| ObjectiveToxoplasmosis is an anthropozoonosis that can seriously harm human health and the promotion of good prenatal and postnatal care. ELISA is currently the conventional detection method to diagnose the infection of toxoplasma. It can determine the infection in the body mainly through the detection of specific IgG (index to indicate the immunity level) and IgM (index to indicate recent infection). However, the method can not detect IgG and IgM antibody simultaneously; furthermore, it requires a long period of detection and consumes a large amount of reagent with complicated operation procedures.Quantum dot is a newly-developed material used in fluorescence labeling and it possesses the following advantages compared with traditional fluorescent dyestuff: it can arouse a wider range of wavelength, the light with the same wavelength can arouse different sizes of quantum dot; the emission wavelength is narrow and symmetrical, the overlapping part between emission spectrum is small when quantum dots with different spectral characteristics are used simultaneously; the quantum dots with different particle diameters and compositions have different emission wavelength. Therefore, the application of quantum dots with different emission wavelength in the labeling of material to be detected and analyzed can detect several indexes at the same time.The quantum dot labeling technique and immune magnetic bead technique were combined together in this research. A detection system which can detect anti-toxoplasma IgG and IgM antibody simultaneously was developed. The method can be operated conveniently and rapidly with high level of specificity and sensitivity. It can be used in the screening of toxoplasma infection. Method:1. Antigen solidified magnetic microsphereThe carbodiimide crosslinking method was adopted to solidify the recombined toxoplasma antigen onto the surface of the carboxyl magnetic microsphere. The carboxyl on the surface of the magnetic microsphere was activated by the EDC and NHS solution. Subsequently, the activated carboxyl reacted with the amino-group on the antigen in order to solidify the toxoplasma antigen onto the surface of magnetic microsphere and in order to obtain the immune magnetic bead. A stable and highly-efficient solidification method for immune magnetic microsphere can be established through the optimization of solidification conditions, including the particle diameter of the magnetic microsphere and the concentration of activating agents such as EDC/NHS.2. Labeling of second antibody using quantum dotThe carboxylated CdS/ZnS quantum dot and antibody were labeled using the carbodiimide crosslinking method. The quantum dots with the emission wavelength of 580nm and 618nm were used to label the sheep anti-human IgG antibody and the sheep anti-human IgM antibody. The effect of pH value, the application concentration of second antibody and the reaction time on the irradiancy of quantum dot was investigated. Quantum dot which was suitable for the synchronizing detection system for anti-toxoplasma IgG and IgM antibody was prepared in order to label the second antibody reagent.3. Establishment of independent detection methods for anti-toxoplasma IgG and IgM antibodyThe detection methods for anti-toxoplasma IgG and IgM antibody were established according to the theory of indirect method by taking the magnetic microsphere that had solidified the toxoplasma antigen as the solid phase carrier and by taking the second antibody that had been labeled by quantum dot as the detection antibody. Besides, the detection conditions were optimized. The standard samples of human anti-toxoplasma IgG and IgM were detected. The linear scope, sensitivity, repeatability and accuracy of this method were observed. Clinical samples were collected and were detected using this method and imported ELISA kit. The results were compared in order to determine if there was statistical difference between the results obtained using the two methods.4. Establishment of the synchronizing detection system of anti-toxoplasma IgG and IgM antibodyBased on the established detection method used in single antibody, the key detection conditions were unified and optimized. The synchronizing detection system of anti-toxoplasma IgG and IgM antibody was established. The standard samples of human anti-toxoplasma IgG and IgM were detected simultaneously. The linear scope, sensitivity, repeatability and accuracy of this system were observed. 280 clinical samples were detected simultaneously using the established synchronizing detection system and the results were compared to the results obtained using the imported ELISA kit. The feasibility of the synchronizing detection of several indexes was discussed.Main results1. Establishment of methodology(1) Solidification of magnetic microsphere by antigen The carboxyl magnetic microsphere with a particle diameter of 3μm was selected as the solid phase carrier. EDC (6mg/ml) and NHS (4mg/ml) were used for activation. The optimal solidification concentration for anti-toxoplasma was 50μg/mL with a solidification efficiency of 54.2%.(2) Labeling of second antibody by quantum dotThe experimental results show that quantum dots marking second antibody the optimum reaction pH value of 6.0, sheep anti-human IgG, IgM concentrations were optimal labelling concentration 40μg/mL, 50μg/mL,respectively.