Comparison of oriented and random antibody immobilization in immunoaffinity chromatography of cytokinins

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Abstract

Immunosorbents for the plant hormones cytokinins prepared by random antibody immobilization (to Affi-Gel 10) and by oriented approach via oxidized carbohydrate moieties on the Fc region (to Affi-Gel Hz or hydrazide derivative of Perloza MT 200) have been compared. Both approaches yielded immunosorbents with high dynamic capacity (ca. 5–10 nmol ml gel−1). Oriented antibody immobilization did not exhibit crucial effects in the case of low-molecular-mass cytokinins. Antibodies immobilized via a spacer to Affi-Gel 10 have probably enough conformational freedom to enable good accessibility to cytokinins. The sorbents were used in analysis of endogenous cytokinins in maize seeds. In phosphatase treated samples trans-zeatin and its riboside were predominant.

Introduction

Cytokinins are plant hormones which exhibit various crucial biological activities, as e.g., promotion of cell division, induction of bud formation and development, reduction of apical dominance and delay of senescence. Natural cytokinins are derivatives of adenine substituted at the N6 position either with isoprenoid or benzyl moiety which may be further modified. Cytokinins exist as free bases, ribosides, nucleotides, glyco-, acetyl- and alanyl-conjugates (e.g., Ref. [1]and references therein).

In plants, cytokinins occur in very low amounts, usually at levels below 30 pmol per g of fresh weight (FW) (e.g., Ref. [2]), together with other much more abundant and often structurally similar substances. Commonly used analytical procedures for cytokinin analysis are based on immunoassays of high-performance liquid chromatography (HPLC) fractions of prepurified plant extracts. However, when a large number of background contaminants is present at high concentrations, the HPLC separation methods, which are effective in fractionation of standards, need not be reliable in the case of plant samples [3]. This problem may be overcome by use of immunoaffinity chromatography (IAC) prior to HPLC fractionation. Highly purified cytokinin preparations containing only traces of other UV-absorbing material could be obtained in this way.

Until now immunosorbents for cytokinin analysis have been prepared by random antibody (Ab) attachment using primary amino or carboxyl groups. A number of chromatographic matrices have been used for immobilization of cytokinin antibodies, especially cyanogen bromide (CNBr)-activated cellulose 4, 5, 6, 7, 8. Davis et al. [3]tested CNBr–Sepharose, Fast-Flow Sepharose CL-4B, glycophase silica and Affi-Gel 10. Affi-Gel 10 was used in some other studies (e.g., Ref. [9]).

Antibody binding to solid supports through the ε-amino group of the amino acids results in multiple antibody orientation on the gel surface which may affect accessibility of the antigen binding site. Using binding of antibodies via their carbohydrate moieties located on the Fc region (heavy chain) remote from the antigen binding site Hoffman and O'Shannessy [10]achieved oriented antibody immobilization with excellent steric accessibility of antigen binding site. Oriented immobilization proved to be superior in a number of cases 11, 12.

Cytokinin immunosorbents prepared by random and oriented immobilization techniques using two different carriers (Affi-Gel and cellulose) are compared in this paper.

Section snippets

Chemicals

Unlabelled cytokinins were obtained from Sigma–Aldrich. Tritium-labelled cytokinins, prepared by alkylation of [2-3H]adenosine with appropriate alkylbromides, were synthesized by Dr. Jan Hanuš, Isotope Laboratory, Institute of Experimental Botany, Prague, Czech Republic. Affi-Gel 10 and Affi-Gel Hz were purchased from Bio-Rad Labs. (Richmond, CA, USA). Bead cellulose Perloza MT 200 (water-swollen, particle size 80–100 μm, bed volume 13.3 ml swollen sorbent g dry matter−1, water content 90%) was

Antibody characterization

Monoclonal antibodies 16B7 raised against isopentenyladenosine (conjugated with bovine serum albumin) were purified by repetitive precipitation with ammonium sulphate as described in Section 2.2. As the antibody titre in ascites was high (ca. 6 mg ml−1), the IgG fraction was directly used for immobilization. The antibody cross-reactivity was characterized by competitive radioimmunoassay (RIA) by determination of the concentration of cross-reactant which inhibited the binding of the primary

Discussion

When IAC is used as purification step before final separation and quantitation relatively broad antibody specificity towards the group of closely related substances (e.g., cytokinins) is advantageous. The choice of antibodies does not seem to be restricted to polyclonals, as various monoclonals exhibit considerably differing cross-reactivities [17]. The broad cross-reactivity is probably inherent characteristic of the whole antibody population and does not reflect the effect of small

Acknowledgements

This work was supported by grants of the Grant Agency of Czech Republic No. 204/96/1424 and No. 206/96/K188. The authors are grateful to Dr. Jan Hanuš for providing of radioactive cytokinins and to Dr. Richard Vytášek for supplying of monoclonal antibodies.

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