Use of immunodotting to select the desorption agent for immunochromatography.
In immunochromatography, a technique of increasing use, the sample containing the antigen (Ag) to be purified or determined is introduced into a chromatographic column containing an antibody (Ab) bound to the packing material. The antigen is retained based on antigen-antibody recognition. To reuse the immunocolumn for subsequent assays, the antigen has to be eluted without causing irreversible damage of antibodies. Selection of conditions for performing immunochromatography is usually made by trial and error. This way of working is time consuming and it may ruin the column. In this article, the feasibility of using immunodotting to select the conditions to be employed in one immunochromatographic assay is shown. An immunodotting method is developed to select the best desorption agent for an enzyme-linked immunoaffinity chromatography (ELIAC) assay to determine beta-lactoglobulin (beta-LG). The effect of several factors on the immunodotting performance is studied. The way of performing solvent exchange to treat the antibody with different solutions considered as potential desorption agents to check their effect is shown. Effectiveness of the solution chosen by immunodotting (4 M MgCl(2) in 20 mM Tris, pH 5.9) as desorption agent is demonstrated by immunochromatographic assays. The immunodotting and solvent exchange methods developed should be useful to choose solvents and conditions for any other kind of assay.
Puerta
Angel
A
Institute of Organic Chemistry (CSIC), Madrid, Spain.
Diez-Masa
Jose C
JC
de Frutos
Mercedes
M
D000906:Antibodies / Q000737:chemistry; D000937:Antigen-Antibody Reactions; D002846:Chromatography, Affinity / Q000592:standards*; D004797:Enzyme-Linked Immunosorbent Assay / Q000592:standards*; D005506:Food Contamination / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting / Q000379:methods*; D007782:Lactoglobulins / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification; D012996:Solutions; D012997:Solvents / Q000737:chemistry
D000906:Antibodies; D007782:Lactoglobulins; D012996:Solutions; D012997:Solvents