Comparative efficiency of ammonium sulfate alone and ammonium sulfate-octanoic acid combined methods in the purification potential of antibodies

Comparative efficiency ammonium alone and acid combined methods the Abstract The use of antibodies in the biopharmaceuticals has been increased from therapeutic to prevention through vaccination. However, the purification processes adopted by the industries make them expensive to use. Ammonium sulfate precipitation is an old and easy way to purify the antibodies from the serum and egg yolks. The purity is somewhat compromised with ammonium sulfate precipitation, therefore, a combination of chromatographic techniques is adopted. Presently, ammonium sulfate alone and in a combination with Octanoic acid was used for the purification of antibodies from caprine serum and the egg yolk. The results of purified antibodies in terms of their yield and purity were compared through statistical analysis. The overall study results indicated a better yield and purity of antibodies from the caprine serum through a combined treatment of ammonium sulfate with Octanoic acid, whereas, ammonium sulfate alone gave a better yield but less pure antibodies from the egg yolk. Ammonium sulfate-Octanoic acid combined precipitation of caprine serum yielded 6.67 mg/mL while sole ammonium sulfate yielded 4.49 mg/mL purified antibodies. In case of IgY Ammonium sulfate-Octanoic acid combined precipitation of yielded only 8.90 mg/mL while sole ammonium sulfate yielded 13.80 mg/mL purified antibodies. A combination of ammonium sulfate and Octanoic acid is a good choice for high yield and purity of antibodies from serum. The purity of antibodies from the egg yolk was enhanced through ammonium sulfate in combination with Octanoic acid but the overall yield was poor.


Introduction
Antibodies are the immunoglobulins secreted by the immune cells in response to invading pathogens. The specific function of antibodies is to opsonize the pathogens to facilitate their engulfment by the phagocytic cells of the immune system [1]. Presently, the demand for antibodies has been increased because of their biopharmaceutical uses and applications in certain therapeutic strategies [2]. The applications of immunoglobulin G (IgG) in the field of biomedical science are increasing for therapeutic and prevention through drug and vaccine preparations [3]. The commercially available IgG is purified through chromatographic techniques, although it increases the purity of the antibodies but at the same time makes them more expensive and limit their use [4]. Another important immunoglobulin employed in the biomedical field is egg yolk immunoglobulin Y (IgY) from the hens [5]. The IgY has more advantages over the mammalian IgG because of its phylogenetic distance. The IgY does not react with rheumatoid factor, fragment crystallizable (Fc) receptors of the bacteria and mammalian complement system [6]. IgY is able to induce more strong immune response compared to IgG as IgY can recognize more epitopes of highly conserved mammalian proteins. The IgY are also employed in the treatment and prevention of various animal disease as in mastitis, bovine viral diarrhea [7]. Recently, a new concept of IgY based vaccines preparations has been introduced because of their potential therapeutics. The limited use of IgY is attributed to the time consuming and difficult practices of purifying antibodies. Purification of antibodies from the serum and egg yolk is carried out through various procedure including chromatographic procedures, soluble phase water dilutions, filtrations and temperature and pH changes [8]. Ammonium sulfate precipitation is an efficacious and easy way to purify antibodies. Antibody purification through ammonium sulfate precipitation involves a high concentration of charged ammonium sulfate ion that competes with the protein particles to bind the water molecules [9]. The water molecules from the proteins are removed by the ammonium sulfate salt resulting precipitation of the antibodies. A variable concentration from 40% to 50% of ammonium sulfate is used for this precipitation. It is not a single step, however, the precipitation of protein through ammonium sulfate may yield impure antibodies. Further steps are combined with ammonium sulfate precipitation for complete purification and high yield [10]. Caprylic acid a short chain fatty acid that may precipitate the serum proteins other than IgG. This purification process gives a high yield of proteins, the drawback of caprylic acid is impurities that can be reduced through combined methods [11]. The present study was performed to evaluate the potential of ammonium sulfate and octanoic acid precipitation. Ammonium sulfate alone and in combination with octanoic acid was employed for the precipitation of antibodies from the caprine serum and egg yolk.

