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Time resolved fluoroimmunoassay (TRFIA) is an immunoassay technology developed on the basis of the unique fluorescence properties of rare earth elements. TRFIA combines the advantages of radioimmunoassay, enzyme-linked immunoassay and common fluorescence immunoassay. It has high sensitivity, strong specificity, good stability, wider measurement range, long fluorescence life, simple operation and non-radiation, and shows a good prospect in the field of immunoassay. In this paper, several common TRFIA materials are discussed based on the latest research progress of time-resolved fluorescence in immunoassay. The application of TRFIA in immunodiagnosis, food detection, environmental monitoring and so on is elaborated, and its development direction and application are prospected.
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【Objective】 To investigate NAT non-reactive results implicated in HBsAg ELISA reactive voluntary blood donors in Shenzhen. 【Methods】 HBsAg ELISA+ but NAT-blood samples were collected, and HBsAg was further retested by TRFIA, Roche ECLIA and neutralization test. HBV DNA of individual donation was detected by commercial Roche MPX and Uultrio Elite, and virus nucleic acid was extracted via 2.5 mL. Molecular characterizations of HBsAg+ /NAT-samples were determined by quantitative polymerase chain reaction(qPCR) and nested PCR amplifification of the precore and core promoter regions and HBsAg(S) region. HBV serological markers were detected, and the samples with suspicious results were followed up and detected by multi-assay. 【Results】 Among 67 602 samples, 73(0.11%) HBsAg ELISA+ and NAT-blood samples were enrolled in the study. 15(20.5%, 15/73) were confirmed HBsAg+ by TRFIA, ECLI and five alternative DNA assays, and the other 2(2.7%, 2/73) were further identified as HBsAg+ by follow-up study. In 17 confirmed HBsAg+ samples, the viral loads ranged undetectable to 378 IU/mL, with the median of 10.1 IU/mL. Weak correlation was found between HBsAg and HBV DNA load(R2=0.394 4). 【Conclusion】 Some Hepatitis B virus infected blood samples may miss even with different HBsAg assays. Multi-assays with high sensitivity should be combined for blood screening to ensure blood safety..The inconsistent results should be followed up and further tested for hepatitis B serological markers to assist the confirmation.
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Objective To evaluation the performance of double antigen sandwich time-resolved fluoroimmunoassay(TRFIA)for specific total antibody to Treponema pallidum (TP).Methods Specific total antibody to TP was detected by a double antigen TRFIA based on recombinant multi epitope chimeric antigen.The methodological precision,low limit of detection,accuracy,linearity,reference standard coincidence rate and other analytical performance indicators were evaluated,and clinical comparison research trials were completed.The x2 test was used for the difference between two methods results,the P ＜0.05 which represents the difference was statistically significant.Results The intra-assay and inter-assay coefficients of var iation (CV) were both less than 10％ respectively.The low limit of detection was 0.05 mIU/ml.The relative deviation of de tecting the national standard was not exceed 10％.The linear range was 1.50～ 155.00 mIU/ml and the linear correlation co efficient could be reached 0.999 9.The performance of detection national reference could meet the national accreditation requirements.The consistent rate was 100 ％ when the TRFIA methodology detected the standardized serum plate.The parallel test of TRFIA and treponema pallidum gelatin agglutination test (TPPA) were completed,the total coincidence rate was 99.56％,and the Kappa index was 0.990 6.Conclusion Their result showed that the TRFIA methodology is high sensitivity,accuracy,wide linear range,and highly clinical coincidence rate,which is valuable for clinical application.
