Time-resolved Fluorescence Immunoassay Research Articles

Commercially Bottled Purified Water as an Alternative Instrument Feed Water in Automated Time-Resolved Fluorescent Immunoassay for TSH, 17-OHP and IRT in Neonatal Screening

Objective. The study was undertaken to determine if commercially bottled purified water can be used as substitute instrument feed water for three (3) newborn screening immunoassays. Methdology. A total of 294 control samples and 300 patient samples were included in this study. Accuracy and precision studies using control samples, and parallel testing using patient samples, were done to compare the use of clinical laboratory reagent water (CLRW) and commercially bottled purified water (CBPW) in the performance of automated time-resolved fluorescent immunoassay of thyroid stimulating hormone (TSH), 17α-OH-progesterone (17-OHP) and immunoreactive trypsinogen (IRT). Results. The use of CBPW as instrument feed water for measurements of TSH, 17-OHP and IRT levels by automated time-resolved fluorescent immunoassay using AutoDELFIA (Perkin-Elmer) in NBS has an acceptable accuracy and precision compared to using CLRW. The parallel testing using patient samples showed that, overall, the performance of using CBPW in automated time-resolved fluorescent immunoassay for TSH, 17-OHP, and IRT is acceptable, compared with using CLRW as instrument feed water. Conclusion. Commercially bottled purified water can be used as substitute when setting up a laboratory water purification system is too expensive for a laboratory, or as back up to clinical laboratory reagent water when there is breakdown of the installed water purification system to be used as instrument feed water in automated time-resolved fluorescent immunoassay of TSH, 17-OHP and IRT in NBS using AutoDELFIA (Perkin-Elmer).

Time-resolved fluorescent immunoassay-based combined detection of procalcitonin, C-reactive protein, heparin binding protein, and serum amyloid A1 to improve the diagnostic accuracy of early infection.

Serological tests are indispensable in the diagnosis of early infection. At present, only procalcitonin (PCT) and C-reactive protein (CRP) are commonly used in clinical practice. Recently, serum amyloid A1 (SAA1) and heparin binding protein (HBP) have been shown to be new biomarkers, because SAA1 is highly sensitive and specific for viral infections, and HBP is predictive for septic shock. In this study, PCT, CRP, HBP, and SAA1 were detected in different combinations to improve the diagnostic accuracy of early infection using the biotin-avidin amplifying system-based time-resolved fluorescent immunoassay (BA-TRFIA). A time-resolved fluorescent immunoassay for PCT, CRP, HBP, and SAA1 was developed and then tested in a clinical setting. All experiments were carried out using the DR6608 time-resolved fluorescent immunoassay analyzer. The cutoff values of PCT, CRP, HBP, and SAA1 were 0.05μg/L, 5.59mg/L, 3.83μg/L, and 1.56mg/L, respectively. The area under the ROC curve (AUC) showed that PCT˃SAA1˃CRP˃HBP>0.8. A methodological comparison of the results showed that a combination of the four biomarkers had the highest accuracy for the diagnosis of infectious diseases. The time-resolved fluorescent immunoassay-based combined detection of PCT, CRP, HBP, and SAA1 was shown to significantly improve the diagnostic accuracy of early infection. Thus, our results indicate that combined detection based on BA-TRFIA may represent a promising strategy in the clinical diagnosis of infection.

Eu3+ /Sm3+ dual-label time-resolved fluoroimmunoassay for measurement of hepatitis C virus antibodies.

To develop a new immunoassay based on the time-resolved fluorescence immunoassay (TRFIA) system for the simultaneous measurement of IgM and IgG antibodies to HCV. Coated recombinant HCV antigens and Eu3+ -labeled IgM and Sm3+ -labeled IgG antibodies were prepared. HCV-IgM/IgG TRFIA was established and optimized, followed by a methodological assessment. Data were expressed as +SD and analyzed using the SPSS 13.0 software. The percentile method was used to calculate cutoff values. The detection sensitivities of HCV-IgM and HCV-IgG were 0.06 S/CO and 0.15 S/CO, respectively. There was a good linear response from 1:40 to 1:40960 for HCV-IgM and 1:20 to 1:40960 for HCV-IgG, when samples strongly positive for HCV-IgM and HCV-IgG were serially diluted from 1:10 to 1:81920. The average intra-assay coefficients of variation (CV) for HCV-IgM and -IgG were 3.45% and 3.71% and the inter-assay coefficients of variation (CV) were 6.49% and 6.79%, respectively. When HCV-negative/positive sera were tested by ELISA and using established kits, the negative, positive, and total conformity rates for HCV-IgM were 93.3% (28/30), 100% (25/25), and 96.4% (53/55), and those for HCV-IgG were 93.3% (28/30), 100% (35/35), and 96.9% (63/65), respectively. Additionally, the established kit exhibited good stability, with declines in fluorescence values to 11.1% and 9.5%, respectively, after storage at 37°C for 7days. We established a dual-label HCV-IgM/IgG TRFIA assay with a wide detection range, high specificity, high sensitivity, good stability, and good clinical value for the simultaneous measurement of HCV-IgM and HCV-IgG titers in a single test.

