Micro-plate Chemiluminescence Enzyme Immunoassay for Clinical Determination of Progesterone in Human Serum

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CHINESE JOURNAL OF ANALYTICAL CHEMISTRYVolume 36, Issue 6, June 2008 Online English edition of the Chinese language journal Cite this article as: Chin J Anal Chem, 2008, 36(6), 729734. Received 18 September 2007; accepted 20 November 2007 * Corresponding author. Email: wang-x@mail.tsinghua.edu.cn This work was supported by the National Key Technology R & D Program of China (863 Program, No. 2006AA02Z4A8) and the Innovative Research Team of the University of China (No. IRT0404). Copyright 2008, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved.RESEARCH PAPERMicro-plate Chemiluminescence Enzyme Immunoassay for Clinical Determination of Progesterone in Human Serum REN Shi-Qi1, Wang Xu2,*, TANG Bao-Jun3, HU Guo-Mao3, LI Zhen-Jia3, CHEN Guo-Nan1,LIN Jin-Ming21 College of Chemistry and Chemical Engineering, Fuzhou University, Fuzhou 250002, China 2 Department of Chemistry, Tsinghua University, Beijing 100084, China 3 Beijing Chemclin Biotech Co. Ltd., Beijing 100094, China Abstract: A high-throughput, simple and rapid chemiluminescence enzyme immunoassay (CLEIA) was developed for the clinical determination of progesterone in human serum, using luminol-hydrogen peroxide as chemiluminescence system catalyzed by horseradish peroxidase (HRP). The solid phase of anti-progesterone antibody was prepared by immunoreaction between anti-progesterone polyclonal antibody and donkey anti-rabbit IgG, i.e. second antibody, which had been physically absorbed on thewells of polystyrene microplate and was used as a universal solid phase. The effect of various factors, such as the dilution ofimmunoreagents, chemiluminescence substrate, chemiluminescence reaction time and incubation condition were examined and optimized. The optimal dilutions of anti-progesterone antibody and HRP-progesterone conjugate were 1:10000 and 1:15000, respectively. The II substrate was chosen and the luminescence was determined after 10 min incubation. The immunoreacted samplewas incubated in water bath at 37C for 1 h. The assay was evaluated with sensitivity as low as 0.08 g l1. The relative standard deviation (RSD) was less than 15% for both intra- and inter-assay precision. The recoveries of three different spiked concentrationsamples were 101%, 101% and 94.4%, respectively. This proposed method has been successfully applied to the clinical evaluation ofprogesterone in 36 human sera. The results showed a good correlation with the accredited radioimmunoassay (RIA) with a correlativecoefficient of 0.9502. Key Words: Progesterone; Human serum; Chemiluminescence enzyme immunoassay 1 Introduction Progesterone is a natural steroid hormone that is secreted by ovary and placenta, with a molecular weight of 314 Daltons. It acts in conjunction with estrogen for the maintenance of menstrual cycle and female sexual characteristics[1]. The dynamic monitoring of progesterone in serum has been used to identify ovulation, evaluate ovarian function and investigate the mechanism of various steroid prophylactics and anti-early-pregnancy drugs[2,3]. In addition, it has been indicated recently that the reproductive function was just one part of the functions of progesterone and that progesterone can be also synthesized and secreted partly by nervous system and affect the neural structure and function[4].The neuroprotective function of progesterone on brain injury and its action mechanisms has become one of the popular topics now[5].Progesterone has been lucubrated in various regions such as medicine, biology, environment and food safety, and several types of analytical methods have also been developed for the determination of progesterone according to its significant effects on the well being of human body and mind. Current methods for the quantification of progesterone can be mainly divided into two categories: chromatography methods and immunoassay methods. Chromatography methods[6,7], as the reference methods for the measurement of progesterone in REN Shi-Qi et al. / Chinese Journal of Analytical Chemistry, 2008, 36(6): 729734 human serum, have numerous predominant advantages of high accuracy, selectivity, reproducibility and sensitivity. However, chromatography methods have some disadvantages such as the requirement of expensive instrument, intensive labour and complicated pretreatment of samples. Therefore, the chromatography methods could not meet the