Development and evaluation of an improved method for screening of amphetamines.
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We developed a homogeneous immunoassay method to eliminate false-positive amphetamine results caused by cross-reactive substances, including over-the-counter allergy and cold medications. This method uses a neutralizing antibody that binds to amphetamines but does not bind to the labeled amphetamine conjugate used in the assay. The amount of neutralizing antibody is sufficient to reduce the assay signal resulting from authentic amphetamine and methamphetamine, but not the signal resulting from cross-reactants. This concept was implemented using the CEDIA DAU Amphetamines assay on Hitachi 747 and 717 clinical chemistry analyzers. Urine samples were tested using the standard, unmodified reagents in one channel and reagents containing the neutralizing antibody in a second channel. The difference in rate between the two tests was calculated by the analyzer; true-positive samples showed a significantly greater decrease in assay signal in response to neutralizing antibody as compared with false-positive samples. The neutralization method was evaluated in two studies using 448 samples that tested positive in the initial CEDIA DAU Amphetamines screening test. The samples were separated into categories of 154 true-positive samples and 294 false-positive samples based upon a secondary screen with the Abbott FPIA Amphetamines assay followed by gas chromatography-mass spectrometry (GC-MS) testing using the HHS (SAMHSA) cutoff criteria. The CEDIA neutralization test successfully identified all 154 of the GC-MS confirmed positive samples. The test successfully identified as false positive 251 out of the 294 (85.4%) samples that failed to confirm by GC-MS. (+info)
Urinary excretion profiles of 11-nor-9-carboxy-delta9-tetrahydrocannabinol: a delta9-THCCOOH to creatinine ratio study.
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Monitoring the major cannabinoid metabolite (delta9-THCCOOH) to creatinine ratio (M/C) has been used to predict new drug use. According to Huestis and Cone, the best accuracy (85.4%) for predicting new marijuana use was a ratio > or = 0.5 from two urine specimens collected at least 24 h apart. Manno et al. recommended an M/C ratio of > or = 1.5. Subjects with a history of chronic marijuana use were screened for cannabinoid use by immunoassay (50-ng/mL cutoff), and presumptive positives were confirmed by gas chromatography-mass spectrometry for delta9-THCCOOH (15-ng/mL cutoff). Creatinine was analyzed with a cutoff concentration of 25 mg/dL. The study objective was to apply the criteria from both groups of workers to determine if consecutive urine specimens (collected at least 24 h apart) positive for cannabinoids could be used to differentiate new marijuana use from the excretion of residual cannabinoid metabolite (delta9-THCCOOH) in an uncontrolled setting. Serial urine specimens (826) were collected from 26 individuals. Huestis and Cone and Manno et al. ratios indicated new drug use in 83% and 33% of serial urine specimens collected at least 24 h apart, respectively. Clinically, the Huestis and Cone ratio is recommended because of a lower false-negative rate (7.4%) than the Manno et al. false-negative rate (24%). In legal situations, we recommend using the Manno et al. ratio because of its lower false-positive rate (0.1%) as stated by Huestis and Cone. (+info)
Tamoxifen-DNA adduct formation in rat liver determined by immunoassay and 32P-postlabeling.
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Tamoxifen (TAM), a nonsteroidal antiestrogen used as a chemotherapeutic and chemopreventive agent for breast cancer, induces liver tumors in rodents and covalent DNA adduct formation in hepatic DNA. Here, we report the development and validation of highly sensitive and specific immunoassays for the determination of TAM-DNA adducts. Rabbits were immunized with calf thymus DNA, chemically modified with alpha-acetoxytamoxifen to 2.4 adducts per 100 nucleotides, and the resulting antisera were characterized by competitive dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA) and chemiluminescence immunoassay (CIA). Compared with DELFIA, the CIA has a much lower background and a 20-fold increase in sensitivity. For the immunogen TAM-DNA, 50% inhibition was at 2.0 +/- 0.11 (mean +/- SE, n = 18) fmol of (E)-alpha-(N2-deoxyguanosinyl)tamoxifen (TAM-dG) adduct in TAM-DNA by DELFIA. For TAM-DNA modified to 4.8 adducts in 10(6) nucleotides, 50% inhibition was at 20.6 +/- 6.6 (mean +/- SE, n = 8) fmol of TAM-dG in TAM-DNA by DELFIA and at 0.92 +/- 0.11 (mean +/- SE, n = 10) fmol of TAM-dG in TAM-DNA by CIA. No inhibition was observed in either assay with up to 20 microg (62.5 nmol of nucleotides) of unmodified DNA. The individual adducts TAM-dG and (Z)-alpha-(N2-deoxyguanosinyl)tamoxifen and the individual compounds TAM and 4-OH-TAM gave DELFIA 50% inhibitions at 828, 2229, 5440, and 8250 fmol, respectively. For assay validation, TAM-dG levels were determined by DELFIA, CIA, and 32P-postlabeling in TAM-DNA samples modified in vitro to different levels, and comparable values were obtained in all three assays. Further validation was obtained in vivo in rat liver. DNA adducts of TAM were measurable in rat liver 24 h after a single i.p. dose of 45 mg TAM/kg body weight and after daily p.o. dosing for 7 days with 5.0, 10.0, and 20.0 mg TAM/kg body weight. In addition, TAM-DNA adducts disappeared slowly over 21 days in rats on a control diet that were first given p.o. TAM at 45 mg/kg/day for 4 days. In the rat experiments, TAM-DNA adduct levels determined by CIA compared well with those determined by 32P-postlabeling, although the CIA gave an underestimation at the highest doses. For rat liver samples, the detection limit by CIA was 3 adducts per 10(9) nucleotides (0.2 fmol of adducts per 20 microg of DNA). (+info)
Activated protein C resistance: automated detection of the factor V Leiden mutation by mismatch hybridization.
