One-Step Homogeneous Immunoassay for Small Analytes - Analytical

The anti-morphine and the anti-IC antibodies were labeled with a pair of fluorescence ... Analytical Chemistry 2011 83 (1), 246-253 ... The Journal of...
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Anal. Chem. 2005, 77, 2637-2642

One-Step Homogeneous Immunoassay for Small Analytes Timo Pulli,*,† Matti Ho 1 yhtya 1 ,‡ Hans So 1 derlund,† and Kristiina Takkinen†

VTT Biotechnology, Tietotie 2, P.O. Box 1500, FI-02044 VTT, Espoo, Finland, and Medix Biochemica, Asematie 13, FI-027700, Kauniainen, Finland

We have developed a one-step, homogeneous noncompetitive immunoassay for small analytes using recombinant antibodies and morphine as the model analyte. A highly specific antibody against the immune complex (IC) formed between an anti-morphine antibody and morphine was selected from a naı1ve scFv phage display library. The in vitro phage library selection procedure avoids the difficulties associated with the production of anti-IC antibodies by animal immunization. The anti-morphine and the anti-IC antibodies were labeled with a pair of fluorescence resonance energy transfer (FRET) fluorophores. In the FRET assay the labeled antibodies were incubated with saliva samples spiked with morphine, codeine, or heroin. Within 2 min, 5 ng/mL morphine, which is clearly under the recommended cutoff level, was detected without cross-reactivity to codeine or heroin. This assay principle is also widely applicable to other small analytes. Abused drugs are an important and increasingly growing area of analytes. Competitive immunoassays performed in microtiter wells or dip sticks are fast and relatively simple methods for the detection of abused drugs. However, due to the unspecific nature of competitive immunoassays, these tests generate many false positives.1 Therefore, all positive drug findings by immunoassay must be confirmed using a more specific technique such as gaschromatography-mass spectrometry (GC/MS). Commercial immunoassay tests for morphine and heroin cross-react with, e.g., codeine, which is a widely used substance in cough medicines. Thus, those tests are considered as opiate specific, and confirmation of the morphine abuse must always be confirmed by other techniques. This is not only a technical issue but raises also ethical concerns. Noncompetitive immunoassays have many advantages over competitive ones, such as improved speed, sensitivity, and specificity. A noncompetitive immunoassay is based on the use of two antibodies that bind to two different epitopes of the analyte. This works well for high molecular weight analytes. However, when the analyte is small, there is not enough space for simultaneous binding of the two different antibodies. Therefore, for the detection of small molecules the competitive immunoassay * Corresponding author. Fax:. +358-20-722 7071. E-mail: [email protected]. † VTT Biotechnology. ‡ Medix Biochemica. (1) Kerrigan, S.; Phillips, W. H., Jr. Clin. Chem. 2001, 47, 540-547. 10.1021/ac048379l CCC: $30.25 Published on Web 03/10/2005

