A quantitative ELISA for monitoring the secretion of ZZ-fusion proteins using SpA domain as immunodetection reporter system.
A sandwich-type enzyme-linked immunosorbent assay (ELISA) was established for monitoring the secretion of ZZ-fusion proteins. Two antibodies, a monoclonal mouse anti-human proinsulin and a rabbit anti-bovine IgG (strongly binding to the ZZ-domain), were used to quantify the secretion of recombinant human ZZ-proinsulin to the growth medium of Escherichia coli cultures. The method here reported conjugates the advantages of sandwich-type ELISA assays, namely, high sensitivity, specificity, and throughput, with the possibility of quantifying small protein molecules (e.g., peptides). A further advantage of gene fusion techniques integrating both downstream processing and product detection and quantitation is highlighted. The method is capable of detecting levels of 0.05 ng of ZZ-proinsulin.
Mergulhão
F J
FJ
Centro de Engenharia Biológica e Química, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisbon, Portugal.
Monteiro
G A
GA
Cabral
J M
JM
Taipa
M A
MA
D000818:Animals; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D017930:Genes, Reporter; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000235:genetics
D011993:Recombinant Fusion Proteins