Dual-label Time-resolved Fluorescence Immunoassay Research Articles

Simultaneous Detection of Prolactin and Growth Hormone Using a Dual-label Time-resolved Fluorescence Immunoassay.

Prolactin (PRL) and growth hormone (GH) are two important hormones secreted by the pituitary gland, and their abnormal levels are often related to disease status. This study aimed to establish a new dual-label time-resolved fluorescence immunoassay (TRFIA) to quantitatively measure PRL and GH levels in serum. A sandwich TRFIA was optimized and established: anti-PRL/GH antibodies immobilized on 96-well plates captured PRL/GH and then banded together with anti-PRL/GH paired antibodies labeled with europium(III) (Eu3+)/samarium(III) (Sm3+) chelates. Finally, a time-resolved analyzer measured the Eu3+/Sm3+ fluorescence values. Clinical serum samples were used to evaluate the detection performance of this method. The sensitivities of this dual-label TRFIA were 0.35 ng/mL and 0.45 ng/mL, respectively, and the detection range was between 0.1 and 1000 ng/mL. All the cross-reactivities were lower than 1.07%. The intra-assay and interassay coefficients of variation were 2.18-7.85% and 2.25-7.30%, respectively. Compared with the registered TRFIA kits, a high Pearson coefficient (r = 0.9626 and 0.9675) was observed. This dual-label TRFIA has high sensitivity, accuracy and specificity with good clinical detection performance, representing a suitable alternative to existing methods for determining PRL and GH levels, and is expected to be used in the clinic in the future.

A new method for screening acute/chronic lymphocytic leukemia: dual-label time-resolved fluorescence immunoassay

BackgroundLymphocytic leukemia (LL) is a primary malignant tumor of hematopoietic tissue, which seriously affects the health of children and the elderly. The study aims to establish a new detection method for screening acute/chronic LL using time-resolved fluorescence immunoassay (TRFIA) via quantitative detection of S100 calcium binding protein A8 (S100A8) and leucine-rich alpha-2-glycoprotein 1 (LRG1) in serum.MethodsHere a sandwich TRFIA was optimized and established: Anti-S100A8/LRG1 caputre antibodies immobilized on 96-well plates captured S100A8/LRG1, and then banded together with the anti-S100A8/LRG1 detection antibodies labeled with Europium(III) (Eu3+)/samarium(III) (Sm3+) chelates. Finally time resolved fluorometry measured the fluorescence intensity.ResultsThe sensitivity of S100A8 was 1.15 ng/mL(LogY = 3.4027 + 0.4091 × LogX, R2 = 0.9828, P < 0.001, dynamic range: 2.1–10,000 ng/mL), and 3.2 ng/mL for LRG1 (LogY = 3.3009 + 0.4082 × LogX, R2 = 0.9748, P < 0.001, dynamic range: 4.0–10,000 ng/mL). The intra-assay and inter-assay CVs were low, ranging from 5.75% to 8.23% for S100A8 and 5.30% to 9.45% for LRG1 with high specificity and affinity in serum samples. Bland–Altman plots indicated TRFIA and ELISA kits have good agreement in clinical serum samples. Additionally, the cutoff values for S100A8 and LRG1 were 1849.18 ng/mL and 588.08 ng/mL, respectively.ConclusionThe present TRFIA method could be used for the quantitative detection of S100A8 and LRG1 in serum, and it has high sensitivity, accuracy and specificity. Clinically, this TRFIA method could be suitable for screening of LL via the quantitative detection of S100A8 and LRG1.

Time-resolved Fluorescence Immunoassay (TRFIA) for the Simultaneous Detection of MMP-9 and Lp-PLA2 in Serum.

Currently, atherosclerosis accounts for the majority of cardiovascular morbidity and mortality worldwide, and predicting the stability of atherosclerotic plaque is the main method to prevent atherosclerotic death. This study aims to establish a dual-label time-resolved fluorescence immunoassay (TRFIA) of matrix metalloprotein-9 (MMP-9) and lipoprotein-associated phospholipaseA2 (Lp-PLA2) to predict atherosclerotic plaque stability. A dual-label TRFIA was introduced for the simultaneous quantification of MMP-9 and Lp-PLA2 using fluorescent lanthanide (Eu3+ and Sm3+) chelates. The performance (sensitivity, specificity, accuracy, precision and reference intervals in different subjects) of this TRFIA was evaluated and compared with commercial kit. The sensitivity of the TRFIA for MMP-9 was 0.85 ng/mL and for Lp-PLA2 was 0.68 ng/mL with high affinity and specificity. The average recoveries were 94.58% to 109.82%, and 104.32% to 109.26%, respectively. All intra- and inter-assay CVs ranged from 3.10% to 5.46%. For the normal subjects, the cutoff value was 160.70 ng/mL for MMP-9 and 183.73 ng/mL for LP-PLA2; for the subjects with stable plaque, the cutoff value was 181.98~309.22 ng/mL for MMP-9 and 194.73~337.89 ng/mL for LP-PLA2; for the subjects with unstable plaque, the cutoff value was 330.43 ng/mL for MMP-9 and 343.23 ng/mL for LP-PLA2. This TRFIA detection results agreed well with the results of commercial kit (R2=0.9567 and R2=0.9771, respectively) in clinical serum samples. The TRFIA developed has a wide detection range and good sensitivity for the high-throughput simultaneous detection of MMP-9 and Lp-PLA2 in serum, which provides a new method for predicting the stability of atherosclerotic plaque.

