Application of an ELISA-elution Assay to Dissociate Digoxin-antibody Complexes in Immunoaffinity Chromatography

Abstract

In this study, we used a modified enzyme‐linked immunosorbent assay (ELISA)‐elution technique as a screening tool to select specific elution conditions. We examined 12 different elution conditions for the removal of antibodies from a complex on an ELISA plate; 0.2 mol/l glycine‐HCl (pH 2.5), 1.0 mol/l acetic acid (pH 2.5), 25% methanol (pH 2.5) and 3 mol/l NaSCN showed a higher elution efficiency. We conducted affinity chromatography with these four conditions for the purification of anti‐digoxin antibodies from hyperimmune sera with a digoxin‐specific column using ω‐aminoalkyl derivatives of Sepharose 4B, whose elution efficiency was similar to that of ELISA. We also monitored the relative specific activities during elution from the digoxin‐specific column. The optimum, general‐purpose dissociation reagent for this immunoaffinity system was identified as 25% methanol (pH 2.5) with an elution efficiency and relative specific activity of 88.40% and 62.25%, respectively. The high purity of the purified antibodies was demonstrated with sodium dodecyl sulphate‐polyacrylamide gel electrophoresis.

Dissociation of Antigen-Antibody Complexes for some Plant Viruses in DAS-ELISA Tests

“…This could be attributed to the elimination of blocking elements at the binding sites as agreed with the cited information by Hull (2002) or exposing nonsurface antigenic sites in the reaction media. Theses results are in agreement with data obtained by many researchers (Gase, 1990;Kim et al, 1990, Hassan, 1999, Stanker et al, 2008and Xu et al, 2010 and almost disagree with Jana and Ali (1999) who mentioned that a competition between virus and non-viral protein cannot be eluted.…”

“…From each 5 strips used for each virus, a single strip was used to study a single pH value. Values were recorded after absorbance, then all microtiter plates were emptied, washed twice with PBS-Tween and filled (700 µl) with 0.1 M glycine -HCl buffer (Xu et al, 2010) adjusted to pH values between 2.0 and 4.0 with 0.5 intervals. The strips were incubated at room temperature overnight and washed twice with PBS-Tween.…”

Immunoaffinity purification of bluetongue virus group specific antibody using recombinant protein adsorbed to polystyrene wells

“…An erythropoietin (EPO) specific immunoaffinity microplate was used for purification of human erythropoietin (hEPO) from human plasma sample (Mallorquí et al, 2010). An ELISA based elution procedure was used for dissociation of digoxin-antibody complexes in immunoaffinity chromatography (Xu et al, 2010). In order to purify the group-specific antibody for BTV, we used recombinant protein VP7 that carries group specific epitope.…”

“…Purification of antibodies in small scales in microtitre plate using synthetic peptide/recombination antigen has been tried in case of rubella (Pullen et al, 1986) Plasmodium falciparum (Brahimi et al, 1993) foot and mouth disease (Bayry et al, 1999) digoxin (Xu et al, 2010) and erythropoietin from human plasma (Mallorquí et al, 2010). In the present study, a method for efficient purification of group specific polyclonal antibody against BTV was optimized using recombinant VP7 protein adsorbed to polystyrene wells.…”

Development of Indirect Competitive Enzyme-Linked Immunosorbent Assay and Lateral-Flow Immunochromatographic Strip for the Detection of Digoxin in Human Blood

“…Therefore, the conventional dose of DIG sometimes also causes poisoning, and the clinical manifestations of overdosing and underdosing are similar. Consequently, the incidence of poisoning during its clinical application is high. , …”