Protein Protocols Protein Protocols

796 Goodarzi, Fotinopoulou, Turner
Table 1
Specificity of Different Lectins
Lectin Abbreviation Specificity
Concanavalin A ConA Mannose α1–3 or mannnose α1–6
Datura stramonium agglutinin DSA Gallactose β1–4 N-acetylglucosamine
Aleuria aurantia agglutinin AAA Fucose α1–6 N-acetylglucosamine
Lens culinaris agglutinin LCA Mannose α1–3 or mannose α1–6
(Fucose α1–6 GlcNAc is also required)
Lotus tetragonolobus agglutinin LTA Fucose α1–2 galactose β1–4
[Fucose α1–3] N-acetylglucosamine
Sambucus nigra agglutinin SNA N-acetylneuraminic acid α2–6 galactose
Maackia amurensis agglutinin MAA N-acetylneuraminic acid α2–3 galactose
Peanut agglutinin PNA Galactose β1–3 N-acetylgalactosamine
on O-linked chains
frequently use large amounts of lectin, which is expensive, requires considerable technical
skill, cannot handle large numbers of specimens, and provides only semiquantitative
results. For a more detailed discussion of these methods the reader should refer to a
previous review (3).
Another approach is to use lectins in the familiar sandwich enzyme-linked
immunosorbent assay (ELISA) technology in multiwell plates. Procedures of this type
have been described for the measurement of particular carbohydrate structures in
α-fetoprotein (10), glycodelin (11), haptoglobin (HP) (12), human chorionic gonadotrophin
(hCG) (13), α1-acid glycoprotein (AGP) (14), fibronectin (15), c-erb-B2 (16),
immunoglobulins (IgG) (17), mucins (18), plasminogen (Pg) activator (19), and transferrin
(TF) (20). In this method, the lectin is used either to capture the molecule of
interest or to identify it. An antibody is used as the other partner in the sandwich and
the degree of binding is measured by the presence of an enzyme label on the identifier.
Both configurations suffer from the disadvantage that the lectin may bind to carbohydrate
determinants on the immunoglobulin used as the antibody. Furthermore, using
the lectin as the capture molecule, the immobilized lectin may bind to glycans of other
glycoproteins in the sample and these will compete with the molecule of interest for the
available binding sites. When immobilized antibody is used as the capture molecule,
care must be taken to ensure that the antibody is not binding to the same determinant as
that reacting with lectin.
The lectin binding assay (LBA) described herein (21) overcomes many of the disadvantages
with the previous lectin immunoassays. It was developed from a previously
reported procedure (22). A purified glycoprotein is absorbed onto the plastic surface of
a well in a microtiter plate. After the unbound protein is removed by washing, uncoated
sites on the plate are blocked using a non-ionic detergent. A lectin labeled with
digoxigenin (DIG) or biotin is added and allowed to interact with the carbohydrate on
the absorbed glycoprotein. Unbound lectin is removed by further washing and the
amount of bound lectin is measured by adding an anti-DIG antibody or streptavidin
conjugated to an enzyme. Streptavidin has a very high affinity for biotin. Following
further washing, the bound enzyme is used to develop a color reaction by the addition