| Owing its specificity to bacterial infections,PCT has been proposed as a pertinent marker in the rapid diagnosis of bacterial infection,especially for use in hospital emergency departments and intensive care units.The serum PCT concentration can be used to effectively determine whether the patients have bacterial infections,and to guide the use of antibiotics in clinic.As the detection of the use of a research tool,PCT antibody,has been in short supply in China and has become an important obstacle to the research and clinical application of domestic reagents.This study intends to improve the monoclonal antibody preparation technology to obtain high affinity PCT paired antibodies.The preparation procedures of monoclonal antibodies mainly include animal immunization,cell fusion,hybridoma positive clone screening and cloning,and antibody preparation in large quantities.During the immunization process,the specific immune response of the antigen is enhanced by adding an immunoadjuvant to obtain more sensitized B lymphocytes.Cell fusion refers to the mixing of syngeneic myeloma cells and mouse spleen cells in a certain ratio,and the addition of a fusogenic agent polyethylene glycol.Due to the action of polyethylene glycol,a lymphocyte can be fused with a myeloma cells to form a hybrids.Only a small number of hybridoma cells growing in HAT medium can secreting a predetermined specific monoclonal antibody,so it is necessary to further screen for positive clones by ELISA,and then clone the hybridoma cells by limiting dilution method.Finally,the immunoglobulin types,subclasses,specificities,affinities,epitopes and molecular weights of antigens secreted by cell lines will be identified.Although the standard procedure for the production of monoclonal antibody has been used to date,in the various technical aspects of animal immunization,cell fusion,and positive clone selection,a number of bottlenecks are retained making antibody preparation time-consuming and labor-intensive.Therefore,in view of these significant problems in the preparation of monoclonal antibodies,this study started from three aspects:animal immunization,cell fusion and hybridoma cell cloning.Technical updates were made to simplify the preparation steps and save preparation time,and thus increase the production efficiency of cloned antibodies.We had prepared and screened anti-procalcitonin paired antibodies by using the modified monoclonal antibody preparation technology.Besides,we innovatively synthesized a novel graphene-gold nanoparticle nanomaterials,and combined with terminal detection signal amplification system to build a new efficient PCT immunoassay system.Some HPVs(Human papilloma viruses),are etiologically linked with several types of cancer,such as skin cancers,head and neck cancers,oropharyngeal cancers,lung and respiratory cancers and anogenital cancers.The most common and dangerous HPV type is HPV 16.Due to the difficulty of cervical cancer treatment and the unsatisfaction of clinical outcomes,the survival rate of women after suffering from cervical cancer is extremely low.Thus,detection of high-risk HPV16 is considered to be potentially useful in three clinical applications:first,as a primary screening test to detect cervical cancer precursors;further as a triage test to select women needing referral for diagnosis and treatment and,finally,as a follow-up test for women treated for high-grade intraepithelial lesion with local ablative or excisional therapy to predict cure or failure of treatment.To date,many approaches to diagnose HPV infections have been developed relying on detection of viral nucleic acids(DNA or RNA),e.g.However,methods aiming to detect nucleic acids(DNA,mRNA)have also obvious shortcomings,including very complicated operations and the associated high cost,need of amplification,and involvement of varies of instruments that may not able to reflect the actual viral load relevant to the patient’s course of disease as well as the risk of virus transmission.Therefore,there is an urgent need to develop a method which could overcome these shortcomings.To overcome these problems,we applied gold nanomaterials,RT-LAMP and enzyme catalysis technology to construct a new high-efficiency detection system for HPV16 mRNA E6/E7.The design strategy of the technology is not only reflect the state of infection,but also realize the possibility of absolute quantification.The specific research mainly focus on the following five parts:1.Expression and purification of PCTThe prokaryotic expression system has many advantages such as convenient operation,quickness,short time,large expression,and suitable for industrial production.In this part,pET32a-PCT vector was used to express PCT in E.coli.IPTG was added to induce protein expression while cells were grown to OD600 nm of 0.6-0.8.We has explored the main factors including the yield and efficiency of protein expression,and the induction temperature,time,and concentration of the inducer,that have a greater influence on