Establishment And Evaluation Of Chemiluminescence Quantitative Immunoassay Of HE-4

Objective:Establish a chemiluminescence quantitative detection method of HE-4(Human epididymis protein4) based on the automated immunoassayanalyzer, and evaluate the performances of this method.Method:1. Using protein A affinity purification column deal with anti-HE4monoclonal antibodys, and identifying antibodys with electrophoresis;2. Labeling anti-HE4antibodys with biotin and horseradish peroxidase,and selecting the best word ratio;3. Coating the biotin-antibodys on the Streptavidin beads, andselecting the best Coated amount;4. The antibody cross-matching, screening out the highest potency andthe best matching pair of antibodies；5. Reference EP17-A Document, determining the LOB、LOD、LOQ ofthis method;6. Using this method detect high concentration commom tumor marker (AFP、CEA、CA199、CA125、CA153), identify there is crossreaction or not;7. Adding different concentration of Hemoglobin, bilirubin,chylomicrons, biotin (Vitmin H) into samples, identify them haveinterference or not;8. Adding some HE4recombinant antigen into clinical sample, anddetecting them with this method, then calculating the adding recovery;9. Diluting a sample (approximately1700pmol/L) to a series gradientconcentration, and detecting them with this method, then calculating thecorrelation of concentration and dilut ratio;10. Detecting high、median、low concentration samples over time,identify this method’s repeatability and inter-day precision;11. Storing the comments of this method in4℃and37℃, observingthe change of signals after7days;12. Using this method (CLIA)、Fujirebio (ELISA) detect clinicalsamples, analysising consistency and correlation of2kits.Result:1. Monoclonal antibodys（1#-9#） have a purity of95%，can be usedby the further experiments;2. Contrast the different ratio of antibody/biotin (1/10、1/20、1/40), theactivated is decrease when the biotin amout increase. We choose1/10asthe biotin labeling ratio; 3. Contrast the different ratio of antibody/HRP (1/2、1/4、1/6)，theresult show1/4have the highest activated. So we choose1/4as the HRPlabeling ratio;4. The result of cross-pairing experiment show the3#have the highestactivated;5. We choose1#as the capture antibody, and3#as detection antibody,there is no signals when we use the pair antibodys detect some clinicalsamples. We replace1#with H6from Makerbio, the result show thismethod have a good correlation with Fujirebio；6. This method is provided with a limit of blank is1.014pmol/L, a limitof detection is5.252pmol/L, a limit of quantitation is10.568pmol/L;AFP、 CEA、 CA125、 CA199have no significant cross-reactivity; ameasure range from20pmol/L to1700pmol/L; the intra repeatability is1.5%-3.6%and the total precision is3.8%-6.4%; the recovery is98.36%-99.14%; Hemoglobin, bilirubin, chylomicrons, biotin; the relativedeviation after7days (store at37℃) is-0.94%～8.34%;7. Compare with ELISA kit from Fujirebio, this method have a goodcorrelation with Fujirebio, y=1.023x-12.280，r=0.9897（P <0.001）.Conclusion:1. After antibody purification、identifying、labeling、cross-pairing、optimization, this method have the ability to detect the concentration ofHE4. 2. Through evaluate the performance and comparison with other kis,this method have ability to detect HE4of clinical sample, it is a effectivetool for diagnosis early ovarian cancer.