Recombinant protein detection in crude extracts by flow-injection immunoassay.
An immunofluorescence method that allows detection and quantification of recombinant proteins in complex mixtures (e.g. crude cell extracts and fermentation media) is described. The method is based on a non-denaturing chromatographic analysis which is performed after the addition of a fluorescently labelled antibody specific for the target protein. HPLC analysis through a size-exclusion column allows to separate and quantify the immunocomplex and the excess antibody, which are the only fluorescent species in the mixture. The method was applied to the analysis of recombinant human Fabs expressed in bacteria. Preliminary validation data are reported.
Bartolomeo
Maria Paola
MP
Bracco Imaging SpA, Centro Ricerche Bracco, via Ribes 5, 10010 Colleretto Giacosa (TO), Italy.
Maisano
Federico
F
D000906:Antibodies; D001671:Biochemistry / Q000379:methods; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D045424:Complex Mixtures / Q000737:chemistry; D017022:Flow Injection Analysis; D005455:Fluorescent Antibody Technique; D006801:Humans; D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins / Q000032:analysis; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D000906:Antibodies; D045424:Complex Mixtures; D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins