Evaluation of an Electrochemiluminescent SARS-CoV-2 Antibody Assay.

BACKGROUND: Little is known about the performance of the Roche novel severe acute respiratory syndrome coronavirus 2 antibody (anti-SARS-CoV-2) assay. We provide an extensive evaluation of this fully automated assay on Cobas e801/e602 immunoassay analyzers. METHODS: We assessed the linearity, precision, and throughput of the Roche anti-SARS-CoV-2 assay. Sensitivity was calculated from 349 SARS-CoV-2 polymerase chain reaction (PCR) positive samples; specificity was determined from 715 coronavirus disease 2019 (COVID-19)-naive samples. We examined cross-reactivity against other antibody positive samples [syphilis, rheumatoid factor (RF), antinuclear antibody (ANA), double-stranded DNA (ds-DNA), influenza, dengue, hepatitis B (HBV), hepatitis C (HCV)] and the anti-SARS-CoV-2 kinetics. RESULTS: The assay cut-off index (COI) was linear up to 90.8. The interassay precision was 2.9% for a negative control (COI = 0.1) and 5.1% for a positive control (COI = 3.0). Assay time is 18 min and results are available 1 min later; throughput for 300 samples was 76 min. Only 1 case positive for HBsAg tested falsely positive; specificity was 99.9%. The assay has a sensitivity of 97.1% 14 days after PCR positivity (POS) and 100% at ≥21 days POS; 48.2% of cases had anti-SARS-CoV-2 within 6 days POS. In 11 patients in whom serum was available prior to a positive antibody signal (COI ≥1.0) the interval between the last negative and first positive COI (time to "seroconversion") on average is 3 days (range 1-6 days) and 4 more days (range 1-7) for the anti-SARS-CoV-2 to plateau. CONCLUSION: The Roche anti-SARS-CoV-2 assay shows excellent performance with minimal cross-reactivity from other viral and confounding antibodies. Antibody development and seroconversion appears quite early.

Impact of the Sofia® Influenza A+B FIA rapid diagnostic test in a pediatric emergency department.

OBJECTIVE: The objective of this study was to evaluate the impact of a rapid diagnostic test for influenza (the Sofia® Influenza A+B FIA rapid diagnostic test [RDT]) in a pediatric emergency department (PED). METHODS: A retrospective, observational, cross-sectional study was conducted in the PED of the Lille University Hospital between 2013 and 2015. All patients under 18 years of age for whom influenza RDT was administered were included. Clinical data, management, and related hospitalizations were compared between positive and negative RDT groups. The length of stay in the PED (main outcome) and the number of additional tests (biological and radiographic tests) between the two groups were compared. RESULTS: A total of 238 tests were reported: 119 positive, 110 negative, nine invalid. The mean length of stay in the PED was significantly lower in the positive RDT group: 4.0h vs. 7.4h (P<10-6). Patients with positive RDT had significantly fewer biological tests (20% vs. 56%; P<10-7) and radiographs (23% vs. 52%; P<10-5). The prevalence of hospitalizations in a short-stay unit was significantly lower in patients with positive RDT (0.8% vs. 9.1%; P=0.009). CONCLUSIONS: This study showed a significant medical impact of the use of Sofia® Influenza RDT A+B FIA in a PED regarding the length of stay and the number of additional explorations.

Performance of a time-resolved fluorescence immunoassay for measuring varicella-zoster virus immunoglobulin G levels in adults and comparison with commercial enzyme immunoassays and Merck glycoprotein enzyme immunoassay.

