Camels produce several immunoglobulin classes in their serum, colostrum and milk, including IgM, IgA and IgG (IgG1, IgG2 and IgG3) [1-3]. Camels are special animals in antibody production as they possess a unique type of antibody in their serum which lack light chains as well as the heavy chain constant domain “CH1”, so-called heavy-chain antibodies . IgG1 occurs in the classical structure form of antibodies, while IgG2 and IgG3 represent heavy-chain antibodies , and they form about 75% of the IgG in camel serum . Heavy-chain antibodies in camels are significantly effective in antigen recognition; in spite of losing their light chains [6-8], also, they have lower molecular weight than conventional antibodies. In addition, these antibodies are less immunogenic than other mammalian antibodies , that means when they are injected into experimental models, it will be less likely to induce adverse reactions during the course of treatment . The above pharmaceutical features recommend the camel antibodies as a substitute for other animals’ therapeutic antibodies [11, 12], or alternatively, it can be converted to a complete humanized antibody .
Antibody purification from different biological fluids such as serum, ascites and milk is an important step in the production of antibodies used for diagnostic, therapeutic, and research applications . There are different methods for purification and the first reported method for purifying camel IgG from serum, by Hamers-Casterman et al. , used protein A-Sepharose and protein G-Sepharose. More recently, Yao et al.  reported the purification of heavy-chain antibodies from bactrian camels’ milk using a similar procedure. However, this method involved the use of buffers of low pH, which may be deleterious to antibody activity, and it could not be applied on a large scale. For a rapid, inexpensive purification, precipitation is adequate; the majority of antibody purification protocols are based on ammonium sulfate precipitation [14, 16, 17]. Nevertheless, as well as being time-consuming, these procedures have disadvantages including contamination with some proteins like albumin, enhanced production of antibody aggregates [14, 18], and the need for the product to be recovered and re-solubilized. Besides, they may comprise a loss of significant antibody potency due to the drastic acidic elution buffer used . In addition, there is occurrence of endotoxin contamination and the difficulty of scaling up this process and maintaining sterility . Consequently, an efficient, cost-effective, and simple antibody purification methodology is needed for laboratories and industry downstream. Now, caprylic acid fractionation is used in purification of antibodies, and can be applied in the industrial manufacturing of whole IgG antibodies [14, 21]. Caprylic acid and its salts are implicated in precipitating non-antibody proteins from mammalian sera and leaving the antibodies in soluble form [14, 22, 23]. This technique is very simple, quick and yields an antibody preparation adequate for many uses without additional purification.
As Camelidae antibodies comprise heavy-chain antibodies, which have unique biophysical properties, they offer advantages in a variety of medical and biotechnological applications. So, further analysis is needed to establish the biological importance of these antibodies that are most likely created against various antigens . Thus, in this study, we describe a simple method that could be scaled up based on one precipitation step using caprylic acid to obtain a high yield of purified IgG from camel serum.
2 Materials and Methods
2.1 Camel sera and VACSERA anti-sera products
Camel blood samples were obtained from healthy and normal camels from slaughterhouses from different governorates in Egypt such as Alexandria, El-Fayoum, and Banha. At room temperature, the blood was left to coagulate, and then the serum was collected by centrifugation at 10,000 rpm for 15 min at 4°C. Finally, it was stored at –20°C until being used. Purified polyvalent Anti-Scorpion serum, polyvalent snake venom antiserum, and tetanus antitoxin, all in liquid form, were obtained from VACSERA Egyptian Organization for Biological Products and Vaccines (Agouza, Cairo, Egypt).
2.2 Chemicals and reagents
The organic acid, caprylic acid, was purchased from Riedel-de Haën (Wunstorfer Str., Seelze, Germany). Fluitest ALB-BCG kit for Albumin concentration determination was purchased from Biocon Diagnostik (Vöhl/Marienhagen, Hesse, Germany). Protein electrophoresis reagents were obtained from Bio-Rad (Alfred Nobel, Hercules, CA 94547, U.S.A.). Nile Red (9-diethylamino-5H-benzo[α] phenoxazine-5-one) was purchased from Sigma (Buchs, Switzerland).