© 2005 American Chemical Society

format has been used almost exclusively despite many fundamental problems with respect to specificity and sensitivity. There are only a few publications where the development of a noncompetitive immunoassay for a small analyte has been reported.2-4 An antibody recognizing an immune complex (IC) of an antibody and tetrahydrocannabinol (THC) has been described.2 The anti-IC antibody was obtained by immunizing with a complex of anti-THC antibody and THC. The binding of an antiIC antibody was enhanced in the presence of ∆9THC. The same principle has been used in preparing anti-IC antibodies to detect digoxin.4 A sandwich immunoassay for the hapten angiotensin II, wherein the immunization involves tolerization with uncomplexed primary antibody prior to immunization with the IC to obtain antiIC antibodies, has also been reported.3 The anti-IC antibodies used in noncompetitive immunoassays for small analytes have been so far conventional polyclonal or monoclonal antibodies obtained by immunization. The assays contain labeling of the primary and immobilization of the secondary antibody or vice versa and separate washing steps. A so-called “idiometric” noncompetitive immunoassay5 is based on two types of anti-idiotypic antibodies which recognize different epitopes within the hypervariable region of the analyte-specific primary antibody. The two secondary antibodies compete for binding to the primary antibody, which is dependent on the presence of the analyte. Recombinant antibodies enriched from a phage display library have been used for development of a competitive anti-idiotypic immunoassay for cortisol and aldosterone.6 An open sandwich immunoassay, based on a phenomenon that in some antibodies antigen is promoting the association of separated VH and VL, has also been utilized for the detection of small analytes.7,8 Despite the significant benefits of the noncompetitive immunoassay format, anti-IC antibodies have been exploited only in the few above-mentioned examples for the detection of small analytes. (2) Ullman, E. F.; Milburn, G.; Jelesko, J.; Radika, K.; Pirio, M.; Kempe, T.; Skold, C. Proc. Natl. Acad. Sci. U. S. A. 1993, 90, 1184-1189. (3) Towbin, H.; Motz, J.; Oroszlan, P.; Zingel, O. J. Immunol. Methods 1995, 181, 167-176. (4) Self, C. H.; Dessi, J. L.; Winger, L. A. Clin. Chem. 1994, 40, 2035-2041. (5) Mares, A.; De Boever, J.; Osher, J.; Quiroga, S.; Barnard, G.; Kohen, F. J. Immunol. Methods 1995, 181, 83-90. (6) Raats, J.; van Bree, N.; van Woezik, J.; Pruijn, G. J. Immunoassay Immunochem. 2003, 24, 115-146. (7) Yokozeki, T.; Ueda, H.; Arai, R.; Mahoney, W.; Nagamune, T. Anal. Chem. 2002, 74, 2500-2504. (8) Suzuki, C.; Ueda, H.; Mahoney, W.; Nagamune, T. Anal. Biochem. 2000, 286, 238-246.

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Figure 1. Schematic illustration of the principle of the one-step, noncompetitive FRET immunoassay for morphine. Eu-labeled antimorphine and Cy5-labeled anti-IC Fab fragments are added into a saliva sample. FRET occurs only when two fluorophores are close to each other; i.e., anti-IC Fab is specifically bound to IC. The star represents the analyte, and dotted patterns represent variable regions of the Fab fragment.

Due to the self-antigenicity and low stability of ICs, production of anti-IC antibodies by traditional immunization-based techniques has been problematic9 and prevented the wider use of anti-IC antibodies for the detection of small analytes. Here the problem has been circumvented by selecting the IC-specific antibody by the antibody phage display library technique, enabling isolation of antibodies to self-antigens10 and furthermore with predetermined specificity.6,11,12 Fluorescence resonance energy transfer (FRET) has been widely used in studies of biomolecular interactions, structure, and dynamics (reviewed in ref 13). The distance between the FRET pair fluorophores should be between 20 and 60 Å for the signal development. Thus, the distance between two antibody Fab fragments forming an IC binding pair is ideal for a FRET assay. Anti-morphine and anti-IC antibody fragments were enriched from phage display antibody libraries and labeled with donor (Eu) and acceptor (Cy5) fluorophores, respectively, for the FRET assay (Figure 1). This assay format is rapid, sensitive, specific, and easy to perform and is also widely applicable to other small analytes. EXPERIMENTAL SECTION Immunization of Mice and Construction of the Antibody Phage Display Library. All basic recombinant DNA methods were done essentially as described.14 Four 6-week-old female Balb/c mice were immunized in 3-week intervals with a morphineBSA conjugate (Fitzgerald Industries International, Inc., MA) in (9) Maruyama, H.; Sperlagh, M.; Zaloudik, J.; Liang, S.; Mizuki, K.; Molthoff, C.; Herlyn, D. J. Immunol. Methods 2002, 264, 121. (10) Huie, M. A.; Cheung, M. C.; Muench, M. O.; Becerril, B.; Kan, Y. W.; Marks, J. D. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 2682-2687. (11) Hemminki, A.; Niemi, S.; Hautoniemi, L.; Soderlund, H.; Takkinen, K. Immunotechnology 1998, 4, 59-69. (12) Saviranta, P.; Pajunen, M.; Jauria, P.; Karp, M.; Pettersson, K.; Mantsala, P.; Lovgren, T. Protein Eng. 1998, 11, 143-152. (13) Selvin, P. R. Nat. Struct. Biol. 2000, 7, 730-734. (14) Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular Cloning: A Laboratory Manual, 2nd ed.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 1990.