(3) Establishment of the detection method for anti-toxoplasma IgG and IgM antibodyThe detection methods used for anti-toxoplasma IgG and IgM antibody were established separately. Based on the optimization of the experiment, the optimal operation concentration of quantum dot sheep anti-human IgG and IgM was 1:50 and 1:100, respectively. The optimal dilution rate for the serum to be tested was 1:100. Based on the study on the detection of the serum samples from 200 healthy people, the Cutoff value of anti-toxoplasma IgG and IgM antibody was 0.45 and 0.44, respectively. (4) Establishment of the detection system for the synchronizing detection of anti-toxoplasma IgG and IgM antibodyBased on the synchronizing detection of anti-toxoplasma IgG and IgM antibody, the optimal operation concentration for the labeling of sheep anti-human IgG and IgM by quantum dot was 1:200 and 1:100, respectively. The optimal dilution rate for the serum to be tested was 1:100. Based on the study on the detection of the serum samples from 200 healthy people, the Cutoff value of anti-toxoplasma IgG and IgM antibody was 0.55 and 0.51, respectively. It took only 30min to detect the anti-toxoplasma IgG and IgM antibody while the conventional ELISA method can only detect one index each time. It usually took 4h to complete the two indexes.2. Methodological evaluation(1) Methodological evaluation on the detection method of anti-toxoplasma IgG and IgM antibodyStandard preparations of anti-Toxoplasma IgG and IgM antibody were taken to detect repeatedly for ten times with the coefficients of variation inter the batch were 5.2%, 5.4%, the minimum detection limit were: 3.04 IU/ml, 3.11 IU /ml. The standard preparations above were taken o detect once everyday , a total of ten times with the coefficients of variation between the batches were 6.5%, 6.6%. The accuracy of experimental coefficients of variation were: 6.0%, 6.3%. The 20 cases of other positive serum samples were no cross-reaction by these methods. Blocking experiments showed that IgG and IgM Toxoplasma gondii antibodies in the blocking rate of more than 50%.(2) Methodological evaluation of the synchronizing detection method for anti- toxoplasma IgG and IgM antibodyStandard preparations of anti- toxoplasma IgG and IgM antibody were taken to synchronizing detect repeatedly for ten times at the same time with the coefficients of variation inter the batch were 6.0%, 6.2%, the minimum detection limit were: 4.21IU/ml, 4.32IU/ml. The standard preparations above were taken o detect once everyday ,a total of ten times with the coefficients of variation between the batches were 7.2%, 7.4%. The accuracy of experimental coefficients of variation were: 7.6%, 7.2%. 3. Methodological comparison(1) Methodological comparison on the detection method of anti-toxoplasma IgG and IgM antibodyAfter the collection of clinical samples, the samples were detected using the established detection method for anti-toxoplasma IgG and IgM antibody and the results were compared to the results obtained using the imported ELISA kit. The coincidence rate for the detection results of anti-toxoplasma IgG and IgM antibody was 96.5% and 96.8%, respectively. The data were analyzed according to the pairedχ2 test using the SPSS10.0 software. All the P values were larger than 0.05. No significant difference was found between the detection results of anti-toxoplasma IgG and IgM antibody using the two methods.(2) Methodological comparison of the synchronizing detection method for anti-toxoplasma IgG and IgM antibodyAfter the collection of clinical samples, the samples were detected using the established synchronizing detection system for anti-toxoplasma IgG and IgM antibody and the results were compared to the results obtained using the imported ELISA kit. The coincidence rate for the detection results of anti-toxoplasma IgG and IgM antibody was 96.4% and 97.1%, respectively. The data were analyzed according to the pairedχ2 test using the SPSS10.0 software. All the P values were larger than 0.05. No significant difference was found between the detection results of anti-toxoplasma IgG and IgM antibody using the synchronizing detection system.Conclusion1. If the magnetic microsphere was use as solid phase carrier, its surface successfully solidify the recombined toxoplasma antigen with the highest solidification efficiency of 54.2%. By taking advantage of the rapid enrichment of magnetic bead, not only the detection speed was accelerated but also the detection sensitivity was enhanced greatly. It only took 30min to detect anti-toxoplasma IgG and IgM antibody simultaneously using this method; it was much faster than that of ELISA which took 2.5h to complete the whole process. 2. The sensitivity, detection scope, repeatability and stability of the established detection method for single index of anti-toxoplasma IgG and IgM antibody had meet the requirements of preliminary clinical application. That had laid the foundation for the synchronizing detection system.3. Different second antibodies were labeled using quantum dots with different emission wavelengths. The same exciting light was used in the arousing process and the light with two different wavelengths was emitted. The synchronizing detection of several indexes was realized and that provided technical support for the synchronizing detection of more antibodies based on the quantum dot labeling.4. The established synchronizing detection system for anti-toxoplasma IgG and IgM antibody had excellent specificity, high speed, convenient operation and high level of sensitivity. It created the opportunities to simplify the operation procedure and to shorten the report time. It also provided a new technical methods to further develop the synchronizing detection for several pathogen infected by TORCH.
|
PDF Full Text Request