2.3 Purification of camel serum IgG with caprylic acid and ammonium sulfate
2.3.1 Ammonium sulfate fractionation of camel serum
Ammonium sulfate at a final concentration of 40% was added slowly to 5 ml of serum with stirring [1, 2]. The mixture was stirred for 30 min at 4°C, and then centrifuged for 30 min at 4°C at 15,000 rpm. The pellet was resuspended in saturated ammonium sulfate to reach a final concentration of 40%, and then the mixture was centrifuged for 30 min at 4°C at 15,000 rpm. The pellet was resuspended in equal volume of 1x PBS pH 7.4 to original serum volume. Finally, the mixture was dialyzed against 1x PBS pH 7.4 for 24 h at 4°C.
2.3.2 Caprylic acid fractionation of camel serum
Camel serum fractionation with caprylic acid was carried out as described previously , with minor modifications. Serum was diluted with 60 mM acetate buffer pH 4.0 at a ratio of 1:4 (serum: acetate buffer), and then the pH of the diluted serum was adjusted to 4.5 with 1 M Tris-HCl. Caprylic acid at a final concentration of 2.5% in diluted serum was then added dropwise with vigorous mixing. After stirring for 30 min at room temperature, the mixture was centrifuged at room temperature for 30 min at 15,000 rpm. The supernatant was ultrafiltered and the pH of the filtrate was adjusted to 7.4 by 1 M Tris-HCl. Finally, the mixture was dialyzed against 1x PBS pH 7.4 for 24 h at 4°C.
2.4 Standardization of caprylic acid fractionation protocol of camel serum
Four variances were screened to evaluate the optimal conditions of caprylic acid fractionation of camel serum: caprylic acid concentration, pH, stirring time, and stirring intensity.
2.4.1 Caprylic acid concentration
Aliquots of 5 ml camel serum were adjusted to pH 4.5 by 1.76 N acetic acid. The organic acid (caprylic acid) was added to each aliquot at final concentrations of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12% and 16% (v/v) slowly with constant stirring. After stirring for 1 h at room temperature, the mixtures were then centrifuged at room temperature for 30 min at 15,000 rpm, followed by filtration. The filtrates were dialyzed for 24 h at 4°C against dH2O, and then dialyzed against 1x PBS pH 7.4 for 24 h at 4°C.
2.4.2 PH gradient
Aliquots of 5 ml camel serum were adjusted to pH values of 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, and 6.5 using 1.76 N acetic acid. Then caprylic acid was added to each aliquot at a final concentration of 8% (v/v) slowly with constant stirring. After stirring for 1 h at room temperature, the mixtures were then centrifuged at room temperature for 30 min at 15,000 rpm, followed by filtration. The filtrates were dialyzed for 24 h at 4°C against dH2O and then dialyzed against 1x PBS pH 7.4 for 24 h at 4°C.
2.4.3 Stirring time
Aliquots of 5 ml camel serum were adjusted to pH 5.5 with 1.76 N acetic acid. Then octanoic acid was slowly added to each aliquot at a final concentration of 8% (v/v). The samples were stirred constantly for different time intervals, 30 min, 60 min, 90 min and 120 min. The mixtures were centrifuged at room temperature for 30 min at 15,000 rpm, and then the supernatant was filtrated. The filtrates were dialyzed for 24 h at 4°C against dH2O, and then dialyzed against 1x PBS pH 7.4 for 24 h at 4°C.
2.4.4 Stirring intensity
Samples of 5 ml camel serum were adjusted to pH 5.5 by adding 1.76 N acetic acid. Octanoic acid was slowly added to each aliquot to final concentration of 8% (v/v). The aliquots were stirred for 90 min under 4 different conditions of stirring, vigorous stirring (750 rpm), moderate stirring (450 rpm), mild stirring (150 rpm) and no stirring (0 rpm). The mixtures were centrifuged at room temperature for 30 min at 15,000 rpm, and then the supernatant was filtrated. The filtrates were dialyzed for 24 h at 4°C against dH2O, then against 1x PBS pH 7.4 for 24 h at 4°C.