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Freund’s adjuvant. The mouse with the highest antibody response to morphine-BSA conjugate when compared to BSA was sacrificed, and the total RNA was isolated from the spleen cells using the RNagents Total RNA Isolation System (Promega Co., WI). The mRNA pool of the total RNA was isolated with the Oligotex mRNA Kit (QIAGEN Inc., Germany). The cDNA was synthesized from the mRNA with oligo-dT priming. Genes encoding antibody Fab fragments were amplified by PCR using antibody κ light-chain and heavy-chain variable region and constant region specific primers. Antibody light-chain PCR products were pooled and digested with NheI and AscI restriction enzymes, purified by preparative agarose gel with the QIAquick Gel Extraction Kit (QIAGEN). The purified DNA was ligated into the Fab phagemid vector phagemid9 derived from pComb315 and transformed into the Escherichia coli (E. coli) XL1-Blue bacteria (Stratagene) by electroporation. Plasmid DNA was isolated with the QIAGEN Plasmid Midi Kit (QIAGEN) from the o/n culture. PCR products encoding the Fd region of the heavy chain were pooled and digested with SfiI and NotI restriction enzymes, purified from the agarose gel, and ligated to the phagemid vector containing the light-chain DNA. The phagemid vector encoding both the heavy and light chains of the Fab fragments was transformed into the E. coli TOP10F’ bacteria (Invitrogen Inc., CA) by electroporation. Transformed cells were incubated o/n at +37 °C on a shaker, and plasmid DNA was isolated with the QIAGEN Plasmid Midi Kit. The diversity of the antibody library was ensured by sequencing partial VL- or VH-gene regions of individual clones. A phage display antibody library was made as follow: 4 µg of antibody library plasmid DNA was transformed into E. coli TOP10F’ by electroporation in two parallel transformations. After transformations cells were suspended into 2.8 mL of SOC medium and incubated for 1 h at +35 °C on a shaker. A 7-mL aliquot of prewarmed (+37 °C) SB medium, 20 µg/mL carbenicillin, and 10 µg/mL tetracycline were added. After 1 h incubation at +35 °C on a shaker, 30 µg/mL of carbenicillin was added and the incubation was continued for 1 h, after which 1 mL (∼1011 pfu) of helper phage VCS-M13 (Stratagene) was added and the phages were let to infect the bacteria for 20 min at +35 °C with a slow shaking. The parallel transformations were joined together, and 80 mL of prewarmed (+37 °C) SB medium with 50 µg/mL carbenicillin and 10 µg/mL tetracycline was added. After 2-h incubation at +35 °C on a shaker, 70 µg/mL kanamycin was added and the incubation was continued o/n. Cells were centrifuged for 15 min at 4000g at +4 °C. Phages were precipitated with 20 mL of 20% PEG, 2.5 M NaCl (PEG/ NaCl) for 30 min on ice. PEG-precipitated phages were centrifuged at +4 °C with 13000g for 20 min. The pellet was resuspended in 2 mL of PBS and transferred into two eppendorf tubes. Bacterial cell debris was removed by centrifugation at +4 °C with 13 000 rpm for 5 min, and phages were reprecipitated by adding 200 µL of PEG/NaCl to the supernatant. After 15 min incubation on ice, phages were pelleted by centrifugation at +4 °C with 13 000 rpm for 5 min. The phage pellet was resuspended in 1 mL of PBS + 1% BSA, and 1 µL of 20% sodium azide was added as a preservative. The phage display antibody library was stored at +4 °C. (15) Barbas, C. F., 3rd; Kang, A. S.; Lerner, R. A.; Benkovic, S. J. Proc. Natl. Acad. Sci. U. S. A. 1991, 88, 7978-7982.

Selection of the Anti-morphine Library. Morphine-specific antibodies were selected from the phage display antibody library as follows: A microtiter well was coated o/n at +4 °C with 1 µg of the morphine-BSA conjugate in 100 µL of 100 mM sodium bicarbonate buffer, pH 9.8. Another well for monitoring the background binding was coated with 1 µg of BSA. The wells were washed two times with PBS and blocked with 0.5% BSA in PBS for 1 h at +37 °C. A 100-µL aliquot of the phage library was incubated in the wells for 1 h at room temperature (RT) with shaking. Unbound phages were removed, and the wells were washed 22 times with PBS. Bound phages were eluted with 100 µL of 100 mM HCl, pH 2.2, for 15 min in a plate shaker, and the elution solution was neutralized with 1 M Tris. A 3-mL aliquot of fresh E. coli XL1-Blue cells (OD600 ≈ 1) grown in SB supplemented with 10 µg/mL tetracycline was infected with the eluted phages at +35 °C for 15 min. Aliquots of the infected bacteria were plated on Luria-ampicillin plates to titer the amount of eluted phages. The eluted phages from the control BSA well were used only for tittering, and they were not processed further. To amplify the phages eluted from the morphine-BSA well, 7 mL of prewarmed SB (+37 °C) containing 20 µg/mL carbenicillin and 10 µg/mL tetracycline was added to the infected bacteria and the culture was incubated for 1 h at +35 °C on a shaker. A 30 µg/mL amount of carbenicillin was added, and incubation was continued for 1 h. A 1-mL aliquot of helper phage VCS-M13 was added to the culture. Incubation was continued for 15 min with slow shaking. The culture was diluted with 90 mL of prewarmed (+37 °C) SB containing 50 µg/mL carbenicillin and 10 µg/mL tetracycline. After 2-h incubation at +35 °C on a shaker, 70 µg/mL kanamycin was added and the incubation was continued o/n. Purification of the amplified phages was performed by PEG precipitation as described above. Purified phages were used in further enrichment rounds. The enrichment of the morphine-specific phages was monitored by comparing the amount of the eluted phages from the morphineBSA-coated well to the amount of eluted phages from the background BSA-coated well. Characterization of Individual Clones. After the fourth panning round, phagemid DNA was isolated with the QIAGEN Plasmid Midi Kit. The plasmid was digested with NheI and NotI restriction enzymes to isolate the Fab gene fragment. The agarose gel isolated DNA was ligated to the expression vector pKKtac.16 Ligated DNA was transformed into the E. coli XL1-Blue cells. Individual clones were picked and miniprep DNA was extracted with the QIAprep Spin Miniprep Kit (QIAGEN). Fab fragments were expressed in 3-mL cultures. A periplasmic fraction of the cells was isolated by freezing and thawing the cells three times in PBS. The binding of individual Fab clones to morphine was tested by ELISA in morphine-BSA-coated microtiter wells: Wells were coated with 200 ng of morphine-BSA in 0.1 M sodium bicarbonate buffer, pH 9.8, o/n at +4 °C. Control wells were coated with 200 ng of BSA in 0.1 M sodium bicarbonate buffer, pH 9.8, o/n at +4 °C. After coating, wells were washed three times with PBS and blocked with 0.5% BSA in PBS for 1 h at RT. Wells were washed (16) Takkinen, K.; Laukkanen, M. L.; Sizmann, D.; Alfthan, K.; Immonen, T.; Vanne, L.; Kaartinen, M.; Knowles, J. K.; Teeri, T. T. Protein Eng. 1991, 4, 837-841.