Materials and methods
Ammonium sulfate precipitation method has been used since long for the precipitation of antibodies. Presently, a comparative study was performed for antibody separation and purifications form the serum and egg yolks. Serum separation from the goats A 10 mL of blood was aseptically collected from the teddy goat. The blood was centrifuged at 10,000 rpm for 5 min to obtain fresh caprine serum. The serum was stored at -20 o C for further studies. Separation of serum antibodies through ammonium sulfate precipitation Acetate buffer was prepared using Sodium acetate and Glacial acetic acid (SIGMA-ALDRICH, Acetic acid glacial, ≥ 99.85%) and the pH was adjusted at 4.5. The serum was diluted with an acetate buffer at 1: 3 ratios. The serum was added with 0.24 g/mL of ammonium sulfate and incubated for half an hour at 25 o C with continuous stirring. Then the samples were centrifuged at 10,000 Xg under 4 o C for 12 min and the supernatant was discarded. The pellets were added with a small volume of 2 M ammonium sulfate solution and re-incubated for half an hour at 25 o C following the centrifugation at 10,000 Xg under 4 o C for 12 mins. Lastly, the supernatant was discarded and the pellet was re-suspended in a small volume of phosphate buffer saline (PBS) [12]. The serum was dialyzed against 70 volumes of PBS for a period of 48hrs, the completion of dialyzation was confirmed using 0.1 M silver nitrate (Silver nitrate crystal. extra pure, Merck). The dialyzed serum was placed in a water bath at 60 o C for 30 mins and then subjected to final centrifugation at 2800 Xg for 20 mins. The pellet was dissolved in 0.5 mL of PBS and proteins were quantified through Nanodrop (Thermo Scientific, NanoDrop 8000) quantification technique.

Separation of serum antibodies through ammonium sulfate-octanoic acid precipitation
The serum was diluted through the previously described method and a 25 µL/mL concentration of Octanoic acid (OA) was dropwise added to the diluted serum with continuous stirring for a period of 30 mins. After then the serum was centrifuged for 20 mins at 11200 Xg and the supernatant was added with one part of PBS. The pH was adjusted to 7.4 and serum was cold to 4 o C, in the very next step 1mL of 40% ammonium sulfate (SIGMA-ALDRICH, ≥ 99.0%) was added dropwise to the serum. The fractionated serum proceeded to centrifugation at 2800 Xg for a period of 20min and supernatant was discarded. The pellet was re-dissolved in a small volume of PBS and dialyzed with 70 volumes of PBS for 48hrs.

Collection of egg yolks
Fresh table eggs from layer (Novogen white) were collected. The eggs were cleaned with a cotton swab dipped in 70% ethanol solution. The egg yolks were aseptically separated from the egg whites and placed in sterilized falcon tubes for further research studies. The egg yolk was separated from the egg white and washed twice with distilled water at 4 o C to remove After proper washing the yolk was separated from the membrane and proceeded for further studies. Pre-treatment of egg yolk and separation of antibodies A 2mL of egg yolk was diluted 9 folds with distilled water at 4 o C and pH was adjusted to 5.0 with the addition of IN HCl. The pH was readjusted at 4.0 after the addition of 0.01 % charcoal. The diluted egg yolks were subjected to overnight freezing at -20 o C. The next day, the diluted sample was thawed at 4 o C and centrifuged for 10 mins at 8000 Xg under 4 o C. The supernatant was used for antibody separation [13]. The egg yolk antibodies were separated through both of the previously described methods for serum antibody separation. Purity Assessment of the antibodies The purity of caprine IgG and egg yolk IgY was assessed through Sodium Dodecyl Sulphate-Poly Acrylamide Gel Electrophoresis (SDS-PAGE). A discontinuous buffer system was applied for the experiment [14].

Results Precipitation of caprine serum antibodies (IgG)
The serum containing the highest percentage of antibody titer proceeded to combined Octanoic acid-ammonium sulfate and sole ammonium sulfate precipitation separately. The initial treatment resulted in precipitation of the lipids and non-IgG proteins within half an hour of incubation. The centrifugation of the Eppendorf tubes resulted in pellet formation as shown in (Figure 1a). The supernatant was collected, the secondary treatment resulted in the formation of a clear white pellet of IgG proteins as shown in (Figure 1b).