Ratiometric quantification of β2-microglobulin antigen in human serum based on elemental labeling strategy

Ratiometric quantification for competitive immunoassay based on internal standard element detection utilizing inductively coupled plasma mass spectrometry (ICP-MS) as multiplex readout has been demonstrated. The Beta-2-microglobulin (β2-MG) associated with clinical diseases was detected by Y-labeled capture antibody used as internal standard probes and Sm-labeled antigen used as report probes via antigen-antibody reaction. The ratiometric quantification was achieved by taking the signal ratio of Sm/Y. The ratiometric method could compensate for the particle loss and suppress the signal fluctuation, which improved the precision of the quantitative result. Under the optimized conditions, the calibration curves for β2-MG antigen was linear in the range of 0.25–8.0 μg/mL with a detection limit of 0.17 μg/mL (3σ, n = 11). The recoveries of 96.5%–132% were obtained for serum samples spiked with different concentration standards, and the relative standard deviation (RSD) was less than 10%. The β2-MG results in serum samples obtained by the proposed method correlated well with those obtained by time-resolved fluorescence immunoassay (r = 0.947). This proposed method provides a simpler labeling strategy for ratiometric quantification of immunoassay.

A Time-Resolved Fluorescence Immunoassay of Golgi Protein 73 and Its Application in Hepatitis B Virus Related Liver Cirrhosis

A Time-Resolved Fluorescence Immunoassay of Golgi Protein 73 and Its Application in Hepatitis B Virus Related Liver Cirrhosis

Development of Time-Resolved Fluoroimmunoassay for Detection of Cylindrospermopsin Using Its Novel Monoclonal Antibodies

Cylindrospermopsin (CYN) is a cyanotoxin that is of particular concern for its potential toxicity to human and animal health and ecological consequences due to contamination of drinking water. The increasing emergence of CYN around the world has led to urgent development of rapid and high-throughput methods for its detection in water. In this study, a highly sensitive monoclonal antibody N8 was produced and characterized for CYN detection through the development of a direct competitive time-resolved fluorescence immunoassay (TRFIA). The newly developed TRFIA exhibited a typical sigmoidal response for CYN at concentrations of 0.01–100 ng mL−1, with a wide quantitative range between 0.1 and 50 ng mL−1. The detection limit of the method was calculated to be 0.02 ng mL−1, which is well below the guideline value of 1 μg L−1 and is sensitive enough to provide an early warning of the occurrence of CYN-producing cyanobacterial blooms. The newly developed TRFIA also displayed good precision and accuracy, as evidenced by low coefficients of variation (4.1–6.5%). Recoveries ranging from 92.6% to 108.8% were observed upon the analysis of CYN-spiked water samples. Moreover, comparison of the TRIFA with an ELISA kit through testing 76 water samples and 15 Cylindrospermopsis cultures yielded a correlation r2 value of 0.963, implying that the novel immunoassay was reliable for the detection of CYN in water and algal samples.

