Homogeneous time-resolved fluoroimmunoassay of thyroxin in serum

We describe a rapid, simple nonseparation fluoroimmunoassay for determination of thyroxin in serum. The assay is based on the labeling of thyroxin directly with a fluorescent europium chelate, the fluorescence of which is quenched on binding to an antithyroxin antibody. With the assay buffer we used, maximum quenching is 90%. The rapid achievement of equilibrium in the assay solution, regardless of the sequence of reagent additions, allows fast measurement of thyroxin. Precision was good (CV less than 5%) within the clinical range for total thyroxin (50-300 nmol/L), and results correlated well with those by a commercial radioimmunoassay.     

Direct measurement of antigens in serum by time-resolved fluoroimmunoassay

A long-lived fluorescence label (Tb3+) has been attached to the antigen of interest by using a bifunctional chelating agent 1-(p-benzenediazonium)-EDTA. A nonequilibrium competitive-binding immunoassay protocol, in conjunction with time-resolved detection of the long-lived fluorescence label, allows the antigen to be analyzed directly in samples containing diluted human serum. Results obtained for immunoglobulin G with this simple and rapid procedure correlated well (r = 0.93) with those by a commercially available fluorescence immunoassay method.     

Determination of desmosine in bronchoalveolar lavage fluids by time-resolved fluoroimmunoassay

BACKGROUND: Urinary excretion of desmosine has been reported to be increased in patients with pulmonary fibrosis; however, several investigators have pointed out that measuring urinary desmosine is not a very useful indicator of lung wall destruction. We developed a sensitive time resolved fluoroimmunoassay (TR-FIA) to identify trace amounts of desmosine in bronchoalveolar lavage fluid (BALF), and applied this method to analyse BALF samples from healthy subjects and patients with interstitial lung diseases. METHODS: In the proposed TR-FIA, a polystyrene strip was coated with desmosine-conjugated gelatin. The strip was then incubated with rabbit anti-desmosine antibody and the test solution. The desmosine bound to the solid phase and free desmosine in the sample or standard solution were allowed to compete to bind to the anti-desmosine. The solid-phase antibody was detected by Eu-complex conjugated anti-rabbit IgG. RESULTS: The detectable limit of desmosine was 50 fmol/ml in the TR-FIA developed in this study. TR-FIA showed low cross-reactivity against amino acids. BALF desmosine levels were significantly higher in patients with idiopathic fibrosis and sarcoidosis compared with healthy subjects. CONCLUSIONS: Desmosine levels in BALF may be useful to investigate lung disease.     

Assessment of a time-resolved fluoroimmunoassay for thyrotropin in routine clinical practice

We assessed the use of a dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA) system to measure thyrotropin (thyroid-stimulating hormone, TSH) in routine clinical practice. The assay is simple and precise, with intrabatch CV of less than 10% down to 0.1 milli-int. unit/L. When compared with free thyroxin, total thyroxin, and triiodothyronine measurements in 142 patients, the present assay most sensitively indicated hyperthyroidism and, in conjunction with free thyroxin, most sensitively indicated hypothyroidism. Free thyroxin was the most specific assay (lowest number of falsely increased or decreased results) in detecting thyroid disorders, with a specificity of 93.6% as compared with 85.1% for TSH, 81.9% for thyroxin, and 77.6% for triiodothyronine.     

