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Production and characterization of scFV Monoclonal antibody against Cry2B and scFv Polyclonal antibody against Cry1Ac through phage display technology

KrishiKosh

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Title Production and characterization of scFV Monoclonal antibody against Cry2B and scFv Polyclonal antibody against Cry1Ac through phage display technology
 
Creator Chidanand.A.Rabinal
 
Contributor Narayana.Moger
 
Subject Plant Biotechnology
 
Description Cry2B and Cry1Ac protein were used to produce single chain fragment variable
(scFv) monoclonal antibody for Cry2B and scFv polyclonal antibody for Cry1Ac. The
Tomlinson I library was amplified and the scFv phages were made displayed on their surface.
They were incubated with immobilized antigen in immunotube for further amplification.
Such four rounds of biopanning process were carried out for selection of scFv polyclonal
antibody against both the proteins. The ELISA test results indicated that the fourth biopan
phage particles had higher binding affinity to the target antigens. In case of Cry2B, forty-five
clones were selected from fourth biopan and the same were subsequently used to produce
monoclones. In case of Cry1Ac, because of their poor affinity with antigen during affinity
selection process only polyclonal antibody was produced. The two clones viz., pscFvCry2B19
and pscFvCry2B43 of Cry2B were selected based on monoclonal phage ELISA and further
confirmed by the cross specificity test with random mutant Cry2B proteins. The selected
clones were sequenced with LMB3 forward and pHEN reverse primer and characterized. The
scFv fragment of pscFvCry2B19 and pscFvCry2B43 is 746 bp and 747 bp long and they
shared homology to each other at DNA and amino acid level, except at C? terminal end.
According to the sequence analysis the clone pscFvCry2B19 consist of 55.09% G+C and
44.91% A+T. Similarly pscFvCry2B43 has 54.89% of G+C and 45.91% of A+T. Both the
clones have similar conserved domains in Ig super family between 47 to 119 positions. The
homology search with BLASTn algorithm indicated 96% identity to Homo sapiens partial
scFv genes. These observations indicated that both the scFv antibodies bind to a single
epitope.
 
Date 2016-11-07T18:25:40Z
2016-11-07T18:25:40Z
2010
 
Type Thesis
 
Identifier http://krishikosh.egranth.ac.in/handle/1/84296
 
Format application/pdf
 
Publisher UAS, Dharwad