Establishment of liquid phase double antibody radioimmunoassay system for in-vitro determination of erythropoietin hormone in human serum

The aim of study was the production of polyclonal antibody of erythropoietin hormone (Anti-EPO) to determine EPO in human serum by liquid-phase radioimmunoassay (RIA). Production of anti-EPO was performed by immunizing Balb/C mice with EPO antigen using primary immunization and four booster doses. EPO tracer (125I-EPO) was prepared using Chloramine-T method. Additionally, EPO standards were mad. Furthermore, the assay was optimized and validated in additional studies. In conclusion, the elaborated EPO-RIA system exhibits a simple, accurate, specific and sensitive method for determining EPO concentration in human serum and might be suitable for clinical diagnosis of myeloproliferative diseases.


Introduction
The glycoprotein hormone erythropoietin (EPO) has 165 amino acids and forms a single polypeptide chain with two intra-chain disulfide bonds. It also has four potential glycosylation sites, three of which are N-linked and one O-linked [1][2][3]. EPO has a molecular mass of 30-34 kDa, although the peptide chain has a much smaller molecular mass of just about 18 kDa. Accordingly, the glycosylated EPO molecule is composed of carbohydrates to a degree of around 40%, mainly in the form of N-linked complex-type glycans [4][5][6]. EPO is synthesized by the peritubular cells of the kidney and modulates erythropoiesis by stimulating the proliferation and maturation of erythroid progenitors [7][8][9]. EPO is also produced in small quantities in the spleen, liver, bone marrow, lung, and brain. The partial pressure of oxygen (pO 2 ) directly regulates EPO production, when pO 2 is low, the production of EPO increases. Similarly, low hemoglobin levels stimulate the production of EPO [10]. The evaluate of EPO in human serum is an effective diagnostic test to differentiate between the many types of polycythaemia and anemia.
Most anemic patients have high serum EPO levels according to the type and severity of their anemia. Also, patients with chronic renal failure and anemia have abnormally low serum EPO levels because the kidneys are producing less EPO [3]. Distinguishing primary from secondary polycythaemia through serum EPO levels is crucial. Patients with primary polycythaemia, such as polycythaemia rubra vera, typically have low or sub-normal serum EPO levels, in contrast, secondary polycythaemia caused by chronic hypoxaemia, such as in cyanotic congestive heart failure or chronic obstructive pulmonary disease, has normal or elevated serum EPO levels [11].
Radioimmunoassay (RIA) technique is used to measure a broad spectrum of clinically and biologically significant materials. This technique can be performed quickly and easily while ensuring accuracy, specificity, and sensitivity, which have a considerable impact on medical diagnosis [12]. RIA technique, as the name implies, achieves sensitivity through the use of radionuclides and specificity which is uniquely associated with immunochemical reactions. Also, RIA is based on the isotope dilution principle, along with the use of a specific antibody to bind to a portion of the substance (antigen) that needs to be measured. When an antigen is mixed with a specific antibody to that antigen, an interaction will occur, forming an antigen/antibody complex that is chemically different from either the antigen or the antibody [13][14][15].

3
The main basic component of RIA is the pure antigen (Ag) which used to produce antibodies (Ab) and prepare each of radiolabeled antigen (Ag*) and standards (unlabeled antigen Ag) in different concentrations. In addition, the separation method is necessary to separate bound from unbound antigen. This can be achieved by using polyethylene glycol (PEG) and second antibody technique as a separating agent in liquid-phase [16,17]. The quality of these components and the selection of an appropriate separation technique both play a significant role in improving assay sensitivity [18]. The specificity and sensitivity of RIA system depends on the affinity of the antigen-antibody reaction and the highly specific binding sites of the utilized antibodies. For the production of a highly specific antiserum, the molecular weight of the antigen must be more than 4000 Da and possess a unique antigenic determinant [19]. The usual protocol for producing antibodies involves injecting the antigen into a number of suitable animals emulsified with Freund's adjuvant, which slows down the antigen's absorption and increases the antigenic stimulation [20].
The label criteria for radioiodine are usually fulfilled; however, Iodine-125 is preferred over Iodine-131 due to its longer half-life, easier handling, and higher counting efficiency. Because the amount of substance to be evaluated generally has low concentrations, the labeled antigen (Ag*) utilized as a tracer must typically be present in low concentrations. To react qualitatively and quantitatively with the antibody, the labeled antigen must be of high specific activity and, after being labeled, maintain the same properties as the unlabeled antigen [20]. A method of producing radioiodinated proteins was described by Hunter and Greenwood using chloramine-T as oxidizing agent [21,22]. Other techniques have been created, including lactoperoxidase [23] and iodine monochloride electrolyte labeling [24].
To interpolate values of samples to be measured, standards are required. A substance intended to be a standard should have specific properties, as: stability, availability in large amounts, free from chemicals that could interfere with the assay, high purity, and availability in a form that enables easy and precise preparation for RIA [25].
The main objective of the present study was to prepare and develop EPO-RIA kit to detect EPO using the locally prepared primary reagents, in order to detect hormone concentration in human serum.
Animals Four female Balb/C mice with body weight about ~ 20 g and 8-10 weeks old were housed under controlled conditions (room temperature, 22 ± 2 °C; artificial illumination, 12 h per day). Mice were obtained from Animal House in labeled compound department, Hot Labs Center-Egypt.

