Establishment And Clinical Application Of Magnetic Microparticles-assisted Time-resolved Fluoroimmunoassay

Labeled-immunoassays, by definition, is to analyze and determine biochemical substances by combining labeling technology and immunological technique. The core of Labeled Immunoassays is the integration of high sensitivity in multiple-labeled tracer technique and high specificity of antigen-antibody reaction in medical immunology. It is a general term of a large class of ultra-sensitive, highly specific detection technology, and it has been widely used in the field of detection of trace materials in food environment, basic medical research and therapy observation of clinical diseases because of its numerous unique advantages.Modern labeled immunoassay was first started by two American scholars, Berson and Yallow, who first used radioimmunoassay in 1959. Since the 1960s, with the development and perfection of radioimmunoassay technology, immunological parameters have been greatly improved in its detecting sensitivity. This traditional analytical method has high specificity and high sensitivity, but the adverse effect of radioactive isotope on biological and laboratory environment, attenuation cycle and other factors, which greatly limit the life of the agent, constraint the development of this technology to some extent.In the 1970s, Swedish scientists first established enzyme-labeled immunoassay, using enzyme to replace isotope. It avoids environmental pollution and physical harm, while reagent has a long period of validity, so that labeled immunoassays are able to obtain a long-term development on the road of non-radioactive labeling immunoassay.So far, many developed immunoassays have emerged, with the progress of tag technology and continuous findings of new markers, such as Colloidal gold immunoassay tagging by colloidal gold, Fluorescence immunoassay (FIA) using fluorescein as label, enzymatic, chemiluminescent substrate-based Chemiluminescent immunoassay (CLIA), Electrochemiluminescence immunoassay (ECLIA) as well as lanthanide chelate labeled time-resolved fluoroimmunoassay (TRFIA). The principles are basically the same, but the final measurement signals vary depending on different labeled markers. Development of innovative labeled immunoassay technology is inseparable from the continuous discovery of new markers, and the application of monoclonal antibodies and genetically engineered antibody technology extensively expands the development of the immune amplification technology, which also significantly improves the specificity and sensitivity of immunological detection. It is relying on the continuous emerge of new technology and methods that labeled immunoassay has become a class of biochemical and immunological detection of trace or ultra-trace bioactive substances. Labeled immunoassay is widely used in the area of food safety, testing of bacteria, viruses, funguses, toxins parasites, drug residues, pesticide residue in the field of environmental hygiene, as well as broader clinical diagnostics involving tumor markers, infectious diseases, diabetes, hormones, prenatal screening, accessory diagnosis and monitoring of drugs and their metabolites during treatment.Time-resolved fluoroimmunoassay (TRFIA), established in early 1980s, is the most promising non-radioactive labeling immunoassay and a new milestone of labeling development after radioimmunoassay, which has become the most promising analytical tools in biomedical research and clinical ultramicro-biochemical detection. Time-resolved fluorescence immunoassay (TRFIA), which employs trivalent-rare-earth ions europium (Eu3+), samarium (Sm3+), terbium (Tb3+) and dysprosium (Dy3+) and or its chelate as tracers, instead of traditional substance such as enzyme, isotope, fluorescein, chemiluminescent and bioluminescent, is used to label proteins, polypeptides, hormones, antibodies, nucleic acid probes or bioactive cell. When detecting reactions includes the antigen-antibody immune reaction, the biotin-streptavidin affinity reaction, nucleic acid probe hybridization reactions begin, after using time resolved fluorescence measurements and spectral separation techniques to exclude non-specific interference in sample, we can measure the final determination of the fluorescence intensity of the product, so that effectively improve the detecting sensitivity and its limit of detection can exceed 10-8 mol. TRFIA technology is based on rare-earth ions, with special fluorescence properties that have wide excitation spectrum, sharp emission peak, large stokes’shift, long fluorescence half-life, high fluorescence intensity, simple labeling preparation, stable physicochemical properties, long storage time, no radioactive contamination, wide range of standard curve, no excitation interference from natural fluorescence and repeatable measurements. It can also enable multi-label for the simultaneous detection of multiple analytes and automation of measurement, so it is much favored by biomedical workers. Our country is a big country full of rare earth elements, going deep into the research of TRFIA to promote the industrial development of its scientific research will help promoting the development and competitiveness of biomedical detection technology.At present, immunoassays at home and abroad are usually based on microplate, which is the main carrier for routine immune reaction and analysis of signal detection. Antigens, antibodies and other biological molecules adsorb to the surface of the carrier through a variety of mechanisms, including by hydrophobic bond, passive adsorption of running water or ionic bond, covalent binding of an amino group and carbon-base by introducing other active groups, and by hydrophilic bond after the surface modification. With the requirements by clinical diagnosis of immunoassay technology continuing to increase, defects of polystyrene plates as solid-phase carrier are as followed:(1) Inefficient bonding of antigen and antibody by physical adsorption on the coated microplate, thus waste raw materials; (2) a certain surface area of micropore which result in a limited amount of antigen-antibody-coating, limited