The low reproductive performance of the ewe may affect the efficiency and benefit cost of ewe production systems in Iran (1). Generally in Iran, sheep raised under semi-intensive system with a high mobility which are less fertile and less prolific compared to sheep raised under intensive systems (2). It is well-documented that the ovulation rate and litter size have a major impact on the reproductive efficiency of goats and ewes. Indeed, the goal of ewe production system is to have a high ovulation rate and litter size. Estrus synchronization is the key events in the breeding programs (3). In small ruminant, equine chorionic gonadotropin (eCG) injection is commonly used in association with a progestagen treatment to synchronize estrus and have a high ovulations during breeding and nonbreeding seasons (4).
There are numerous applications for eCG treatment in domestic species including induction of puberty, reversal of anestrus, superovulation and improvement of fertility and litter size (5). eCG treatment possessing both FSH and LH activities in ruminant. The long half-life (i.e., 5 days) and availability in large quantities make this unique gonadotropin a convenient exogenous hormone to induce estrus synchronization in small ruminants (4, 5). However, eCG treatment may causes the postovulatory of follicular waves (6) along with a high level of steroid production (3). It is proposed that an injection of anti-eCG antibody 1-2 days following eCG injection may minimize the adverse effect of eCG by drop the half-life of eCG in systemic circulation which may had beneficial effect on the ovulation rate, pregnancy rate and quality of embryos (7-10).
Equine chorionic gonadotropin previously known pregnant mare serum gonadotropin (PMSG) is secreted by trophoblastic cells to the blood of pregnant mare between the 36th and 120th days of gestation (11, 12). The structure of eCG is composed of two dissimilar subunits with a no covalent association which is required for the biological activities. Each subunit is composed of a peptidic part connected to a glycan moiety (N- and O-chains) (6, 13). eCG is the most heavily glycosylated glycoprotein hormone, with a majority of biantenna glycans ending mainly in sialic acids that play an important role in the long half-life of hormone in systemic circulation (5). Glycan chains are also involved in the stability of the heterodimeric structure of hormone and are necessary for the efficiency of signal transduction activity (14).
There are numerous productions of eCG reported in the literature (14-16) and also available in market (Bioniche Animal health (A/Asia) Pty Ltd, Australia). Generally, eCG may purify from pregnant mare serum with the classical purification scheme in two main steps (14, 17). First, precipitation and filtration steps removing the main contaminants like albumin to generate an intermediate product. Second, the generation of a final extract by chromatographic steps. However in recent publication, isolation of biomolecules directly from crude suspensions using Solid-Phase Immune Extraction (SPE) has been known in different areas of biotechnology. Small scale bioseparation experiments applying SPE have been reported to produce acrylamide in food (18), recombinant human myelin basic protein (rhMBP) in milk of transgenic cows (19) and plant sulfolipids (20).
Therefore, the purpose of the current study was to develop an accurate and fast method for purification of eCG from pregnant mare serum using SPE method with polyclonal antibody of eCG which in turn produced in rabbits.
3. Materials and Methods
3.1. Production of Polyclonal Antibodies
3.1.1. Immunization of Rabbit
Four male rabbits, about 4 months old and 2 kg weights, were chosen for preparing antibody against eCG. A basic immunization was done by injecting 25 IU of eCG (Bioniche Animal health (A/Asia) Pty Ltd, Australia) as the immunogen, emulsified with complete Freund’s adjuvant, into each rabbit by a multipoint subcutaneous implantation. After 10-days growth, 8 enhancing immunization was further carried out as described above but using incomplete Freund’s adjuvant, with 7 days interval between each implantation. Inoculations have been performed by high purity eCG of Bioniche Animal health (A/Asia; Pty Ltd, Australia). In the end of immunization protocol, rabbits’s heart blood was taken using Ouchterlony assay (Double immunodiffusion test). Volume and number of injections are according to Antibody Production Canadian Council on Animal Care & UIC Polyclonal Antibody Production, Guidelines/ACC 2012 (21-23).
