Anal. Chem. 2004, 76, 6166-6171
Correspondence
Preparation of Steroid Antibodies and Parallel Detection of Multianabolic Steroid Abuse with Conjugated Hapten Microarray Hongwu Du†,‡,§ Yuan Lu,‡ Weiping Yang,‡ Moutian Wu,| Jun Wang,⊥ Shan Zhao,‡ Mangeng Pan,‡ and Jing Cheng*,†,‡,§,#
Department of Biological Sciences and Biotechnology and Institute of Biomedicine, Tsinghua University, Beijing 100084, P.R. China, National Engineering Research Center for Beijing Biochip Technology, Beijing 102206, P. R. China, China Doping Control Center, Beijing 100061, P. R. China, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, College of Chemistry, Peking University, Beijing 100871, P. R. China, and State Key Laboratory of Biomembrane and Memborance Biotechnology, Beijing 100084, P.R. China
A conjugated hapten microarray based on miniature immunoassay for fast and multiplex detection of anabolic steroids is reported for the first time. This preliminary study investigated the possibility of using a microarray technology as a multisteroid detection assay. The microarray system used eight monoclonal antibodies raised against three steroid conjugates, 4-androsten-4-chloro17β-ol-3-one, 1,5r-androsten-1β-methyl-17β-ol-3-one, and 5β-androsten-1-en-17β-ol-3-one, which were conjugated to BSA by the active ester method. In addition to 4 commercial conjugated haptens, 18 steroid-BSA conjugates were synthesized and from all these a conjugated hapten microarray was fabricated. The analyzed substances included 42 types of anabolic steroid reference materials and 28 positive urine samples. Of these, 24 anabolic steroids and 12 positive urines were successfully detected. All anabolic steroids are synthetic derivatives of testosterone. In 1935, testosterone was first isolated and discovered to be capable of increasing protein synthesis in target tissues.1 This discovery has led to the synthesis of many testosterone derivatives. Although testosterone and its derivatives were originally used for therapeutic diagnosis, such as stress and hirsutism,2,3 more and more elite athletes and teenagers abuse them to improve sport performance and to modify physique. In the last six Summer Olympic Games, nearly 53% (26/49) of all positive cases of * Corresponding author. Tel.: +86-10-80726868. Fax: +86-10-62773059. E-mail:
[email protected]. † Department of Biological Sciences and Biotechnology, Tsinghua University. ‡ National Engineering Research Center for Beijing Biochip Technology. § Institute of Biomedicine, Tsinghua University. | China Doping Control Center. ⊥ Peking University. # State Key Laboratory of Biomembrane and Memborance Biotechnology. (1) Ruzicka, L.; Wettstein, A. Helv. Chim. Acta 1935, 18, 986. (2) Farta, L. I.; Kushlinskii, N. E. Probl. Endokrinol. 1986, 32, 25-30. (3) Akyuz, S.; Pince, S.; Hekin, N. J. Clin. Pediatr. Dent. 1996 20, 219-223.
