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BACKGROUNDMeasurement of prostate-specific antigen (PSA) in prostate cancer patients following radical prostatectomy (RP) has been hindered by the limit of quantification of available assays. Because radical prostatectomy removes the tissue responsible for PSA production, postsurgical PSA is typically undetectable with current assay methods. Evidence suggests, however, that more sensitive determination of PSA status following RP could improve assessment of patient prognosis and response to treatment and better target secondary therapy for those who may benefit most. We developed an investigational digital immunoassay with a limit of quantification 2 logs lower than current ultrasensitive third-generation PSA assays.METHODSWe developed reagents for a bead-based ELISA for use with high-density arrays of femtoliter-volume wells. Anti-PSA capture beads with immunocomplexes and associated enzyme labels were singulated within the wells of the arrays and interrogated for the presence of enzymatic product. We characterized analytical performance, compared its accuracy with a commercially available test, and analyzed longitudinal serum samples from a pilot study of 33 RP patients.RESULTSThe assay exhibited a functional sensitivity (20% interassay CV) CONCLUSIONSThe robust 2-log improvement in limit of quantification relative to current ultrasensitive assays and the validated analytical performance of the assay allow for accurate assessment of PSA status after RP.

The need for assay methods of sufficient sensitivity to determine the low concentrations of hormones present in body fluids led to the original development of immunoassays and analogous “binding” (or “ligand”) assays in the late 1950s and early 1960s. These methods depend on the use of a binding agent (also commonly referred to by other terms such as “receptor,” “binding reagent,” and “analyte-specific reagent”), a substance used to recognize and bind the target analyte. Typical binding agents include antibodies, antigens, cell receptors, and serum binding proteins. Immunoassays still constitute the most widely used class of binding assays, although microarray-based nucleic acid assays, employing oligonucleotides as binding agents, are rapidly increasing in popularity. A principal objective in this field since the emergence of these assays has been to increase their sensitivities, especially in their application to certain analytes. Typifying such attempts, Rissin et al. (1) have recently reported a new approach to the further improvement of immunoassay sensitivities, claiming that with the use of an ELISA-type system, they were able to “detect serum proteins at subfemtomolar concentrations” and to increase the sensitivity of measurements “using a typical ELISA plate reader by a factor of about 68 000.” But before discussing the novel features of Rissin et al.'s approach, we should briefly examine the concept of sensitivity and the meaning of the term “sensitive” to describe the performance of a binding assay—or indeed that of any measurement system. Many workers in this area, including Rissin et al., identify sensitivity with the lower limit of detection (LoD)3 of an assay. However, certain bodies, including the American Chemical Society and the International Union of Pure and Applied Chemistry, have formally defined sensitivity as the slope of the dose–response curve [or the response/dose (R/D) ratio—an intrinsically meaningless concept with which we strongly disagree (see …

Background: Quantitative, miniaturized nucleic acid assays and immunoassays can be developed with single microparticles, microfluorometric detection, and intrinsically fluorescent lanthanide chelates in a multiple assay format to decrease reagent consumption, cost, and assay time. We used recombinant Fab fragments to capture and detect free and total prostate-specific antigen (PSA) from serum in a submicroliter volume single-particle immunoassay.Methods: Genetically engineered thiol-Fab or thiolated monoclonal antibodies (mAbs) were covalently attached onto uniformly sized 60-μm maleimide-activated microparticles. Free and total PSA were detected with europium- or terbium-labeled Fab fragments on a single microparticle using a microfluorometer in a time-resolved mode.Results: The detection limit of the free- and total-PSA assays (mean + 3 SD of zero calibrator) was 0.35 μg/L, with a total volume of 330 nL per particle. An excellent correlation was found in microparticle and microtiter-well assays for 21 serum samples: slopes for free and total PSA were 1.06 ± 0.03 and 1.03 ± 0.02, respectively (Sy|x = 0.084 and 0.057 μg/L), with intercepts of 0.013 ± 0.018 and 0.013 ± 0.017 μg/L (R >0.99). Furthermore, the particle-immobilized Fab fragment had a PSA binding capacity 1.5-fold higher than the intact mAb capacity on a single microparticle. Capacity, kinetics, and sensitivity of the Fab fragment and intact mAb assays in the microparticle and microtiter well formats are discussed.Conclusions: With site-specific (cysteine tail) covalent attachment of Fab fragments on a microparticle, subattomole amounts of PSA can be detected quantitatively.

