Takeo M, Taguchi S, Odagaki S, Baranwal P, Kimura K, Negoro S. Simple ELISA using Polyclonal Antibody and Lectin for Determination of Soluble Chitosan - Like Polysaccharide in Bacterial Culture. Int J Bioanal Methods Bioequival Stud. 4(1), 76-81. 76 OPEN ACCESS http://scidoc.org/IJBMBS.php International Journal of BioAnalytical Methods & BioEquivalence Studies (IJBMBS) ISSN 2470-4490 Takeo M 1* , Taguchi S 1 , Odagaki S 1 , Baranwal P 1 , Kimura K 2 , Negoro S 1 1 Department of Applied Chemistry, Graduate School of Engineering, University of Hyogo, Shosha, Himeji, Hyogo, Japan. 2 Hyogo Analysis Center Co., Ltd., Seimondori, Hirohata, Himeji, Hyogo, Japan. Introduction Several microbial polysaccharides such as xanthan, levan, and curdlan have been practically produced in industrial scales and used for the production of foods, medicines, and cosmetics [1- 3]. To reduce the production costs, researchers have made their best efforts to improve the productivity, which has been evaluated as the wet weight or the dry weight of the target polysaccharide produced per liter after the recovery as solid from the culture supernatants [2, 3]. However, the recovered materials may still contain large amounts of impurities. In addition, their biological and physiological activities do not always respond to the weight measured. Therefore, it is very useful if the major structures of polysaccharides or structures essential for their activities can be directly and easily evaluated in culture supernatant or solution. We are studying a chitosan-like polysaccharide (CP) with the aver- age molecular weight of >1.66 MDa produced from acetate by Citrobacter strains, which has strong locculating activity for sus- pended solids in water, and thus, can be used as a locculant [4-6]. This biopolymer can be recovered as solid by ethanol precipita- tion and re-solubilized with acidic solutions for the use. Analyses of the dry weight and the sugar content of the recovered solid may provide good information on the amount of the CP in the original culture, but these analyses are not speciic to the target polysaccharide as described above. In addition, they require long analytical time, laborious operational steps, and considerable amounts of the culture. Therefore, in the production of other microbial polysaccharides, more rapid and selective assays based on speciic protein binding have been employed to quantify them [7-11]. Enzyme-linked immunosorbent assay (ELISA) is one of such methods [12, 13] and can detect a biomolecule of interest sensitively even in the presence of impurities, because it is based on speciic antigen-antibody reaction. ELISA is well automated with the use of 96-well microtiter plates, a plate washer, and a plate reader, and thus, it has been used in many laboratories and facilities as a conventional analytical method [12]. Only the bottle neck to use ELISA is preparation of speciic antibodies, which is a laborious work and very costly. Instead of antibody, lectins, which are glycan-speciic binding proteins without enzyme activi- ty [14], have also been used in similar assays [called enzyme-linked lectin or lectin sorbent assay (ELLA)] for the detection of poly- Abstract We have developed a simple enzyme-linked immunosorbent assay (ELISA) for the direct determination of chitosan-like polysaccharide (CP) produced in the cultures of Citrobacter strains. CP in sample solutions was reacted with anti-CP rab- bit polyclonal antibody immobilized in a 96-well microtiter plate and sandwiched with horseradish peroxidase-conjugated lectin, followed by color development by the enzyme activities. Using standard chitosan solutions, a good standard curve (R 2 =0.977) was established between 0 and 250 μg l -1 with the average coeficient of variation of 7.0%, and CP in the diluted culture supernatants could be directly determined. This method can apply for the direct determination of other polysac- charides in solution by selecting an inexpensive custom-made polyclonal antibody and an appropriate lectin, both of which may be commercially available. Keywords: ELISA; Chitosan; Biopolymer; Polysaccharides; Lectin; Citrobacter. *Corresponding Author: Masahiro Takeo, Department of Applied Chemistry, Graduate School of Engineering, University of Hyogo, 2167 Shosha, Himeji, Hyogo 671-2280, Japan. Tel/Fax: 81 (0) 79 267 4893 E-mail: takeo@eng.u-hyogo.ac.jp Received: November 07, 2017 Accepted: December 05, 2017 Published: December 07, 2017 Citation: Takeo M, Taguchi S, Odagaki S, Baranwal P, Kimura K, Negoro S. Simple ELISA using Polyclonal Antibody and Lectin for Determination of Soluble Chitosan - Like Polysac- charide in Bacterial Culture. Int J Bioanal Methods Bioequival Stud. 4(1), 76-81. doi: http://dx.doi.org/10.19070/2470-4490-170009 Copyright: Takeo M © 2017. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited. Simple ELISA using Polyclonal Antibody and Lectin for Determination of Soluble Chitosan - Like Polysaccharide in Bacterial Culture Research Article