A Washing-free Gold Nanoparticles Based Plasmonic Immunoassay For Mouse Sendai Virus

Objective Sendai virus（SeV）is one of the pathogens that must be excluded from the national standards of experimental animal quality control（GB/T14926.23-2008）of Specific Pathogens Free（SPF）or above levels of experimental mice.SeV is often recessive infection in experimental mice.Traditional serological methods could not detect the pathogen in time,which affected the results of animal experiments.Therefore,it is urgent to establish a detection method for SeV pathogen.Therefore,in this study,a SeV pathogen detection method was established by using no-wash nano plasma immune technology,which has the advantages of simple use,convenient operation,high sensitivity and strong specificity.It can detect SeV pathogen accurately and quickly,and realize large-scale,rapid and timely monitoring of SeV.Methods According to the codon bias of Escherichia coli,major SeV immunogenicity genes HN and F（Gene Bank: NC₀01552.1）were selected for codon optimization,respectively,and prokaryotic expression recombinant plasmids p GEX-6p-1-HN,p ET-32a-HN,p ET-28a-F and p GEX-6p-1-F were constructed.Ten female BALB/c mice aged 4 to 6 weeks were randomly divided into two groups with 5 mice in each group inoculated with purified GST-HN and His-F recombinant proteins for 6 times.The antibody titers of mice were monitored by indirect ELISA assay using recombinant proteins GST-F and His-HN as coated proteins from the third immunization.After the sixth immunization,the mice with the highest titers were selected for enhanced immunization and cell fusion was performed.The positive hybridoma cells obtained by subclonal screening were cultured and injected into the abdominal cavity of mice to prepare monoclonal antibody to ascites,which was purified by caprylic acid-ammonium sulfate method.Matching test was conducted to determine the best antibody combination.Colloidal gold nanoplasma solution was prepared by lemon tri-sodium reduction method,and the p H value of the reaction and the amount of respective antibody labeling were optimized.SeV wasser-free nanoplasma immunoassay method was established,and its specificity,sensitivity and repeatability were evaluated.And compared with RT-PCR method.Results In this study,recombinant expression plasmids p GEX-6p-1-HN,p ET-28a-F,p GEX-6p-1-F and p ET-32a-HN were successfully expressed in the prokaryotic system.Mice were immunized with purified recombinant proteins GST-HN and His-F respectively,and the titer reached 1:16000.Then,three hybridoma cell lines with stable anti-Sev HN antibody were obtained by using hybridoma cells to prepare monoclonal antibody technology.They were named as 2F9,5D8,10H2 and two hybridoma cell lines with titers that could secrete antibodies against SeV F protein,named as 1H4 and 3E2,respectively.The five cell lines could secrete antibodies stably.The hybridoma cells were injected into the abdominal cavity of mice,and ascites monoclonal antibodies against SeV virus were successfully prepared and purified.The titer of purified antibody reached1:16000.After subtype and specificity identification,the results showed that the four monoclonal antibodies of 2F9,5D8,10H2 and 3E2 were Ig G1 subtype and1H4 was Ig G2 a subtype,all of which could bind specifically to SeV.The results of pairing experiment showed that 2F9 and 5D8 had the best pairing effect,the optimal p H of reaction was 8.0 and 8.5,and the optimal dosage of antibody labeling was 0.6 μg and 0.4 μg,respectively.The optimal dosage of chlorauric acid and hydroxylamine were 0.6 μL（concentration 3.9 μg/μL）and 1.0μL（concentration 1.078 mg/μL）,respectively.Based on the above optimized reaction conditions,washing-free gold nanoparticles based plasmonic immunoassay for mouse Sendai virus was established,and the process of color development could be completed in 2 min.Specificity experiments showed that this method only had specific binding reaction to SeV,and had no cross reaction with other mouse viruses in GB/T14926.23-2008,showing good specificity.In addition,the minimum virus volume of the method was 1.46×104 copies/μL,showing good sensitivity.The results of repeatability test are the same,indicating that the method has good repeatability.The coincidence rate of this method applied in clinical detection and PCR detection results was 98.3%.Conclusions1 、 3 strains of monoclonal antibodies against HN were prepared by hybridoma cells,and 2 strains of monoclonal antibodies against F protein.The titer of purified antibodies reached 1:16000.2 、 Using the best paired monoclonal antibody as the reactant,SeV washerless nano plasma immunoassay was successfully established.The method is simple in operation,rapid in response,the results can be identified by the naked eye,and has good specificity,repeatability and sensitivity,realizing the rapid detection of SeV pathogen.