Determination of Free Triiodothyronine by Chemiluminescent Enzyme Immunoassay

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  • CHINESE JOURNAL OF ANALYTICAL CHEMISTRY Volume 36, Issue 5, May 2008 Online English edition of the Chinese language journal

    Cite this article as: Chin J Anal Chem, 2008, 36(5), 609613.

    Received 15 July 2007; accepted 15 November 2007 * Corresponding author. Email:; Tel/Fax: +86 10-62792343 This work was supported by the National Project of Scientific and Technical Supporting Programs of China (No. 2006BAK02A13). Copyright 2008, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved.


    Determination of Free Triiodothyronine by Chemiluminescent Enzyme ImmunoassayLIN Zhen1,2, WANG Xu2, REN Shi-Qi1,2, CHEN Guo-Nan1, LI Zhen-Jia3, LIN Jin-Ming2,* 1 Department of Chemistry, Fuzhou University, Fuzhou 350002, China 2 Department of Chemistry, Tsinghua University, Beijing 100084, China 3 Beijing Chemclin Biotech Co., Ltd. Beijing Academy of Science and Technology, Beijing 100094, China

    Abstract: The methodology based on chemiluminescent enzyme immunoassay was established for the determination of free Triiodothyronine (FT3). The HRP-luminol-H2O2 chemiluminescent system with high sensitivity was chosen as the detection system. The linear range was 0.9080 pg ml1. Under the selected conditions, the CVs of intra- and inter- assays were less than 15%. When the method was applied to detect free triiodothyronine in human serum, the diagnostic accordance rate of the method for hyperthyroidism was 83.3%. Compared with the commercial kit, the correlation coefficient was 0.9005. All the conditions chosen were to maintain the equilibrium between the free and the bound hormones to improve the validity of the experimental result. Key Words: Free triiodothyronine; Chemiluminescent; Enzyme immunoassay; Analog

    1 Introduction

    Triiodothyronine (T3) is the one of the main thyroid hormones that acts on the body to increase the basal metabolic rate, affect protein synthesis and fat or carbohydrate metabolism. 65% of triiodothyronine is secreted by the thyroid gland. 35% of triiodothyronine is produced from peripheral conversion of thyroxine. Most of the thyroid hormone circulating in the blood is bound to carrier proteins (such as thyroid binding globulin (TBG), albumin and pre-albumin). Only a very small fraction of the circulating hormone is free and considered to be biologically active. Hence, measuring concentrations of free thyroid hormones is of great diagnostic value.

    Elevated concentration of triiothyronine indicates hyperthyroidism and Gravess disease. As for the measurement of free triiodothyronine (FT3) is concerned, there are two main difficulties encountered. One is that the high sensitivity is needed owing to the low concentration of the free hormone in human blood. The other is the exclusion of some interference factors from the blood matrix. Those

    interference factors were from the carrier proteins[1,2], auto-antibodies[3,4], as well as some drug interferences.

    At present, the main methods for FT3 measurement include equilibrium dialysis or ultrafiltration[5], two-step method[6], as well as one-step, and labeled-antibody assay[7,8]. The reference methods such as equilibrium dialysis or ultrafiltration are first utilized as a physical process to separate free hormone from protein-bound hormone, then measured the free hormone by radio immunoassay. These methods are time-consuming and are unsuitable for clinical practice. Antibody immune extraction is another method to eliminate the interference of the serum proteins. In this method, the specific antibody is immobilized on the tubes or wells. Then the hormone is extracted from the serum. After a washing step, the various proteins in the serum are removed. The tracers are added into the tubes or wells subsequently. It is easier for operation than equilibrium dialysis. However, the precision of the method is not good enough especially when large amounts of samples are measured. The method, which utilizes labeled antibody, needs large amounts of antibody and one kind of antibody with high purity is necessary.

  • LIN Zhen et al. / Chinese Journal of Analytical Chemistry, 2008, 36(5): 609613

    A method combining the specialty of the antibody and the high sensitivity of chemiluminescent was developed to meet the need of clinical practice. T3 in the sample was competitive with HRP labeled T3 derivative for the limited site of the anti T3 antibody immobilized on the solid phase. The horseradish peroxidase enzyme-luminol-H2O2 chemiluminescent system with high sensitivity was chosen as the detection system. The light intensity developed was in reverse proportion to the T3 present in the samples. T3 was decorative with large molecular protein to avoid its binding with carrier protein. Compared with other assay, this method had a good correlation. The method developed was stable and precise enough for clinical practice. 2 Experimental 2.1 Apparatus and reagents

    96-well micro-plate was from Shenzhen Jincanhua Industry Co. Ltd. (Shenzhen, China). A chemilunescence micro-plate reader was purchased from Hamamatsu photons Technology Co. Ltd. (Beijing, China). Automatic plate washer was obtained from Topu Analytical Instruments Co. Ltd. (Type: DEM-, Beijing, China). Electric homoiothermic water bath tank was from Changan Scientific Equipment Co. (Beijing, China). Single-channel volume-adjustable pipettor (20 to 200 l, Finland) plus tips was used in the study. Ultraviolet-visible spectrophotometer was purchased from Jinghua Instrument Co. Ltd. (Shanghai, China). The shaker (Xinjingke Biotechnology Co. Ltd., Beijing, China) was used to mix the solutions.

