| Malignant tumors are such diseases that seriously threaten human health, and theincidence rate is increasing year by year worldwide. However, due to the limited diagnostictechnology, many patients with cancers are diagnosed in the late stage, and the properopportunity for treatment was missed. As a result, a sensitive, fast, convenient, andnon-invasive detection method attracts much concern to researchers. The protein quantitativedetection of serum tumor markers to screen various types of tumors has been reported athome and abroad, and the value in the detection of various types of tumors has been affirmed.MUC1, a novel tumor marker, has been widely studied in recent years. Patients with manykinds of adenocarcinomas, such as breast cancer and lung cancer, were detected an evaluatedsoluble MUC1protein level in peripheral blood than normal control. Recent studies haveshown that cells of hematologic malignancies express MUC1protein levels higher thannormal control. However, the expression level of soluble muc1mucin in peripheral bloodhas not been reported.At present, enzyme-linked immunosorbent assay (ELISA) is the most common methodof detecting the soluble MUC1protein in peripheral blood. Monoclonal antibodies are usedto prepare the commercial ELISA kits. Although monoclonal antibodies can greatly improvethe specificity of antigen recognition, they only bind to certain epitopes. Differentindividuals with cancers have different antigen epitopes, and therefore, the detection ratio isdecreased. Taking the limitations of commercial kit into account, in the present study, weprepared a new kit of double antibody indirect sandwich ELISA by combined anti-humanpolyclonal antibodies with GP1.4monoclonal antibodies. The kit was evaluated by detectingMUC1in serum of health subject and patients with breast cancer and lung cancer, and thenwe attempted to analyze the expression level of MUC1mucin in peripheral blood of patientswith hematologic malignancies.Methods and Results: 1. Preparation of MUC1-GST fusion protein Frist,culture large numbers ofpGEX-MUC1/BL21which express target proteinMUC1-GST. Then,the bacteria werebroken by sonicate and removed supernatant containing fusion protein.Pure MUC1-GSTfusion protein was obtained after Glutathione Sepharose4B affinity layer andNative-Polyacrylamide Gel Electrophoresis.2. Antibody preparation and titer assay MUC1-GST fusion protein was used toimmunize rabbit in order to prepare polyclonal antibodies. We detected the titers of rabbitanti-MUC1polyclonal antibody.the antibody titer was320000.3. Antibody purification Purified rabbit anti-MUC1Polyclonal antibodies wereobtained after precipitated by saturated ammonium sulfate, purified by protein A andabsorbed by anti-GST antibody.4. Preparation of standard sample MUC1-GST fusion protein was digested withthrombin to obtain MUC1standard.5. Establishment of double antibodies indirect sandwich ELISA A double antibodyindirect sandwich ELISA method was successfully established after screening differentcombinations of anti-human MUC1polyclonal antibody and GP1.4monoclonal antibody.The coated antibody was GP1.4monoclonal antibody; rabbit anti-human polyclonal MUC1antibodies were the detected antibodies, and the enzyme-labeled antibody was HRPAffiniPure Goat Anti-Rabbit IgG.6. Determination of the optimum working conditions for double antibodies indirectsandwich ELISA The antibody concentration of coated antibody, detection antibody andHRP-labeled antibody was chose by checkerboard titration method. The optimum workingconcentration of coated antibody and detection antibody were1ug/ml and15μg/ml,respectively. The optimum dilution ratio of HRP AffiniPure Goat Anti-Rabbit IgG was1:5000. The MUC1standard sample was diluted as follow:0.5ng/ml,1ng/ml,2ng/ml,4ng/ml,8ng/ml,16ng/ml,32ng/ml, and64ng/ml. The sensitivity of the kit was0.5ng/ml.7. Detection of MUC1protein levels in peripheral blood After establishment of thedouble antibody sandwich ELISA,225patients were enrolled for detection of MUC1proteinof peripheral blood, including13cases of breast cancer,60cases of lung cancer,48cases ofacute myeloid leukemia,26cases of acute lymphoblastic leukemia,24cases oflymphatictumor,17cases of multiple myeloma,20cases of healthy individuals,7cases oflung benign disease, and10cases of benign hematological diseases. The resultsdemonstrated that patients with breast cancer and lung cancer had significantly higher MUC1protein levels in peripheral blood compared to the healthy control (P<0.01). However,no significant difference of MUC1protein levels was observed between patients withhematologic malignancies and healthy controls (P>0.05). Cutoff value of MUC1protein wasdetermined by ROC curve analysis.1.92ng/ml was the cutoff value for patients with breastcancer and healthy control, and the sensitivity and specificity were100%and100%,respectively. For patients with lung cancer,1.88ng/ml was the cutoff value, and thesensitivity and specificity were63.3%and100%, respectively. For the patients of lungcancer and benign lung diseases,1.98ng/ml was the cutoff value with a sensitivity of61.7%and a specificity of100%.8. Comparing between double-antibody sandwich ELISA and CA15-3kit The sameblood sample from breast cancer and health subject were assayed by ELISA and CA15-3.After drawing ROC curves, the results showed that double-antibody indirect sandwichELISA had a significantly higher accuracy in diagnosis of breast cancer compared withCA15-3kit.Conclusions1. In this study, we develop a double antibody sandwich ELISA method which has abetter sensitivity and specificity.2. The sensitivity and specificity are greatly improved for diagnosis of lung and breastcancer using the present ELISA kit which is expected to develop conventional kit for clinicaldiagnosis.3. The detection of MUC1protein level in peripheral blood of patients withhematologic malignancies remains to be further explored. |
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