Highly sensitive and specific, quantitative assays are needed to detect varicella-zoster virus (VZV) immunoglobulin G in human sera, particularly for determining immune status and response following vaccination. A time-resolved fluorescence immunoassay (TRFIA) has been developed, and its performance was compared to that of two commercial enzyme immunoassays (EIAs) and Merck glycoprotein EIA (gpEIA). The TRFIA had equivalent sensitivity (97.8%) and high specificity (93.5%) in relation to gpEIA. A commercial (Behring) EIA compared favorably with TRFIA in terms of sensitivity (98.4%) but had lower specificity (80.7%). Another commercial EIA (Diamedix) had high specificity (97.1%) but low sensitivity (76.4%) compared to TRFIA if equivocal test results were treated as negative for VZV antibody. A novel feature of the TRFIA was that the cutoff was generated using population mixture modeling and was expressed in mIU/ml, as the assay was calibrated using the British standard VZV antibody.

Specific immunofluorimetric assay detecting the chemotactic epitope of the urokinase receptor (uPAR).

The urokinase plasminogen activator receptor (uPAR) fragments D1 and D2D3 are often found in biological fluids from normal individuals and patients of cancer and other diseases. The D2D3 fragment may possess chemotactic activity depending on its N-terminal sequence. We have developed a sensitive and specific immunoassay for the chemotactic form of D2D3 and show that its level can be measured with high specificity and sensitivity in human serum and urine. Synthetic peptides (residues 84-92) derived from the linker region between domains 1 and 2 of uPAR were used as immunogens to generate mouse monoclonal antibodies. Recombinant soluble uPAR (D1D2D3(1-277)) was used to immunize rabbits to obtain polyclonal antibodies. A sandwich-type immunofluorimetric assay was developed with these antibodies. The assay specifically measures D2D3 containing the 84-88 residues, has a detection limit of 0.25 ng/ml and shows no cross-reactivity with D2D3(93-274). The assay is linear at 0-30 ng/ml, with an intra-assay CV of 10% (n=20), inter-assay CV of 15% (n=9) and a recovery of D2D3(84-274) added to urine samples of between 94% and 105%. A statistically significant difference level of D2D3(84-274) was found in two groups of tumor patients versus healthy volunteers (p

Measurement of vasoactive intestinal peptide using a competitive fluorescent microsphere immunoassay or ELISA in human blood samples.

The concentration of Vasoactive Intestinal Peptide (VIP) as measured by recycling immunoaffinity chromatography (RIC) has been reported to be elevated in the blood of patients with autism as compared with normal subjects. In this study, we have developed a "Competitive Fluorescent Microsphere Immunoassay" (cFMI) in which VIP competes with biotinylated VIP in binding to polyclonal antibodies on microspheres. The results were obtained using the Luminex100 system. We measured VIP in serum, plasma, and material eluted from dried blood spots on filter paper with both the cFMI and an ELISA procedure. We found that a purification procedure was necessary for obtaining useful results from plasma and serum, however, a preincubation step was required with the blood eluates. This newly developed cFMI was more sensitive (2.5 vs. 20.0 pg/ml), and more reproducible than the ELISA. To get accurate measurements of VIP in eluted material high sensitivity is especially important. Thus, the cFMI using the Luminex system has definite advantages over a conventional ELISA including the possibility that samples can be assayed at higher dilutions. We have determined that the VIP concentrations of serum, plasma, and dried blood spot eluate specimens as measured with the cFMI assay system were similar to those measured with ELISA. Thus, the new cFMI using Luminex system may be useful for detection of VIP in human blood samples.

A sensitive time-resolved fluorescent immunoassay for metallothionein protein.

Metallothioneins (MTs) are a family of low molecular weight metal-binding proteins induced by a broad range of stress conditions, including exposure to transition metal ions. Biochemical and immunological methods to measure MT protein levels in tissues and cultured cells have been reported, but accuracy and sensitivity is impeded by high background levels, low specificity of currently available reagents, and relatively laborious and time-consuming multistep procedures. To address these difficulties, a protocol has been developed to measure MT protein levels using a competitive solid phase assay based on dissociation enhanced lanthanide fluoroimmuno (DELFIA) detection of anti-MT monoclonal antibody bound to solid phase MT. This assay allows time-resolved detection of antibody binding, based on binding and exchange of different lanthanide chelates followed by fluorescent detection, designed to reduce background fluorescence and increase sensitivity. The method allows measurement of low MT levels that are undetectable using current radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) protocols, and yields reproducible results with low background over a wide range of MT concentrations. Improved sensitivity of MT protein detection is of value in toxicological measurement of stress responses and assessment of MT expression and function.