requirements of clinical measurement such as rapidity, convenience, low cost and high throughput. In contrast, immunoassay methods are specific, easy to use and cost-effective and have been widely used for the clinical determination of progesterone. Radioimmunoassay (RIA)[8], enzyme linked immunoabsor- bent assay (ELISA)[9], fluoroimmunoassay (FIA)[10], time resolved fluoroimmunoassay (TRFIA)[11] and chemilumine- scence immunoassay (CLIA)[12] have also been reported for the evaluation of progesterone in human serum. RIA has the drawbacks of environmental contamination, short useful-life of reagents and tardiness of determination with the use of radioisotopes, which restrict its further application. The sensitivities of ELISA and FIA are limited and they cannot be used for the measurement of low concentration of progesterone in serum. The TRFIA and CLIA overcome the disadvantages of the forementioned immunoassay methods and are automated completely abroad. However, these imported diagnostic reagents generally require the specially matching instrument; both the reagents and instruments are expensive and their patent protection also restricted their applications. Chemiluminescence enzyme immunoassay (CLEIA), which integrates the advantages of immunoassay and chemiluminescence determination such as high specificity and throughput, rapidity and convenience in operation and relatively simple and inexpensive instrumentation[1316], is a type of clinical diagnostic technique, which is currently applied worldwide. To the best of our knowledge, the study on the methodology of progesterone CLEIA has barely been reported previously. One of the reasons is that progesterone comes under the drug type of small molecule, and displacement reagent should be used for the release of progesterone from its binding proteins during the determination of total progesterone, as most of the progesterone exists in the protein-bound forms and only about 1% amount exists in the free form in human serum[17].Another reason is that the preparation and selection of the specific antibody and enzyme conjugate to the hormones of small molecule compared with the proteins of big molecule. In the present study, using the luminol-H2O2 chemilumine- scence system catalyzed by HRP, a CLEIA for the determination of progesterone in human serum was developed with the optimization of immunoreagents dilution and immunoreaction conditions, and the methodology of CLEIA was also evaluated. This proposed method has been applied for the clinical evaluation of progesterone in human sera and obtained a good relation with the commercially available progesterone RIA kits.2 Experimental 2.1 Apparatus and chemicals A chemiluminescence microplate reader (Luminis Light BHP9504 device from Beijing Hamamatsu Photon Techniques Inc., China) was used in this study. The washing step was accomplished using an automated microplate washer (DEM-3, Beijing Tuopu Analytical Equipment Co. Ltd., China). A vortical mixer (XW-80A, Beijing Xinjingke Biotechnology Co. Ltd., Beijing, China) was used to prepare solutions. The microplate shaker (WZ-2A, Beijing Xinjingke Biotechnology Co. Ltd., Beijing, China) was employed to blend the solutions in microwells. The incubation procedures were carried out using electric homoiothermic water bath tank (HH. W21-Cr000, Beijing Changan Scientific Equipment Co., China). The single-channel volume-adjustable micropipette (20 to 200-l, Dalong Medical Equipment Co. Ltd., Shanghai, China) plus tips were also used in this study. Horseradish peroxidase-labeled progesterone conjugate was purchased from Fitzgerald Industries International, Inc. (USA). Donkey anti-rabbit IgG, polyclonal anti-progesterone antibody, chemiluminescence substrate solutions, steroid-free human serum and progesterone RIA kits were provided by Beijing Chemclin Biotech Co. Ltd. (Beijing, China). Progesterone, testosterone, 17-estradiol, estriol, cortisol, bovine serum albumin (BSA), hydrolysed gelatin, 8-anilino-1-naphthalene- sulphonic acid (ANS) and Tween-20 were purchased from Sigma Chemical Corporation (USA). All other chemical reagents from Beijing Chemical Plant (China) were of analytical grade. The quadratic distilled water was used throughout the experiments. The coating solution was 0.06 M citrate salt buffer, pH 4.8. The blocking solution was 0.05 M phosphate-buffered saline (PBS), pH 7.4, with 1% (w/v) BSA and 0.1% (v/v) proclin-300. The washing solution was 0.05 M PBS, pH 7.4, containing 0.05% (v/v) Tween-20. The assay buffer was 0.05 M PBS, pH 7.4, supplemented by 1% (w/v) BSA, 0.8% (w/v) hydrolyzed gelatin and 0.1% (v/v) proclin-300. 