Sensitive, rapid procedure for time-resolved immunofluorometry of lutropin

In this new immunofluorometric method for quantification of lutropin in serum, the "sandwich" principle is combined with time-resolved fluorescence measurements, with the europium chelate 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA) used as label. A monoclonal antibody to the alpha-subunit of lutropin is adsorbed onto the walls of white-opaque microtiter wells to form the solid-phase capture antibody, and a biotin-labeled soluble monoclonal antibody is used for antigen quantification. The detection system is completed with streptavidin, which has been linked to a protein bulking agent labeled with multiple BCPDA residues. In the presence of excess europium, the fluorescence of the final complex attached to captured lutropin molecules is measured on the dried solid phasse with an automated time-resolved fluorometer. The assay can be performed as a rapid (less than 60 min incubation) or regular (150 min incubation) procedure. The rapid assay is well-suited for routine daily monitoring of increasing or ovulatory lutropin concentrations; the regular assay, with its greater sensitivity (0.5 int. unit/L), is a practical procedure for lutropin measurements in hyposecretory states. The assay measures up to 240 int. units/L, and results compare well with those by a commercially available radioimmunoassay, an immunoradiometric assay, and another time-resolved immunofluorometric procedure.     

Time-resolved immunofluorometric assay of 34-kDa somatomedin-binding protein

In this time-resolved immunofluorometric assay for the 34-kDa somatomedin-binding protein (SmBP), affinity-purified polyclonal antibodies are used, along with solid-phase separation of bound and free analyte. The first antibody is bound to polystyrene microtiter wells; the second is labeled with europium(III) chelate. The detection limit of the method is 0.25 microgram/L, much lower than that (about 8 micrograms/L) for radioimmunoassay. By immunofluorometric assay, SmBP is detectable, and could be accurately quantified, in the serum of all 88 individuals we tested, whereas by radioimmunoassay a third of the samples had concentrations below the detection limit. When SmBP was detectable by both methods, the concentrations measured by the two techniques correlated well (r = 0.98).     

Ultrasensitive thyrotropin immunoassay based on enzymatically amplified time-resolved fluorescence with a terbium chelate.

We describe an ultrasensitive, enzymatically amplified time-resolved fluorescence immunoassay of thyrotropin (thyroid-stimulating hormone) in serum with use of a terbium chelate as the detectable moiety. In this assay, thyrotropin is first simultaneously reacted with a solid-phase (microtiter well) monoclonal antibody and a soluble biotinylated monoclonal detection antibody. After washing, a streptavidin-alkaline phosphatase conjugate is added, followed by another washing. Alkaline phosphatase acts on the substrate 5-fluorosalicyl phosphate (FSAP) to produce 5-fluorosalicylic acid (FSA). FSA, but not FSAP, can then form with Tb3+ and EDTA a highly fluorescent ternary complex of long fluorescence lifetime. This complex is quantified with time-resolved fluorometry. The thyrotropin assay is highly sensitive (detection limit approximately 0.003 milli-int. unit/L when a total assay time of 85 min is used), precise, and accurate. The thyrotropin assay can also be completed in less than 30 min (detection limit 0.013 milli-int. unit/L), thus making this procedure a candidate technology for high-throughput automated analyzers.     

Time-resolved immunofluorometric assay of alpha-fetoprotein in serum and amniotic fluid, with a novel detection system

We describe a new "sandwich"-type nonisotopic immunoassay of alpha-fetoprotein (AFP) in serum and amniotic fluid. In the assay, AFP binds to a monoclonal antibody immobilized in a microtiter well and to a polyclonal soluble biotinylated antibody. A fluorescent product is created on the solid-phase after adding streptavidin labeled with a new Eu3+ chelate, 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA), and excess Eu3+. The fluorescent complex, monoclonal antibody-AFP-polyclonal antibody-biotin-streptavidin-BCPDA-Eu3+, is quantified by nitrogen laser excitation at 337.1 nm, the emission at 615 nm being monitored in an especially designed gated fluorometer working in a time-resolved mode. This assay of AFP has a broad dynamic range (up to 1 mg/L), and is precise and accurate. The detection limit is approximately 0.1 microgram/L. Results agree well with those obtained by established techniques.     