时间分辨荧光免疫法定量测定孕妇血清中甲胎蛋白含量

目的 探讨孕妇血清甲胎蛋白（MSAFP）含量与孕周、孕妇体质量和孕妇年龄的关系.方法 自建MSAFP时间分辨荧光免疫测定法（TRFIA）并对其进行评价,采用自建方法定量检测孕妇血清3711例,建立实验室参考值,分析AFP浓度与孕周、孕妇体质量和孕妇年龄的关系.结果 自建AFP-TRFIA方法灵敏度优于1.0 U/ml,线性范围为1.00～1000.00 U/ml,分析内、分析间变异系数（CV）分别不超过10.0%、15.0%,与国外同类试剂比对结果相关性好（r=0.9856）.在13～13周＋6d,14～14周＋6d,15～15周＋6d,16～16周＋6d,17～17周＋6d,18～18周＋6d,19～19周＋6d,20～20周＋6d,21～21周＋6d各孕周的MSAFP含量中位数（M）分别为16.51 U/ml,23.81 U/ml,29.98 U/ml,32.96 U/ml,36.86 U/ml,43.01 U/ml,49.61 U/ml,59.66 U/ml,71.11 U/ml.各孕周MSAFP平均浓度或浓度中位值与孕周呈线性正相关,r分别为0.9766,0.9832.各体质量段MSAFP浓度与孕妇体质量呈负相关,r在-0.9929～-0.9142.同孕周组内,各年龄段间的MSAFP浓度均数差异无统计学意义（P〉0.05）.结论 自建AFP-TRFIA法检测性能能满足临床应用的需要.MSAFP浓度与孕周和孕妇体质量密切相关,在产前筛查时要准确校对以上因素.     

时间分辨免疫荧光法检测乙型肝炎病毒血清标志物的研究

目的：探讨时间分辨免疫荧光法（TRFIA）检测乙型肝炎病毒（HBV）血清标志物的准确性及意义。方法选取2011年1月至2013年1月在该院就诊的乙型肝炎（乙肝）或疑似乙肝患者180例,分别采用TRFIA定量和酶联免疫吸附试验（ELISA）定性两种方法检测患者的临床血清标本的乙型肝炎病毒表面抗体（HBsAb）、乙型肝炎病毒表面抗原（HBsAg）、乙型肝炎病毒e抗原（HBeAg）、乙型肝炎病毒核心抗体（HBcAb）、乙型肝炎病毒e抗体（HBeAb）5个指标,将TRFIA设为治疗组,ELISA设为对照组,通过κ检验对两种方法间的一致程度进行评价。结果采用TRFIA检测HBV血清标志物HBsAg、HBsAb、HBeAg、HBeAb、HBcAb的阳性率分别为47．1％、41．1％、44．4％、58．9％、48．9％,采用ELISA检测的阳性率分别为48．3％、42．2％、42．2％、57．8％、52．2％,两组阳性率差异无统计学意义（P＞0．05）;两种方法检测5个指标的效率均表现高度的一致,其中HBsAb指标表现为极强的一致性,其他指标表现为高度一致。结论在检测HBV血清标志物方面,传统的ELISA可靠性也较高,与TRFIA一致性较高,都具有很高的应用价值。     

Sensitive time-resolved immunofluorometric assay of thyrotropin in serum

We have developed a time-resolved solid-phase immunofluorometric assay for thyrotropin (TSH). The assay is performed in white opaque microtitration wells which are coated with a monoclonal capture antibody. Serum TSH binds simultaneously to the solid phase and to a biotinylated monoclonal detection antibody. The degree of biotinylated antibody binding is quantitated with streptavidin conjugated to thyroglobulin which is heavily labelled with the Eu3+ chelator 4,7-bis [chlorosulfophenyl] -1,10-phenanthroline -2,9-dicarboxylic acid (BCPDA). The final fluorescent complex is measured on the solid phase with time-resolved fluorometry. The assay requires two incubation steps and can be completed in 5 hours. The detection limit is 0.03 milli-int. units/L. The present assay was compared with two immunoradiometric assays and gave satisfactory results.     