2.5 Fractionation of camel serum with caprylic acid and ammonium sulfate; after standardization of caprylic acid fractionation method
Ammonium sulfate fractionation of camel serum was performed as mentioned above. Aliquot of 5 ml serum was adjusted to pH 5.5 with 1.76 N acetic acid. Caprylic acid was added to undiluted serum dropwise with moderate stirring at a final concentration of 8%. After stirring for 90 min at room temperature, the mixture was centrifuged at room temperature for 30 min at 15,000 rpm, and then the supernatant was filtrated. Finally, the filtrate was dialyzed for 24 h at 4°C against dH2O then against 1x PBS pH 7.4 for 24 h at 4°C.
2.6 Comparison of caprylic acid purified camel IgG preparation with anti-sera products of VACSERA
Camel IgG prepared by caprylic acid fractionation of camel serum was compared with the purified polyvalent Anti-Scorpion serum, polyvalent snake venom antiserum and tetanus antitoxin. The comparison was based on determination of protein and albumin contents and albumin/globulin (Alb/Glob) ratio. The most important factor included in this comparison was characterization of aggregates in each sample to evaluate our preparation “camel IgG purified by caprylic acid”.
2.7 Detection of different camel IgG subclasses by ELISA
A 96-well ELISA plate was coated with either 50 μl/well of 50 μg/ml of caprylic acid-purified human IgG (prepared using the optimal conditions determined for camel serum IgG and used as control) or camel IgG preparation. After 24 h of incubation at room temperature, the plate was washed five times with PBS. Then 100 μl of blocking buffer (2% gelatin in PBS) were added to each well for 1 h at room temperature. Next 50 μl of alkaline phosphatase-conjugated goat-antihuman total IgG (1:1,000; Perry Laboratories INC; Court Gaithersburg, Maryland, USA), biotin-labeled goat-antihuman IgG1 (1:1,000; clone 8c/6-39; Sigma-Aldrich; Merck Millipore, Darmstadt, Germany), biotin-labeled goat-antihuman IgG2 (1:15,000; clone HP-6014; Sigma-Aldrich), or biotin-labeled goat-antihuman IgG3 (1:4,000; clone HP-6050; Sigma-Aldrich) were added, followed by an incubation of 1 h at room temperature. After that, the plate was washed five times with PBS. In case of biotin-labeled antihuman IgG1 IgG2, or IgG3, 50 μl of streptavidin-alkaline phosphatase-conjugate in concentration 1:4,000 were added for 1 h at room temperature. p-NPP was added for color development in case of alkaline phosphatase-conjugated goat-antihuman total IgG and absorbance recorded at 405 nm. Unbound antibodies were washed five times with PBS in case of biotin-labeled antihuman IgG1 IgG2, or IgG3 then p-NPP was added.
2.8 Miscellaneous procedures
The following parameters were measured for the purified fractions: total concentration of protein, concentration of albumin, protein aggregates, and electrophoretic profile of the resulted fractions.
2.8.1 Protein and albumin concentrations
2.8.2 SDS Polyacrylamide gel electrophoresis (SDS-PAGE)
Analysis of caprylic acid fractionation and ammonium sulfate precipitation fractions of camel serum was carried out using 12% SDS-PAGE  under nonreducing conditions. SDS-PAGE gels were stained with 0.1% Coomassie blue R250 stain for protein detection.
2.8.3 Characterization of antibody aggregates
Detection of antibody aggregates in the resulted fractions of serum was carried out by staining with the fluorescent hydrophobic probe Nile Red, and then scanning with fluorescence microscopy . Nile Red (8 μg/ml) was dissolved in a mixture of methanol and water (80:20, v/v) . Staining was conducted by addition of 0.3 μl of dissolved Nile Red solution to 10 μl of the fractions. Immediately the mixture was applied to a Haemocytometer slide and examined under fluorescence microscope with 40χ magnification.
2.9 Statistical analysis
The significance of the differences between the mean values of the different parameters measured in different camel IgG preparations from camel serum as well as in IgG preparations before and after standardization was determined by the Student’s t-test . P<0.05 was considered to indicate a statistically significant difference.