Direct solid-phase time-resolved fluoroimmunoassay of 17 alpha-hydroxyprogesterone in serum and dried blood spots on filter paper

We describe a direct, solid-phase time-resolved fluoroimmunoassay (TRFIA) for measuring 17 alpha-hydroxyprogesterone (17OHP) in serum and blood spots on filter paper. We used 17OHP-3-carboxymethyloxime (17OHP3CMO) coupled to polylysine as the label, which enabled incorporation of up to 34 atoms of europium per molecule of 17OHP, for a very high specific activity. The assay is based on competition between labeled 17OHP3CMO and 17OHP in blood specimens for polyclonal rabbit anti-17OHP antibodies. The antibody-label complex is separated by binding to anti-rabbit antibodies coated onto microtiter strips. The assay buffer contains danazol to displace 17OHP from steroid-binding proteins in serum. For serum samples, the assay is accomplished in 1 h of incubation at room temperature. The blood spot assay with filter paper discs involves incubation overnight at 4 degrees C. Results for both types of specimens from the same subjects correlated well. The lowest measurable concentrations of 17OHP (nmol/L) were 0.10 (3 SD) and 0.75 (3 SD) for serum and dried blood on filter paper, respectively. Intra- and interassay CVs were about 5-15% for both types of samples.     

Solid-phase enzymoimmunoassay for osteocalcin in human serum or plasma, with use of a monoclonal antibody

In this solid-phase enzymoimmunoassay on microtiter plates for osteocalcin in serum or plasma, we use an osteocalcin-horseradish-peroxidase conjugate and a monoclonal antibody raised against bovine osteocalcin. We thoroughly standardized the assay for measurement of osteocalcin in both serum and plasma, demonstrating independence of sample volume, and determining the analytical recovery and within-and between-assay CVs. The detection limit was between 0.6 and 1.1 micrograms/L and the ED50 was 16 micrograms/L for a 5-microL sample volume. The intra-assay CV over the range 3 to 74 micrograms/L was less than or equal to 15%. The interassay CV over the range 3.6 to 46 micrograms/L was less than or equal to 16%. Results by this assay and by an in-house radioimmunoassay in which the same monoclonal antibody was used correlated well (r2 = 0.948). Osteocalcin concentrations in serum and plasma as measured with the present assay agreed well with published values.     

Evaluation of a direct solid-phase radioimmunoassay for progesterone, useful for monitoring luteal function

We have evaluated a commercially available, direct, solid-phase radioimmunoassay kit for progesterone determination in serum or plasma. The assay is precise, within-run precision (CV) in the clinically significant ranges being 2.5 to 5.2%, between-run 5.5 to 5.8%. Mean analytical recovery of different concentrations of progesterone added to serum was 99.7% (range 95.3 to 102.7%). Fourteen closely related steroids showed no cross reactivity. The minimum detection limit was 0.5 microgram/L. Luteal-phase progesterone concentrations in serum were increased (greater than 3 micrograms/L) in 19 normal ovulatory menstrual cycles and decreased (less than 1.5 micrograms/L) in two nonovulatory cycles. We found this direct assay for progesterone to be analytically and clinically sound, and useful for assessing luteal-phase function.     

A more specific, simpler radioimmunoassay for carcinoembryonic antigen, with use of monoclonal antibodies

A solid-phase, two-site monoclonal antibody radioimmunoassay for carcinoembryonic antigen in serum or plasma has been developed. Two monoclonal antibodies are used: 3d6, which is immobilized on polystyrene beads, reacts with high-molecular-mass CEA; the other, C4, with apparently restricted reactivity toward gastrointestinal tract and mammary carcinomas, is labeled with 125I. The assay consists of incubating 0.2 mL of serum both with 3d6-coated beads and 125I-labeled C4 at room temperature for 18 h. The CEA standard is calibrated against WHO international standard (73/601). Advantages of this assay include: (a) no heat or acid pre-treatment of samples; (b) linear response over a wider functional range, 0 to 150 micrograms/L, requiring fewer calibration points; and (c) no interference by glycosaminoglycans. Average inter- and intra-assay reproducibilities (CVs) are less than 10%; analytical recovery of CEA was 94 to 107%. CEA of less than 0.5 micrograms/L can be detected. The mean concentration of CEA in serum from healthy individuals is 0.97 (SD 1.18) micrograms/L; only 3% of the sera tested had concentrations greater than 3.0 micrograms/L. On comparing this assay with a polyclonal RIA, we found similar assay sensitivity for colorectal carcinoma but fewer false-positive results for sera from patients with benign liver and bowel diseases.     

