A novel affinity ligand for polystyrene surface from a phage display random library and its application in anti-HIV-1 ELISA system.
In order to develop an affinity ligand for site-directed immobilization of target proteins on polystyrene (PS) surface, a linear 12-mer peptide phage display random library was screened. Phage clones that specifically bound to PS plate were sequenced after three rounds of biopanning. The obtained DNA sequences revealed that there were several aromatic and basic amino acid residues, which may be critical to binding. One of the selected dodecapeptides, named Lig1 (FKFWLYEHVIRG), was genetically fused to the N/C-terminus of recombinant antigen ENV which could be recognized by specific antibodies against human immunodeficiency virus type 1 (HIV-1), to investigate its performance as an affinity ligand. The ligand-fused ENVs overexpressed in Escherichia coli were compared to the original one in terms of the immobilization characteristics on PS plate in enzyme-linked immunosorbent assay (ELISA). The results indicated that the ligand-fused proteins showed a considerably improved affinity to PS surface, and were preferentially adsorbed on PS plate suffering only scarcely from interference by coexisting protein molecules. Anti-HIV-1 ELISA system, which employed Lig1-ENV (Lig1 fused to ENV N-terminus) as immobilization antigen also exhibited sufficiently high sensitivity and specificity in serodiagnosis tests.
Feng
Bo
B
Biological Engineering Department, Xiangtan University, Xiangtan 411105, Hunan Province, China. fengbo@xtu.edu.cn
Dai
Youzhi
Y
Wang
Lu
L
Tao
Nengguo
N
Huang
Shirong
S
Zeng
Hongyan
H
D000915:Antibody Affinity / Q000276:immunology; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D015686:Gene Products, env / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D015483:HIV Antibodies / Q000032:analysis*; D015497:HIV-1 / Q000276:immunology*; D007164:Immunosorbents / Q000138:chemical synthesis* / Q000378:metabolism; D008024:Ligands; D008954:Models, Biological; D019151:Peptide Library; D011137:Polystyrenes / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity; D013499:Surface Properties
D015686:Gene Products, env; D015483:HIV Antibodies; D007164:Immunosorbents; D008024:Ligands; D019151:Peptide Library; D011137:Polystyrenes; D011993:Recombinant Fusion Proteins