microplate chemiluminescent enzyme immunoassay for the quantitative analysis of free...

CHINESE JOURNAL OF ANALYTICAL CHEMISTRY Volume 35, Issue 11, November 2007 Online English edition of the Chinese language journal

Cite this article as: Chin J Anal Chem, 2007, 35(11), 1541–1547.

Received 15 May 2007; accepted 19 July 2007 * Corresponding author. Email: [email protected] This work is supported by the National Natural Science Foundation of China (No. 20621703). Copyright © 2007, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved.

RESEARCH PAPER

Microplate Chemiluminescent Enzyme Immunoassay for the Quantitative Analysis of Free Prostate-Specific Antigen in Human SerumShi Gen1, Tang Bao-Jun2, Wang Xu3, Zhao Li-Xia1, Lin Jin-Ming1,3,* 1State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China

2Beijing Chemclin Biotech Co. Ltd., Beijing 100094, China 3Department of Chemistry, Tingshua University, Beijing 100084, China

Abstract: A microplate chemiluminescent enzyme immunoassay method was established with high sensitivity for the quantitative analysis of free prostate specific antigen (f-PSA) in human serum. The immunoassay used alkaline phosphatase (ALP) as the labeled enzyme and highly sensitive ALP-(3-(2'-spiroadamantane)-4-methoxy-4-(3''-phosphoryloxy) phenyl-1,2-dioxetane (AMPPD) chemilumincence reaction as detection system. Several physicochemical parameters such as incubation time, immunoreaction sequence, and detection time were optimized. The linear range of proposed method for f-PSA was 0.15–20 ng ml–1 with a correlation coefficient of 0.998. The detection limit was 0.01 ng ml–1. Inter-assay and intra-assay C.V. was below 7%. The recovery was 88%–108%. Cross-reactivity of normal tumor markers in human serum, such as CA50, CA125, CA15-3, CA242, CEA and AFP, were also studied. It showed that the specificity was good. The stability of these markers was examined for 3 days/5 days/7 days under 4 °C and 37 °C to ensure the feasibility of commercial use: C.V. was below 6%, liner correlation coefficient was above 0.998. Compared with CLIA kit, which purchased from Monobind Inc., USA, and these markers showed good correlation. Analysis results showed that this method was stable and reliable. It can be used for the development of commercial kit and also in the clinical analysis of f-PSA for the auxiliary diagnosis of prostate cancer. Key Words: Free-prostate-specific antigen (f-PSA); Prostate cancer; Chemiluminescent enzyme immunoassay; Tumor marker

1 Introduction

Prostate specific antigen (PSA) is a 30–33 kDa single-chain glycoprotein secreted by the epithelial cell of human prostate[1–7]. After secreted by the prostate, PSA becomes an important protein-composition of seminal fluid, which exists in free form with the concentrations of 0.5–5.0 g l–1[8]. PSA binds with other protein molecule and forms complexed-PSA (c-PSA)，, it is then released into blood at very low level. It exists in human prostate, seminal fluid, and serum, and very low level PSA had been detected in female serum[3]. PSA level is usually below 4 ng ml–1 in normal male's serum, whereas high PSA level is found in the serum of prostate

cancer (PCa) patients. In addition, with the increase age of normal males or in the serum of benign prostate hyperplasia (BPH) patient, the PSA level is increased. Therefore, the PSA concentration in the range of 4–10 ng ml–1 is considered as a “diagnostic gray zone”[1,5,6,10,11]. To improve the efficiency of detecting methods, different diagnostic indices, such as PSA rate (PSAR), PSA density (PSAD), age-specific reference ranges (ASR-RS)[11,13] ,etc, were posed by experts. But these indices had some limitations in the evaluation of PCa. It was reported that the free-PSA (f-PSA) level in the serum of PCa patient was lower than that in BPH patients or normal people. A novel clinic index of f-PSA percent was recommended, which was more specific for the evaluation of PCa and could