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prohibited substances were identified as steroid abuse. In general, the ratio of steroid abusers varied between 4 and 12% among male and 0.5 and 2% among female adolescents.4 Despite anabolic steroid abuse being a major public health problem, few people take this problem seriously.5 Steroid abuse is claimed to have serious side effects. For example, males who abuse anabolic steroids may have reduced sperm production, have shrunken testicles, and generate the irreversible enlargement of their breasts. Females may develop masculine characteristics, such as deepening of the voice and excessive body hair. Many steroids are now widely available and ∼40 types have been prohibited by the International Olympic Committee (IOC).6 To ensure public safety and to prevent the problem from worsening, it is important to inform the public of the potential adverse effects of steroid abuse along with the introduction of regular screening of these prohibited substances. Immunology and separation assays have been successfully employed for the detection of steroids in the clinical laboratory, but there are some limitations for large-scale screening. Immunoassay has been traditionally performed on an individual sample in respect of a single analyte.7 While a definitive separation assay employing gas chromatography/mass spectrometry (GC/MS) can identify minute amounts of analyte, its use as a screening tool is limited since it requires expensive instrumentation and timeconsuming procedures of sample preparation. In 1991, Ekins suggested that quantitative immunoassays could be developed by the use of microspots of antibodies on nonporous solid supports, which permitted the development of “multianalyte” immunoassay systems integrating ultrasensitivity with simulta(4) Bahrke, M. S.; Yesalis, C. E.; Brower, K. J. Child Adolesc. Psychiatr. Clin. N. Am. 1998, 7, 821-838. (5) Leshner, A. L. Anabolic Steroid Abuse; Research Report Series; National Institute on Drug Abuse: Rockville, MD, 2000. (6) The 2004 Prohibited List. Published by World Anti-Doping Agency, Montreal, Canada. http://www.wada-ama.org/docs/web/ standards_harmonization/code/list_standard_2004.pdf (Accessed June 2004). (7) Engvall, E.; Perlman, P. Immunochemistry 1971, 8, 871-874. 10.1021/ac049159a CCC: $27.50
© 2004 American Chemical Society Published on Web 09/15/2004
neous measurement of multiple analytes.8 Recent advancement in multianalyte biochip technology has made Ekin’s suggestion a reality. More practical examples have been reported in this area including serodiagnosis for autoantibodies,9 detection of infectious diseases,10 clinical evaluation for cancer markers,11 detecting allergen-specific serum immunoglobulins,12 and analysis of rheumatoid diseases.13 For routine screening and high-throughput test of steroids, a conjugated hapten microarray has been produced in our laboratory. Using a contact microarray printer, bovine serum albumin (BSA)-conjugated steroids were immobilized at discrete locations on the surface of homemade mercapto-modified glass slides. Competitive immunoassays were then performed. The bound steroid antibodies were subsequently detected with fluorescentlabeled antibodies. The signal pattern of fluorescent was imaged using a confocal scanner. To date, through careful selection of pairs of conjugated antigens and antibodies, this new microarray can differentiate up to 24 anabolic steroids and 12 urine metabolites. MATERIALS AND METHODS Chemicals. Four conjugates (digoxin-3-BSA, estradiol-3BSA, progesterone-11-BSA, testosterone-3-BSA), and their antibodies were purchased from Fitzgerald (Concord, MA). Eight steroid-BSA conjugates were synthesized. Cy3 goat anti-mouse mAb (1 mg/mL) were purchased from Amersham Pharmacia (Buckinghamshire, England). Forty-two reference materials of standard anabolic steroids and 28 positive urine samples from persons who had consumed steroids were supplied by the China Doping Control Center (CDCC, Beijing, China). Unless otherwise specified, all chemicals used were of reagent grade. Preparation of Mercapto-Modified Slides. Mercapto-modified glass slides were used as the solid supports. The selected slides were cleaned with chromic acid for 6 h, followed by copious rinsing with deionized water. The slides were dried under a stream of nitrogen and then dipped into absolute alcohol for 30 min. The cleaned slides were silanized in a working solution (1% 3-mercaptopropyl trimethoxysilane, 95% ethanol, and 16 mM acetic acid, pH 4.5) for 30 min. After the surface was modified, the solution of 95% ethanol and 16 mM acetic acid was used to rinse the slides three times. The slides were placed in a vacuum oven at 150 °C for 2 h to complete the preparation process (Figure 1). The finished slides can be stored in a desiccation box at room temperature for up to 2 months. Protein Preparation for Chip Printing. Eight steroids were made immunogenic and suitable for immobilization on microscope slides by coupling to a protein carrier (BSA) through the active ester method.14,15 With the help of a bifunctional linker (isobutyl chloroformate), the activated haptens were conjugated to BSA via (8) Ekins, R. P.; Chu, F. W. Clin. Chem. 1991, 37, 1955-1967. (9) Robinson, W. H.; DiGennaro, C.; Hueber, W.; Haab, B. B.; Kamachi, M.; Dean, E. J. Nat. Med. 2002, 8, 295-301. (10) Mezzasoma, L.; Bacarese-Hamilton, T.; Di Cristina, M.; Rossi, R.; Bistoni, F.; Crisanti, A. Clin. Chem. 2002, 48, 121-130. (11) Balachandra, K.; Laisupasin, P.; Dhepakson, P.; Warachit, J.; Jantraraksri, U.; Issaragrisil, S.; Yang, X. L.; Hus, G. X. Asian Pac. J. Allergy Immunol. 2003, 21, 171-178. (12) Jahn-Schmid, B.; Harwanegg, C.; Hiller, R.; Bohle, B.; Ebner, C.; Scheiner, O.; Mueller, M. W. Clin. Exp. Allergy 2003, 33, 1443-1449. (13) Urbanowska, T.; Mangialaio, S.; Hartmann, C.; Legay, F. Cell Biol. Toxicol. 2003, 19, 189-202.