Figure 1(a). Eppendorf tube showing dissolved lipid contents of goat serum pelleted after
Octanoic acid treatment Figure 1(b). The pellets of proteins sediment after ammonium sulfate precipitation step Quantification of caprine serum antibodies (IgG) Five samples were measured for total protein contents of (IgG) through Nanodrop spectrophotometer, (IgG) protein concentration was above 6 mg/mL as shown in (Figure 2). A maximum IgG protein concentration 7.42 mg/mL while a minimum of 6.04 mg/mL concentration was detected in the selected samples. The pooled protein concentration of the IgG contents in the Ammonium sulfate-Octanoic acid (AS-OA) precipitated samples were recorded to 6.67 mg/mL. The comparative IgG contents in the ammonium sulfate precipitated caprine serum was very low. The selected five samples showed a maximum of 5.01 mg/mL protein contents through Nanodrop quantification. Most of the IgG contents in AS precipitated serum were below 5 mg/mL as presented in (Figure 2). Pooled protein contents of 4.49 mg/mL were recorded from selected serum samples. Overall IgG contents of serum proteins precipitated with Ammonium sulfate-Octanoic acid combined treatment was higher than sole ammonium sulfate precipitation.

Figure 2. Comparative Caprine Serum Protein (IgG) Concentrations obtained through sole Ammonium sulfate and Ammonium sulfate-Octanoic acid precipitation a)
b)

The purity of IgG antibodies
The caprine immunoglobulin G was assessed through SDS-PAGE, the bands of 150 kDa in (Figure 3) are indicating the presence of IgG in the serum. Band density of ammonium sulfate-octanoic acid combined treatment is indicating more IgG contents in the lane AS-OA while the lane AS is indicating fewer IgG contents after precipitation with ammonium sulfate alone.

Precipitation of Egg yolk antibodies (IgY)
The charcoal treated egg yolk upon centrifugation formed a pellet at the bottom of the Eppendorf tube. In (Figure 4a) depicts precipitation of lipids and non-IgY proteins present in the egg yolk after primary treatment of diluted egg yolk with ammonium sulfate that resulted in yellow pellet formation upon centrifugation. More lipid contents were observed in primary treatment. The secondary treatment with 2M ammonium sulfate resulted in precipitation of IgY proteins that resulted in the formation of the clear white pellet as in (Figure 4b)). The supernatant was discarded and the pallets were dialyzed overnight against PBS.

Quantification analysis of egg yolk antibodies (IgY)
The IgY protein concentrations obtained from the sole ammonium sulfate precipitated egg yolks were more than 13 mg/mL in most of the samples processed. From the selected five samples a maximum of 14.14 mg/mL protein concentration was obtained while the minimum protein concentration obtained was 13.04 mg/mL as represented in (Figure 5). The pooled protein concentration of ammonium sulfate precipitated egg yolks was 13.80 mg/mL.
A variable protein concentration from 8.64 mg/mL to 9.33 mg/mL was recorded from the selected five egg yolk samples precipitated with AS-OA. However, most of the concentrations were less than 9 mg/mL, a maximum of 9.33 mg/mL protein concentration was observed as indicated in Figure 5. Pooled protein concentration in AS-OA precipitation was 8.90 mg/mL. The IgY contents of the egg yolk precipitated with sole ammonium sulfate were comparatively higher than IgY of egg yolk precipitated with AS-OA combined treatment.

Discussion
The present research was performed to evaluate comparative precipitation potential of the Octanoic acid and ammonium sulfate to purify antibodies from the serum and egg yolk. The caprine serum was used for the purification of IgG proteins through a combined ammonium sulfate-octanoic acid precipitation (AS-OA) and sole ammonium sulfate precipitation. A similar method was employed for the precipitation of IgY from the egg yolk. AS-OA method is an efficient way to purify antibodies from serum. The method is superior to affinity chromatography and HPLC in terms of yield of antibodies  (Barati et al., 2018) where they used the freeze-thaw method with sodium chloride precipitation method for the purification of IgY from the egg yolk [20]. IgY have a protective effect on the animal models and can be easily used for experimentation. Presently, a maximum of 14.14 mg/ml IgY protein contents was measured from sole ammonium sulfate precipitated egg yolks.

Conclusion
The overall study evidenced a combined ammonium sulfate-octanoic acid precipitation was superior to the sole ammonium sulfate precipitation from the serum, however, the reverse was true for the purification of antibodies from the egg yolk. The combined ammonium sulfate-octanoic acid treatment enhanced the purity of the antibodies from both of the sources, however, the yield of antibodies was compromised in when precipitated from egg yolk.