SHI Gen et al. / Chinese Journal of Analytical Chemistry, 2007, 35(11): 1541–1547

reduce the limitations of clinic screening. The methods commonly used for the measurement of t-PSA and/or f-PSA are radioimmunoassay (RIA)[13], immunoradiometric analysis (IRMA), enzyme immunoassay (EIA), enzyme linked immunosorbent assay (ELISA)[5,6], chemiluminescence immunoassay (CLIA), time-resolved fluorescence immunoassay (TRFIA)[1,9,14], and electrochemiluminescence immunoassay (ECLIA)

Chemiluminescent immunoassay was more widely accepted by researchers and clinical analysts because of its high sensitivity, wide linear range, and easy operation and automation. In our previous study, a microplate chemiluminescent immunoassay method for the determination of f-PSA in human serum was developed by combining the high sensitivity of chemiluminescence with the high specificity of immunoreaction, in which alkaline phosphatase (ALP) was used as tracer and AMPPD as substrate[15,16]. This method has been used to detect C-peptide[16] and E2

[17]. The analysis system is stable and reliable and can be used for the development of commercial diagnostic kits, which is highly significant in the clinical analysis of f-PSA in serum and for the diagnosis of PCa. 2 Experimental 2.1 Instruments and reagents

BHP9504 Micro-titer plate chemiluminescent analyzer (from Beijing Hamamatsu Photons Technology Co. Ltd., Beijing, China), DEM-3 micro-plate washer (Tuopu Analytical Instrument Co. Ltd., Beijing, China), DragonMed mechanical pipette (adjustable volume, Dragon Medical Co. Ltd., Shanghai, China), 96-well plate (Shenzhen Jincanhua Industry. Ltd.), and electric heat constant temperature incubator (Beijing Chang'an Science Instrument Company) were used in this experiment. PSA was purified from human seminal plasma in PLA General Hospital. Couple of PSA monoclonal antibodies was purchased from Fitzgerald Co. Ltd., USA. AMPPD was purchased form DPC Co. Ltd., USA. Chemiluminescent immunoassay kit for the analysis of f-PSA was provided by Monobind Inc., USA.

The coating buffer for microplate was 0.06 M citrate buffer (pH 4.8). Blocking buffer was 0.02 M phosphate buffer (PBS) with 1% (w/v) Bovine serum albumin (BSA) and 5% cane sugar. The washing buffer was 0.02 M PBS. The sample dilution buffer was calf serum.

2.2 Principle

The sandwich immunoreaction method was used for the

detection of f-PSA. An anti-PSA antibody was coated on the surface of polyvinyl microplate by physical adsorption to form the solid-phase antibody. Then the antigen and

enzyme-labeled anti-f-PSA antibody were added into the microplate in turns. After immunoreaction, the antibody- antigen-enzyme-labeled antibody complex was formed on the surface of the microplate. The unbinding antigen or enzyme-labeled antibody was removed by washing. Finally, substrate solution was added. The relative light unit (RLU) was measured using BPH9504 analyzer, and the concentration of sample was quantitated.

2.3 Experimental method 2.3.1 Preparation of solid phase antibody

An anti-PSA antibody solution of 100 μl (5 μg ml–1) was added to each well on the microplate and stored at 4 °C overnight. After washing thrice, 300 μl blocking buffer was added per well and incubated at 37 °C for 2 hours, removed the solution in the well, and allowed to stand at room temperature for 24 hours till it dried. The microplate was then sealed and stored at 2–8 °C.

2.3.2 Preparation of enzyme-labeled antibody

The method of glutaraldehyde cross-link[16] was used to label ALP to the anti-f-PSA IgG. Anti-f-PSA IgG of 4.0 mg and ALP of 2.0 mg were used in this experiment. Anti-f-PSA IgG and ALP were added into sodium chloride solution (0.9%, w/w), the mixture volume was controlled at 0.50 ml. And then 0.50 ml glutaraldehyde (1%, w/v) was added, mixed for 15 min with magnetic agitator at room temperature, reacted for 4 hours in the absence of light and finally, 0.10 ml ethanolamine (1.0 M) was added and incubated for 2 hours at room temperature. Dialysis was used to remove free ALP or IgG. The fractions containing HRP-labeled IgG were pooled, equal volume glycerol and 1% (w/v) BSA were added, uniformly mixed, and stored at –20 °C.

