Determination of lectin in root and leaf of rice (Oryza sativa L.) by enzyme-linked immunosorbent assay

Abstract

Lectin was purified from embryos of rice cv RD 7 and RD 25 to the purity of 1,637 and 1 ,632 fold of crude extracts respectively. Production of antibody against lectin was achieved yielding serum titer 16 for cv RD 7, and it was able to cross-react with lectin RD 25 completely by technique of agar gel diffusion. The antibody showed cross-reaction with lectin from 30 cultivars (Oryza sativa L.) 2 wild rice species and WGA (Wheat Germ Agglutinin). The antibody against lectin RD 7 was used in combination with FITC-goat antirabbit immunoglobulin to localize the lectin in root and leaf of rice seedlings on day 4 and day 7 grown under dark condition. Lectin was located on the root cap, root hair tip, and also on epidermal cells. In the leaf, lectin was observed on hydathode and stoma. The indirect ELISA method was developed using lectin RD 7 antibody and goat antirabbit immunoglobulin conjugated with alkaline phosphatase which showed the high sensitivity of 10 ng lectin/ml, and assay range of 10-60 ng/ml , its accuracy was high(92-98 % recovery), with % CV of intra-assay 2-7%and interassay of 2-11%. Lectin can be quantitated in embryo of 28 rice cultivars by ELISA exhibiting the average value of 3218± 1350 nanogram/50 embryos. The quantitative determination of root and leaf lectin in 4-7 day old seedling required modification of ELISA method by albumin precoating which gave the same sensitivity as the first method, but wide assay range of 10-100 ng/ml. The per cent CV of intra-assay and interassay was 4-11% and 2-13% respectively. It was found by this method that seedlings of rice cv RD 7 grown under dark and light condition differed in leaf lectin content. Maximum lectin content in leaf and root were significantly observed on day 4. Comparison of lectin contents in seedlings among 8 rice cultivars, showed the highest value in NMS 4 both in leaf and root (42304 ± 1135 and 92±20 ng/100 plants), and the lowest in KDML 105 (598±151 and 5 ng/100 plants) . Lectin in seedling was significantly decreased both in the leaf and root when grown under 2 and 20 mMNH4Cl. Study on the cross-reaction among 32 rice varieties to antibody against lectin RD 7 , indicated that some varieties contained lectin identical to RD 7, and the others are partially identical, however some varieties contained lectin more than one form . Lectin from all varieties of rice tested and wheat (WGA) shared a common antigenic determinant to lectin RD 7. The quantity of' lectin was comparatively high in embryo, and gradually decreased in developmental seedling. In flowering stage lectin was merely detected in adventitious root tip.