3.1.2. Agarose-Gel Double Immunodiffusion
Ouchterlony assay or double immunodiffusion test was used to assess the titer of antiserum against eCG. For double immunodiffusion, the antiserum was diluted at 1:4 and 1:16 in 1% agarose gel then, incubated with eCG at 37 °C for 48 h.
3.1.3. Farr Assay (Ammonium Sulphate Precipitation) and Dialysis
Immunoglobulins were separated from the whole serum by precipitation with ammonium sulphate solution. If ammonium sulphate is added to diluted serum to 50% saturation, most of the immunoglobulins are precipitated, while other serum proteins such as albumin remain in the solution.
In the first step, whole blood sample was collected by invasion method from rabbit heart and then for access to serum of sample, all those tubes keep at room temperature for an hour and immediately centrifuged using 3000 rpm in 15 °C temperature within 30 min. In the second step, for obtaining the immunoglobulin and elimination of protein contaminations 35% aluminum sulfate were added. Then, the serum was centrifuged with 3000 rpm in 12 °C for 30 min. Subsequently, obtained immunoglobulin pellet dissolved and washed again using 0.01 M Phosphate buffer saline (PBS). For removing added aluminum sulfate, it is shake using dialysis bags for overnight in 4 °C. Then, immunoglobulin solution was lyophilized and stored at -20 °C.
It has been performed Biuret assay for total protein of anti-rabbit antibody using albumin as standard.
3.2. Blood Collection From MARES
Seventeen mares during initial stages of pregnancy were identified using ultrasonography diagnosis method. Blood samples (300 mL) were taken from jugular vein in the tube containing sodium citrate on days 50, 70 and 90 of mare’s pregnancy. The samples immediately were transfer to laboratory and keep in 4 °C for overnight. After red cell precipitation, plasma was siphoned and phenol solution has been added to the plasma. In the final step, collected plasma was mixed together and made ready for next steps of Solid-Phase Extraction of eCG.
3.3. eCG Purification
3.3.1. Solid-Phase Immune Extraction (SPE)
Coating Step: 1:15000 dilution of the antibody in coating buffer (Carbonate Bicarbonate Buffer 50 mM, pH 9.6) prepared and 300 µL of the solution added to the wells of microplate (96 wells). Then, the microplate incubated overnight at 4 °C. After incubation and coating the wells with the antibodies, contents of the wells poured out and the wells washed three times using wash buffer (Phosphate buffer saline with 0.05% tween20).
Blocking step: for blocking the uncoated surface of the wells, 300 µL phosphate buffer saline with 0.1% bovine serum albumin added to allocated wells and incubated for 2 h at 37 °C and finally washed the wells using the mentioned wash buffer.
Sample loading: For immune-separation of the hormone, 300 µL of plasma added to each antibody coated wells and incubated for 2 h at room temperature (RT). Then, the well contents poured out and the wells washed three times using wash buffer. In this step, the hormone bonded to coated antibodies on the inner surface of the well.
Hormone separation: For separating and releasing eCG from immobilized antibodies, the well washed with wash buffer with higher detergent content. So at first, added 300 µL concentrated wash buffer (Phosphate buffer saline with 0.5% tween 20) and incubated for 2 h at RT. The wells content which contain immune separated hormone collected with sampler, lyophilized and stored at -80 °C. The eCG content of SPE end product was determined by the method of Bradford using Human Serum Albumin and gamma globulin as the standard.
3.4. Sodium Dodecyl Sulfate–Poly Acrylamide Gel Electrophoresis (SDS-PAGE)
Briefly, the gel concentration was 12%, condition was none reducing, voltage 200V and time was 3 h, electrophoresis instrument including tank and power supply was Bio-Rad made.
Results based on dual diffusion of antigen and antibody in agarose gel (Agarose-gel double immune-diffusion) showed that the rabbits have been completely immunized. Due to precipitate, the lines were produced from rabbit’s antibodies with injected antigen (Figure 1).
