| The research background and purposeGastric cancer (GC) is one of the most common malignancies worldwide. Recurrence after surgery is one of the main reasons for the high mortality rate of GC. At present, the detection of tumor marker has become one of the commonly used aids for clinical diagnosis and monitoring of GC. Early, convenient, rapid and accurate detection of tumor markers in human blood may be beneficial for diagnosing, treating and improving prognosis of cancer patients. Carbohydrate antigen 72-4 (CA72-4) is a kind of high molecular weight glycoprotein, which is recognized by CC49 and B72.3. It is mainly found in gastrointestinal tumors and ovarian tumors. Numbers of studies show that the positive rate of CA72-4 in GC patients is 16-70%,9% in early stage and 60.6% in advanced stage. Furthermore, the level of CA72-4 is also related to the depth of tumor invasion, metastasis and tumor staging. Especially in patients with advanced GC, the positive rate of CA72-4 is much higher than other tumor markers. Therefore, rapid and highly sensitive detection of CA72-4 will be helpful to the diagnosis, treatment and postoperative monitoring of advanced gastric cancer.At present, the traditional methods for detection of CA72-4 are radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), and electrochemiluminescence immunoassays (ECLIA). Generally, these methods are highly sensitive and specific for the detection of target analytes. But these technologies still have several shortcomings (e.g., they are time-consuming and require expensive equipment and well-trained personnel) that limit their application in primary or community hospitals, especially for applications requiring close monitoring of the disease or large-scale population screening. Because of the miniaturized instruments, simplify operations and timely report, POCT technologies greatly reduce the detection time and report results in a shorter time. So rapid clinical decision-making may become possible. Therefore, to develop a simple and convenient POCT method for CA72-4 detection is also very important.Immunochromatographic assay (ICA) is a new detection method which combined with labelling and chromatography technique in 1980s. It is a common POCT detection technology in the market. ICA could achieve the purpose of qualitative or quantitative detection. The reaction not only has the advantages of specificity, but also innovative to be transferred to the membrane. Furthermore, it does not require separation of binding markers and free markers. This method is rapid (20 minutes for the test results), easy to operate without using complex equipment, making it very suitable for field testing. Now it has been widely used in various detection fields, such as pathogenic microorganisms, human hormone-related protein, parasites, drug concentration in blood and urine, regulation of food, and drugs. As a simple and rapid detection technology, ICA is developing in the direction of quantitative, multiplex detection and high sensitivity. However, the optical detection technology based on colloidal gold and other markers can only reach qualitative or semi-quantitative detection, and sensitivity and consistency of optical detection is low. In recent years, magnetic signal detection based on magnetic nano materials has become a hot spot in the field of ICA quantitative detection.Superparamagnetic nanoparticles (SPMNPs) is a kind of nanometer magnetic particles. It contains magnetic metal oxide (such as iron, cobalt, nickel and its compounds) ultrafine powder. The particles have super paramagnetic properties, which make them have good dispersion and maneuverability, when the particle is smaller than the critical size. The surface can also be coupled with a variety of functional groups with biological activity, which can be combined with protein or other macromolecular substances. It can be applied to a variety of research areas since the biocompatibility. Immunomagnetic beads formed by the combination of antibody and SPMNPs. The size and shape of immunomagnetic beads are uniform, which can be combined with target material quickly and effectively. The generated complex has the same magnetic response in a magnetic field, so as to achieve the purpose of separation, detection, purification of genes, proteins, cells, microorganisms and so on. The strong magnetism and monodispersity of magnetic microspheres have attracted wide attention in the field of immunochromatography.The research contents and methodsIn this paper, the immuno magnetic nanoparticles were prepared by EDC and NHS. Then they were used as detecting probes to construct the magnetic ICA test strip. Various components of the test strip (including conjugate pad, sample pad, and NC membrane) were optimized based on various factors that may affect the performance of the test strip (including the release of the pad, elimination of false positive and improvement of sensitivity). The detection time was determined according to the change of the magnetic signal in the chromatography process. The stability of the test strip was evaluated according to the line color after different storage time. The clinical application value of the test strip was further verified by the clinical serum samples, including the detection sensitivity and specificity, the cross reactivity with other antigens, the lowest detection limit, and the consistent