Biological Detection Limit Research Articles

Conditional optimization and methodological evaluation of liquid protein microarray in detecting cystatin C

To optimize the detection conditions and evaluate of cystatin C(CysC) by liquid protein microarray. CysC was detected by double antibody sandwich method using liquid protein microarray. On the basis of determining the optimal concentration combination of captured antibody and detected antibody, the detection conditions were optimized by determining the biological detection limit and lower detection limit, drawing the S-shaped curve and judging the linear range, and establishing the standard curve and regression equation. Methodsologically evaluate the accuracy, precision, reportable range and analytical specificity of the detection method. The optimal concentration combinations of CycC trapping-detection antibodies were 26.6 μg/mL-1∶800. The lower limits of detection and biologic limits of detection of the CysC were 0.037 and 0.237 ng/mL, respectively. Regression equation were as followes: y=-3.315x~2+283.04x+160.89, R~2=0.9921. The relative bias of CysC which was detected on the liquid protein microarry was 5.81%. The dilution recovery and recovery were 70.35%-84.91%(n=3)and 79.94%-122.41%(n=3)respectively. The correlation coefficient of method ology comparison experiment was r=0.616, P<0.05, and there was no significant difference between the two method(t=0.948, P=0.358); The within-run precision range from 3.54% to 4.03%(n=10); The between-run precision range from 12.07% to 15.05%(D=5, n=3); The reportable range was 0.26-3784.04 ng/mL. The analysis of interference test result showed that the both concentrations of hemoglobin(160.00, 71.11 g/L) had interference to the result of CysC detected on the chip. This study completed the optimization of conditions and methodological evaluation of liquid protein microarray in detecting CysC.

Optimization of platform conditions for simultaneous detection of multiple nutritional marker proteins by liquid protein microarray

To optimize the technical conditions for simultaneous detection of serum ferritin(SF), soluble transferrin receptor(sTfR), C-reactive protein(CRP)and retinol-binding protein four(RBP4)by liquid protein microarray. The trapping antibodies of the four proteins were coupled to magnetic beads with different codes, and the samples were added to the 96-well plate. The antibodies were detected by double antibody sandwich method. The serum of 5 patients were diluted with commercial diluent, 1% albumin from bovine serum(BSA) and phosphate buffer saline(PBS) to detect 4 target proteins, and the results were compared. The antibody specific binding ability was tested by antibody specific validation test. The interference between proteins was verified by the paired t test of the signal values of the single reaction system and the mixed reaction system. The lower limit of detection and the limit of biological detection of each protein were found by using multiple dilution method. The standard curve and regression equation were established. 1%BSA and PBS were selected to replace commercial diluent as diluents for the detection of 4 proteins in this experiment. The cross-reaction rate of the four antigens with other capture antibodies and detection antibodies was less than 2%. There was no significant difference in the signal value of each protein in the single reaction system and the mixed reaction system. The limit of detection and the limit of biological detection of SF were 1.155 and 1.625 ng/mL, respectively. The lower limit of detection and the limit of biological detection of sTfR were 2.682 and 5.208 ng/mL, respectively. The detection limit and biological detection limit of CRP were 0.302 and 0.391 ng/mL, respectively. The lower limit of detection and the limit of biological detection for RBP4 were 1.814 and 3.540 ng/mL, respectively. The standard curve and regression equation of the four proteins within the common linear range were as follows: SF y=172.5x-39.65, R~2=0.9968;sTfR y=60.10x+77.38, R~2=0.9972;CRP y=-6.000x~2+210.3x+246.1, R~2=0.9063;RBP4 y=-0.6998x~2+64.31x+134.8, R~2=0.9748. The conditions of the detection platform for four proteins such as SF, sTfR, CRP and RBP4 were optimized by using liquid protein chip technology.

The linear accumulation of atmospheric mercury by vegetable and grass leaves: Potential biomonitors for atmospheric mercury pollution.