Highly sensitive determination of plasma cytokines by time-resolved fluoroimmunoassay; effect of bicycle exercise on plasma level of interleukin-1 alpha (IL-1 alpha), tumor necrosis factor alpha (TNF alpha), and interferon gamma (IFN gamma).

A highly sensitive time-resolved fluoroimmunoassay of human plasma cytokines is described. The cytokines such as interleukin-1 alpha (IL-1 alpha), tumor necrosis factor alpha (TNF alpha) are known to be acute inflammatory cytokines and it has been reported that these cytokines are secreted into blood by physical exercise. In this study, a sandwich-type immunoassay of cytokines was established using a europium chelate BHHCT-Eu3+ as a powerful labeling material. The minimum detection limits of cytokines, i.e. IL-1 alpha, TNF alpha, and interferon gamma (IFN gamma) were about 1/10 smaller than those of enzyme-linked immunosorbent assay currently used. By this immunoassay we investigated cytokine increase/decrease in plasma which was thought to derive from the myocytes damaged by bicycle exercise. Healthy young men performed two kinds of bicycle ergometer exercises, under conditions of an incremental and a constant loading. Blood samples were taken before, during, and after exercises, and the concentration levels of plasma IL-1 alpha, TNF alpha, and IFN gamma were determined. In the case of incremental exercise, IL-1 alpha increased significantly at the first stage but decreased to the basal level from the second stage, in spite of heavier exercise. In the case of 30 min constant exercise, the level of plasma IFN gamma increased in recovery period, 2 h after the light-exercise. TNF alpha level was significantly higher in a heavy-exercise. The concentration of IL-1 alpha peaked at the early stage of the incremental exercise; this fact has not been reported in previous studies. This cytokine is unique in showing a sudden increase during the early stage, while others increase after the exercise. Our highly sensitive assay made it possible to detect a slight change in plasma cytokines.

Continuous-flow fluoro-immunosensor for paclitaxel measurement.

A fluorescence-based continuous-flow immunosensor for sensitive, precise, accurate and fast determination of paclitaxel was developed. The sensor utilizes anti-paclitaxel antibody immobilized through its Fc region and crosslinked by dimethylpimelimidate to protein A attached covalently onto the silanized inner walls of a glass capillary column followed by saturation of the paclitaxel-binding sites with rhodamine-labeled paclitaxel. The assay is based on the displacement and detection downstream of the rhodamine-labeled paclitaxel, by a flow-through spectrofluorometer, as a result of the competition with paclitaxel introduced as a pulse into the stream of carrier buffer flowing through the system. The peak height of the fluorescence intensity profile of the displaced rhodamine-labeled paclitaxel was directly proportional to the concentration of paclitaxel applied and was a function of the carrier buffer flow rate. The sensitivity of the immunosensor response ranged from 0.31 relative fluorescence units (RFU)/ng/ml at a flow rate 0.1 ml/min to 0.52 RFU/ng/ml at 1 ml/min, while the lower detection limit ranged from 1 ng/ml at 0.1 ml/min to 4 ng/ml at 1 ml/min. The immunosensor response was very reproducible (RSD=4.8%; n=10) and linear up to 100 ng/ml. The assay time ranged from 2 min at 1 ml/min to 8 min at 0.1 ml/min. A technique developed to resaturate the antigen binding sites of the immobilized antibody with rhodamine-labeled paclitaxel was successful in regenerating the capillary column without affecting its performance, thus enhancing the economic viability of the immunosensor. The immunosensor was successfully applied for the determination of paclitaxel in human plasma.

Zeptomole detection sensitivity of prostate-specific antigen in a rapid microtitre plate assay using time-resolved fluorescence.