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Objective To detect the serum levels of calpainin (S100A11) using nanomagicbeabs sorting-time resolved fluoroimmuno assay (NMBS-TRFIA) and evaluate its diagnostic value in pancreatic carcinoma.Methods 88 patients with pancreatic carcinoma,50 patients with acute pancreatitis,10 patients with pancreatic cyst and 20 healthy controls were selected as the study subjects.The human peripheral serum blood was sorted with S100A11 antibody coupled nanomagicbeabs,and the concentration of S100A11 was detected by TRFIA method.The area under the receiver operating characteristic (ROC) curve (AUC) was used to determine the cut-off level for diagnosis of pancreatic carcinoma,in order to evaluate the sensitivity and specificity for diagnosis of pancreatic carcinoma.Results S100A11 showed a linear relationship within the range of 6.08～ 500 ng/ml using NMBS-TRFIA method,intraassay CV≤6.35％,inter-assay CV≤7.12％,and the average recovery rate was 104.7％.The serum levels of S100A11 in patients with pancreatic carcinoma,patients with acute pancreatitis and patients with pancreatic cyst were 185.53 ± 161.19,106.06±113.83 and 68.99± 47.83 ng/ml respectively.Compared with the normal control group (37.98±25.14 ng/ml),the differences were statistically significant (t=-8.065,-3.375,-2.266,all P ＜0.01).The serum levels of S100A11 in patients with pancreatic carcinoma was significantly higher than those in patients with acute pancreatitis and patients with pancreatic cyst (all P＜0.05).According to the ROC curve,ROCAUC=0.985 (95％ CI:0.972 ～ 0.997),the best cut-off level for the diagnosis of pancreatic carcinoma was 89.5 ng/ml (sensitivity 81.8 ％,specificity 67.5 ％).Conclusion NMBS-TRFIA can enrich S100A11 in serum and improve the detection sensitivity of serum S100A11,and the method is simple and easy to be popularized.Serum S100A11 has a high sensitivity and specificity in the diagnosis of pancreatic carcinoma,and is a new serum marker for the diagnosis of early pancreatic carcinoma.
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Objective To establish the time-resolved fluoroimmunoassay(TRFIA)of CA199 based on Mag-netic Microspheres(Nano-TRFIA).Methods Based on a sandwich-type immunoassay format,analytes in sam-ples were captured by magnetic particles coated with anti-CA199 antibody B1 and"sandwiched"by anti-CA199 antibody B7 labeled with europium chelates.A total of 90 serum samples were analysed by this new method. Results The sensitivity was 0.2 U/mL,the intra.and inter.assay CV of the Nano-TRFIA were 4.84% and 8.32% respectively,and the average recovery rate was 97.91%.The cross-reacting rates with alpha fetopro-tein and CA125 were negligible.The labeled B1 with Magnetic Microspheres was at least stable for three months at 4 ℃.Serum samples from patients and healthy blood donors were analyzed,the linear correlation of TRFIA and ECLIA measurements was positive(Y = 0.969 8X+ 4.015 3).As the gold standard of ECLIA, Nano-TRFIA had two false positive.Conclusion The newly developed Nano-TRFIA based on Magnetic Mi-crospheres technique was highly sensitive,stable and specific in the immuno-determination of serum CA199. The results showed that the methods of Nano-TRFIA based on Magnetic Microspheres could be used for the clinic.
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A novel polydimethylsiloxane ( PDMS)-poly-L-lysine ( PLL) microfluidic chip, comprised of 24 reaction channels with 2. 5 μL volume of each reaction channel only, was proposed for quantitative analysis of methamphetamine ( MET) based on time-resolved immunoassay techniques. The chip utilized the adsorption characteristics of PDMS and PLL towards proteins to immobilize MET complete antigens ( MET-BSA) on the surface of reaction channel. Then the competition reaction could happen between MET-Ag in the sample solution and MET-BSA on the inner surfaces of the reaction channels with MET-Ab in the reagent. The surface of latex microspheres was labeled by lanthanide, which could emit red fluorescence under the exposure of ultraviolet ( UV ) . Based on the principle of competitive immunoassay, the more MET-Ag, the less the fluorescence intensity in the reaction channel. The detection results of this chip were acquired using UV-irradiation fluorescence imaging method. With this method, 24 samples could be detected and analyzed simultaneously on a chip by just taking the fluorescence image of the chip. The method allows the detection of MET antigens ranging from 100 ng/mL to 3000 ng/mL, with less sample consumption and high-throughput. This chip is suitable for the police preliminary screening work and has a good application prospects.