One question in the use of plants as biomonitors for atmospheric mercury (Hg) is to confirm the linear relationships of Hg concentrations between air and leaves. To explore the origin of Hg in the vegetable and grass leaves, open top chambers (OTCs) experiment was conducted to study the relationships of Hg concentrations between air and leaves of lettuce (Lactuca sativa L.), radish (Raphanus sativus L.), alfalfa (Medicago sativa L.) and ryegrass (Lolium perenne L.). The influence of Hg in soil on Hg accumulation in leaves was studied simultaneously by soil Hg-enriched experiment. Hg concentrations in grass and vegetable leaves and roots were measured in both experiments. Results from OTCs experiment showed that Hg concentrations in leaves of the four species were significantly positively correlated with those in air during the growth time (p < 0.05), while results from soil Hg-enriched experiment indicated that soil-borne Hg had significant influence on Hg accumulation in the roots of each plant (p < 0.05), and some influence on vegetable leaves (p < 0.05), but no significant influence on Hg accumulation in grass leaves (p > 0.05). Thus, Hg in grass leaves is mainly originated from the atmosphere, and grass leaves are more suitable as potential biomonitors for atmospheric Hg pollution. The effect detection limits (EDLs) for the leaves of alfalfa and ryegrass were 15.1 and 22.2ngg(-1), respectively, and the biological detection limit (BDL) for alfalfa and ryegrass was 3.4ngm(-3).

Biomonitoring of airborne mercury with perennial ryegrass cultures

A biomonitoring network with grass cultures was established near a chlor-alkali plant and the mercury concentration in the cultures were compared with the average atmospheric total gaseous mercury (TGM). Biomonitoring techniques based on different exposure periods were carried out. When comparing the mercury concentration in the grass cultures, both the average atmospheric TGM concentration during exposure and the exposure time determined to a large extent the accumulation rate of TGM. The maximum tolerable level of mercury in grass (≅110 μg kg −1 DM) corresponds with an average TGM concentration of 11 ng m −3 for 28 days exposure. The background concentrations in grass were on an average 15 μg kg −1 DM and the effect detection limit (EDL) was 30 μg kg −1 DM. This value corresponds with an average TGM concentration of 3.2 and 4.2 ng m −3 for 28 and 14 days exposure, respectively, which is in turn the biological detection limit (BDL) of ambient TGM. Exposures for 7 days were less appropriate for biomonitoring.

Determination of biochemical relapse after radical prostatectomy by biological detection limit (BDL) after cystoprostatectomy using two PSA assays

The monitoring of PSA values following prostatectomy demands for the use of highly sensitive tests with low detection level. The possibilities to use the EIA Dialab test to monitor the PSA values after radical prostatectomy for early detection of persistent diseases were investigated by determining the biological detection limit (BDL) in serum of patients who unrewent cystoprostatectomy. The obtained values were compared with Abbott Imx test for PSA determination. A good correlation between the two studied methods was establisahed, r = 0.9827 with the regression curve Yx = 0.20463 + 0.96277. Test indicated that there was no significant difference (p < 0.001) between the investigated methods.

Bayer immuno 1™ PSA assay: An automated, ultrasensitive method to quantitate total PSA in serum

The Bayer Immuno 1 PSA Assay measures total PSA in human serum and demonstrates excellent performance with an interassay CV < or = 3.4% and a biological detection limit of 0.03 microgram/L. No significant interference from common hormonal and chemotherapeutic drugs, kallikrein, prostatic acid phosphatase, and trypsin, or elevated levels of total bilirubin, hemoglobin, triglycerides, and IgG was observed. The 95th percentile values for healthy individuals increased with age from 3.0 micrograms/L for males 50-59 years and 3.3 micrograms/L for males 60-69 years, to 4.6 micrograms/L for males > or = 70 years. Clinical studies with retrospective samples demonstrated correspondence between serial measurements of PSA and clinical outcome for 98% of 159 prostate cancer patients. Clinical sensitivity for patients with clinical evidence of disease, untreated at the time of specimen draw, increased with increasing stage from 77.5-100%. Specificity of 60-70% for BPH and other benign urogenital diseases was consistent with previous findings. Bayer Immuno 1 PSA Assay values for 2131 specimens from healthy subjects and patients with prostate cancer, BPH, and other malignant and nonmalignant diseases correlated well with the Abbott IMx PSA Assay over the range 0.0-6,238 micrograms/L (Y = 1.10 x + 0.02). The Bayer Immuno 1 PSA Assay provides automated ultrasensitive, precise, and equimolar measurement of total PSA in human serum.