An UV/VIS spectrophotometric assay technique was developed that was able to routinely monitor peptidylprolyl cis/trans isomerase (PPIase) activity of biological fluids in 96-well microtiter plates. The assay, based on monitoring the cis-to-trans isomerization of succinyl-Phe-cisPro-Phe-4-nitroanilide as substrate in a chymotrypsin-coupled reaction, yields a throughput of 96 samples per 30 min. The assay’s capacity was exemplified by dealing with the PPIase activity in several normal and pathological human sera. Reference values of 151 healthy subjects (83 females, 69 males, 17 to 60 years old) were found to possess significant sex-specific differences. PPIase activity factor K of the sera was significantly greater in males (5th, 50th, 95th percentiles: 17, 36, 55 K) than females (14, 30, 48 K). PPIase activities of sera from healthy donors (n = 151) were significantly higher (Mann–Whitney rank-sum test P

Widespread evidence supports the existence of an endogenous digitalis-like compound in mammals. We report here the development of a novel enzyme immunoassay for ouabain that, in conjunction with a detailed HPLC study, identifies a ouabain-like compound (OLC) in extracted human plasma. The assay is sensitive—minimum detection limit for OLC 37 pmol/L (11 pmol/L in plasma)—and has a working range (between-assay CV

A specific and sensitive assay for quantifying the immunosuppressant FK506 and its metabolites in blood and urine was developed. 32-O-Acetyl FK506 was synthesized and used as internal standard. FK506 and its metabolites were purified from the samples by solid-liquid extraction and were injected into a high-performance liquid chromatographic (HPLC) system linked to a mass spectrometer (MS) by particle-beam interface. The FK506 derivatives were separated from interfering material by use of a 100 x 4 mm C8 analytical column and water/acetonitrile or water/methanol gradient elution; they were detected by negative chemical ionization with methane as reagent gas. The limit of detection was 25 pg in a standard solution, and the limit of quantification in blood was 250 pg (extracted from 1 mL of blood). The CV was 11.3% at 5 ng, and no interferences with other drugs were found.

Estimation of the extent and location of infarct is important for the prognosis and hence therapeutic strategy in patients with acute myocardial infarction (AMI). Because cardiac myosin is the major structural protein of the myocardium, and may thus reflect the extent of injured tissue, we established a new sensitive immunoradiometric assay, using a pair of monoclonal antibodies (Mabs) that specifically bind the myosin heavy chain fragments liberated from the myocyte into plasma after a heart attack. A first Mab is linked to a magnetic solid phase. A second Mab, radiolabeled with 125I, is used to detect myosin trapped on the solid phase by the first Mab during a 3-h incubation. This assay can detect 10 micro-units of myosin per liter and is highly reproducible.

The nitric oxide radical (NO·) plays an important role as a physiological messenger (1). NO is formed from l-arginine (2) by NO synthase (NOS; EC 1.14.13.39), which exists in several isoforms (3). Constitutive calcium-dependent isoforms (cNOS) modulate the control of vascular tone in endothelial cells or the neurotransmission in neurons, whereas inducible calcium-independent isoforms (iNOS) are located in macrophages, chondrocytes, and hepatocytes and are induced by cytokines and endotoxin (4)(5). Pathological conditions associated with increased release of cytokines and endotoxin, e.g., inflammation or sepsis (6), can therefore increase NO production. NO is a very unstable, short half-life gas that breaks down rapidly into the stable products nitrate and nitrite (7). Upon coming into the bloodstream, nitrite reacts immediately with oxyhemoglobin to form methemoglobin. Consequently, most NO produced is detected in serum as the remaining product, nitrate (8). Recently, several reports focused on methods to measure nitrate concentrations in biological fluids (9)(10)(11). One of the most commonly used methods is based on the reduction of nitrate to nitrite by cadmium or nitrate reductase, the nitrite produced being determined by Griess reaction (9)(12)(13). Other methods for monitoring NO production are based on chemiluminescence (11)(14), enzymatic assay with an internal standard (10), or chromatographic procedures (8) for nitrate (15)—all of which are time-consuming for routine application in clinical chemistry laboratories. We describe a rapid semiautomated method based on the Griess reaction, involving a shortened incubation period of nitrate with cadmium. The method is applicable to several types of biological fluids. Serum or plasma samples from healthy individuals after a 12-h fast were obtained in accordance with the Medical Ethical Committee of our hospital. Samples were stored at −20 °C and were stable for at least 6 months. The method was …

