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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), 609–613.

Received 15 July 2007; accepted 15 November 2007 * Corresponding author. Email: [email protected]; 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.

RESEARCH PAPER

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 0.90–80 pg ml–1. 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 Graves’s 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): 609–613

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-Ⅲ, Beijing, China). Electric homoiothermic water bath tank was from Changan Scientific Equipment Co. (Beijing, China). Single-channel volume-adjustable pipettor (20 to 200 μl, 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 ml–1.

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 μl 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 μl 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 μl calibrators/samples and 50 μl 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 μl 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


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 µg ml–1. 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 µg ml–1 was chosen.

Concentration of antibody coated (mg l–1)

Fig.1 Effect of concentration of antibody coated

C(FT3) (ng l–1) Fig.2 Ratio of inhibition with different amounts of antibody coated Concentration of antibody coated: a, 2.35 mg l–1; b, 4.71 mg l–1; c, 11.70 mg l–1

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 µg ml–1. 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 M–1 [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


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 0.90–80 pg ml–1. 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 ml–1 serum calibrator in 10 replications. The minimal detected concentration (mean – 2SD of zero calibrator) calculated from the data is 0.33 pg ml–1.

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

Sample

Mean (pg ml–1)

Standard deviation

C.V. (%)

1 4.89 0.58 11.6 2 12.0 1.48 12.1 3 43.6 3.11 7.13

Intra-assay (n = 10)

1 4.73 0.45 9.63 2 13.2 1.97 15.0

Inter-assay (n = 30)

3 43.4 1.82 4.21

specificity of an assay. In the human body, some kinds of compounds are similar in structure. They are the potential factors to interfere with the measurement of free triiodothyronine. The structures of these compounds are shown in Fig.4.

The cross-reactant concentration causing 50% inhibition was used to calculate the cross-reactivity. The cross-reactivity was calculated according to the equation:

Cross-reactivity (%) = (concentration of free triiodothyronine at IC50)/(concentration of cross-reactant at IC50)

Different concentrations of thyronine and reverse triiodothyronine were measured as samples. The IC50 is calculated from the value measured. The cross-reactivity (%) of thyronine and reverse triiodothyronine is less than 0.01% from the experimental result. It could be proved that those cross-reactants had little effect on the free triiodothyronine measurement. 3.2.5 Effect of interferences

Sera were spiked with heparin, EDTA, bilirubin, as well as

triglyceride to study the influences from anticoagulant and serum matria on the measurement. From the experimental result, it was found that there was no significant concentration change (with a concentration deviation within 10%) in these

Fig.4 Structures of cross-reactants


sera spiked with heparin, EDTA, bilirubin, as well as triglyceride within the given concentration (heparin 40 mg ml–1, EDTA 1.25 mg ml–1, bilirubin 22 mg ml–1, as well as triglyceride 1 mmol l–1).

3.2.6 Normal range for free triiodothyronine

Forty normal specimens were obtained from the local

hospital (PLA General Hospital). These samples were tested by the ACS 180 system (Ciba Corning) and proved to be euthyroid samples. The mean value and the standard deviated value of these samples were calculated. The final range (Mean ± 2SD) was 1.4–4.2 pg ml–1. The range was in accordance with reports. It could be proved that there was little interference from the carrier proteins.

3.2.7 Comparison with other method

To compare the proposed method with method established

by Monobind Inc., 88 samples were measured simultaneously with both methods. The serum samples drawn from a population consisted of 40 euthyroid subjects, 24 patients with hyperthyroidism, and 24 patients with hypothyroidism. The relative coefficient obtained for the two methods was 0.9005 (Fig.5). This was a good evidence to prove that the method was able to distinguish the hyperthyroidism from hypothyroidism and suitable for the clinical practice. The diagnostic accordance rate of the proposed method is shown in Table 3.

All the serum samples were measured by local hospital (PLA General Hospital, China) with Ciba Corning ACS:180 (USA) and divided to be hyperthyroidism group, euthyroidism group, as well as hypothyroidism group. Free Triiodothyro- nine, free thyroxine, thyrotropin, total triiodothyronine, total

Fig.5 Correlation between the proposed methods and the method

from Monobind Table 3 Comparison of diagnose accordance rate of two methods

Hyperthyroi- dism group

Euthyroi- dism group

Hypothyroi- dism group

Diagnostic accordance rate of proposed method (%)

83.3 100 25.0

Diagnostic accordance rate of Monobind kit (%)

79.2 92.5 45.8

thyroxine were all taken into consideration. It should be noticed that, free triiodothyronine is a significant marker for the diagnostics of hyperthiroidism. The diagnostic accordance rate of the proposed method for Hyperthyroidism group was 83.3% which was much better than that the method established by Monobind Inc.. Thus, it is useful for clinical practice. The diagnostic accordance rates for hypothyroidism group were low for the two methods. It is because that, the level of free triiodothyronine may be lower[17] in those patients with severe non-thyroidism, or when the inhibition of the conversion process from thyroxine to triiodothyronine happened. So the other four parameteres for thyroid detection should be taken into consideration when these situations happened.

4 Conclusions

Measuring concentrations of free thyroid hormones is of

great importance for the diagnostics of hyperthyroidism. And it is useful in the evaluation of the treatment of the thyroid disease. It is because that the measurement is less affected by the concentration of carrier proteins and the binding capacity of these proteins than total hormone measurement.

A competitive chemiluminescent immunoassay specific for free triiodothyronine was established. 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 HRP-luminol-H2O2 chemiluminescent system with high sensitivity was chosen as the detection system in the purpose of improving the sensitivity of the method. 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 proteins[18]. Hence the carrier proteins had less effect on the detection than the traditional method that used a small analog with low molecular. It was much simpler than that in the labeled antibody mode. It provided a sensitive and useful tool for the measurement of hormones with small molecular proteins.

It should be worth noting that the anti-triiodothyronine auto-antibody[3,4,19] as well as some interference drugs may cause some abnormal result. Hence, it is necessary to combine the other four parameters (thyrotropin, total triiodothyronine, total thyroxine, free thyroxine) into consideration when some unusual situations take place.

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