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* E-mail: jmlin@mail.tsinghua.edu.cn; Tel./Fax: 0086-010-62792343 Received March 11, 2011; revised May 28, 2011; accepted July 4, 2011. Project supported by the National Natural Science Foundation of China (Nos. 90813015, 20935002).

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A Novel Chemiluminescence Immunoassay Using Solid-Phase Antigen for Free 17β-Estradiol in Human Serum

Qi, Yuanyuana,b(祁媛媛) Chen, Huib(陈惠) Lin, Zhenb(林珍) Chen, Guonanb(陈国南) Lin, Jinming*,b(林金明)

a Department of Chemistry, Fuzhou University, Fuzhou, Fujian 350002, China b Department of Chemistry, Tsinghua University, Beijing 100084, China

A high-performance chemiluminescence immunoassay, with long-term durability, good precision and time- saving, was proposed for the detection of free 17β-estradiol (E2) in human serum. Ninety-six microplates were coated with bovine serum albumin conjugated E2 antigen as solid phase for the immunoassay. The E2-BSA antigen coated on the microplate and the E2 antigen in the sample competed for the binding sites on the horseradish peroxi-dase (HRP) labeled anti-E2 antibody. Chemiluminescence reaction was subsequently carried out by HRP catalyzing luminol-H2O2 substrates, and the chemiluminescence intensity was inversely proportional to the amount of analyte in human sera samples. The concentration of immunoreagents, immunoreaction time, and other relevant variable conditions upon the immunoassay were studied and optimized. The proposed method exhibited detection limit as low as 5.94×10－3 µg•L－1 in a linear detection range from 0.01 to 1.00 µg•L－1, good recoveries between 105% and 108%, and high precision with intra- and inter-assay coefficients between 7.9% and 14.3%.

Keywords immunoassay, 17β-estradiol, antigen, indirect competitive immunoreaction, horseradish peroxidase

Introduction

Estrogen is steroid hormones naturally present in human being, and the amount varies with age, sex, diet, exercise, pregnancy and the stage of the menstrual cy-cle.1 Loss of estrogen during menopause alters the sen-sitivity of bone tissue to mechanical load.2 17β-Estradiol (E2), which is a semi-antigen small molecule, with es-trogenic activity can be used to cure some diseases and its level in serum serves as important diagnostic markers in women and men. Specially, E2 exerts both tumor ini-tiating and tumor promoting effects,3 hence it is consid-ered as a carcinogen. Sato et al. reported that chemilun- minescence immunoassay for detectable concentration of E2 in women serum was 150—1000 pmol•L－1, the E2 levels decrease to about 15—51 pmol•L－1 after meno-pause.4 The serum E2 level and its temporal change are followed closely to monitor follicle development and maturation, predict ovulation, prevent ovarian hyper-stimulation in anovulatory women undergoing induction of ovulation and determine postmenopausal status and for clinical evaluation of men with gynecomastia.5 Se-rum E2 levels are also used to assist reproductive tech-niques, such as in vitro fertilization or embryo transfer. Therefore, a high-throughput assay for the diagnosis of certain gynecological and the disorders of the endocrine system was with high value.

There were many methodologies for the determina-

tion of E2 involving gas chromatography-mass spectro- metry (GC-MS),6 high performance liquid chromatog-raphy (HPLC)7 and immunoassay, such as radio immu-noassay (RIA),8 enzyme linked immunosorbent assay (ELISA) 9 and chemiluminescence enzyme immunoas-say (CLIA).10 GC-MS and HPLC are time consuming and require expensive instruments, the detection limit of GC-MS was 0.03 µg•L－1, and the detection limit of HPLC was 23 µg•L － 1. In contrast, immunoassay method is highly sensitive and easy to perform. RIA is sensitive and reliable, but suffers from problems associ-ated with the use of radioisotopes and radioactive waste disposal. Although commercial diagnostic ELISA kits are widely used and the detection limit of the method is 7.5×10－3 µg•L－1, they are not thoroughly validated by the kit manufacturer with respect to assay accuracy, sensitivity and precision. At present, CLIA has already been used to detect E2 in sera samples, using the direct competitive CLIA, the detection limit was 0.01 µg•L－1 but the blood samples need to be extracted with diethyl ether before determinate. So this method was time-cost.11 However, the CLIA kits, which can be used to detect E2 levels in serum have not been produced yet, and no manufacturer optimizes the conditions for it.