Prostate-specific antigen (PSA) was detected in microtitre wells coated with a PSA-specific antibody using biotinylated antibody and streptavidin-coated, highly fluorescent 107 nm nanoparticles, which contained more than 30000 europium ions entrapped by beta-diketones. PSA was monitored directly on the surface of a well without any additional enhancement step. The sensitivity of the assay was 1.6 ng/L, corresponding to 50 fmol/L or 250 zeptomoles (250 x 10(-21) mol/L) of PSA. The high specific activity and low non-specific binding of the streptavidin-coated nanoparticles improved the sensitivity of the PSA assay 100-fold compared to the conventional europium-labelled streptavidin tracer in the same assay format. Additionally, the streptavidin-coated nanoparticle label made very rapid assays possible, due to the high affinity of the streptavidin-biotin complex and a high number of binding sites available for tracing the biotinylated antibody on the surface. Due to the inherent problems of tracing analyte with a complex of biotinylated antibody and streptavidin-coated nanoparticles, the streptavidin-coated nanoparticles reacting with the surface-captured analyte and biotinylated antibody was favoured and factors influencing this are discussed. This universal labelling technology can be applied to detect any biotinylated molecule, either in solution or on a solid phase, in order to improve detection sensitivities in many areas of biochemical analysis, such as cyto- and histochemistry, multianalyte DNA-chip assays and single-particle assays.

Detection of unwanted residues of ivermectin in bovine milk by dissociation-enhanced lanthanide fluoroimmunoassay.

Avermectins are frequently used to control parasitic infestations in many animal species. Previous studies have shown the long-term persistence of unwanted residues of these drugs in animal tissues and fluids. An immunoassay screening test for the detection and quantification of ivermectin residues in bovine milk has been developed. After an extensive extraction procedure, milk samples were applied to a competitive dissociation-enhanced lanthanide fluoroimmunoassay using a monoclonal antibody against an ivermectin-transferrin conjugate. The monoclonal antibody, raised in Balb C mice, showed cross-reactivity with eprinomectin (92%), abamectin (82%) and doramectin (16%). The limit of detection of the assay (mean + 3 SD), calculated from the analysis of 17 known negative samples, was calculated as 4.6 ng/mL. Intra- and inter-assay RSDs were determined as 11.6% and 15.8%, respectively, using a negative bovine milk sample fortified with 25 ng/mL ivermectin. Six Friesian milking cows were treated with ivermectin, three with a pour-on formulation of the drug and three with an injectable solution at the manufacturer's recommended dose rate. An initial mean peak in ivermectin residue concentration was detected at day 4 (mean level = 47.5 ng/mL) and day 5 post-treatment (mean level = 26.4 ng/mL) with the injectable form and pour-on treatment, respectively. A second peak in residue concentration was observed using the DELFIA procedure 28 days post-treatment in both treatment groups (23.1 ng/mL injectable and 51.9 ng/mL pour-on). These second peaks were not confirmed by HPLC and must at this time be considered to be false-positive results. By day 35 after treatment the mean ivermectin residue concentration of both groups fell below the limit of detection of the assay.

Time-resolved fluorometry (TRF)-based immunoassay concept for rapid and quantitative determination of biochemical myocardial infarction markers from whole blood, serum and plasma.

We report the development of a time-resolved fluorometry-based immunoassay concept for the rapid measurement of three cardiac markers from whole blood, serum or plasma. Using a universal all-in-one (AIO) dry reagent concept, all the analyte specific reagents are built into a single microtire well, to which an identical assay protocol is applied. Addition of 5-20 microL sample (whole blood, serum or plasma) together with a universal buffer initiates the reaction, which is brought close to equilibrium in 15 min. After the wash step the Eu chelate-derived signal is measured directly from the dried surface. Application of this concept to the three cardiac markers illustrates its ability to provide rapid, highly sensitive and fully quantitative results over a large dynamic range with good reproducibility. Such a performance, especially when using whole blood specimens, is largely a consequence of the inherently fluorescent and stable Eu-chelate employed in the system. Correlation to commercial assays was excellent for all three analytes, as was between-sample matrix correlation using the AIO assays. The presented assay concept enabling a simple automation is particularly suited for point-of-care applications, where the performance characteristics are fully comparable to state-of-the-art central laboratory immunoassays.