Simultaneous detection of fungal (1,3)-β-d-glucan and procalcitonin using a dual-label time-resolved fluorescence immunoassay.

Neonatal infectious diseases are a serious threat to the health of newborns. The aim was to establish a new detection method for the simultaneous measurement of (1,3)-β-d-glucan and procalcitonin in serum for the early screening and efficacy testing of neonatal infectious diseases. We established a sandwich dual-label time-resolved fluorescence immunoassay (TRFIA): anti-(1,3)-β-d-glucan/procalcitonin antibodies immobilized on 96-well plates captured (1,3)-β-d-glucan/procalcitonin antigens and then banded together with the detection antibodies labeled with europium(III) (Eu3+ )/samarium(III) (Sm3+ ) chelates. Finally, time-resolved fluorometry was used to measure the fluorescence intensity. The linear correlation coefficient (R2 ) of the (1,3)-β-d-glucan standard curve was 0.9913, and the R2 of the procalcitonin standard curve was 0.9911. The detection sensitivity for (1,3)-β-d-glucan was 0.4pg/mL (dynamic range: 0.6-90pg/mL), and the average recovery was 101.55%. The detection sensitivity for procalcitonin was 0.02ng/mL (dynamic range: 0.05-95ng/mL), and the average recovery was 104.61%. There was a high R2 between the present TRFIA method and a commercially available assay (R2 =0.9829 for (1,3)-β-d-glucan and R2 =0.9704 for procalcitonin). Additionally, the cutoff values for (1,3)-β-d-glucan and procalcitonin were 23.95pg/mL and 0.055ng/mL, respectively. The present TRFIA method has high sensitivity, accuracy, and specificity and is an effective method for early screening and efficient testing of neonatal invasive fungal infection.

A dual-label time-resolved fluorescence immunoassay for screening of osteoporosis based on simultaneous detection of C-terminal telopeptide (β-CTX) and aminoterminal propeptide (P1NP) of type I procollagen

Osteoporosis is a disease where increased bone weakness increases the risk of a broken bone. Until a broken bone occurs, there are typically no symptoms. Osteoporosis affects more than 75 million people in the United States, Europe and Japan. The diagnosis of osteoporosis is primarily determined by measuring bone mineral density using dual-energy X-ray absorptiometry, but for men under 50 years of age, premenopausal women should not be made on the basis of densitometric criteria alone. Bone biomarkers are a useful tool in detecting osteoporotic. A two-step dual-label time-resolved fluorescence immunoassay (TRFIA) was developed for the simultaneous detection of serum C-terminal telopeptide (β-CTX) and amino-terminal propeptide (P1NP) of Type I procollagen in a single run. The performance of this assay was first evaluated using clinical serum samples, and then compared with commercialized kits. The sensitivity of this assay for β-CTX was 1 ng/L (dynamic range, 0–1000 ng/L), and the sensitivity for P1NP detection was 1 μg/L (dynamic range, 1–1000 μg/L). High correlation coefficients (R) were obtained between the present dual-label TRFIA and commercially available kits (R = 0.99 for β-CTX and P1NP). The present dual-label TRFIA has high sensitivity, specificity and accuracy in clinical sample analysis. It is a good alternative to the single-label diagnostic methods.

