Preparation of a conjugate of 2',5'-triadenylate 5'-triphosphate and biotinylated firefly luciferase and its use for sensitive bioluminescent enzyme immunoassay

A bioluminescent enzyme immunoassay (BLEIA) for 2',5'-oligoadenylate 5'-triphosphate (pppA2'p5'A2'Ap5'A, 2-5A) was developed using a conjugate of 2-5A and firefly luciferase as a probe. The conjugate was prepared from a 2-5A derivative bearing a thiol group at the 2'(or 3') end, and streptavidin (SA) bearing maleimide via a linker-arm and biotinylated luciferase. The thiol group of the 2-5A derivative was relatively stable under aerobic conditions and remained 100% and 56% intact at 25 degrees C after 1 and 96 h, respectively, without dimerization by aerobic oxidation. The thiol-modified 2-5A was coupled with the SA-maleimide conjugate, followed by complex formation with biotinylated firefly luciferase. BLEIA using this conjugate allowed for the direct analysis of 2-5A in a range of 5-500 fmol (0.1-10 nM in a 50 microL sample) and in a reaction time of 15 min. The measurement of microquantities of 2-5 A from various sources is easy to perform by this BLEIA, because no radioisotope labeled 2-5A was required as a probe.     

Influence of different combinations of antibodies and penicillinase-labeled testosterone derivatives on sensitivity and specificity of immunoassays.

Three antisera raised against bovine serum albumin (BSA) conjugates of testosterone-3-(O-carboxy-methyl)-oxime (T-3-CMO), 11 beta-hydroxytestosterone-11-carboxymethyl ether (T-11 beta-O-CME) and 19-hydroxytestosterone-19-carboxymethyl-ether (T-19-O-CME) were evaluated in enzyme immunoassays (EIAs) in combinations with penicillinase-labeled T-3-CMO, T-11 beta-O-CME, T-19-O-CME, and testosterone-17 beta-hemisuccinate (T-17 beta-HS) for their influence on the sensitivity and specificity of EIAs. Of the various combinations, anti-T-3-CMO antiserum along with T-11 beta-O-CME-penicillinase showed no cross-reaction with any of the closely related steroids, although the same antibody had 21.6% binding to 5 alpha-dihydrotestosterone (5 alpha-DHT) in radioimmunoassay. All the homologous combinations appeared to be less sensitive due to their low affinity for testosterone. It was also apparent that of all the heterologous systems tested, only two combinations, (a) anti-T-19-O-CME antiserum and T-3-CMO-penicillinase and (b) anti-T-3-CMO antiserum and T-11 beta-O-CME-penicillinase, were found to be more sensitive. The former was less specific; it showed 70% cross-reaction with 5 alpha-DHT. The ability of testosterone to displace the hapten-enzyme conjugate and the specificity of the assay appear to depend on the position of the enzyme label on the steroid molecule as well as on the availability of antigenic sites in particular combinations of antibody and hapten-enzyme conjugates.     

Diagnostic value of salivary cortisol in end stage renal disease

OBJECTIVES: Salivary cortisol has been proposed a surrogate marker for free serum cortisol measurements. The aim of this study was to ascertain the diagnostic value of basal and stimulated salivary cortisol for the detection of adrenal insufficiency (AI) in hypotensive end stage renal disease (ESRD) patients. Basal salivary cortisol and basal total serum cortisol were studied in order to determine the accuracy of both biomarkers in predicting AI. PATIENTS AND METHODS: Twenty-nine ESRD patients with sustained hypotension were investigated for possible AI. Salivary cortisol was assessed at baseline and 30min after 25microg ACTH i.m. (LDTs). The dosage of salivary aldosterone was performed in salivary cortisol hypo-responders. Basal blood samples were drawn for steroids, renin and ACTH measurements. RESULTS: A clear separation between patients with normal and impaired adrenal function was obtained through salivary cortisol levels at 30min after ACTH. AI was detected in six cases (21%) through impaired salivary cortisol responses; stimulated salivary aldosterone helped to differentiate primary (n=3) from secondary AI (n=3). ROC curves showed that cutoff values for basal SAF < or =4.4nM and serum cortisol < or =232.0nM suggest AI (sensitivities: 93% and 69%; specificities: 86.4% and 91%, respectively). CONCLUSIONS: We conclude that ACTH stimulated SAF is an accurate biomarker for the diagnosis of AI in hypotensive ESRD patients. Neither basal salivary cortisol nor serum cortisol showed 100% sensitivities for the detection of AI.     

Synthesis of a biotin-conjugate of phosmidosine O-ethyl ester as a G1 arrest antitumor drug

This paper deals with the synthesis of a stable biotin–phosmidosine conjugate molecule 3 that is required for isolation of biomolecules that bind to phosmidosine (1). It was found that introduction of a biotin residue into the 6-N position of phosmidosine could be carried out by reaction of an N⁷-Boc-7,8-dihydro-8-oxoadenosine derivative 13 with phenyl chloroformate followed by displacement with a diamine derivative 6 along with the simultaneous removal of the Boc group and one of the two phenoxycarbonyl groups and the successive condensation with an N-tritylated biotin derivative 5. The condensation of an N-prolylphosphorodiamidite derivative 4 with an appropriately protected 7,8-dihydro-8-oxoadenosine derivative 17 having the biotin residue gave the coupling product 18, which was deprotected to give the biotin–phosmidosine (O-ethyl ester) conjugate 3.     

Determination of bradykinin in rat urine by coupled-column high pressure liquid chromatography with precolumn derivatization with a water-soluble fluorogenic reagent

The green fluorescent protein (GFP) and its mutants have been extensively used to study various cellular processes and, more recently, as labels in binding assays. We have employed a mutant of GFP, an enhanced GFP (EGFP), in the development of homogeneous assays for biotin and cortisol. To demonstrate the feasibility of using EGFP as a label with different kinds of binders in the development of homogeneous assays, we employed the biotin-avidin and an antigen-antibody as the binding pairs. Biotin and cortisol were chemically conjugated to EGFP. A quenching of fluorescence intensity of EGFP was observed upon binding of avidin to the EGFP-biotin conjugate. The percentage fluorescence quenching observed decreased as the concentration of free biotin in the sample increased due to the fewer binding sites on avidin available for binding to the EGFP-biotin conjugate. A dose-response curve for biotin was generated by relating percentage fluorescence quenched with free biotin in the sample. To determine whether EGFP can undergo a similar type of homogeneous change when used as a label for antigen-antibody type of binding, cortisol was selected as a model analyte. In the presence of an anti-cortisol antibody the fluorescence signal of the EGFP-cortisol conjugate was quenched. A dose-response curve for cortisol was generated by relating the quenching in the fluorescence signal with varying amounts of free cortisol in the sample. This is the first time that GFP or one of its mutants has been employed as a label in homogeneous assays, thus enhancing the versatility of employing GFP or its mutants in a number of bioanalytical applications, such as clinical analysis and high-throughput screening systems.     

Aequorin-based homogeneous cortisol immunoassay for analysis of saliva samples

Homogeneous assays are attractive because they are performed in only one phase, namely, the liquid phase, and thus, they do not require separation of phases as their heterogeneous counterparts do. As opposed to heterogeneous assays, the signal generation in a homogeneous assay is a direct result of analyte binding, which allows the multiple washing and incubation steps required in an indirect heterogeneous assay format to be eliminated. Moreover, homogeneous assays are usually fast and amenable to miniaturization and automation. In this article, we describe the development of a homogeneous assay for the hormone cortisol using the bioluminescent photoprotein aequorin as a reporter molecule. A cortisol derivative was chemically conjugated to the lysine residues of a genetically modified aequorin in order to prepare an aequorin-cortisol conjugate capable of binding anticortisol antibodies. The binding of anticortisol antibodies to the aequorin-cortisol conjugate resulted in a linear response reflected in the emission of bioluminescence by aequorin. A competitive binding assay was developed by simultaneously incubating the aequorin-cortisol conjugate, the anticortisol antibodies, and the sample containing free cortisol. Dose-response curves were generated relating the intensity of the bioluminescence signal with the concentration of free cortisol in the sample. The optimized homogeneous immunoassay produced a detection limit of 1 x 10 (-10) M of free cortisol, with a linear dynamic range spanning from 1 x 10 (-5) to 1 x 10 (-9) M. Both serum and salivary levels of cortisol fall well within this assay's linear range (3.0 x 10 (-7) M to 7.5 x 10 (-7) M and 1.0 x 10 (-8) M to 2.5 x 10 (-8) M, respectively), thereby making this assay attractive for the analysis of this hormone in biological samples. To that end, it was demonstrated that the assay can be reliably used to measure the concentration of free cortisol in saliva without significant pretreatment of the sample.     