Figure 1. Surface groups of mercapto-modified slide.
Figure 2. Sample matrix and array image after preparation. (A) Sample matrix of the microarray. NI, normalize index, mouse IgG (1 mg/mL); NC, negative control, printing solution; SC1, testosterone3-BSA; SC2, methyltosterone-3-BSA; SC3, 5β-androsten-1-en-17βol-3-one-3-BSA; SC4, testosterone-17-BSA; SC5, methyltestosterone17-BSA; SC6, 5β-androsten-1-en-17β-ol-3-one-17-BSA; SC7, 4-chlorodehydro-methyltestosterone-1-BSA; SC8, 1,5R-androsten-1β-methyl17β-ol-3-one-17-BSA; SC9, 4-androsten-4-chloro-17β-ol-3-one-7BSA; SC10, prosterone-11-BSA; SC11, estradiol-3-BSA; SC12, digoxin-3-BSA; HI, human IgG; SSA, sheep serum albumin (0.5 mg/ mL). (B) Array image after preparation.
a two-step reaction. All these steroid haptens were chosen for conjugation at positions 1, 3, 7, and 17, respectively, to preserve the specific chemical moieties in rings A or D. Preparation of Conjugated Hapten Microarrays. Conjugated haptens, as well as different controls (mouse IgG, BSA, human IgG, sheep serum albumin), were printed on prepared mercapto slides using computer-controlled high-speed robotics (PixSys 5500, Cartesian Technologies, Inc., Irvine, CA). Samples were transferred from the 384-microtiter plates to the glass slides using stainless steel pins (SMP3, TeleChem International, Inc. Sunnyvale, CA). Arrays consisted of a 9 × 9 matrix, which included 12 steroid conjugate haptens printed in 3 replicates. BSA, human IgG, and sheep serum albumin were printed as negative controls (Figure 2). The mouse IgG acted as a reference and a normalization index for the data evaluation, which would help to compare the microarray variance between batches. All printed substances were dissolved in printing solution (50 mM phosphate-buffered saline, 40% glycerol). Cy5-BSA (5 µg/mL) was added to the printing buffer to help assess an equable distribution between the different spots.16 Printing was performed in a chamber at room temperature and 50% humidity. Such conjugated hapten microarrays can be stored at room temperature for up to 3 months. Development of Monoclonal Antibodies. Female Balb/C mice were immunized in a two-week protocol by intraperitoneal (14) Hosoda, H.; Fukuda, K.; Gotoh, Y. Chem. Pharm. Bull. (Tokyo). 1991, 39, 2373-2377. (15) Vincze, I.; Hackler, L.; Szendi, Z.; Schneider, G. Steroids 1996, 61, 697702. (16) Xie, W. Z.; Wang, D.; Du, H. W.; Cheng, J. Prog. Biochem. Biophys. 2002, 39, 311-315.