2.2 Experimental methods 2.2.1 Preparation of calibratorsA total of 5 mg of progesterone was accurately weighed and dissolved in 5 ml ethanol as a stock solution of 1.0 g l1 and stored at 20 C. Subsequently, 5 ml working solution of 10 mg l1 was prepared by diluting 50 l stock solution with 50% (v/v) ethanol water-solution. Finally, the calibrators (60, 21, 7.0, 2.0 and 0.5 g l1) were prepared by the dilution of working solution with steroid-free human serum, which was also used as zero calibrator. The prepared calibrators were stored at 4 C. REN Shi-Qi et al. / Chinese Journal of Analytical Chemistry, 2008, 36(6): 729734 2.2.2 Purification of donkey anti-rabbit IgG The saturated ammonium sulfate (SAS) precipitation method was used for the purification of donkey anti-rabbit IgG. Certain volume of donkey anti-rabbit antiserum was diluted with physiological saline and placed on the blender of magnetic force; the same volume of SAS solution was slowly added to make the saturation of 50% and the last admixture of antiserum and SAS solution was placed at room temperature for 2 h. A centrifuge of low-temperature was used at 4 C for 30 min and the supernate was discarded. The obtained deposition was dissolved in physiological saline and SAS solution was slowly added to make the saturation of 35%. The centrifugation and dissolution steps were repeated, and then the SAS solution was slowly added to make the saturation of 33%. The centrifugation step was performed further. The deposition obtained at this time was dissolved in 0.02 M PBS (pH 7.4), dialyzed and then centrifuged for the removal of slight deposition. The final product was the solution of purified donkey anti-rabbit IgG. Its absorbency was determined using ultraviolet (UV) spectrophotometer at 280 nm with 1 cm of photic distance, and the concentration of IgG was calculated according to the molar adsorption coefficient method. An in-split charging product was obtained after the addition of 0.05% (v/v) biological preservative of proclin-300 and stored at 20 C. 2.2.3 Preparation of solid-phase anti-progesterone antibody The indirect immobilizing method was used for the preparation of solid phase anti-progesterone antibody. In brief, the wells of the microplates were coated with 150 l donkey anti-rabbit IgG (as secondary antibody, 8 mg l1) in coating solution. The sealed plates were allowed to stand at 4 C for 18 h. Then, the secondary antibody coating solution was removed and the plates were blocked with 350 l blocking solution for 3 h at room temperature. Afterwards, the blocking solution was discarded and 150 l anti-progesterone polyclone antibody in assay solution (dilution ratio of 1:100000) was added, followed by overnight incubation at 4 C. After another washing step, the plates were tapped against the absorbent paper and dried for 24 h at room temperature. Finally, the dried plates were vacuumized and stored at 4 C. 2.2.4 Immunoassay procedure The prepared microplate coated with anti-progesterone antibody was utilized. A total of 50 l calibrator solutions or serum samples and 100 l HRP-P conjugate were added into the wells of the microplate, shook for 0.5 min to blend those reagents well, and then incubated at 37 C for 1 h. After the competitive immunoreaction, it was washed five times using the automated microplate washer and the remnants solution was discarded by tapping the plates against the absorbent paper. A total of 100 l solution of chemiluminescence substrate was added and incubated in the dark place for 10 min at room temperature. Then, the microplate was placed into the chemiluminescence microplate reader and the emitted photons were measured. 2.2.5 Data management The attached software was used to process those data obtained by BHP9504 chemiluminescence microplate reader. Calibration curves were drawn by plotting logitY against the logarithm of X and fitted to the linear equation of logitY logX, in which the value of logitY was calculated according to the formula: logitY = ln[X/(1X)]Where, Y = B/B0, B0 is the CL intensity of the zero calibrator, B is the CL intensity of other calibrators or samples, and X isthe concentration of analyte. 2.3 Sample collection The human serum samples were collected from both inpatients and outpatients of Chinese Military Hospital of 301. The blood sampled by venipuncture was transferred into tubes containing coagulant. After centrifugation, the sera obtained were stored at 20 C until required. 