Production and characterization of anti-human IgG F(ab’) 2 antibody fragment

Abstract

In present study an optimized protocol for the separation of antibodies into antigen-binding fragments F(ab’)2 using pepsin digestion was investigated. The production of these fragments is a consequential step in the development of medical research, treatment and diagnosis. For production of polyclonal antibody rabbit received antigen in four steps. The rabbit serum at 1/128000 dilution showed high absorbance in reaction with human IgG at the designed ELISA method. Rabbit IgG was purified by Ion-Exchange Chromatography (IEC) method. Purity was assessed by SDS-PAGE method. In non-reduced condition only one band was seen in about 150 kDa MW position and in reduced form, two bands were seen in 50 and 25 kDa MW positions. Rabbit IgG was digested by pepsin enzyme. The antibody fragments solution was applied to Gel filtration column to isolate the F(ab’)2. Non-reduced SDS-PAGE for determining the purity of F(ab’)2 fragment resulted in one band in 100 kDa corresponds to F(ab’)2 fragment and a band in 150 kDa MW position corresponds to undigested IgG antibodies. The activities of FITC conjugated F(ab’)2 fragment and commercial ones were compared using flowcytometry method. The activity results implied that the FITC conjugated- anti human F(ab’)2 fragment worked as efficiently as the commercial one.

Keywords

Polyclonal antibody F(ab’)2 fragment FITC conjugation immunization enzyme digestion

1. Introduction

Because of their extreme importance to human health, it is necessary study more about the structure and function of antibodies than practically any other molecule [1]. More than 100 years of investigation into the structure and function of immunoglobulin has only served to emphasize the complex nature and several applications of this protein [2]. Some published literature has been described the application of enzymes to fragment IgG molecules. Pepsin digests IgG into an Fc fragment and a F(ab’)2 (single dimeric fragment) that can cross-link, as well as bind, antigens. The Fab fragment contains1 complete L chain in its entirety and the V and CH1 portion of 1 H chain [2, 3]. F(ab’)2 is frequently preferred to intact IgG in some medical applications due to lower immunogenicity and faster onset of action [4]. The Ig purification is the first step for obtaining labeled or conjugated antibodies. Antibody fragments are often used in a labeled form [5, 6]. IgG F(ab’)2 fragment have many therapeutic and diagnostic applications such as in diagnostic kits of rheumatoid arthritis and tumors. These fragments can be used for treatment of envenoming and as passive immunotherapy for influenza and radio immunotherapeutic agent for leukemia [7, 8, 9, 10]. In present study an optimized protocol for the efficient separation of antibodies into antigen-binding F(ab’)2 fragments using pepsin digestion was investigated. This product should comprise only highly pure immunoglobulin fragments since Fc and other contaminating fragments may lead to side effects [3]. The production of these pure fragments is a consequential step in the development of medical research, treatment and diagnosis towards self-sufficiency of the country.

2. Materials and methods

2.1 Immunization protocol and screening of immunized rabbit

Antibody production was performed on a New Zealand white rabbit which was seven-month–old at the first injection. These procedures were done according to the Animal Laboratory guidelines and approved by the Regional Medical Sciences Research Ethics Committee of Tabriz University of Medical Sciences. Rabbit received antigen in four steps. The first injection was done by 300 $\mu$ g/300 $\mu$ l of human IgG (Sigma, Deisenhofen, Germany) with the same volume of freund’s complete adjuant (Sigma, Deisenhofen, Germany). Immunization followed by 2 boosters. Inoculations of antigen in a freund’s incomplete adjuant (Sigma, Deisenhofen, Germany) emulsion was administered intramuscularly on days 22 and 36. Final immunization was done without any adjuant on day 60. After each immunization, rabbit was monitored daily for side effects.

Exsanguination was performed by heart puncture under general anesthesia. Produced antibody titer was investigated by ELISA test. Rabbit serum was collected and precipitated at the final concentration of 50% saturated ammonium sulfate. It is important to add the ammonium sulphate slowly on stirrer. Dialysis was done and the protein concentration determined by UV spectrophotometer (pharmacia, Uppsala, Sweden) at 280 nm.

2.2 Rabbit IgG purification

Rabbit IgG was purified by Ion-Exchange Chromatography (IEC) method on a DEAE-sepharose column (pharmacia, Uppsala, Sweden). The column was equilibrated by 40 mM Tris-phosphate buffer at pH 8.1. IgG was eluted by 75 mM Tris-HCl buffer at pH 8.1. Purity was assessed by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and purified antibodies were visualized in the gel by Coomassie Brilliant Blue G 250 staining [11, 12].

