| Objective:To master the preparation technology of monoclonal antibody against TSLCl,after prepqration of monoclonal antibody agaist TSLCl,we apply immunohis-tochemistry dyeing technology with the sample we have chosen,whih is simple and cheap.taken together,studying the relationship between the occuring and developing of BTCC and the abnormal expression of TSLC1 protein.Methods:At first,we immunize Balb/c female mice,and preparation of monoclonal antibody agaist TSLC1 by spleen cell and myeloma cell fusion technology.We use ELISA to determine the potency.Meantime,we choose 65 BTCC pathology wax objects,40 of 65 wax objects are low malignant tumor,the others are high malignant tumor.Classified by clinical staging,the 25 wax objects are Ta-1 tumors,the 20 wax objects are T2 tumors,the 20 wax objects are T3-4 tumors.And we also choose 20 normal mucous membrane of urinary bladder.Then we start to determine the expression of TSLC1 protein with immunohistochemistry technology(sp method).The pathology doctors help us to analyze the dyeing states.they determine three states:negative(-),weakly positive(+),strongly positive(++).We also use IPP6.0 to quantitative analysis with the positive expression cells.At last,we use SPSS13.0 to analyze the TSLC1 protein expression results of different clinical staging and different grading.Results:We use ELISA to detect serum TSLC1 polyclonal antibody,when dilution is 1:8000,the OD value is 1.355,much higher than the negative group(OD=0.545),it means good effect on mice immune.After cell fusion,we get twenety-two positive clones.Strictly limited dilution to clone,finally retain six hybridoma cell lines,which have the stability of a high titer of McAb secretion.they are AD6,BC10,CB7,CE8,DC5,DF4,respectively.Then we determine antibody subtype,the result reals:AD6 and CE8 belong to IgGl;BC10 and DF4 belong to IgG2a;CB7 and DC5 belong to IgG3.Check AD6 cells cultured for amplification,10~7 cells injected in mice to produce ascites to prepare IgG1,After purification,of the post-test titer was 1:100000.The results of immunohistochemistry technology are the TSLC1 protein expression in cell plasma membrane.High malignant BTCC samples,the weakly positive(+) is only 12%(3/25),most others are negative(22/25);Low malignant BTCC samples,the weakly positive is 75%(30/40),the others are negative(10/40),the difference of two groups has statistical meaning (P<0.05.what is more,with all normal mucous membrane of urinary bladder,the TSLC1 protein express in all samples,and they are strongly positive(++),therefore,the normal mucous membrane of urinary bladder is obviously different from the BTCC samples.We also find that the TSLC1 protein expression has relationship with the clinical staging,the weakly positive of Ta-1 is 92%(23/25),the weakly positive of T2 is 60%(12/20),the weakly positive of T3-4 is 10%(2/20),the differences of three groups also have statistical meaning(P<0.0167).Meantime,we use IPP6.0 to quantitatively analyze them,in the grading,the PI of low malignant samples is 68.55±10.50%,the PI of high malignant samples is 20.65±5.52%;PI and pathological grading of BTCC show a negative correlation(r= -0.905,P=0.000).in clinical staging,PI of the Ta-1 samples is 82.15±5.12%,PI of the T2 samples is 60.55±10.25%, PI of the T3-4 samples is 18.25±6.58%,PI and clinical staging of BTCC also show a negative correlation(r=-0.932,P=0.000).Conclusion:We mastered the preparation technology of monoclonal antibodies against TSLC1,and which may be used to detect the biological behavior of BTCC,the staining results are satisfactory.By immunohistochemistry,we consider the low TSLC1 protein expression or null is associated with the happening and developing of BTCC,and the positive expression of TSLC1 protein(PI) in BTCC showed a negative correlation with pathological grade and clinical stage,so in determining the malignant degree of BTCC,it has important clinical value,and can also be used to evaluate the prognosis of BTCC.It is a valuable marker in the biological behavior of BTCC.
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