    Anti-triiodothyronine antibody was purchased from Chinese Institute of Biological Products. T3 antigen was purchased from Sigma Chemical Co. (USA). Substrates including luminol, enhancer, as well as H2O2 were obtained from Monobind Inc. (USA).

    The coating buffer was 0.06 M citric acid buffer (pH 4.8). The washing buffer was Tris-HCl with 0.5% (v/v) Tween-20. The blocking buffer was 0.02 M PB (pH 7. 4) with 1% (w/v) BSA and 2.5% (w/v) cane sugar.

    2.2 Experimental methods 2.2.1 Standard preparation

    The calibrators were prepared by spiking different amounts

    of T3 (which was dissolved in DMF solution) into the hormone free serum. The calibrators were stored at 4 C to maintain the equilibrium between the free and the bound hormone. Then the concentration was calibrated against national calibrators five times.

    2.2.2 Purification of anti-T3 antibody

    The antibody was mixed with 35% (v/v) saturated (NH4)2SO4. The precipitation was separated from the solution by centrifugation. Then the precipitation was dissolved in salt solution (containing 0.9% (w/v) NaCl). The antibody was purified again with 35% (v/v) saturated (NH4)2SO4. Subsequently, the solution was dialysed and the concentration of the antibody was measured by ultraviolet-visible spectrophotometer. The final concentration of the antibody was 5.50 mg ml1.

    2.2.3 Immobilization of anti-triiodothyronine antibody on the micro-plate

    Anti-triiodothyronine antibody was diluted by the coating

    buffer. The solution with a volume of 100 l was pipetted into each well on the plates. The plate was placed still for one night to make sure that the antibody was immobilized on the plate. 300 l of the blocking buffer was added into each well and the plate was put at 37 C for 1 h to block the active sites on the plate. Subsequently, the solution in the well was aspirated and the plate was made dry. Finally, the plate was vacuumized and stored for further use.

    2.2.4 Chemiluminescent immunoassay for free triiodothyronine in human serum

    50 l calibrators/samples and 50 l horseradish peroxidase

    enzyme labeled triiodothyronine analog were added into the wells of the coated plate and incubated at 37 C for 45 min. All unbound components were aspirated by the washer and washed 5 times using washing buffer. After having been washed, the plates were inverted and hit on absorbent paper until no moisture appears. 100 l of substrate (luminol solution with H2O2 and enhancer) for HRP was added to each well subsequently. After an enzymatic reaction, the light emitted from the chemiluminescent reaction was measured by the chemilunescence micro-plate reader and was proportional to the free triiodothyronine content in each well.

    2.2.5 Data analysis

    Dose responsive curves were obtained by plotting the logit

    of chemiluminescence intensity (in relative light unit, RLU) against the logarithm of analyte concentration:

    Logit = ln[Y/(1 Y)] Where, Y in the formula is calculated from the other formula (Y = B/B0). B0 and B stands for the binding rate of the zero calibrator and other calibrators (or analytes)[11]. 3 Results and discussion 3.1 Optimization of the conditions of chemiluminescent immunoassay

  • LIN Zhen et al. / Chinese Journal of Analytical Chemistry, 2008, 36(5): 609613

    3.1.1 Kinetics of immunoassay

    30 min, 45 min, 60 min and 180 min were chosen as the immunoassay time for the method. Taking both RLUmax and IC50 into consideration, the immune reaction almost reached equilibrium when the incubation time was longer than 45 min due to the little change of the light intensity. So, 45 min was chosen as the best incubation time to improve the efficiency of the assay.

    3.1.2 Effect of concentration of coated antibody on measurement

    Generally speaking, the amount of antibody in a

    competitive immunoassay should be appropriate. And the relative light intensity of one method should be taken into consideration. The effect of different concentration of the coated antibody was investigated. The RLUmax was measured at different conditions. The relationship between the concentration of the antibody and RLUmax is shown in Fig.1. It can be seen that the RLUmax changed little when the concentration of the coated antibody was higher than 2.35 g ml1. And there was no obvious difference in the inhibition ratio (the inhibition ratio = RLUs/RLUmax) of all the calibrators when the higher concentration of the coated antibody was used (Fig.2). RLUmax and RLUs referred to the light intensity of the zero calibrators and the other calibrators, respectively. So the concentration of the coated antibody of 2.35 g ml1 was chosen.