We developed and evaluated a method for the determination of microgram/L concentrations of individual arsenic species in urine samples. We have mainly studied arsenite [As(III)], arsenate [As(V)], monomethylarsonic acid (MMAA), and dimethylarsinic acid (DMAA) because these are the most commonly used biomarkers of exposure by the general population to inorganic arsenic and because of concerns over these arsenic species on their toxicity and carcinogenicity. We have also detected five unidentified urinary arsenic species resulting from the metabolism of arsenosugars. We combined ion pair liquid chromatography with on-line hydride generation and subsequent atomic fluorescence detection (HPLC/HGAFS). Detection limits, determined as three times the standard deviation of the baseline noise, are 0.8, 1.2, 0.7, and 1.0 mu/L arsenic for arsenite, arsenate, MMAA, and DMAA, respectively. These correspond to 16, 24, 14, and 20 pg of arsenic, respectively, for a 20-muL sample injected for analysis. The excellent detection limit enabled us to determine trace concentrations of arsenic species in urine samples from healthy subjects who did not have excess exposure to arsenic. There was no need for any sample pretreatment step. We used Standard Reference Materials, containing both normal and increased concentrations of arsenic, to validate the method. Interlaboratory studies with independent techniques also confirmed the results obtained with the HPLC/HGAFS method. We demonstrated an application of the method to the determination of arsenic species in urine samples after the ingestion of seaweed by four volunteers. We observed substantial increases of DMAA concentrations in the samples collected from the volunteers after the consumption of seaweed. The increase of urinary DMAA concentration is due to the metabolism of arsenosugars that are present in the seaweed. Our results suggest that the commonly used biomarkers of exposure to inorganic arsenic, based on the measurement of arsenite, arsenate, MMAA, and DMAA, are not reliable when arsenosugars are ingested from the diet.

A glass microchip is described in which reagents and serum samples for competitive immunoassay of serum theophylline can be mixed, reacted, separated, and analyzed. The device functions as an automated microfluidic immunoassay system, creating a lab-on-a-chip. Electroosmotic pumping was used to control first the mixing of 50-fold-diluted serum sample with labeled theophylline tracer in a 1:1 ratio, followed by 1:1 mixing and reaction with anti-theophylline antibody. The 51-nL on-chip mixer gave the same concentration as dilution performed off-chip, within 3%. A 100-pL plug of the reacted solution was then injected into an electrophoresis separation channel integrated within the same chip. Measurements of free and bound tracer by fluorescence detection gave linear calibration curves of signal vs log[theophylline] between 0 and 40 mg/L, with a slope of 0.52 +/- 0.03 and an intercept of -0.04 +/- 0.04 after a 90-s reaction time. A detection limit of 0.26 mg/L in serum (expressed before the dilution step, actual concentration of 1.3 micrograms/L at the detector) was obtained. Recovery values were 107% +/- 8% for 15 mg/L serum samples.

We developed a rapid, competitive enzyme immunoassay (EIA) for measuring 25-hydroxyvitamin D3 [25(OH)D3] in serum. The EIA was based upon 25(OH)D3-3-hemisuccinate covalently coupled to secondary amino groups grafted onto the polystyrene surface of microtiter wells. Optimal coupling conditions were established, and we found that inclusion of 40 μmol/L chloramine T, an agent not previously described for use in coupling to these plates, resulted in both more reproducible coupling as well as more than a twofold increase in the coupling efficiency. Before EIA, 25(OH)D3 was extracted from the serum samples by acetonitrile, and the redissolved extract was incubated with polyclonal rabbit antibody raised against 1,25-dihydroxyvitamin D3-3-hemisuccinate conjugated to bovine serum albumin. Peroxidase-labeled antibody raised in goat against rabbit immunoglobulins was used for detection. The detection limit of the EIA was 4.4 μg/L; recovery 102%; on-plate CV 11%; within-run CV including extraction 12%, and between-run CV 15%. There was no clinically important cross-reactivity with other vitamin D metabolites, and results obtained by the EIA were compared with results obtained by a previously described RIA.

Fluid and electrolyte assessment during the first week of life is complicated by rapid changes in fluid and electrolyte balance during the transition from fetal to neonatal life and by the newborn's small size. A physiologic decrease in extracellular water volume, as well as a transient increase in serum potassium and transient decreases in plasma glucose and total plasma ionized calcium concentrations must be taken into account. In general, the more immature the newborn, the greater the changes that can be expected. The use of plasma creatinine as an indicator of glomerular filtration rate is limited because it is a function of maternal renal function at birth and because of non-steady-state conditions in the immediate postnatal period. Guidelines for monitoring schedules are provided on the basis of these physiologic considerations and the author's experience. Method of blood sampling and time to separation of serum are important considerations in interpreting results. Minimization of sample volume is critical to minimize blood transfusion requirements. Clinicians should be aware of the analytical error associated with these measurements in their own institutions. Reference ranges are provided.

Binary-system capillary electrophoresis (CE) with head-column field-amplified sample stacking permits determination of amiodarone and desethylamiodarone in 20-microL serum samples. The assay is characterized by a detection limit for both compounds of 80 nmol/L and by excellent linear response over the recommended therapeutic range for amiodarone (1.5-4 mumol/L). Intra- and interday reproducibilities (CVs) between 3% and 6% and run times of approximately 10 min are comparable with those for conventional HPLC. Besides excellent sensitivity, attractive features of the assay include low operating costs, high separation efficiency, rapid drug extraction, consumption of almost negligible amounts of organic solvents, and simple operation. If appropriate microvessels and liquid-handling facilities are available, the same assay can be performed with 2 microL of serum, and if the serum is not diluted but rather is concentrated during extraction,1 nmol/L of amiodarone can be detected.

