Sensitive Time-resolved Fluoroimmunoassay Research Articles

Highly sensitive quantitation of methamphetamine by time-resolved fluoroimmunoassay using a new europium chelate as a label.

A simple and highly sensitive time-resolved fluoroimmunoassay of methamphetamine (MA) using a new fluorescent europium chelate (BHHCT-Eu3+) as a label is described. Two variations of competitive immunoassay were attempted. In the first (one-step) assay, microtiter plates coated with anti-MA were used, and the new label was bound to a conjugate of bovine serum albumin and N-(4-aminobutyl)-MA (MA-BSA). In the second (two-step) assay, instead of the labeled MA-BSA, biotinylated MA-BSA and BHHCT-Eu3+-labeled streptavidin-BSA were used. The lowest measurable concentrations of MA for the one-step and the two-step methods were 1 ng/mL (25 pg/assay) and 1 pg/mL (25 fg/assay), respectively. These were 10 to 1000 times superior to the detection limits of MA in any other immunoassay. Intra-assay coefficient of variation was approximately 2-8% at eight different concentrations (n = 4). Analysis of 34 urine samples with the new method and conventional gas chromatography showed a good correlation (r = 0.954). The high detectability of the present assay also enabled segmental hair analysis with a few centimeters of a hair.

Sensitive Time-Resolved Fluoroimmunoassay of Human Thyroid-Stimulating Hormone by Using a New Europium Fluorescent Chelate as a Label

Sensitive Time-Resolved Fluoroimmunoassay of Human Thyroid-Stimulating Hormone by Using a New Europium Fluorescent Chelate as a Label

Highly Sensitive Time-Resolved Fluoroimmunoassay of Human Immunoglobulin E by Using a New Europium Fluorescent Chelate as a Label

A new europium fluorescent chelate, 4,4′-bis(1″,1″, 1″,2″,2″,3″,3″-heptafluoro-4″,6″-hexanedione-6″-yl)-chlorosulfo-o-terphenyl (BHHCT)–Eu3+, was used as a label for highly sensitive time-resolved fluoroimmunoassay of human IgE. Two assay formats were employed in the analysis. In the first format, an immunoconjugate of rabbit anti-human IgE antibody–human IgE-biotinylated goat anti-human IgE antibody–BHHCT- Eu3+-labeled SA (or BHHCT–Eu3+-labeled BSA–SA; BSA, bovine serum albumin; SA, streptavidin) was used for measurement. The method gives the detection limits of 3.6 × 10−2IU/ml (labeled SA) and 1.1 × 10−2IU/ml (labeled SA–BSA). In the second format, an immunoconjugate of goat anti-human IgE antibody–human IgE-rabbit anti-human IgE antibody–biotinylated goat anti-rabbit IgG antibody–BHHCT–Eu3+-labeled SA (or BHHCT–Eu3+-labeled BSA–SA) was used for measurement. The detection limits of these methods are 3.0 × 10−3IU/ml (labeled SA) and 1.5 × 10−3IU/ml (labeled BSA–SA). The above detection limits are one to two orders of magnitude lower than those of the conventional radioimmunoassay and enzyme immunoassay. The CV of the present method is less than 7%, and the recovery is in the range of 85–105% for serum samples.

Synthesis of a new tetradentate β-diketonate europium chelate and its application for protein labeling and highly sensitive time-resolved fluoroimmunoassay

Synthesis of a new tetradentate β-diketonate europium chelate and its application for protein labeling and highly sensitive time-resolved fluoroimmunoassay

Time-resolved fluoroimmunoassay for pituitary adenylate cyclase activating polypeptide 27 (PACAP27) using europium (III) ion chelate labeled streptavidin-biotin complex

Pituitary adenylate cyclase activating polypeptide (PACAP) is a novel peptide hormone and has a variety of biological action. In studies of the physiological behaviour of endogenous PACAP, the determination of PACAP levels in biological materials require a highly sensitive and specific method. Therefore, we developed a sensitive time-resolved fluoroimmunoassay (TR-FIA) for PACAP27 which is the biologically important fragment of PACAP. Accordingly, we developed TR-FIA using a biotinylated PACAP27 (b-PACAP27) as a tracer and europium (III) chelate labeled streptavidin-biotinylated bovine serum albumin complex as a detection of biotin on solid phase. A measurable range of PACAP27 was 7.8–1000 pg ml −1 by the proposed TR-FIA. For measurement of biological samples, the samples were purified to eliminate substances which interfered with the TR-FIA. The mean recovery of PACAP27 using commercially reversed phase column was 74.7% ( n = 12). The various tissues, extracts and plasma concentrations of rat could be measured by the proposed TR-FIA.

