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POTENTIAL$USE$OF$LECTINS$

!
2.6$Lecitin$meassures$

! Ligands! of! sera! from! the! experimental! groups! were! analysed! by! their!
reactivity!with!15!lectins/lectin!derivatives!in!comparison!to!control!sera.!

! To! quantify! specific! carbohydrate! structures! carried! on! serum!
glycoconjugates,!we!coated!ELISA!plate!wells!with!serum!samples;!100!µl!of!serum!
diluted!with!PBS!buffer/well!were!incubated!at!4!°C!overnight.!After!washing!with!
PBS/Tween!20,!the!biotinylated6lectin!solution!was!added!and!allowed!to!bind!for!
1! hour! at! 37! °C.! The! excess! lectin! was! removed! by! 3! washes! with! PBS/Tween! 20.!!
Then,! an! ExtrAvidin®6Peroxidase6complex! containing! solution! was! added! and!
allows! binding! for! 30! min! before! removing! the! excess! complex.! The! ExtrAvidin®6
Perixodase! bound,! and! therefore! the! lectin! bound,! was! quantified! by! measuring!
the! peroxidase! reaction! using! ABTS! as! substrate.! Other! details! can! be! found!
elsewhere!(16).!

! We!optimised!the!experimental!conditions!to!avoid!non6specific!fixation!of!
the!lectins!and!to!use!convenient!concentrations!of!both!lectins!and!sera.!

! Comparison! between! groups! was! performed! using! analysis! of! variance!
(ANOVA).! The! Scheffé! and! Fischer! testy! were! employed! for! comparison! among!
groups! using! Stat! View! SE+GraphicsTM! 1.03! software! for! Macintosh;! P! values! =!
0.01!were!considered!highly!significant.!Correlation!analysis!was!realized!using!the!
same!software.!All!results!were!expressed!as!mean!±!S.E.M.!

3.)RESULTS)))
The!15!lectins!whose!abbreviated!and!detailed!names!are!indicated!in!Table!

1!were!assayed.!For!each!lectin!we!used!3!different!concentrations!and!determined!
their! fixation! on! a! serum! dilution! series! (from! 1/50! to! 1/102400! in! PBS).! The!
results!for!Lycopersicon+esculentum!lectin!(LEL)!and!for!Lotus+tetragonolobus!lectin!
(LTL)! are! shown! in! figure! 1! and! figure! 2,! respectively.! Similar! results! were! found!
for!the!other!assayed!lectins!(data!not!shown).!

The! optimal! experimental! conditions! for! all! the! lectins! assayed! are!
summarised!in!Table!I.!

In! addition,! we! found! that! the! optimal! concentration! for! the! ExtrAvidin?6
Peroxidase! complex! is! 2! µg/ml! diluted! in! PBS.! Thus,! non6specific! binding! of! the!
complex! to! the! ELISA! plate! was! minimal,! with! no! decrease! in! fixation! capacity! to!
the! biotinylated! lectin.! After! final! adaptation! of! the! method,! we! measured! the!
fixation!capacity!of!the!biotinylated!lectins!to!ligands!from!pathological!and!normal!
sera.!The!results!are!shown!in!Table!II.!

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123$

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