A novel and robust homogeneous fluorescence-based assay using nanoparticles for pharmaceutical screening and diagnostics.

We have established a new type of homogeneous immunoassay based on nanoparticles (nanoparticle immunoassay, or NPIA) being analyzed using fluorescence intensity distribution analysis (FIDA). This method allows the characterization of single fluorescently labeled molecules or particles with respect to their molecular brightness and concentration. Upon binding of conjugates to molecules coupled to the nanoparticle surface, the brightness of the complex scales with the number of bound conjugates. The complexes can then be distinguished accurately from free conjugate and concentrations of free and bound molecules can be determined reliably. In this study we present various examples of NPIAs where capture antibodies were linked to the nanoparticles, which were either artificial beads or bacteria. Two assay formats have been developed; first, direct labeling of the conjugate was used to quantitate free antigen through competition experiments, and second, an antigen-directed antibody was labeled to establish an assay similar to a sandwich ELISA setup. The major advantages of a NPIA are the robustness and high signal-to-noise ratio at short measurement times, as demonstrated with a miniaturized experiment in a Nanocarriertrade mark holding a volume of 1 microl/well. In addition to the good data quality, NPIAs are straightforward to perform because they require no washing steps. NPIAs open new dimensions for high throughput pharmaceutical screening and diagnostics. Assay development times can be reduced significantly because of a simple toolbox principle that is applicable to most types of assays.

Development of an offline noncompetitive flow immunoassay for the determination of interleukin-8 in cell samples.

A noncompetitive flow immunoassay system (FIA) for the analysis of interleukin-8 (IL-8) in cell samples was developed. Affinity interaction assays based on offline incubation of excess labeled antibodies and antigen (IL-8) were carried out. The residual unbound labeled antibody was trapped in an immunoaffinity column with immobilized IL-8 while the immunocomplex, labeled antibody/IL-8, was detected by a fluorescence detector. Two fluorophores, FLUOS and Cy5.5, were conjugated with IL-8 antibody. Optimization and comparison between the two fluorescent labeled antibodies were performed with regard to pH, antibody concentration, flow rate, injection volume, and association time. Additionally, a horseradish peroxidase enzyme label was used for the conjugation to the anti-IL-8. The enzyme substrate reaction was optimized with respect to temperature and length of the substrate reaction coil. The detection limits were found to be 200 amol using the FLUOS-labeled anti-IL-8 and 1 fmol using the Cy5.5 fluorescence label. The developed FIA technique was applied for the analysis of IL-8 in cell samples. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to identify IL-8 in the cell samples.

Highly sensitive and specific time-resolved fluoroimmunoassay (TR-FIA) of cyproterone acetate and free cyproterone.

We have developed a non-isotopic TR-FIA for Cyproterone acetate and Cyproterone in plasma. Synthesis of the biotinylated tracers, biotinylated Cyproterone acetate, and Cyproterone, as well as the preparation of anti-Cyproterone acetate and anti-Cyproterone antisera are reported. The specificity of anti-Cyproterone acetate antiserum resulting from the coupling of link bridge (link bridge between steroid and BSA), on the 3-position on the steroid skeleton, allowed to carry out the Cyproterone acetate assay directly on extracted plasma (without chromatography). On the other hand Cyproterone assays require a purification step, including extraction plus chromatography, because the plasma Cyproterone acetate concentrations in Cyproterone acetate-treated women are 200 times higher than for Cyproterone. Theses plasma TR-FIA of Cyproterone acetate and Cyproterone presented the advantage of needing only small doses of radioactivity for recovery controls, and better practicability related to the only existing RIA described to date.

Time-resolved fluorescence immunoassay of thyroxine in serum: immobilized antigen approach.

With T(4)-bovine IgG as a solid-phase antigen, we have developed a direct competitive-type immunoassay for serum total thyroxine (TT(4)), which depends on the competitive distribution of europium-labeled anti-T(4) monoclonal antibody between solid-phase-bound T(4) and the T(4) in the sample or standard. The captured fraction of the tracer was measured after a dissociation-enhancement step. Four different T(4) protein conjugates were synthesized, of which T(4)-bovine IgG was selected as the most favorable for the preparation of solid-phase antigen. The sensitivity was 3.5 ng/ml with a sample volume of 20 microl. T(4) values obtained by this procedure agreed well with those obtained by RIA (r = 0.967, n = 38) and EG&G Wallac TRFIA (r = 0.926, n = 64). All other quality criteria was also fulfilled with respect to precision, accuracy, and dynamic range.

Differences in urinary excretion patterns of the hLH beta core fragment in premenopausal, perimenopausal, and postmenopausal women.

OBJECTIVE: The heterodimeric luteinizing hormone beta core fragment (hLH beta cf) is a highly stable urinary analyte reflective of circulating hLH. It is measured easily because of its high molar content and has none of the multiple isoforms and subunit dissociation problems of LH urinary measurements. As part of a long-term effort to develop new biochemical assays to stage women during the perimenopausal transition, we have examined the patterns of urinary excretion of this metabolite of hLH in premenopausal, perimenopausal, and postmenopausal women. DESIGN: We measured the concentration of the hLH beta cf in 10 consecutive first morning void urine specimens from premenopausal, perimenopausal, and postmenopausal women. Day 1 of collection was the first day of menses in the cycling women. RESULTS: Postmenopausal women exhibited a widely fluctuating pattern of LH beta core fragment excretion, which is not correlated with hLH measured by immunofluorometric assay or with follicle-stimulating hormone measured by immunofluorometric assay. The postmenopausal group was easily distinguished from premenopausal women on the basis of an area-under-the-curve concentration function. Perimenopausal women displayed intermediate hLH beta cf concentrations; some clearly were in postmenopausal ranges, and others were in the premenopausal ranges. CONCLUSIONS: The pattern of excretion and concentrations of the hLH beta cf is significantly different between premenopausal and postmenopausal women. Perimenopausal women exhibited intermediate changes. The capability to measure this type of stable urinary metabolite as a reflection of changes in dynamics of its parent circulating hormone offers new possibilities in the development and application of large-scale testing that does not require blood sampling.

Evaluation of single- and dual antigen delayed fluorescence immunoassay in comparison to an ELISA and the in vivo toxin neutralisation test for detection of diphtheria toxin antibodies.

An evaluation of the delayed fluorescence immunoassay (Delfia) against an ELISA method for determination of diphtheria antitoxin levels in serum was performed. The Delfia was also validated in the in vivo toxin neutralisation test (Txn) in rabbits. Two variants of the Delfia were studied, a single-antigen Delfia (sDelfia) with only the diphtheria toxin included and a dual-antigen Delfia (dDelfia) with tetanus toxoid included for simultaneous detection of antibodies against two antigens. The diphtheria antitoxin cut-off levels in the sDelfia and the dDelfia were 0.004 and 0.002 AU/ml, respectively, which is lower than the internationally accepted level showing any protection against diphtheria (0.01 IU/ml). Both Delfia variants showed good correlation with the ELISA procedure above the ELISA cut-off level of 0.02 AU/ml. Results from samples assayed in the in vivo Txn assay indicated that the low antitoxin levels detected by the Delfia were valid. These results show that the Delfia could be considered as an in vitro reference method for detection of diphtheria antitoxin in seroepidemiological surveys and vaccine studies.

[Lanthanide immunofluorescent analysis: virus detection and the serodiagnosis of viral infections]. / Lantanidnyi immunofliuorestsentnyi analiz: indikatsiia virusov, serodiagnostika virusnykh infektsii.

This communication summarizes 10-year experience gained by the author in developing and using the lanthanide immunofluorescence assay (LIFA) for the laboratory diagnosis of viral infections. The bulk of studies has been conducted on natural focal viruses, including Venezuelan equine encephalitis, tick-borne encephalitis, Crimean Congo hemorrhagic fever, California serogroup, and other viruses. Moreover, test systems have been developed for diagnosis of infections caused by herpes simplex and cytomegaloviruses. The studies performed have demonstrated the sensitivity of LIFA in the indication of viruses in the laboratory materials and the samples from natural foci is 10-100 times higher than that of enzyme immunoassay and it is close to that of the biological isolation assay; the specificity of LIFA is comparable to that of the neutralization reaction, but it is more accessible in practice due to the fact it does not require the use of living viruses and biological models. The results of detection of herpes viruses in the clinical samples by LIFA are shown to rather well correlate with the data of virus isolation in the cultured cells, with other diagnostic methods and with the clinical manifestations of diseases. LIFA is recommended for use in large-scale studies involving the monitoring of infection foci and the screening of risk population groups for social infectious diseases.

Development of a magnetic microplate chemifluorimmunoassay for rapid detection of bacteria and toxin in blood.

A magnetic microplate chemifluorimmunoassay (MMCIA) is described using an immunomagnetic separation and a fluorescent microplate technique for rapid detection of low-level Escherichia coli O157:H7, Bacillus subtilis var. niger spores, and Staphylococcal enterotoxin type B from whole blood. In general, the MMCIA has at least several-fold more sensitivity than the conventional enzyme-linked immunosorbent assay. In addition, the assay sensitivities using direct fluorochrome label as the reporter, or alkaline phosphatase (AP) with various assay substrates, such as pNPP and AttoPhos, were assessed.

A time-resolved fluorescence immunoassay for the measurement of testosterone in saliva: monitoring of testosterone replacement therapy with testosterone buciclate.

Monitoring of testosterone replacement therapy requires a reliable method for testosterone measurement. Determination of salivary testosterone, which reflects the hormone's biologically active plasma fraction, is a superior technique for this purpose. The aim of the present study was to establish a new sensitive time-resolved fluorescence immunoassay for the accurate measurement of testosterone levels in saliva and to validate it by monitoring testosterone replacement therapy in eight hypogonadal men. A clinical phase I-study with the new ester testosterone buciclate was performed to search for new testosterone preparations to produce constant serum levels in the therapy of male hypogonadism. After two control examinations eight male patients with primary hypogonadism were randomly assigned to two treatment groups (n = 2 x 4) and given single doses of either 200 mg (group I) or 600 mg (group II) testosterone buciclate intramuscularly. Saliva and blood samples were obtained 1, 2, 3, 5 and 7 days post injection and then weekly for three months. The time-resolved fluorescence immunoassay for salivary testosterone shows a detection limit of 16 pmol/l, an intra-assay CV of 8.9% (at a testosterone concentration of 302 pmol/l), an inter-assay CV of 8.7% (at a testosterone concentration of 305 pmol/l) and a good correlation with an established radioimmunoassay of r = 0.89. The sample volume required by this method is only 180 microliters for extraction and duplicate determination. The assay procedure requires no more than three hours. In group I (200 mg) testosterone did not increase to normal levels either in saliva or in serum. However, in group II, androgen levels increased significantly and were maintained in the normal range for up to 12 weeks with maximal salivary testosterone levels of 303 +/- 18 pmol/l (mean +/- SE) and maximal testosterone levels of 13.1 +/- 0.9 nmol/l (mean +/- SE) in serum in study week 6 and 7. The time-resolved fluorescence immunoassay for salivary testosterone provides a useful tool for monitoring androgen status in men and women and is well suited for the follow-up of testosterone replacement therapy on an outpatient basis. The long-acting ester testosterone buciclate is a promising agent for substitution therapy of male hypogonadism and in combination with testosterone monitoring in saliva offers an interesting new perspective for male contraception.