Immunoassays are widely used in many fields rang-ing from medical diagnostics to environmental moni-toring. The principal advantage of immunoassays is the specificity of detection, which derived from the selec-

Chemiluminescence Immunoassay for Free 17β-Estradiol in Human Serum

Chin. J. Chem. 2011, 29, 2520—2524 © 2011 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.cjc.wiley-vch.de 2521

tion of antibody-antigen binding. CLIA has been inten-sively used in antigen-antibody detection, due to the sensitivity of CLIA is much higher than that of the tra-ditional ELISA. CLIA is a clinical diagnostic method with the basic principle of radioimmunoassay.12 It pos-sesses both the advantages of chemiluminescence and immunoassay with high-throughput and high selectivity, respectively.13 Therefore, to determine E2 concentra-tions in human sera, a high-throughput immunoassay method should be developed.

In this study, we reported a CLIA for E2 based on indirect competitive reaction using E2-BSA as solid phase and HRP-labeled anti-E2 antibody conjugation. The competition assay took place in a conventional 96-well microplate. E2 is a small molecule and more than 75% of which in human serum is binding with protein. Therefore it is necessary to block protein com-bination before determination. Up to now, the semi-antigen coated on the well as solid phase has not been reported at home and abroad. Compared with other reported results, we achieved the best sensitivity and detection limit.

Experimental

Apparatus and chemicals

Chemclin Micro-titer plate chemiluminescent ana-lyzer (Chemclin Biotech Co., Beijing, China), DEM-3 micro-plate washer (Tuopu Analytical Instrument Co., Beijing, China), Dragon Med mechanical pipette (Dragon Medical Co, Shanghai, China), 96-well plate (Shenzhen Jincanhua Industry), XW-80A blending shaker (Shanghai Jingke Industry Co, China), and HH.W21-Cr000 Electric homoeothermic water bath tank (Beijing Chang’an Science Instrument Company) were used in this experiment.

E2 antigen, bovine serum albumin (BSA), Tween-20, 1-ethyl-3-(3-dimethyllaminopropyl) carbodiimide hy-drochloride (EDC) and 8-(phenylamino)-1-naphthalene- sulfonicacimonosodiumsalt (ANS) were purchased from Sigma (USA). HRP-labeled anti-E2 antibody and the CL substrate solutions were obtained from Beijing Chem-clin Biotech, China.

Buffers and calibrators

The coating buffer was 0.01 mol•L－ 1 carbonate buffer (CB), pH 9.6. The blocking buffer was 0.01 mol•L－1 phosphate buffered saline (PBS) buffer con- taining 0.5 mg•mL－1 BSA and 1.0 mg•mL－1 NaN3, pH 7.4. The washing buffer was 0.05 mol•L－1 phosphate solution with 0.05% Tween-20 (PBST). Dilution solu-tion for horseradish peroxidase (HRP) labeled E2-Ab was 0.05 mol•L－1 PBS containing 15 mg•mL－1 BSA and 0.2% ANS, pH 7.4. The chemiluminescence sub-strates were luminol in 0.1 mol•L－1 Tris-HCl of pH 8.5 and H2O2 in citrate buffer of pH 4.5.

For calibration, E2 standard was prepared in PBS. The serial concentration of calibrators were determined

as 0, 0.01, 0.03, 0.10, 0.30 and 1.00 µg•L－1, assigned to S0, S1, S2, S3, S4 and S5, respectively. The prepared cali-brators were stored at 4 ℃ for further use.

Preparation of E2-BSA

A 3.5-mg BSA was dissolved in 1.0 mL of 0.9% physiological saline, firstly. Then 136 µL of 1000 mg• L－1 E2 solution and 5 mg of EDC were added into 1.0 mL of 1% ethanol. The dissolved BSA solution was added slowly into this solution, the mixture was incu-bated at room temperature for 4 h in dark. At last, the resulted solution was dialyzed with 0.01 mol•L－1 PBS (pH 7.4) under 4 ℃ for 24 h.

Preparation of solid phase antigen

E2-BSA antigen solution of 150 µL (4000 µg•L－1) was added to each well on the microplate and stored at 4 ℃ overnight. After washing twice, 350 µL of ethanol was added per well and incubated at room temperature for 30 min, then 400 µL of blocking buffer was added per well and incubated at room temperature for 3 h, re-moved the solution in the well, and allowed to stand at room temperature for 24 h till it dried. The microplate was then sealed and stored at 2—8 ℃.