2.3.3 Preparation of calibration samples

Calf serum was used as the matrix serum, which was inactivated at 56 °C for 30 min. f-PSA was added to matrix serum to prepare the calibration, the series concentrations were 0(S0), 0.15(S1), 0.6(S2), 1.7(S3), 3.3(S4), 8.3(S5), and 20(S6) ng ml–1. The calibration was calibrated by national standard calibration (15044-200702), subpackaged, and stored for use.

2.3.4 Immunoassay procedures

The calibration/sample, solid-phase antibody, and enzyme-labeled antibody were fetched from refrigerator and put at room temperature for 15 min. The calibration of 50 μl or sample and 50 μl labeled-antibody solution were added into


the solid-phase antibody in turns and mixed well (distributed by the oscillator). It was washed for 5 times after incubation under 37 °C for 60 min. The substrates of 50 μl were added into each well. After 30 min incubation in the absence of light, the RLU was detected using chemiluminescence analyzer, and the f-PSA level of the serum was calculated according to the standard curve.

3 Results and discussion 3.1 Effect of coating concentration

The coating concentration was an important factor in

chemiluminescence immunoassay. The antibody was diluted into 2.0, 4.0, 5.0, 6.0 and 10 μg ml–1 with 0.06 M citrate buffer (pH 4.8). The coating procedure is performed as in section 2.3.1.

The effect of different coating concentration on the RLU is shown in Table 1. The RLU of S6 increased with the increase of coating concentration in the range of 2–5 μg ml–1, decreased after the coating concentration of 5 μg ml–1, and the value of S1/S0 and S6/S0 was max at this coating condition. Thus, the coating concentration of 5 μg ml–1 was selected.

3.2 Effect of enzyme-labeled antibody

Enzyme-labeled antibody was diluted at the ratio of 1:200, 1:500, 1:1000, and 1:2000 with buffer. The effect of the diluted ratio of enzyme-labeled antibody on the RLU was studied. When the diluted ratio was 1:500, RLU was about 300000, which could be fit in with our CL analyzer, S/N was 3.45, and correlation coefficient was above 0.998. Therefore, 1:500 ratios were selected in our research.

Table 1 Effect of coating concentration

RLU Coating concentration (μg ml–1) S0 S1 S6 S1/S0 S6/S0

Correlation coefficient

2 317 15567 414357 49 1307 0.9290 4 309 45697 698536 148 2260 0.9966 5 323 53516 773537 165 2395 0.9989 6 386 44893 748116 116 1938 0.9973 10 506 51183 685771 133 1355 /

3.3 Effect of detection time

Different times were selected to detect the RLU of S6 after the addition of substrate into the microplate. As shown in Fig. 1, the RLU increased in the range of 5–30 min during 30–90 min, then observed a platform of RLU, and slowly decreased after 90 min. Therefore, 30–90 min detection time was selected in this experiment.

Fig.1 Kinetic curve of AMPPD-ALP chemiluminescence system 3.4 Effect of immunoreaction step

One-step and two-step immunoreaction procedures had significant effect on the sensitivity and reaction time in chemiluminescent enzyme immunoassay. The operation procedure of one-step method is described in section 2.3.4. The procedure of two-step method is as follow: (1) first, the solid-phase antibody, enzyme-labeled antibody solution, sample, and calibration were collected to equilibrate for 15 min at room temperature; (2) a total of 50 μl sample or

calibration was added into microplate, mixed with shaking, and incubated for 60 min at 37 °C; (3) it was washed for 5 times with PBS (0.02 M, pH 7.4); (4) a total of 50 μl enzyme-labeled antibody solution was added into the microplate, mixed with shaking, and incubated for 60 min at 37 °C; (5) it was again washed for 5 times with PBS; (6) a total of 50 μl substrate (AMPPD) was added into the microplate and incubated for 30 min at room temperature in the absence of light; (7) finally, RLU was detected using the analyzer. Analytical result was obtained according to the standard curve.