Immunization
The immunization schedule for the production of anti-EPO polyclonal antibody was carried out according to Thorell 1978 andMason-Garcia et al. 1990 [26,27], with some modifications. Four female Balb/C mice (M 1 , M 2 , M 3 and M 4 ) were immunized via intraperitoneal injection with EPO antigen, 40 µg in 500 µl sterile saline was emulsified with 500 µl of Freund's adjuvant complete (FAC) for the first immunization at a ratio 1:1 (10 µg EPO/250 µl emulsion per mouse).
All mice received three boosters at one-week intervals with the same dose (10 µg/mouse) emulsified with Freund's adjuvant incomplete (FAI). Finally, all mice received fourth booster via intravenous injection into the tail with EPO antigen (10 µg/mouse) in 100 µl normal saline. The blood was harvested from the mice via cardice puncture, 3 days after the fourth booster [28]. The next step, serum was separated from the blood by centrifugation at 4000 rpm for 20 min. Antisera was stored at frozen in − 20 °C until use and assess the characterization of the optimum titre, displacement and immunoresponse by RIA technique.

Characterization of the obtained Anti-EPO polyclonal antibody for EPO-RIA
Titre and displacement of anti-EPO polyclonal antibody were assessed by diluting the obtained serum from 1/50 to 1/128,000-fold in assay buffer (0.05 M phosphate, pH 7.4, containing 0.15 M NaCl, 0.01 M EDTA 0.1% BSA and 0.1% (NaN 3 ) sodium azide). Titre and displacement were estimated by incubating 100 μl of different dilutions of EPO antisera (1/50, 1/100, 1/500, 1/1000, 1/2000, 1/4000, 1/8000, 1/16000, 1/32000, 1/64000, 1/128000) as a (1st Ab) with 100 µl of the EPO standards zero or high concentrations (0.0 or 250 mIU/ml), with 100 μl of 125 I-EPO tracer for 3 h at room temperature. The separation of the bound and free fractions was achieved by adding separating formula as a follow: 100 μl goat anti-mouse IgG (2nd Ab) (1/10), 100 μl non-immune mouse serum NMS (1/100) and 0.5 ml of 8% polyethylene glycol (PEG-8000) into all assay tubes. After incubation for 30 min at room temperature, the tubes were centrifuged at 4000 rpm for 30 min at 4 °C. The precipitates containing the bound fractions were counted in a multi-crystal gamma-counter (Auto Gamma Counter, Cobra II, Packard Instrument, USA). The binding percentage anti-EPO for different dilutions for each bleeding for each mouse were calculated using logit-log graph paper. By creating various anti-EPO dilution curves with zero and high levels of EPO standards (0.0 and 250 mIU/ml), the displacement percent between the maximum binding percent (Bo%, 0.0 mIU/ml) and the minimum binding percent (B H %, 250 mIU/ml) for the harvested antisera for each mouse was calculated by this equation: Antiserum EPO with the optimum titre and displacement was partially purified using the sodium sulfate/ammonium sulphate precipitation method, followed by dialysis against phosphate-buffer saline (0.02 M) according to Goding 1986 and Maboudi et al., 2016 [28,29]. The IgG content of EPO was measured using a UV spectrophotometer at an absorbance of 280 nm [30]. The concentration of IgG-EPO was calculated using two equations derived from the Beer-Lambert Law [29,31]. The purified anti-EPO was divided into aliquots and stored either at 4 °C for short-term storage or at − 20 °C for long-term storage.