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Table 1. Antibody Clones and Their Corresponding Antigen Conjugates Analyzed by Chip hapten-carrier conjugates
code of clone (total, 18)
4-androsten-4-chloro-17β-ol-3-one-7-BSA 5β-androsten-1-en-17β-ol-3-one-17-BSA 4-androsten-17R-methyl-17β-ol-3-one -17-BSA
C19F1-C19F4, C2D11, C1E2, C1E6 D21A1-D21A5 M6B31-M6B36
injection of steroid-BSA conjugates. All mice, which showed positive responses against target steroids (ELISA dilutions >80 000), were used for the cell fusion experiments. The mice were killed, and their splenocytes were isolated and fused with myeloma cells. Positive cells were obtained by screening four separate analytes through ELISA assay. The criteria were as follows: target steroid (+); carrier protein (-); steroid-doping-free female urine (-) and steroid-doping-free male urine (-). All positive clones were used for the production of monoclonal antibodies, and aliquots of the positive clones were frozen in liquid nitrogen. Competitive Immunochemical Assays. The experiment procedure was as follows: The prepared microarrays were immersed into blocking solution (10% sheep serum, 90% phosphatebuffed saline) at room temperature for 30 min to block out the unused sites and then rinsed three times with phosphate-buffed saline, 0.05% Tween 20, pH 7.4 (PBST). A mixture of first antibodies and steroid reference were added onto the surface of the microarray, which was then maintained at 37 °C in a humid box for 30 min. Next, the microarray was rinsed three times with PBST, the second antibody (Cy3 goat anti mouse IgG) was added, and this chip was kept at 37 °C. Half an hour later, the microarray was rinsed again and ready for analysis. The capability of the antibody to shift the binding equilibrium of immobilized conjugated hapten to free steroid was reported by the Cy3 fluorescence. If a tested sample contains any of the prohibited steroids, the fluorescent signal will decrease at the corresponding location of immobilized conjugated steroid. The decrease in fluorescent signal was directly proportional to the amount of steroid in the detected sample. Suitable Selection for the Dilution of Antibody. Antibodies of serial dilution (from 1/1000 to 1/50 000 in PBST, 20 uL) were added into separate arrays and followed the experiment procedure described above. The suitable dilution of antibody was chosen for later study based on the suitable fluorescent signal (10 00030 000 units, PMT 70, laser power 70, GenePix 4000B, Axon, Union City, CA). Detection of Steroid References. To analyze the 42 reference materials, the selected dilutions of the corresponding antibody (10 µL) were mixed with different steroid reference materials (1 µg/mL in doping-steroid- free urine, 10 µL) and then added to separate arrays, and the procedure described above was followed. The immobilized steroid/antibody combinations that showed an inhibition of absorbance in the presence of extrinsic steroids higher than 10% were chosen for further studies. For each material selected, serial dilutions of different types of standard steroids (1 pg/mL-1 µg/mL in doping-steroid-free urine) were analyzed. Detection of Steroid Positive Urine. To further the study, 28 positive urine samples of known steroids were tested by both the chip and GC/MS methods. Because most anabolic steroids are usually metabolized in the human body, differentiating the steroid6168 Analytical Chemistry, Vol. 76, No. 20, October 15, 2004