Parameters Relating to Ultrasensitive Prostate-Specific Antigen Assays

Several recent papers have dealt with the performance of prostate-specific antigen (PSA) assays at very low concentrations (1)(2)(3). In these papers, no less than 5 different parameters have been discussed: the lower limit of detection (LLD), the biological detection limit (BDL), functional sensitivity (FS), the residual cancer detection limit (RCDL), and the minimal detectable concentration (MDC). One may wonder whether such a profusion of terms is meaningful or necessary. However, I believe that, although these parameters are to some extent presented or used as alternatives to each other, fundamental differences exist among them. Here, I aim at distinguishing two groups of parameters, each of which has a specific use. Two types …

An ultrasensitive immunoassay for prostate-specific antigen based on conventional colorimetric detection

Objective: Development of an ultrasensitive immunoassay for serum PSA involving conventional detection probes. Design and Methods: The assay involves a polyclonal antibody immobilized in microtitration Wells and a monoclonal antibody labeled with horseradish peroxidase. In a one-step assay, the enzymatic activity of the bound detection antibody is monitored by the addition of hydrogen peroxide/tetramethylbenzidine substrate reagent followed by spectrophotometric quantification of the conversion product. Results: The assay has a lower detection limit of 0.003 μg/L, biological detection limit of 0.009 μg/L, and intra- and inter-assay CVs of 8.2% and 10.5% at PSA concentrations of 0.022 and 0.065 μg/L, respectively. The recovery of the assay averaged 104% and it demonstrated a dilution linearity down to at least 0.01 μg of PSA/L. Results of comparison data correlated well with those obtained by a well established enzyme immunoassay. The serum PSA concentrations were <0.012 μg/L in the majority of patients (53.8%) who had undergone radical prostatectomy. Conclusions: This assay is well suited for post-surgical monitoring of PSA in patients with prostate cancer.

Original Articles: Prostate Cancer: Measurement of Serum Prostate Specific Antigen Levels in Women and in Prostatectomized Men With an Ultrasensitive Immunoassay Technique

ABSTRACTSerum prostate specific antigen (PSA) was measured in 89 prostate cancer patients with no evidence of relapse after radical prostatectomy, in 387 hospitalized women with various diseases and in 674 apparently healthy female blood donors, using an ultrasensitive time resolved immunofluorometric assay with a biological detection limit of 0.01 micrograms./l. Of the prostatectomized cancer patients 50% had measurable PSA (0.010 micrograms./l. or greater) and 21% had levels of 0.050 micrograms./l. or greater. Postoperative PSA was not associated with year of surgery, preoperative PSA or histological grade of the tumors. The distribution of PSA in 1,064 female sera is also reported, of which 83% had no detectable serum PSA, and in 15% PSA was between 0.010 and 0.049 micrograms./l. Only 27 subjects (2.5%) had PSA levels of 0.050 micrograms./l. or greater, including 16 (1.5%) with PSA of 0.10 micrograms./l. or greater. High serum PSA in women was associated with age 50 years or older. Further studies are ...

Original Articles

Serum prostate specific antigen (PSA) was measured in 89 prostate cancer patients with no evidence of relapse after radical prostatectomy, in 387 hospitalized women with various diseases and in 674 apparently healthy female blood donors, using an ultrasensitive time resolved immunofluorometric assay with a biological detection limit of 0.01 microgram./l. Of the prostatectomized cancer patients 50% had measurable PSA (0.010 microgram./l. or greater) and 21% had levels of 0.050 microgram./l. or greater. Postoperative PSA was not associated with year of surgery, preoperative PSA or histological grade of the tumors. The distribution of PSA in 1,064 female sera is also reported, of which 83% had no detectable serum PSA, and in 15% PSA was between 0.010 and 0.049 microgram./l. Only 27 subjects (2.5%) had PSA levels of 0.050 microgram./l. or greater, including 16 (1.5%) with PSA of 0.10 microgram./l. or greater. High serum PSA in women was associated with age 50 years or older. Further studies are needed to examine if the difference in PSA levels between women and prostatectomized men is due to residual or recurrent tumor or to other reasons associated with gender.

