Preparation, Characterization And Application Of Monoclonal Antibodies Against HMW Glutenin Subunits 1Bx13 And 1By16 In Wheat

Bread-making quality of wheat is related not only to quantity but also to compositions of wheat storage proteins. Measuring of breadmaking quality usually needs expensive instruments such as Extensograph, Mixograph and Farinograph etc. Therefore, the wide utilization of these instruments is difficult for most breeding departments. Furthermore, measurements using these instruments need relatively large amounts of materials which are usually not adequate in the early stages of breeding program. Althougt some methods are economical such as sedimentation value and so on they are time-consuming. Recently, enzyme-linked immunosorbent assay (ELISA) with its significant advantages of low capital, low running costs, small sample amount needed and simple sample handling, has been used for quality predicting and screening. For the use of this method to measure wheat quality, preparing of suitable antibodies, especially monoclonal antibodies is highly important Previous studies showed that HMW-GS 1Bx13 and lByl6 had positive effects on wheat quality, and therefore, preparation of monocloanal antibodies to the two subunits could be more valuable in breeding application!. Additionally, as coding genes of the two subunits have not been isolated, monoclonal antibodies against 1Bx13 and lByl6 could be used as useful probes to screen cDNA expressing libraries and to clone their coding genes and other storage protein genes.In this study, high molecular weight glutenin subunits (HMW-GS) of spelt wheat (Triticum spelta L.) cultivar Somiedo were separated and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), acid polyacrylamide gel electrophoresis (A-PAGE), A-PAGEx SDS-PAGE, high performance capillary electrophoresis (HPCE), high performance liquid chromatography (HPLC) and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). The 1Bx13 and lByl6 subunits were collected and purified by SDS-PAGE method. The two subunits were used as antigen to immune BALB/C mice, and monoclonal antibodies (McAb) were prepared by using hybridomas method. Correlation coefficients between monoclonal antibody and quality parameters of wheat were analyzed by indirect ELISA and SPSS software. Applications of the antibodies on predicting of breadmaking quality and characterization of special subunits were studied.1. Separation and identification of HMW-GSAt present, there are many techniques which can be used to detect different HMW-GS. PAGE was the most economical method while the resolution of 2-DE was the highest. Single HMW-GS can be separated and purified by RP-HPLC, but it is very expensive. Otherwise HPCE was much more economical method that can avoid using poisonous chemicals. In this study, five HMW glutenin subunits (1, 13+16, 2+12) of Somiedo can be well separated and identified by all the...