Magnetic Beads-based Time-resolved Fluoroimmunoassay For The Determination Of Carcinoembryonic Antigen And Simultaneous Determination Of α-fetoprotein And The Free β-subunit Of Human Chorionic Gonadotropin

Since the 1960s, when immunoassay and antibody labeling technology emerged, various labeling techniques have been introduced. By combining the high selectivity of the immunological reaction with the high sensitivity of various labeling technology, numerous labeling strategies have been widely used in clinical diagnosis, therapy observation and basic medicine research, which improved the clinical and biochemical analysis from macroanalysis to trace and ultramicro analysis. In a solid-phase immunoassay, an analyte reacts with an immobilized antibody forming a complex. This capturing antibody is immobilized on a surface actively via its functional groups, by means of an antibody capturing protein (or antibody), or passively by hydrophobic interactions. A solid-phase immunoassay provides a convenient means of separating unbound reactants. Such a separation is possible owing to the high specificity and affinity of the analyte to the antibody. The analyte concentration is assessed in a non-competitive immunoassay by monitoring the quantity of a second, labeled antibody bound to the analyte on the surface. In recent years, the development of new theories and new techniques has enormously improved the techniques in labeling immunoassay, such as radioimmunoassay(RIA), fluorescence immunoassay(FIA), enzyme linked immunosorbent assay(EIA), chemiluminescence immunoassay(CLIA), electrochemiluminescence immunoassay (ECLIA) and time-resolved fluoroimmunoassay(TRFIA), have been proposed to meet the clinical analysis. Among the methods mentioned above, time-resolved fluorescence immunoassay (TRFIA) possessed the advantages of high sensitivity and specificity, wide range of applications, high stability, no radioisotope pollution, better reproducibility, not sensitive to natural fluorescence interference. TRFIA has attracted considerable attention their outstanding physical and chemical properties that the application scope of TRFIA has been extended to medicine, biology, environmental science, food security and many other fields.Time-resolved fluorescence immunoassay (TRFIA), which employs the lanthanide chelate labels with special fluorescence properties that have wide excitation spectrum, sharp emission peak, large stokes’shift, long fluorescence half-life and high stability. As the chelates of these lanthanides have fluorescence peaks at different wavelengths and are clearly distinguished from one another, the combined use of several chelates enables double or even multiple-label immunoassays to simultaneously quantitate several indicators contained in a single sample. These advantages enable an effective assay with short analysis time, broad dynamic range, and low reagent and labor consumption and show broad application prospects in the aspect of clinical immunology test. Increasing attention has recently been focused on the development of immunoassay methods for quantifying two or more analytes simultaneously in one sample in a single assay to improve the diagnostic sensitivity and specificity. Such as combination of Human epididymis protein 4(HE4) and Cancer antigen 125(CA125) predicting ovarian malignant tumor; thyroid hormone concentration changes associated with different thyroid diseases in thyroid function tests; Serum C peptide and insulin level classification diagnosis of diabetes; Usually need to measure a variety of infectious diseases such as Human immunodeficiency virus(HIV), Hepatitis C virus(HCV), Treponemiapallidum(TP), Hepatitis b virus (HBV), TORCH and other disease. Hence, this strategy has significant promise and could be further developed for practical clinical detection of further important biomarkers.Magnetic nanoparticles is one kind of nanoparticles, which is a novel functional materials developed in the 1950 s, Magnetic particles construct of transition metals such as iron, cobalt and nickel oxide offer high technological potential since they can be conveniently collected with an external magnetic field. Magnetic particles have attracted considerable interest in the field of biomedical field due to their interesting physico-chemical properties, high specific surface area and good mechanical stability. Immunomagnetic beads, the efficient separation and enrichment function of magnetic beads combination with the highly specific immunological reaction has became a new immunology technology developed in recent years, which have gradually been applied to the immune detection, cell separation and the extraction of biomolecules such as DNA,RNA,or mRNA. Besides this, Magnetic beads combined immune fluorescence immunity analysis (FIA), chemiluminescence immunoassay applied to food safety and environmental monitoring aspect has been provided by several recent studies, however few utilize magnetic particles in TRFIA which applied to the research on some biomarkers detection of clinical disease.Carcinoembryonic antigen(CEA), an acidic glycoprotein with a molecular weight of approximately 200 kDa, which was first described by Gold and Freedman in 1965, as it was found only in the digestive adenocarcinomas. Under normal circumstances, CEA was expressed highly by the gastrointestinal epithelium, pancreas and hepar of fetuses. The CEA is an oncofetal antigen and is produced in volume by the normal developing fetus and in some cancers, but only in trace amounts by normal adult human cells. CEA is the organ specific tumor associated antigen, divided by the endoderm cell, formed in the cytoplasm, cross a cell membrane into the body fluids, which could be detected in the variety of body fluids. CEA is a broad-spectrum tumor marker, High serum CEA