Early Detection of Residual Prostate Cancer After Radical Prostatectomy by an Ultrasensitive Assay for Prostate Specific Antigen

We evaluated the usefulness of an ultrasensitive immunoassay for prostate specific antigen (PSA), modified from the standard Yang Pros-Check PSA test and with a biological detection limit for PSA in serum of 0.07ng./ml., to detect residual prostate cancer at an earlier date. We studied retrospectively serial frozen serum samples from 22 prostate cancer patients after radical prostatectomy who later had residual cancer with detectable PSA levels of 0.3ng./ml. or more by the standard PSA test. As controls we studied 33 cystoprostatectomy patients (for bladder cancer) without histological evidence of prostate cancer and 23 patients after radical prostatectomy who had the highest probability of cure of the cancer.All control patients without cancer had PSA values (282 of 283 samples, 99.6%) of less than 0.1ng./ml This value was called the residual cancer detection limit. In the 22 patients with recurrent cancer the ultrasensitive assay detected cancer recurrence (PSA 0.1ng./ml or more) much earlier (median 202 and mean 310 days) than the standard assay (PSA 0.3ng./ml or more). On screening 187 current post-radical prostatectomy patients without evidence of cancer by the standard assay the ultrasensitive assay detected 21 (11.2%) with evidence of residual cancer, that is PSA level of 0.1ng./ml or more. We conclude that an ultrasensitive assay for PSA can detect residual cancer after radical prostatectomy much earlier than current immunoassays for PSA. Earlier detection of residual cancer may improve long-term survival by allowing for earlier institution of adjuvant therapy.

Evaluation of the Abbott IMx® Automated Immunoassay of Prostate-Specific Antigen

We detail the performance characteristics of the new IMx PSA immunoassay developed by Abbott Laboratories, addressing PSA recovery, assay reproducibility, standard curve storage, lower limit of detection, dilution linearity, and correlation with the Hybritech Tandem-R PSA immunoassay. We analyzed 686 sera for PSA retrospectively, testing 555 of these concurrently with the IMx and the Tandem-R immunoassays. The IMx PSA standard curve was linear from 0 to 100 micrograms/L, and curve storage was maintained for 4 weeks. The lower limit of detection of the IMx PSA assay was < or = 0.03 microgram/L; allowing for the assay precision yielded a biological detection limit of 0.06 microgram/L. We conservatively set the clinical threshold at 0.1 microgram/L. Regression analysis of dilution linearity involving 10 samples (0.44-200 micrograms/L) resulted in coefficients of correlation ranging from 0.9972 to 1.000. Reproducibility studies with 18 specimens within the range of 0.39-413.67 micrograms/L gave intra- and interassay CVs < 6.5%. The interassay 95% confidence interval for a specimen containing 0.06 microgram of PSA per liter was 0.03-0.09 microgram/L. Correlation between IMx and Tandem-R PSA assay results was excellent: r = 0.9909 and slope = 0.95. Overall, the IMx PSA immunoassay, with the conveniencies of automation, curve storage, and nonisotopic handling, provided an improved lower limit of PSA detection, which allows for earlier indication of residual or recurrent disease after radical prostatectomy.

Sensitivity and Reproducibility of Urinary C‐peptide as Estimate of Islet B‐cell Function in Insulin‐treated Diabetes

The aims of the present study were to evaluate the ability of urinary C-peptide determination to demonstrate presence of residual insulin secretion, and to evaluate the reproducibility of urinary C-peptide excretion in 125 insulin-treated diabetic patients. C-peptide was determined in two consecutive 24-h urine specimens and related to plasma C-peptide 6 min after the intravenous injection of 1 mg glucagon. The detection limit of C-peptide in plasma was defined analytically (greater than or equal to 0.02 nmol l-1) and from pancreatectomized patients (greater than or equal to 0.06 nmol l-1), and in urine only analytically (greater than or equal to 0.1 nmol l-1). If the analytical detection limit of plasma C-peptide was used as indicator of residual insulin secretion, islet B-cell function was preserved in all patients. In patients with stimulated plasma C-peptide levels from 0.02- less than 0.06 nmol l-1 no increase was found in plasma C-peptide values after stimulation with glucagon. This unresponsiveness of islet B-cells is in good agreement with the existence of a biological detection limit of C-peptide in plasma of 0.06 nmol l-1. Using this biological plasma C-peptide detection limit, 49 of 125 patients were without residual insulin secretion. In contrast to this, only 7 patients were diagnosed as C-peptide nonsecretors using the analytical detection limit of urinary C-peptide. Eighty-four per cent of patients considered to have Type 1 (insulin-dependent) diabetes with a duration of diabetes of more than 15 years had detectable C-peptide in the urine.(ABSTRACT TRUNCATED AT 250 WORDS)