positive urine sample from the ordinary negative one is more actual application. Data Evaluation. After the immunoreactions were performed, the prohibited substances in the sample would bind to the corresponding antibodies, other antibodies would bind to the immobilized steroids, so each discrete spot generated a fluorescent signal. The fluorescent signals were detected by a laser confocal scanner (GenePix 4000B, Axon Instruments. Inc. Union City, CA), and then digitally processed by GenePix Pro 3.0 (Axon Instruments. Inc.). The spatial alignment of the array consisted of small spots (100-120 µm in diameter) of proteins in a gridlike arrangement. Three spots of each steroid-BSA conjugate were immobilized on one array. For data evaluation, mouse IgG were printed at the fringe and middle of the array. The fluorescence median of 27 spots of mouse IgG within the same array was calculated and selected as a normalization index to adjust the nonanticipated data difference between different arrays and chips. On each chip, a steroid-dope-free sample was used as a negative calibrator. RESULTS Synthesis of Conjugated Haptens. Eight anabolic steroid conjugates, including testosterone-17-BSA, methyltestosterone-3BSA, methyltestosterone-17-BSA, 4-chlorodehydromethyltestosterone-1-BSA, 5β-androsten-1-en-17β-ol-3-one-3-BSA, 5β-androsten-1en-17β-ol-3-one-17-BSA, 4-androsten-4-chloro-17β-ol-3-one-7-BSA, and 1,5R-androsten-1β-methyl-17β-ol-3-one-17-BSA, have been synthesized and used for antibody production and chip immobilization. All the hapten-carrier conjugates were confirmed by mass spectrometry and nuclear magnetic resonance analysis. For these conjugates, the number of moles of steroid per mole of carrier (BSA) was between 22 and 35. Antisteroid Monoclonal Antibodies. Haptens are not immunogenic and cannot stimulate antibody response by themselves. They have to conjugate with a carrier to become immunogenic. Although all of the conjugated haptens have a common steroid nucleus structure, the coupling position at the ring has an important effect on the production of antibody. Generally, C1, C3, C7, and C17 positions were chosen for modifying and coupling, but not all these conjugates can elicit a similar immune response. Position 3 of 5β-androsten-1-en-17β-ol-3-one-3-BSA cannot elicit antibody response whereas position 17 (5β-androsten-1-en-17β-ol3-one-17-BSA) raised immunoreactions successfully. The C7 position (4-androsten-4-chloro-17β-ol-3-one-7-BSA) and C17 position (1,5R-androsten-1β-methyl-17β-ol-3-one-17-BSA) antigens can stimulate the production of antibodies (Table 1). Binding Pattern of Steroid Antibodies. It is necessary for antibodies and antigens to have complementary atomic groups and shapes in order to form high-affinity complexes. Classical noncovalent bonds, including hydrogen bonds, charge-charge interactions, hydrophobic bonds, and van der Waals forces, hold
the antigen to the antibody binding sites and confine their threedimensional structure. Because all steroids come from the parent steroid nucleus molecule, their chemical structures are similar. As a result, most of the steroid-carrier conjugates are highly similar. Though all of the antibodies were designed against the different positions and groups of the steroid nucleus, the immunoreactions in vivo were very complicated and difficult to control. Some antibodies studied can recognize other conjugates. It is perhaps that the coupling reaction to the steroid nucleus may change the conformation of the parent molecule to some degree. How the antibody recognized these antigens remains to be elucidated. If the accurate relationships between chemical structure and antibody formation or cross-reactivity can be better understood, measures could be taken to resolve this problem by subtle modulation of antibody-binding properties through protein engineering methods. Figure 3 shows the binding characteristics of three clones. Anabolic Steroid Substances Detected by the Microarray. This microarray is a qualitative screening tool for some exogenous steroids. The selected cutoff is 100 ng/mL, and we use this criterion to judge which steroid can be identified and which cannot. The average chip variance is 11% within batch and about 13-14% between batch. Most anabolic steroids are usually metabolized in the human body, and therefore, their presence can be verified by detecting these metabolites in urine. Metabolites in urine are measured by comparison with samples of known chemical compositions, i.e., reference standards. Anabolic steroid reference materials