three times with PBS, and 1:10 diluted periplasmic fractions in 100 µL of 0.5% BSA in PBS were added into the wells. After shaking for 1 h at RT in a plate shaker, wells were washed three times with PBS and 100 µL of alkaline phosphatase conjugated anti-mouse Fab specific antibody (Sigma, A-1293) diluted 1:2000 in 0.5% BSA-PBS was added. Wells were shaken for 1 h at RT and washed three times with PBS. A 100-µL aliquot of alkaline phosphatase substrate solution (2 mg/mL p-nitrophenyl phosphate disodium salt in diethanolamine-MgCl2-buffer) was added to the wells, and absorbance was measured at 405 nm. Fermentation and Purification. Fab fragments were expressed in E. coli RV308 strain by fed-batch fermentation in a BioFlow IV fermenter (New Brunswick) and purified by the Sepharose SP ion-exchange or immobilized metal affinity chromatography (IMAC) and protein G chromatography (Pharmacia). The purity of the Fab fragment was checked by SDS-PAGE. Specificity of M1 Fab. Cross-reactivity of the M1 antimorphine Fab fragment with codeine and heroin was tested in a competitive ELISA. Microtiter plate wells were coated with 500 ng of morphine-BSA in 100 µL of sodium bicarbonate buffer, pH 9.8, for o/n at +4 °C. The wells were washed three times with PBS, blocked with 0.5% BSA in PBS for 1 h at RT, and washed again three times with PBS. Two parallel 100-µL samples containing morphine, codeine, or heroin (0-160 ng/mL) in PBS with 5 ng of purified M1 Fab were added into the wells and incubated for 30 min at RT on a shaker and washed three times with PBS. A 100-µL aliquot of the alkaline phosphatase conjugated anti-Fab antibody (Sigma, A-1293) diluted 1:2000 in PBS-0.5% BSA was added to the wells and incubated for 30 min at RT on a shaker. Wells were washed three times with PBS, 100 µL of alkaline phosphatase substrate solution was added, and A405 was measured. Development of an Anti-IC Antibody Specific to the Immune Complex of M1 Fab and Morphine. The M1 Fab fragment was biotinylated with ImmunoPure Sulfo-NHS-LC-Biotin Kit (Pierce). Biotinylated antibody was purified and buffer changed to PBS with Econo-Pac 10DG Columns (Bio-Rad, CA). A 200-µL aliquot of a naı¨ve human scFv phage display library (κ or λ light chain) in 0.5% BSA-PBS was preincubated with 10 µL of streptavidin-coated magnetic beads (Dynal, M-280) and 0.5 µg of biotinylated M1 Fab for o/n at +4 °C. The naı¨ve human scFv phage display library has been constructed from pooled lymphocytes of 50 healthy individuals. The size of the library has been estimated to be 1 × 108 clones. The naı¨ve human scFv phage display library contains the IgM-specific VH genes combined either to the κ- or λ-specific VL genes (Takkinen et al., unpublished data). Unbound phages were separated from the beads, and 100 µL of the phage library was incubated with 100 ng of morphine, 500 ng of biotinylated M1 Fab, and 5 µL of streptavidin-coated magnetic beads for 1 h at RT on a shaker. The beads were washed five times with 0.5 mL of PBS, and bound phages were eluted with 100 µL of HCl, pH 2.2, for 30 min. Eluted phages were neutralized with 1 M Tris. Phages were amplified by infecting E. coli XL1Blue cells and purified as described above. Conversion of K11 scFv to Fab. The CH1 domain of human IgG and the constant domain of human κ light chain were amplified separately by PCR from another Fab clone. VL and VH domains of K11 scfv were also amplified by PCR. VH and CH1, or VL and CL PCR products were assembled by overlapping PCR. Analytical Chemistry, Vol. 77, No. 8, April 15, 2005

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Figure 2. Amino acid sequences of the M1 (A) and K11 (B) antibody Fab fragments. CDR regions are underlined and shown in bold.

