitated with 40% Ammonium Sulfate for purification with ion exchange chromatography and conjugation with HRP. The result of purification confirmed with SDS-PAGE (Fig. 2) and Immunoblotting. The result of conjugation with HRP showed that 1/10,000 dilution of conjugate had absorbance approximately 1.3 with human IgA but no cross reactivity with IgM & IgG. The immunoblotting of the monoclone, showed sharp band in IgA position and did not show any band in IgM & IgG positions (Fig. 3). The supernatant of the appropriate clone and its ascitic fluid were investigated for class and subclass by mouse isotyping kit (ZYMED).Subclass of monoclonal antibody was IgG1 and its light chain was kappa (Table 2). The titer of ascitic fluid was assessed by ELISA and the results showed that its 1/100,000 dilu-

F. Ezzatifar et al. / Development and characterization of monoclonal antibodies against human IgA in Balb/c mice




160 kDa

Fig. 3. Immunoblotting of monoclonal antibodies from IgM, IgG, IgA. Only one specific band was seen in 160 kDa in IgA. (Colours are visible in the online version of the article; http://dx.doi.org/ 10.3233/HAB-140277)

tion has high absorbance with IgA (above 1) but has no absorbance with IgM & IgG (Table 3). So this titer demonstrates higher concentration of the produced antibody in mouse peritoneum compared with in-vitro one.

4. Discussion The current era of monoclonal antibodies, (mAb) was initiated with the hybridoma technology developed by Kohler and Milstein in 1975. This revolutionary technique presented that any chosen specificity could be fused with a myeloma cell line that leads to enormous production of monoclonal antibodies carrying that specificity. Currently, monoclonal antibodies play important roles in diagnosis applications, disease monitoring, identifying prognostic markers and therapy. Meanwhile each antibody is very specific for a particular antigen; this characteristic feature of antibodies has led to their routine use in diagnostic kits [18]. In infectious diseases, IgA is produced against pathogen and for its detection, we necessitate monoclonal anti–human IgA conjugate. Therefore, the production of monoclonal antibody can have important applications in the diagnostic kits of infectious diseases. The most immune mouse was selected for the fusion and the final injection from vein play a significant role in the localization of B cells in the spleen. So immune mouse serum had absorbance above 1.68 with 1/64000 dilution. The monoclones with high absorbance, which did not show cross reactivity with IgM & IgG, were considered as appropriate clones. A monoclone (3-D5) was selected for the large-scale production of antibody. The result of SDS-PAGE was confirmed by immunoblotting. Appearance of one band


in IgA position and no band in IgM & IgG positions showed that 3-D5 monoclone had no cross reactivity with IgM & IgG. The appropriate clone among them was 3-D5 that was selected for high production in culture media and ascitic fluid. In large-scale production, reducing the expenses as well as reducing the supernatant contamination to FCS proteins are important factors, consequently the percentage of FCS was reduced from 20% to 1% in 5 times of passage [19]. In production of ascitic fluid, because of well priming with Pristane and the presence of appropriate hybridoma cells, the produced ascitic fluid with 1/100000 dilution had absorbance above 1 with IgA. Therefore, monoclonal antibodies of 3-D5 clone produced in-vitro and in-vivo was collected and after precipitation with Ammonium Sulfate and purification with ion exchange chromatography, they were conjugated with HRP [20,21]. The mouse anti-human IgA conjugate with 1/10,000 dilution displayed high absorbance with IgA and did not show any cross reactivity with IgM & IgG. Therefore, it can be useful in diagnostic kits of infectious diseases. MAbs may demonstrate to be important tools for quantitative measurement of human IgA subclasses in biological samples. In a study, Abramson et al. produced hybridomas against NALM-6-M1 (acute lymphoblastic leukemia cell line) by fusing spleen cells from hyperimmunized mice with NS-1 mouse myeloma cell. They concluded that monoclonal antibody BA-1 was valuable in the study of early stages of human B lymphocyte development [23]. In the other study, Baradaran et al. produced a monoclonal antibody against human epidermal growth factor receptor (EGFR) which could be used in diagnosis and treatment of tumors with membranous EGFR. A431 cells were used for immunization that possess large amount of EGFR [24]. In this study, we report the development and characterization of monoclonal antibodies directed against human IgA. For immunization of mice, human IgA was administered each time. Upon such procedure, we observed high immunologic response in the immunized mouse, whose serum resulted in absorbance above 1 with 1/64000 dilution and the most immune mouse was chosen for the fusion. Class and subclass of monoclonal antibodies in supernatant of the desired clones and mice ascetic fluid was IgG1 class with Kappa (κ) light chain. The production of monoclonal antibody in the ascitic fluid is commercially cost effective for large-scale production in comparison with expensive and time-consuming culture methods. So, the monoclone with high ab-


F. Ezzatifar et al. / Development and characterization of monoclonal antibodies against human IgA in Balb/c mice

sorbance was selected for the large scale production of antibody. In this study, we achieved 5 mg monoclonal antibody which purified from ascites by ion-exchange chromatography. In accordance with conformity ELISA results, purified mAb could recognize human IgA. The result of ELISA was confirmed by immunoblotting. Appearance of one band in IgA position indicated that monoclones had reactivity with IgA. In conclusion, anti-IgA monoclonal antibody could apply to diagnosis infectious disease. Upon our findings, it can be proposed that this particular approach for production of an anti-human IgA antibody is feasible and cost-effective. Further, this study clearly indicates that the produced antibodies can be utilized in research, diagnosis as well as clinic if produced in chimeric form.

Acknowledgement This work was financially supported by the Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran, and the manuscript was written based on a dataset of Master thesis, registered in Tabriz University of Medical Sciences.

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Immunoglobulin A (IgA) is the second immunoglobulin in human serum. Monoclonal antibodies have many potential uses in diagnosis, treatment and purific...
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