are used to detect the presence of prohibited steroid in urine samples commonly tested by mass spectrometry. Similarly, steroids and their metabolites in urine samples can be detected and identified by comparison with the reference materials on a chip. To identify the antibody, reference materials of known concentration were added to steroid-doping-free urine samples and the results evaluated. The binding pattern of each antibody was obtained through analysis of 42 types of anabolic steroid reference materials. The chip performance was further proved by using 28 standard steroiddrug-positive urine samples collected from Chinese athletes by CDCC over the period November 1992 to December 2002. The identity of any steroid in the urine samples was further confirmed by GC/MS assay. In addition to the anticipated binding, all the antibodies from the three conjugated haptens bind to a range of reference materials (Figure 4). These results demonstrated that the “monoclonal” antibodies directed against the described haptens might have more than one reactivity. With the chip, 24 standard materials and 12 positive urine samples were analyzed. The highly conservative structure and relatively low molecular weight of steroids means that the effective antigenic determinant of steroid is limited, and the preparation of an antibody against a specific steroid is difficult. Among hundreds of steroid family members, only 30-50 types of antisteroid antibodies are commercially available. This was one reason for the selection of a few antibodies of low specificity that showed marked affinity toward other steroids. In this way, the chip can detect a large number of family members simultaneously when their mass concentration is above 100 ng/mL. Other tested materials, including 11β-oh-an, 11β-oh-etio, 17-R-methyl-an, 5R,3R,17β-diol, 5R,3β,17β-diol, 5β,3R,17β-diol, bolasterone, bolasterone (m1), clostebol, clostebol
Figure 3. Chip-bound conjugates that bind with C2D11 (A), D21A34 (B), and M6B31 (C).
(m1), dehydroepiandrosterone (DHEA), dianabol (m2), epioxandrolone, epitestosterone, fluoxymesteron (m1), formebolone (m), mesterolone (m1), and oral turinabol (m1), cannot be so identified. Some positive urine samples, including boldenone, dehydrochlormethyltestosterone, dianabol, drostanolone, fluoxymesterone, formebolone, mesterolone, metenolone, methyltestosterone, nandrolone, oral turinabol, oxymetholone, stanozolol, and trenbolone, can be identified by the chip. DISCUSSION It is difficult for a compound to be identified by chip immunological techniques because of the similarity in steroid Analytical Chemistry, Vol. 76, No. 20, October 15, 2004
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Figure 4. Representative microarray assay results. Table 2. Anabolic Steroid Reference Materials (24) That Can Be Detected by the Chipa compound name 1, 4-androstadien-17R-methyl-17β-ol-3-one 1, 4-androstadien-17β-ol-3-one 1, 5R-androsten-1β-methyl-17β-ol-3-one 17R-methyl-5R-androstane-3R,17β-diol 17R-methyl-9R-fluoro-6β,11β,17β-hydroxy-androst-4-en-3-one 17β-methyl-5β-androsten-1-ene-3R,17R-diol 1-methyl-5R-androstan-3R-ol-17-one 2-formyl-17R-methyl-androsta-1,4-diene-11R,17β-diol-3-one 2R-methyl-5R-androstan-3R-ol-17-one 4, 9, 11-estratrien-17β-ol-3-one 4-androsten-4-chloro-17β-ol-3-one 4-androsten-9R-fluoro-17R-methyl-11β, 17β-diol-3-one 4-chlorodehydromethyltestosterone 5R-androsten-17R-methyl-3β, 17β-diol 5R-androsten-2-hydroxymethylene-17R-methyl-17β-ol-3-one 5R-androstan-17R-methyl-17β-ol-[3,2-c]pyrazole 5R-androstan-17R-methyl-17β-ol-[3-ol,2-c]pyrazole 5R-androstan-17β-ol-3-one 5R-androstan-1R-methyl-17β-ol-3-one 5R-androstan-2R-methyl-17β-ol-3-one 5R-androstan-3R-ol-17-one 5β-androstan-3R-ol-17-one 5β-androsten-1-en-17β-ol-3-one nandrolone (m1-4, mix) a
SC1
SC3
58
45
62
63 60 53
SC4
39
SC6
25 47 52
SC7
34 62
SC8
SC9
54
60 60 28 69 65
48
SC11 66 16 23 52
66 52 73
71 24
73
65
52 72
74 67
48
71
51
72 64
74 63 6
74% 69
28 61 65 60
11
41 9
28 14
48 73
52 73
61 60 9 73
All values in percent.
