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  • Common enzyme quantitative methods include enzyme linked imm

    2020-11-20

    Common enzyme quantitative methods include enzyme-linked immunosorbent assay [,] (ELISA), chemiluminescence (CLIA) [, , ] and immunochromatography []. The advantages of ELISA are cost reduction and automation, but non-specific reaction caused by HOOK effect, false positive caused by cross-contamination, low sensitivity, low detection rate of window phase is its inherent defect. As a new detection method in recent years, CLIA method [,] has good specificity for immunological reaction and high sensitivity for the detection of luminescence method. Compared with ELISA, CLIA is a heterogeneous immunoassay, so that antigen and antibody can bind efficiently without the shortcomings of traditional one-step method. At the same time, CLIA results are more stable, and the detection time is shorter. However, few reports have been found on the clinical application of DNMT1 using the CLIA without the participation of enzyme. In this study, we developed a novel magnetic enhanced chemiluminescence enzyme immunoassay (MCLEIA) to achieve the simple, economical and easily commercialized DNMT1 detection in serum samples, and this method was compared with the purchasable ELISA kits. The experimental conditions, including the reaction time of DNMT1 and [email protected], the dilution ratio of PcAb, and the dilution ratio of HRP-second-Ab were examined and optimized. The methodology parameters including precision, accuracy and specificity were also evaluated. By comparing the established two methods, the results indicated that MCLEIA has shorter total reaction time, fewer capture antibodies, and lower detection limit. Therefore, the proposed MCLEIA could be used for the high-throughput detection of DNMT1 in clinical (). The experimental section, including materials and instruments, Adenosine Kinase Inhibitor hydrate receptor and solutions, immobilization of antibody on magnetic beads, characterization of the [email protected], theory and program of MCLEIA immunoassay and theory and program of ELISA immunoassay can be found in Supporting information. We first conducted characterization of [email protected] The coupling rate determined by coomassie brilliant blue staining was more than 75%. As shown in Figs. A and B, the [email protected] surface morphology has changed; the surface is rough and covered with a layer of white substance, presumed to be McAb. Therefore, the results show that the coupling is successful. The C showed that the 20 times dilution of immunomagnetic beads has the highest absorbance compared with the other two dilutions and there is a gradient between the three groups. It was shown that the carboxylate magnetic particle surface was successfully modified with McAb and maintained the immune activity of the antibody, which can be used in subsequent experiments. The magnetism characterization of [email protected] were presented in D, from which we could see in the left figure, the magnetic bead solution was brown, the solution was well dispersed, and no precipitation occurred within a few hours without the effect of external magnetic field. In the right figure, the immune magnetic beads were rapidly enriched with good magnetic response, and the solution was partially clarified in magnetic field. Therefore, the prepared [email protected] still had good magnetic response. Next, we optimized MCLEIA method. [email protected] with different dilution ratios was optimized (A). The concentration of DNMT 1 was 100 ng/mL. When the dilution ratio of [email protected] was 1:40, the RLU intensity reached the maximum, and the difference between the experimental group and the control group was the largest. Therefore, 1:40 was chosen as the optimal dilution ratio of [email protected] Results as shown in B, the RLU value was relatively high when the reaction time between [email protected] and DNMT1 was 90 min and 120 min, but the RLU value did not change significantly after 90 min, indicating that the balance of antigen antibody complexes has reached stabilization. Thus, the optimal reaction time was 90 min for further experiments. After different dilutions of PcAb, the chemiluminescence values of DNMT1 (100 ng/mL) were determined. RLU was determined with 0 ng/mL as blank control, and the difference value RLU (RLU − RLU) was used to dependent variable (C). When the dilution ratio of PcAb was 1: 2000, the RLU intensity was the strongest. The result indicated the best dilution ratio of PcAb was 1: 2000. After different dilutions HRP-second-Ab, the chemiluminescence values of DNMT1 (100 ng/mL) were determined. RLU was determined with 0 ng/mL as blank control, and the difference value RLU (RLU − RLU) was used to dependent variable (D). When the dilution ratio of HRP-second-Ab was 1: 4000, the RLU intensity was the strongest. The result indicated the best dilution ratio of HRP-second-Ab was 1: 4000.