| OBJECTIVE:1. To establish an up-converting phosphor technology-based ten-channel lateral flow (UPT-TC-LF) assay using up-converting phosphor(UCP)as a marker and UPT-LF single target strips as the experimental platform.2. To detect ten kind antibody targets in serum samples of plague animals and patients using the UPT-TC-LF assay, and to search the antibodies maybe used as the sublimentary plague diagnostic markers besides F1.METHODS:Firstly, eight proteins of Y. pestis were cloned and expressed using an E. coli expression system, purified with the Ni-NTA affinity chromatography, and renatured the inclusion body expressed proteins using a urea gradient dialysis method. The polyclonal antibodies corresponding to ten proteins were prepared by immunizing New Zealand white rabbits using the conventional method. Bloods were obtained from rabbits for collection of sera and purification of specific immunoglobulin G (IgG) with a caprylic acid-saturated ammonium sulfate precipitation method.Secondly, using the antigen covalently conjugated with UCP and the paired antibody as matiarals, 10 kind UPT-LF single target strips were developed with the double-antigen sandwich mode to detect the antibodies. And their performances, including the linearity, sensitivity, reproducibility, and stability, were evaluated through the simulated samples detection.Then, the uniform sample-treating procedure was obtained through the systematic optimization of experimental conditions corresponding to each kind strip. 10 kind strips were integrated into a UPT-TC-LF disc for simultaneously detection of 10 different antibodies under the same experimental conditions. A biosensor with a scanning function was also developed to acquire the sample results. The concistency performances of the UPT-TC-LF assay and the UPT-LF single target strip assay were evaluated through the simulated samples detection. Finally, 118 copies (13 monkeies, 54 rabbits, and 51 patients) actual serum samples infected with Y. pestis were detected using the UPT-TC-LF assay, and the qualitative results were compared with those of ELISA assay. Western Blot (WB) assay was used as the confirmatory method to validate any discrepant results of monkey sera samples.RESULTS:1. Eight proteins were successfully expressed and purified in E. coli as soluble (YPO1613, YopD) or inclusion body (YPO1089, YPO1303, YPO1435, YPO2131, YPO2118, YPO3827) form. The proteins expressed as inclusion body were all refolded.2. Ten kind specific polyclonal antibodies paired corresponding proteins were accquired. The ELISA titers and quantities (mg/ml) were as follows: 1:51,200 and 9 for YPO3827, 1:204,800 and 20 for YPO2131, 1:204,800 and 40 for LcrV, 1:109,600 and 12 for YPO1089, 1:25,600 and 22 for YPO1303, 1:51,200 and 25 for YopD, 1:204,800 and 29 for YPO1435, 1:204,800 and 17 for YPO1613, 1:204,800 and 12 for FI, and 1:51,200 and 17 for YPO2118,respectively.3. Ten kind UPT-LF single antibody target strips were developed. The detection results of simulated samples showed that they all had good performances. The linearity fitting coefficient of determination (R ) of them is between 0.93 and 0.99. The sensibility is comparable with that of ELISA for 5 kind strips and slightly lower for the other 5. The stabity phase of them is between 10 and 21 days at 37°C. The CV value of the reproducibility index is between 6.4% and 15.2%.24. The UPT-TC-LF system to test 10 targets simultaneously, including the UPT-TC-LF disc and TC-UPT biosensor, were successfully developed. The detection results of simulated samples showed that the performance of the UPT-TC-LF disc and that of the single-target UPT-LF strips had good consistency, including the samples detection values and the coefficients of variations (CV). So the performance of the UPT-TC-LF system including the linearity ability, the sensitivity, the reproducibility and the stability all can use those of the strips.5. The antibody profiling results of true sera samples infected Y. pestis detected by UPT-TC-LF assay and ELISA assay had some discrapency according different samples genus. Took every antibody taget in every serum sample as one copy sample, the qualitative results of all copies detected by the two assays were compared.Among 130 copies of 13 monky serum samples, 112 are accordance and 18 are different, without statistical difference. Using the result of WB assay for the 18 different copies as the gold standard, 11 results of UPT assay and 7 results of ELISA were correct. The positive ratio of 6 kinds antibody targets among 10 targets in monkey sera samples are higher than 50%, which is F1-Ab(100%), YopD-Ab(100%), LcrV-Ab(92%), YPO1303-Ab(64%), YPO2118-Ab(55%), and YPO3827-Ab(50%).Among 510 copies of 51 rabbit serum samples, 349 are accordance and 191 are different. To calculate the positive ratio of antibody targets with the uniform qualitative results detected by two methods, there have 3 kinds antibody targets in rabbit serum samples higher than 50%, which is F1-Ab(100%), YPO1089(75%), and YopD-Ab(71.9%).Among 510 copies of 51 patient serum samples, 391 are accordance and 119 are different. To calculate the positive ratio of antibody targets same as that of the rabbit sera, there have 4 kinds antibody targets in rabbit serum samples higher than 50%, which is F1-Ab(100%), YopD(83%), YPO1435(61%), LcrV(54%).CONCLUSIONS:1. The study successfully developed a new UPT-TC-LF assay platform. It is a versatile system that could be not only used for multi-antibody detection, but also adapted for multi-pathogen examination in microbiology or other research fileds. With the development of new UPT-LF strips, this system could also be adapted for many biodetection fields, such as disease's markers test, or food safety control, or water recource detection, et al.2. The 10 kind antibodies against Y. pestis in the serum samples of plague animals and patients were detected by the UPT-TC-LF system, and the antibody spectrum in samples showed some change according different genus sera. Yet the positive ratio of the F1–Ab was all 100% in different plague animals and patient sera, which vetified once again that F1 was the most reliable markers for plague diagnosis. In addition, the YopD-Ab also showed high positive ratio in all different genus sera, it implied that YopD maybe take as the potential sublimentary diagnostic marker of plague besides F1. |
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