structures, which in turn gives rise to reduced specificity. GC/ MS confirmation will be needed to identify the specific steroid being used. The chip can play an important role as a screening procedure since antibodies showing cross-reactivity were deliberately selected. The chip utilizes this cross-reactivity to detect new steroid drugs. For example, tetrahydrogestrinone (THG) is a steroid that was brought to the attention of the sporting world in October 2003. If an antibody of low specificity against gestrinone or trenbolone (the steroids structurally related to THG) is available, THG and other structurally related steroid analogues might be identified and then their structures can be confirmed by GC/MS. 6170
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Anabolic steroids can be divided into two groups: exogenous and endogenous. Endogenous refers to a substance that is capable of being produced by the body naturally. There are many types of endogenous steroids in the human body, and some of them are present at high concentrations, e.g., etiocholanolone (0.72.5 µg/mL) and 17-ketogenic steroid (6-20 µg/mL). These endogenous steroids will markedly interfere with the affinity of the steroid antibody. For the chip to work well, such steroids must be tested for any cross-reactivity, and if possible, such crossreactivity should be eliminated or modulated in some way. To eliminate all the potential clones that can bind with all of the endogenous steroids, urine samples from 200 healthy male
and 120 female of ages between 18 and 30 were collected. All these samples were analyzed using standard drug-testing procedures of GC/MS and were reported free of exogenous steroids. These samples were mixed according to gender and named “steroid-doping-free female urine” or “steroid-doping-free male urine”. Aliquots of these reference samples were stored at -20 °C before use. In the process of hybridoma selection, all the supernatant fluids were analyzed using a standard method and with the specific requirement that they must not bind with the steroid-doping-free references. Using this criterion, among more than 1000 hybridomas tested, only 18 clones were identified as fulfilling this requirement. Exogenous steroids, unlike endogenous ones, are generated artificially and do not occur naturally in the human body. While most prohibited steroids are exogenous, there are exceptions, such as DHEA, androstenedione, and dihydrotestosterone. These steroids will result in a problem for the chip system, since it is difficult to distinguish between administered and those naturally occurring ones. All potential clones that can bind to such steroids were eliminated in the process of hybridoma selection. Protein microarray may play an important role for the functional analysis of cellular activity and protein-protein interaction (17) Cahill, D. J. J. Immunol. Methods. 2001, 250, 81-91. (18) MacBeath, G.; Schreiber, S. L. Science 2000, 289, 1760-1763. (19) Haab, B. B.; Dunham, M. J.; Brown, P. O. Genome Biol. 2000, 1, 1-22.
as well as for clinical diagnostics.17 The biggest advantage is perhaps its potential for the high-throughput function analyses,18 and such systems have been used to detect and quantify specific target protein in complex solution.19 Indeed, many protein microarray assays continue to take advantage of the proven utility of ELISA, in which the antigen is immobilized or in the sandwich format. Despite some shortcomings, the chip approach still displays several distinct advantages over routine assays. In conclusion, anabolic steroid abuse is a serious problem for public health and has become increasingly prevalent among school students. To safeguard the health of adolescents, systemic and routine screening for steroids among schoolchildren on a large scale with the conjugated hapten microarray might be a possible solution. ACKNOWLEDGMENT We thank Dr. Brian Caddy and Qiang Zheng for their critical comments and suggestions on the manuscript. This work was supported by Beijing Natural Science Foundation of China (H010210640121) and the National Hi-Tech Program of China (2002AA2Z2011). Received for review June 8, 2004. Accepted September 1, 2004. AC049159A
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