detection range of high concentration; (3) small contact area of the solid-liquid phase reaction in the microplate, long reaction time; (4) for the whole strip of microplate, detection of clinical samples cannot be tested in time, detect sample needs to be accumulated to a certain amount.Magnetic nanoparticles are one kind of nanoparticles, which is a novel functional materials developed in the 1950s. Since magnetic nanoparticles can be conveniently collected with an external magnetic field, they have attracted considerable interest in the field of biomedical field due to their interesting physical-chemical properties, high specific surface area and good mechanical stability. Immuno-magnetic beads, the efficient separation and enrichment function of magnetic nanoparticles combination with the highly specific immunological reaction has become a new immunology technology developed in recent years, which have been more and more widely applied to cell sorting, immunological detections and diagnosis, cancer treatment, food safety, environmental microbial testing and other areas. Through external modification of functional groups, magnetic beads can bind to active proteins, as a carrier of antigen and antibody. When the antibody adsorbed on magnetic beads react with the corresponding microorganisms or specific antigen substance, it forms "bead-antibody-antigen complexes", which has a higher magnetic response. By moving directionally in the force of the magnet, these complexes are separated from other substances, to achieve purposes of separation, concentration, as well as purification from microorganisms or specific antigenic material. Specifically, as a carrier throughout the experiment and tests, magnetic beads have far more merits compared to conventional microplate. The advantages points are as followed:(1) larger surface area of spherical micro-beads, which makes it more capable of binding to protein molecules; (2) by means of covalent coupling, protein is conjugated to chemical group on surface of magnetic beads in a way much solider, more stable than physical adsorption of polystyrene micropore, and thus saving reagent materials; (3) the immuno-magnetic beads evenly suspended in the reaction environment, so that greatly increase the chance of contacting with the analytes. It needs less sample required for the reaction, and achieve reaction dynamic balance quicker, thereby speeding up the reaction rate and reducing the reaction time; (4) combined application of magnetic beads coated with different fluorescence probe and various tracer of lanthanides in time-resolved fluorescence immunoassays, which makes the idea that the same samples can be tested for different analytes possible. Beside this, it is easily automatized and experimental samples can be tested at any time.According to the literature and market research, magnetic beads-based enzyme-linked immunosorbent assay and magnetic beads-based chemiluminescence immunoassay, which are a combination of magnetic labeling technology and conventional ELISA and CLIA technology, has been developed in immune-analytical industry with more mature reagents and industrialization bringing out. However, the survey found that most reagents of time-resolved fluorescence immunoassays at current market are still based on traditional microplate. Meanwhile, we are lack of comprehensive and accurate systematic study of the application in detection of markers, in clinical disease, combined with magnetic beads and time-resolved fluorescence immunoassay.Aiming at all kinds of protein molecules in serum, including macromolecule such as antigen and antibody, haptens and small monovalent antigen, established reaction system of TRFIA using magnetic beads as solid support magnetic beads as solid support has series of properties:a fast, small sample size, wide detection range, high sensitivity, miniaturization, and other characteristics. These properties meet the needs of clinical follow-up testing, and would provide it with a broader clinical application.In view of this, this study evaluates the clinical application value of the new carrier-based magnetic immunoassay platform comprehensively and accurately. According to the specific characteristics of analytes and detection conditions, different detection methods have been designed, including double-antibody sandwich method, double-antigen sandwich method, competition and etc. In this study, hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg), antibody to hepatitis B surface antigen (anti-HBs) and free thyroxine (FT4), these three typical types of serum markers are representative of double-antibody sandwich method, double-antigen sandwich method and competition method respectively for feasibility analysis of such response patterns. The experiment contents on this subject is mainly divided into three parts:(1) to develop magnetic-bead based TRFIA reagents of HBsAg and HBeAg by reaction mode of double-antibody sandwich; (2) to develop magnetic-bead based TRFIA reagents of anti-HBs by reaction mode of double-antigen sandwich; (3) to develop magnetic-bead based TRFIA reagents of FT4 by reaction mode of direct competition. Each experiment content above covers establishment and optimization of the reaction system, by optimizing the reaction system to evaluate comprehensively and integrally every property of the reagents, including accuracy, sensitivity, dose-response curve, specificity, precision, anti-interference analysis and stability. We also compare self-made reagents with similar imported reagents to test the feasibility for clinical diagnosis.The first experiment:(1) The result showed that under the optimum condition, for HBsAg, the analytical sensitivity was 0.02 IU/mL and the linear range was 0.2-700 IU/mL; The intra-assay coefficients of variation were between 4.7%-8.7% and the inter-assay coefficients of variation were between 3.8%-7.5%; Analytical recovery was between 92.5%～106.7%,the reproducibility and recoveries of the proposed assay in our cases were acceptable; Use a certain concentration of HBeAg, HBcAg, HCV, HIV, TP as a sample of the reagent to measure the cross-reactivity