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Objective To develop europium (Ⅲ) [Eu (Ⅲ)] chelated microparticles for homogeneous immunoassay.Methods Anti-human PCT antibodies were labeled with Eu (Ⅲ) chelated nanoscale microparticles as the detection antibody,and another anti-human PCT antibody was labeled with biotin as the solid-phase antibody.Magnetic microspheres labeled with streptavidin were used to separate the complexes of Eu-IgM-PCT-IgM-Biotin.Results In the homogeneous immunoassay,the standard curve fit was not linear.The quadratic curve was Y =19170.12 + 75493.74X-26.00X2(r =0.9986).According to the standard curve,the limit of detection for PCT was 0.04 ng/ml.Conclusion The homogeneous immunoassay which uses Eu (Ⅲ) chelated microparticles is highly sensitive for detection of PCT recombinant antigens and may serve as a promising method to measure serum PCT levels in the future.
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Objective To explore the clinical value of magnetic resonance time-resolved imaging of contrast kinetics (MR-TRICKS)in the assessment of lower extremity venous lesions in Klippel-Trenaunay syndrome (KTS).Methods Image data of 20 patients (total 21 affected limbs)diagnosed with KTS in our hospital were analyzed retrospectively.All patients underwent MR-TRICKS angiography and ascending phlebography of the lower extremities.The images acquired by these two methods were graded and compared.Kappa test was used to examine the consistency between the results of two inspection methods.Results The image quality of MR-TRICKS angiography and ascending phlebography graded on excellent proportion were 85.71％(18/21)and 80.95％(17/21)for the deep veins,95.24％(20/21)and 90.48％(19/21)for the superficial veins,and 90.48％(19/21)and 85.71％(18/21) for venous malformation,respectively.MR-TRICKS demonstrated that the deep veins were normal in 61.90％ (13/21),hypogenetic in 25.57％(7/21),atretic in 4.86％(1/21).Superficial vein diseases including varicosis of the great saphenous vein accounted for 25.57％(6/21), varicosis of the small saphenous vein accounted for 14.29％ (3/21 ),while marginal veins in venous malformation accounted for 100％ (21/21).Ascending phlebography of the lower limbs showed that the deep veins were normal in 57.14％(12/21)and dysplasia in 39.10％(8/21),while the other results were consistent with those of TRICKS.Consistency between results from the two methods were excellent (Kappa >0.75).Conclusion MR-TRICKS can accurately describe the angioarchitecture of the lower extremity vena in patients with KTS,and has an important clinical value in providing support for the assessment and treatment of KTS.
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Objective To explore the clinical value of magnetic resonance time-resolved imaging of contrast kinetics (MR-TRICKS)in the assessment of lower extremity venous lesions in Klippel-Trenaunay syndrome (KTS).Methods Image data of 20 patients (total 21 affected limbs)diagnosed with KTS in our hospital were analyzed retrospectively.All patients underwent MR-TRICKS angiography and ascending phlebography of the lower extremities.The images acquired by these two methods were graded and compared.Kappa test was used to examine the consistency between the results of two inspection methods.Results The image quality of MR-TRICKS angiography and ascending phlebography graded on excellent proportion were 85.71％(18/21)and 80.95％(17/21)for the deep veins,95.24％(20/21)and 90.48％(19/21)for the superficial veins,and 90.48％(19/21)and 85.71％(18/21) for venous malformation,respectively.MR-TRICKS demonstrated that the deep veins were normal in 61.90％ (13/21),hypogenetic in 25.57％(7/21),atretic in 4.86％(1/21).Superficial vein diseases including varicosis of the great saphenous vein accounted for 25.57％(6/21), varicosis of the small saphenous vein accounted for 14.29％ (3/21 ),while marginal veins in venous malformation accounted for 100％ (21/21).Ascending phlebography of the lower limbs showed that the deep veins were normal in 57.14％(12/21)and dysplasia in 39.10％(8/21),while the other results were consistent with those of TRICKS.Consistency between results from the two methods were excellent (Kappa >0.75).Conclusion MR-TRICKS can accurately describe the angioarchitecture of the lower extremity vena in patients with KTS,and has an important clinical value in providing support for the assessment and treatment of KTS.
