ABSTRACT
An efficient and inexpensive laboratory approach for the generation and the purification of polyclonal antibodies to human antigen CD34 was developed. It was shown that cloned refolded and purified from Escherichia coli recombinant extracellular fragment of CD34 antigen retained immunogenic determinants of cell-surface expressed CD34. Immunization of mice with unglycosylated truncated recombinant protein elicit polyclonal antibodies specific for the native human antigen CD34. The antibodies generated are applicable for phenotyping of CD34+ cells using immunocytochemistry and flow cytometry assays.
Subject(s)
Antibodies/isolation & purification , Antigens, CD34/immunology , Animals , Antibodies/immunology , Antigens, CD34/genetics , Blotting, Western , Cell Line, Tumor , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Escherichia coli/genetics , Female , Genetic Vectors , Humans , Immunohistochemistry , Mice , Mice, Inbred BALB C , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/isolation & purificationABSTRACT
A panel of single-chain antibodies (ScFv's--single-chain Fv-antibodies) against recombinant human interferon beta 1b (rhIFN-beta1b) has been obtained from immune and naive combinatorial cDNA libraries of the mouse variable immunoglobulin genes. ScFv's were expressed into Escherichia coli cells. For producers isolated from the immune library a difference in production yield of ScFv's in periplasm and incubation medium as well as their expression stability in passages and storage stability have been demonstrated. After sequencing of target DNA the multiple alignment and structural analysis of ScFv's sequences with different primary structures were carried out and significant difference in both complementarity-determining (CDR) and framework (FR) regions of their variable domains has been shown. For the ScFv's isolated from the immune library, specificity of their binding with native and denatured rhIFN-beta1b in ELISA and Western-blotting as well as their high storage stability have been shown. The affinity constants for each representatives of the ScFv's panel were in the range from 1.96 x 10(-8) to 1.69 x 10(-9) M.
Subject(s)
Antibodies, Monoclonal/immunology , Immunoglobulin Fragments/immunology , Immunoglobulin Light Chains/immunology , Immunoglobulin Variable Region/immunology , Interferon-beta/immunology , Animals , Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/genetics , Antibody Affinity/immunology , Antibody Specificity/immunology , Blotting, Western , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Escherichia coli/genetics , Gene Library , Humans , Immunoblotting , Immunoglobulin Fragments/biosynthesis , Immunoglobulin Fragments/genetics , Immunoglobulin Light Chains/biosynthesis , Immunoglobulin Light Chains/genetics , Immunoglobulin Variable Region/biosynthesis , Immunoglobulin Variable Region/genetics , Interferon beta-1b , MiceABSTRACT
A combinatorial library of single-chain antibodies (ScFv) from mice immunized with human alpha2b interferon (hIFN-alpha2b) was constructed. For obtaining of phage display antibodies the DNA fragments of ScFv were cloned into phagemid vector pCANTAB-5E and rescued from Escherichia coli cells by infection with bacteriophage M13. Bacterial clones synthesizing specific ScFv against hIFN-alpha2b were isolated by several rounds of affinity selection of phage library. After isolation and affinity purification of ScFv-IFN from bacteria cells a high ability to binding of the hIFN-alpha2b has been shown. The sequencing of isolated ScFv DNA and analysis of the data obtained have been carried out. The data of expression stability of obtained E. coli producers such as some features of ScFv expression are also discussed.