Radiolabeled 125 I-EPO
Iodination of EPO with radioactive 125 I was prepared using the Chloramine -T method, as described by Greenwood et al. in 1963 andMason-Garcia et al. in 1990 [22, 27] with slight modifications. The phosphate buffer 0.05 M, pH 7.4 was used as a diluent for all reagents, except for the EPO antigen diluent, which was in 0.5 M phosphate buffer. 125 I-EPO tracer was prepared as follows: 100 µl EPO antigen (10 µg) in 0.5 M phosphate buffer pH 7.4 was added to an Eppendorf tube, followed by 2 µl of Na-125 I (250 µCi = 9.25 MBq) and 20 µl of Chloramine-T (20 µg). The reaction was allowed to proceed for one minute at room temperature and was terminated with the addition of 20 µl of Sodium metabisulfite (20 µg) and 100 µl of potassium iodide (100 µg). 125 I-EPO tracer was separated and purified using a chromatography column PD-10 (Sephadex G-25) with an elution buffer (0.05 M phosphate buffer containing 0.1% BSA). The flow rate was adjusted using a pump at 0.5 ml/min per tube, and the counting was done with a radioisotope dose calibrator. The elution profile of the 125 I-EPO was constructed by plotting radioactivity count (µCi) against fraction numbers. The percentage of radioactivity was estimated for each of 125 I-EPO and free iodide. The radiochemical yield, immunoreactivity, non-specific binding, radiochemical purity, and specific activity of the 125 I-EPO tracer were evaluated. The 125 I-EPO tracer was stored at − 20 °C in aliquots in Eppendorf tubes according to Mason-Garcia et al. 1990 [27].

Optimization of liquid-phase double antibody EPO-RIA
To obtain the best optimize process for EPO-RIA, various affecting factors were extensively studied, including the effect of standard or sample volume, incubation time and separating agent (concentration of second antibody, normal mouse serum (NMS) and polyethylene glycol (PEG)).
The assay was performed using 100 μl of anti-EPO with optimum titre and highest displacement, along with either EPO standard or sample volume (50, 100, 200 µl). A fixed volume of prepared tracer (100 μl 125 I-EPO) with count ~ 25,000 cpm was added, followed by 200 μl of assay buffer. The reaction was allowed to proceed with different incubation time (1, 2, 3, 24 h). At the end of incubation time, 100 μl different dilutions of goat anti-mouse-IgG (1/5, 1/10, 1/15, 1/20, 1/30, 1/50) with 100 μl of different dilutions of NMS (1/50, 1/100, 1/200) were added and the reaction mixture was incubated for 30 min. After this incubation, 0.5 ml different concentrations of PEG-8000 solution (4%, 8%, 12%) in 0.1 M sodium phosphate buffer pH 7.4 was added to accelerate the precipitation of the formed ligand (mouse anti-EPO-IgG/goat anti-mouse-IgG with unlabeled EPO and labeled 125 I-EPO). The tubes thoroughly vortexed and centrifuged at 4000 rpm for 15 min at 4 °C. The supernatants were aspirated, the tubes were counted using multi-crystal gamma counter. The results of optimization studies of the liquid-phase RIA system for EPO standard curve were constructed.

Performance characteristics of the EPO-RIA system
Studies were conducted to ensure the validity, sensitivity, precision (intra and inter assay), accuracy (recovery and dilution tests), and method comparison for liquid-phase double antibodies of EPO-RIA. All the obtained optimum results were written in bold in all tables.