and the result shows no significant cross-reactivity; Triglycerides, bilirubin, hemoglobin were added in corresponding controls and no interferences were detected; HBsAg levels of 399 blood samples were measured by our developed immunoassay and compared with those detected by a commercially available CLIA kit from Abbott LabsTM, and coincidence rate of clinical sensitivity and specificity is 100%. By paired chi-square test,P=1.000, Kappa=1.000. Then 231 of double-positive blood determination results are set horizontal and vertical coordinates to do regression analysis, correlation equation:Y=1.182X-0.017, correlation coefficient r=0.989, P <0.001. The above statistical results showed that the coincidence rate of these two methods was statistically significant, result of clinical blood test were significantly correlated. (2) The result showed that under the optimum condition, for HBeAg, the analytical sensitivity was 0.021 PEI U/mL and the linear range was 0.4-160 PEI U/mL; The intra-assay coefficients of variation were between 2.8%～4.3% and the inter-assay coefficients of variation were between 4.2%-6.0%; Analytical recovery was between 98.0%-99.7%,the reproducibility and recoveries of the proposed assay in our cases were acceptable; Use a certain concentration of HBsAg, HBcAg, as a sample of the reagent to measure the cross-reactivity and the result shows no significant cross-reactivity; Triglycerides, bilirubin, hemoglobin were added in corresponding controls and no interferences were detected; HBeAg levels of 257 blood samples were measured by our developed immunoassay and compared with those detected by a commercially available CLIA kit from Abbott LabsTM, and coincidence rate of clinical sensitivity and specificity is 95.8% and 98.9%, respectively. By paired chi-square test, P=1.000, Kappa=0.951. Then 184 of double-positive blood determination results are set horizontal and vertical coordinates to do regression analysis, correlation equation:Y=1.002X-0.333, correlation coefficient r=0.974, P<0.001 The above statistical results showed that the coincidence rate of these two methods was statistically significant, result of clinical blood test were significantly correlated.The second experiment:The result showed that under the optimum condition, for anti-HBs, the analytical sensitivity was 0.45 mIU/mL, and the linear range was 2-600 mIU/mL; The intra-assay coefficients of variation were between 2.4%-4.3% and the inter-assay coefficients of variation were between 3.5%-5.0%; Analytical recovery was between 96.4%-101.8%, the reproducibility and recoveries of the proposed assay in our cases were acceptable. Use a certain concentration of HBeAb, HBcAb, as a sample of the reagent to measure the cross-reactivity and the result shows no significant cross-reactivity; Triglycerides, bilirubin, hemoglobin were added in corresponding controls and no interferences were detected; anti-HBs levels of 269 blood samples were measured by our developed immunoassay and compared with those detected by a commercially available CLIA kit from Abbott Labs. and coincidence rate of clinical sensitivity and specificity is 96.3% and 98.1%, respectively. By paired chi-square test, P=0.687, Kappa=0.932. Then 210 of double-positive blood determination results are set horizontal and vertical coordinates to do regression analysis, correlation equation:Y=0.934X+2.511, correlation coefficient r=0.988, P<0.001. The above statistical results showed that the coincidence rate of these two methods was statistically significant, result of clinical blood test were significantly correlated.The third experiment:The result showed that under the optimum condition, for FT4, the analytical sensitivity was 0.47 pmol/L and the linear range was 2-200 pmol/L; The intra-assay coefficients of variation were between 2.7%-4.3% and the inter-assay coefficients of variation were between 4.0%-7.0%; Analytical recovery was between 93.4%-97.3%,the reproducibility and recoveries of the proposed assay in our cases were acceptable; Use a certain concentration of T3、rT3, as a sample of the reagent to measure the cross-reactivity and the result shows no significant cross-reactivity; Triglycerides, bilirubin, hemoglobin were added in corresponding controls and no interferences were detected; FT4 levels of 202 blood samples were measured by our developed immunoassay and compared with those detected by a commercially available CLIA kit from Abbott LabsTM, Then determination results are set horizontal and vertical coordinates to do regression analysis, correlation equation:Y=0.986X+0.037, correlation coefficient r=0.980, P<0.001 The above statistical results showed that the coincidence rate of these two methods was statistically significant, result of clinical blood test were significantly correlated.In summary, a novel time-resolved fluoroimmunoassay (TRFIA) protocol using magnetic beads for the measurement of HBsAg、HBeAg、anti-HBs and FT4, four different serum markers is successfully developed based on the different reaction modes double-antibody sandwich, double antigen sandwich and competition. After evaluation, the indicators of magnetic bead-based TRFIA reagents, including accuracy, sensitivity, precision, specificity, interference and other indicators are all meet the requirements of clinical testing reagents. Compared with the same imported reagents, its clinical specificity and sensitivity of them have no significant difference and regression analysis show a significant correlation. This paper comprehensively and accurately describes the magnetic beads as solid support can improve the detection sensitivity, expand linear range, shorten the analytic time and improve detection efficiency, etc. It indicates that the feasibility of the platform of TRFIA based on magnetic beads to apply in the detection of clinical disease markers. Also, it manifests that this developed immunoassay is expected to be applied by further optimization into production. It’s very important to develop clinical immunoassay reagents in need in molecular diagnostics and detection of trace substances in food environment and expand the applications of labeled immunoassay.