Gas-liquid chromatography of phencyclidine in serum, with nitrogen-phosphorus detection

We developed a two-step assay of phencyclidine (PCP), in which 2.5 mL of serum is adsorbed onto a disposable solid-phase extraction column and the eluted drug is determined by gas chromatography with nitrogen-phosphorus detection. Methapyriline is the internal standard. The detection limit of this technique is 0.5 microgram/L and the linear range exceeds 200 micrograms/L. Precision (CV) ranges from 30 to 10% with increasing concentration. No interferences were encountered in more than 400 clinical samples. The assay permits serial observation of low concentrations of the drug in serum for pharmacokinetic study and for quantitative clinical correlation with the Brief Psychiatric Rating Scale.     

Sensitive time-resolved immunofluorometric assay of thyrotropin in serum

We have developed a time-resolved solid-phase immunofluorometric assay for thyrotropin (TSH). The assay is performed in white opaque microtitration wells which are coated with a monoclonal capture antibody. Serum TSH binds simultaneously to the solid phase and to a biotinylated monoclonal detection antibody. The degree of biotinylated antibody binding is quantitated with streptavidin conjugated to thyroglobulin which is heavily labelled with the Eu3+ chelator 4,7-bis [chlorosulfophenyl] -1,10-phenanthroline -2,9-dicarboxylic acid (BCPDA). The final fluorescent complex is measured on the solid phase with time-resolved fluorometry. The assay requires two incubation steps and can be completed in 5 hours. The detection limit is 0.03 milli-int. units/L. The present assay was compared with two immunoradiometric assays and gave satisfactory results.     

Immunofluorometric demonstration and quantification of placental protein 5 in the absence of pregnancy

This time-resolved immunofluorometric assay (IFMA) developed for measurement of placental protein 5 (PP5) involves two antibodies: a monoclonal anti-PP5 antibody attached to a solid phase and an europium(III) chelate-labeled polyclonal anti-PP5 antibody as a tracer. The measuring range is 0.05-100 micrograms/L and the detection limit is 20 times lower than that of a PP5 radioimmunoassay (RIA) performed with the same polyclonal antiserum. By IFMA, PP5 could be detected and quantified in all plasma and serum samples of nonpregnant and pregnant individuals, whereas PP5 was undetectable by RIA in serum of healthy men and nonpregnant women. The mean concentration of PP5 in sera from men was 0.43 micrograms/L (SD 0.13, range 0.19-0.75, n = 47) and in sera from nonpregnant women 0.49 micrograms/L (SD 0.19, range 0.20-0.90, n = 41). PP5 concentrations in serum showed no systematic variation during the menstrual cycle. In serum samples from 60 pregnant women the results obtained by IFMA and RIA correlated well (r = 0.97).     

Competitive solid-phase immunoassay of gastrin in serum using time-resolved fluorometry

Abstract

Background. A competitive solid-phase assay for the measurement of gastrin in serum using time-resolved fluorescence was developed as an alternative to conventional radioimmunoassay (RIA) technology. Methods. The assay depends on the competitive binding of unlabelled versus Eu-labelled gastrin to specific gastrin antibodies – bound to anti-rabbit IgG immobilized on polystyrene microtitration strips. The bound Eu³⁺-label was dissociated from the bound gastrin and converted to a fluorescent β-diketone chelate which was measured by fluorometry with time-resolution. Results. Using a sample volume of 50 μl the lower limit of detection was below10 pmol/L. Dilution of samples showed an excellent linearity. Spiking with gastrin-17 in known concentrations showed a recovery of 103% indicating that there is no bias inherent in the assay. The method correlated fully with the routine in-house radioimmunoassay in the concentration range 10–400 pmol/L (slope = 0.98 with r² = 0.98). Thus the reference interval for clinical samples does not require modification when changing from one method to the other. Conclusion. We have described a convenient and accurate competitive assay for measurement of gastrin in serum based on solid-phase technology using time-resolved fluorometric detection as a realistic alternative to the established state-of-the-art RIA-technology.     

Immunofluorometry of choriogonadotropin by time-resolved fluorescence spectroscopy, with a new europium chelate as label

We describe a new "sandwich"-type non-isotopic immunoassay for human choriogonadotropin (hCG) in serum. In the assay, hCG is captured by a beta-subunit-specific monoclonal antibody, which is immobilized in a white microtiter well. The sandwich is completed by adding a second biotinylated monoclonal antibody specific for the whole hCG molecule. The degree of binding of biotinylated antibody, which is proportional to the amount of hCG present in the sample, is quantified by adding streptavidin labeled with the europium chelate 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA), in the presence of excess Eu3+. The fluorescent complex formed on the solid-phase [monoclonal antibody-hCG-monoclonal antibody-biotin-streptavidin-BCPDA-Eu3+] is measured by excitation at 337.1 nm with a nitrogen laser and monitoring the emission at 615 nm in a specially designed gated fluorometer working in a time-resolved mode. A two-step procedure is proposed for routine use to avoid the "high-dose hook effect" of the simpler and faster one-step procedure. The hCG assay described has a dynamic range of 1 to 500 int. units/L, and is precise and accurate. Results agree well with those obtained with a commercially available immunoradiometric and a time-resolved immunofluorometric procedure.     