The resulting light chain and VH-CH1 part of the heavy chain was cloned into the expression vector pKKtac. Homogeneous Fluorescence Resonance Energy Transfer Immunoassay for Morphine. The anti-morphine M1 and the anti-M1 + morphine IC K11 Fab fragments were labeled with europium by the LANCE Eu-W1024 ITC chelate Kit (Wallac) and Cy5 by the FluoroLink-Ab Cy5 labeling kit (Amersham Pharmacia Biotech), respectively. The buffer of the labeled M1 was changed to 50 mM Tris, pH 7.8, 0.9% NaCl. The mouth of a nonaddict individual was rinsed with water, and after 5 min saliva was collected. Saliva was spiked with various dilutions of morphine or other drugs. After 2-min centrifugation three parallel 50-µL samples were pipetted into the black microtiter wells (Cliniplate, Labsystems). Six parallel nonspiked samples were also analyzed as negative controls. A 0.5-µg amount of europium-labeled M1 Fab and 1 µg of Cy5 labeled K11 Fab were added into the wells in 50 µL of BSA/PBS. The microtiter plate was incubated at RT, and FRET was measured after various time points by VictorV fluorometer (Wallac). RESULTS Development of an Anti-morphine Antibody. Mice were immunized with the morphine-BSA conjugate in order to develop anti-morphine Fab fragments (the primary antibody of the sandwich assay). A phage display antibody library was constructed from the spleen cells of the mouse serum sample which gave the best response to morphine-BSA. Anti-morphine Fab fragments were selected by panning the library in the morphine-BSA-coated microtiter wells. After four selection rounds morphine-specific antibody phages were enriched over 1000 times in comparison to BSA binding phages (background). Genes encoding Fab fragments were cloned into an expression vector pKKtac for soluble Fab fragment production, and individual clones were characterized by sequencing. Two morphine-specific Fab clones, named M1 and M2, were isolated. Sequences of these two clones are quite similar, and seven amino acid differences can be found 2640

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Figure 3. Cross-reactivity of the anti-morphine M1 Fab to codeine and heroin in a competitive ELISA. M1 Fab with various amounts of morphine, codeine, or heroin in PBS was added into morphine-BSA coated wells. After washings the bound Fab was detected with alkaline phosphatase conjugated anti-Fab antibody.

in the CDR regions of these clones (data not shown). Fab fragments were expressed in 3-mL cultures of E. coli strain RV308. Periplasmic fractions of the cells were isolated, and the performance of the clones was tested by ELISA. M1 Fab showed higher response in the assay (data not shown) and was therefore produced and purified in a large scale. The sequence of M1 Fab is shown in Figure 2A. The M1 Fab was expressed in a 4-L fermenter and purified by ion-exchange and protein G chromatographies with a yield of about 10 mg/L. The performance of M1 Fab was tested by competitive ELISA using samples spiked with various amounts of morphine, codein, or heroin. A positive result could be achieved with 5 ng/mL of morphine, as shown in Figure 3. However, M1 Fab has a high cross-reactivity to codein and heroin, which are structurally very similar to morphine. Enrichment of an Anti-IC Antibody from a Naive Human scFv Phage Display Library. Anti-IC antibodies were enriched by panning from naı¨ve human scFv phage display libraries. Two libraries, containing either κ or λ light-chain variable regions, were used in separate selections using biotinylated antibodies and streptavidin beads. Before panning with the IC, the library was

Figure 5. Effect of codeine and heroin on the performance of the assay. Saliva was spiked with equal amounts of morphine, codeine, and heroin or with morphine alone, and FRET was measured after 2-min incubation. Error bars represent standard deviations of three parallel measurements.

Figure 4. One-step, noncompetitive FRET immunoassay for morphine with spiked saliva samples. FRET was measured after 5 and 30 min from saliva samples spiked with morphine, codeine, or heroin. The 5 ng/mL concentration of morphine (the cutoff detection level of opiates in oral fluids is 40 ng/mL as recommended by SAMSHA) was detected with 5-min incubation without cross-reactivities to codeine or heroin. Error bars represent standard deviations of three parallel measurements.