3.1 Comparison of fractionation of camel serum with caprylic acid and ammonium sulfate; before standardization of caprylic acid fractionation method
Table 1 presents a comparison between camels’ IgG purified by ammonium sulfate and caprylic acid fractionation. It is clear that precipitation of IgG by caprylic acid gave better results than ammonium sulfate in all aspects; lower albumin concentration regardless of lower protein content associated with good yield of IgG. Also, lower albumin/globulin ratio and protein aggregates besides shorter production time. In addition, SDS-PAGE analysis of the purified IgG (Fig. 1A) showed that IgG purified with caprylic acid has high purity and is less turbid.
Analysis of antibody aggregates of each fraction with Nile Red dye (Fig. 1B) showed that fractionation of camel serum with ammonium sulfate results in higher protein aggregates than fractionation with octanoic acid (Table 1).
3.2 Determination of the optimal conditions for fractionation of camel serum with caprylic acid
3.2.1 Caprylic acid concentration
The results of fractionation of serum using a gradient of caprylic acid concentrations are shown in Table 2. It was observed that the protein content of IgG and albumin concentration were slightly decreased when caprylic acid concentrations ranging from 1% to 4% were used. Considering the other parameters, it was observed that using caprylic acid at concentration of 8% gave the best results regarding protein content of IgG, albumin concentration and albumin/globulin ratio as well as SDS-PAGE analysis of the resulted fractions (Fig. 2A). Assessment of antibody aggregates (Table 2) using Nile Red fluorescence showed that caprylic acid concentration of 8% gave lower aggregates than the other caprylic acid concentrations.
3.2.2 PH gradient
Considering the effect of pH on the purification step, the results of serum fractionation at different pH values are indicated in Table 3. The protein and albumin contents were decreased at very low pH until pH 4.5 and then increased at pH 5.0. The best results were obtained after adjusting the pH of the serum to 5.5 using 1.76N acetic acid, giving a lower albumin/globulin ratio with low turbidity, very low albumin concentration and good protein concentration in the purified IgG as shown in SDS-PAGE analysis (Fig. 2B).
Antibody aggregates of resulted fractions were also analyzed (Table 3); the fraction of pH 5.5 gave the lowest amount of aggregates. Therefore, it was the best pH with regard to the preceding factors like protein and albumin content and aggregates amount.
3.2.3 Stirring time
Stirring the mixture for different time periods producted little difference in protein content and albumin concentration of the resulting fractions (Table 4). However, stirring for 30 min and 90 min gave good results concerning protein content, albumin concentration and albumin/ globulin ratio. Also, analysis of antibody aggregates of the fractions confirmed this result. However, as shown in Fig. 2C the electrophoretic pattern of the resulted fractions of purified IgG from camel serum indicated that the best time for stirring was 90 min.
3.2.4 Stirring intensity
The stirring status of the purification was determined to be the moderate stirring condition (450 rpm) as it gave the best results regarding IgG content, albumin content and albumin/globulin ratio as presented in Table 5. However, SDS-PAGE analysis of fractions (Fig. 2D) showed little variation in the protein content of IgG in all conditions of stirring intensity. Antibody aggregates were characterized for each fraction (Table 5), stirring the reaction mixture at intensity of 450 rpm (“moderate stirring”) had slightly elevated aggregates, but its appearance in SDS-PAGE was better than the other fractions.
In summary, the optimal conditions for good purification of IgG from camel serum by caprylic acid fractionation have been determined as follows: optimal pH of the serum was 5.5 without dilution and the best concentration of caprylic acid was identified to be 8%. While the conditions of stirring the mixture throughout the precipitation were at 450 rpm for 90 min.
3.3 Comparison of fractionation of camel serum with caprylic acid and ammonium sulfate, after standardization of caprylic acid fractionation method
Comparing the results of purification by these methods revealed that caprylic acid purification gave better results than ammonium sulfate precipitation as indicated in Table 6 and Fig. 3. Camel IgG purification by ammonium sulfate resulted in high protein and albumin concentrations. However, ratio of albumin/globulin was low. The appearance of the clarified protein solution was turbid indicating the presence of protein aggregates. While, in the case of caprylic acid, after adding caprylic acid to undiluted serum with the precipitation procedure optimized, a high yield of IgG with low aggregates was obtained. Purification of IgG by this protocol resulted in optimal protein concentration with low albumin concentration.
