Table II.

Interaction of antibodies with WIBS and fractions or modifications

A. longipes WIBS and chemically modified WIBS were blotted onto nitrocellulose and screened against supernatant from four cloned (AL.C1–AL.C4) and two uncloned (AL.E1 and AL.E2) hybridoma colonies.A. coffeaeformis WIBS was also blotted and screened against the antibodies for cross-reactivity. Detection was by peroxidase secondary anti-mouse antibody with digital imaging of the developed blot. Numbers represent averaged, background-subtracted density values that have been scaled to WIBS.

Isolated/Modified FractionsAL.C1AL.C2AL.C3AL.C4AL.E1AL.E2
%
A. longipesWIBS100100100100100100
A. longipes WIBS size-exclusion fractions
 F1 125101100100104122
 F2 6610015095113111
 F3 5040131634370
Modified A. longipes WIBS
 Desulfated14010010050195260
 -COOH reduction560460833483355720
 Uronic acid site cleavage91601911316858
 Periodate oxidation1-a 1096818771678
 Sodium acetate buffer1-b 77104109177174124
Hydrolyzed A. longipes WIBS
 1 m TFA (80°C), 30 min502073314222
 1 m TFA (80°C), 1 h00180163
 1 m TFA (80°C), 5 h000030
A. coffeaeformisWIBS53907120
  • F1-a Periodate oxidation was carried out in 50 mm sodium acetate buffer, pH 4.5.

  • F1-b Treatment with 50 mm sodium acetate (pH 4.5) served as a control to rule out loss of antigenicity by periodate oxidation as a result of exposure to low pH.