A dual-label time-resolved fluorescence immunoassay for the simultaneous determination of cystatin C and β2-microglobulin in urine

Background: Contrast media is widely used in clinical diagnostic and interventional procedures, but may cause damage to the kidney, that is, contrast-induced nephropathy. This study was to establish a dual-label time-resolved fluorescence immunoassay (TRFIA) for the simultaneous determination of renal function markers cystatin-C (Cys-C) and β2-microglobulin (β2-MG) for the early diagnosis and follow-up surveillance of contrast-induced nephropathy.Methods: A sandwich immunoassay was used to detect the concentration of Cys-C, and the competitive immunoassay was used to detect the concentration of β2-MG in 50 samples of urine. The performance of this dual-label TRFIA was evaluated and compared with commercial assays.Results: The sensitivity for Cys-C detection was 1.26 ng/ml, the average recovery was 99.36%; The sensitivity for β2-MG detection was 2.13 ng/ml, the average recovery was 100.18%. Bland–Altman analysis showed that the dual-label TRFIA method and the commercial kits had a good agreement, suggesting they can be used interchangeably in clinical urine analysis.Conclusion: The present dual-label TRFIA has high sensitivity, specificity and accuracy in clinical sample analysis. This method can be used for the early diagnosis and follow-up surveillance of the contrast-induced nephropathy.

A dual-label time-resolved fluorescence immunoassay for the simultaneous determination of ferritin and β2 -microglobulin.

Lymphocytic leukemia is a kind of primary malignant tumor of hematopoietic tissue. The aim was to establish a dual-label time-resolved fluorescence immunoassay (TRFIA) for the simultaneous determination of ferritin (FER) and β2 -microglobulin (β2 -MG) for the early screening and follow-up surveillance of lymphocytic leukemia. The sandwich immunoassay was used to detect the concentration of FER, and the competitive immunoassay was used to detect the concentration of β2 -MG in serum. FER in serum was captured by anti- FER antibody immobilized on microtiter wells, and then banded together with another anti- FER labeled with europium(III) Eu3+ chelate, followed by fluorescence measurement using time-resolved fluorometry (TRF). Sm3+ labeled β2 -MG and β2 -MG samples were added to compete with a certain amount of anti-β2 -MG antibody, followed by fluorescence measurement using TRF. The performance of this dual-label TRFIA was evaluated using the clinical blood and compared with the commercial assays. The linear correlation coefficient (R2 ) of the FER and β2 -MG standard curves were 0.9914 and 0.9927, respectively. The sensitivity for FER detection was 8ng/mL (dynamic range 0-1000ng/mL), the average recovery was 100.51%; The sensitivity for β2 -MG detection was 1ng/mL (dynamic range 0-1000ng/mL), the average recovery was 101.02%. High correlation coefficients (R2 ) were obtained between the commercial assays (R2 =.9966 for FER, and R2 =.9897 for β2 -MG). The present dual-label TRFIA has high sensitivity, specificity, and accuracy in clinical sample analysis. It is an effective detection method for the early screening and follow-up surveillance of the acute and chronic lymphocytic leukemia.

Development of a Dual-Label Time-Resolved Fluorescence Immunoassay (TRFIA) for Screening of Bladder Cancer based on Simultaneous Detection of BLCA-4 and NMP52 in Urine

Bladder cancer is a heterogeneous disease and occupies highest incidence in developed country. Time-resolved fluoroimmunoassays (TRFIA) is a new detection technique with a feature of sensitive, simple and inexpensive. The aim of this study is to establish a dual-label TRFIA for the simultaneous detection of BLCA-4 and NMP52 in urine in a single run. The sandwich immunoassay was used to detect the concentration of BLCA-4 and NMP52 in urine. BLCA- 4 and NMP52 in urine were captured by anti-BLCA-4 and anti-NMP52 antibodies immobilized on microtiter wells, and then banded together with another anti-BLCA-4 and anti-NMP52 labeled with europium(III) Sm3+ and samarium(III) Eu3+ chelates, followed by fluorescence measurement using time resolved fluorometry. To assess the performance of this assay, clinical urine samples were used, and then commercialized kits were compared. The sensitivity for BLCA-4 and NMP52 detection of this assay were 2 U/mL (dynamic range, 5-300 U/L) and 1 μg/ml (dynamic range, 2-150 μg/ml) respectively. The correlation coefficients (R) between the present dual-label TRFIA and commercially available kits were high. R-values were 0.99 for BLCA-4 and NMP52. The cross-reactivity seems not to influence the results. The present dual-label TRFIA, allowing the simultaneous detection ofBLCA-4 and NMP52, has high sensitivity, specificity, and accuracy in clinical sample analysis. Therefore, it has good prospects of application.

