Development Of Up-converting Phosphor Technology Based Lateral Flow Assay For Detection Of Zaire Ebolavirus

Background and ObjectiveEbola haemorrhagic fever is a highly infectious disease caused by Ebola virus,which is identified by WHO as one of the most serious viruses that have been harming humans with high pathogenicity and high mortality.Five genotypes of Ebola virus was discovered in the world.Zaire ebola virus,with the mortality of 53%～90%,cause the greatest harm,which have killed more than 11,000 Africans in 2014-2015.Ebola virus,a virus of Filoviridae familiy,is an enveloped single negative strand RNA virus with the genome of 18.9 Kb,encoding 7 structural proteins and a non-structural protein.The structural proteins,nucleoprotein and glycoprotein have many specific epitopes,so they are the first target for the rapid detection of Ebola virus.Many scientists had invented various rapid detection,espcially using the ucleoprotein and glycoprotein as a target.Some of them have applied to the clinical diagnosis of Ebola in Africa.The up-converting Phosphor Technology is using Up-Converting Phosphor connected to antigen or antibody.UPT has the advantages of no background,no quenching.It can improve the sensitivity,specificity,stability and quantitative detection ability of traditional immunochromatography.It has been applied to detection of aflatoxin,ricin and Foodborne pathogenic bacteria.This study will use the nucleoprotein as an antigen.The gene fragments of ZEBOV nucleoprotein willbe synthesized.Then it will be converted to the E.coli BL21/DE3,and induced to express fusion protein,named recombinant nucleoprotein.The rNP is going to be purified as an antigen to prepare and identify the specific monoclonal antibody.A double-antibody-sandwich model based Up-converting Phosphor Technology based Lateral Flow assay strips for detecting ZEBOV was established.Specificity of this method were evaluated by detecting the proteins of dengue virus type 1(DV1),chikungunya virus(CHIKV),zika virus(ZIKV),Japanese encephalitis virus(JEV)and the E.coli cells strain BL21/DE3.Sensitivity was evaluated by detecting gradient dilution of rNP samples,and a standard curve was produced.It will provide a new choice for rapid,specific and sensitive and quantitative detection of Zaire Ebolavirus.Methods1.Bioinformatics analysis of ZEBOV structural protein.Using bioinformatics analysis software of Protean,Clustal_X and BLASTN,gene and amino acid sequences of ZEBOV collected from Genebank database was analysised.Antigen fragment of epitope and homology was clarified,and the codon of open reading frame was optimized.2.Preparation of ZEBOV recombinant nucleoprotein.The rNP encoding gene fragment of ZEBOV was synthesized.The recombinant plasmidrNP encoding gene fragment was inserted into the expression vector of pET-28(a).And then the recombinant plasmid was transformed into BL21/DE3 engineering bacteria to construct recombinant rNP bacteria.The expression was induced,and the rNP was purified by His-tag and liquid chromatography.3.Preparation of monoclonal antibodies against ZEBOV recombinant nucleoprotein.McAb was generated by immunizing 7 weeks female Balb/c mice with purified recombinant nucleoprotein.The mice spleen cells with anti-serum titer were fused to the murine myeloma.Hybridomas were successively selected using ELISA and limiting dilution.Ascitic fluid was generated by injecting hybridoma cells into Balb/c mice.McAb was purified with affinity chromatography.Indirect ELISA was used to detect ascites titer.4.Assembly and optimization up-converting phosphor technology based lateral flow assay.Two monoclonal antibodies against ZEBOV recombinant nucleoprotein was picked out in the last step.One of them was labeled with up-converting phosphor,and the other one was immobilized on nitrocellulose membrane as capture antibody.The suitable working concentration of antibodies was determined respectively.A double-antibody-sandwich model based UPTLF for detecting ZEBOV was established.5.Specificity and sensitivity of up-converting phosphor technology based lateral flow assay was evaluated.Specificity of this method were evaluated by detecting the proteins of dengue virus type 1(DV1),chikungunya virus(CHIKV),zika virus(ZIKV),Japanese encephalitis virus(JEV)and the E.coli cells strain BL21/DE3.Sensitivity was evaluated by detecting gradient dilution of rNP samples,and a standard curve was produced.Results1.Using bioinformatics analysis method,a ZEBOV nucleoprotein fragment was selected.From the literature and Clustal_X,BLASTN analysis,a fragment of amino acid sequence located between 412 to 605aa was compared with the amino acid sequence of Marburg virus,Ebola virus gene amino acid sequence.Extremely low sequence homology,highly concentrated epitope of nucleoprotein,β-folding suggest that it is an ideal target antigen.2.The expression of recombinant nucleoprotein.The nucleic acid sequence of nucleoprotein located between 1234 to 1815 bp were optimized and chemically synthesized.The rNP was expressed in the E.coli cells strain BL21/DE3 and purified.After purification,the size of rNP was about 48 KDa,and there was an abnormal shift with the estimated position of 23.2 KDa.According to the sequencing,open reading frame codon analysis and His-tag resistance analysis,rNP has been expressed normally according to the preset steps,but the antigen fragment has a highly acidic region,which will cause its abnormal size.3.Monoclonal antibodies against recombinant nucleoprotein were prepared.The monoclonal antibodies were generated via immunization mice with recombinant NP,and evaluated.Two monoclonal antibodies,mAb-rNP-land mAb-rNP-2 were examined.The concentration of the antibody samples was 5 mg/mL,and the titers of the two antibodies were 10ng/mL.The titers of the two antibodies met the requirements of the double-antibody-sandwich model based UPTLF.4.A double-antibody-sandwich model based UPTLF for detecting ZEBOV was assembled and optimized.Two monoclonal antibodies named mAb-rNP-1 and mAb-rNP-2 were singled out for the preparation of double-antibody-sandwich model based UPTLF.The mAb-rNP-1 was the labeled antibody,and covalently coupled to form UCP labeled McAbs.MAb-rNP-2 is a capture antibody and immobilized on the nitrocellulose membrane with the concentration of 2mg/mL.The double-antibody-sandwich model based UPTLF was optimized and excluded interference factors.5.The up-converting phosphor technology based lateral flow assay performed good capability of specificity and sensitivity.The result of specificity indicated that this method is specific to rNP but not proteins of dengue virus type 1,chikungunya virus,zika virus,Japanese encephalitis virus and the E.coli cells strain BL21/DE3.Its sensitivity can reach rNP concentration of 0.1ng/μL.And it had a good linear response with the linear fitting coefficient of determination(r=0.98776),and took 15min for detection.ConclusionThis study will use the nucleoprotein as an antigen.The gene fragments of ZEBOV nucleoprotein willbe synthesized.Then it will be converted to the E.coli BL21/DE3,and induced to express fusion protein,named recombinant nucleoprotein.The rNP is going to be purified as an antigen to prepare and identify the specific monoclonal antibody.A double-antibody-sandwich model based Up-converting Phosphor Technology based Lateral Flow assay strips for detecting ZEBOV was established.The result of specificity indicated that this method is specific to rNP but not proteins of dengue virus type 1,chikungunya virus,zika virus,Japanese encephalitis virus and the E.coli cells strain BL21/DE3.Its sensitivity can reach rNP concentration of 0.1ng/μL.And it had a good linear response with the linear fitting coefficient of determination(r=0.98776),and took 15min for detection.The developed UPTLF provides a new choice for rapid,specific and sensitive and quantitative detection of Zaire Ebolavirus.