Immunoreaction procedures

The calibration/sample, solid-phase antigen, and en-zyme-labeled antibody were fetched from refrigerator and put at room temperature for 15 min. 25 µL of calibrators or serum samples and 100 µL of HRP-labeled anti-E2 antibody solution were added into the solid-phase antigen in turns and mixed well (distributed by the oscillator). It was washed for 5 times after incubation under 37 ℃ for 90 min. The substrates of 100 µL were added into each well. After 5 min incubation in the absence of light at room temperature, the relative light unit (RLU) was detected using chemiluminescence analyser.

Data analysis

Standard and samples were measured in double wells, and CL intensity values were integrated. Standard curves were obtained by plotting CL intensity against the logarithm of analyte concentration and fitted to a linear equation.

Samples

The serum samples were collected from Beijing 301 Hospital (Beijing, China). After vein blood sampling, the blood was centrifugated and the sera were aspirated, subpackaged, and stored at －20 ℃ for further use.

Results and discussion

Principle

As shown in Figure 1, the E2 in sera competed for the HRP-labeled anti-E2 antibody against the E2-BSA coated on the microplate, so the amount of HRP-labeled E2 antibody combined with the solid-phase E2-BSA was

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inversely proportional to the amount of the examined E2. The concentration of immunoreagents, immunoreac-tions time and other relevant variable conditions upon the immunoassay were optimized.

Figure 1 Preparation of E2-BSA and principle of this novel method.

Optimization of CL immunoassay

In order to obtain a high-throughput determination of E2, the effect of ethanol on the preparation of E2-BSA coated solid phase, the concentration of E2-BSA, dilu-tion ratio of HRP-labeled anti-E2 antibody, protein in-

hibitor, immunoreaction time, and detection time were studied and shown in Figure 2.

Ethanol made the E2-BSA protein denatured to fix more firmly on the board during the process of coating. As can be seen in Figure 2a, the RLU was enhanced after adding ethanol. The coated E2-BSA treated by ethanol is more stable, not easily fell off, and deterio-rated by outside temperature or humidity. This treated microplate can be stored in the room temperature for a long time.

The coating concentration was an important factor in CLIA. Before coating, E2-BSA (stock solution of 1 g• L－1) was diluted by CB into 1000, 2000, 4000, 8000, and 10000 µg•L－1, respectively. The effect of different coating concentration on RLU was shown in Figure 2b. In a competitive reaction, the inhibition ratio, namely RLUS1/RLUS0 (84%) and RLUS5/RLUS0 (10%), was appropriate, indicating high sensitivity and wide liner range. Thus, the coating concentration of 4000 µg•L－1

was selected. An appropriate dilution ratio of HRP-labeled anti-E2

antibody conjugate is important for obtaining an accu-rate standard curve. Moreover, the sensitivity of a com-petitive immunoreaction is also influenced by the dilu-tion ratio of reagents. The concentration of HRP-labeled anti-E2 antibody was diluted into 1∶250, 1∶500, 1∶1000 and 1∶2000, respectively. We found that when

Figure 2 Influence of adding ethanol when the E2-BSA coated on the well (a), the coating concentration (b), the dilution ratio of HRP-labeled anti-E2 antibody (c), and the immunoreaction time (d).

Chemiluminescence Immunoassay for Free 17β-Estradiol in Human Serum

Chin. J. Chem. 2011, 29, 2520—2524 © 2011 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.cjc.wiley-vch.de 2523

the dilution ratio of HRP-labeled anti-E2 antibody was increased from 1∶250 to 1∶2000 (lower dilution ratio corresponds to higher concentration), the RLU was de-creased. Furthermore, effects of the dilution ratios on the RLU were studied. When the diluted ratio was 1∶250, inhibition ratio was lower than 1∶500 (shown in Figure 2c). So dilution ratio of 1∶500 was selected finally.

E2 is a small molecule, more than 75% E2 combined with sex hormone binding globulin in human serum. Therefore, it is necessary to fully block its binding with protein when determine the total amount of E2. So the ANS was added into the dilution solution for HRP-la-beled anti-E2 antibody.

The influence of CL reaction time was investigated by recording RLU in 0.5, 1.0, 1.5 and 2 h at 37 ℃. As can be seen in Figure 2d, RLU increased with increasing incubate time, but 2 h is time-cost, and the RLU was not much higher than 1.5 h, so incubation for 1.5 h at 37 ℃.