抗心磷脂抗体IgG时间分辨荧光免疫分析法的建立

目的建立高灵敏度、宽量程的定量检测患者血清中的抗心磷脂抗体(anticardiolipin antibody,ACA)IgG的时间分辨荧光免疫分析方法。方法采用ACA抗原(心磷脂+β2糖蛋白I)和鼠抗人IgG抗体分别作为固相抗原和铕标记抗体,如果样本中存在ACA抗体,则形成ACA抗原-ACA抗体-铕标记鼠抗人IgG抗体复合物,加入解离增强液解离铕离子,检测荧光强度,样本中ACA-IgG抗体含量与荧光强度成正比;对建立的时间分辨荧光免疫(time-resolved fluoroimmunoassay,TRFIA)法检测ACA抗体的线性范围、精密度、检测范围进行分析;对25例ACA-IgG抗体阳性血清标本分别利用TRFIA及ELISA法检测ACA-IgG抗体,分析其相关性;收集50例健康献血者,利用TR-FIA检测其血清中的ACA-IgG抗体,计算临床特异度。结果 利用SPSS 13.0统计软件进行统计学分析,TRFIA法检测高、中、低3种浓度混合血清的批内(n=20)精密度分别为2.34%、3.25%和3.87%,批间(n=8)精密度分别为2.89%、3.67%和4.50%;方法的灵敏度为0.1GPL U/ml;TRFIA法检测健康献血者,临床特异度为98%;TRFIA与ELISA 2种方法检测结果的一致性检验采用相关性分析,相关系数为0.987;TRFIA比ELISA的检测范围更宽,稳定性能好。结论首次建立稳定的高灵敏度和宽检测范围的TRFIA法检测人血清中的ACA-IgG抗体,对早期诊断自身免疫性疾病及监测疗效具有重要意义,该方法以其优势有望在各检验科室得以普遍应用。     

Thyroid screening of neonates without use of radioactivity: evaluation of time-resolved fluoroimmunoassay of thyrotropin

We evaluated the usefulness, in routine newborn screening for congenital hypothyroidism, of a time-resolved fluoroimmunoassay kit (DELFIA Neonatal TSH) for the determination of thyrotropin (TSH) in dried blood spots. A total of 11 531 dried blood samples from newborns were tested in parallel in each of two Swiss screening laboratories, by RIA and DELFIA. Six cases of confirmed congenital hypothyroidism were detected during the study period. The rate of false-positive results, after single TSH determination in the DELFIA assay, was 0.16%. Correlation of RIA and DELFIA results for TSH was very good in both laboratories (0.959 and 0.97, respectively). The new method fulfills the criteria for precision and sensitivity of a screening assay. Screening results are usually available the day after the sample arrives in the laboratory, thus favoring early diagnosis and allowing treatment to begin by the seventh or eighth postnatal day.     

Two new two-step immunoassays for free thyroxin evaluated: solid-phase radioimmunoassay and time-resolved fluoroimmunoassay

We measured concentrations of free thyroxin (FT4) in serum by using two new two-step FT4 assays--a solid-phase two-step radioimmunoassay. Spectria, and a time-resolved fluoroimmunoassay. Delfia--and compared the results with those by a two-step FT4 assay (RIA-gnost), a one-step FT4 analog assay (Amerlex-M), and FT4 measured after equilibrium dialysis. The new FT4 assays classified 30 hypothyroid and 43 hyperthyroid patients (untreated) well. In 138 patients with nonthyroidal illness (NTI) and in late pregnancy (n = 36), fewer subnormal FT4 values were reported by Spectria (P less than 0.001), Delfia (P less than 0.001), and RIA-gnost (P less than 0.01) than by Amerlex-M. The results of the Spectria and Delfia methods correlated with the results of the dialysis method (r = 0.76) in NTI patients and pregnancy, and were in better agreement with the clinical state than was FT4 by Amerlex-M. The FT4 values by Amerlex-M, but not by other methods, correlated with albumin concentration. We conclude that these new two-step methods present good alternatives for FT4 analysis.     

An evaluation of serum CA 50 levels in cancer using a time-resolved fluoroimmunoassay

The CA 50 levels in serum samples from 440 patients were estimated using a dissociated enhanced lanthanide immunofluorimetric assay. The distribution was similar to CA 50-RIA assays. Raised levels (greater than 14 U/ml) were present in 95% pancreatic cancer, 68% hepatoma, 54% advanced colorectal cancer, 58% advanced breast cancer and 48% lung cancer. High values were observed in adenocarcinoma of the lung, and were related to tumour mass in small cell lung cancer. CA 50 is independent of CEA. The marker is of considerable potential in pancreatic cancer where the majority of patients express the Can 50 Ag.     

