Preparation and Validation of Double Antibody Radioimmunoassay for Thyroglobulin (Tg) using Balb/C Mice as Host Animals

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1 Preparation and Validation of Double Antibody Radioimmunoassay for Thyroglobulin (Tg) using Balb/C Mice as Host Animals I.Y. Abdel-Ghany, H.M. Shafik and N.R.A. El-Mouhty Labelled Compounds Dept, Hot Labs. Center, Atomic Energy Authority, P.No , Cairo, Egypt. Received: 19/1/2014 Accepted: 26/2/2014 ABSTRACT The preparation and development of primary reagents of thyroglobulin (Tg) radioimmunoassay technique with low cost is considered to be the main objective of present study. The production of polyclonal antibodies of thyroglobulin was undertaken by immunizing three bulb/c mice intraperitonial through primary injection and two booster doses. The preparation of 125 I-Tg radiotracer was carried out using chloramine-t as oxidizing agent. The preparation of thyroglobulin standards were carried out. Optimization and validation of the assay were studied out. The results obtained provide a highly sensitive, specific and accurate RIA system for thyroglobulin based on liquid phase separation. In conclusion, this assay could be used for diagnosis of thyroid cancer. Key Words: Thyroglobulin / Immunization / Liquid phase / Radioimmunoassay. INTRODUCTION Thyroglobulin (Tg) is a large glycoprotein of molecular weight Dalton (1). It consists of two dimmers and contains about 5170 amino acid molecules of which are tyrosine and about 10% of the total weight is carbohydrate. For every 2 or 3 thyroglobulin molecules there are two T4 molecules and one T3 molecule (2). Thyroglobulin is discovered in the lymph secreted from the thyroid gland of monkey and rat (3). Serum thyroglobulin levels are raised in some pathological condition such as differentiated thyroid carcinoma, hyperthyroidisms (4), toxic and non toxic adenomas, subacute thyroiditis. Measurement of thyroglobulin in the serum provides very important clinical information about patients who have surgical or additional radioiodine therapy for differentiated thyroid carcinoma. The presence of thyroglobulin in the circulation after total thyroidectomy indicates recurrence or metastatic spread of thyroid cancer (5). Thus estimation of thyroglobulin useful in monitoring the course of disease during follow up after thyroidectomy or radioiodine therapy. RIA is a very sensitive technique and detects as low as ng quantity of Tg in circulation. To cover the very wide range of concentration associated with the great variety of pathological processes (6), the current liquid phase radioimmunoassay is a simple and powerful diagnostic tool for the detection of Tg in human sera. MATERIALS AND METHODS For preparation of RIA system for estimation of Tg in human sera a number of reagents were used. Purified human Tg was obtained from Tata Momoreial Hospital, NaI 125 (5 mci /50 µl), ph 7-11 (Izotop, Hungary). Carrier free and reductant free solution. Complete and incomplete Freund s adjuvant, Chloramine-T, Sodium metabisulphite, Polyethylene glycol 6000 (PEG). Local second antibody (sheep anti-mouse serum) and local normal mice serum. Bovine serum albumin was purchased from Sigma Chemical Co., USA. All other chemical reagents were of analytical grade obtained from reputed manufacturers. The present research plan was achieved through the local preparation of the following: 233