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We attempted to evaluate maternal thyroid function in a new self-sequential longitudinal reference interval (SLRI) which we established recently. By this method, we analysed the correlation between pregnancy outcome, neonatal thyroid stimulating hormone (TSH) level and maternal thyroid diseases. A total of 1744 pregnant women participated in the study and 1747 babies were born from those women (three bore twins). The levels of TSH, free thyroxine (FT4) and thyroid peroxidase antibodies (TPO-Ab) of mothers were quantified by electrochemistry immunoassay (ECL). The levels of neonatal blood TSH were detected by time-resolved fluorescence immunoassay (TRFIA). All data were collected and statistically analysed by SPSS 13.0 software. With our new SLRI method, we found that 0.11%-3.84% pregnant women would get thyroid diseases. Subclinical hypothyroidism was the most common maternal thyroid disorder. Being positive for thyroid peroxidase antibodies was a significant risk factor of subclinical hypothyroidism during pregnancy. The median, P2.5-P97.5, and interquartile range (IQR) of neonatal TSH (N-TSH) of 1747 babies were 2.72 mIU/L, 0.10-8.01 mIU/L and 2.62 mIU/L, respectively; 28.6% of pregnant women with thyroid diseases developed pregnancy complications. The prevalence was significantly higher than in the normal thyroid function group (p < 0.001). The levels of N-TSH were low correlated with maternal TSH levels (p < 0.05), but there were no significant correlations between N-TSH and maternal FT4 and maternal TPO-Ab (p > 0.05). Thyroid disorders, especially subclinical hypothyroidism, are common in pregnant women. These disorders are associated with pregnancy and fetal outcome. Routine maternal thyroid function screening is important and should be recommended.

Ultrasensitive detection of Shiga toxin 2 and its variants in Shiga toxin-producing Escherichia coli strains by a time-resolved fluorescence immunoassay.

A rapid and sensitive two-step time-resolved fluorescence immunoassay (TRFIA) was developed for the detection of Shiga toxin 2 (Stx2) and its variants in Shiga toxin-producing Escherichia coli (STEC) strains. In sandwich mode, a monoclonal antibody against Stx2 was coated on a microtiter plate as a capture antibody. A tracer antibody against Stx2 labeled with europium(III) (Eu3+ ) chelate was then used as a detector, followed by fluorescence measurements using time-resolved fluorescence. The sensitivity of Stx2 detection was 0.038ng/ml (dynamic range, 0.1-1000ng/ml). The intra- and inter-assay coefficients of variation of the assay were 3.2% and 3.6%, respectively. The performance of the established assay was evaluated using culture supernatants of STEC strains, and the results were compared to those of a common HRP (horseradish peroxidase) labeling immunosorbent assay. A polymerase chain reaction (PCR) for the detection of genes encoding Stx1 and Stx2 was used as the reference for comparison. Correlation between the Stx2-specific TRFIA and PCR was calculated by the use of kappa statics, exhibiting a perfect level of agreement. The availability of the sensitive and reliable Stx2-specific TRFIA method for quantifying Stx2 and its variants in STEC strains will complement bacteria isolation-based platform and aid in the accurate and prompt diagnosis of STEC infections.

Fluorescence ELISA based on glucose oxidase-mediated fluorescence quenching of quantum dots for highly sensitive detection of Hepatitis B

Herein, we present a novel sandwich fluorescence enzyme linked immunosorbent assay (ELISA) for highly sensitive detection of Hepatitis B virus surface antigen (HBsAg) based on glucose oxidase (GOx)-induced fluorescence quenching of mercaptopropionic acid-modified CdTe quantum dots (MPA-QDs). In this system, hydrogen peroxide (H2O2) sensitive MPA-QDs was used as a signal output, and glucose oxidase (GOx) was used as label which can generate H2O2 via catalytic oxidation of glucose. The proposed method showed dynamic linear detection of HBsAg both in the range of 47pgmL−1 ~ 380pgmL−1 and 0.75ngmL−1 ~ 12.12ngmL−1. The detection limit of the proposed fluorescence ELISA was 1.16pgmL−1, which was approximately 430-fold lower than that of horseradish peroxidase (HRP)-based conventional ELISA. The average recoveries for HBsAg-spiked serum samples ranged from 98.0% to 126.8% with the relative standard derivation below 10%, thus indicating acceptable precision and high reproducibility of the proposed fluorescence ELISA for HBsAg detection. Additionally, the developed method showed no false positive results analyzing 35 real HBsAg-negative serum samples, and exhibited excellent agreement (R2=0.9907) with a commercial time-resolved fluorescence immunoassay (TRFIA) kit for detecting 31 HBsAg-positive serum samples. In summary, the proposed method based on fluorescence quenching of H2O2 sensitive QDs is considerably to be an excellent biodetection platform with ultrahigh sensitivity, good accuracy and excellent reliability.

Simple and rapid monitoring of doxorubicin using streptavidin-modified microparticle-based time-resolved fluorescence immunoassay.