3 Results and discussion 3.1 Optimization of dilution of immunoreagents Chessboard titration was applied to screen out the optimal dilutions of anti-progesterone antibody and HRP-progesterone conjugate. Table 1 presents the results. The luminescence intensity of zero calibrator (B0) and the ratio of luminescence intensity between B1 and B0 (B1/B0), B5 and B0 (B5/B0) were mainly considered in this experiment for optimizing the dilution of immunoreagents. In competitive immunoassay, it was demonstrated that the lower the value of B1/B0, the more sensitive the method was. Also, the value of B5/B0 was kept at approximately 15% for the competitive immunoassay, as B5 was so small that the deviation would augment if the value of B5/B0was lower than 10%. Considered the sensitivity, reliability and kinetic range of the assay totally, the dilution of 1:100 000 for the anti-progesterone antibody and the dilution of 1:15 000 for HRP-progesterone conjugate were selected. 3.2 Comparison between different formulas of chemiluminescence substratesThe selection of chemiluminescence system and chemilumin- REN Shi-Qi et al. / Chinese Journal of Analytical Chemistry, 2008, 36(6): 729734 Table 1 Chessboard titration Luminescence intensity (n = 3) Binding percentage (%, n = 3)Dilution of anti-progesterone antibody Dilution of HRP-progesterone B0 B1 B5 B1/B0 B5/B01/10000 826632 809597 444976 98 54 1/10000 1/20000 386635 284509 129363 74 33 1/10000 712980 684978 311260 96 44 1/50000 1/20000 329833 250415 57349 76 17 1/5000 746724 696897 195281 93 26 1/10000 468152 410401 95714 88 20 1/15000 328977 273286 56134 83 17 1/100000 1/20000 215074 180793 32072 84 15 escence substrate is crucial for the development of CLEIA.Luminol-H2O2 chemiluminescence system catalyzed by HRP has been extensively applied. Luminol chemiluminescence substrate is generally composed of two sections, solution A and solution B. Solution A and solution B should be mixed together in fixed proportion just before addition to wells, and the microplate should be kept in dark place after the addition of chemiluminescence substrate to wells. Generally, the strongest chemiluminescence signal appears during 5 min to 10 min and keep constant in a period of time, according to the stability of substrate and the characteristic of analyte. Two different formulas of Luminol chemiluminescence substrates, prepared by the authors of this study and marked as substrate I and substrate II, respectively, were compared with each other. The mixed volume proportion of solution A and solution B was 100:1 for substrate I, 1:1 for substrate II. The drift of the kinetic curve along the luminescence time was examined using substrate I and substrate II, and the results are shown in Fig.1. The kinetic curve was extremely drifted along the luminescence time when substrate I was used (shown in Fig.1a). In this case, the significant intra-assay variation would easily appear if the chemiluminescence reaction time was not in tight control. Using substrate II, the kinetic curves at 5, 10, 15 and 20 min were examined and there was almost no drift. In addition, the luminescence intensity using substrate II was stronger than that with substrate I for the same calibrator at the same time. Theoretically, the higher the luminescence intensity, the lower the relative analytical error was. On the basis of the overall consideration of the above factors, substrate II was chosen for the determination of chemiluminescence in this study. 3.3 Effect of incubation condition on chemiluminescence intensity and precision The three incubation conditions, incubation in water-bath at 37 C, oscillation at room temperature (23 2) C and standing at room temperature, were compared and their effects on chemiluminescence intensity and precision were studied. For all experiments, the incubation time was set as 1 h. It was shown that the luminescence intensity of zero calibrator, for which the incubation was performed in water-bath at 37 C, was higher compared with other incubation conditions such as oscillation at room temperature and standing at room temperature. This is attributed to the fact that immunoreaction can be significantly accelerated by temperature and can be brought into equilibrium more easily. Also, it was presented that the RSD for water-bath at 37 C and that for oscillation at room temperature were quite close to each other and both of them were lower than the RSD for standing at room temperature. It may be because the sensible raising of temperature and gentle shaking can increase the probability of collision between antibody and antigen, and thereby equilibrium of immunoreaction can be quickly achieved.Fig.1 Drift of the kinetic curve along the luminescence time (a) substrate of I (b) substrate of II , 5 min;, 10 min;, 15 min;, 20 minREN Shi-Qi et al. / Chinese Journal of Analytical Chemistry, 2008, 36(6): 729734 Shaking also can improve the uniformity of the wells of the microplate and therefore diminish the variations in measurement. By considering the luminescence intensity and the RSD of the assay, water-bath at 37 C was selected as the optimal incubation condition. 