Preparation Of Antibodies Against Plant Honnone Jasmonates And Anticancer Drug 5-fluorouracil And Establishment Of Enzyme-linked Immunosorbent Assays

Enzyme-linked immunoassay(ELISA)is a qualitative and quantitative analytical method with high sensitivity and specificity.In this thesis,two kinds of ELISA were developed for quantitative analysis of two substances:plant hormone jasmonates and anticancer drug 5-fluorouracil.Jasmonates(JAs),mainly including jasmonic acid(JA)and methyl jasmonic acid(MeJA),are plant hormones which can regulate plant growth and resistance.The content of JAs in plants is very low,and the improper use of exogenous JA or MeJA will also reduce the ecological benefits.And JA and MeJA have certain differences in physiological functions,and at the same time detecting JA and MeJA is still a difficult problem for plant biologists.Therefore,it is necessary to develop highly sensitive and specific analytical method for the detection of JA and MeJA in plant samples.In the first part of this thesis,monoclonal antibody(mAb)against JA was prepared by hybridoma technology.Based on the prepared mAb and two sample extraction methods,a sensitive and specific ELISA for simultaneous determination of JA and MeJA was established.The semi-inhibitory concentration(IC50)and detection limit(LOD)of the ELISA were 2.02 ng mL-1 and 0.201 ng mL-1,respectively.The results from cross-reactivity testing revealed the specificity of the ELISA was high.The recovery of JA from the spiked samples tested by the ELISA was found in the range of 79.6-89.5%.The spiked samples were measured by ELISA and HPLC.There was a good correlation between ELISA and HPLC.Three plant samples was detected by the ELISA and it was found that the content of JA was about 3?5 times higher than that of MeJA.Compared with similar methods,the biggest advantage lies in the establishment of an ELISA method for simultaneous detection of JA and MeJA.5-fluorouracil(5-FU)is one of the broad-spectrum anticancer drugs.However,it exhibits some shortcomings in cancer treatment.It is of great clinical value to develop a real-time.simple and highly sensitive method to detect 5-FU in patient's blood.In the second part of this thesis,a 5-FU polyclonal antibody(pAb)was prepared and an ELISA was established for the detection of 5-FU.The IC50 and LOD of the ELISA were 72.2 ng mL-1 and 10.3 ng mL-1,respectively.It seemed that the sensitivity of the ELISA was insufficiently high enough to meet the clinical application.The results of the cross-reactivity experiment showed the cross-reaction rates of the ELISA with uracil,cytosine and thymine were within 5.56-17.4%,which indicated the specificity of the ELISA was not so high.It seems the polyclonal antibody is unsuitable for the detection of 5-FU in patient's blood.To develop an immunoassay for 5-FU,the preparation of an anti-5-FU monoclonal antibody has been going on.A hybridoma cell line which is able to secret the antibody with high affinity binding with 5-FU has been obtained.It is possible in the future that a monoclonal antibody-based ELISA will be develoed for the detection of 5-FU in blood samples.