The sensitivity of both immunoreaction procedures was approximately the same. In one-step immunoreaction procedure, the reaction time was 90 min, S/N was 6.20, the RLU of S6 was 279010, and correlation coefficient was 0.9994. In two-step immunoreaction procedure, the reaction time was 150 min, S/N was 6.21, the RLU of S6 was 297256, and correlation coefficient was 0.9994. The reaction time of two-step immunoreaction procedure was 60 min higher than that of one-step immunoreaction procedure, so one-step immunoreaction procedure was chosen. 3.5 Effect of incubation conditions

Two incubation conditions, shaking at room temperature and without shaking at 37 °C, were investigated. The RLU was little higher under the conditions of shaking e.g. the RLU of S6 was 280434 and 240486. The incubation time for both conditions was 60 min, the S/N was 4.62 and 4.41, and the correlation coefficient was 0.9989 and 0.9989. The reaction


rate of antibody and antigen depended on the rate of diffusion of antibody and antigen to the surface of the microplate, while the speed of diffuse depended on the concentration of antibody/antigen and the concentration grads between the medium and the surface of the microplate without shaking. Relative movement of solution and microwell surface that sped up during shaking resulted in the increase of immunoreaction rate. In this experiment, 37 °C without shaking was chosen as the incubation condition because of easy control and good reproducibility. 3.6 Methodology parameters 3.6.1 Standard curve and sensitivity

After all the parameters were optimized, 0.15–20 ng ml–1 concentration was chosen as the detected range to fit in with the clinical needs. The standard curve based on the logarithm of the concentration of calibration and RLU was set. The

linear equation was log[Y] = a + log[X], correlation coefficient was above 0.998, S/N was about 6.20, and the RLU of S6 was about 280000.

The detection limit of f-PSA, defined as the minimal dose that can be distinguished from zero and the minimum detected concentration (mean + 2SD of zero standard, 10 replicates), was 0.01 ng ml–1, which was lower than that of RIA (the sensitivity of BNIBT f-PSA KIT was 0.1 ng ml–1) and ELISA (the sensitivity of Monobind f-PSA ELISA was 0.05 ng ml–1). 3.6.2 Precision

The intra- and inter- CV were studied by analyzing three different concentrations of quality control (QC) serum in the range of 1.07–1.61 ng ml–1, 3.51–5.27 ng ml–1, and 15.15–22.72 ng ml–1, respectively. Once with 8 duplicates was for intra-CV and nine times in four days was for inter-CV. The intra- and inter-CV shown in Table 2 were all below 7%.

Table 2 Inter-assay and Intra-assay variability (C.V.) for f-PSA

1.07–1.61 (n = 8) 3.51–5.27 (n = 8) 15.15–22.72 (n = 8) QC range (ng ml–1) Average (ng ml–1) C.V. (%) Average (ng ml–1) C.V. (%) Average (ng ml–1) C.V. (%)

1 1.38 3.76 4.57 2.43 19.73 3.52 2 1.38 2.29 4.44 1.74 18.63 2.22 3 1.34 4.55 4.36 1.29 18.89 2.24 4 1.30 2.43 4.13 0.89 17.71 2.06 5 1.25 4.50 4.26 5.91 17.26 6.42 6 1.32 4.57 4.38 5.61 17.87 6.92 7 1.30 4.01 4.27 3.90 19.50 1.52 8 1.39 3.43 4.50 3.33 21.40 1.45 9 1.32 3.58 4.33 3.18 20.13 1.44

Total Average (ng ml–1) 1.33 4.36 19.01

C.V. (%) 3.47 3.06 6.94

3.6.3 Accuracy

The recovery experiment was operated through tow evaluations: (A) The serum sample and calibrator (S1, S4 and S6) were mixed in equal volume and detected the f-PSA level in duplicate; (B) The different concentration of f-PSA was

added into serum and detected in duplicate. The recovery was calculated by the ratio of detected value to the expected value of mixed sample. The results in Table 3 indicate that the recovery was 88%–108%. The recovery in experiment B was slightly lower than that in experiment A, which was probably resulted from the different matrices.