Bioluminescence immunoassay for cortisol using recombinant aequorin as a label

The analysis of hormones in saliva is a powerful tool in the assessment of a patient's endocrine function, since it allows multiple noninvasive samplings. Since salivary levels of most hormones are 10 to 50 times lower than plasma levels, accurate and highly sensitive assays are needed for saliva measurements. Herein, we describe the development of a solid-phase competitive immunoassay for cortisol in saliva, in which a mutant of the photoprotein aequorin has been used as a label. We have chemically conjugated cortisol to aequorin at different molar ratios. The various cortisol-aequorin conjugates were characterized in terms of bioluminescent activity and affinity for the anti-cortisol antibody. The conjugate that gave the best analytical performance was used for the development of the immunoassay and the analysis of cortisol in saliva samples. The conjugates were stable for at least 6 months when stored at 4 degrees C. The method fulfilled all the standard requirements of precision and accuracy. The optimized immunoassay gave a detection limit of 300 fmol/tube, corresponding to 3 nmol/L, with a linear dynamic range of 10-1000 nmol/L. Therefore, cortisol can be detected down to 0.1 ng in 100 microl of saliva sample using this assay, without any sample pretreatment. This detection limit is almost one order of magnitude lower than the physiological levels of salivary cortisol, which are reported to be 10-25 nmol/L. This allows the quantification of salivary cortisol to be performed in the linear range of the calibration curve, which is most reliable for quantification purposes.     

Label-free, chemiresistor immunosensor for stress biomarker cortisol in saliva

Salivary cortisol is commonly used as a bioindicator of the psychobiologic response to environmental and psychological stressors. Current analytical approaches rely on immunoassays performed at distant, centralized laboratories and involve an elaborate specimen collection-processing-transportation-storage-analysis-reporting cycle. To facilitate point-of-use measurement of salivary cortisol levels, we describe the development and proof-of-concept testing of an ultrasensitive, label-free immunosensor based on a single-walled, carbon nanotube-based chemiresistive transducer. Carbon nanotubes were functionalized with a cortisol analog [cortisol-3-CMO-NHS ester] and a monoclonal anti-cortisol antibody was ligated to this receptor. Addition of phosphate buffer as well as artificial saliva spiked with varying cortisol concentrations displaced the anti-cortisol antibody producing corresponding decreases in the resistance/conductance of the nanotube-biomolecule hybrid. The immunosensor demonstrated an ultralow detection limit of 1 pg/ml and excellent binding selectivity for cortisol even in the presence of structurally similar steroids such as 21-hydroprogesterone. The nanotube immunosensor offers attractive prospects for the development of highly sensitive biosensor for rapid, label-free measurement of salivary cortisol in a variety of clinical and research settings.     

Validation and application of a highly specific and sensitive ELISA for the estimation of cortisone in saliva, urine and in vitro cell-culture media by using a novel antibody

It is generally acknowledged that local tissue concentrations of cortisol and cortisone are modulated by site-specific actions of 11β-hydroxysteroid dehydrogenase (11β-HSD) isoenzymes 1 and 2. Cortisone, the inactive metabolite of cortisol is produced by 11βHSD type 2. To assess 11β-HSD types 1 and 2 activities, the cortisol/cortisone ratio has to be accurately determined. Immunoassays to measure cortisone levels are not widely available and tend to lack specificity. The aim of this project was to develop a highly specific and sensitive ELISA method for the estimation of free cortisone levels in urine, saliva and in vitro media samples without chromatographic separation. Antibodies against cortisone were raised in rabbits using cortisone-3-CMO-KLH as immunogen. HRP-goat anti-rabbit IgG conjugate was used as enzyme tracer. Cross-reactivities of the untreated cortisone antiserum with major interfering steroids were minimal except for cortisol (3.15%). However, following an immune-affinity purification of the antibodies using CNBr-activated sepharose-cortisol-3-CMO-BSA, cross-reactivity of the purified cortisone antibody with cortisol was reduced to 0.27%. The minimum detection limit of cortisone ELISA was 28 pg/mL (77.7 pM). The validity of the cortisone ELISA was confirmed by the excellent correlation obtained before and after an HPLC fractionation step (Y=1.09X-0.21, R2=0.98). Intra-assay and inter-assay imprecision were 5.5-11.7% and 8.7-12.8% CV, respectively. Using this assay, salivary cortisone levels showed a circadian rhythm in men and women (11.2±7.3 nM at 08.00 h and 5.1±3.6 nM at 18.00 h), and the levels were reduced following liquorice ingestion. In media of adrenocortical H295 cell line incubations, basal cortisone levels were 4.24±0.22 nM that increased to 8.6±1.2 nM post forskolin treatment. Urinary free cortisone excretion levels in healthy subjects were 56.66±36.9 nmol/day. In human volunteers following ingestion of green coffee bean extract for 2 weeks, urinary free cortisol excretion reduced significantly from 66.67±22.3 to 42.66±17.5 nmol/day (p=0.02) and the cortisol/cortisone ratio from 2.04±1.33 to 1.49±1.13, p=0.05. In conclusion, a simple and highly specific and sensitive ELISA has been developed and applied to estimate cortisone levels in biological fluids and culture media.     