level is related to some digestive cancers cancers such as colon cancer, hepatic carcinoma, pancreatic cancer, more than 90% of primary colorectal carcinomas produce CEA. It has been reported that the serum from individuals with breast cancer, lung cancer and other malignant tumors had higher levels of carcinoembryonic antigen than healthy individuals. CEA is a broad spectrum multi-tumor marker for clinical diagnosis and it can be used as an auxiliary diagnosis index for evaluating curative effect judging recrudescence or metastasis as a marker. Thus, detection of the serum CEA level plays an important role in an initial diagnostic evaluation and any follow-up examination during therapy.Down’s syndrome (DS), also called Trisomy 21 or innate clumsy, is one of the most common autosomal diseases, which the main feature is a serious congenital mental retardation, distinctive craniofacial features, and accompanied by a variety of congenital malformations. Prenatal screening aims to identify women with a higher risk for carrying a fetus with chromosomal abnormalities to provide individual pregnant women an option to decide upon the continuation of their pregnancy. At present there is no effective treatment for the disease, so prenatal screening and diagnosis of trisomy 21 and neural tube defects in order to prevent and reduce its birth rate is the particularly important. in improving the health of newborns in prenatal disgnosis in our country. In recent years, many reaserchers have found that the abnormal of pregnant women serum a-fetoprotein(AFP), free β-human chorionic gonadotrophin(Free β-hCG), pregnancy-associated plasma protein-A(PAPP-A) and unconjugated estriol(uE3) that generated by fetal placenta were closely related to trisomy 21 and neural tube defects and have be used in prenatal screening. While these serum markers exists low detection rate, false positive, and false negative when used alone, most centres use two or more markers to increase the accuracy of the test. The double-marker analysis for Free β-hCG and PAPP-A of maternal serum screening for Down syndrome in first-trimester, and in the second trimester of pregnancy measurement of AFP and Free β-hCG, in conjunction with factors such asmaternal age and gestational age, have found clinical utility when screening for pregnancies complicated by Down syndrome, and prospectively to identify 60-80% of Down syndrome cases for a 5% false-positive rate. Then with the development of high resolution immunoassay, serum marker is used more and more common in prenatal screening and prenatal diagnosis and meet the demand for clinical applicationIn spite of many advances in the field of TRFIA, there is still a paucity of novel approaches for improving the simplicity, selectivity, and sensitivity of clinical immunoassays, in order to respond to the demands and needs of modern medical diagnostics and biomedical research applications. A novel immunoassay for the determination of tumor markers in human serum was established by combining a time-resolved fluoroimmunoassay (TRFIA) and immunomagnetic separation. Based on a sandwich-type immunoassay format, analytes in samples were captured by magnetic beads coated with one monoclonal antibody and "sandwiched" by another monoclonal antibody labeled with europium chelates. The immunocomplex was separated and washed by exposure to a magnetic field and treatment with enhancement solution; fluorescence was then measured according to the number of europium ions dissociated. This method not only possess the advantage such as high sensitivity, long storage time, no radioactive contamination, a wide range of standard curve that the TRFIA have, but also greatly reducing the reaction time and improve detection sensitivity by the use of immunomagnetic beads uniformly suspended in the solution makes the combined surface area fully expanded. Unlike conventional TRFIA, a specific anti-analyte antibody was covalently coupled to the surface of magnetic beads rather than immobilized on the surface of 96-well microplates by physical adsorption. The available sites for antigen binding are symmetrical and the mass transfer distance of analytes to the immobilized antibody is greatly reduced, with minor spatial hindrance, by this approach, and consequently, antibody-antigen binding equilibrium can be achieved rapidly. Magnetic beads modified with chemically functional groups provide a relatively large surface area, which enables more antibodies to be coupled efficiently to the surface. Also, the immunocomplex can be separated conveniently from unbound components by application of a magnetic field. These advantages enable an effective assay with short analysis time, broad dynamic range, and low reagent and labor consumption. In addition, exploring the new strategies for sensitive and convenient multianalyte detection with high throughput in clinical diagnosis is of great interest.In this study we investigated a novel immunoassay uses magnetic beads as an immobilization matrix and means of separation, while the luminescent europium and samarium chelates are used as probes. Based on a single-step, sandwich-type, we developed of a novel TRFIA methodology for the rapid and sensitive detection of tumor markers in human serum using CEA biomarker as a model and the simultaneous determination of a-fetoprotein(AFP) and the free β-subunit of human chorionic gonadotropin(Free β-hCG) in human serum. This study is divided into three parts of as the test content:Firstly, use different sizes and different chemical groups modified magnetic beads covalent binding to antibody, select the magnetic beads and optimized coupling scheme, and labeling of antibody with lanthanide chelates; Secondly, Establishment and optimization of reaction system and evaluation of parameters such as analytical sensitivity, linearity, reproducibility, recovery and specificity; Finally, Compare with similar foreign-made kit diagnostic reagents for clinical and evaluated the feasibility of its initial application in clinical laboratories.The result showed that under the optimum condition, for CEA, the detection limit was 0.5 ng/mL and the analytical range was 1.0-1000.0 ng/mL; The intra-assay coefficients of variation were 2.722%～3.252% and the inter-assay coefficients of variation were 3.157%～3.765%; Analytical recovery was 90.1%～99.2%, the reproducibility and recoveries of the proposed assay in our cases were acceptable; The specificity of the assay for CEA was evaluated by measuring the cross-reactivity with four possible interfering compounds. There was no cross-reaction to AFP, CA125, CA19-9 and HSA, clearly verifying the high specificity of the proposed immunoassay; Triglycerides, bilirubin, hemoglobin were added in CEA controls and no interferences were detected; The reagent showed good stability proved by performance evaluation after stored at 37℃ for days and 4℃ for 12 months. To further evaluate the feasibility of the new TRFIA method for clinical applications, 239 serum samples from patients with different cancerous diseases were recruited. CEA levels were measured by our developed immunoassay and compared with those detected by a commercially available TRFIA kit and CLIA kit. Linear regression analyses revealed good correlations between the developed method and TRFIA and CLIA. The correlation coefficient was r= R2=0.970, P<0.001 and R2=0.951, P <0.001 between our proposed method and the commercially available TRFIA or CLIA kits. The equation of the regression curve was Y= 1.049 X+1.696 or Y= 0.873 X-3.802, respectively, where X is the CEA concentration estimated with the proposed method and Y is the CEA concentration from TRFIA or CLIA methods. Our proposed method had a good correlation with TRFIA or CLIA methods. This finding indicated that the proposed assay could serve for clinical determination of CEA in human serum.The result showed that under optimized experimental condition, The new TRFIA method was validated in terms of the response linearity and limits of detection using a one-step immunoassay format:a series of AFP/free β-hCG standards with different concentrations The calibration curves for AFP showed a linear relationship over the concentration range of 0.1-750 ng/mL, with a lower detection limit of 0.05 ng/mL for AFP. And the concentration range of 0.16-400 ng/mL, with a lower detection limit of 0.08 ng/mL for Free β-hCG. The reproducibility of the immunoassay method was estimated by intra- and inter-assay coefficients of variation using maternal serum controls at three concentrations. It was found that the intra-assay variations were 2.6%～5.1% for AFP and 4.1%～4.7% for Free β-hCG, while the inter-assay variations were 1.9%～5.1% for AFP and 4.7%～5.0% for Free β-hCG, demonstrating an acceptable reproducibility; Recovery was evaluated using given amounts of AFP or Free P-hCG standards (50 ng) by spiking into the above-mentioned maternal serum controls at various analyte levels. the recoveries of the analyte varied from 96.0 to 106.0%, indicating that the recovery of the proposed immunoassay was satisfactory. The specificity of the TRFIA was evaluated over a wide spectrum of possible interferents, namely, CEA, CA125, CA15-3, CA19-9, LH, FSH TSH and hCG. The results show that the presence of the potential interferents at relatively high concentrations had negligible effects on the simultaneous assay for AFP/free β-hCG, clearly verifying the high specificity of the proposed immunoassay. Triglycerides, bilirubin, hemoglobin were added in AFP/free β-hCG controls and no interferences were detected; The reagent showed good stability proved by performance evaluation after stored at 37℃ for days and 4℃ for 12 months. The reliability of the immunoassay system for clinical applications was further investigated by analyzing eleven clinical serum samples from second trimester pregnancies. Results were compared with those obtained from a commercially available TRFIA kit using linear regression analysis, as shown in Figure 6A and B. The correlation between the two methods was investigated using t-tests for comparison, and no significance differences were observed. A good agreement between both analytical methods was observed, with R2 values of 0.970 for AFP and 0.966 for free (3-hCG. The regression line was fitted to y= 0.961+1.554x, P<0.001 or y= 1.044x-1.421, P<0.001, respectively, where x stands for the AFP or free P-hCG concentrations estimated by the proposed method and y stands for those obtained using the TRFIA kit. The results indicate that the magnetic particle-based TRFIA has good potential application for the simultaneous determination of AFP and free β-hCG in real sample analyses.In this paper, a novel time-resolved fluoroimmunoassay (TRFIA) protocol using magnetic beads for the measurement of CEA in human serum, and the simultaneous determination of a-fetoprotein (AFP) and the free β-subunit of human chorionic gonadotropin (free β-hCG) in human serum is described. Satisfactory specificity, reproducibility, and recovery of the immunoassay were demonstrated. Good correlations were obtained in the analysis of human serum samples between the proposed method and a commercial available kit. The range of linearity was broadened appreciably and the analysis time was shorter considerably. This assay platform revealed such advantages as higher sensitivity, time efficiency, and less consumption of reagents compared with the conventional method. These results demonstrate the feasibility and potential of the new method as a rapid and highly sensitive immunoassay that could be developed into a platform for multianalyte determinations in clinical practice.