    Concentration of antibody coated (mg l1)

    Fig.1 Effect of concentration of antibody coated

    C(FT3) (ng l1) Fig.2 Ratio of inhibition with different amounts of antibody coated Concentration of antibody coated: a, 2.35 mg l1; b, 4.71 mg l1; c, 11.70 mg l1

    3.1.3 Effect of concentration of HRP-labeled analog

    The competition between analog and FT3 in the sample is very important for an immunoassay. So the concentration of the HRP-labeled analog should be investigated carefully. In the experiment, the HRP-labeled analog was diluted by the potassium phosphate buffer with 1 BSA and 0.5 nM EDTA when the concentration of the coating antibody was 2.35 g ml1. The data are shown in Table 1.

    The different RLUs got with different dilution of the HRP-labeled analog were 1208681, 862421, 700281 and 400126, respectively. RLU and the inhibition percentage were both taken into consideration when the conditions of the experiment were optimized. Hence, HRP-labeled analog dilution of 1:100 was chosen as the final concentration for the method.

    3.1.4 Effect of carrier proteins

    The main proteins that can bind with triiodothyronine were

    thyroxine binding globulin[12,13], thyroxine binding pre- albumin, as well as human serum albumin. Although the concentration of human serum albumin is 2000-fold more than thyroxine binding globulin, the binding capability (Ka = 1 109 M1 [14]) of thyroxine binding globulin with triiodothyronine is much stronger than human serum albumin. It was reported that 75% of triiodothyronine was bound with thyroxine binding globulin. Thus, the binding between triiodothyronine and thyroxine binding globulin should be taken into consideration first when the structure of triiodothyronine analog was designed.

    The binding between triiodothyronine and proteins can be reduced through the modification of the structure of the analog[15]. The immune reaction occurred at 37 C [12,16] with the buffer (pH 7.4), which was the physical temperature and pH. Under these conditions, there was little interference on the equilibrium between the bound and free hormone. Thus, it can assure the accuracy of the experimental result.

    3.1.5 Effect of concentration of Tween-20

    Tween-20 is a kind of surfactant which can reduce

    non-specific interactions. So it is commonly used in immunoassay. However, when the concentration of the surfactant is too high, it may be inhibit the immune reaction that takes place between the analyte and the antibody.

    Table 1 Effect of concentration of HRP labeled T3 analog Diluted proportion 1:100 1:200 1:400RLUS1/RLUS0 0.83 0.78 0.68 RLUS2/RLUS0 0.67 0.54 0.45 RLUS6/RLUS0 0.25 0.16 0.08

  • LIN Zhen et al. / Chinese Journal of Analytical Chemistry, 2008, 36(5): 609613

    The effect of different concentration of Tween-20 was investigated. When the concentration of Tween-20 0.01% was higher than 0.01%, the RLU of the calibrators was reduced. Thus the concentration of Tween-20 0.01% was selected as the optimal concentration.

    3.2 Evaluation of the method

    3.2.1 Dose-responsive curve

    Under the optimal conditions, dose-responsive curves

    obtained with chemiluminescence detection are presented in Fig.3. The higher the concentration of the free triiodothyronine in samples, the less amount of the horseradish peroxidase enzyme labeled analog binding with the antibody immobilized on the solid phase. Thus, the lower relative light intensity was obtained. The linear ranger of the method was 0.9080 pg ml1. 3.2.2 Sensitivity of the method

    The sensitivity is defined as the minimal dose that can be

    distinguished from zero. The minimum detected concentration of FT3 was ascertained by determining the variability of the 0 pg ml1 serum calibrator in 10 replications. The minimal detected concentration (mean 2SD of zero calibrator) calculated from the data is 0.33 pg ml1.

    3.2.3 Precision of the method

    The precision is of great importance for a qualitative

    analysis. The within and between assay precision of the method was determined by the analysis on three different concentrations of the specimens. Each specimen was analyzed 10 times. The mean values, the standard deviation as well as the coefficient of variation for each sample are presented in Table 2. 3.2.4 Specificity

    The importance of specificity for a method has often been

    stressed. The specificity of the antibody will greatly affect the

    Fig.3 Dose response curve for FT3 chemiluminescent enzyme immunoassay

    Table 2 Precision of microplate chemiluminescent immunoenzyme- tric FT3 assay


    Mean (pg ml1)

    Standard deviation



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