Influence of hydrophobic and hydrophilic spacer-containing enzyme conjugates on functional parameters of steroid immunoassay

Introduction of spacers in coating steroid antigen or enzyme conjugates or immunogen is known to exert an influence on the sensitivity of steroid enzyme immunoassays. We have introduced hydrophobic and hydrophilic spacers between enzyme and steroid moieties and studied their effects on functional parameters of enzyme immunoassays, using cortisol as a model steroid. Cortisol-3-O-carboxymethyloxime-bovine serum albumin (F-3-O-CMO-BSA) was used as immunogen to raise the antiserum in New Zealand white rabbits. Three enzyme conjugates were prepared using cortisol-21-hemisuccinate (F-21-HS) as carboxylic derivative of cortisol and horseradish peroxidase (HRP) as an enzyme label. These were F-21-HS-HRP (without spacer), F-21-HS-adipic acid dihydrazide-HRP (adipic acid dihydrazide as hydrophobic spacer), and F-21-HS-urea-HRP (urea as hydrophilic spacer). The influence of hydrophobic and hydrophilic spacers on the functional parameters of assays such as lower detection limit, ED50, and specificity was studied with reference to enzyme conjugate without spacer. The results of the present investigation revealed that the presence of a hydrophilic spacer in the enzyme conjugate decreases the lower detection limit, decreases the ED50, and marginally improves the specificity of assays. These improvements in functional parameters of assays may be due to the decreased magnitude of the overall hydrophobic interactions existing between the spacer in enzyme conjugate and the antigen binding site of the antibody.     

Site-specific photobiotinylation of antibodies, light chains, and immunoglobulin fragments

The high affinity of biotin for avidin has been exploited for many antibody-based assays. This requires that biotin is covalently conjugated to the antibody molecule. Several chemically reactive biotinylation reagents are commercially available. Except for the attachment via sulfhydryl groups in the immunoglobulin (Ig) molecule, these reagents attach biotin randomly to various amino acid side chains. Although non-site-specific modification of antibodies does not interfere in most immunoassays, specific application and sensitive antibodies would benefit from site-specific biotinylation. Here we describe an affinity biotinylation technique based on a photoreactive biotin reagent. The design of this reaction was possible from the discovery of a conserved binding site in the variable Ig domain for nucleotides and nucleosides. The described photoaffinity biotinylation offers the advantages of ease, convenience, and production of a reproducible and defined biotinylated antibody preparation.     

Methods of collection for salivary cortisol measurement in dogs

Salivary cortisol has been increasingly used as a measure of stress response in studies of welfare, reaction to stress and human-animal interactions in dogs and other species. While it can be a very useful measure, there are a number of saliva collection issues made evident through studies in the human and animal fields which have not been investigated in the canine species. Collection materials and the volume of saliva that is collected; the use of salivary stimulants; and the effect of food contamination can all dramatically impact cortisol measurement, leading to spurious results. In order to further examine the limitations of the collection method and the effects of collection material and salivary stimulant on salivary cortisol levels, a series of clinical, in vitro and in vivo studies were performed. It was found that there is a large amount of inter- and intra-individual variation in salivary cortisol measurement. Beef flavoring of collection materials leads to unpredictable variability in salivary cortisol concentration. Using salivary stimulants such as citric acid also has the potential to affect cortisol concentration measurement in saliva. Hydrocellulose appears to be a useful collection material for salivary cortisol determination. Recommendations for collection materials and use of salivary stimulants are presented.     