with the results of ECLIA.The main research work including1. Preparation and verification of labeled probes. NHS and EDC were used to form active ester functionalities with carboxyl groups and then CA72-4 monoclonal antibody 1 (mAb1) was conjugated to the surface. The prepared nano particle is 136nm in diameter and has good monodispersity. The coupling effect and basic characteristics of immunomagnetic beads were verified through a variety of ways.2. Preparation of test strips. The test strips were prepared through double antibody sandwich method. We focused on optimizing parameters associated with the type of NC membrane, conjugate pad, sample pad, and components within the pretreating solution to achieve the best results of chromatography.3. Detection of CA72-4 based on double antibody sandwich method. The developed test strip was used to analyze serum samples. After determining the optimal detecting time, we quantified the content of CA72-4 by detecting the magnetic signal intensity.4. Evaluation of clinical application.100 clinical serum samples were detected by magnetic test strip and ECLIA method to verify sensitivity, specificity, lowest detection limit, cross reactivity, and the consistency with ECLIA. The results were used to further evaluate the feasibility of the application of the test strip in clinical detection.5. Stability of test strips. The aging process of strip can be accelerated in 37℃ and the test strips were regularly tested with same sample. The stability of test strips were evaluated through the color comparison of each result. After sample detection the test strip was stored at 60℃.The stability of magnetic signal was assessed through analysis the trend of T/C values in 3,9,12,15 days.The research results1. By optimizing the coupling reaction, EDC/NHS could successfully combined monoclonal antibody (MAb1) with the carboxyl magnetic beads through chemical bond. The coupling rate was 35μg mAbl/lmg SPMNPs. And through comparison of different magnetization, particle size, surface charge, ultraviolet and infrared absorption peak and mobility situation in agarose gel electrophoresis of magnetic beads to further verify the coupling effect, as well as the characterization of immunomagnetic beads.2. In order to improve the sensitivity and reduce the nonspecific reaction for immunochromatographic detection, by optimizing each component of the test strip, the final determination of the type of NC membrane was Pall Vivid 170 with a length of 1.7 cm. The antibody coating concentration was 2 mg/mL in T line and 1 mg/mL in C line. The material of conjugate pad and sample pad was glass fiber. Sample pad was pretreated with borate buffer (0.02 M pH 7.4) containing 2%(w/v) NaCl,5% (w/v) BSA,0.2%(w/v) PVP, and 0.05%(w/v) Triton X-100. Conjugate pad was pretreated with borate buffer (0.02 M pH 7.4) containing 5%(w/v) sucrose+2%(w/v) trehalose+0.05%(w/v) Triton X-100.3. By comparing the changes of magnetic intensity in T line and C line, the test results were proved to reach a stable state after 20 minutes. We observed a proportional change in the color of T line associated with the CA72-4 concentration in serum samples, and furthermore, the qualitative detection sensitivity is 1 IU/mL. The gradual increase in the obtained T/C ratio is proportional to the sample concentration of CA72-4 according to a linear relationship in the range of 0-100 IU/mL. The resulting calibration was a linear model (y= 0.00892x+0.08, R2= 0.9996). The detection limit is 0.38 IU/mL4.100 clinical serum samples were simultaneously detected by magnetic test strip and ECLIA method. The results of ECLIA method were used as the standard. The detection sensitivity and specificity of immunochromatography is 99% and 93% and no statistical difference exists in the two methods(x2= 0.004; P= 0.95).Detection with other antigens (CA199, CEA, HCG) proved that the test strips had no cross reaction with other antigens. Sample testing recovery rate of the method was 98.4%-102%(SD 3.5%-8.2%), which indicates that in the course of the immune chromatography, other substances in the serum will not interfere the detection of CA72-4.5. The T line and C line were still be clearly visible in the 150th days after the accelerated aging; Magnetic nanoparticles on the NC membrane could remain stable at 60 "C, which guarantee the stability of repeated measurement.The research conclusionIn this study, we established magnetic lateral flow immunoassay strip with SPMNPs as markers to initially realize the rapid detection of CA72-4. Immunochromatographic test strip not only provide qualitative analysis, but also quantitative result. The detection sensitivity can reach the requirement of clinical application. Compared with conventional detection methods, the operation time is shortened, the operation steps are simplified, and the detection cost is reduced greatly without affecting the accuracy of qualitative and quantitative results. ICA has achieved rapid, simple and accurate detection of blood tumor markers, and provides a new fast and efficient detection method for clinical detection of CA72-4. The method is also applicable to primary hospital or as a means of self-examination to facilitate the monitoring of GC patients. ICA has good application prospects in improving survival rate and life quality of patients with gastric cancer. |
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