We investigated the efficiency, accuracy, and reliability of the ultrafiltration/dipstick methodology commonly used to diagnose myoglobinuria. Twenty-five myoglobin-containing urine specimens were filtered by centrifugation for 15 min at 1500g through a Centricon-30 membrane filter. Both the original specimen and filtrate were assayed for myoglobin. The amount of myoglobin recovered subsequent to filtration varied from1-38%. This poor and variable recovery was independent of sample matrix or precentrifugation of the specimens. This was most critical for urine specimens with myoglobin concentrations60 000 microg/L. Fourteen of 18 such filtrates had concentrations350 microg/L, a concentration below which a negative result would be obtained by using conventional dipstick methods. Thus, the use of this procedure has the potential to misdiagnose patients with myoglobin concentrations associated with increased risk of subsequent renal dysfunction, in particular when urine myoglobin concentrations are60 000 microg/L.

Amrinone (AMR), a bipyridine derivative, is receiving increasing use in postoperative cardiac patients as an inotrope and vasodilator. The hemodynamic response to amrinone in adults is linearly related to AMR concentrations, warranting therapeutic drug monitoring. We report a rapid microsample HPLC method for monitoring AMR and its principal metabolites, N-acetyl (N-ac) and N-glycolyl (N-gly) AMR. Serum was precipitated with acetonitrile, and the supernatant fluid was then injected into a C18 narrow-bore column. The mobile phase consisted of a 0.1 mol/L sodium phosphate buffer (pH 6) with a gradient of acetonitrile going from 50 to 100 mL/L of eluent. Detection with a diode-array detector (DAD) concurrently monitored the absorbances at 320 and 345 nm. Monitoring 320 nm allows optimal quantification of AMR, N-gly, and N-ac. Patients often receive concurrent cephalosporin therapy, which is detectable at 320 nm but not 345 nm. Because cephalosporins coelute with AMR or metabolites, monitoring at 345 nm allows separation of these antibiotics from AMR and metabolites while retaining a detection limit of 0.5 mg/L.

Previously, using recombinant human progastrin-releasing peptide (ProGRP)(31-98), we developed a RIA for ProGRP(31-98) and demonstrated that the determination of serum ProGRP(31-98) was a reliable marker for small-cell lung carcinoma (SCLC) (Miyake et al., Cancer Res 1994;54:2136-40). Aiming for a more convenient assay system, we have now developed and evaluated a highly sensitive and specific ELISA for ProGRP(31-98). Only 50 microL of nonextracted serum is needed, and results are obtained in only 2 h. Intraassay and between-day CVs were 1.7-4.6% and 4.2-6.8%, respectively. The log-log calibration curve was linear to 1000 ng/L, and analytical recovery was 91.5-108.7%. The detection limit of this assay, 1.9 ng/L, means that basal concentrations of ProGRP(31-98) were detectable in all healthy subjects. The cutoff value, based on the mean + 3 SD of concentrations in 247 healthy subjects, was set to 45.1 ng/L. Serum concentrations exceeded this value in 18 of 25 SCLC patients, similar to the frequency of increased values found by RIA previously. In contrast, the frequency of increased serum ProGRP(31-98) in patients with nonmalignant pulmonary diseases or non-SCLC was quite low: 0% and 5.0%, respectively. Such results may justify a clinical trial for evaluating this ELISA for the diagnosis and monitoring of SCLC patients.

We used an ultrasensitive prostate-specific antigen (PSA) assay with a detection limit of 0.02 microgram/L for long-term monitoring of PSA changes in 5 patients who were cured by radical prostatectomy and in 10 patients who had failed prostatectomies; 5 patients who underwent cystoprostatectomy were also evaluated with one sample after surgery. Relapse-free periods, determined on the basis of criteria designed specifically for the ultrasensitive assay or proposed for other currently available PSA assays, were calculated for the patients with failed prostatectomies. Tumor-doubling times were also calculated, postsurgery, according to a model that assumes exponential tumor growth over time. We found that prostate cancer relapse, on average, could be diagnosed 420 or 883 days earlier with the ultrasensitive assay than with assays having detection limits of 0.1 or 0.3 microgram/L, respectively. Tumor-doubling times, calculated after radical prostatectomy, ranged from 67 to 568 days among the 10 patients. We also present evidence that even more-sensitive PSA assays might be able to further reduce the relapse-free periods in approximately 50% of the prostate cancer patients who ultimately relapse.