2 Production of Anti-Thyroglobulin Polyclonal Antibody: Production of polyclonal anti-tg was carried out according to the immunization schedule described by Maiolin et al., (7). This study involved through Balb/C mice. The mice were maintained under standard laboratory conditions of relative humidity (55±5%), temperature (25±2 o C) and fed on standard laboratory diet and water was supplied ad libitum. The production of polyclonal antibody was carried out through primary immunization and two boosters. The preparation of immunogen was undertaken by mixing and emulsifying µg of highly purified Tg antigen in 250 µl saline and 750 µl of complete freund s adjuvant using Hamilton double-hub syringes. The mixing between the aqueous phase (Tg) and oil phase (adjuvant) was performed until stable emulsion was produced for primary immunization, the emulsion volume was assigned to immunize three Balb/C mice. Each one received 25 µg of Tg in 250 µl emulsion. Mice were injected intraperitonial. Booster doses were given in the same way but using incomplete Freund s adjuvant. The first booster immunization was given after 2 weeks from first immunization. Also, the second one was given after 2 weeks from 1 st booster. The antisera of the immunized mice were tested after the second booster by RIA system in terms of titre and displacement. These tests were carried out as follow: The blood spot was taken from each immunized mouse from the end of the tail and left to dry at room temperature (blood spot 2x3 mm contains about 2µl serum), 300 µl of assay buffer were added to each blood spot and incubated for 2-3h at room temperature, (the initial dilution 1 : 150). Serial dilutions were taken from the first dilution (1/150) to reach 1 : 450, 1 : 1350, 1 : 4050, 1 : 12150, 1 : 36450, with zero standard (Bo, assay buffer) and the other set with the highest Tg standard (300 ng/ml, Bs). Assay tubes were set up in duplicates containing µl of two Tg standards (zero and 300 ng/ml), µl of each of working Tg antibody and 125 I-Tg. The content was mixed and incubated 3 hrs at room temperature. After that 200 µl of local second antibody separating agents (sheep antimouse, normal mice serum prepared according to Shafik et al. (8) and 1 ml of 4 % PEG 6000) were added to all tubes. The tubes were mixed and incubated for 1hr at room temperature, then centrifuged at 4 o C for 20 min at 3000 rpm. Then the tubes were decanted and counted using gamma counter. Bo%, Bs% (highest standard), (Bo-Bs) / Bo% were calculated for each dilution for all mice. Two curves were plotted between each of Bo and displacement percent and different dilutions of antibody. Preparation of Radiolabeled 125 I-Tg Tracer: The preparation of radiolobeled Tg with radioactive I 125 was performed using chloramine-t oxidation method described by Hunter and Greenwood (9). To an eppendorf vial, 15 µg of Tg antigen in 10 µl of 0.5 M phosphate buffer were added. This mixture was mixed with 5 µl of NaI 125 (500 µci, 18.5 MBq). Reaction was initiated by addition of 10 µl of Ch-T (2mg/ml) in 0.05 M phosphate buffer. The reaction was allowed to proceed for 1 min and quenched by addition of 10 µl of sodium metabisulphite (30 mg/ml). Finally, µl of 2% potassium iodide in 0.05 phosphate were added as carrier. The iodination yield was estimated by paper electrophoresis. The purification was carried out by transferring the iodination mixture to the top of PD-10 column (Pharmacia). The elution was performed using 0.05M phosphate buffer containing 0.3% BSA solution, and the flow rate was adjusted to give 0.5 ml/3min. Elution pattern was monitored by counting radioactivity of fractions in a well type NaI(II) scintillation counter. Elution profile was constructed by drawing counts against fraction number. The 125 I-Tg tracer produced was characterized in terms of radiochemical yield, specific activity and non-specific binding. The tracer was tested by radioimmunoassay (RIA) technique. Preparation of Tg Standards: The preparation of working standards for Tg RIA system (ranged from ng/ml) was done by diluting Tg antigen using an assay buffer. The assay buffer contained per ml distilled 234

3 water, 5 ml phosphate buffer (0.5 M, ph 7.4), 5 ml NaCl (3M), 0.1 ml 10% triton x-, 0.1 gm sodium azide and 10 ml bovine serum albumin solution (BSA). Formulation of RIA System for Tg: The assay design can be summarized as follow: µl of Tg standards or unknown samples and µl of 125 I-Tg tracer were incubated with µl of Tg antibody (1 : 450) 3 hrs at room temperature. The separation of bound and free fractions were carried out by adding 200µl of second antibody (µl of normal mice serum 1 : 200, µl of sheep anti-mouse 1 :) and 1ml of PEG (4%) into all assay tubes. The tubes were incubated at room temperature for 1 hr and decanted carefully after centrifugation. The bound fraction was counted using gamma counter and the results were calculated using Logit-Log graph paper. Performance Characteristics of RIA System: Some studies were carried out to assure the validity of the assay such as sensitivity, precision, accuracy and method comparison. RESULTS AND DISCUSSION Three components were prepared and characterized to obtain a valid and accurate assay system. These components were polyclonal anti-tg antibody, 125 I-Tg tracer and Tg standards. The production of polyclonal anti-tg was carried out by immunization three Balb/C mice with 25µg/250µl emulsion. After an interval of 2 weeks mice were immunized with 2 booster doses of Tg immunogen. The results revealed that all mice gave positive results. The data illustrated in Fig.(1) reveal very consistent findings that can be summarized as follow: Mouse (3) gave the best immune response followed by mouse (2). Mice 1 gave the lower immune response. The titre of an antiserum was estimated by determining the percent binding of a fixed quantity of tracer with different dilutions ranged from 1/150 : 1/36450 of harvested antisera of each mouse with zero and highest standard (300ng/ml). The results illustrated in Fig.(2) show that mouse (3) gave the best displacement at dilution 1/450. % Tg Bound Mouse 1 Mouse 2 Mouse 3 1:150 1:450 1:1350 1:4050 1: :36450 Dilution of antiserum Fig. (1): Immunoresponse curve of the polyclonal antibody produced. 235

4 35 Mouse1 Mouse2 Mouse3 30 Displacement(%) :150 1:450 1:1350 1:4050 1: :36450 Dilution of antiserum Fig. (2): Displacement percent of polyclonal antibody produced. Preparation of Radiolabeled 125 I-Tg Tracer: Radiolabeled 125 I-Tg tracer was prepared using chloramine-t method and purified from radioiodination reaction mixture using PD-10 column. The purification profile was illustrated in Fig.(3). The figure shows two peaks, one shows large and somewhat sharp for 125 I-Tg with radiochemical yield 55% and radiochemical purity 96% and the other for free radioactive iodine with relatively small peak. 125 I-Tg 80 Radioactivity, Ci I Fraction Number Fig (3): Purification profile of 125 I-Tg on PD

