Comparative analysis of carbohydrate-binding specificities of two anti-glycogen monoclonal antibodies using ELISA and surface plasmon resonance Sachiko Nakamura-Tsuruta a , Michiko Yasuda a , Toshiyuki Nakamura a , Eri Shinoda b , Takashi Furuyashiki c , Ryo Kakutani c , Hiroki Takata c , Yoji Kato d , Hitoshi Ashida a,b,⇑ a Organization of Advanced Science and Technology, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan b Graduate School of Agricultural Science, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan c Institute of Health Sciences, Ezaki Glico Co., Ltd, 4-6-5, Utajima, Nishiyodogawa-ku, Osaka 555-8502, Japan d School of Human Science and Environment, University of Hyogo, 1-1-12, Shinzaike-honcho, Himeji 670-0092, Japan article info Article history: Received 24 November 2011 Received in revised form 26 December 2011 Accepted 30 December 2011 Available online 8 January 2012 Keywords: Natural source glycogen Enzymatically-synthesized glycogen Monoclonal antibody Surface plasmon resonance ELISA abstract For immunological experiments on glycogens, anti-glycogen antibodies are indispensable to capture, detect, and visualize sugar molecules. An anti-glycogen monoclonal antibody (IV58B6) and newly con- structed antibody (ESG1A9mAb) have a common immunoglobulin type (IgM) and binding ability to glyc- ogens, but overall possess different binding features. Therefore, they may prove useful for the construction of an advanced system of quantitative ELISA based on their molecular structures. For this purpose, detailed information on the carbohydrate-specificities of ESG1A9mAb and IV58B6 is first required, but their fine specificities for various types of glycogens have not been elucidated. To overcome this problem, we performed interaction analysis by ELISA of ESG1A9mAb and IV58B6 toward 15 glucose polymers, that is, 5 enzymatically-synthesized glycogens (ESGs), 6 natural source glycogens (NSGs), 3 enzymatically digested glycogens (EDGs), and soluble starch. To provide a more detailed analysis, we determined the association constants (K a ) of the two antibodies toward these glycogens by surface plas- mon resonance. The results indicated that the carbohydrate-binding properties toward NSGs of ESG1A9- mAb and IV58B6 were similar, but markedly differed for ESGs and EDGs. ESG1A9mAb showed significant affinity for all the ESGs and NSGs tested, whereas IV58B6 had only slight affinity for ESGs, although the affinities were increased when the ESGs were enzymatically digested. This information should be helpful for the design of both in vitro and in vivo immunological assays. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction Glycogen is a major storage polysaccharide of glucose, which is composed of Glca1, 4Glc units with Glca1, 6Glc branches with high molecular weight, from 1000 to 1,000,000 kDa. 1,2 In addition to its role as an energy source in animals and microorganisms, nat- ural source glycogens (NSGs) are known to have immunostimulat- ing activity. 3 Recently, we developed a novel process to obtain structurally uniform glycogens using plant starch as a starting material, and designated them as enzymatically-synthesized glyc- ogens (ESGs). 4 Their molecular weights were controllable in the range from 3000 to 30,000 kDa and they were shown to have var- ious beneficial effects for health promotion, including prolonging of the survival time for tumor-bearing mice, immunostimulating activity in immune cells, and dietary fiber-like activity in mice. 5– 7 Moreover, the safety of ESGs was supported by toxicological stud- ies, 8 and together these findings suggest that ESG can be expected to be a novel functional food factor. However, the underlying mechanisms of these functions and bioavailability, and their metabolism were not fully clarified, and thus, further studies are needed. Quantification of glycogens is generally complicated and involves measuring the glucose content after hydrolyzing in HCl. 9 Anti-glycogen antibodies are a powerful tool for constructing a quantification system for glycogens. An anti-glycogen monoclonal antibody (IV58B6) was previously constructed by Baba et al. 10 using mandibular condylar cartilage as the antigen, and classified as an IgM type. By means of electron microscopic immunocyto- chemical observation and dot blot analysis, IV58B6 was estimated as a glycogen or glycogen-related sugar chain-specific antibody. Although IV58B6 has played an important role in the detection of 0008-6215/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.carres.2011.12.029 Abbreviations: ESG, enzymatically-synthesized glycogens; NSGs, natural source glycogens; EDGs, enzymatically digested glycogens. ⇑ Corresponding author. Tel./fax: +81 78 803 5878. E-mail address: ashida@kobe-u.ac.jp (H. Ashida). Carbohydrate Research 350 (2012) 49–54 Contents lists available at SciVerse ScienceDirect Carbohydrate Research journal homepage: www.elsevier.com/locate/carres