3.4 Comparison of caprylic acid purified camel IgG preparation with anti-sera products of VACSERA
Table 7 indicated the differences between caprylic acid-purified camel IgG and some products of VACSERA (Purified polyvalent Anti-Scorpion serum, polyvalent snake venom antiserum and tetanus antitoxin). The most important factor in this comparison was aggregate characterization in these samples. As indicated in Table 7, caprylic acid-purified camel IgG had an aggregate value within the range of VACSERA product’s aggregate characterization values.
3.5 Detection of different camel IgG subclasses by ELISA
ELISA results showed that anti-human IgG (total), IgG1 IgG2, and IgG3 were reactive toward caprylic acid-purified camel IgG but with significantly (P<0.05) lower reactivity than that towards corresponding antibody; human IgG preparation (Table 8). Interestingly, anti-human IgG2 and IgG3 were able to react but to a significantly (P<0.05) lesser extent than anti-human IgG (total) and IgG1 toward caprylic acid-purified camel IgG preparation, though camel IgG2 and IgG3 represent about 75% of the IgG in camel serum, this may be explained by the fact that these heavy-chain antibodies differ structurally from classical antibody camel IgG1 or human IgG2 and IgG3 so could not be detected by anti-human IgG2 and IgG3.
Camel milk and its ingredients have been claimed to have significant in vitro and in vivo activity against cancer, jaundice, hepatitis B and C, diabetes, dropsy, tuberculosis, spleen problems, anemia, piles, food allergies, high cholesterol in the blood and asthma (31-41). One of these unique ingredients is the camel heavy-chain antibodies which have advantages because of their low molecular weight, while their light chains are absent; they should bind their antigen by one single domain, the variable domain of the heavy chain, which is referred to as “VHH”, a nanobody of only about 12 to 15 kDa molecular weight allowing its access to small spaces not accessible for conventional antibodies [42, 43]. These antibodies are adjustable to phage display and other screening techniques for rapid and simple isolation of antibodies with high affinity . Reports have clearly confirmed that the VHH is soluble and stable after exposure to extreme conditions such as low pH and high temperature . These biochemical characters of the VHH domain make it an active binder for certain enzymes’ active sites, and as competitors’ enzymatic inhibitor [46, 47]. SDS-PAGE analysis designated two bands; one at ∽160 kDa corresponds to the classical antibody IgG1; it yields 50 KDa heavy chains and 30 KDa light chains after reduction. The other band appears at ∼100 kDa and corresponds to heavy-chain antibodies (IgG2 and IgG3), which yields only heavy chains of 46 and 43 KDa respectively after reduction .
Camilidae antibodies (IgG) are also less immunogenic than most mammalian antibodies , which reduces the risk of adverse effects. This should lead to improving current camel antibody purification and production methods, as the main feature of an immunotherapeutic agent like antibodies is immunogenicity, which should be low as possible. Consequently, camel immunoglobulins should be purified by a method that will be efficient, simple, inexpensive, and can be applied on a large scale with high yield in laboratories and industry .
Using affinity and/or immunoaffinity chromatography to downstream of therapeutical proteins normally faces obstacles such as loss of activity, aggregations and the fact that they are time consuming as well high-cost. Camel IgG have previously been purified from serum using affinity chromatography using protein A-Sepharose and protein G-Sepharose , but this method can affect the activity of the purified antibody by the low pH of elution buffers used. Furthermore, it is a very expensive, time-consuming method and unsuited to industrial scaling up. Srdic-Rajic et al.  reported purification of (IgG)-reactive IgG fraction from IgG of normal human serum by absorption onto (CNBr)-activated Sepharose 4B linked to intact IgG molecules: an expensive and complicated procedure.