preincubated with the biotinylated M1 Fab fragment and streptavidin beads without morphine in order to remove background binders. IC-specific enrichment was observed with the κ library after five panning rounds. The enrichment of specific binders to the IC was also clearly detected when the eluted phage pools from the different selection rounds were tested by a phage ELISA (data not shown). Individual phage clones were isolated and sequenced. All the IC-specific clones had the same sequence. Since scFvs are prone to aggregation during expression and purification, the IC-specific clone K11 was converted to a Fab fragment (Figure 2B) by cloning the gene regions of human κ and IgG1 CH1 constant regions to the VL and VH genes, respectively. A C-terminal tag containing six histidine residues was inserted into to the C-terminus of the CH1 for immobilized metal affinity chromatography (IMAC) purification. K11 Fab was cloned into the expression vector pKKtac and transformed into E. coli RV308 cells. The K11 Fab was expressed by a 4-L fed-batch fermentation and purified by IMAC and protein G chromatographies. The purification yield was 8,6 mg/L. Development of a Homogeneous Noncompetitive FRET Immunoassay. To develop an easy, sensitive, specific, and fast test for the detection of morphine in saliva, anti-morphine M1 Fab and anti-IC K11 Fab were labeled with europium and Cy5, respectively, for the FRET-based homogeneous immunoassay. The saliva of a nonaddict individual was spiked with various amounts of morphine, codeine, or heroin. A 50-µL amount of the samples was added into microtiter wells followed by 50 µL of 0.5% BSA in PBS containing labeled M1 and K11 Fabs. No further steps before measurement of FRET were needed. FRET was measured after various time points by a fluorometer (Figure 4). After 5-min incubation, 5 ng/mL of morphine in saliva was detected without cross-reactivities to codeine or heroin. The K11 Fab is able to

discriminate completely M1 and morphine IC from the ICs between M1 and heroin or codeine, which gave fluorescent values corresponding to the background level. No cross-reactivities to tetrahydrocannabinol or amphetamine were observed (data not shown). Immunoassays may suffer from lot to lot variations. Therefore, other lots of purified Fab fragments were labeled with Eu and Cy5. No change in the specificity or sensitivity of the morphine IC assay performed with the Fab fragments from different production, purification and labeling experiments was observed (data not shown). The effect of codeine and heroin on the performance of the assay was studied further by spiking saliva samples with morphine only or with morphine plus codeine and heroin (Figure 5). A 5 ng/mL amount of morphine was detected already after 2-min incubation. These two opiates neither cross-react nor interfere in the assay. DISCUSSION We have developed a FRET-based fast and homogeneous sandwich immunoassay for small analytes. In this immunoassay an antibody is specifically recognizing the immunocomplex formed between the primary antibody and hapten. The small size of recombinant antibody fragments, Fabs and scFvs, makes them ideal for FRET assays, since the distance between fluorophores (20-60 Å)13 is critical in FRET. Furthermore, small analytes do not considerably extend the distance between the antibodies in sandwich type assays, which makes the anti-IC antibody based FRET assays a tempting format. Due to the self-antigenicity of the primary antibodies, it is difficult to develop anti-IC mono- or polyclonal antibodies by immunizing animals. Furthermore, ICs are labile and tend to dissociate during immunization, which further complicates the development of anti-IC antibodies. For example Ullman et al.2 found only one anti-IC monoclonal antibody out of five fusions. The phage display technique combined to a naı¨ve antibody library allows isolation of antibodies with predetermined specificity without immunization and can thus be applied to isolate antibodies, e.g., against self-antigens. By preincubating the antibody library with the primary antibody before selection with the immunocomplex, those antibodies binding to the primary antibody without the analyte were efficiently removed. This kind of preabsorption of phage libraries has been successfully utilized for selection of Analytical Chemistry, Vol. 77, No. 8, April 15, 2005