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Purpose To establish a new method for detecting p16INK4a in cervical tissues with time-resolved fluoroimmunoassay (TRFIA).Methods 126 cases of paraffin imbedding tissues of cervix were selected for immunohistochemistry (IHC) of EnVision two-step and TRFIA.Results There were 20 cases of no intraepithelial lesion or malignancy,24 cases of low-grade squamous intraepithelial lesion (LSIL),53 cases of high-grade squamous intraepithelial lesion (HSIL) and 29 cases of squamous cell carcinoma (SCC).In the groups of no intraepithelial lesion or malignancy,LSIL,HSIL and SCC,p161NK4a positive was seen in 1,19,53 and 28,respectively.TRFIA test results displayed p16INK4a positive in 3,17,50 and 27 cases,respectively.Positive of p16 using by TRFIA in no intraepithelial lesion or malignancy,LSIL,above HSIL was 15.00％,70.83％ and 93.90％,respectively (P ＜ 0.01).Conclusion TRFIA is suitable for detecting of p16INK4a protein and demand low detection equipment,p16INK4a expression detected by TRFIA may helpful for large scale detection in various clinical institution.
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Using Eu3+as a tracer,a sandwich?type assay was established. HE4 in serum specimens from 225 patients were detected by TRFIA. Serum levels of HE4 in pelvic tumors were quantitatively analyzed. From receiver operating characteristic (ROC) curves,the reference values was calculated for endometrial cancer,cervical cancer and ovarian cancer,respectively. Results The working range of serum HE4 for TRFIA was 10~10000 pmol/L with a sensitivity of 7.5 pmol/L. The method offered less interaction with CA125 and CA199,and also provided a better correlation with ECLA,while the CV of intra?assay was below 10%. HE4 levels were significantly higher in endometrial cancer,cervical cancer and ovarian cancer groups than in healthy volunteer group,while did not differ significantly between uterine fibroid group and healthy women group. As aging,the risk for developing pelvic tumors increased. The area of ROC for malignant tumors was all above 0.5 and the accurate interpretation of HE4 was 60 pmol/L. Conclusion A stable,precise HE4 TRFIA is well established,which is helpful in diagnosis of gynecologic cancers.
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Objective To evaluate the accuracy and feasibility of time-resolved immunofluorometric assay (TRI FA) for detection of HBsAg based on Abbott automated chemiluminescence immunoassay(CMIA),so as to carry out this project in primary hospitals,and provide reference for individual antiviral strategy and prediction of therapeutic effect.Methods Serum of 157 patients infected with hepatitis B virus were detected with CMIA and TRIFA,specimens with HBsAg titers exceeding the detection limit were firstly diluted,then performed quantitative analysis.HBsAg levels were divided into 4 groups:≤100 IU/mL,101-1 000 IU/mL,1 001-20 000 IU/mL,and ＞ 20 000 IU/mL,quantitative correlation between two methods was analyzed.Results The linear regression equation of two methods was Y=2.323X-896.3,correlation coefficent r=0.943,P＜0.001.CMIA was as a reference,4 groups were divided for analysis,results showed that when detected specimens was at low concentration of HBsAg,TRIFA value was low compared with CMIA method,while detected specimens was at high concentration of HB sAg,CMIA value was high,two reagents had good consistency in the detection of different concentrations of HBsAg(both P＜0.05),when concentration was at 1 001-20 000 IU/mL,consistency was the best.Conclusion The accuracy of two reagents in the quantitative detection of HBsAg is similar,and the best correlation of detection value is 1 000-20 000 IU/mL.TRIFA assay has wide application for its low-cost and easy to be operated,which is especially suitable for primary hospitals.