Purification of immunoglobulins with ammonium sulfate resulted in precipitation of undesirable proteins besides IgG [1, 2, 24]. Additionally, these two methods cannot be applied on a large scale. Recently, Khamehchian et al.  combined ammonium sulfate precipitation and ion-exchange chromatography for preparation of therapeutic camel antivenom (IgG) against Naja Naja Oxiana and recommended this combined process for effective removal of residual proteins in the final preparation of camel IgG and also for large-scale refinement of therapeutic camel antivenoms. Caprylic acid precipitation of non-IgG proteins in serum or plasma was previously proved to be appropriate method in several laboratories for equine-derived antivenom production [51-53]. Thus, in this study we have developed a simple, quick, inexpensive and efficient method for separation of camel antibody from serum, that is, caprylic acid purification which is proved to be more effective and safe than ammonium sulfate method in simplicity, purity, and lower non-IgGs protein concentration in the final preparation with lower protein aggregates.
The obtained data demonstrate that caprylic acid fractionation of immunoglobulin-G from serum is preferable compared to purification using ammonium sulfate fractionation. Camel antibodies were purified from serum by ammonium sulfate at 40% final concentration [1, 2, 24], this method has a lot of disadvantages despite of high protein content of the purified product, it yields IgG contaminated with some undesirable proteins and albumin, and produces high aggregates that may cause loss of IgG activity. While IgG purified by caprylic acid appeared good in SDS-PAGE, its IgG content is suitable for further studies without any additional purification. In addition, caprylic acid is capable of purifying both types of IgG from camel sera. Also, the identity of different camel IgG subclasses in caprylic acid-purified camel IgG preparation was confirmed by ELISA using anti-human IgG (total), IgG1, IgG2, and IgG3.
In the present study, we purified IgG from camel serum with caprylic acid, which precipitates most of the proteins from serum except IgG, all of that occurred in specific physiochemical conditions, leaving high IgG concentration in the solution. Four variables were defined affecting purification of IgG by caprylic acid: caprylic acid concentration, pH of the serum sample, and stirring conditions (time and intensity). Comparison was done by measuring some factors for each variable: protein and albumin content, albumin/globulin ratio, SDS-PAGE analysis of the purified fractions and analysis of aggregates presence using Nile Red fluorescence dye. Working with different concentrations of caprylic acid to cover a wide range of concentrations resulted in defining the best concentration, which was 8% (v/v) of caprylic acid added directly to undiluted camel serum. The best pH of camel serum that will be used in purification was 5.5. Finally, optimal stirring time and intensity were determined to be stirring for 90 min at moderate stirring intensity (450 rpm). In contrast to the method described by Mckinney and Parkinson , in which serum was diluted with acetate buffer before addition of the organic acid, we added caprylic acid directly to undiluted serum as dilution of serum would introduce an additional step, that of concentrating the resulting fraction.
Proteins, especially immune-proteins, are the most widely-used biopharmaceutical drugs on the market. However, the activity of protein drugs can easily be affected by instability. Aggregate presence are a factor in instability of protein drugs such as antibodies and antisera. In this work, we compared caprylic acid-purified camel serum IgG with some products of VACSERA. This comparison mainly built on aggregation characterization in camel IgG preparation against the selected VACSERA products. Aggregation characterization was conducted using Nile Red/fluorescence microscopy as it allows characterization of turbid and concentrated protein solutions, like vaccines or subcutaneous administration, respectively . It was found that our preparation of camel IgG by caprylic acid fractionation of camel serum resembles the selected VACSERA products in aggregation characterization.
The current study implies a simplified and highly efficient protocol at biological and economical levels for fractionation of camel serum, which is suitable for antibody production in industries and laboratories. The methodology of serum fractionation with caprylic acid is the best and its optimal conditions were determined.
This work has been financially supported by master fellowship grant from the egyptian academy of scientific research and technology at the city of scientific research and technological applications “srta-city”.
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About the article
Published Online: 2017-05-04
Conflicts of interest: Authors state no conflict of interest
Citation Information: Open Life Sciences, Volume 12, Issue 1, Pages 143–155, ISSN (Online) 2391-5412, DOI: https://doi.org/10.1515/biol-2017-0017.
© 2017 Asmaa Shawki et al.. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License. BY-NC-ND 3.0