A new method (enzyme-ligand immunoassay, ELIA) is described for the estimation of estrogen (ER) and progesterone (PR) receptors in microsamples of human breast cancer tissue. The technique, based on the nonisotopic measurement of receptor-bound estradiol and progesterone, involves three steps: (a) simultaneous saturation of active receptors with their respective authentic ligands, (b) heat treatment of the cytosol to release the steroids from their cognate receptors before or after absorption with dextran-coated charcoal, and (c) measurement of both steroids present in the cytosol by a modified competitive-inhibition enzyme immunoassay. The useful range of the method was 10-4000 pmol/L for ER and 6.5-1000 pmol/L for PR. The correlation coefficient (r) between the one-point and Scatchard plot analysis was 0.95 for ER and 0.99 for PR. Comparison of the one-point ELIA and expected values with the radioligand binding assay (RLBA) results for EORTC samples gave r = 0.88 and 0.99 for ER and PR, respectively. Further comparison of the one-point ELIA with RLBA and with a commercial enzyme immunoassay, in blind testing of cancer tissue microsamples from 70 patients, gave good agreement for ER with r = 0.95-0.97 and concordance of 92.9-94.4% (cutoff, 15 pmol/g protein) against the other two methods. The results were more disperse in all three methods for PR estimation, the assay correlating perhaps better with the enzyme immunoassay (r = 0.90) at a concordance of 89.4% (same cutoff value).

High-performance liquid chromatography with fluorescence detection has been utilized for the rapid determination of total homocysteine, cysteine, and cysteinylglycine in human serum and plasma. Our earlier procedure (Anal Biochem 1989;178:208), which used monobromobimane to specifically derivatize thiols, has been extensively modified to allow for rapid processing of samples. As a result,80 samples a day can be assayed for total homocysteine, cysteine, and cysteinylglycine. The method is sensitive (lower limit of detectionor = 4 pmol in the assay) and precise (intra- and interassay CV for homocysteine, 3.31% and 4.85%, respectively). Mean total homocysteine concentrations in plasma and serum were significantly different, both from healthy male donors (9.26 and 12.30 mumol/L, respectively; P0.001) and healthy female donors (7.85 and 10.34 mumol/L, respectively; P0.001). The differences in total homocysteine between sexes were also significant (P = 0.002 for both plasma and serum). Similar differences were found for cysteine and cysteinylglycine. We found a significant inverse correlation between serum cobalamin and total homocysteine in men (P = 0.0102) and women (P = 0.0174). Serum folate also inversely correlated with total homocysteine in both sexes.

We developed a sandwich-type enzyme immunoassay to measure cholesteryl ester transfer protein (CETP) mass in human plasma. A specific monoclonal antibody (TP-4) that recognizes an epitope located in the C-terminal domain was used for antigen capture and an anti-CETP peptide antibody directed against the 290-306 residue was used for detection. Bound antibodies were revealed with an antibody-peroxidase conjugate specific for rabbit IgG. The presence of 10 mL/L Triton X-100 in the incubation buffer increased antigen exposure of CETP in plasma. The curves for CETP in standard plasma and partially purified CETP were parallel. This technique is rapid (results within 6 h), accurate, precise (mean intra- and interassay CVs 3.6% and 8.4%, respectively), and simple to perform. Assay sensitivity is at microgram concentrations, with a working range of 20-200 micrograms/L. In 40 normolipidemic healthy subjects, the mean CETP concentration in plasma was 1.1 +/- 0.4 mg/L. A strong correlation between CETP concentration and CETP activity (r = 0.91, n = 42) was observed. In plasma, the bulk of CETP was found in high-density lipoprotein fractions. Therefore, this assay may be a useful tool for investigations of CETP and its significance in relevant diseases.

A nonextraction, competitive, solid-phase enzyme immunoassay with a monoclonal antibody was developed and validated for measuring progesterone in saliva. The antibody was raised against 11 alpha-hydroxyprogesterone hemisuccinate-bovine serum albumin conjugate and was indirectly immobilized to the walls of microtiter wells. The labeled analyte (progesterone-horseradish peroxidase conjugate) was homologous with the immunogen. The lower detection limit (concentration equivalent to B0-3 SD) was 38 pmol/L of saliva sample. We validated the assay with studies to establish the independence of the concentration determined from the volume of saliva assayed, quantitative recovery of progesterone added to saliva, interference from possible cross-reactants, and agreement with a similar assay that incorporated an extraction step. In addition, we determined luteal-phase concentrations of salivary progesterone in normal women and compared the results with those of an older assay involving a polyclonal antibody.

Analysis of minute sample volumes is a major analytical challenge that requires an understanding of fluid flow in microstructures. Accordingly, flow dynamics of biological fluids and cell suspensions in straight glass-capped silicon microchannels (40 to 150 microns wide, 20 and 40 microns deep) were studied. We demonstrated that these microstructures are appropriate components for microfluidic analytical devices. Different fluids were easily manipulated in the microchannels, and measurements of flow rate as a function of pressure for whole human blood, serum, plasma, and cell suspensions revealed non-Newtonian behavior. By means of micromachined filters (5 microns) located in channels, blood cells and microparticles were effectively separated from nanoliter-sized samples, clearly indicating the future role of microstructures for a variety of analytical purposes.