Developing a simple analytical method suitable for therapeutic drug monitoring in a clinical setting is key to establishing guidelines on accurate dose administration and the advancement of precision medicine. We devised a simple rapid analytical method through the combination of streptavidin-modified microparticles and a time-resolved fluorescence immunoassay for therapeutic drug monitoring. The analytical performance of this method was investigated and validated using clinical samples. By determination of doxorubicin concentration, the proposed assay has shown a satisfactory linear range of detection (3.8–3000 ng mL−1) with a limit of detection of 3.8 ng mL−1 and an IC50 of 903.9 ng mL−1. The intra and inter-assay coefficients of variation were 4.12–5.72% and 5.48–6.91%, respectively, and the recovery was acceptable. The applicability of the proposed assay was assessed by comparing the determined results with those measured by LC-MS/MS, presenting a satisfactory correlation (R2 = 0.9868). The proposed assay, which shows satisfactory analytical performance, has great potential for application in the field of TDM in the future.

Relationships of plasma insulin-like peptide 3, testosterone, inhibin, and insulin-like growth factor-I concentrations with scrotal circumference and testicular weight in Japanese Black beef bull calves.

This study was conducted to clarify the relationships of plasma concentrations of insulin-like peptide 3 (INSL3), testosterone, inhibin, and insulin-like growth factor-I (IGF-I) with scrotal circumference and testicular weight in Japanese Black beef bull calves (n = 20), from birth to pre-puberty. Monthly blood sampling (0 to 7 months) and scrotal circumference measurements (0 to 7 months) were performed. Testicular weight was recorded immediately after castration at 7 months. Plasma INSL3, testosterone, inhibin, and IGF-I concentrations were measured either by enzyme immunoassay or time-resolved fluorescence immunoassay. The correlation coefficients of these hormonal concentrations with scrotal circumference were significant (P < 0.0001) and it was higher for INSL3 (r = 0.647) than for testosterone (r = 0.597), IGF-I (r = 0.400), and inhibin (r = –0.453). Calves with heavier testes (> 60 g) at castration (7 months) had higher (P < 0.05) plasma INSL3 (from 3 to 7 months) and inhibin (from 1 to 4 months) concentrations than those with lighter testes (< 60 g). The calves with heavier testes at castration had larger (P < 0.05) scrotal circumference than those with lighter testes from 3 to 7 months. In conclusion, blood INSL3 concentrations may be the best functional indicator among the hormones analyzed for determining total testicular volume during pre-puberty in bull calves. In addition, inhibin and INSL3 concentrations in early calfhood may be functional predictors for testicular weight at pre-puberty.

A Homogeneous Time-Resolved Fluorescence Immunoassay Method for the Measurement of Compound W

Objective:Using compound W (a 3,3′-diiodothyronine sulfate [T2S] immuno-crossreactive material)-specific polyclonal antibodies and homogeneous time-resolved fluorescence immunoassay assay techniques (AlphaLISA) to establish an indirect competitive compound W (ICW) quantitative detection method.Method:Photosensitive particles (donor beads) coated with compound W or T2S and rabbit anti-W antibody were incubated with biotinylated goat anti-rabbit antibody. This constitutes a detection system with streptavidin-coated acceptor particle. We have optimized the test conditions and evaluated the detection performance.Results:The sensitivity of the method was 5 pg/mL, and the detection range was 5 to 10 000 pg/mL. The intra-assay coefficient of variation averages <10% with stable reproducibility.Conclusions:The ICW-AlphaLISA shows good stability and high sensitivity and can measure a wide range of compound W levels in extracts of maternal serum samples. This may have clinical application to screen congenital hypothyroidism in utero.

Ultra-sensitive method based on time-resolved fluorescence immunoassay for detection of sulfamethazine in raw milk

ABSTRACT A novel lateral flow assay (LFA) was developed by introducing Eu (III)-doped polystyrene nanoparticles (EuNPs) for rapid and ultra-sensitive detection of sulfamethazine (SM2) in raw milk. The limit of detection and linear range of the proposed method were 0.0045 and 0.05–10 ng/mL, respectively. The recovery of LFA for the detection of SM2 in raw milk was 96.1–108.2%. The proposed LFA provides a rapid and convenient strategy for fast and ultra-sensitive screening of SM2 in raw milk. EuNP-LFA may be a remarkable method for the detection of other targets at low concentrations to ensure food safety.