3.4 Optimization of chemiluminescence reaction timeA platform of chemiluminescence lighted by HRP-Luminol- H2O2 system appears with the stabilizing agent and chemiluminescence intensifier. The intensity of chemiluminescence was the highest and can keep constant during the range of the platform. There was a disparity in the period of the chemiluminescence platform according to the category and formula of reagent from different substrate manufacturers. The effect of chemiluminescence reaction time was examined, using the substrate II. It can be seen that the intensity of chemiluminescence sharply increased with the chemiluminescence reaction time in the range of 010 min. From 10 min to 20 min, a platform of chemiluminescence intensity appeared and during this period of reaction time, chemiluminescence intensity was higher and quite stable. After 20 min, the chemiluminescence intensity rapidly decreased. Therefore, 10 min of reaction time was chosen as the optimal reaction time for the purpose of rapid analysis. 3.5 Examination of the stability of calibratorsThe accelerating experiment was performed at 37 C to examine the stability of calibrators. The chemiluminescence intensity of every calibrator and the measured value of concentration in all samples were considered for the comparison of these calibrators stored at 4 C all the time with those stored at 37 C for 3, 5 and 7 days, respectively, and the results showed no obvious differences in terms of chemiluminescence intensity and measured concentration. Thus, the characteristics of the calibrators will remain stable for a long time at 4 C. 3.6 Evaluation of the assay 3.6.1 Calibration curvesOn the basis of the previous optimized conditions, the CLEIA for the determination of progesterone in human serum was developed. The concentrations of the calibrators were 0.5, 2.0, 7.0, 21 and 60 g l1, respectively. Calibration curves were obtained by plotting logit Y against the logarithm of X and fitted to the linear equation of logit Y = 1.6412logX + 1.1514. The linear correlative coefficient was 0.9972, indicating that the proposed method can be used for the accurate quantification of progesterone. 3.6.2 Sensitivity A total of the chemiluminescence intensity measurement of 10 replicates was performed and the mean value and the standard deviation were calculated. The sensitivity was determined by calculating the minimum amount of progesterone that could be markedly distinguished from S0 (mean binding at S0 2SD). The sensitivity experiment was repeated five times, and the mean value was 0.08 g l1.3.6.3 Recovery The proposed method was applied for the determination of progesterone in human serum and the accuracy was studied through a recovery experiment. Different amounts of P were added to the mixed male human serum and the theoretical concentration of progesterone for these samples were 5.0, 10 and 20 g l1, respectively. These samples and the mixed male human serum were simultaneously analyzed using the proposed CLEIA. This experiment was repeated five times and the average recoveries of low, middle and high concentration samples were 101%, 101% and 94.4%, respectively. 3.6.4 Precision Three spiked samples of different concentrations were involved and every sample was determined 10 wells inside a plate at one time. This experiment was repeated thrice and the variations in intra-assay and inter-assay were evaluated. As shown in Table 2, the RSD of intra-assay and inter-assay for all these samples were lower than 15%. 