2.3 IgG digestion

Rabbit IgG was digested by pepsin enzyme. The optimum concentration of pepsin and antibody was used to give a ratio of 30 mg IgG per 0.5 mg of enzyme.

For the large-scale production of F(ab’)2 the optimum pH for pepsin digestion was chosen (pH: 3.2). At the end 200 $\mu$ l Na2HPO4 (0.5 M) was added to raise the pH to $\sim$ 8.0 to stop the reaction. Prepared solution was dialyzed overnight against PBS (pH, 7.2) at 4 ${}^{\circ}$ C.

2.4 F(ab’)2 purification

The antibody fragments solution was applied to Gel filtration with G100 matrix column to isolate the F(ab’)2 from the other digestion products. Samples were diluted with the appropriate running buffer and centrifuged before loading, run at a flow rate of 0.5 ml/min over 1 h. The eluted protein was measured by absorbance at 280 nm. Purity of the eluted antibody fragments was demonstrated by reducing SDS-PAGE method. The process was carried out according to laemmli method using 12% polyacrylamide with SDS (Merck, Darmstadt, Germany). Samples were boiled with SDS (2%) (Merck, Darmstadt, Germany) for 10 min and 0.2 $\mu$ g of each sample was loaded for each sample well on to an electrophoresis gel in a vertical chamber.

2.5 FITC Conjugation of F(ab’)2

The antibody fraction was dialyzed against 500 Mm Carbonate (pH, 9.2) as reaction buffer in 24 hours. One mg fluorescein isothiocyanate (FITC) (Sigma, Deisenhofen, Germany) was dissolved in 1cc anhydrous DMSO and 8 $\mu$ l of obtained FITC solution was added to 0.1 mg antibody. The tubes were wrapping in foil and incubated at room temperature for 1 hour. Unreacted FITC was removed and exchanged the antibody into Storage Buffer (150 mM NaCl, 10 mM Tris, 0.1% NaH ${}_{3}$ , pH $=$ 8.2) [13].

2.6 Flow cytometric analysis

Human IgG were added to different tubes and incubated with FITC-conjugated and commercial (Dako) anti-human F (ab’)2 antibodies separately for 1 h. After thorough washes with PBS, the reactivity of antibodies was analyzed with a FACS Calibur flowcytometer (Becton- Dickinson, Mountain View, CA). The data were processed using Cell Quest 3.1 software (Becton-Dickinson) [13].

3. Results

The rabbit serum at 1/128000 dilution showed high absorbance in reaction with injected antigen (human IgG) at the designed ELISA method.

Figure 1.

Purification of rabbit polyclonal IgG by ion-exchange chromatography. a: Isolation of IgG by 75 mM Tris-HCl buffer at pH 8.1.

Figure 2.

SDS page analysis of rabbit polyclonal antibody A: non-reduced condition; M; marker 1; washed materials, 2,3; purified antibody B: reduced condition; 1; purified antibody M; marker.

Figure 1 shows results of the purification of polyclonal antibody from rabbit serum. 30 mg protein was loaded on the Ion exchange chromatography column. 12 mg rabbit polyclonal anti-human IgG antibody reached in purification by Ion exchange chromatography. Results of SDS-PAGE method for analysis of purified rabbit anti-human IgG are shown in Fig. 2. In non-reduced condition (Fig. 2A) only one band was seen in about 150 kDa MW position and in reduced form (Fig. 2B), two bands were seen in 50 & 25 kDa MW positions. These results demonstrate a purity level of over 95%. 50 mg rabbit IgG was digested with enzyme. Gel filtration separation of digested materials yielded 32 mg F(ab’)2 fragment (Fig. 3). Reduced SDS-PAGE for determining the purity of F(ab’)2 fragment resulted in one band in 100 kDa corresponds to F(ab’)2 fragment and a band in 150 kDa MW position corresponds to undigested whole IgG antibodies (Fig. 4).

Figure 3.

The step wise elution of digestion products by Gel filtration column. a: separation of hole rabbit IgG antibody; b: elution of F(ab’)2 fragment; c: elution of P(Fc) fragment.

Figure 4.

SDS page analysis for purity determination of purified rabbit F (ab’)2 fragment. Lines 1, 2: purified F(ab’)2 M: Marker.

The pattern of reactivity of FITC-conjugated antibodies in comparison to the commercial one revealed that human IgG antigen reacted 1.1 by FITC-conjugated F(ab’)2 antibodies and 3.32 by commercial one using flowcytometry analysis (Fig. 5).

Figure 5.

