Screening Specific Antigen Of Fetal Nucleated Red Blood Cell Based On Label-free Quantification

Background and Objective:Birth defect is an important factor affecting social development.The purpose of prenatal diagnosis is to reduce birth defects through appropriate testing and monitoring methods,and to minimize the adverse effects of birth defects on pregnant women and newborns.In the current situation of high risk of invasive prenatal diagnosis and high false positive rate of non-invasive prenatal testing,fetal nucleated red blood cells(FNRBC)in the pregnant women’s peripheral blood become the most potential cell for non-invasive prenatal diagnostic because it contains all the fetal genome.However,FNRBC lacks specific antigens.The specificity of the antigens currently used for sorting FNRBC is not good,resulting in a low efficiency of sorting FNRBC in pregnant women’s peripheral blood,which limiting its application in noninvasive prenatal diagnosis.Therefore,this study using label-free quantification technology with small sample requirements,relatively simple operation,and small experimental errors to screens for specific proteins of FNRBC sorted in umbilical cord blood by magnetic activated cell sorting(MACS),and performs bioinformatics analysis and immunofluorescence verification to find the specific proteins or antigen that can be used to sort FNRBCs,and to meet the needs of clinical non-invasive prenatal diagnostic.Methods1.Obtaing FNRBC from umbilical cord blood as experimental group by magnetic activated cell sorting method(MACS),using Ficoll separation medium to obtain peripheral blood mononuclear cell(PBMC)from healthy adults as control group,and perform verification using flow cytometry;2.Using label-free technology to identify,quantify,and analyze significant difference between the two groups of cell protein,determine the target protein,including differentially expressed proteins and specifically expressed proteins;3.Perform bioinformatics analysis on the target protein obtained from label-free quantification,and combine the quantitative results to initially screen for FNRBC specific proteins;4.Using immunofluorescence technique(IF)to verify the specificity of the above-mentioned initially locked FNRBC specific protein,and clarify its cell localization and expression.Results1.Successfully constructed a method for sorting cord blood FNRBC by MACS,which effectively obtained three tubes of cord blood FNRBC,approximately 1×106 cells/tube;Using Ficoll separation effectively obtained three tubes of healthy adult PBMC,approximately 1×107 cells/tube,and successfully passed the flow cytometry validation;2.A total of 4392 proteins were identified using label-free technology,and 2235 and 3611 proteins were identified in FNRBC and PBMC,respectively.Among them,203 and 369 differentially expressed proteins were highly expressed in FNRBC and PBMC,respectively;FNRBC and PBMC specific proteins were 280 and 1,147 respectively;3.Bioinformatics analysis of the target protein,combined with the label-free quantitative results,CD59,RTN-3,LAMTOR2,MEDD4,SLC44A2 were screened as FNRBC specific proteins and will be verified;4.IF verification showed that CD59 was highly expressed in FNRBC membrane and cytoplasm;LAMTOR2,SLC44A2 was highly expressed in PBMC membrane and cytoplasm;MEDD4 was not expressed in FNRBC,but lowly expressed in PBMC;RTN-3 was not significantly expressed in both group cells.ConclusionsUsing MACS and media separation can effectively obtain FNRBC and PBMC,and provide experimental materials for label-free;And label-free quantification can be used as an effective method to study FNRBC and PBMC proteomics;the bioinformatics analysis can obtain molecular functions,cell components,biological processes,participation pathways of protein identified by label-free and further screened for FNRBC specific proteins;after IF verification,CD59 was considered to be FNRBC specific protein and could be used as a specific antigen for sorting FNRBC;LAMTOR2 and SLC44A2 were specific proteins for PBMC,that can be used for sorting FNRBC,and provides research conditions and technical support for optimizing non-invasive prenatal diagnostic technology.