Direct solid-phase time-resolved fluoroimmunoassay of 17 alpha-hydroxyprogesterone in serum and dried blood spots on filter paper

We describe a direct, solid-phase time-resolved fluoroimmunoassay (TRFIA) for measuring 17 alpha-hydroxyprogesterone (17OHP) in serum and blood spots on filter paper. We used 17OHP-3-carboxymethyloxime (17OHP3CMO) coupled to polylysine as the label, which enabled incorporation of up to 34 atoms of europium per molecule of 17OHP, for a very high specific activity. The assay is based on competition between labeled 17OHP3CMO and 17OHP in blood specimens for polyclonal rabbit anti-17OHP antibodies. The antibody-label complex is separated by binding to anti-rabbit antibodies coated onto microtiter strips. The assay buffer contains danazol to displace 17OHP from steroid-binding proteins in serum. For serum samples, the assay is accomplished in 1 h of incubation at room temperature. The blood spot assay with filter paper discs involves incubation overnight at 4 degrees C. Results for both types of specimens from the same subjects correlated well. The lowest measurable concentrations of 17OHP (nmol/L) were 0.10 (3 SD) and 0.75 (3 SD) for serum and dried blood on filter paper, respectively. Intra- and interassay CVs were about 5-15% for both types of samples.     

Development of an improved time-resolved fluoroimmunoassay for simultaneous quantification of C-peptide and insulin in human serum

Objectives

Diabetes mellitus is a chronic disease affecting millions of people globally and resulting in significant death rates each year. A fast, inexpensive alternative to traditional testing and monitoring techniques is desirable, since secretion of insulin and C-peptide is impaired in diabetes mellitus.

Design and methods

A highly sensitive immunoassay was developed for the simultaneous measurement of C-peptide and insulin levels in human serum, utilizing dual-label time-resolved fluoroimmunoassay (TRFIA) and magnetic particle technologies. This assay was characteristic for a single-step sandwich-type immunoassay, wherein antibody-coated magnetic particles were used as the solid phase and Eu^3 + and Sm^3 + chelate labels were used for detection.

Results

Antibody-coated magnetic particles in a TRFIA format performed well in addressing a number of quantitative needs.

Conclusions

The results of this assay correlated well with commercial chemiluminescence assays and provided a number a advantages, including reduced sample volume, reduced reagent and personnel costs and reduced assay time, while maintaining the required clinical sensitivity.     

Measurement of estrone-3-glucuronide in urine by rapid, homogeneous time-resolved fluoroimmunoassay

We describe a liquid-phase nonseparation time-resolved fluorescence immunoassay for measuring estrone-3-glucuronide in undiluted urine. The sensitivity, specificity, and accuracy are similar to those for a conventional separation fluoroimmunoassay or radioimmunoassay, but the speed, convenience, precision, reliability, and clinical utility of the new method are more advantageous. The labeled antigen, a fluorescent europium chelate covalently linked to estrone-3-glucuronide, is incubated for 10 min with a limited concentration of polyclonal or monoclonal antibodies to estrone-3-glucuronyl-6-bovine serum albumin and 10 microL of standard or sample (undiluted urine) in microtiter wells. The fluorescence emanating from the antibody-free label, which is proportional to the concentration of estrone-3-glucuronide in the standard or sample, is then measured in a time-resolved fluorometer. The method is useful for monitoring ovarian function in women.     

The detection and enumeration of Leptospira interrogans serovar hardjo by time-resolved fluoroimmunoassay

Laboratory diagnosis of leptospirosis is hampered by difficulties in growing the organism and identifying the isolate. Previous studies using an ABEI-labelled chemiluminescent method indicated that it provided a rapid and accurate method of detecting leptospires. The present study outlines efforts to improve both the sensitivity and specificity of the technique. Time-resolved fluoroimmunoassay (TR-FIA) with a solid-phase indirect sandwich technique incorporating a europium-labelled swine anti-human IgG was developed. Antibodies raised in rabbits against Leptospira hardjo were immobilized onto the walls of a strip microtitration plate. Varying concentrations of L. icterohaemorrhagiae, L. canicola and L. hardjo were added. The mixture was incubated at 30°C for 30 min and then washed. Leptospires (in this case L. hardjo) will adhere to the walls of the wells in the presence of its homologous antisera, whereas heterologous leptospires such as L. icterohaemorrhagiae will not. Human anti-L. hardjo serum was then added and the whole incubated again at 30°C for 30 min. After washing to remove excess antibody the europium-labelled swine anti-human IgG was reacted with the bound antigen-human antiserum complex. The mixture was then washed and enhancement solution added to promote the rapid dissociation of europium cations from the labelled antibody where they form highly fluorescent chelates. Fluorescence was measured in a time-resolved fluorometer. Calculation and interpretation of results were based on the signal values of positive and negative controls. The results obtained using TR-FIA show that the background fluorescence is greatly reduced and that L. hardjo can be detected at very low numbers (1 × 10²). Neither L. icterohaemorrhagiae nor L. canicola were detected using L. hardjo antisera, thus making this method both sensitive and specific.     

