The Research And Development Of Lung Cancer Diagnosis Reagents And Molecular Imaging Reagent Of Lung Cancer

Objective: To develop the in vitro diagnostic ELISA kit of lung cancer usinglung cancer-related markers NSE and CYFRA21-1. That is，this study was designedto develop a new molecular in vivo imaging diagnostic reagent by marking thepolypeptides screened and obtained previously with nuclide [¹⁸F]，thus providing amore accurate in vivo diagnostic method for early clinical diagnosis anddetermination of pathological type of lung cancer.Methods: Cancer-related various markers were screened on nitrocellulosemembrane using dot immuno-blot assay. The combination of two markers of NSE andCYFRA21-1contributes to the highest diagnostic rate of lung cancer. The antibodiesspecific to NSE and CYFRA21-1were packed to the end of the microtiter plates at acertain percentage of combination. To make the whole double-color ELISA kit morestable and have higher diagnostic rate，the factors of coating buffer，packet time，thepacket temperature，and the coated protective solution，as well as the liquid formulacomposition of protection liquid of detection antibody and color liquid reactionwere all taken into account for obtaining the efficient and stable microtiter plates.The modified compound （ZP） of polypeptide was synthesized by chemical methodsand marked by FITC. The positive diagnostic rate of lung cancer of ZP was identifiedby microarrays of different lung cancer，lung diseases and normal lung tissues. ZPwas injected into nude mice via the tail vein. Then，the heart，liver，spleen，lung，kidney，brain and tumor tissues were made frozen sections and the fluorescenceintensity of ZP in different tissues was observed to determine its in vivo targeted distribution. Finally，ZP was marked with [¹⁸F] and injected into nude mice. The invivo distribution and metabolism of [¹⁸F]-ZP was determined by PET/CT.Results: The NSE and CYFRA21-1-based lung cancer double-color diagnosticELISA kit was stored at4℃，and the validity time was6months. The optical densityof CYFRA21-1-based ELISA kit is between0and0.38478. The normal range of theCYFRA21-1-based ELISA kit is0to24.08ng/mL. The sensitivity，specificity andaccuracy of this diagnostic ELISA kit were83.8%（129/154），53.8%（57/106） and71.5%（186/260），respectively.The ZP compounds were synthesizes with high yield rate and high purity. ZP wasdetected by lung cancer tissue microassy of ZP-FITC with the sensitivity of50%（35/70），specificity of60%（3/5） and accuracy of50.7%（38/75）. The distribution ofZP-FITC at lung squamous carcinoma and adenocarcinoma tissues was obviouslymuch wider than that at normal tissues. The technology of labeling ZP with nuclide[¹⁸F] is reliable and mature. Then the compound was injected into the nude mice andimaged by PET/CT. After injection，tumor imaging is the most obvious.Conclusion: The NSE and CYFRA21-1-based lung cancer double-colordiagnostic ELISA kit has the potential of developing into a new diagnostic molecularimaging reagent for lung cancer，identified using serum of lung cancer patients，otherlung diaeases and normal persons. The targeting of [¹⁸F]-ZP is confirmed using lungcancer tissue microarray，tissue sections of nude mice and in vivo imaging of [¹⁸F]-ZP.This study lays a foundation for the further development about diagnostic reagents oflung cancer.