One of the first observations of antigen-antibody reaction was their ability to precipitate when combined in proportions at or near equivalence. By performing these reactions in agar gels, it is possible to distinguish separate antigen-antibody reaction produced by different populations of antibody present in the serum (Figure 1). The precipitin arcs formed between the antibodies and the antigen fuse indicating that the antibody is precipitating identical epitopes (24). The purity and composition of the various preparations were assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE analysis of commercial and purified equine gonadotropin preparations were showed in Figure 2.
Comparison of loaded samples beside of standard commercial hormone can demonstrate concentration and evidence of isolated hormone in the present study (Figure 2). In other hands, these results may support effectiveness of extracted antibody against eCG and also accuracy of the suggested method for extraction and purification of the hormone.
Total protein of Anti-eCG antibody has been calculated via Biuret assay (25). Immunoglobulin’s concentration has been determined (3g.dl-1).
The eCG content of SPE end product was determined by the method of Bradford using Human Serum Albumin and gamma globulin as the standard (R2 =0.996) (25). Therefore, eCG concentration has been evaluated (5250 mg.ml-1).
There are various methods were recorded for purification of eCG from pregnant mare serum during 40 to 120 days of gestation. Cartland and Nelson (15) collected 16.5 liter of plasma from 49 mares around day 65 of pregnancy. They made fractional precipitation, using ethanol and acetone, and in each step they removed the impurity. Aggarwal et al. (14) separated eCG from two groups of pregnant mares with a high and a low serum titers. From biochemical perspective, the structure of eCG in both groups showed different amino acid and carbohydrate compounds. Owen Reed (16) patented a method using molecular ultrafiltration for separation. With an assumption of 30000 Dalton molecular weight for eCG, they used the membranes cut off for metabolites smaller than 20000 and larger than 40000 Dalton to separate the hormone. It is reported that is not possible directly apply ion exchange adsorbents, due to the high salt (conductivity: 10-13 mS.cm-1) and protein (60 mg.ml-1) content of mare serum. Accordingly, it is documented that several precipitation steps are needed in order to reduce the foreign protein content and the conductivity of pregnant mare serum (26). Classical purification of eCG from serum includes pH fractionation with metaphosphoric acid, two ethanol precipitation steps as well as dialysis followed by fixed-bed chromatography. In the present study, it is suggested the new method for purification of eCG.
High specificity of antibodies are valuable markers for recognition, purification and quantitative measurement of their own antigens. Since, it is possible to produce in vivo antibodies for macromolecules and small chemical materials, the methods based on using antibody were developed (24). Previously, using antibody in the purification methods was depend on the ability of antibody and its specific antigen to build a big immune complex (either in solution or in gel) for recognizing by optical methods. These methods have been replaced by simpler ones for fixing antibodies and antigens on solid surface, like the standard method of SPE (27, 28).
Solid-phase extraction has been known as one of the most important methods of extraction and pre-concentration (28). It is suggested that SPE is preferred to other purification and concentration methods. The advantages of SPE are the fast and easy operation, high pre-concentrate factor, various solid phases, small usage of organic solvents, absorbance of interested materials on solid phase with high stability, and low recognition level (29). SPE is a method which separate and concentrate analyte by absorbing on solid phase. This process continues washing analyte with one or some appropriate solvents to prepare for instrumental analysis (27, 29). Different factors should be optimized in the process of absorption and reabsorption of analyte. Results of SDS-PAGE analysis in the present study showed that eCG has been precisely extracted and purified.
In the current study using anti-eCG antiserum, a simple and rapid technique for the isolation and characterization of eCG is proposed; this could be superior to other methods that use multiple precipitation and salting out steps. The current method only uses one step purification which results in eCG recovery in one fraction without significant contamination.
In conclusion, the extraction of eCG with polyclonal antibody using SPE method and production of anti-eCG antiserum in rabbits is suitable and may be a cost effective method for large scale production of eCG and anti- eCG antiserum in Iran.
I would like to show my gratitude to Dr Naser Mohammad poor, Miss Laleh Hoghoughi and Dr Arash Javanmard for their insightful comments and their helps during laboratory activities and preparation of the present manuscript.
All authors were participated equally in the manuscript preparation.
There is not any conflict of interest between the authors.
This research was supported by University of Tehran Department of Animal Science.