Development of monoclonal antibodies for an assay of cardiac troponin-I and preliminary results in suspected cases of myocardial infarction.

To improve the specificity of biochemical markers of myocardial infarction (MI), we have developed a double monoclonal "sandwich" enzyme immunoassay to measure cardiac troponin-I (cTnI) in serum. We produced eight IgG monoclonal antibodies against human cardiac troponin-I (cTnI) and tested them against human and animal (canine, bovine, and rabbit) troponins. Five antibodies were cardiac-specific; none of the antibodies were species-specific. Two of the five cTnI-specific monoclonal antibodies were utilized in an immunoassay. Standards were made by adding purified human cTnI to affinity-stripped cTnI-free human sera to cover the range 0-100 micrograms/L for cTnI. The dose-response curve was nonlinear but reproducible. Total assay imprecision (CV) varied between 11% and 21%. The upper limit of the reference range (nonparametric 95% interval) was established as 3.1 micrograms/L by measuring cTnI concentration in sera of 159 hospitalized patients without evidence of cardiac disease. Purified human skeletal TnI up to 10,000 micrograms/L did not affect the assay (calculated cross-reactivity < 0.1%). Diagnostic sensitivities of creatine kinase MB isoenzyme (CK-MB) and cTnI were evaluated retrospectively in 49 consecutive patients with proven MI. In the 30 patients for whom sufficient information was available to establish an accurate time course, CK-MB was more sensitive during the first 4 h after the onset of chest pain, but thereafter the sensitivities were similar up to 48 h. However, cTnI is more cardiac-specific than is CK-MB and remains increased longer than does CK-MB.     

Circulating antibodies to mouse monoclonal immunoglobulins in normal subjects--incidence, species specificity, and effects on a two-site assay for creatine kinase-MB isoenzyme

With a two-site CK-MB assay, we screened serum samples from 1008 blood donors for the presence of antibodies to mouse monoclonal immunoglobulin. These antibodies were capable of cross-linking the labeled antibody with the solid-phase antibody in the two-site assay, thus generating a falsely high apparent CK-MB concentration. In 92 (9.12%) of the blood donors tested, apparent CK-MB concentrations of 10-1000 micrograms/L decreased to less than 3 micrograms/L when re-assayed with non-immune mouse serum (10 mL/L) included in the assay reagent. We tested the ability of non-immune sera from other animal species to lower the concentration of apparent CK-MB in 58 of the 92 samples. Bovine and ovine serum were almost as effective as mouse serum; feline, canine, and rabbit serum were less effective. Of the samples tested, 12% (1.1% of the original population screened) showed apparent CK-MB values that either were not depressed by bovine serum or were only partly depressed. We discuss the possible etiology of these antibodies in normal subjects and recommend that all mouse monoclonal two-site assays should contain non-immune mouse serum (or a suitable "irrelevant" mouse monoclonal antibody) to prevent false-positive results.     

"Sandwich" enzyme immunoassay for serum ferritin with polypropylene test tubes as the solid phase

A "sandwich" enzyme immunoassay for ferritin in human serum is described, in which horseradish peroxidase-labeled antibody and a very sensitive chromogen, 2,2'-azino-di(3-ethyl-benzthiazolin-6-sulfonate), are used. The solid phase is polypropylene test tubes treated with glutaraldehyde; immunologic reactions are developed during constant rotation of the tubes. The assay requires 20 microL of serum per assay and takes less than 3 h. The standard curve is linear between 0 and 400 micrograms of serum ferritin per liter. Analytical recoveries of various amounts of standard added to a serum sample ranged from 92 to 98.6%. The sensitivity is 2 micrograms/L, which translates to 0.4 ng per assay tube. Within-run CV ranges from 1.6 to 5.4% and between-run from 4.2 to 7.8% according to the ferritin concentration. Results by competitive radioimmunoassay correlated well (r = 0.98). No "hook effect" was observed for concentrations up to 13 mg/L. The reference interval is 40-240 micrograms/L for men, 8-100 micrograms/L for women.     

