Rapid detection of proteins by enzyme-linked immunofiltration assay after transfer onto nitrocellulose membranes.
Enzyme-linked immunofiltration assay (ELIFA) for labeling transferred proteins is an interesting and powerful technique for the rapid specific detection (15 min) of proteins immobilized on nitrocellulose or nylon membranes (0.20 and 0.45 micron). ELIFA does not require fastidious handling of the membranes. Saturation, specific labeling and washing procedures are achieved by filtration, controlled by a monitoring unit which regulates the flow rate and ensures excellent specificity, repetition and reproducibility. The recycling by closed circuit or by repetitive inversion of the flow direction offers the advantage of reducing the volumes of expensive reagents while simultaneously increasing the sensitivity of the technique. The detection limit is at least as low as 1-5 ng using directly or indirectly enzymatically labelled probes. ELIFA may be extended to the identification of glycoproteins using specific ligands such as lectins or to the immunocapture of an antigen using specific antibodies immobilized on an activated membrane. ELIFA complements fast separation, by e.g., isoelectric focusing, polyacrylamide gel electrophoresis, or sodium dodecyl sulfate-polyacrylamide gel electrophoresis and accelerated electrotransfer to membranes with rapid detection reducing the total time for separation transfer and detection to less than 2 h.
Laboratoire de Parasitologie-Mycologie C.H.R.U., Reims, France.
J M
JM
Pinon
D
D
Puygauthier-Toubas
H
H
Lepan
C
C
Marx
A
A
Bonhomme
J
J
Boulant
R
R
Geers
H
H
Dupont
D000818:Animals; D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan / Q000032:analysis; D001710:Biotin; D003101:Collodion; D005374:Filtration; D006023:Glycoproteins / Q000032:analysis; D006046:Gold; D006801:Humans; D007124:Immunoenzyme Techniques / Q000592:standards; D007202:Indicators and Reagents; D051379:Mice; D011506:Proteins / Q000032:analysis*; D011786:Quality Control; D013194:Staining and Labeling; D014122:Toxoplasma / Q000276:immunology; D014123:Toxoplasmosis / Q000276:immunology
D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D006023:Glycoproteins; D007202:Indicators and Reagents; D011506:Proteins; D001710:Biotin; D006046:Gold; D003101:Collodion