Table 3 Comparisons between the results of two different recovery experiment s for f-PSA

Recovery experiment A (%) Recovery experiment B (%) Low level Middle level High level Low level Middle level High level

1 103.8 109.6 104.0 96.5 93.4 94.4 2 111.7 95.1 97.5 99.7 83.7 85.3 3 / / / 91.8 87.6 90.3

Average (%) 107.8 102.4 95.9 96.0 88.2 90.0

3.6.4 Specificity

Six normal tumor markers in human serum, such as CA50, CA125, CA15-3, CA242, CEA and AFP, were added into the serum at the different concentration of 10–100 folds normal level in human serum to detect their cross-reaction with f-PSA. The result in Table 4 shows that the f-PSA had either little or

no cross-reaction with the other tumor marker.

Table 4 The analytic results for the specificity of f-PSA CLIA Detected marker Labeled level (U ml–1) Detected level (ng ml–1)CA50 140 N/A CA125 2500 0.03 CA15-3 500 0.01 CA242 600 N/A CEA 162 ng ml–1 N/A AFP 1600 ng ml–1 N/A


3.6.5 Soundness

A serum (concentration is known) was fold-diluted by the S0 for 5 times, and the serially diluted serums were detected. The regression analysis of the detected value and expected value showed that the linear correlation coefficient was above 0.999, which indicated the soundness of this study.

3.6.6 Stability

The components of a kit, such as solid-phase antibody, enzyme-labeled antibody solution, calibration, washing buffer, and substrate were separately placed under the conditions of 4 °C and 37 °C n for 3, 5 and 7 days for the investigation of stability. The result in Table 5 shows that all the components of the kit were stable during the investigation period.

Table 5 Stability test results of analytic system at 4°C and 37 °C

Item RLU C.V. (%) Temp. (°C) Time (h) S0 S1 S6

S/N Sensitivity (ng ml–1)

correlation coefficient Low level Middle level High level

72 492 3876 243052 7.9 0.01 0.9996 2.91 2.87 2.60 120 279 2993 234112 10.7 0.01 0.9989 5.99 2.49 4.00

4 168 422 4197 242697 10.0 0.02 0.9996 4.94 3.39 5.38

72 456 3198 275882 7.0 0.01 0.9995 3.58 3.93 2.14 120 230 2979 276212 13.0 0.01 0.9995 2.07 2.80 2.07

37

168 183 1914 197315 10.5 0.01 0.9986 5.29 4.85 4.07

3.6.7 Comparison of different quantitative analysis kit for f-PSA

Compared with the RIA, ELISA, CLIA, and ECLIA kits, the sensitivity of the proposed method was 0.01 ng ml–1, as

shown in Table 6 (refer the inserts of the kit). The f-PSA level in normal human serum was below 2.0 ng ml–1, which was about 5–40% of the total-PSA level. The detection range of 0.2–20 ng ml–1 was available for clinical application.

Table 6 Comparison of different methodology parameters for f-PSA

Factory Assay Principle Tracer Solid phase Sensibility (ng ml–1) Linear range (ng ml–1)Beijing North Institute of Biological Technology

RIA Sandwich 125I Tube 0.1 0.3–15

Institute of isotopes IRMA Sandwich 125I Tube 0.02 0.1–50 BioCheck Inc. EIA Sandwich horseradish peroxidase (HRP) Microplate 0.1 1.0–25 Monobind Inc. ELISA Sandwich horseradish peroxidase (HRP) Microplate 0.052 0.5–10 Monobind Inc. CLIA Sandwich horseradish peroxidase (HRP) Microplate 0.025 0.5–10 DiaSorin CLIA Sandwich Isoluminol magnetic particle / / Beijing Chemclin bio-tech. Inc.

CLIA Sandwich ALP Microplate 0.01 0.2–20

3.6.8 The analytical results of the methodology comparison of clinical serum

The analytical results obtained for the clinical serum of 98 cases and 40 cases (provided by the 301 hospital) using the proposed method were compared with Piaison f-PSA ECLIA and Monobind f-PSA, respectively. The linear equation was Y = 1.5081x + 0.1065 and Y = 1.1953x – 0.6485, and correlation coefficient was 0.976 and 0.933, respectively.

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