Generation of polyclonal antibodies against recombinant human glucocerebrosidase produced in Escherichia coli.
Deficiency of the lysosomal glucocerebrosidase (GCR) enzyme results in Gaucher's disease, the most common inherited storage disorder. Treatment consists of enzyme replacement therapy by the administration of recombinant GCR produced in Chinese hamster ovary cells. The production of anti-GCR antibodies has already been described with placenta-derived human GCR that requires successive chromatographic procedures. Here, we report a practical and efficient method to obtain anti-GCR polyclonal antibodies against recombinant GCR produced in Escherichia coli and further purified by a single step through nickel affinity chromatography. The purified GCR was used to immunize BALB/c mice and the induction of anti-GCR antibodies was evaluated by enzyme-linked immunosorbent assay. The specificity of the antiserum was also evaluated by western blot analysis against recombinant GCR produced by COS-7 cells or against endogenous GCR of human cell lines. GCR was strongly recognized by the produced antibodies, either as cell-associated or as secreted forms. The detected molecular masses of 59-66 kDa are in accordance to the expected size for glycosylated GCR. The GCR produced in E. coli would facilitate the production of polyclonal (shown here) and monoclonal antibodies and their use in the characterization of new biosimilar recombinant GCRs coming in the near future.
Novo
Juliana Branco
JB
Centro de Biotecnologia, Instituto Butantan, Av. Vital Brasil, 1500, São Paulo, SP, 05503-900, Brazil. jubranco@butantan.gov.br
Oliveira
Maria Leonor Sarno
ML
Magalhães
Geraldo Santana
GS
Morganti
Ligia
L
Raw
Isaías
I
Ho
Paulo Lee
PL
D000818:Animals; D000917:Antibody Formation; D001483:Base Sequence; D015153:Blotting, Western; D019556:COS Cells; D002522:Chlorocebus aethiops; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics*; D005962:Glucosylceramidase / Q000276:immunology*; D006367:HeLa Cells; D006801:Humans; D007106:Immune Sera; D051379:Mice; D008807:Mice, Inbred BALB C; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
D017931:DNA Primers; D007106:Immune Sera; D011994:Recombinant Proteins; D005962:Glucosylceramidase