We evaluated the EMIT Cyclosporine Assay, designed for specific quantification of cyclosporine (CsA) in whole blood. The assay was run on the Roche Cobas Mira or Cobas Mira S analyzer. Analytical recovery from both normal donor whole blood and transplant patients' samples was within 17% of the standards. Measurement of diluted out-of-range samples also yielded excellent recovery (101-105%). Within-run, between-run, and total CVs wereor = 8.1%, 10.9%, and 12.1%, respectively. The detection limit was32 micrograms/L. The assay was linear from 0 to 500 micrograms/L. No significant cross-reactivity was observed for CsA metabolites AM1, AM19, and AM4N. Slight cross-reactivity occurred with metabolite AM9; 670 micrograms/L AM9 increased the measured CsA concentration by 49 micrograms/L. High concentrations of bilirubin, uric acid, triglycerides, and cholesterol, as well as 54 commonly coadministered drugs did not interfere with CsA quantification. Similarly, neither extreme values of hematocrit nor choice of anticoagulant affected CsA recovery. Sample extract stability was4 h, and assay calibration was stable for at least 2 weeks. Patients' samples analyzed by the EMIT assay showed strong correlation with both HPLC and 125I-RIA (r0.97). We conclude that the EMIT Cyclosporine Assay provides a convenient, accurate, and precise method for specific quantification of CsA in whole blood.

We modified the Hybritech Tandem-E prostate-specific antigen (PSA) assay by increasing the sample volume, increasing enzyme-substrate incubation time, and using diethanolamine buffer. Our modified method has a detection limit of 0.009 microgram/L (P0.01). The assay curve is linear from 0.01 to 1.0 micrograms/L and has an overall assay time of about 4 h. Linear plots are obtained when the 1.0 micrograms/L standard is diluted with either matrix buffer or serum from men containing PSA0.01 microgram/L. Recovery of PSA (0.10 microgram/L) added to serum from men averaged 94%. Interassay CVs were 13%, 7%, and 4% at PSA concentrations of 0.04, 0.07, and 0.30 micrograms/L, respectively (n = 33). This assay should be useful in the detection of early recurrence of prostate cancer after radical prostatectomy.

Molecular nanotechnology involves the ability to manufacture objects to precise atomic specifications. A central postulate is that any structure that can be specified and that does not violate physical law can be built. Three pathways to molecular nanotechnology are proximate probe technology (the use of improvements of the scanning tunneling microscope, STM), biotechnology, and supramolecular chemistry. Combinations of these technologies appear particularly powerful. The biotechnological approach should make it possible to use in vitro translation systems to manufacture polymers containing at least 10 times as many different artificial monomers as there are natural amino acids. These polymers could further adsorb various other molecular devices, and the use of STMs should enable the complexes to be arranged into sophisticated machines, including molecular computers. The implications include pocket superautomated analyzers and the ability to base medical therapy on the biochemical individuality of specific patients.

Flow injection-inductively coupled plasma mass spectrometry has been evaluated for determining the distribution profile of trace elements along a single strand of hair. Hair was cut into several mm long sections from follicle to the distal end. Each section was solubilized in a capped 1.5-mL polypropylene tube with small volume of nitric acid (typically 50 microL) at room temperature. After dilution an aliquot (50 microL) was introduced into the mass spectrometer by flow injection. The limit of determination was typically 5-50 pg with 5-10% precision (CV), depending on the element examined; this corresponds to sub-microgram/g concentrations of these elements in hair segments. Recent exposure and intake history of individuals to thallium or mercury could be reconstructed by this system.

Enzyme amplification has proved to be a highly sensitive quantification technique for immunoassays. We have shown that by using a fluorescent end-point, even more sensitive enzyme amplification assays can be generated than hitherto reported. We describe some general properties of this system and demonstrate its application in an assay for human proinsulin in plasma. The detection system can be used to measure less than one thousandth of an attomole (1 zeptomole) of alkaline phosphatase, equivalent to about 350 molecules of alkaline phosphatase per well of a microtiter plate. We have used this system to construct a proinsulin assay with a sensitivity of 0.017 pmol/L.

FK506, a promising new immunosuppressant, is currently under clinical investigation. Because dose-dependent toxicity is possible, blood concentrations of FK506 should be monitored. We improved the original ELISA of FK506 by shortening the incubation time. With some modification of materials, results are obtained within 6 h instead of 2 days, with similar or even better precision. Internal and external quality-control programs showed that our results correlated satisfactorily both with values determined by the original method and the theoretical expected values. Either plasma (detection limit 0.1 microgram/L) or whole-blood (detection limit 1 microgram/L) samples can be used. The sensitivity of the method makes it particularly useful for accurate pharmacokinetic studies or measurement of low blood concentrations. Twenty-four drugs and nine biological variables showed no significant interference on the assay. Study of the concentration- and temperature-dependent distribution of FK506 shows that the drug is largely bound to erythrocytes (ratio of blood to plasma concentrations is 10-40); as the erythrocytes become saturated, more of the drug is found in the plasma. Plasma concentrations may vary according to the blood temperature. We conclude that whole blood should be used for FK506 monitoring, as it is for monitoring cyclosporine.