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Objective To investigate the effect of different degree lipidemia on time-resolved fluoroimmunoassay(TRFIA)for determination of unconjugated estriol(uE3).Methods Mixed serum was prepared by collecting different levels of lipidemia samples which were normal and chylous appearance from male and by mixing with definite value serum of uE3.The levels of uE3 in the sam-ples were measured by TRFIA and the effect of lipidemia on TRFIA for determination of uE3 was evaluated.Results For the ap-pearance of chylous specimens,mild lipidemia increased uE3,mid-or hiper-lipidemia samples reduced uE3 and the effect of both was considerable.Conclusion The chylous lipidemia has variant degree of influence to TRFIA for determination of uE3,then the results effect accuracy of Down′s screening.
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Objective To develop a kit of time?resolved fluoroimmunoassay(TRFIA)for detection of Schistosoma japonicum protein SjP38,and evaluate its effectiveness. Methods The anti 9G7 SjP38 monoclonal antibody was used as the capture anti?body coated with 96?hole plate,and the Eu3+labeled 1A6 monoclonal antibody was used as the detection antibody to establish the TRFIA SjP38 kit. In addition,the accuracy,sensitivity,precision,stability and coincidence rate to pathogenic diagnosis of the kit were evaluated. Results This established kit possessed high accuracy,wide linear range from 2 to 1 250 ng/ml,high sensitivity with the minimum detectable concentration of 0.14 ng/ml,and good precision(the coefficient variation of the intra?and inter?assay were 3.6%to 4.6%and 5.1%to 6.7%,respectively). The stability tests showed that the reagents could be stable for six months at 4℃,7 d at 37℃. The positive and negative corresponding rates to the pathogen detection method were 95%and 100%respectively. Conclusion All the performance and detection indicators of the kit have reached the requirements of clinical test,but its clinical application still needs further validation.
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HSP90 is widely expressed in cells with the main function in assisting the maturation of other proteins that are called clients. Many clients play critical roles in the occurrence and development of cancer. Inhibition of HSP90 can lead to degradation of the oncogenic proteins, and result in potent anti-cancer effects. HSP90-HOP interaction is critical for the chaperone function of HSP90, thereby disruption of the HSP90-HOP interaction is a novel strategy in the inhibition of HSP90. Based on the technology of homogeneous time-resolved fluorescence (HTRF), we developed a new assay for the identification of new inhibitors of HSP90-HOP interaction. This method was evaluated in the study of the HSP90-HOP inhibition activity of the pentapeptide MEEVD from HSP90 C-terminal and its derivatives. This study can provide a basis for the screening and discovery of novel HSP90-HOP disruptors.
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To detect furaltadone metabolite 3-amino-5-morpholinomethyl-2-oxazolidinone ( AMOZ ) in fish sample, an Eu3+ labeling time-resolved fluoroimmunoassay ( TRFIA ) was developed. The effects of experimental conditions including AMOZA-OVA concentration, dilution of antibody, and reaction time on the sensitivity of TRFIA were explored. The results showed that the optimized assay conditions were as follows:the AMOZA-OVA concentration was 0. 25 μg/mL; the antibody was diluted 5í104 folds, and the competitive reaction time was 50 min. Under optimal conditions, the method showed a detection limit of 0. 01 ng/mL, an IC50 of 0. 26 ng/mL and a linear range (IC20-IC80) of 0. 025-2. 83 ng/mL. The recoveries of AMOZ in fish at three spiked levels ranged from 78 . 0% to 86 . 0%, and the relative standard deviations were less than 15%. Good correlation between the ic-TRFIA and high performance liquid chromatography-tandem mass spectrometry was obtained for spiked food samples. The proposed ic-TRFIA method was suited for the determination of AMOZ residue in food samples.