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BACKGROUND: A single point mutation in the factor V gene has been demonstrated to be the cause of factor Va resistance to proteolytic cleavage by activated protein C. Knowledge of the patient's genetic disposition is of great importance in situations such as pregnancy, surgery, use of oral contraceptives, and immobilization. METHODS: We have developed a rapid, automated test for the detection of the factor V mutation that makes use of differences in thermal stability between perfect-match and non-perfect-match hybrids. A DNA fragment spanning the mutation is amplified with a biotin-labeled primer. Ruthenium-labeled oligonucleotides, perfectly matching either the biotinylated wild-type strand or the biotinylated mutation strand, are added. Heating to 95 degrees C and subsequent cooling lead to the formation of double-stranded DNA. Under the conditions chosen, ruthenium-labeled oligonucleotides form stable, double-stranded DNA with the biotinylated strand only if both strands perfectly match each other. The ruthenium signal is measured on a modified Elecsys 1010 system (Roche Diagnostics). RESULTS: The ratio between the signals obtained with perfectly matching and non-perfectly matching oligonucleotides reflects the genetic status. Analyzed samples can be divided into three nonoverlapping groups based on these ratios. We confirmed the reliability of the method by analyzing several samples of known genetic status; the results were identical in every single instance. CONCLUSIONS: The test discriminates unambiguously between the heterozygous and the homozygous states. Because of its low costs and easy handling, the assay is suitable for use in routine laboratories of clinical chemistry. (+info)
New electrochemiluminescent immunoassay for the determination of CYFRA 21-1: analytical evaluation and clinical diagnostic performance in urine samples of patients with bladder cancer.
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BACKGROUND: A new electrochemiluminescent immunoassay (ECLIA) has been developed for the determination of cytokeratin 19 (CYFRA 21-1) in the Elecsys 2010 immunoassay system. Urinary CYFRA 21-1 might have a role in the diagnosis of bladder cancer. METHODS: We performed an analytical evaluation of the CYFRA 21-1 ECLIA for serum and urine samples. The clinical value of urinary CYFRA 21-1 for the detection of bladder cancer was evaluated through its measurement in 226 urine samples from symptomatic and asymptomatic controls. RESULTS: At concentrations of 2-30 microg/L, within-assay imprecision (CV) was below 2.1% for sera and 3.3% for urines, with interassay CVs below 3.3% for sera and 4.9% for urines. The day-to-day CV was <20% at concentrations >0.2 microg/L (functional sensitivity). Measurement of diluted samples showed that the assay estimated CYFRA 21-1 between 98% and 103% for sera and 98% and 105% for urines. Recovery of added CYFRA 21-1 was 99-105% for sera and 96-115% for urines. We separately compared serum and urine CYFRA 21-1 ECLIA results with those obtained with an IRMA (CIS bio international). Regression analysis for sera was: CYFRA 21-1 (ECLIA) = 0.520 + 1.018 CYFRA 21-1 (IRMA); [95% confidence interval (CI) (y-intercept), -0.260 to 1.309]; 95% CI (slope), 0.978-1.060; n = 100; S(y|x) = 3.242; r(2) = 0.987. For urine samples it was: CYFRA 21-1 (ECLIA) = 0.716 + 0.966 CYFRA 21-1 (IRMA); 95% CI (y-intercept), 0.009-1.422; 95% CI (slope), 0.956-0.976; n = 100; S(y|x) = 4.136; r(2) = 0.986. In urine samples voided by patients with and without bladder cancer, the best ROC analysis discrimination provided 81.0% (95% CI, 72.7-87.7%) sensitivity and 97.2% (95% CI, 90.2-99.6%) specificity at a threshold value of 5.7 microg/L. CONCLUSIONS: Our initial evaluation showed reliable analytical performance for urinary CYFRA 21-1, which might assist urologists in the detection of bladder cancer as a noninvasive adjunct to cystoscopy. (+info)
Two-site expression immunoassay using a firefly luciferase-coding DNA label.
