| BackgroundTuberculosis (TB) is a chronic disease spreaded through the respiratory tract, mainly infect lung. It is also an important worldwide infectious disease caused by single pathogen with highest mortality. Therefore, a simple, fast and accurate laboratory examination of tuberculosisas is very important for early diagnosis to control this disease. At present, the diagnosis of TB is principally through bacteria examination and culture. But the culture needs relatively long time and the sensitivity of bacteria examination is only approximately 50%. After tuberculosis infection, the antigens appear firstly. So, the detection of mycobacterium tuberculosis (MTB) antigens plays a very important role in the early diagnosis. The antigens can be regarded as the direct evidence for MTB existence. Meanwhile, compared to MTB antibody detection, antigens detection can avoid the false negative resulting from low immune responses of humoral immune or cellular immune, and it is more likely to become the early rapid sensitive specific new diagnostic method. The earliest MTB antigen is aquired from the MTB culture in liquid medium containing glycerol, known as the old tuberculin. Since 1932, purified protein derivation of tuberculin (PPD) is obtained via further purified by purification from the old tuberculin. It is mainly used for skin test. Because of the antigen composition of MTB is complex and it share components with Non-Tuberculo-Mycobacterium and BCG, so the specificity is low, and it can not distinguish between infected and BCG vaccinated at the same time. Furthermore, it is not sensitive to open late phase and systemic tuberculosis.Culture filtrate protein 10 is a 10KDa antigen, which was found in the culture filtrate of MTB in recent years. The protein is highly specific to M.tuberculosis and M.bovis, absent from BCG strains and Non-Tuberculo-Mycobacterium, with satisfactory immunogenicity. In a word, CFP10 can be used as a specific candidate antigen for diagnosis of tuberculosis. But usually, levels of CFP10 in tuberculosis patients' serum or body fluids (phlegm pleural effusion in cerebrospinal fluid (CSF) joint effusion, etc.) are too low to be detected by ELISA or colloidal gold immunochromatography.Time-resolved fluorescence immunoassay (TRFIA) is a kind of new marker analysis technique developing since the 1980s. Compared to other immune marker analysis technology, TRFIA has its unique advantages. It overcomes the radioactive immune analysis (RIA) pollution caused by a radioactive isotope, and the unstable shortcomings of enzyme immune process (EIA). Besides, TRFIA can well eliminate interference of background fluorescence, which makes the sensitivity much higher than normal fluorescence method. High sensitivity (10 to 18 mol/hole), convenient operation, easy automation, a wide range standard curve, free from sample natural fluorescence interference, simple marker, stability, without radioactivity pollution, multi-label. All the characteristics make it become a highly potential clinical application for ultra trace analysis.In the study, we prepared recombinant proteins CFP10 and CFP10/SA by genetic engineering techniques. After purification, renaturation and identification, the proteins were evaluated by double-antibody sandwich TRFIA in sensitivity, linear range and stability. Then the optimal reference standard for detection of CFP10 by TRFIA was screened. At the same time, we developed double-antibody sandwich TRFIA for CFP10 preliminarily.ObjectiveTo prepare recombinant proteins CFP10 and CFP10/SA, screen the best as the reference standard for detection of CFP10 by TRFIA and develop double-antibody sandwich TRFIA for CFP10 preliminarily.Methods1. Construction of prokaryotic recombinant plasmidsThe DNA encoding CFP10 were required by PCR with different primers, and cloned into the vectors PET21a-SA, PET24b and PET24b-SA. Then, the expression plasmids were verified by DNA sequencing.2. Expression and purification of recombinant proteinsE.coli cells transformed with the expression plasmids were cultured in 3ml Luria broth medium with antibiotics (kanamycin or ampicillin). When OD600 of the culture reached 0.4-0.6, lmmol/L IPTG was added to induce the expression of fusion proteins. Expression conditions including incubation temperature and time were optimized. The cells were harvested by centrifugation. The pellet was disrupted in cell lysis buffer by sonication. The expressed proteins were analyzed by SDS-PAGE. After washing, the pellets were dissolved in 8M urea, filtered by 0.22μm membrane and purified by Ni-NTA. The soluble protein was filtered directly by 0.45μm membrane and purified by Ni-NTA as well.3. Refolding of recombinant proteins and Western blottingThe purified fusion proteins were adjusted to 0.2mg/ml and refolded by dialysis against 50 vol buffer (20 mmol/L Tris-HCl,6 M Urea,0.5 M L-Arg,0.1% PEG8000, 0.5 M EDTA) with slowly stirring at 4℃. Solution was replaced every 4 hours to final urea concentration 0M. At last, the refolding system was dialyzed against PBS for 12-24 hours. The dialyzed product was centrifugated to remove the sediment and filtered by 0.22μm membrane, stored in -20℃. The purified and refolded fusion protein was separated by SDS-PAGE and electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked for 2 hour at 37℃in phosphate buffered saline with 0.5% Tween-20 (PBST) supplemented with 5% skim milk. Then it was incubated with monoclonal anti-Mouse CFP10 antibody. After washing, the membrane was incubated with goat anti-mouse antibody conjugated with horseradish peroxidase in PBST for 1 h. After extensive washing of the membrane, the blot was developed with DAB display liquid kit. 4. Characterization of reference standards for time-resolved fluoroisnmunoassay of culture filtrate protein 10Paired anti-CFP10 monoclonal antibodies and CFP10 antigen standard were used to establish double-antibody sandwich TRFIA. CFP10 and CFP10/SA were evaluated by optimized TRFIA reaction model in sensitivity and stability.Results1. Construction of recombinant plasmidsThe nucleotide sequence of CFP10 and SA were confirmed by DNA equencing. It was identical with standard nucleotide sequence.2. Preparation of recombinant proteinsThe fusion proteins SA-CFP10 and CFP10-SA were expressed at about 25% and 30% respectively of total cellular protein, both in the form of inclusion body in E.coli. The protein CFP10 expression was about 35% of total cellular protein in soluble form. The inclusion body was washed and dissolved with 6M urea. After membrane filtration, The proteins CFP/SA and CFP10 were purified via Ni-NTA column, proteins purity could reach 90%. The CFP10/SA fusion protein polymers could be obtained after renaturation. Soluble CFP10 was also refolded after light denaturation in 2M urea. The refolded proteins were stored at-20℃.3. Optimal reference standard for time-resolved fluoroisnmunoassay of culture filtrate protein 10Antibody pairing results showed that the best coated antibody concentration was 3ug/ml and the labelled antibody was 0.8ug/ml. The optimal reference standard CFP10/SA was confirmed by TRFIA in linearity, sensitivity and stability. The linear range is 0.1-80ng/ml; the minimum detection concentration can reach 0.02ng/ml. Meanwhile, maximum degradation rate were less than 8% after CFP10-SA respectively placed 7,14,21 and 28 days in 37℃. The system had good accuracy with intra-assay coefficients of variation below 6.0% and the inter-assay coefficients of variation below 5.0%. The experiment on the recovery rate revealed that the. average recovery rate is 87.2%. Double logarithm curve showed correlative coefficient R2=0.9997, also has better repeatability.Conclusions1. CFP10/S A and CFP10 recombinant proteins were prepared sucessfully 2. Double-antibody sandwich TRPIA for CFP10 was established preliminarily andthe optimal reference standard for TRFIA of CFP10 antigen is-CEP10-SA... |
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