A novel enzyme-linked immunosorbent assay for ethynylestradiol using a long-chain biotinylated EE2 derivative

Ethynylestradiol (EE2) is one of the most potent endocrine disrupting compounds capable to induce estrogenic effects even at trace level concentrations in the aquatic environment. Methods for detecting EE2 in such concentrations are generally based on GC or HPLC coupled to at least one mass spectrometer. Another approach are immunoassays and sensor systems but for most designs, derivatives of EE2 are required (e.g. for coupling to carrier proteins, enzyme or fluorescent labels, etc.). Here we present the straightforward synthesis and complete characterization of a new long-chain biotinylated EE2 derivative. The new EE2 derivative is used as tracer in a direct competitive enzyme-linked immunosorbent assay (ELISA) for the determination of EE2. With pure water, the limit of detection (LOD, signal-to-noise ratio, S/N = 3) and the test midpoint were found to be 14 and 136 ng l(-1), respectively. Cross reactivity (CR) was tested for 10 endogenous steroids and the BSA-conjugate used for immunization, as well as a synthetic precursor of the conjugate. Among the naturally occurring compounds, CR was determined to be maximum for metabolites of EE2 conjugated at ring-position 3 (17% and 37% for 3-glucuronide and 3-sulphate, respectively). Assay stability was tested against humic substances and organic solvents. Increasing amounts of organic solvents in the sample caused a clear decrease in sensitivity, presence of humic substances lead to an overestimation of EE2.     

Biotin carboxyl carrier protein Co-purifies as a contaminant in core-streptavidin preparations

We have successfully cloned and expressed core-streptavidin in Escherichia coli. Core-streptavidin was expressed in shaker flask culture as a soluble protein, isolated by periplasmic extraction, purified by immobilized metal affinity chromatography column, and analyzed for its size, thermal stability, and biotin-binding activity. In Western blots using streptavidin-horseradish peroxidase (HRP) as a probe, we identified a contaminant that co-purified with core-streptavidin, identified as biotin carboxyl carrier protein (BCCP). Although BCCP cannot be detected on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it appears as a prominent band in Western blot when probed with streptavidin peroxidase conjugate. Based on the results from in vitro gel digestion, mass spectrometry and Mascot database search results, we confirmed the presence of BCCP. It was found that BCCP can complex with core-streptavidin and can dissociate when heated above 80°C. BCCP could be successfully removed and recovered by using core-streptavidin immobilized magnetic beads under mild conditions. In addition, the enriched fractions of core-streptavidin oligotetramers were separated, which may be the by-products of BCCP binding to core-streptavidin in various ratios. Finally, enzyme linked immunosorbent assay results have shown that the amount of biotin-HRP binding to core-streptavidin was higher compared to commercially available streptavidin.     

Colorimetric competitive inhibition method for the quantification of avidin, streptavidin and biotin.

A colorimetric competitive inhibition assay for avidin, streptavidin and biotin was developed. The method for avidin or streptavidin was based on the competitive binding between avidin or streptavidin and a streptavidin-enzyme conjugate for biotinylated dextrin immobilized on the surface of a microtitre plate. For biotin quantitation the competition is between free biotin and the immobilized biotin for the streptavidin-enzyme conjugate. The limits of detection which was determined as the concentration of competitor required to give 90% of maximal absorbency (100% inhibition) was approximately 20 ng/100 microl per assay for avidin and streptavidin and 0.4 pg/100 microl per assay for biotin. The methods are simple, rapid, highly sensitive and adaptable to high throughput analysis.     

Enzymatic amplification detection of DNA based on "molecular beacon" biosensors

We described a novel electrochemical DNA biosensor based on molecular beacon (MB) probe and enzymatic amplification protocol. The MB modified with a thiol at its 5' end and a biotin at its 3' end was immobilized on the gold electrode through mixed self-assembly process. Hybridization events between MB and target DNA cause the conformational change of the MB, triggering the attached biotin group on the electrode surface. Following the specific interaction between the conformation-triggered biotin and streptavidin-horseradish peroxidase (HRP), subsequent quantification of DNA was realized by electrochemical detection of enzymatic product in the presence of substrate. The detection limit is obtained as low as 0.1nM. The presented DNA biosensor has good selectivity, being able to differentiate between a complementary target DNA sequence and one containing G-G single-base mismatches.     