Sample processing approach for detection of ricin in surface samples

With several ricin contamination incidents reported over the past decade, rapid and accurate methods are needed for environmental sample analysis, especially after decontamination. A sample processing method was developed for common surface sampling devices to improve the limit of detection and avoid false negative/positive results for ricin analysis. Potential assay interferents from the sample matrix (bleach residue, sample material, wetting buffer), including reference dust, were tested using a Time-Resolved Fluorescence (TRF) immunoassay. Test results suggested that the sample matrix did not cause the elevated background fluorescence sometimes observed when analyzing post-bleach decontamination samples from ricin incidents. Furthermore, sample particulates (80mg/mL Arizona Test Dust) did not enhance background fluorescence or interfere with ricin detection by TRF. These results suggested that high background fluorescence in this immunoassay could be due to labeled antibody quality and/or quantity issues. Centrifugal ultrafiltration devices were evaluated for ricin concentration as a part of sample processing. Up to 30-fold concentration of ricin was observed by the devices, which serve to remove soluble interferents and could function as the front-end sample processing step to other ricin analytical methods. The procedure has the potential to be used with a broader range of environmental sample types and with other potential interferences and to be followed by other ricin analytical methods, although additional verification studies would be required.

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.

Development of a Time-Resolved Fluorescence Immunoassay for the Diagnosis of Hepatocellular Carcinoma Based on the Detection of Glypican-3.

Glypican-3(GPC3), an oncofetal protein, is a potential novel marker for hepatocellular carcinoma (HCC). In this study, we attempted to establish a new method to detect serum GPC3 using the antibodies identified in our previous research, and then evaluated its clinical application for the diagnosis of HCC. Herein, a sandwich time-resolved fluorescence immunoassay (TRFIA) for detecting serum GPC3 was developed. The detection limit, analytical recovery, specificity and precision of the proposed TRFIA assay were satisfactory. A total of 415 patients were collected and divided into seven groups: hepatocellular carcinoma (101), colorectal cancer (67), gastric cancer (44), esophageal cancer (15), cirrhosis (55), hepatitis (61), normal liver (72). Using this proposed method, the concentration of serum GPC3 in these clinical samples was detected. The results demonstrated that the levels of GPC3 in serum from HCC patients were significantly higher than that in others. Compared with the results of chemiluminescence immunoassay (CLIA), a high consistency (Kappa =0.84) was observed. Thus, an effective, sensitive and reliable TRFIA-GPC3 kit for diagnosing HCC was successfully developed. It offers a suitable alternative to existed methods of determining GPC3 and is expected to be used in clinic in the future.

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.

Changes of plasma concentrations of insulin-like peptide 3 and testosterone, and their association with scrotal circumference during pubertal development in male goats

Insulin-like peptide 3 (INSL3) has been used as a testis-specific biomarker for puberty in several species, but the secretory profile of INSL3 during pubertal development in small ruminants is unknown. Here we sought to determine the age-related changes in the plasma concentrations of INSL3 and testosterone and their association with scrotal circumference during pubertal development in five male Shiba goats. Blood samples and scrotal circumference measurement were taken every 2 weeks from week 10 to week 52 of each goat's lifespan. Based on the changes in scrotal circumference, data were grouped into early pubertal (10–22 weeks), late pubertal (22–34 weeks) and post-pubertal (34–52 weeks) categories. The plasma concentrations of testosterone and luteinizing hormone (LH) were measured by enzyme-immunoassays (EIAs), and we used a time-resolved fluorescence immunoassay (TRFIA) to measure plasma INSL3. The biweekly sampling showed that the plasma INSL3 secretions maintained a moderate increase during and after puberty, whereas the plasma testosterone secretions fluctuated over the same period. The comparison of the three age categories revealed a significant increase (p < 0.01) in the mean plasma INSL3 concentrations during the late and post-pubertal periods compared to the early pubertal period. There was no difference in the mean plasma testosterone concentrations between the early and late pubertal periods, but a significant increase (p < 0.01) was observed during the post-pubertal period compared to early and late pubertal periods. The mean plasma LH concentrations increased significantly (p < 0.05) from the early pubertal to late pubertal and from the late pubertal to post-pubertal periods. A significant increase (p < 0.05) in the mean scrotal circumference from the early pubertal to late pubertal and from the late pubertal to post-pubertal periods was observed. The R2 value of the best regression curves between scrotal circumference and INSL3 (0.513; p < 0.001) was higher than that between scrotal circumference and testosterone (0.162; p < 0.01) from 10 to 52 weeks of age. In conclusion, in male goats, plasma concentrations of INSL3 increased continuously during and after puberty, whereas testosterone secretions were fluctuated. The scrotal circumference was more highly correlated with the INSL3 concentrations than with testosterone, implying that INSL3 is superior as a biomarker of testicular total Leydig cell volume.

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.