Construction of cell surface-engineered yeasts displaying antigen to detect antibodies by immunofluorescence and yeast-ELISA.
In order to detect monoclonal antibodies (MAbs) from insufficient and unavailable human proteins, yeast cells were engineered to display human antigens on their surface and consequently endowed with the ability to specifically bind antibodies. Thus, a fusion gene for the expression of the human proteasome subunit alpha 6 (hPSA6) and human profilin I (hProI) were assembled, respectively, with a His.tag marker at the C-terminal and displayed on yeast surface. With anti-His.tag MAb as the primary antibody and the fluorescein isothiocyanate-conjugated goat anti-mouse Immunoglobulin G as the second antibody, the surface display of hPSA6 and hProI were verified by immunofluorescence labeling. The antigen-displayed yeast particles were used for MAbs detection from ascites through both immunofluorescence and yeast-enzyme-linked immunosorbent assay (ELISA) methods. The results were verified by Western blotting and indirect ELISA. By improving the sensitivity, the novel MAbs detection can be applied in the generation and screening of positive hybridoma. It is suggested that by combining the DNA immunization, the present study can evolve into a quick and protein-free way of MAbs production for insufficient and unavailable antigen.
School of Bioscience and Bioengineering, South China University of Technology, Panyu, Guangzhou 510006, China.
Yu Qian
YQ
Tang
Shuang Yan
SY
Han
Hong
H
Zheng
Lin
L
Wu
Mitsuyoshi
M
Ueda
Xiao Ning
XN
Wang
Ying
Y
Lin
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000941:Antigens / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods*; D019084:Fluorescent Antibody Technique, Indirect / Q000379:methods; D015870:Gene Expression; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D051379:Mice; D051304:Profilins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics / Q000276:immunology* / Q000378:metabolism
D000906:Antibodies; D000941:Antigens; C495738:PFN1 protein, human; D051304:Profilins; D011993:Recombinant Fusion Proteins; C465641:PSMA6 protein, human; D046988:Proteasome Endopeptidase Complex