Luminescent immunoassay using isoluminol derivatives

Isoluminol derivatives (ID) were early employed in the preparation of tracers for immunoassay. Their wide use was mainly due to their high quantum efficiency, low molecular weight, well-known chemical structure, low cost and high stability. Moreover the light efficiency of some ID may be modified by specific binding to the antibody, thus allowing the development of homogenous immunoassays requiring no bound/free separation step. Some ID are commercially available under both the amino terminal and carboxyl terminal form for conjugation to respectively carboxy derivative of steroid and amino residues of protein. The linking reaction can be commonly carried on via active esters chemistry and can be easily accomplished within one day. Steroid conjugates can then be rapidly purified by silica gel thin layer chromatography, and protein conjugates by gel filtration on a short disposable column. In the field of steroid studies, isoluminol derivative conjugates were prepared for the immunoassay of almost all compounds of clinical interest. When dealing with protein, both antigens and antibody were labelled in this way, resulting in highly specific activity tracers for competitive and non-competitive immunoassays. Recently the labelling of streptavidin with amino-buty-ethyl-isoluminol allowed the development of very sensitive immunoassay methods which take advantage of the biotin-avidin system.     

Design, synthesis, and evaluation of 5'-S-aminoethyl-N(6)- azidobenzyl-5'-thioadenosine biotin conjugate: a bifunctional photoaffinity probe for the es nucleoside transporter

A bifunctional biotinylated photoaffinity label for the nitrobenzylmercaptopurine riboside (NBMPR)-sensitive (es) nucleoside transporter (ENT1) has been synthesized and evaluated. This new probe,5'-S-aminoethyladenosine-N(6)-azidobenzyl-5'-thioadenosine biotin conjugate (SAEATA-14-biotin), exhibited high-affinity binding to the es transporter in K562 cells as determined by flow cytometry, with a K(i) of 2.69 nM in competition against 5-(SAENTA)-x8-fluorescein. It also exhibited covalent linking to the es transporter in BeWo cell membranes upon UV irradiation. This new bifunctional probe is a potential tool for determining the amino acid residues involved in ligand binding at the NBMPR-binding site of the ENT1 nucleoside transporter, as well as for the purification of the transporter.     

Use of a biomimetic peptide in the design of a competitive binding assay for biotin and biotin analogues

A competitive binding assay for biotin, biocytin, and desthiobiotin utilizing a genetically engineered enzyme-ligand conjugate is described herein. This assay is unique in that the enzyme-ligand conjugate consists of the streptavidin binding peptide Strep-tag II, which mimics the binding of biotin to streptavidin, rather than biotin itself. This allows for the construction of a well-defined, oligosubstituted enzyme-ligand conjugate for which the site of attachment of the ligand on the enzyme is known precisely. The assay has detection limits of 5 x 10(-8) M for biotin, 1 x 10(-7) M for biocytin, and 2 x 10(-6) M for desthiobiotin, and it serves as a model system in that it demonstrates the feasibility of using enzyme-ligand conjugates in which a peptide mimic of the analyte ligand is genetically fused to the enzyme. This avoids the problems associated with covalent attachment of the ligand to the enzyme, such as multiple substitution of the ligand and variability of the site of attachment. To our knowledge, this is the first example of using an enzyme-peptide mimic conjugate to detect a nonpeptide analyte.     

Competitive Chemiluminescent Immunoassay for Estradiol Using an N-Functionalized Acridinium Ester

We have compared three competitive chemiluminescent immunoassays (CLIA) for estradiol (E2) using an N-functionalized acridinium ester (AE). The assays were a standard competitive assay using immobilized antibody and directly labeled antigen (type A), an immobilized antibody and indirectly labeled antigen (type B), and an immobilized antigen and labeled antibody (type C). In an antibody-immobilized system, the assay using both AE- and E2-labeled thyroglobulin as a tracer (type B) was more sensitive than that using AE directly coupled with E2 (type A). Subsequently, a comparison of the antibody-immobilized system (type B) and an antigen-immobilized system (type C) showed that the latter was slightly more sensitive than the former. The sensitivity of the CLIA (type C) was similar or superior to commercially available CLIA or radioimmunoassays for E2. Thus, the N-functionalized AE proved to be a useful labeling reagent for a competitive CLIA with high sensitivity.