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BACKGROUND: We report the first two-site, "sandwich type" expression immunoassay using as a label an expressible DNA fragment encoding firefly luciferase. METHODS: The DNA label consisted of a T7 RNA polymerase promoter, a firefly luciferase-coding sequence, and a poly(dA/dT) tail. The 3' end of the DNA label was biotinylated and complexed with streptavidin. A sandwich immunoassay for prostate-specific antigen (PSA) was developed in which the antigen was first bound to an immobilized monoclonal antibody and then reacted with a biotinylated polyclonal antibody. The streptavidin-luciferase-coding DNA complex was then bound to the immunocomplex. The DNA label was subsequently expressed in vitro by coupled transcription and translation. The generated luciferase was measured by its characteristic bioluminescent reaction. RESULTS: The bioluminescence was linearly related to the concentration of PSA in the sample. As low as 30 ng/L PSA was measured (12.5-microL sample) with a signal-to-background ratio of 2.3, and the linear range extended to 3 microg/L. The results obtained from the proposed assay agreed well to those determined by IMx immunoassay (y = 0.98x + 0.74 microg/L; r = 0.971; n = 44). CONCLUSIONS: The use of the newly developed DNA label in a two-site immunoassay was demonstrated for the first time. The assay was applied successfully to the measurement of serum PSA. (+info)
The combination of human glandular kallikrein and free prostate-specific antigen (PSA) enhances discrimination between prostate cancer and benign prostatic hyperplasia in patients with moderately increased total PSA.
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BACKGROUND: Prostate-specific antigen (PSA) is the most reliable tumor marker available and is widely used for the diagnosis and management of prostate cancer. Unfortunately, PSA cannot distinguish efficiently between benign and malignant disease of the prostate, especially within the range of 4-10 microg/L. Among the refinements developed to enhance PSA specificity is the free/total PSA ratio, which is useful in discriminating between the two diseases within the diagnostic "gray zone". Recent data indicate that human glandular kallikrein (hK2), a protein with high homology to PSA, may be an additional serum marker for the diagnosis and monitoring of prostate cancer. METHODS: We analyzed 206 serum samples (all before treatment was initiated) from men with histologically confirmed benign prostatic hyperplasia (n = 100) or prostatic carcinoma (n = 106) with total PSA in the range of 2.5-10 microg/L. Total and free PSA and hK2 were measured with noncompetitive immunological procedures. Statistical analysis was performed to investigate the potential utility of the various markers or their combinations in discriminating between benign prostatic hyperplasia and prostatic carcinoma. RESULTS: hK2 concentrations were not statistically different between the two groups of patients. There was a strong positive correlation between hK2 and free PSA in the whole patient population. hK2/free PSA ratio (area under the curve = 0.69) was stronger predictor of prostate cancer than the free/total PSA ratio (area under the curve = 0.64). At 95% specificity, the hK2/free PSA ratio identified 30% of patients with total PSA between 2.5-10 microg/L who had cancer. At 95% specificity, the hK2/free PSA ratio identified 25% of patients with total PSA between 2.5 and 4.5 microg/L who had cancer. CONCLUSIONS: Our data suggest that hK2 in combination with free and total PSA can enhance the biochemical detection of prostate cancer in patients with moderately increased total PSA concentrations. More specifically, the hK2/free PSA ratio appears to be valuable in identifying a subset of patients with total PSA between 2.5 and 4.5 microg/L who have high probability of cancer and who should be considered for biopsy. (+info)
Monoclonal gammopathy may disturb oestradiol measurement in the treatment and monitoring of in-vitro fertilization: case report.
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A 31 year old woman had her treatment for infertility by in-vitro fertilization (IVF) cancelled because a highly elevated serum concentration of oestradiol was detected, contrary to the clinical picture and that observed by vaginal ultrasound. The immunoassay for measuring oestradiol had been affected by circulating heterophilic antibodies in the form of an elevated immunoglobulin (Ig) G-kappa M component. This may often be associated with a haematological malignancy of lymphoid origin, but this patient had a benign monoclonal gammopathy. Monoclonal gammopathy has not been described in IVF patients previously, nor has monoclonal gammopathy been reported as a cause of erroneously elevated oestradiol concentration. This sort of interference in oestradiol analysis is probably very rare, but may lead to unnecessary cancellation of the treatment. A highly elevated oestradiol that is not in accordance with the clinical course may indicate heterophilic antibody interference, and the cause should always be investigated. (+info)