expression conditions.The optimum condition for expression of PCT is 30℃,induction time of 6 h,and IPTG 0.4 mM.The protein was purified and analyzed by SDS-PAGE and western blot for purity>90%.2.Improvement of monoclonal antibody preparation technologyIn view of these significant problems in the preparation of monoclonal antibodies,this study started from three aspects:animal immunization,cell fusion and hybridoma cell cloning.Technical updates were made to simplify the preparation steps and save preparation time,and thus increase the production efficiency of cloned antibodies.The specific research mainly focus on the following three parts:(1).Construct a new strategy of animal immunization to improve the specific immune responses to antigenIn order to improve the acquisition rate of sensitized B lymphocytes with higher specificity,we first reconstructed the immunization strategy in the part of animal immunization.Based on the classical immune combination strategy,we have tried a variety of formula combinations.While using Freund’s adjuvant stimulation,we designed to use cytokine stimulation to synergistically improve the efficiency of immune response.After multiple rounds of screening,we obtained a synergistic combination of cytokines.The Mix recipe was finally built.The adjuvant stimulation was used as a control group(group P).By analyzing the results of these two immunopotency results,we found that the primary immunization titer of the mice,in the Mix immunization group,was higher than that in the group P.The titers of the Mix group was higher than group P.The results of the two groups of immunization showed that the Mix formula can effectively increase the acquisition rate of specific sensitized B lymphocytes,and the titer after the third immunization can reach more than 80000,and the titer of cell fusion can be achieved at this time.(2).Screening and enriching B cell clones with higher antigen specificity to improve the efficiency of hybridoma cell fusionBinding of the antigen with a B cell’s specific antigen receptor triggers activation and differentiation of the B cell into antibody-secreting plasma cells or memory B cell.Although the immune system can respond to a large number of antigens,only thousands of B cells can express receptors specific for the given epitopes,and the frequency of cells producing a particular antigen is typically less than 1%.The classical cell fusion process is to isolate all cells in the spleen and make a single cell suspension for cell fusion.During the fusion process,a large number of non-antigen-specific cells interfere with each other,forming a large number of null clones and increasing the workload at screening.To address this problem,we designed two protocols for antigen-specific B cell isolation and enrichment to obtain high-purity antigen-specific B cell suspensions.The specific protocol is as follows:Firstly,the BCD22+CD5-cell is isolated and used for subsequent fusion.The main advantage of this method is that,the sorted cells can be clearly distinguished between its developmental and differentiation stage based on the expression pattern of a particular cell surface marker.We used another separation method based on magnetic bead sorting system.First,the biotin-labeled antigen is incubated with the spleen single cell suspension.Second,the antigen-bound B cells are captured by the avidin-labeled magnetic beads.This method is highly accurate and reproducible and can capture antigen-specific B cells directly.Moreover,the separation of specific B cells accounts for about nine ten thousandths of the total cells,and has the advantages of simple operation and short time.The results of the two sortings are as follows:Although the number of cells sorted by FACS is very high(33%-44%of spleen),subsequent fusion results indicate that the FACS sorting system has a large damage to the cell membrane and is not suitable for cell fusion.Although the magnetic bead sorting system only separates 104 antigen-specific B cells(0.9‰ of total cells),the positive rate of hybridoma cells after subsequent fusion is higher.(3).High content screening improves the screening rate of positive clone of hybrid tumor cellsA considerable proportion of unrelated cell fusions can be produced during cell fusion,so the hybridoma positive clones need to be screened and cloned.The commonly used method ELISA for screening hybridoma positive clones has the advantages of high sensitivity,high specificity.But it still has the disadvantages of cumbersome steps,long time-consuming,and high volume of samples required for detection.In response to the above problems,we designed two new screening protocols.The first protocol was use the SP2/0 cells with green fluorescent protein(EGFP-SP2/0)to replace the original SP2/0 for fusion.The successfully hybridized hybridoma cells can secrete antibodies and display green fluorescence,which can be further screened by high content screening.The second option is to use antigen-labeled magnetic beads.After incubation