3.6.5 Dilution test A dilution test was used to verify whether the calibrators had the same matrix with these samples to evaluate the reliability of the proposed assay. In this experiment, a serum, containing high-content progesterone, was diluted 2-, 4-, 8-, 16- and 32-fold with steroid-free human serum and then the samples were analyzed using the proposed CLEIA. X axis represents the dilution of these samples, and Y axis represents the measured concentration of progesterone. A curve was obtained by plotting Y against X and fitted to the linear equation of Y = 19.591X +Table 2 Precision of proposed progesterone chemiluminescence enzyme immunoassay Sample Times of replicate determination (n)Found of progesterone concentration (g l1)RSD (%)Intra-assay 1 10 5.3 10.2 2 10 9.3 11.0 3 10 15.1 11.7 Inter-assay 1 30 5.2 11.7 2 30 9.2 11.8 3 30 15.4 11.0 REN Shi-Qi et al. / Chinese Journal of Analytical Chemistry, 2008, 36(6): 729734 5050Concentration of progesterone at ICCross-reactivity %Concentration of cross-reactant at IC0.6722. The coefficient of linear correlation was 0.9962, indicating that the linear correlation between the dilution and the measured concentration was good and the analytical results obtained by this developed CLEIA was reliable. 3.6.6 Specificity Specificity of the immunoreaction should be one of the most important indices to evaluate the immunoassay and was usually represented by cross-reactivity. The lower the cross-reactivity, the more specific the antibody was, consequently the immunoassay becomes more reliable and acceptable. The cross-reactivity of anti-progesterone antibody was assessed by the criteria of Abraham[18]. Some clinical samples, which concentration of these related steroids was much higher than that of the normal physiological concentration, was obtained by spiking several structural analogues of progesterone into the steroid-free human serum and then the progesterone and its analogues concentration causing 50% inhibition were determined by the proposed CLEIA. The cross-reactivity of the anti-progesterone antibody with these structural analogues of progesterone was calculated, according to the equation: The results are shown in Table 3 and the cross-reactivity with all the examined steroids was less than 1%. Therefore, this selected anti-progesterone antibody was sufficiently specific for the accurate measurement of progesterone in human serum. 3.7 Quantitative determination of progesterone in human serum: correlation with RIA Thirty-six human sera obtained from the Chinese Military Hospital of 301 were determined by both the accredited RIA and the proposed CLEIA, and the results are shown in Table 4. The value measured by the proposed CLEIA was marked as Xaxis. The value measured by the accredited RIA was marked as Y axis. The correlative curve for the values of progesterone concentration measured by the two methods was plotted and the equation of linear regression was Y = 1.0809X 0.1537. The coefficient of correlation was 0.9502, i.e. r = 0.9502. 4 Conclusions A high-throughput, simple and rapid CLEIA was developed for the clinical determination of progesterone in human serum, using HRP-H2O2-Luminol chemiluminescence system. The indirect immobilization method was used for the preparation of solid phase anti-progesterone antibody. The dilution of the immunoreagents and the incubation condition were optimized in the experiments. The assay was evaluated and appears to be quite sensitive, specific and accurate. This proposed method has been successfully applied for the clinical evaluation of progesterone in 36 human sera. The results showed a good correlation with the accredited radioimmunoassay, and it was demonstrated that the proposed assay could meet the demands in clinical measurement. Table 3 Cross-reaction of some steroids with anti-progesterone antibody in proposed CLEIA Steroids Cross-reactivity (%) Progesterone 100 Testosterone < 0.06 Androstenedione 0.03 Dihydrotestosterone 0.02 Cortisol 0.26 Estrone 0.10 17-estradiol < 0.10 Estriol < 0.01 Table 4 Comparison between results measured by accredited RIA and proposed CLEIA Concentration of progesterone (g l1) Concentration of progesterone (g l1) Concentration of progesterone (g l1)Sample CLEIA RIA Sample CLEIA RIA Sample CLEIA RIA U1 0.94 0.45 U13 7.99 2.85 U25 1.81 1.41 U2 0.69 0.38 U14 4.16 4.13 U26 4.81 3.60 U3 1.35 1.19 U15 17.74 13.60 U27 8.08 8.54 U4 0.78 1.02 U16 6.09 1.69 U28 11.12 8.66 U5 5.07 6.12 U17 4.72 4.76 U29 6.22 6.75 U6 16.19 18.60 U18 1.44 2.55 U30 3.30 0.82 U7 2.29 3.01 U19 26.22 16.90 U31 20.93 19.10 U8 0.90 0.60 U20 0.82 0.47 U32 17.96 16.90 U9 22.52 21.50 U21 1.20 1.25 U33 2.58 1.32 U10 0.54 1.23 U22 1.15 0.95 U34 1.04 2.09 U11 1.27 1.30 U23 2.51 2.07 U35 4.59 5.91 U12 0.97 0.71 U24 24.51 24.60 U36 1.56 1.29 References[1] Li Z J, Wang R Z. 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