Development of a Time-Resolved Fluoroimmunoassay (TR-FIA) for Testosterone: Measurement of Serum Testosterone Concentrations after Testosterone Treatment in the Rainbow Trout (Oncorhynchus mykiss)

A sensitive time-resolved fluoroimmunoassay (TR-FIA) for testosterone was developed, and the assay system was used for measuring serum testosterone concentrations in rainbow trout. Testosterone-3-(O-carboxymethyl)oxime-bovine serum albumin (T-3-CMO–BSA) was immobilized by physical adsorption to the wells of microtiter plates. A competitive assay using two antibodies was performed among T-3-CMO–BSA in the solid-phase, unknown amounts of testosterone, testosterone antibodies, and europium labeled secondary antibodies, followed by measurements using a time-resolved fluorometer (DELFIA system). The TR-FIA had a sensitivity of 0.075 pg/50 μl sample (1.5 pg/ml), and the range of the assay system was between 1.5 pg/ml and 25 ng/ml. The intra- and interassay coefficients of variation for the testosterone TR-FIA were satisfactorily low, and were between 1.62 and 6.38% and 2.96 and 8.29%, respectively. The assay system was applied to measure the serum testosterone concentrations after an injection of testosterone dissolved in saline, propyleneglycol, or coconut oil. Among the three solvents, the coconut oil group showed continuously high serum testosterone levels. In contrast, the saline and propyleneglycol groups had maximum concentrations 24 hr after the injection, but their levels were significantly lower than that of the coconut oil group. The testosterone TR-FIA method is sensitive, repeatable, and is as accurate as conventional RIAs. It is very good for measuring serum testosterone concentrations.

Sensitive time-resolved fluoroimmunoassay of salmon calcitonin

A two-site assay was developed by use of the "dissociation and enhancement lanthanide fluoroimmunoassay" (DELFIA) technique for determination of salmon calcitonin (SCT) in serum after administration to osteoporotic patients. Polyclonal antibodies were produced in rabbits immunized with SCT coupled to ovalbumin. After affinity purification, the antibodies were used both as immobilized capture antibodies and as Eu-chelate-labeled signal antibodies. A sensitive assay with a detection limit of 1.1 pmol/L was achieved, and no cross-reaction with human calcitonin was observed. The intra- and interassay CVs were < 12% (n = 10) and < 15% (n = 4), respectively. Analytical recovery of SCT added to serum was 91% +/- 3% (mean +/- SD, n = 4). SCT was measurable in all the samples from eight osteoporotic patients after subcutaneous SCT administration. We conclude that this new sensitive and specific two-site DELFIA can reliably measure SCT in serum.

Time-resolved Fluoroimmunoassay for Secretin and Cholecystokinin using Europium chelate-labeled Streptavidin

We have developed a sensitive time-resolved fluoroimmunoassay for secretin and cholecystokinin (CCK). Europium chelate-labeled streptavidin (Eu-SA) was prepared by labeling Euchelate [N'-(p-isothiocyanatobenzyl)diethylenetriamine-N, N N ', N-tetraacetato]europium (III)]. The assay procedure was as follows: A standard secretin or CCK-8 solution (100 ul), rabbit anti-human secretin or anti-human CCK anti-serum solution (50 pl) and biotinylated secretin or CCK-33 solution (50 ul) were added to the well of microtiter plate coated with coated with goat anti rabbit IgG antibody and incubated for 16 or 72 h at 4 °C. Then, the wells were washed with 0.05 mol/L Tris-HC1 buffered saline containing 0.05% Tween 20(pH 7.7). The solution of Eu-SA (100 pl) was added to the biotinylated secretin or CCK-33bound to the well. After incubation for 90 minutes, the well was washed and then enhancement solution (Pharmacia Co. Ltd. ) (200 AO was added to each well and incubated at room temperature for 10 minutes. The time-resolved fluorescence intensity was measured by Arcus 1230 fluorometer (Pharmacia Co. Ltd. ). A measurable range and fifty percent value of inhibition concentration (IC 50) for secretin and CCK-8 were from 25 to 1000 pg/m/ and 520 pg/m/ and from 62.5 to 32000 pg/m/ and 1450pg/m/, respectively, and the coefficients of variation for within-run was 1.7 -3.30% (n= 8)and 2.34-7.56% (n = 6). Further, the sensitivity of IC 50 value and detection limit for CCK-8 with ELISA amplification system (Daiichi kagakuyakuhin Co. ) was 18.5-fold and 31.3-fold better than that was obtained by without ELISA amplification system.539

Quantification of carbonic anhydrase III and myoglobin in different fiber types of human psoas muscle.

Carbonic anhydrase (CA III) and myoglobin contents from isolated human muscle fibers were quantified using a sensitive time-resolved fluoroimmunoassay. Human psoas muscle specimens were freeze-dried, and single fibers were dissected out and classified into type I, IIA and IIB by myosin ATPase staining. Fiber typing was further confirmed by SDS-PAGE. CA III and myoglobin were found in all fiber types. Type I fibers contained higher concentrations of CA III and myoglobin than type IIA and IIB fibers. The relative concentrations of CA III in type IIA and IIB fibers were respectively 24% and 10% of that in type I fibers. The relative concentrations of myoglobin in type IIA and IIB fibers were 60% and 28% of that in type I fibers. Anti-CA III immunoblotting results from fiber-specific pooled samples agreed well with quantitative measurements. The results indicate that CA III is a more specific marker than myoglobin for type I fibers.