Flowcytometry results A: FITC-conjugated anti-human IgG F(ab’)2 antibody fragment; B: Commercial (FITC-conjugated anti-human IgG F(ab’)2 antibody fragment).

4. Discussion

Until now, a large series of F(ab’)2 antibody fragments have been studied and developed [14]. In published literature several methods for production of F(ab’)2 fragments have been reported [15]. In 2002 R. Jones purified antibody F(ab’)2 fragments in high yield from ovine serum [3]. In 2010 L.wang discussed a continuous two-stage cascade ultrafiltration system for production and purification of F(ab’)2 fragment [4]. Yeast and bacterial cells are good hosts for the production of F(ab’)2 and various antibody fragments. Recently, there have been reports of substantial progress in genetic engineering and biotechnology, results in development of potential hosts [16].

In this study, we report the development and characterization of a high affinity polyclonal anti-human F(ab’)2 antibody fragment using immunization and conjugation strategies. For this, rabbit was immunized with human IgG as antigen. Rabbit was used as host animal for polyclonal antibody production, because it has a convenient size, is easy to handle and bleed and produce adequate volumes of high-titer, high-affinity antiserum [17].

Produced rabbit polyclonal antibody was separated by IEC method. Due to high binding capacity and cost benefits, IEC was well established in our laboratory for rabbit polyclonal antibody purification. By changing the mobile phase, the polyclonal antibody was eluted. According to the SDS-PAGE results rabbit IgG obtained with purity near 100%. Purification by this method yielded about 12 mg of rabbit polyclonal antibody (loaded amount: 30 mg). Purity and amount of Product and output of this study was higher compared to Ezzatifar, Eivazi and Maleki studies [12, 17, 18, 19]. Due to the high quality of injected antigen (human IgG) and used adjuvant, different immunization routes and boosting protocol, immunization was very effective and produced polyclonal Ab had high titer. It showed excellent immunoreactivity with immunizing human IgG in ELISA assay as well as suggesting its functionality. Obtained polyclonal antibody titer (titer: 1/128000) could be beneficial for many types of detection methods and shows the high quality of the product. z, a noticeable amount of anti-IgG antibody could be obtained, which would meet many research and educational medical requirements [18]. Produced polyclonal antibody can be used in designing of ELISA kits. Guidelines on the production of pAbs have been published by various organizations, for example the Scientists Centre for Animal Welfare [17]. Proteolytic fragmentation of polyclonal antibody using pepsin took place at pH 3.2. The combination of enzyme concentration, incubation time and pH that provided the most efficient digestion of the antibody into F(ab’)2 was used in present study. To purify the F(ab’)2 fragment, gel filtration chromatography (GFC) was used. Digestion by pepsin generates F(ab’)2 fragments that encompasses the two Fab_ regions linked by the hinge region and the numerous small peptides of the Fc region. Following SDS-PAGE electrophoresis a peak at 100 kDa was observed on the gel that represented the F(ab’)2 fragment and a single band at 150 kDa which corresponded to the full-length antibody. The highest yield was obtained (95%) without any important degradation products. The anti-human F(ab’)2 recognized human IgG appropriately, so it seems that purified F(ab’)2 fragment can be applied for human IgG detection. The activities of FITC conjugated anti-human F(ab’)2 fragment and commercial ones were compared. The activity results implied that the FITC conjugated- anti human F(ab’)2 fragment worked as efficiently as the commercial one.

Purified antibody fragments can be used as immunogen and for evaluation of polyclonal immune sera [5]. It is applicable for conjugation with enzymes and radiolabels. It can be used as ligands for the development of immunoaffinity purification columns [20]. This product is highly specific and functional in biomedical research and diagnosis applications such as immunoassay tests for example: ELISA, western blot, immunofluorescence, immunohistochemistry, immunoelectrophoresis, western blot and WBC X match and flowcytometric tests. F(ab’)2 fragments are recommended for staining of cells or tissues which contain Fc receptors. The spatial expression of an antigen relative to an individual cell can be analyzed with these antibody fragments [21, 22, 23, 24, 25]. F(ab’)2 fragments are used widely throughout the world as antitoxins and anti-venoms for the treatment of envenoming for example snake envenoming [3].

5. Conclusion

In the current study, we aimed to produce cost-effective F(ab’)2 antibody fragments against human IgG with high affinity to be used as diagnostic agents.

Footnotes

Acknowledgments

This work was financially supported by the Immunology Research Center, Tabriz University of Medical Sciences. The manuscript was written based on a dataset of a Master’s thesis registered in Tabriz University of Medical Sciences.

Conflict of interest

The authors report no conflicts of interest in this work.

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