We developed a method for determining individual free fatty acids in serum by using a modified one-step Dole extraction, derivatization, and a new high-performance liquid chromatographic (HPLC) separation. Sample handling is minimized to a single transfer of the fatty acids (upper layer of the Dole extract), which are readily derivatized at 85 degrees C with p-bromophenacyl bromide without significant hydrolysis of esterified fatty acids. The derivatization mixture is directly injected into the HPLC apparatus. The new method, which uses C6 (3-microns particle) column material and an isocratic acetonitrile-water eluent, separates nearly to baseline 12 of the physiologically most abundant long-chain fatty acids (C12-C22) in20 min with a detection limit of approximately 2 pmol. It is therefore suitable for routine analysis even with basic HPLC equipment and can easily analyze a series of 10-20 samples in about 2 h including extraction until first results are available. The method is also applicable to other matrices than serum, e.g., for determination of precursor fatty acids such as arachidonic acid in platelets or of fatty acid patterns liberated by lipases or phospholipases A1/A2 in test systems.

We report microchromatographic measurement of fetal hemoglobin (HbF) proportions in a 36-year-old African-American multigravida woman. At 34 weeks she delivered a 630-g male infant who subsequently did well. Hemoglobin electrophoresis of the hemolysate revealed nearly 100% HbF without HbA, an extremely unusual naturally occurring sample. Family studies revealed a combination of hereditary persistence of fetal hemoglobin (HPFH) and beta zero-thalassemia minor. Southern blot technique confirmed heterozygous alpha 2 thalassemia and HPFH but failed to identify the beta thalassemic lesion. The absence of HbA and the very high amounts of HbF led us to measure HbF by several methods to confirm the accuracy of microchromatography of HbF at values approaching 100%. HPLC revealed a 14% F1 suggestive of microchromatographic underestimation due to glycated HbF. We conclude that cation-exchange microchromatography and the Betke method of alkali denaturation underestimate HbF values as they approach 100% and do not recommend these procedures in this rare situation.

Pergolide, a synthetic ergoline with potent dopaminergic activity, is used to treat Parkinson disease. The low plasma concentrations of pergolide achieved during therapy complicate the development of a method for its analysis. Because radioimmunoassay successfully measures other structurally related ergolines in physiological fluids, we undertook the development of a radioimmunoassay of pergolide. The detection limit of the radioimmunoassay is 21 ng/L with an optimal working range from 100 to 1000 ng/L. We maximized assay specificity by using a monoclonal antibody that displayed low cross-reactivity with pergolide sulfoxide, a major metabolite found in animals. The radioimmunoassay has performed acceptably for2 years during toxicology studies with rats and rhesus monkeys and in clinical studies involving patients with Parkinson disease. We consider the radioimmunoassay a valid method for quantifying therapeutic concentrations of pergolide in plasma.

We examined an instrument-free test for C-reactive protein (CRP) in whole blood. The NycoCard CRP Whole Blood test uses a cell-solubilizing dilution liquid, a membrane-bound antibody that binds CRP, and a gold-conjugated antibody for making visible the bound CRP. We obtained essentially identical dose-response curves in citrate-, heparin-, and EDTA-treated blood. CVs were 6.7-12.5% within series and 10.1-14.7% between series. The detection limit was 12 mg/L. Intralipid added to blood increased measured CRP by 10-20%, whereas no change was seen with added bilirubin, added serum amyloid P component, or the presence of rheumatoid factor. In 234 patients' blood samples the results of the NycoCard Whole Blood test correlated well (r = 0.96) with those of a turbidimetric serum method. The test allows reliable measurement of CRP from a small volume of whole blood (25 microL) without using expensive equipment; it should be useful for decentralized testing in hospital departments, emergency units, and primary health care centers.

We describe an amplified enzyme-linked immunoassay of human proinsulin in serum that detects intact proinsulin and both the 32/33 and 65/66 split forms. The method uses the IgG fraction of a polyclonal antibody raised in a guinea pig against intact proinsulin, which we used to coat plastic microtiter plates. A sandwich was formed with proinsulin by using a monoclonal antibody against C-peptide labeled with alkaline phosphatase. We quantified the reaction by using the enzyme amplification procedure, which detected as little intact proinsulin as 0.1 pmol/L. We found no cross-reactivity with C-peptide in the assay, and decreased recovery attributable to the presence of insulin could be demonstrated only with a 30-fold excess of this hormone over proinsulin.

