| BackgroundLung cancer is the leading cause of cancer-related death worldwide,which brings on a disaster to human health.Annually,there are 400 000 deaths caused by lung cancer,whereas 10-year survival rate of patients diagnosed at early stage can reach up to 92%.Therefore,the effective method to reduce the mortality of patients with lung cancer is early diagnosis and thus early treatment.Compared with the imaging examination,laboratory detection of tumor markers is a more promising way to achieve early diagnosis of lung cancer.However,there is no biomarker of lung cancer with high sensitivity and specificity currently.Members in our laboratory had successfully prepared a monoclonal antibody(McAb)designated NJ001 by immunization with SPC-A1(human lung adenocarcinoma cell line).The corresponding antigen SP70 to McAb NJ001 was demonstrated both tissue and cell type specificity to non-small cell lung cancer(NSCLC)by immunohistochemisty.And SP70 was proofed to be located in the cytoplasm and on the surface of NSCLC cells with indirect immunofluorescence and Western bolt assays.ObjectiveTo evaluate whether the specific antigen SP70 existed in serum of NSCLC patients and could be used as an early serum biomarker for the diagnosis of NSCLC.MethodsWestern blot was preliminary conducted to identify whether targeting antigen SP70 existed in serum of NSCLC patients.Polyclonal antibody(PcAb)was prepared by immunizing New Zealand rabbit with SPC-A1 cells.Sandwich ELISA was carried out by using newly-prepared PcAb coating assay plates,McAb NJ001 and HRP goat anti-mouse antibody as primary antibody and labeling antibody respectively.After optimizing the experiment conditions and determining the cut-off value,1089 serum samples were examined,including 479 cases of lung cancer(306 lung adenocarcinoma,134 squamous cell lung cancer and 39 small cell lung cancer(SCLC)),50 cases of benign lung disease(BLD),20 cases of breast cancer,20 cases of gastric cancer,20 cases of colon cancer and 500 healthy controls(HC).Meanwhile,the classical biomarkers of lung cancer such as CEA,NSE and CYFRA21-1 were measured for comparison.ResultsWestern blot analysis showed the expression level of the targeting antigen SP70 in NSCLC group was obviously higher than HC,which approved that the targeting antigen SP70 was existed in serum of NSCLC patients.A polyclonal antibody to SPC-A1 was obtained,and the titer of the purified antibody was 1:160 000.With the newly prepared PcAb to SPC-A1,McAb NJ001 and the secondary antibody,we successfully established the sandwich ELISA for the detection of SP70.In this method,the final concentration of coating antibody was 0.63 ?g/ml,and the optimal dilution of NJ001 was 1:4 000.Besides,the best reaction time was 2 hours and temperature was 37?.Positive rates of SP70 in lung adenocarcinoma,squamous cell lung cancer,SCLC and BLD were 63.1%,59.7%,20.5%and 18.0%,respectively.They were significantly higher than HC(8.8%,P<0.05).NSCLC(Adenocarcinoma 63.1%,Squamous cell cancer 59.7%,62.0%)had obviously higher positive rate of SP70 than both SCLC(62.0%vs 20.5%,P<0.01)and BLD(62.0%vs 18.0%,P<0.01).Among 276 cases of NSCLC patients who had known the staging,the positive rates at the early(?/?)stage reached up to 72.7%(64/88).Positive rates of SP70 in NSCLC group were significantly higher than in breast cancer,gastric cancer and colon cancer group(25.0%,10.0%and 15.0%),P<0.01.Besides,positive rates of CEA,NSE and CYFRA21-1(40.7%,17.7%and 37.5%)were significantly lower than the targeting antigen to McAb NJ001 in NSCLC;the combined determination of SP70,CEA,NSE and CYFRA21-1 had obviously higher positive rate than the combination of CEA,NSE and CYFRA21-1(increased by 20%).ConclusionIt showed high positive rates of SP70 in serum of NSCLC patients,compared with CEA,NSE and CYFRA21-1;and SP70 can be detected at early stage of NSCLC.So it approved that SP70 was a potential valuable biomarker for the early diagnosis of NSCLC. |
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