Preparation And Preliminary Application Of The Monoclonal Antibody Against Liriomyza Trifolii

Purpose Prepare the monoclonal antibody against Liriomyza trifolii, and research the characters of the monoclonal antibody using the modern biological technology. Optimize the optimum experiment conditions of indirect ELISA method to detect L. trifolii. Confirm the advantages and disadvantages of the indirect ELISA method by comparing ELISA detection and DNA barcoding. Samples which collected from April to November in2012around Hangzhou city in Zhejiang Province were detected based on indirect ELISA method to realize the dynamics of L. trifolii and provide reference for controlling of L. trifolii.Method①Prepared monoclonal antibody against L. trifolii based on hybridoma technique.②Based on modern biological technology such as Western blot, ELISA, the characters of monoclonal antibody against L. trifolii including target proteins, titer, specificity, sensitivity, best concentration, subclasses and so on were studied.③Used the indirect ELISA method to confirm the best dilution multiple of L. trifolii larval, pupa and adult.④COI and ITS1sequences were amplified using the DNA extracted from L. trifolii raised in lab, and compared with COI and ITS1sequences of L. trifolii, Liriomyza sativae, Liriomyza huidobrenisis and Chromatomyia horticola from Genbank after sequencing and assembling. The genetic analysis was performed. Compared the advantages and disadvantages between DNA barcoding and ELISA methods to identify L. trifolii.⑤Collected insect samples from green vegetables, celery, peas, green beans, lettuce, tarragon and mustard in Yuhang, Binjiang, Xihu and Fuyang regions from April to November in2012to detect the positive rates of the samples reacted with the monoclonal antibody based on indirect ELISA method. Provide reference for controlling of L. trifolii.Result①Obtain a stable hybridoma cell strain named4C9F2, which could product monoclonal antibody stably. Prepared about100mL ascite antibodies.②When the larval, pupa and adult of L. huidobrenisis were responsed with monoclonal antibody against L. trifolii, the optical densities (ODs) were0.267,0.233and0.271respectively in490nm test by the indirect ELISA. When the larval, pupa and adult of C. horticola were responsed with monoclonal antibody against L. trifolii, the ODs were0.221,0.204and0.229respectively. When L. trifolii were responsed with monoclonal antibody against larval, pupa and adult of L. trifolii, the ODs were0.760,0.880,0.698. The titer of the ascites monoclonal antibodies was8.192×106. The sensitivity of this antibody was45.3μg/L. The OD was1.021, when ascite antibody was diluted2×104times. The subclass of the antibody was IgA. It was demonstrated by western blot that the antibody could response with122kDa and68kDa protein of L. trifolii.③The ODs were0.933,1.129,1.047respectively, when the larval, pupa and adult samples were homogenated with16mL,1mL and500μL coating solution respectively and100μL of them were used in indirect ELISA.④Both DNA barcoding and indirect ELISA method can be used to identify the L. trifolii. For COI sequence, the genetic similarity between L. trifolii and L. huidobrenisis was0.68, which was the highest among4species of leafminers. For ITS1sequence, the genetic similarity between L. trifolii and L. sativae was the hightest. It was0.885. The method of DNA barcoding based on COI sequence shows higher confidence than that based on ITS1sequence.⑤The detection results of field samples showed that the positive rates were between70%～80%in Yuhang, Binjiang, Xihu and Fuyang regions from September to November in2012. All of the positive rates of L. trifolii collected from Greens, black-eyed peas, green beans, lettuce, and tarragon were more than50%. But the proportion of L. trifolii had a large difference from different vegetables. The positive rate of L. trifolii collected from celery was the highest. It was92.4%.Conclusion①Obtain a stable hybridoma cell strain named4C9F2, which could product monoclonal antibody stably.②This antibody had high titer, strong specificity and high sensitivity. On this basis, we build an indirect ELISA method to detect L. trifolii. The larval, pupa and adult samples were homogenated with16mL,1mL and500μL coating solution respectively and100μL of them were used in indirect ELISA. The monoclonal antibody diluted20000times, the second antibody diluted4000times.③L. trifolii can be identified with DNA barcoding based on COI and ITS1sequences and the indirect ELISA method. Compared with ELISA detection, DNA barcoding is suitable for using in laboratory research. The ELISA detection only needs to add samples, and requires low technical difficulty in the whole process, so it is suitable for a large number of samples testing in the field. It provides the possibility to achieve the dynamic L. trifolii.④In the closely related species, September-November is the season of L. trifolii. Green vegetables, celery, peas, green beans, lettuce and tarragon are the host plants of L. trifolii and it has preferences for celery.