We describe a method for the collection and microanalysis of the volatile organic compounds in human breath. A transportable apparatus supplies subjects with purified air and samples their alveolar breath; the volatile organic compounds are captured in an adsorptive trap containing activated carbon and molecular sieve. The sample is thermally desorbed from the trap in an automated microprocessor-controlled device, concentrated by two-stage cryofocusing, and assayed by gas chromatography with ion-trap detection. Compounds are identified by reference to a computer-based library of mass spectra with subtraction of the background components present in the inspired air. We used this device to study 10 normal subjects and determined the relative abundance of the volatile organic compounds in their alveolar breath. The breath-collecting apparatus was convenient to operate and was well tolerated by human volunteers.

Of eight methods examined for measuring plasma hemoglobin in micromolar concentration, all exhibited acceptable linearity, reproducibility, and concurrence except when specimens were icteric or lipemic or contained methemoglobin or methemalbumin. Measurement of absorbance at 578 nm with an Allen correction permits precise assay of plasma oxyhemoglobin concentration as low as 0.01 g/L (1 mg/dL, 0.16 mumol/L), unaffected by hyperlipidemia or hyperbilirubinemia. Discrepancies between methods occurred in 11.6% of a consecutive series of 50 nonicteric patients' plasma specimens. Examination of absorption spectra is helpful when discrepancies are observed between methods. The presence of methemalbumin or methemoglobin in plasma is not recognized by methods that measure only oxyhemoglobin. Increased ceruloplasmin or beta-carotene does not significantly affect results.

Featured Article: Pesce MA, Strande CS. A new micromethod for determination of protein in cerebrospinal fluid and urine. Clin Chem 1973;11:1265–7.1 This paper described a dye-binding micromethod for measuring protein concentrations in cerebrospinal fluid and urine. The method involved coprecipitation of the proteins with a Ponceau S dye trichloroacetic acid solution, dissolving the protein precipitate in dilute sodium hydroxide, and measuring the absorbance at 560 nm. This method was rapid, easy to use, and required a specimen volume of 5–50 μL. I began my career as a clinical chemist in 1971 in the pediatric microchemistry laboratory at Columbia Presbyterian Medical Center. This laboratory served the newborn, infant, and child population of Babies Hospital, …

This is a sensitive method, suitable for measuring subanogram amounts of cholesterol. Cholesterol and the internal standard, epicoprostanol (5-beta-cholestan-3-alpha-ol), are derivatized with pentafluorobenzoyl chloride and detected by electron-capture gas chromatography. The pentafluorobenzoyl esters of cholesterol and the internal standard are easily formed and possess excellent chromatographic and electron-capturing properties. The lower limit of detection of the method, approximately 100 pg injected, is about 500-fold as sensitive as chromatographic methods involving flame-ionization detection. Within-day and between-day coefficients of variation were 4.2% and 8.2%, respectively, for determinations of a urinary cholesterol concentration of 570 micrograms/L (1.47 mumol/L). Such sensitivity permits analysis for cholesterol in (e.g.) physiological fluids, tissue samples, and cell cultures that contain very low concentrations of cholesterol.

Cholesterol (C) concentrations in the two major subfractions of high-density lipoproteins (HDL2-C and HDL3-C) in sera from both sexes, ages ranging from newborns to adults, were measured by use of a micromethod for combined precipitation-ultracentrifugation. Sera were obtained from 91 boys, 68 girls, 15 healthy men, and 14 women. The HDL2-C concentration was higher in women than in men; the HDL3-C concentration was similar in these two groups. This sex-related difference, generally seen in adults, was found to begin at ages 11-15 y. The value of HDL2-C in females increased with age in a stepwise manner, whereas that in males increased up to ages 6-10 y but tended to decline thereafter. The HDL3-C concentration was higher in the adults than in the children. This micromethod for separating operationally defined HDL subfractions is of value for lipoprotein research in children.

To select the best tracer for use in a competitive immunoassay, we conjugated human somatomedin C (SmC) to various chemiluminescent compounds via two different synthetic pathways. Naphthylhydrazides and arylhydrazides, used as the labels, were incorporated via their imidate or their succinimide esters. Conjugating the carboxy terminal of (amino ethyl)ethyl-isoluminol to SmC via a succinimide linkage supplied the most sensitive detection limit and the most immunoreactive conjugate. We developed an immunoassay based on the use of this conjugate, and evaluated dextran-coated charcoal, second-antibody precipitation, and solid-phase immunoprecipitation for separating bound and free label. This chemiluminescent method has a detection limit of 16 pg per tube, and it is accurate and precise. Correlation studies with a conventional radioimmunoassay (x) for SmC gave the following regression equation: y = 0.66x + 3.76 (r = 0.953, n = 30); the slight discrepancies between the two methods are probably ascribable to the use of different antibodies. We thus propose this chemiluminescence immunoassay as an inexpensive and sensitive alternative to radioimmunoassay for measuring SmC in serum or in extracts of serum.