Measurement of free desipramine in serum by ultrafiltration with immunoassay.

We developed an ultrafiltration method for assaying free desipramine (DMI) in serum. An ultrafiltrate of DMI-containing serum was prepared by centrifugation through an Amicon Centrifree micropartition filter. Syva DMI solid-phase extraction (SPE) columns were used to extract the DMI from the serum and ultrafiltrate. The Syva monoclonal EMIT assay was used to quantify the DMI in the extract. In some experiments, the percent free DMI was quantified with radioactivity. Nonspecific losses of DMI in serum to the ultrafilter system were low (recoveries > 91%). Extraction of [3H]DMI from phosphate-buffered saline (to mimic serum ultrafiltrate) with the Syva SPE system was quantitative (recoveries of 98.4% +/- 4.6%). Free DMI concentrations, derived from serum containing 2.5-2500 micrograms/L DMI, were determined by ultrafiltration; results agreed well with values determined by equilibrium dialysis, the average percent of free DMI being 18.4% +/- 0.25% and 15.9% +/- 0.51%, respectively. To increase the sensitivity of the free DMI assay in the therapeutic range (total DMI 125-300 micrograms/L), we increased fourfold the ultrafiltrate volume applied to the SPE column. For free DMI at 11-130 micrograms/L, the within-run and between-run CVs for the ultrafiltration method were < 9% and < 15%, respectively. Binding of DMI to serum proteins decreased over the pH range 6.0-8.0, although temperatures between 20 and 28 degrees C did not affect binding. The ultrafiltration assay is fast, accurate, simple, and adaptable to standard laboratory instrumentation.     

Abbott IMx evaluated for assay of prostate-specific antigen in serum.

We evaluated a new fully automated procedure for quantitative measurement of prostate-specific antigen (PSA) by the Microparticle Enzyme Immunoassay (MEIA) technology developed for the Abbott IMx automated immunoassay system. The performance characteristics of the Abbott IMx PSA assay (y) were evaluated and compared with those of the Hybritech Tandem-E PSA assay (x), a solid-phase two-site immunoenzymometric assay. PSA values for both assays were well correlated (r = 0.99); regression analysis yielded the equation y = 0.92x - 0.23 micrograms/L. The Abbott assay proved reliable and reproducible, as shown by the intra- and interassay coefficients of variation (2.0-3.4% and 3.1-4.7%, respectively). The assay gave a linear standard curve up to 100 micrograms/L and was very sensitive (detected PSA < 0.1 microgram/L). This analytical sensitivity was comparable with that of the Tandem-E PSA assay. Overall, the IMx PSA assay demonstrated the accuracy, precision, linearity, and intermethod correlation required for monitoring patients with prostate cancer.     

ImmunoConcentration--a new format for solid-phase immunoassays

A new format for solid-phase immunoassays has been developed in which a monoclonal antibody-coated membrane, incorporated into a cylindrical, disposable device, regulates sample and reagent delivery. We illustrate the method with a two-site, immunoenzymometric assay that can detect human choriogonadotropin at less than 50 int. units/L (4 micrograms/L) in urine and less than 25 int. units/L (2 micrograms/L) in serum and takes less than 5 min to perform. The solid-phase antibody is located in a circular area in the center of the membrane so that in the presence of the hormone, after addition of substrate, a blue enzyme product is generated in this circular area. The high ratio of surface area to volume within the microporous matrix of the membrane assures short diffusion distances and therefore rapid binding of liquid-phase reagents to the solid phase. Pseudo-first-order reaction kinetics describe the binding of antigen to immobilized antibody and the binding of enzyme-labeled antibody to immobilized antigen. The speed and simplicity of this format may facilitate testing for many analytes, both soluble and particulate, as well as serological testing.     

Improved micromethod for mezlocillin quantitation in serum and urine by high-pressure liquid chromatography.

A rapid, sensitive, and specific method of analysis for mezlocillin in serum and urine by high-pressure liquid chromatography is described. A solid-phase extraction column was used to remove interfering substances from samples before chromatography. Quantitation included the use of an internal standard, nafcillin. Mezlocillin was chromatographed with a phosphate buffer-acetonitrile (73:27) mobile phase and a C-18 reverse-phase column and detected at a wavelength of 220 nm. The assay had a sensitivity of 1.6 micrograms/ml and a linearity of up to 600 micrograms/ml and 16 mg/ml in serum and urine, respectively, with only 0.1 ml of sample. The interday and intraday coefficients of variation for replicate analyses of spiked serum and urine specimens were less than 6.5%.