Results and discussion
The production of polyclonal anti-EPO was undertaken by immunization four Balb/c mice (M 1 , M 2 , M 3 and M 4 ) with EPO antigen (10 µg/250 µl emulsion)/mouse for primary immunization with FCA. The same dose (10 µg/250 µl emulsion) was used for each booster (3 boosters) with FIA, except the final booster (fourth booster), where each mouse was boosted intravenously with EPO antigen (10 µg/mouse) in 100 µl normal saline.
The results obtained lead to the following conclusion: mouse 1 (M 1 ) provided the best results for the binding, immunoresponse and displacement study (73.4%) between the zero and highest standards (0.0 and 250 mIU/ml) at initial dilution titre 1/8000 (final dilution titre 1/40000) as shown in Fig. 2.
The purity of the antibody purified by the ammonium sulphate precipitation method was measured by UV spectrophotometry at λ 280 nm, and the concentration of IgG-EPO was calculated according to equations derived from Beer-Lambert Law [29] using the following methods: First method IgG-EPO concentration was calculated using this equation: So, concentration of IgG-EPO = 1 X 1.29/1.4 = 0.921 mg/ ml.
Second method The concentration of IgG-EPO can be determined by substituting the molecular weight, extinction coefficient and λ max into a derived form of the Beer-Lambert Law, where the λ max is the wavelength at which it experiences the strongest absorbance, typically at 280 nm [31].
Concentration of IgG-EPO was calculated according to the following equation:  The obtained results show that the concentration of IgG-EPO is 9.21 mg/ml, which is in good and agreement with Goding, 1986 [28], who stated that the concentration of total IgG in serum ranges from 5 to 20 mg/ml. Also, he reported that the antibody concentration in the serum of mice ranged from 2 to 10 mg/ml. The purification step for neat antiserum, which contains produced antibodies, leads to improvement of the binding percentage. It is essential to obtain antibodies with satisfactory specifications without denaturing their components.
The purified antibodies were used to assemble a diagnostic liquid-phase double antibody EPO-RIA system to evaluate EPO concentrations in human sera.
EPO was successfully radioiodinated using Chloramin-T as an oxidizing agent. The 125 I-EPO tracer was obtained was purified using gel chromatography on a PD-10 (Sephadex G-25) column, and the elution pattern was created by plotting the activity (μCi) versus the fraction numbers as shown in Fig. 3. The graph displays two peaks that correspond to 125 I-EPO (76%) and free 125 I (18.1%) respectively. The specific activity obtained was 23.2 µCi/ µg. The pool of fractions containing of 125 I-EPO tracer was stored at − 20 °C until use in the liquid phase double antibody EPO-RIA according to Mason-Garcia et al. 1990 [27]. 125 I-EPO tracer was stable for up to eight weeks after iodination. When applying liquid-phase double antibody EPO-RIA, the immunoreactivity which includes: maximum binding of the zero standard (Bo%), high standard 250 mIU/ml (B H %) and non-specific binding (NSB%) was 50.3%, 13.2%, and 2.9%, respectively. These findings are consistent with the study conducted by Butt et al. 1983 [32]. Radiochemical purity of the 125 I-EPO tracer was = 1.29 210, 000 M −1 Cm −1 × 1 Cm × 150, 000 g∕mol × 10 = 9.21 mg∕ml tested over a period up to 8 weeks via electrophoresis. The results showed an appropriate radiochemical purity of 98%, which decreased to 92% within 8 weeks.

Optimization of the liquid-phase EPO-RIA
The ideal assay conditions were achieved by studying the following parameters, such as: sample volume, incubation time and separating agent formula, to optimize the liquidphase EPO-RIA system under investigation.

Sample volume
According to the findings in Table 1, the sample volume of 100 μl gave the maximum displacement percentage between the various EPO standards' zero, low and high concentrations (0.0, 3.75, and 250 mIU/ml).

Incubation time
Studying the effect of incubation time on the locally prepared liquid-phase EPO-RIA was conducted over a period of time ranging from 1 to 24 h, all at 37 °C. The assay was carried out using three distinct concentrations of EPO standards: zero, low and high (0.0, 3.75, 250 mIU/ml) respectively. As illustrated in Table 2, the highest displacement % was revealed by the results for 3 h incubation and nearly similar to 24 h incubation.

Separating agents
To obtain the optimum conditions for separating agent formulas, the assay was carried out with goat anti-mouse IgG (2nd Ab) at dilutions ranged from 1/5 to 1/50, non-immune   Tables 3, 4 and 5, the results obtained for separating agent formula indicated that: 100 μl goat anti-mouse (2 nd Ab) at dilution (1/10), 100 μl NMS at dilution (1/100) with 0.5 ml of PEG-8000 at concentration (8%) are optimum and more effective in separating the bound fractions. The results revealed high displacement percent between zero and high levels EPO standards, low and high levels EPO standards and gave low non-specific binding percent. Based on the results discussed above, the optimum conditions used to establish the assay was: 100 μl of purified anti-EPO at initial dilution of 1/8000 (final dilution of 1/40,000) (M 1 ), 100 μl of standards or samples, 100 μl of 125 I-EPO tracer (25,000 cpm) and 200 μl assay buffer. Using a gentle vortex, the reaction mixture was incubated at 37 °C for 3 h. After that, the following separating agents were added: 100 μl of goat anti-mouse IgG (1/10) with 100 μl of NMS (1/100) and tubes incubated for 30 min. Following that, 0.5 ml of PEG-8000 (8%) was added to each tube, and the mixture was vortexed and centrifuged at 4000 rpm for 15 min at 4 °C. Assay tubes were decanted and the antibodyantigen ligand was counted using a multi-crystal gamma counter. The obtained results of optimization studies of the liquid-phase RIA system for EPO standard curve was constructed as illustrated in Fig. 4.