A dual-label time-resolved fluorescence immunoassay (TRFIA) for screening of Coronary atherosclerosis based on simultaneous detection of Lp-PLA2 and HsCRP

Atherosclerosis is the underlying cause of most coronary events. The conventional method for coronary atherosclerosis detection is morphological examination or coronary arterionyraphy. These methods are complex and time-consuming. In this study a two-step dual-label TRFIA was developed for the simultaneous detection of Lp-PLA2 and hsCRP in a single run. The performance of this assay was first evaluated using clinical serum samples, and then compared with commercialized kits. The sensitivity of this assay for Lp-PLA2 detection was 1ng/mL (dynamic range, 0–1000U/L), and the sensitivity for hsCRP detection was 1mg/L (dynamic range, 1–1000mg/L). High correlation coefficients (R) were obtained between the present dual-label TRFIA and commercially available kits(R=0.99 for LP-PLA2 and hsCRP). The present dual-label TRFIA has high sensitivity, specificity, and accuracy in clinical sample analysis. It is a good alternative to the single-label diagnostic methods.

Dual-Labeled Time-Resolved Immunofluorometric Assay for the Simultaneous Quantitative Detection of Hepatitis B Virus Antigens in Human Serum.

In this paper, a novel time-resolved fluorescence immunoassay (TRFIA) is described that allows the simultaneous quantitative detection of hepatitis B virus surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) in human serum to aid the diagnosis and monitoring of hepatitis B virus infection. The proposed method was developed based on a two-step sandwich immunoassay protocol in which monoclonal antibodies against HBsAg and HBeAg were co-coated in 96 microtitration wells, then tracer polyclonal antibodies against HBsAg labeled with samarium and tracer monoclonal antibodies against HBeAg labeled with europium chelates were used for detection. The detection range was 0.1-150IU/mL for HBsAg and 0.5-160 PEIU/mL for HBeAg, and the detection limits were 0.03IU/L and 0.09 PEIU/ml, respectively. The intra- and inter-assay coefficients of variation were below 8% for both virus antigens. The dilution linearity and accuracy of the assay were satisfactory. No statistically significant differences were observed in sensitivity or specificity for the serum samples between the dual-label TRFIA and a commercial single-label TRFIA. These results demonstrate that an effective, reliable and convenient HBsAg/HBeAg dual-label TRFIA was successfully developed that may be clinically applicable for blood screening to monitor the course of hepatitis B virus infection and predict treatment responses.

The development of dual-label time-resolved fluorescence immunoassay (TRFIA) for screening of ovarian cancer based on simultaneous detection of human epididymis protein-4 and cancer antigen 125

ABSTRACTA two-step dual-label TRFIA was developed for the simultaneous detection of human epididymis protein-4 and cancer antigen 125 in a single run. The performance of this assay was first evaluated using clinical serum samples, and then compared with commercialized kits. The sensitivity of this assay for cancer antigen 125 detection was 0.5 U/mL (dynamic range, 0–1400 U/L), and the sensitivity for human epididymis protein-4 detection was 1 pM (dynamic range, 1–900 pM). High correlation coefficients (R) were obtained between the present dual-label TRFIA and commercially available kits (R = 0.99). The present dual-label TRFIA has high sensitivity, specificity, and accuracy in clinical sample analysis. It is a good alternative to the single-label diagnostic methods.

A Dual-Label Time-Resolved Fluorescence Immunoassay for the Simultaneous Determination of β-Human Chorionic Gonadotropin and Progesterone

A dual-label time-resolved fluorescence immunoassay (TRFIA) for the simultaneous determination of β human chorionic gonadotropin and progesterone was developed for the early diagnosis of ectopic pregnancy. The performance of this assay was evaluated using clinical serum and then compared with standard procedures. The sensitivity for β human chorionic gonadotropin detection was 1 U/L (linear dynamic range, 0–8000 U/L), and the sensitivity for progesterone detection was 0.05 ng/mL (linear dynamic range, 0–50 ng/mL). High correlation coefficients (R2) were obtained between the reported immunoassay and standard methods (R2 = 0.99 for β human chorionic gonadotropin, R2 = 0.97 for progesterone). The present dual-label TRFIA has high sensitivity, specificity, and accuracy in clinical analyses and is a suitable alternative to the single-labeled diagnostic methods.

Simultaneous detection of sulfamethazine and sulfaquinoxaline using a dual-label time-resolved fluorescence immunoassay

A dual-label time-resolved fluoroimmunoassay (TRFIA) was introduced for the simultaneous quantification of sulfamethazine (SM2) and sulfaquinoxaline (SQX). Lanthanide (Eu3+ and Sm3+)-labelled antibodies were used because lanthanides have higher stabilities and narrower emission spectra than most fluorescent dyes. The sensitivity of the TRFIA for SM2 was 0.02 ng ml−1, and the average recoveries and the intra- and inter-assay CVs were 77.2–107.6%, 5.4–10.5%, and 6.0–11.2%, respectively. The sensitivity of the TRFIA for SQX was 0.04 ng ml−1; and the average recoveries and the intra- and inter-assay CVs were 74.1–102.8%, 4.6–10.9%, and 8.7–11.2%, respectively. The method was used to analyse chicken tissue and egg samples, and the results agreed well with the results of HPLC and enzyme-linked immunosorbent assay (ELISA) analyses, with correlation coefficients (R2) of 0.9415–0.9724. The TRFIA developed is a simple, fast and sensitive method for the high-throughput simultaneous screening of SM2 and SQX in edible animal tissues.