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antibodies against complex and rare antigens, e.g., antigen mixtures and whole cells.17 Antibodies used in the immunoassays for morphine are crossreacting with other opiates, such as codeine and heroin, and are therefore considered as opiate-specific only.1,18 The anti-morphine Fab fragment M1 is also opiate-specific, cross-reacting with codeine and heroin (Figure 3). The specificity of the anti-morphine M1 Fab could be improved by antibody engineering techniques.11,19 However, the possibility of isolating immunocomplexspecific antibodies by the phage display library technology enables not only improvement of the assay specificity but also the use of a noncompetitive, one-step assay format as shown here with the anti-IC Fab K11. This Fab is only recognizing the immunocomplex formed between the M1 Fab and morphine and not the immunocomplexes between M1 Fab and heroin or codeine (Figure 4). Furthermore, a saliva sample spiked with morphine, codeine, and heroin is giving the same values as a sample containing only morphine, showing that these two other opiates are not interfering with the assay (Figure 5). The structures of codeine and heroin are very similar to morphine (Figure 3), and the complexity of opiate metabolism is further making the analysis of opiates challenging. Heroin has a short half-life after administration, and it is rapidly deacetylated to 6-monoacetylmorphine and further to morphine.20 Codeine is predominantly metabolized by glucuronidation to codeine-6glucuronide. Minor metabolic pathways include N-demethylation to norcodeine and O-demethylation to morphine.21,22 Morphine is further metabolized to morphine-3- and morphine-6-glucuronides.23 All opiate positive results, despite the specificity of the developed immunoassay, need to be confirmed by other methods such as GC/MS.24 We have shown here that the immunocomplex assay for morphine is highly specific using spiked saliva samples. However, the performance of the assay has to be further evaluated with clinical samples with GC/MS-validated opiate levels. (17) Hoogenboom, H. R.; de Bruine, A. P.; Hufton, S. E.; Hoet, R. M.; Arends, J. W.; Roovers, R. C. Immunotechnology 1998, 4, 1-20. (18) Dillon, P. P.; Daly, S. J.; Manning, B. M.; O’Kennedy, R. Biosens. Bioelectron. 2003, 18, 217-227. (19) Moghaddam, A.; Borgen, T.; Stacy, J.; Kausmally, L.; Simonsen, B.; Marvik, O. J.; Brekke, O. H.; Braunagel, M. J. Immunol. Methods 2003, 280, 139155. (20) Nakamura, G. R.; Thornton, J. I.; Noguchi, T. T. J. Chromatogr. 1975, 110, 81-89. (21) Yue, Q. Y.; Hasselstrom, J.; Svensson, J. O.; Sawe, J. Br. J. Clin. Pharmacol. 1991, 31, 635-642. (22) Chen, Z. R.; Somogyi, A. A.; Reynolds, G.; Bochner, F. Br. J. Clin. Pharmacol. 1991, 31, 381-390. (23) Brunk, S. F.; Delle, M. Clin. Pharmacol. Ther. 1974, 16, 51-57. (24) Jones, J.; Tomlinson, K.; Moore, C. J. Anal. Toxicol. 2002, 26, 171-175.

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The U.S. Substance Abuse and Mental Health Services Administration (SAMSHA) recommends that the detection level of morphine in oral fluids is 40 ng/mL. This sensitivity was reached already after 2-minutes incubation time with the noncompetitive FRET assay. The sensitivity of the assay was not significantly increased with the function of time, which demonstrates well the high rate by which the equilibrium between free and bound antibodies is reached. When compared to the competitive assay with the M1 Fab, the sensitivity remained approximately the same. This indicates high specificity of the anti-IC K11 Fab fragment. It binds specifically to the IC, and thus the sensitivity of the assay is dependent on the anti-morphine M1 Fab. The sensitivity and the speed of the assay may be further improved by optimizing assay conditions and the labeling ratio of the antibodies. The short assay time combined with the simplicity of performance makes this assay format attractive for point-of-care and even field use. CONCLUSIONS This homogeneous noncompetitive immunoassay is fast and very simple to perform. These characteristics are looked for, e.g., by the police for field use. In addition, the small volume of the saliva required in the assay simplifies the collection and analysis of the sample. To further simplify the use, the reagents can be kept in dry form in a vessel. The saliva sample is added, and the results can be read after a couple of minutes by a portable fluorometer. The method can be easily introduced in routine analysis. The only equipment required is a fluorometer capable of FRET measurements. Furthermore, the production of recombinant antibody Fab fragments by fermentation can be easily scaled-up, enabling inexpensive manufacturing of large amounts of reagents. As a whole, there is a big demand for a sensitive, specific, and fast assay method for many small molecules, e.g., pharmaceuticals, drugs of abuse, steroids, and toxins. Because in principle the immunoassay described herein can be applied to any kind of small analytes, it offers a novel solution to this need. ACKNOWLEDGMENT We thank Armi Boman for skilfull technical assistance and the National Technology Agency of Finland (Tekes) for financial support.

Received for review November 3, 2004. Accepted February 5, 2005. AC048379L