| Objective: Lewis Y antigen (LeY) is one kind of blood group related antigens and some glycoproteins and glycolipids contain LeY as terminatio oligosaccharide.LeY is abnormally expressed by 60~90% percent of cancers of epithelial origin.The LeY containing glyproteins or glylipids may secrete or shedd into the blood.It's correlates with the growth and metastasis of tumour. The goal is to establish one kind of serological diagnosis method being fit for the clinical examination and provide the basis for the clinical tumor diagnosis.Method: The method LeY is examined in the person blood serum which the experiment plans to establish to be as follows:First:Case choiseChoose different malignant tumor patients 169 examples include breast cancer 30 examples, ovarian cancer 15 examples, rectal cancer 32 examples,lung cancer 36 examples,stomach cancer 38 examples, carcinoma of urinary bladder 18 examples;Take normal persons blood serum 200 examples as the negative comparison.Second:the ELISA pair anti-contains sandwich method establishment1. ELISA general steps: (1) BG-8 antibody coating; (2)3%BSA sealing up; (3) Adding the detected blood serum, Installing the blank ,negative and positive control; (4) Adding the enzyme labelled antibody; (5) Adding the substrate coloration; (6) Adding the termination fluid; (7) Determining the OD450nm values.2. The dilution determination of coating antibody: Diluting the BG-8 antibody to 1:2000, 1:1000, 1:500, 1:200, 1:100 different levels, according to the ELISA steps, determining the OD450nm values and the best concentra- tion.3. The best density determination of enzyme labelling antibody (UEA-1): Preparing enzyme labelling UEA-1 1μg/ml, 2.5μg/ml, 5μg/ml, 10μg/ml, 20μg/ml levels, according to the ELISA steps, determining the OD450nm values and the best concentration.4. The choice of reaction time: According to the ELISA steps, change each step reaction time, compared with the OD450nm values of different time, determine the suitable reaction time.5. Positive control: The person transitional epithelial tumer cell of endometrium protein decomposition fluid A431 ,the degree of dilution to determine: Diluting A431 fluids to 1:400, 1:100, 1:50, 1:10, 1:25 levels, according to the ELISA step, determining the OD450nm values and the best concentration.Third: Determination: According to the condition which determined ELISA sandwich method ,detecting LeY OD450nm values of different tumor patients blood serum. Analyze LeY glycoprotein expression spectrums of the normal blood serum groups and the patient groups with Westen blot method.Results: First:Condition optimization: The definite coating antibody and the enzyme labelled agglutinin working concentrations respectively are: 1:500, 2.5μg/ml; The positive control A431 cell decomposition fluid working level is 1:50; The coating condition is 4℃and 36 hours; The sero-reaction time is 2 hours; The sealing time is 1 hour; The staining time is 20 minutes. Second: Result analysis: (1) The manufacture of the normal persons examine curve: Dilute LeY antigen standard to the levels as 1000ng/ml, 500ng/ml, 250ng/ml, 125ng/ml, 62.5ng/ml, 31.25ng/ml, 15.625ng/ml, titer mapping, making the specification curve after the regression analysis.Regression coefficient r=0.966. Substitute the OD450nm values of testing samples means to specification curve regression equation, the LeY concentration values are obtained.(2) To determine the values between the normal persons and the different tumor patients,carry on statistics comparison with Spss11.5 software, analyze linear relationship and the relevance.Third:Each groups of blood serum LeY determination results is: breast cancer group blood serum Ley level is: (156.81±46.93) ng/ml, the ovarian cancer group: (176.90±50.71) ng/ml, Rectal Cancer group: (119.70±38.82) ng/ml, the lung cancer group: (55.58±35.02) ng/ml, the stomach cancer group: (66.40±33.51) ng/ml, the carcinoma of urinary bladder group: (79.44±41.28) ng/ml, the normal person group: (35.17±22.49) ng/ml. The experimental groups tumor blood serum LeY levels is higher than the normal groups obviously, the difference has statistics significance (p<0.01); And compare the blood serum of the breast cancer, the ovarian cancer group ,the rectal cancer group with the normal person groups, LeY levels are different more obviously from other tumor groups. The Westen blot method demonstrated that the detection rate of cancer groups are higher than the normal groups,and the breast cancer, the ovarian cancer and rectal cancer bands detection rates are more higher (70%), obviously are higher than other cancer groups and the normal groups.Conclusion: 1st: LeY exists the expresses in the tumor patients peripheral blood, Within the breast cancers, the ovarian cancers, rectal cancers, the lung cancers, the stomach cancers,the first three cancers expresses are more obvious. 2nd: The difference trendency of the Westen blot method qualitative test are concordant with determination of ELISA sandwich method. 3rd, this method sensitivity is high, the specificity is good, and it has far-reaching significance in the partial tumors clinical diagnosis. |
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