5 Optimization for Tg using Liquid Phase RIA System: For achieving reliable assays, the general principals of the assay optimization must be followed: The development of RIA system for thyroglobulin was carried out by studying incubation time and sample volume. - Incubation time: The effect of incubation time on this system was carried out throughout 1 hr, 3 hrs and 24 hrs all at room temperature. The bound radioactivity was increased with increasing incubation time and reach the optimum after 3 hrs incubation. Therefore, it can be concluded that the optimum incubation time is 3 hrs to get a valid determination for Tg. - Sample volume: The assay procedures were performed using variable volumes of Tg standards. The results obtained show that the highest difference in binding between the different levels of Tg were found using µl of standards. Performance Characteristics of the RIA System for Tg: To assure the validity and reliability of the suggested assay, some performance characteristic studies, including sensitivity, precision (intra and inter-assay), accuracy (recovery and dilution tests), and method comparison were carried out. Sensitivity: Sensitivity of the assay or the minimal detectable concentration of the lignand (MDC) which can be distinguished from zero dose. For this purpose zero standard was set up in 20 replicates along with the other standards. The mean and standards deviation (S.D) of the counts were calculated. Then the dose corresponding to mean 2S.D (~95%) was read off the standard curve. This value was taken as sensitivity of the proposed assay. The sensitivity of this local Tg-RIA was 0.53 ng/ml. (Table 1). These data are in good agreement with that of El-Mouhty et al. (10). Table (1): The sensitivity of the assay. Mean (x) - SD of 20 Mean (x) - 2SD B/Bo % Approximate tubes sensitivity, ng/ml % 0.53 Precision: Precision is a statistical index of the ability of an assay to yield the same result when the assay is repeated on the same sample in a single assay or different assays. a- Intra assay precision (within-run): This is the ability of a method to give the same result in the same assay. It was determined for Tg from the mean of 20 pairs of tubes in a single assay, each for three polled human serum samples and the statistics were calculated for each sample. The results are illustrated in Table (2). b- Inter-assay (run to run): This is the ability of a method to give the same results for separate determinations. It was determined from the pairs of tubes in 20 different assays with the same three pooled human serum samples. The results are summarized in the same Table (2). These results are in accordance with pervious studies (11, 12) which stated that the intra-assay coefficient of variation (CV) should be less than 10% while in case of inter-assay, they reported that CV of interassay should be less than 15%. 237

6 Table (2): Intra-assay and Inter-assay precision for Tg RIA. Sample Tg ng/ml No. Intra assay Inter assay Mean SD CV % Mean SD CV % ng/ml ng/ml Accuracy: Accuracy is defined as the degree of agreement between the measured value and the true value. The assay accuracy was checked by recovery and dilution tests. Recovery Test: Recovery test measures the concentration in human specimens before and after adding known amounts of pure analyte (Tg). The difference between two measurement is expressed as a percentage of the added mass. The optimal recovery is % (13). According to Pillai and Bhandarkar, the recovery (14, 15) of an assay should be ± 15 %. The recovery data of the present study in Table (3) are in good agreement with the data of Pillai et al. (14). The data obtained reveal normal recoveries. This indicates that the matrix of standards and the sample were identical. Table (3): Recovery test for Tg RIA. Samples number Endogenous Tg (ng/ml) Standard added ng/ml Expected value (E) Observed value (O) O / E% Dilution Test: The results in Table (4) show the concentration of three human serum samples, undiluted and at various dilutions in the matrix of the assay (zero standard) to assess the linearity of the assays. Edwards (13) reported that non-linearity indicates inaccurate calibration or an inappropriate matrix or both. The results obtained maintains good linearity under dilution (11, 16). Method Comparison: The statistical analysis was carried out to compare Tg results of 20 different human serum samples obtained by commercially available kit (Siemens Medical Solutions Diagnostics) to those obtained by the present system. It was found that the present method gave clinically acceptable results. The statistical analysis showed good correlation between the results obtained from the present system and the commercially available kit, r = (Fig. 4). 238

7 Table (4): Dilution test for Tg RIA. Samples number Tg undiluted ng/ml Dilution Factor : 2 1 : 4 1 : : 2 1 : 4 1 : : 2 1 : 4 1 : 8 Expected value (E) Observed value (O) O / E % Local method, ng/ml a= b= r= Siemens method, ng/ml Fig (4): Linear regression equation and correlation coefficient (r) between the results of Tg local method and the reference method (Siemens). In conclusion, the technical simplicity of this sensitive, precise and accurate method may suggest that Tg-RIA technique should be suited for routine laboratory use and can be used as a decisive diagnostic marker for thyroid gland diseases. So that the present study establishes the possibility of preparing all the basic reagents for Tg-RIA of good quality at low cost. REFERENCES (1) L.E. Braverman and R.D. Utigen; "Fundamental and Clinical Text" 7 th Ed., Lippincott-Raven, Philadelphia (1996). (2) R.P. Ekins; Br. Med. Bull; 30, 3 (1974). (3) P.M. Daniel, M.M. Gale, L.G. Plaskett and O.E. Pratt; J. Physiol.;165, 65 (1973). 239

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