with the cloned wells,they were incubated with FITC-labeled goat anti-mouse secondary antibody and screened for clones using a high-content system.The experimental results of the two screening schemes are as follows.In the first protocol,only the successfully hybridized hybridoma cells survive after screening with the HAT selection medium.Surviving hybridoma cells all show green fluorescence,and the high content screening allows for easy and rapid screening of cloned wells.However,hybridoma cells with green fluorescence gradually lost the ability to secrete antibodies during the subsequent cloning process.At last,positive clones secreting antibodies were not obtained,and the specific mechanism needs further exploration.The second screening protocol can identify positive clones simply and quickly by using the high content screening analysis system.4.Synthesis of novel graphene-AuNPs nanomaterials and the signal amplification effect of terminal detection based on themIn healthy individuals the PCT content is at a low level(<0.1 ng/mL),the conventional detection method is almost undetectable or the detection value is very low,so a relatively sensitive detection method is required.The commonly used detection method in clinical practice is the immunochemiluminescence method developed by Roche.Due to the extensive clinical application of PCT,more detection methods need to be developed to meet the application of different fields and scenarios.For the first time,we successfully synthesized rGO-AuNPs nanocomposites by electrostatic adsorption,and obtained nanocomposites with immobilized antibodies with stable and excellent performance,which significantly improved the response performance of the sensor interface of the immunosensor.Secondly,due to the excellent characteristics of AuNPs,the electrochemical immunoassay technology and tyramine signal amplification(TSA)technology are skillfully combined to construct a highly sensitive and highly specific PCT analysis and detection system.The detection limit of the detection system reaches 0.1pg mL-1,and the linear range is 0.05-100 ng mL-1.Compared with the equivalent detection method,not only is the minimum detection limit 10 times higher,but also the required sample volume is reduced by 5-10.Multiple(10 μL).5.Combined with gold nanomaterials,RT-LAMP,enzyme catalytic technology to construct a new system for detection of HPV16 E6/E 7 mRNACurrently,screening targets for clinical cervical cancer are mainly targeted at HPV nucleic acids(DNA and RNA).These detection methods include hybrid capture signal amplification methods such as HC2(Qiagen/Digene),real-time quantitative PCR methods such as Cobas HPV(Roche),restriction enzyme amplification methods such as Cervista(Hologic),and NASBA methods such as Aptima(Gen-Probe).However,these detection methods have many drawbacks.On the one hand,due to the transient and repetitive infection characteristics of HPV,the detection method of HPV DNA has a high probability of false positives and false negatives,which may cause misdiagnosis and missed diagnosis.On the other hand,due to the current detection of HPV RNA.There are a few methods,and the amplification of the target sequence requires a high-performance nucleic acid amplification instrument,which not only greatly increases the cost input,prolongs the detection time,but also does not reflect the true viral load,so it cannot be used for clinical HPV infection.The exact stage of the disease.Furthermore,E6/E7 is small in cervical cells and is easily cleared by immune cells.Therefore,there is an urgent need to develop a highly sensitive,highly specific,and reliable new detection method for improving the deficiencies in cervical cancer screening.In response to these shortcomings,we used the ssDNA probe-modified gold nanoparticles to capture the HPV16 E6/E7 mRNA product amplified by the loop isothermal nucleic acid amplification technology,and combined with the new detection system of enzyme catalytic signal amplification to achieve the target HPV16 E6/E7 mRNA highly sensitive and highly specific detection.The detection system has three advantages:First,RT-LAMP can work efficiently at a constant temperature,greatly simplifying the requirements of the reaction equipment.Secondly,the amplified product can be captured by a DNA probe modified on gold particles(AuNPs),thereby specifically enriching the RT-LAMP amplification product onto the graphite electrode(GE),further enhancing the specificity of the biosensor.Sex and sensitivity.Finally,the enzymatic reaction has high catalytic activity,high efficiency and good selectivity.The introduction of more HRP to the electrode by the biotin-streptomycin affinity system not only enhances the sensitivity of the detection system,but also reduces the detection cost.After triple signal amplifications,the detection sensitivity of the detection system is as low as 0.08 fmol/mL,and the linear range spans 6 orders of magnitude. |
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