Preparation And Identification Of Chicken Egg Yolk Immunoglobulins Against EV71 And CA16 For Diagnosis Of Hand-foot-and-mouth Disease

Objective:Hand,foot and mouth disease?HFMD?is a common infectious disease caused by various enteroviruses.The main pathogen of hand,foot and mouth disease prevailing in China is enterovirus 71?EV71?and coxsackievirus A16?CA16?.Due to the lack of effective antiviral drugs in clinical,the early and rapid monitoring of EV71 and CA16pathogens has become one of the hot issues in public health research.Traditional detection methods have some disadvantages such as complicated operation,time-consuming,low sensitivity and poor specificity.Therefore,there is an urgent need to establish a rapid,sensitive and specific method for HFMD detection.The aim of this study is to prepare EV71 and CA16 egg yolk immunoglobulin Y?IgY?and identify it's purity,titer and specificity.IgY was generally immobilized on the AuNPs surface through electrostatic interaction,followed by IgY antibodies-gold nanoparticles?IgY-AuNPs?conjugates,obtained as capture probes to recognize EV71 or CA16.In this study,we established a rapid detection method for HFMD based on fluorescence resonance energy transfer?FRET?technology and construct EV71 and CA16 two kinds of hand,foot and mouth disease pathogen detection kits.In the actual detection work,the pre-treated samples were added to different fluorescence quenching systems,one step detection can be realized.And applied detection kits to clinical samples,then the detection effect of this method was evaluated.Methods:EV71 and CA16 IgY was prepared,then the purity,protein content,titer and specificity of IgY were identified;A rapid detection method for HFMD was established based on IgY and FRET technology,and applied it to clinical samples to evaluate the effect of the established detection method.The details are as follows:1.The inactivated virus was used as an antigen to immunize SPF hens,eggs was collected.Then,specific IgY was separated and extracted from egg yolk by PEG precipitation method;Sodium dodecyl sulfate–polyacrylamide gel electrophoresis?SDS-PAGE?analysis and bicinchoninic acid?BCA?protein assay was used to identify the purity and concentration of IgY.Indirect enzyme-linked immunosorbent assay?iELISA?was used to represent the titer and specificity of IgY,respectively.2.The IgY-AuNPs was generated based on electrostatic self-assembly of IgY onto the surface of GNPs and employed as capture probes to recognize EV71 or CA16.Composition,size,topography and performance of IgY-AuNPs was characterized by Fourier transform infrared spectroscopy?FTIR?,ultraviolet visible spectroscopy?UV-Vis?,transmission electron microscopy?TEM?,dynamic light scattering?DLS?and ZETA potential?Zeta potential?.3.A rapid detection method for HFMD clinical samples based on IgY and FRET technology was established in this study.IgY-AuNPs and quantum dots?QDs?were applied to form a fluorescence quenching system.The fluorescence detection of the complex was carried out by means of a fluorescence spectrophotometer.By optimizing the detection system IgY-AuNPs dosage,NaCl dosage,fluorescence recovery time and other indicators,the sensitivity and specificity of the detection method were evaluated,and the EV71 and CA16 two hand,foot and mouth disease pathogen detection kits were constructed.In the actual detection work,the pre-treated samples were added to different fluorescence quenching systems to achieve qualitative and quantitative detection of the two pathogens,which meets the actual needs of rapid screening and detection of clinical samples of hand,foot and mouth disease.Results:1.The EV71 and CA16 IgY prepared in this experiment has good purity,clear bands and no other obvious bands;The average protein content of EV-71-IgY is 26.60mg·L^-1;The highest titer of EV-71-IgY is 1:512000.The average protein content of CA-16-IgY is 12.15 mg·L^-1;The highest titer CA-16-IgY is 1:128000.Both virus chicken yolk antibodies have good specificity,which can specifically recognize the target virus instead of the interferent.2.A rapid detection method for EV71 type hand,foot and mouth disease pathogen based on EV-71-IgY and FRET technology was established in this study.After the experimental conditions were optimized,the optimal dosage of EV-71-IgY-AuNPs was determined to be 350?L(1.3×10^-4 g·mL^-1),and the optimal fluorescence recovery time was 60 min.Under the optimized detection conditions,the response signals linearly correlated with the concentration of EV71 over the ranges of 10⁴⁵×10⁶ PFU·mL^-1.The standard curve was I_638nm=24.364C-59.168,R²=0.9836,the limit of detection?LOD?was caculated to be 10⁴ PFU/mL.The specificity of this method is good,which has no cross-reactivity with CA16 virus.Compared with the clinical detection“gold standard”qRT-PCR,the agreement rate reached 94.44%.The detection method established in this experiment can be applied to the detection of clinical samples.3.A rapid detection method for CA16 type hand,foot and mouth disease pathogen based on CA-16-IgY and FRET technology was established in this study.After the experimental conditions were optimized,the optimal dosage of CA-16-IgY-AuNPs was determined to be 400?L(1.3×10^-4 g·mL^-1),and the optimal fluorescence recovery time was 90 min.Under the optimized detection conditions,the response signals linearly correlated with the concentration of CA16 over the ranges of 10⁴²×10⁶ PFU·mL^-1.The standard curve was I_525nm=15.452IgC-9.746,R²=0.9932,the limit of detection?LOD?was caculated to be 10⁴ PFU/mL.The specificity of this method is good,which has no cross-reactivity with EV71 virus.Compared with the clinical detection“gold standard”qRT-PCR,the agreement rate reached 93.75%.The detection method established in this experiment can be applied to the detection of clinical samples.Conclusion:1.EV71 and CA16 IgY with good quality was successfully prepared in this study.2.AuNPs-IgY was successfully prepared and it's size,composition,topography and performance was characterized.3.EV71 and CA16 hand,foot and mouth disease detection methods were successfully established,and prepared EV71 and CA16 two kinds of hand,foot and mouth disease pathogen detection kits.In the actual detection work,the pre-treated samples were added to different fluorescence quenching systems to achieve qualitative and quantitative detection of the two pathogens,which meets the actual needs of rapid screening and detection of clinical samples of hand,foot and mouth disease.