A direct antigen-binding assay for detection of antibodies against native epitopes using alkaline phosphatase-tagged proteins.
We describe a simple and efficient method to detect antibodies against native epitopes following immunization with denatured proteins and peptides. With this method, soluble antigens genetically fused with placental alkaline phosphatase (AP) are used as probes to detect antibodies immobilized on nitrocellulose membranes. The AP-tagged proteins can be produced in sufficient amounts using transient transfection of eukaryotic cells with an appropriate cDNA fragment in a commercial AP-tag vector. The intrinsic thermo-stable phosphatase activity of a tagged protein obviates the need for its purification. To evaluate the method, three recently identified proteins of the FcR family, FCRLA, FCRL1, and FCRL4, were fused with AP and tested in a reaction with various polyclonal and monoclonal antibodies raised by immunization with bacterially produced antigens and peptide conjugates. All the three probes demonstrated high specificity in analysis of immune sera and hybridoma supernatants. Sensitivity of the assay varied depending on antibody tested and, in some cases, was in the subnanogram range. The results obtained show that AP-tagged proteins are useful tools for discrimination of antibodies against native epitopes when production of antigen in its native conformation is laborious and expensive.
Baranov
Konstantin
K
Laboratory of Immunogenetics, Institute of Cytology and Genetics, Siberian Division of the Russian Academy of Sciences, Lavrentyev ave. 10, Novosibirsk 630090, Russia.
Volkova
Olga
O
Chikaev
Nikolai
N
Mechetina
Ludmila
L
Laktionov
Pavel
P
Najakshin
Alexander
A
Taranin
Alexander
A
D000469:Alkaline Phosphatase / Q000235:genetics / Q000276:immunology*; D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D015153:Blotting, Western; D002478:Cells, Cultured; D000939:Epitopes / Q000276:immunology*; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D011961:Receptors, Fc / Q000235:genetics / Q000276:immunology*; D011971:Receptors, Immunologic / Q000235:genetics / Q000276:immunology*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology
D000911:Antibodies, Monoclonal; D000939:Epitopes; C423544:FCRL4 protein, human; C452186:FCRLA protein, human; D011961:Receptors, Fc; D011971:Receptors, Immunologic; D011994:Recombinant Proteins; D000469:Alkaline Phosphatase