Simultaneous detection of deoxynivalenol and zearalenone by dual-label time-resolved fluorescence immunoassay

The health risks of deoxynivalenol (DON) and zearalenone (ZEN) necessitate the development of analytical methods for widespread food and feed screening. We sought to establish a rapid, economic and sensitive dual-label time-resolved fluoroimmunoassay (TRFIA) to detect DON and ZEN simultaneously. Eu(3+)- and Sm(3+)-labelled antibodies were used, as lanthanides are more stable and have narrower emission spectra than most fluorescent dyes. The limit of detection was 0.0194 ng mL(-1) for DON (range: 0.0194-100 ng mL(-1)) and 0.37 ng mL(-1) for ZEN (range: 0.37-50 ng mL(-1)). DON recovery in spiked cereal samples was 88-107%, and for ZEN was 83-108%, with both intra- and inter-assay coefficients of variation (CVs) less than 5%. The dual-label TRFIA results correlated well with ELISA results (correlation coefficients: 0.9733 for DON and 0.9784 for ZEN). The dual-label TRFIA is a simple, fast and sensitive method for high-throughput screening of DON and ZEN in food and feedstuff.

Simultaneous Measurement of Plasma Concentrations of Proinsulin and C-Peptide and Their Ratio with a Trefoil-Type Time-Resolved Fluorescence Immunoassay

When the concentrations of 2 or more substances are measured separately, their molar ratios are subject to the additive imprecisions of the different assays. We hypothesized that the cumulative error for concentration ratios of peptides containing a common sequence might be minimized by measuring the peptides simultaneously with a "trefoil-type" immunoassay. As a model of this approach, we developed a dual-label time-resolved fluorescence immunoassay (TRFIA) to simultaneously measure proinsulin, C-peptide, and the proinsulin-C-peptide ratio (PI/C). A monoclonal antibody captures all C-peptide-containing molecules, and 2 differently labeled antibodies distinguish between proinsulin-like molecules and true C-peptide. The trefoil-type TRFIA was capable of measuring plasma C-peptide and proinsulin simultaneously without mutual interference at limits of quantification of 48 and 8125 pmol/L, and 2.1 and 197 pmol/L, respectively. Within-laboratory imprecision values for the trefoil-type TRFIA ranged between 8.4% and 12% for the hormone concentrations. Unlike the hormone results obtained with separate assays, imprecision did not increase when PI/C was calculated from trefoil assay results (P < 0.05). Peptide concentrations were highly correlated with results obtained in individual comparison assays (r(2) > or = 0.965; P < 0.0001). The total error for PI/C obtained with the trefoil-type TRFIA remained < or = 25% over a broader C-peptide range than with separate hormone assays (79-7200 pmol/L vs 590-4300 pmol/L C-peptide). Preliminary data indicate little or no interference by heterophile antibodies. The developed trefoil-type TRFIA is a reliable method for simultaneous measurement of proinsulin, C-peptide, and PI/C and provides proof of principle for the development of other trefoil-type multiple-label immunoassays.

Simultaneous detection of two verotoxin genes using dual-label time-resolved fluorescence immunoassay with duplex PCR.

Verotoxin (VT) produced by several Escherichia coli serotypes causes haemorrhagic colitis and has been associated with haemolytic uraemic syndrome in humans. Two types of verotoxin are known. Conventional diagnosis of verotoxin-producing Escherichia coli (VTEC) is conducted after isolation of bacteria from clinical specimens, followed by serological determination and identification of VTs. This method is complicated and time-consuming. Recently, rapid, direct immunological methods for identification of VTEC, i.e. immunochromatography and latex agglutination, have been developed. However, these techniques continue to suffer from limited sensitivity and a lack of specificity. These difficulties arise from the fact that the antibody used in these procedures reacts exclusively with the O157 antigen; moreover, VTEC strains with non-O157 antigens, such as O26, O103 and O111 antigens, exist. These VTEC groups did not react with anti-O157 antibody. Consequently, it is necessary to diagnose the VT gene in these bacteria. Therefore, we have designed a sensitive and specific method for the detection of two VT genes simultaneously, utilizing duplex PCR with time-resolved fluorescence immunoassay (TRFIA).