Antibody-based surfaces: rapid characterization using two complementary colorimetric assays.
Finding a general solution for optimizing the grafting of antibody on solid surfaces is difficult due to the variety of material, grafting principles and chemistries or surface formats available (beads, microplates, fibers, etc.). Pre-screening methods able to assess grafting efficiency (GE) and specific activity (SA) are required. In this context, we present here two colorimetric assays that can be used on a wide variety of surface format, chemistry, etc. The first one, ADECA (Amino Density Estimation by Colorimetric Assay) allows a rapid estimation of grafted antibodies and allows calculating the GE. The second one, A(2)HRP (Antibody Anti-HorseRadish Peroxidase) provides a measure of the amount of active antibody, which, combined to ADECA, is used to determine the SA of grafted antibody. Analytical parameters (limit of detection, repeatability, linearity, etc.) of these two colorimetric assays are presented. Using two commercially available microplates, we demonstrated that, when used in parallel, these rapid and sensitive methods are well adapted to pre-screening of antibody grafting performances.
Institut des Biomolécules Max Mousseron (IBMM), Unité Mixte de Recherche 5247, Centre National de la Recherche Scientifique, Université de Montpellier 1, Université de Montpellier 2, place E. Bataillon, CC17006, 34095 Montpellier cedex 5, France. tmoreau@univ-montp2.fr
Thomas
T
Moreau
Clément
C
Faye
Mickael
M
Baqué
Isabelle
I
Desvignes
Gaëlle
G
Coussot
Robert
R
Pascal
Odile
O
Vandenabeele-Trambouze
D000818:Animals; D055910:Antibodies, Immobilized / Q000737:chemistry* / Q000276:immunology; D000918:Antibody Specificity; D003124:Colorimetry / Q000379:methods*; D050091:Dendrimers / Q000737:chemistry; D006735:Horseradish Peroxidase / Q000276:immunology; D011522:Protons; D015203:Reproducibility of Results; D012394:Rosaniline Dyes / Q000737:chemistry; D013499:Surface Properties; D013997:Time Factors
D055910:Antibodies, Immobilized; D050091:Dendrimers; D011522:Protons; D012394:Rosaniline Dyes; D006735:Horseradish Peroxidase; C004692:coomassie Brilliant Blue