The records of RLU in 1—17 min interval after adding the CL substrate were compared. The CL inten-sity was increased with adding CL substrate into the microplate cell until 7 min, kept at stable intensity in the range of 7—13 min, and then began to decrease. There-fore, after adding the substrate solution, incubation for 8 min at room temperature was selected before CL detec-tion.

Methodology evaluation

Under the optimal reaction conditions, a standard curve of lg Y＝－1.8502lg X＋3.4235 (r＝－0.9981) was obtained.

The detection limit, defined as the minimal dose that can be distinguished from zero, the minimum detected concentration (mean－2S.D. of zero standard, 10 repli-cates) of E2 was 5.94×10－3 µg•L－1.

Each serum was measured ten times within one as-say to obtain the intra-assay precision. Inter-assay pre-cision was calculated by measuring three serum samples in three times assays. Good precisions were obtained. The intra- and inter-assay coefficients of variation (CV) were 7.9%, 13%, 8.8% and 14.3%, all less than 15%.

The specificity of the immunoassay depends on the antibody’s specificity. So to estimate the specificity of the anti-E2 antibody, some compounds, such as proges-terone, testosterone, estrone and estriol (E3), which have analogous structure to E2, were tested. The cross-reac- tivity (CR%) of anti-E2 antibody was calculated by the formula as following: CR%＝(concentration of E2 at IC50)/(concentration of cross-reactant at IC50). IC50 means the concentration causing 50% inhibition of RLUmax. Results showed that there was no cross-reac-tivity with them. The cross-reactivity with the related compounds was all less than 5%. Therefore, the anti-E2 antibody used in this study has high specificity for the determination of E2.

As shown in Table 1, the recoveries were measured by adding 0.04, 0.08 and 0.16 µg•L－1 E2 into a mix male serum, and then these samples were detected by this assay for three times. The recoveries for the low, medium and high concentrations of three samples were 105%, 106%, and 108%, respectively.

Table 1 Recovery of E2 spiked to the serum samples (n＝3)

Known/ (µg•L－1)

Spiked amount/ (µg•L－1)

Found/ (µg•L－1)

Recovery/%

0.0207 0.04 0.0628 105

0.0154 0.08 0.101 106

0.0291 0.16 0.203 108

Dilution test was used to verify whether the calibra-

tors had the same matrix effect with these samples to evaluate the reliability of the proposed assay. A serial dilution of serum E2 in high-value was prepared in ma-trix of E2 standard solution by dilution ratio of 1/2, 1/4, 1/8 and 1/16, respectively. Then the samples were ana-lyzed using the proposed CLIA. X axis represents the dilution ratios, and Y axis represents the measured con-centration of E2. The linear equation for E2 was Y＝724.9X－37.57, and linear correlation coefficient R was 0.9994. The good linear correlation suggested that there was no matrix effect between the sample and the stan-dard solution in the case of high concentration of E2. It shows that results obtained by this CLIA were reliable.

For the stability study, solid phase coated E2-BSA, calibrators, HRP-labeled anti-E2 antibody, buffers, and chemiluminescence substrate were stored at 4 and 37 ℃ for 3 and 7 d, respectively. After that, they were used to perform the assay. As shown in Table 2, there was no obvious effect on the assay results.

Table 2 Stability of the reagents

Time/d Temperature/℃ RLUS1/RLUS0 r CV/%

4 87.6% 0.9985 3.0 1

37 86.2% 0.9991 7.6

4 87.0% 0.9970 4.1 3

37 85.5% 0.9944 5.9

4 86.6% 0.9982 4.3 7

37 85.3% 0.9977 5.2

Application

Concentration of E2 in the random serum samples was measured with this novel chemiluminescence im-munoassay, and the results obtained were compared with that from a commercial ELISA kit. A good corre-lation was obtained with a satisfied R2 of 0.9717, as shown in Figure 3. It indicated that the proposed method was a convenient, economical and superior method for detecting E2 in human sera.

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Figure 3 Comparison between results measured by the CLIA and ELISA.

Conclusions

A high-throughput detection for E2 was constructed. The conditions of the antibody labeling, and the pa-rameters of the competitive immunoreactions were op-timized. The luminol-H2O2-HRP system with high sen-sitivity was utilized as the detection system. Both the recoveries and precision were satisfied. Compared with other methods, this technique only required small vol-umes of samples and did not need expensive equipment. Furthermore, it exhibited high-throughput, and low de-tect limits. Sera samples can be detected directly with-out pretreatment, which indicates its potential use in clinical diagnosis.

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(E1103111 Li, L.)