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Objective To compare the advantages and disadvantages of 3D BRAVO and TRICKS for detecting cerebral venous sinus thrombosis (CVST) and to explore the MR scanning methods on CVST.Methods A retrospective analysis was conducted on 40 patients who were diagnosed with CVST in the Fourth Affiliated Hospital of Kunming Medical University from January 2011 to June,2014.All the patients underwent TRICKS scan and 3D BRAVO examination and were classified into 3 groups based on dural venous sinuses,deep cerebral vein and superficial cerebral vein.Detection rates of TRICKS and BRAVO were evaluated and the results were analyzed.Result Detection rates of dural venous sinuses by TRICKS and BRAVO were 98.0％ and 100％ respectively.Detection rates of deep cerebral vein were 92.9％ and 100％ respectively.No significant difference was found (P＞0.05).Detection rates of superficial cerebral vein were 66.7％ and 100％ respectively.Significant difference was found between the two (P＜0.05) Conclusion BRAVO technology can clearly demonstrate the details of CVST,especially in superficial cerebral vein.With the combination use of TRICKS sequences,it will be of higher diagnostic value.
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Objective To theoretically study the non-invasive measurement method of blood hemoglobin in vivo based on the time-resolved approach combined with occlusion spectroscopy.Methods The dynamic dual wavelength time-resolved transmission was analyzed based on the artificial blood flow kinetics condition on a human finger model.The sensitivity of hemoglobin measurement was improved by Laplace transforming of time-domain data.The method was validated using hemoglobin with a mass concentration range of 6～16 g/dl.Results The simulation results showed that compared with the continuous wave method,the time-resolved method could provide higher detection sensitivity using Laplace transform parameter p =5 × 1010 s-1.Conclusions The sensitivity of hemoglobin measurement can be enhanced when early arriving photons are emphasized by Laplace transformingthe time-resolved transmission with a small positive parameter.
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Objective To explore significance of alpha‐fetoprotein isoform L2(AFP‐L2) in the screening of Down syndrome in pregnant women ,so as to provide references for clinical application .Methods A total of 250 healthy pregnant women and 22 preg‐nant women with Down syndrome were enrolled in this study .Serum specimens were collected and AFP‐12 was separated and cap‐tured by using the magnetic bal ,time‐resolved fluorescence immunoassay was used to detect levels of AFP and AFP‐L2 ,and the percentage of AFP‐L2 (AFP‐L2% ) was calculated .Results The serum level of AFP of pregnant women with Down syndrome [(20.2±4.2)ng/mL]was lower than that of healthy pregnant women[(46.7±19.9)ng/mL],and had statistically significant difference(P<0 .05) .Serum AFP‐L2% of pregnant women with Down syndrome was higher than that of healthy pregnant women , and had statistically significant difference(P<0 .05) .Conclusion Detection of AFP level and AFP‐L2% could be an indicator for Dow n syndrome screening .
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A new type of surface plasmon resonance ( SPR) spectroscopy system was designed and built. Here, a kind of dual photocell sensor was developed as a detection device to achieve a rapid measurement of SPR angle within a certain range. This SPR system was combined and integrated with electrochemical workstation to obtain a new type of electrochemistry-time-resolved SPR ( EC-TR-SPR ) instrument via instrumental technique. This EC-TR-SPR instrument was used to characterize the electrochemical polymerization process of aniline to validate the spectroscopic characteristics. Applications of transient electrochemical characterization methods, including chronoamperometry and differential pulse voltammetry, confirmed the time resolution and the applicability of this instrument system toward the steady state and transient electrochemical methods upon small molecular reactions. The experiment results showed that this EC-TR-SPR possessed the time resolution up to 10000 times per second (0. 1 ms), and could be used to real-time investigate the doping and de-doping of polymerization process of aniline monomer as well as the prepared polyaniline film, which could not be discriminated on a conventional electrochemical current-time curve. .