Sensitive time-resolved fluoroimmunoassay of nerve growth factor and the disappearance of nerve growth factor from rat pheochromocytoma PC12 cell culture medium

A sensitive time-resolved fluoroimmunoassay of nerve growth factor (NGF) has been developed. The method is based on the unique property of the lanthanides for delayed fluorescence, which reduces substantially the endogenous fluorescence of biological substances, because the excitation of the sample and detection of the fluorescence signal are separated in time and in wavelength. Using the europium-conjugated antibodies to the NGF from Vipera lebetina (snake) venom and to the βNGF from mouse submandibular gland in a solid-phase quantitative two-site fluoroimmunoassay, we obtained a maximal sensitivity of 10 pg/ml (0.38 pM)for mouse NGF and 40 pg/ml (1.2 pM) for snake NGF. Using this method, we investigated the disappearance of NGF from rat pheochromocytoma PC12 cell culture medium. Mouse βNGF (5–10 ng/ml) disappeared completely after 12 h of incubation, whereas snake NGF was not substantially internalized even after 48 h.

Comparison of monoclonal time-resolved fluoroimmunoassay with monoclonal capture-biotinylated detector enzyme immunoassay for adenovirus antigen detection

A sensitive time-resolved fluoroimmunoassay (TR-FIA), adapted from TR-FIA procedures already described, was developed with monoclonal antibodies and compared with several enzyme immunoassays (EIAs) for detecting adenovirus antigens in clinical specimens. The most sensitive EIA was an all-monoclonal assay with biotin-labeled detector antibody and streptavidin-peroxidase conjugate. All tests were evaluated with nasopharyngeal aspirate specimens from respiratory illness, with tissue homogenates from patients with systemic infection, and with stool specimens from gastrointestinal illnesses. For respiratory and tissue specimens, the TR-FIA detected adenovirus in 85% of the specimens positive by culture, which was a sensitivity similar to those of the all-monoclonal biotin-avidin EIA (79%) and the polyclonal-capture biotin-avidin EIA (88%). For stool specimens, the TR-FIA detected adenovirus in 100% of the specimens positive by culture, which was a decidedly higher sensitivity than either EIA format (78 and 75%, respectively). The TR-FIA was shown to be an efficient, flexible, and specific test for large numbers of clinical specimens.

Determination of thyrotropin in serum by time-resolved fluoroimmunoassay evaluated.

We evaluated a new, highly sensitive time-resolved fluoroimmunoassay for thyrotropin (TSH) in serum. This direct immunometric "sandwich"-type assay involves two monoclonal antibodies against TSH, one immobilized, the other labeled with europium. Extremely high specific activity of the label and the use of labeled antibody in large excess make the method sensitive enough to measure TSH values falling below the normal reference interval. The standard curve is nearly linear over a wide range of TSH concentrations (standard concentrations range from 0.25 to 324 milli-int. units/L). The lowest concentration detectable was 25 micro-int. units/L. The CV for the assay was less than 6% at 0.5 milli-int. unit/L or higher, 11.3% at 0.1 milli-int. unit/L. For a CV of 10% the lower limit of the working range would be around 0.1 milli-int. unit/L. The interassay CV was 6.7 to 11.8% for TSH concentrations of 0.31 to 19.6 milli-int. units/L. The 95% confidence interval for sera from 111 healthy persons was 0.6-3.8 (range 0.3-3.8) milli-int. units/L. For hyperthyroid patients and thyroid cancer patients treated with thyroxin after thyroidectomy, serum TSH values were all below the reference interval (most were less than 25 micro-int. units/L).

Immunoreactive phospholipase A2 in serum in acute pancreatitis and pancreatic cancer.

Immunoreactive phospholipase A2 (EC 3.1.1.4) was measured by a new sensitive time-resolved fluoroimmunoassay in the serum of 58 healthy subjects and 103 patients with acute pancreatitis. Patients with acute pancreatitis were grouped according to the etiology and clinical severity of the disease. The mean phospholipase A2 concentration in the reference (healthy) group was 5.5 (SD 1.9) micrograms/L. In acute pancreatitis the mean phospholipase A2 concentration was increased on the first day after hospital admission in all groups, and returned to normal somewhat more slowly than did serum amylase, especially in the patients with severe alcoholic pancreatitis. In this latter group the mean concentration of serum phospholipase A2 on the first day was 42.6 (SD 29.5) micrograms/L. In patients with pancreatic cancer, serum phospholipase A2 was 29.2 (SD 21.3) micrograms/L. The phospholipase A2 and amylase values were closely associated in all groups. The clinical sensitivities were 90.9% for severe alcoholic pancreatitis and 87.5% for pancreatic cancer. Immunochemical determination of phospholipase A2 in serum provides fast and specific detection of injury to pancreatic acinar cells. In addition to the early diagnosis of acute pancreatitis, follow-up determinations of phospholipase A2 seem to be useful in differentiating between mild and severe forms of pancreatitis.