Performance characteristics of the EPO-RIA
Some studies on performance characteristic were carefully examined to ensure the validity and reliability of the proposed assay.

Sensitivity
Ultra-sensitive assay is required to provide parallel improvement in biochemical discrimination between EPO levels observed in myeloproliferative disorder. As indicated in Table 6, the sensitivity or minimum detection limit for EPO-RIA was calculated by the interpolation of the mean minus 2 standard deviation (SD) of 10 replicates of the zero mIU/ml EPO standards [33]. The sensitivity of the proposed assay was determined by the dose that was red off the standard curve and corresponded to mean minus 2 SD (mean-2 SD). The sensitivity value obtained in this study was 1.1 mIU/ml.

Precision
Precision is a statistic that measures an assay's ability to produce consistent results when repeatedly run on the same sample. Precision studies of the assay system were   conducted by determining intra-assay and inter-assay variations. The mean of 10 replicates (n = 10) for three pooled human serum samples with low, medium, and high EPO concentrations (3.5, 30, 85 mIU/ml) respectively were used to evaluate the intra-assay precision (within-run) and interassay precision (run to run). For each sample, statistics were calculated, and the outcomes were tabulated in Table 7. The data from the present study on intra-assay and inter-assay precision proved the consistency of the results obtained by the current technique. The findings of the present procedure agree with those reported by Schlagerter et al., 1990 [34], who indicated that the CV % of the observed ligand concentration should be less than 10% in the case of the intra-assay (within assay), whereas they stated that the CV% of interassay (between assays) variance should be less than 15%.

Accuracy assessment
The degree of agreement between the measured value and the true value is known as accuracy. In this study, recovery and dilution tests were carried out to evaluate the accuracy of the assay.

Recovery test
The recovery test measures the concentration in three human serum samples or standards of different diagnostic states of EPO level (3.5, 30 and 92.5 mIU/ml) before and after adding a known amounts and concentrations of pure analyte of EPO levels (7.5, 30 and 62.5 mIU/ml). The value of the recovery test as show in Table 8 ranged from 96.1 to 101.8%. The recovery results from the current study for EPO are in good accordance with the data provided by Pillai and Bhandarkar 1998 [33], who reported that the RIA system's recovery should be 100 ± 15%. The observed data showed typical recoveries, indicating that the standards and the human samples were completely compatible. Therefore, the results obtained confirmed that the local liquid-phase

Dilution test
Dilution (parallelism) is carried out by diluting a sample or standard with the suitable diluent and performing the assay [35]. The observed results of dilution test in the cerrent study revealed the concentrations of three levels (15,30 and 125 mIU/ml) of EPO samples or standards at different dilutions in zero standrad to evaluate the linearity of the local assays. As shown in Table 9, which illustrates the concentration, the dilution test for EPO ranged from 93.3 to 104.2%. The results demonstrate that the technique of the current study for EPO had good linearity with dilution. The results are in good agreement with Edwards 1996 [36], who explained that the non-linearity pointed to either an unsuitable matrix or poor calibration. In conclusion, the results of the recovery and dilution tests showed precise calibrations and an adequate matrix.

Method comparison
In this study, correlation coefficient is used to measure the strength of the relationship between two variables. The linear correlation coefficient is known as Pearson's "r", which reflects the direction and strength of the linear relationship between the two variables x and y. It returns a value between + 1 and − 1, where + 1 indicates a strong positive correlation and − 1 indicates a strong negative correlation. If the result is zero, it indicates no correlation, which is also known as zero correlation. The results of the statistical analysis of the linear regression and correlation coefficient "r" between the 20 different human serum samples of EPO measured using commercially available kits (ELISA) and the locally prepared technique EPO-RIA system show a strong linear positive correlation (r = 0.996) as shown in Fig. 5.

Conclusion
In conclusion, this study aimed to produce primary reagents of liquid-phase EPO-RIA system: Anti-EPO polyclonal, 125 I-EPO tracer and EPO standards. The results and observations of the present study were successfully used to prepare sensitive, precise, accurate and stable system to detect EPO concentrations in human sera. This system can be used as a diagnostic marker in some disorders such as anemia, primary and secondary polycythemia, and chronic renal failure.
Funding Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).

Conflict of interest
The authors declare no conflict of interest.
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