C-肽时间分辨荧光免疫分析试剂盒的研制

目的研制血清C-肽的时间分辨荧光免疫分析（TRFIA）试剂盒。方法采用双抗体夹心法建立C-肽TRFIA试剂盒,并进行方法学评价。结果C-肽可测量范围为0．4-20ng／mL;分析敏感性为0．23ng／mL;分析批内、批间精密度分别为7．99％、11．97％;C-肽回收率为99．57％;与胰岛素和胰岛素原无交叉反应。稳定性试验表明,试剂可以贮存在4℃稳定9个月,37℃稳定7d;该试剂盒测试200份正常人血清样本,其参考范围为0．358-3．590ng／mL;收集122份血清样本,用该试剂盒与国外其他方法的同类试剂盒同时检测,其相关系数为0．9432,线性方程为Y=0．9854X＋0．5063。结论C-肽TRFIA试剂盒各项指标均达到临床检测要求,适合临床推广使用．     

C-肽时间分辨荧光免疫分析试剂盒的研制

目的研制血清C-肽的时间分辨荧光免疫分析(TRFIA)试剂盒。方法采用双抗体夹心法建立C-肽TRFIA试剂盒,并进行方法学评价。结果C-肽可测量范围为0.4~20 ng/mL;分析敏感性为0.23 ng/mL;分析批内、批间精密度分别为7.99%、11.97%;C-肽回收率为99.57%;与胰岛素和胰岛素原无交叉反应。稳定性试验表明,试剂可以贮存在4℃稳定9个月,37℃稳定7 d;该试剂盒测试200份正常人血清样本,其参考范围为0.358~3.590 ng/mL;收集122份血清样本,用该试剂盒与国外其他方法的同类试剂盒同时检测,其相关系数为0.9432,线性方程为Y=0.9854X+0.5063。结论C-肽TRFIA试剂盒各项指标均达到临床检测要求,适合临床推广使用。     

血清透明质酸时间分辨荧光免疫分析法的反应条件优化及其临床应用

目的优化血清透明质酸(HA)时间分辨荧光免疫分析法(TRFIA)的反应条件,并探讨其临床应用价值。方法采用TRFIA检测血清中HA的含量,确定参与HA-TRFIA的各反应成分的浓度。结果最佳反应条件为包被浓度10 mg/L、HA结合蛋白(HABP)1∶500稀释、铕标记HA(Eu3+-BSA-HA)1∶200稀释。敏感性为5.17μg/L,批内变异系数(CV)为2.21%~3.78%,批间CV为3.73%~5.40%,平均回收率为100.4%。该法HA测定值与放射免疫测定值显著相关,且与临床结果一致。结论本研究建立的血清HA-TRFIA是一种灵敏准确的分析方法,适合临床应用。     

血清透明质酸时间分辨荧光免疫分析法的反应条件优化及其临床应用

目的优化血清透明质酸（HA）时间分辨荧光免疫分析法（TRFIA）的反应条件,并探讨其临床应用价值。方法采用TRFIA检测血清中HA的含量,确定参与HA-TRFIA的各反应成分的浓度。结果最佳反应条件为包被浓度10 mg/L、HA结合蛋白（HABP）1∶500稀释、铕标记HA（Eu^3＋-BSA-HA）1∶200稀释。敏感性为5.17μg/L,批内变异系数（CV）为2.21%-3.78%,批间CV为3.73%-5.40%,平均回收率为100.4%。该法HA测定值与放射免疫测定值显著相关,且与临床结果一致。结论本研究建立的血清HA-TRFIA是一种灵敏准确的分析方法,适合临床应用。